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Home , Blastomere, Fertilisation, Human fertilization, Human reproduction, Wojciech Giertych

THE BEFORE IMPLANTATION SCIENTIFIC ASPECTS AND BIOETHICAL CONSIDERATIONS

PROCEEDINGS OF THE TWELFTH ASSEMBLY OF THE PONTIFICAL ACADEMY FOR LIFE

Vatican City, 27 February -1 March 2006

Edited by :

ELIO SGRECCIA

JEAN LAFFITTE

LIBRERIA EDITRICE VATICANA 2007

Presentation H.E. Msgr. ELIO SGRECCIA, Msgr. JEAN LAFFITTE

Discourse of the Holy Father BENEDETTO XVI

Final Comuniqué

CONTRIBUTIONS OF THE TASK FORCE

- H.E. R. Card. J. LOZANO BARRAGÁN, The culture of against the culture of life in the teaching of Evangelium Vitae

- Prof. M.ZERNICKA-GOETZ, Cells of the early mouse embryo follow biased and yet flexible development

- Prof. R. COLOMBO, The process of fertilization and its steges. From parental to a developing one- embryo

- Prof. G. SICA, The development of pre-implantation embryo

- Prof. G. SICA, The embrio-maternal dialogue and preparation for implantation

- Prof. C. BELLIENI, Pre-implantation diagnosis, prenatal diagnosis

- Prof. K. FITZGERALD, PGD: bio-medical insights and ethical considerations

- Prof. M.O. RETHORE', Prenatal and pre-implantation diagnosis from the parents' viewpoint

1 - H.E. Msgr. W.J. EJIK, I The criteria of overall individuality and the bio-anthropological status of the embryo before implantation

- Prof. M. PANGALLO, The philosophy of Saint Thomas on the human embryo

- Prof. P. IDE, Is the human embryo a person? Status questionis and determination

STATEMENTS IN THE ROUND TABLE ""Is the Embryo a Person""

- Prof. A. GIL LOPES, The pre-implantation embryo between and philosophy: the individual being

- Msgr. I. CARRASCO DE PAULA, The embryo before implantation: between nature and person

- Prof. R. SPAEMANN, When does the human being to be a person?

- Dr. J.-M. LE MENE', Why is it a duty to protect by law the pre-implantation embryo?

- Rev. P. WOJCIECH GIERTYCH, Begotten, not made

- Prof. P. SERGEJ FILIMONOV, Si può considerare l'embrione come persona?

2 ELIO SGRECCIA, JEAN LAFFITTE

PRESENTATION

In successive waves in the world of scientific research, the field ofadvanced medicine and in ethical, political and juridical debate, the identityand the status of the human embryo has been a renewed subject for discussion.In recent times the periods of greatest vivacity in this sense have beenthree in number. The first wave took place in the 1970s, at a time when inEurope the forceful campaigns for the legalization of , campaigns thatwere ideological in character and funded by internationally known pressuregroups, were being developed. These campaigns, underpinned as they wereby the so- called “ sexual revolution ”, secured permissive laws in nearly all ofthe countries of Europe, with the exception of Ireland, Malta and SanMarino. Of those States where abortion was legalized only Poland went backto establishing a prohibition on abortion, and it did this by repealing theabortionist law that had been passed (by the Sentence of the ConstitutionalCourt of 29 May 1997). At that time a copious literature in favour of the legalization of abortioncame into being. First of all an attempt was made to emphasize the principleof the autonomy of the (the right to choose, pro- choice). As a result,the full value of the foetus came to be acknowledged beginning with its acceptanceby the mother. This acceptance, therefore, was seen as the real constitutiverelationship of the new human individual. In the view of others, on theother hand, the human value of the embryo was to be recognized only beginningwith the acquisition by the foetus of a figure (a human physiognomy).The ratio philosophica (the autonomy of women) and psychological sensibilitywere associated to deny the full human dignity of the embryo and itsright to life from the onset of fertilization. A second wave took place with the use of artificial fertilization, starting inthe 1980s, and in particular with the publication of the Warnock Report inthe United Kingdom (1984). This was the time of the fifteenth day, thefamous claimed boundary between the so-called pre-embryo and the embryo,which was said to correspond to the pre-implantation period of the developmentof the embryo. Prior to implantation, in fact, the embryo was said“ not yet to exist ” whereas after that time it was said to posses a biologicallywell-defined itinerary. Here various theories were brought into play such asthose on the uncertainty of implantation, on the possibility of twinning(within the first fifteen days of development), and on the necessary presenceof the first elaboration of nervous as an announcement of the possibilityof thinking in a human way. During those years we were often obliged to discuss and rebut the argumentsof the fifteenth day, and the Warnock Report itself confessed that thedevelopment of the embryo, beginning with fertilization, is constant and thatthe date of two weeks of development was a sort of conventional threshold,the outcome of a decision that was needed to end the concerns of thoseengaging in experiments. Recent years have witnessed the “ third wave ”, whose principal axis continuesto revolve around the event of fertilization and the first days of thedevelopment of the embryo within the mother’s body or in a laboratory.First of all, there has been the new fact of the agamic embryo, that is tosay an embryo that is not the outcome of an encounter between two gametesbut which derives from the transfer of the nucleus of a cells into aovule which has had its nucleus removed: in other words, from a procedureof cloning. Is this a real human being? Then there was the discovery of the “ stem cells ” that are present in thehuman organism, a real resource for regenerative medicine, which has openedup a new page in the history of medicine. It was specifically this discoverythat led some advanced researchers to think that the use of stem cells takenfrom could provide “ more effective ” results. Thus was opened upthe front of the fight between some researchers who were the exponentsof the use of somatic stem cells from an organism – stem cells whichhad been shown, according to the first promising experiences in this field, tobe able to regenerate tissues in a sick organism – and other researchers whowere the exponents of a 3 hypothetical use of stem cells taken from embryosfertilized in vitro or cloned or frozen. Unfortunately, the extraction of embryocells from the internal cell mass inevitably involves (given present-day technicalpossibilities) the dissection of the embryo (that is to say its elimination) atthe stage. In this hypothesis, people came to dream of, and to popularize, a final victoryover grave diseases such as Parkinson’s, Alzheimer’s, diabetes and verymany others for tens of millions of infirm people. This was a flight ofthe imagination that was located on the borders of “ science fiction ”, withoutscientific grounding and even sufficient experimentation on animals. There are those who ask for funds for the first line of research and thosewho seek them for the second; there are those who posit research to produceembryo stem cells without producing an embryo with various genetic contrivancesand there are those who accuse the Church, which constantly refersto the illicit character of eliminating an embryo by invoking its dignity as anindividual human being, of obscurantism and of a sadistic approach to unfortunatesick people who cannot be healed without the elimination (somethingthat is truly sadistic and useless) of embryos that have been cloned or fertilizedin vitro so as to then be broken up and used as a form of miraculousmedicine or panacea. This pseudo-science took on an authentic anti-scientific and very roughmadness and recently it has also been connected with fraud. In order to justify the whole of this rush to the miraculous embryo whichwas to be exploited, cloned, patented and commercialized with internationaljoint ventures, it was necessary to proclaim that the embryo is nothing elsebut a bunch of cells, forsaking calling it a pre-embryo as well. The approachto the beginning of life is thus changed by abandoning the perspective offinalism according to which the beginning should be assessed by taking intoaccount its natural and autonomous outcome, and ends up by judging it solelyon the basis of its present quantity or perhaps on what it can produce to theadvantage of those who exploit it, thereby reducing it to a commodity andending up by eliminating it. Another initiative, that of the day after pill and the even more deadlyRU486, has followed this line: the first is interceptive (it impedes the implantationof a possible embryo in the case of a fertile sexual act); the second, onthe other hand, is able to extirpate the embryo after implantation as well andfor up to more than forty days after fertilization. Those who are in favour of the privatisation of abortion – with consequentsavings for the treasury of a “ socialised ” state which for some time has legalizedsurgical abortion and made it free – have swelled the ranks of those whoaffirm that the embryo is something and not yet someone. Thus, they say, chemicalabortion is not a crime and can be dealt with as a private matter. Lastly, but the story does not finish here, the neologisms ootide and prezygotehave been recently imported from the United States of America todefine (indeed to disqualify) the embryo at the beginning of the process offertilization when the has penetrated the pellucid membranebut has not yet brought about the complete remixing and reordering of itsown genetic material with the genetic material of the . And this, it is said, should allow the freezing of the ootide – the fertilizedovule that is said to be not yet an embryo – with a view to its subsequenttransfer into the , with the idea proposed that in this way one does notdestroy an embryo, a real and authentic , but only a pre-embryo, somethingthat is not yet someone. One may leave to one side references to other forms of research on thefrontier of the first stages of fertilization, such as, for example, ,which is obtained without the use of male gametes and which is said togive rise to the so-called parthenote…And what would this be? Or to put thequestion better: who would it be? The need is felt to generate clarity, first and foremost in the domain ofscience but also in the domain of ethics. This is the impelling reason why thePontifical Academy for Life organized the Congress with the title: “TheHuman Embryo Before Implantation. Scientific Aspects and Bioethical Considerations”.

4 The task was to know whether a human being exists or does not existduring the stage before implantation, with its human qualifications and withan anthropologically real identity which is to be identified with that individualitywhich, structured and animated by a spiritual vitality, is already in possessionof full human dignity – that dignity peculiar to a being that is notsimply a biological product, nor merely the work of a and a , butwhich refers to the Creator, to His dignity, to His , and to His protection,even before the incumbent protection of the parents and the law. The programme of this Congress envisaged, after the opening sessionwhen the President of the Pontifical Academy for Life and the President ofthe Pontifical Council for Health Pastoral Care, His Eminence Cardinal JavierLozano Barragán, both spoke, two scientific sessions devoted to a descriptionof the processes of fertilization and the very first stages of the development ofthe embryo before implantation. During this part of the Congress specialistsin biology and genetics from various countries (M. Zernicka-Goetz from theUnited Kingdom; R. Colombo, G. Sica and C. Bellieni from Italy; K. FitzGeraldfrom the United States of America; and M.O. Rethoré from France) allgave papers. The third session presented the anthropological contents of the subject.A reflection on the criteria of the individuality of an organism and on the bioanthropologicalstatus of the embryo (W.J. Eijk, Holland) was followed byreference to the classical and medieval theories on the human embryo(M. Pangallo, Italy) and by an exposition of the theories on the humanembryo as “ not yet a man ” (P. Ide, France). The fourth session dealt with concluding questions of an anthropological-8 Presentationethical character. The following took part in the Round-Table on: “ Is theEmbryo a Person? ”: the Brazilian scientist, A. Gil Lopes; the famous thinkerin the field of moral philosophy, R. Spaemann (Germany); the moral theologianand director of the Institute of Bioethics of the Catholic University of theSacred Heart of Rome, I. Carrasco De Paula; and J.M. Le Méné, a jurist andmagistrate, and the director of the Lejeune Foundation (Paris). During the Audience granted to the Academicians and those taking partin the Congress, the Holy Father Benedict XVI confirmed with his Magisteriumthat: “ The love of God does not make a distinction between thenewly conceived embryo in the womb of its mother, or the , or the youngperson, or the adult man or the elderly person. It does not make a distinctionbecause in each one of them He sees the impress of His own image and likeness(Jn 1, 26). He makes no distinction because in them all He sees reflectedthe face of His only Begotten Son, he chose us in him before the foundation ofthe world…He destined us…to be his sons…according to the purpose of hiswill ” (Eph 1, 4-6).

5 BENEDICT XVI

DISCOURSE TO THE PARTICIPANTS OF ITS 12th GENERAL ASSEMBLY OF THE PONTIFICAL ACADEMY FOR LIFE Clementine Hall 27 February 2006

Venerable Brothers in the Episcopate and in the Priesthood, Distinguished Ladies and Gentlemen,

I address a respectful and cordial greeting to everyone on the occasion ofthe General Assembly of the Pontifical Academy for Life and the InternationalCongress on: “ The Human Embryo Before Implantation ”, which hasjust begun. I greet in particular Cardinal Javier Lozano Barragán, President of thePontifical Council for Health Pastoral Care, as well as Bishop Elio Sgreccia,President of the Pontifical Academy for Life, whom I thank for the kindwords with which he has presented clearly the special interest of the themestreated on this occasion, and I greet Cardinal-elect Carlo Caffarra, a longstanding friend. Indeed, the study topic chosen for your Assembly: “ The Human EmbryoBefore Implantation ”, that is, in the very first days subsequent to conception,is an extremely important issue today, both because of the obvious repercussionson philosophical-anthropological and ethical thought, and also becauseof the prospects applicable in the context of the biomedical and juridical sciences. It is certainly a fascinating topic, however difficult and demanding it maybe, given the delicate nature of the subject under examination and the complexityof the epistemological problems that concern the relationship betweenthe revelation of facts at the level of the experimental sciences and the consequent,necessary anthropological reflection on values. As it is easy to see, neitherSacred Scripture nor the oldest Christian Tradition can contain anyexplicit treatment of your theme. St. Luke, nevertheless, testifies to the active,though hidden, presence of the two . He recounts the meeting of the Mother of Jesus, who had conceived himin her virginal womb only a few days earlier, with the mother of John theBaptist, who was already in the sixth month of her : “When Elizabethheard Mary’s greeting, the baby leapt in her womb ” (Lk 1, 41). St. Ambrose comments: Elizabeth “ perceived the arrival of Mary, he(John) perceived the arrival of the Lord the woman, the arrival of theWoman, the child, the arrival of the Child ” (Comm. in Luc. 2, 19, 22-26). Even in the absence of explicit teaching on the very first days of life of theunborn child, it is possible to find valuable information in Sacred Scripturethat elicits sentiments of admiration and respect for the newly conceivedhuman being, especially in those who, like you, are proposing to study themystery of human procreation. The sacred books, in fact, set out to show God’s love for every humanbeing even before he has been formed in his mother’s womb. “ Before I formed you in the womb I knew you, and before you were bornI consecrated you ” (Jer 1, 5), God said to the Prophet Jeremiah. And thePsalmist recognizes with gratitude: “You did form my inward parts, you didknit me together in my mother’s womb. I praise you, for you are fearfuland wonderful. Wonderful are your works! You know me right well ”(Ps 139[138], 13-14). These words acquire their full, rich meaning when onethinks that God intervenes directly in the creation of the soul of every newhuman being. God’s love does not differentiate between the newly conceived stillin his or her mother’s womb and the child or young person, or the adult andthe elderly person. God does not distinguish between them because he seesan impression of his own image and likeness (Gn 1, 26) in each one. Hemakes no 6 distinctions because he perceives in all of them a reflection of theface of his Only-begotten Son, whom “ he chose…before the foundation ofthe world…He destined us in love to be his sons…according to the purposeof his will ” (Eph 1, 4-6). This boundless and almost incomprehensible love of God for the humanbeing reveals the degree to which the human person deserves to be loved inhimself, independently of any other consideration – intelligence, beauty,health, youth, integrity, and so forth. In short, human life is always a good, forit “ is a manifestation of God in the world, a sign of his presence, a trace ofhis glory ” (Evangelium Vitae, n. 34). Indeed, the human person has beenendowed with a very exalted dignity, which is rooted in the intimate bondthat unites him with his Creator: a reflection of God’s own reality shinesout in the human person, in every person, whatever the stage or condition ofhis life. Therefore, the Magisterium of the Church has constantly proclaimed thesacred and inviolable character of every human life from its conception untilits natural end (Ibid., n. 57). This moral judgment also applies to the originsof the life of an embryo even before it is implanted in the mother’s womb,which will protect and nourish it for nine months until the moment of : “ Human life is sacred and inviolable at every moment of existence, includingthe initial phase which precedes birth ” (Ibid., n. 61). I know well, dear scholars, with what sentiments of wonder and profoundrespect for the human being you carry out your demanding and fruitful workof research precisely on the origin of human life itself it is a mystery on whosesignificance science will be increasingly able to shed light, even if it will bedifficult to decipher it completely. Indeed, as soon as reason succeeds in overcoming a limit deemed insurmountable,it will be challenged by other limits as yet unknown. Man willalways remain a deep and impenetrable enigma. In the fourth century, St. Cyril of Jerusalem already offered the followingreflection to the catechumens who were preparing to receive Baptism: “Whoprepared the cavity of the womb for the procreation of children? Whobreathed life into the inanimate within it? Who knit us together withbones and sinews and clothed us with skin and flesh (Jb 10, 11), and as soonas the child is born, causes the to produce an abundance of milk? Howis it that the child, in growing, becomes an adolescent, and from an adolescentis transformed into a young man, then an adult and finally an old man,without anyone being able to identify the precise day on which the change occurred? And he concluded: “O Man, you are seeing the Craftsman you are seeingthe wise Creator ” (Catechesi Battesimale, 9, 15-16). At the beginning of the third millennium these considerations still apply.They are addressed not so much to the physical or physiological phenomenonas rather to its anthropological and metaphysical significance. We have madeenormous headway in our knowledge and have defined more clearly the limitsof our ignorance but it always seems too arduous for human intelligence torealize that in looking at creation, we encounter the impression of the Creator.In fact, those who love the truth, like you, dear scholars, should perceive thatresearch on such profound topics places us in the condition of seeing and, asit were, touching the hand of God. Beyond the limits of experimental methods,beyond the boundaries of the sphere which some call meta-analysis,wherever the perception of the senses no longer suffices or where neither theperception of the senses alone nor scientific verification is possible, begins theadventure of transcendence, the commitment to “ go beyond ” them. Dear researchers and experts, I hope you will be more and more successful,not only in examining the reality that is the subject of your endeavour, butalso in contemplating it in such a way that, together with your discoveries,questions will arise that lead to discovering in the beauty of creatures a reflectionof the Creator. In this context, I am eager to express my appreciation and gratitude to thePontifical Academy for Life for its valuable work of “ study, formation andinformation ” which benefits the Dicasteries of the Holy See, the localChurches and scholars attentive to what the Church proposes on their terrainof scientific research and on human life in its relations with ethics and law.

7 Because of the urgency and importance of these problems, I consider thefoundation of this Institution by my venerable Predecessor, John Paul II,providential. I therefore desire to express with sincere cordiality to all of you,the personnel and the members of the Pontifical Academy for Life, my closenessand support. With these sentiments, as I entrust your work to Mary’s protection, Iimpart the Apostolic Blessing to you all. © Copyright 2006 - Libreria Editrice Vaticana

8 FINAL COMMUNIQUÉ

It can escape no one that the contemporary bioethical debate, especiallyin recent years, has focused mainly on the reality of the human embryo, consideredin itself or in relation to how other human beings behave towards it.This is only natural since the multiple implications (scientific, philosophical,ethical, religious, legislative, financial, ideological, etc.) connected to theseareas inevitably catalyze different interests, as well as attract the attention ofthose in search of authentic ethical action. The need to ask the basic question: “Who or what is the human embryo”,has therefore become unavoidable, in order to draw from a relevant, consistentanswer to it criteria for action that fully respect the integral truth of theembryo itself. To this end, in accordance with a correct bioethical methodology, it isnecessary first of all to look at the data that the most up-to-date knowledgeputs at our disposal today, enabling us to know in great detail about the differentprocesses through which a new human being begins its existence.These data must then be subjected to an anthropological interpretation inorder to highlight their significance and the emerging values to which to referin the last place, to derive the moral norms for practical action and standardprocedures.

HUMAN LIFE BEGINS AT CONCEPTION Consequently, in light of the most recent discoveries of , it ispossible to establish certain universally recognized points: the moment thesperm penetrates the is when the existence of a new “ human being ”begins. Fertilization induces a whole series of consecutive events and transformsthe cell into a “zygote”. In the human species, the nucleus of thespermatozoid (contained in the head) and a (which will play a determiningrole in the formation of the mitotic fusus in the act of the first cellulardivision) enter the oocyte; the plasmatic membrane remains on the outside.The male nucleus undergoes profound biochemical and structural changesthat depend on the ovular cytoplasm in preparation for the role that the malegenome will immediately begin to play. Here we are witnessing the decon-densation of the chromatin (induced by factors synthesized in the final phasesof ovogenesis) that makes transcription of the paternal genes possible. After the penetrates the oocyte, it completes its second meioticdivision and expels the second , reducing its genome to a haploidnumber of in order to associate with the chromosomes broughtby the male nucleus the karyotype characteristic of the species. At the sametime, it encounters an “ activation ” from the metabolic viewpoint, with a viewto the first . It is always the cytoplasmatic environment of the oocyte that inducesthe centriole of the spermatozoon to duplicate itself, thereby constituting thecentrosome of the zygote. This duplicates itself with a view toconstituting the that will make up the mitotic fusus. The two sets of chromosomes find the mitotic fusus already formed andarrange themselves at the equator in a of metaphase. The otherphases of mitosis follow, and finally the cytoplasm divides and the zygotegives life to the first two . The activation of the embryonic genome is probably a gradual process. Inthe single-cell human embryo seven genes are already active; others areexpressed during the passage from the zygote stage to that of two cells. Biology, and more particularly embryology, provides the documentation ofa definite direction of development: this means that the process is “oriented”– in time – to the direction of a progressive differentiation and acquisition of complexity and cannot regress from the stages it has already completed. A further point acquired with the earliest phases of development is the autonomyof the new being in the process of the auto-duplication of genetic material. The characteristics of gradualness (the time needed for the passage froma less differentiated stage to a more differentiated stage) and of the coordinationof development (the existence of mechanisms that 9 regulate the developmentalprocess in a unitary whole) are also strictly linked to the property of continuity. These properties – virtually ignored at the beginning of the bioethicaldebate – are considered more and more important in recent times because ofthe successive discoveries that research offers on the dynamic of embryonicdevelopment also at the “ ” stage, which precedes the formation of blastocytes. All together, these trends already form the basis for interpreting thezygote as being a primordial “organism” (monocellular organism) that con-sistently expresses its potentials for development through a continuous integration,first, among the various internal components and then, among thecells to which it progressively gives rise. Their integration is both morphologicaland biochemical. The research that has been underway for several yearsnow only yields further “proofs” of this reality. These breakthroughs of modern embryology must be submitted to thescrutiny of philosophical and anthropological interpretation in order tounderstand the precious value inherent in and expressed by every humanbeing, also at the embryonic stage. Thus, the basic question of the moral statusof the embryo must be faced squarely. It is well-known that, among the different hermeneutical proposals presentin the current bioethical debate, various moments in the embryonicdevelopment of the human being have been indicated to which a moral statuscan be attributed to the embryo, and claims are put forward based on“ extrinsic ” criteria (that is, starting with factors external to the embryo itself). However, this approach has not proved suitable for truly identifying themoral status of the embryo, since any possible judgment ends by being basedon factors that are wholly conventional and arbitrary. To be able to formulate a more objective opinion on the reality of thehuman embryo and therefore to deduce ethical indications from it, it is necessaryinstead to take into consideration criteria that are “ intrinsic ” to the embryo itself, starting precisely with the data that scientific knowledge puts atour disposal.

IS THE EMBRYO ALREADY A PERSON? It can be concluded from this data that the human embryo in the phaseof pre-implantation is already: a) a being of the human species; b) an individualbeing; c) a being that possesses in itself the finality to develop as a humanperson together with the intrinsic capacity to achieve such development. From all this may one conclude that the human embryo in the pre-implantationstage is really already a person? It is obvious that since this is a philosophicalinterpretation, the answer to this question cannot be of a “ definitekind ”, but must remain open, in any case, to further considerations. Yet, on the precise basis of the available biological data, we maintain thatthere is no significant reason to deny that the embryo is already a person inthis phase. Of course, this presupposes an interpretation of the concept of the personof a substantial type, referring, that is, to itself as such, rich inpotential that will be expressed during the embryo’s development and alsoafter birth. To support this position, it should be noted that the theory ofimmediate animation, applied to every human being who comes into existence,is shown to be fully consistent with his biological reality (in addition tobeing in “ substantial ” continuity with the thought of Tradition). The Psalm states: “ For you did form my inward parts, you did knit metogether in my mother’s womb. I praise you, for you are fearful and wonderful.Wonderful are your works! You know me right well ” (Ps 139[138],13-14), referring to God’s direct intervention in the creation of every newhuman being’s soul. From the moral viewpoint, moreover, over and above any consideration ofthe human embryo’s personality, the mere fact of being in the presence of ahuman being (and even the doubt of this would suffice) would demand fullrespect for the embryo’s integrity and dignity: any conduct that might in

10 someway constitute a threat or an offence to its fundamental rights, and first andforemost the right to life, must be considered as seriously immoral. To conclude, we would like to make our own the words that the HolyFather Benedict XVI spoke in his Address to our Congress: “ God’s love doesnot differentiate between the newly conceived infant still in his or hermother’s womb and the child or young person, or the adult and the elderlyperson. God does not distinguish between them because he sees an impressionof his own image and likeness (Gn 1, 26) in each one. “ He makes no distinctions because he perceives in all of them a reflectionof the face of his Only- begotten Son, whom he chose…before the foundationof the world…He pre-destined us in love to be his sons…according to the purposeof his will ” (Eph 1, 4-6).

(From L’Osservatore Romano, English edition, 26 April 2006, p. 6)

11 JAVIER LOZANO BARRAGÁN

THE CULTURE OF DEATH AGAINST THE CULTURE OF LIFE IN THE TEACHING OF “EVANGELIUM VITAE”

INTRODUCTION

This international Congress organized by the PontificalAcademy for Lifeis dedicated to the human embryo during the stage before implantation. Thescientific aspects and the bioethical dimensions will be analyzed. In this introduction,which I have been asked to engage in from a theological point ofview, it seemed to me that a choice presented itself: either an attempt toengage in a theological reflection on the origin of life or an attempt to presenta cultural, philosophical and theological framework by which to introduce thescientific and bioethical subjects. Given that I had an opportunity to addressthe first subject here at this Academy, I have chosen, on this occasion, the secondpossibility. This, indeed, is a subject that was much addressed by the veneratedJohn Paul II in his Encyclical Evangelium Vitae. Thus it appeared tome to be advisable to analyze certain ideas ‘taking this Encyclical as a startingpoint ‘connected with certain philosophical and theological aspectsaddressed by John Paul II in Evangelium Vitae, and these ideas will lead usto the heart of the thought of Benedict XVI as expressed in his first Encyclical,Deus Caritas Est.

THE CURRENT SITUATION I believe that it is not difficult to realise that we live in a world that is characterized by a Malthusian mentality. There is the fear, above all in the First World, that consumer goods are not sufficient in number for everyone. Hence the idea of eliminating the new partakers at the banquet of life, in particular if they come from the Third World, who thereby place even more at risk the advantages that the members of the First World believe they possess in their own world of privileges. Thus we find that everywhere there have increased in number campaigns for the control of through, in addition, the use of contraceptives and of sterilization, the legitimation of abortion through state laws, murders carried out by criminals, and corruption. This also takes place at a state level, as is the case in China with the murder of baby , where, after the state imposed on couples the possibility of having only one child, if a baby is born she is killed so that there will then be an opportunity to have a male child. And the same may be said of wars, which are never absent, or of euthanasia, which is increasingly widespread. Other ways of eliminating life are the use of embryos for scientific purposes, where they are seen as mere material for experiments, and in general all the kinds of manipulation which are possible because of genetic engineering, where what is engaged in is done without any respect for life as such.

THE CULTURE OF DEATH In the face of these facts, when we reflect on why they exist we find their origins in the culture of death which, in its turn, has its roots in secularism. John Paul II, in the above mentioned Encyclical, points out that this culture has its roots in wrath, greed, irresponsibility, lies and materialism. In the recent Encyclical Fides et Ratio, he spoke to us about the banning of metaphysics, of relativism, and of the flight from truth. I will now attempt to guide my paper along these lines.

Cartesianism and secularism Secularism, as a form of Western culture that then spread throughout the world, has, in part, I believe, its origin in the thought of the French philosopher René Descartes. To resolve the question of doubt and 12 to arrive at irrefutable certainty, he thought that one could accept only that which is base upon clear and distinct certainty. Thus this French philosopher upheld the existence of only three realities by which one could affirm such certainty, namely God, man, and the world. God, described as such, man as described res cogitans and the world described as res extensa. But for these realities to be accessible as clear and distinct ideas it was necessary for their existence not to pre-suppose any other reality, and he called this independence substance. He thus defined substance as what exists and does not need anything else or anybody else to exist. From this point of view, what confers clarity and distinction on thought, and thus certainty, is autonomy. Something that exists in itself. The problems that arose were varied but above all there was that of how to relate these three entities. Subsequently, not needing any of the others to exist, it was easy to arrive at the elimination of God, and thus it was thought that the clearest thing was matter. On the one hand, he who thinks identifies with matter; on the other, he affirms the existence only of what he thinks. Before Descartes reality was conceived as that which exists; from Descartes onwards it was conceived as what is thought: thus it exists to the extent that it is thought. From here two philosophical-cultural tendencies were to develop: materialism and idealism, which would have various repercussions and forms of influence on each other.

Secularism and evolutionism First of all secularism was established, and subsequently there was a separation from God. Man came to consider himself as closed up within himself, in his own creations, which appeared to be prolongations of his own being. His understanding was now reduced, as has been said, to thought or to matter, opening up space to a different conception of the meaning of man in his cultural creations. Reduced to res extensa by a current of thought ‘positivism, to begin with, and then neo-positivism ‘man asked himself about his origins and found an answer in evolutionism, whose fundamental element is the struggle for life. In this approach man is seen as the result of the struggle for survivalof a higher species against lower species. It is important to observe that inthis approach life appears as a consequence of struggle, a struggle untodeath, which even means the wish to destroy the other, or hatred, whichis the same thing. To return to man, life is seen as being experiencedwithin this framework of reference; life is seen as conquest, as the result ofthe hatred which has led to the elimination of the other. Within the contextof this intense battle (life), everything is allowed, given that the objectiveis to live and to survive. This everything refers, as John Paul II says in Evangelium Vitae, to wrath, greed, irresponsibility, lying, and total materialism.Life touches man and is perpetuated in him as the outcome ofhatred and destruction. Such is life: the strongest win, the weakest areeliminated both as individuals and as groups. Thus the culture of deathcomes to be established.

Secularism, individualism and idols In this culture of conquest one arrives at a situation of strong individualism;man closed himself up in himself and in what is convenient to him inorder to go on living. This individualism of closure, the outcome of conquest,projects him into his desires, which we may call idols and which are what isadvantageous to him in the form of the idols of having, of power, and of pleasure:he lives to possess; the more he has the better he is. He needs power, heneeds to take from others, he needs to subject them. He feels that he is, andhe struggles to be, the despotic master of everything that exists. In this doeshe experience his greatest satisfaction, above all in the field of what he canexperience in the realm of the senses. Thus the external world exists to be dominated; everything takes on valuefor him alone, within his own ‘extension ‘or materiality. Thus nature is notseen as something that is sacred, as something that must be respected andbrought to fulfilment: it is man who becomes the end of nature, manipulatingit without any limits and preying upon it at his pleasure and according to hisadvantage. Matter appears to man as 13 something that can be manipulated,without exceptions, and man sees other men and other forms of life in thesame way. For this reason, the origin of life, as well, is an element that can bemanipulated totally on the basis of the whim of the manipulator. In the fieldof genetic engineering there are no limits except the self-interest and the willof those who work in this field. Technology, therefore, appears to conform toa single law: that which is possible. The end of man as such remains excluded,unless it is understood as the end of this concrete man, of the technician, ofthe scientist, of the politician, and all the rest, who manipulates nature as heso wishes, which is the sole limit and the only law. In relation to the sexual sphere, as well, this is the only law: everything isinterpreted from the point of view of pleasure and an attempt is made to avoideverything that can cause pain. When in sexual activity shared life is comeacross, given that the sharing of life does not matter if this does not meet therequirements of this concrete man who dominates this technical or economicsphere, life is simply shared to the extent that this is in some way convenientfor that individual. Thus is born the Malthusian mentality and one comes tovarious ways by which to limit, or better to eliminate, birth, when, indeed, thisis not completely subjected to the selfish whim of this concrete man. Who is this concrete man? He who has conquered the world with technology,politics and economics and because of this conquest dictates the lawsto which those who have not attained this conquest will be subordinated,those who remain on the ladder of lower beings and, whatever the case, thosewho have been defeated. This man, as I have already pointed out in thispaper, does indeed live in a community but he lives in a community that ismade up of selfish individualities that seek only their own self- interest and areunconcerned about the welfare of those who have not reached the top,namely those who have been the defeated. This is the culture of death.

THE CULTURE OF LIFE Faith in God the Creator and Redeemer In a completely opposed position to this culture of death we find the cultureof life. The culture of life is the outcome of faith in God, the spring oflife, the Creator, the Word made flesh, Christ who died and rose again. It isfaith in a full openness to God that reveals to us the mystery of His existenceand to whom we turn in reverent fashion faced with the Mystery of His Transcendencemade intimacy for man in the Incarnation of the Word. This is aposition that is diametrically opposed to the position previously outlined, justas life and death themselves are also opposed. Life has a paradigm and it isGod, our Lord, with whom we can enter into contact through the Incarnationof the Word, his death and redemptive resurrection. This is the real paradigmof life. In this way the life of man opens up to a fundamental relationship: he isa creature, he is created by God. Life is not acquired by conquering it as aright to pleasure but by receiving it freely as an ineffable gift which meanscreation but which at the same time from the Word made Flesh acquires newand indeterminable horizons that are projected towards eternal life. Godbecame man so that man could become God, the Holy Fathers declared.Specifically human life has a path to follow that will never end. It is a qualitythat is constantly growing towards its horizon, which is eternal life ‘God.And it is eternal life that became history in Christ, the Word made Flesh.Thus life is born from the love of God and it is love in itself. Here we enterthe heart of the Encyclical Deus Caritas Est of Benedict XVI. Following thethought of the Pope, life is, as a result, a mere relationship of gratitudebetween man and God and therefore has a purpose: to reflect this relationshipof free giving in service to others, not as a conquest but as sharing, andThe culture of death against the culture of life in theâ?¦ 25especially with those who do not have anything; thus life comes from creation,from nothing.

14 Participation in the life of the Most Holy Trinity

The fullness of life has an eternal finality; it is a receiving that goes beyondthe very nature of being a creature of man and raises him to the status ofbeing a son of God. Its contingent dimension will be defeated by eternity, byeternal life. It is a participation of the One and Triune God, whose life is aninfinite giving from the Father to the Son and from the Son to the Father, agiving that in full love is the Holy Spirit. Thus human life should be a givingwithout limits for others; far from being closed and individualistic, it shouldbe openness and living always for other people. Man is always a being in arelationship, just as God is the Being in Relation; from the relationship withGod derives the relationship between men and between man and creatures.In this sense can we understand the definition that Boethius gives of eternitywhen he defines it as interminabilis vitae tota simul atque perfecta possessio.

Dominion over the Creation

Man will always have dominion over the other beings of the Creation butalways in a way that respects the purpose for which they were created. Thusthe horizon of science and technology is not only what is possible; it is alsothe finality of things. It is true that nature must be manipulated but thisshould be done in a way that respects her principal laws which come fromGod and contribute to the welfare of man himself. In the deepest part ofevery created being shines a finality willed by God the nature of which hasbeen revealed to God so that he can secure it. In this way all created beingsconverge in man, the only being in the Creation who was willed for himselfand who can use all the other creatures as an instrument.

The origins of human life

If we now consider the origin of human life and we refer to the sexual act,this should be seen as an authentic act of giving of love, of agape, accordingto Benedict XVI, as the profound vital symbol of the relationship of love fromwhich flows life in the image of the giving of love which is the life itself ofGod. Being a love of total giving, the sexual act appears as a totalizing expres-sion of love which is given only in this totality of a personal relationship thatbears the name of and family. Genetic engineering applied to human generation should have one aim:that of facilitating and completing this total personal giving and not that ofimpeding it or destroying it. The whole field of genetic engineering, when itis connected with human life, does not have as its subject something that canbe freely manipulated, but something to which respect is due in line with fullquality of life, namely the human and divine life of every person. Human life, therefore, implies an ethic, an ethos, a tendency to open itselfto its own finality. This finality is received from its model, that is to say thedivine life of the Word made Flesh, which was realized in history with thedeath and resurrection of Christ. As a consequence, human life is a life oftotal giving until death. This is its Ethic. Instead of closing itself within a totalindividualism, it opens itself fully and gives itself to other people until death,as a path towards divinization, which is the resurrection. Thus this giving doesnot exclude suffering and pain but includes them in the cross of Christ. Lifeis full life, it is truth, but it is not without difficulties, pain, and death, and yeta transitory death which in the loving remission of the Spirit of Love in thehands of the Father of Heaven sprouts into an eternal life, which is true lifeand is about to be achieved. In this way, life enters the sphere of divinity andit is enclosed in the Christian mystery, which is worshipped and contemplated,more than in something that is to be dominated and manipulated.

15 The quality of life

This is authentic quality of life. Quality of life justifies the respect due tolife itself in every sphere, and it is evident that the quality of human life is notonly measured on the basis of economic coefficients, and indeed not only onthe basis of social or ecological ones. Its quality is essentially determined bythe paschal eternity that characterizes and specifies human life. The contemplationof this quality of life is the specific aptitude by which to accede to itand to really establish the culture of life. Its foundation is the relationshipbetween this life and the life of the Word made Flesh, a relationship that isnot merely external but which is projected in the very essence of human lifebecause of the fact that man is raised to the dignity of being a son of Godthrough the Son of God. There is something intrinsic in human life itself, nota mere reference but something profound which in itself qualifies life andallows us to accede to the mystery of life in full worship of God. It is thus evident that life is not the outcome of a conquest but the gratefulwelcoming of a gift which, as such, is absolutely freely given and comes to usas the beginning of a new logic: the logic of the freely given.

Evolution

In stating that life is not a conquest we do not exactly contradict the possibilitythat the origin of life lies in evolution. It is simply that with the teachingof the Church we hold that in the case of the evolution of life, on theascending scale when we come to man we do not encounter a mere developmentof what is taking place in the lower spheres, where the selection ofspecies and the survival of individuals through struggle and the prevailing ofthe strongest is not denied: man is not simply the result of a struggle, thestrongest primate. We hold that when one comes to this culminating stage oflife, there flows within it a special action of God that is repeated continuallyin every man and is to be identified in the creation of the soul, of his ownvital principle which is divine because of the participation of the life of theWord made Flesh in death and resurrection.

The triumph of the culture of life

As was said at the beginning of this paper, the contemporary world ischaracterized by a culture of death but the culture of life will always emergeas the winner. In the Encyclical Evangelium Vitae, Pope John Paul II employsthree examples to point out to us this certainty of hope, and these are examplesthat I think it is very important to present here: the example of thePharaoh who had the new born children of the Israelites killed, the exampleof Herod who killed all the children of Bethlehem, and the example of themysterious woman of the Apocalypse who is about to give birth to a childwhen a dragon draws near to her in order to devour it ‘however the womanis given wings with which to fly towards the desert and to give birth to herchild in safety. In the contemporary world we see how from all quarters thereare threats to human life from its beginning. However, just as the Pharaoh,Herod and the dragon failed in their missions of death, so also these perilswill not meet with success; life will go on and we will continue to appreciateit as the great gift that God gives to us and which raises us to the dignity ofdivine filiation. The truth of life lies in giving, in the true love that is built up in this rela-tionship with the exercise of freedom: the freedom that edifies life and .Life, love and freedom are related to one another in such a deepway that one cannot exist without the other and they all together build up thetrue culture of life.

16 CONCLUSION

The destruction of the embryo obeys a Malthusian mentality mixed witha mechanistic positivism and an idealism in which subjective thought is thenorm for action. Thus is born the culture of death, which is fully closed to theTranscendent. The only ethical norm for action is a contract as an expressionof the will of the strongest. In the culture of death, science and technology arethe instruments of man understood as the despotic master of nature, includinghuman life. The only limit is what is possible. The culture of life, beginning with the truth of the Creation and theRedemption, is opposed to the culture of death. The authentic culture of lifeis participation in divine life. The background to the culture of life is free giving.Life is the donation of reciprocal love. In the culture of life man appears as the lord of the Creation in order tomeet his own needs. This is the limit of science and technology: to meet theauthentic needs of man. Thus the horizon of the technical is not mere possibilitybut finality. Through the virtue of hope, the Christian opens to optimismand to the knowledge that the culture of death will not prevail over theculture of life. The Most Holy Virgin is our model for the culture of life. Christ came sothat we should have life and that we should have it in abundance. Christcomes to us through Mary. Mary is the source of life. From her, by the workof the Holy Spirit, Christ, God and true man, the only one who gives us truelife, was born. In the struggle against the culture of death it is Mary, thewoman of the apocalypse, who will defend life and who will ensure that theculture of life will impose itself in definitive fashion. May she, from here, from the Pontifical Academy of Life, intercede forthe life of the world so that this world of ours, which is so afflicted by theconsequences of the culture of death, have life and have it in abundance!

17 M.ZERNICKA-GOETZ

CELLS OF THE EARLY MOUSE EMBRYO FOLLOW BIASED AND YET FLEXIBLE DEVELOPMENT

Development of the early mammalian embryo is regulative, which means that development is flexible and responsive to experimental intervention. Such flexibility could be explained if embryogenesis were originally unbiased producing identical cells. Alternatively, regulative development could mean that the embryo has some bias from the beginning, with restriction of cell fate increasing steadily with time. Interest in the early mammalian embryo has undergone a renaissance following discovery of some bias in development of embryonic patterning in the mouse embryo before implantation. This patterning does not restrict developmental flexibility of individual mouse embryo cells or the embryo as a whole. Thus, the patterning and regulative ability of the embryo peacefully co-exist. In many organisms, the polarity of the embryo is determined from the very beginning of development because they inherit spatially localised cytoplasmic factors that act as determinants to guarantee specific cell fate(1) (St Johnston & Nusslein-Volhard, 1992; Gurdon, 1992). However, the development of the mammalian embryo is regulative rather than determinative. This raises the important question of how cell fate develops in the absence of determinative factors. Are the first cell fate decisions taken entirely randomly? Or there is some non-rigid pattern that could bias developmental decisions and yet allow flexibility? In this lecture I had reviewed our studies that show that polarity and cell fate progressively emerge in the developing mouse embryo.

THE FIRST SIGNS OF DIFFERENCE The mouse embryo at the 3rd day of development, named blastocyst, is clearly polarised. To generate this polarity two distinct groups of cells have to be set aside: "inside" cells that will retain pluripotency and give rise to the (ICM) that provides progenitors for the future body and some extra- embryonic cells and the "outside" cells that will differentiate into the trophectoderm that provides only extra-embryonic structures. When cells become positioned inside or outside they acquire different properties either because these alternative environments cause them to differ(2) (Tarkowski & Wroblewska, 1967) or because they inherit either the apical or basal domains as a result of asymmetric divisions of their polarised progenitors(3) (Johnson & Ziomek, 1981). Or both mechanisms operate(4) (for review see Zernicka-Goetz 2005). With the formation of the blastocyst cavity, the ICM becomes asymmetrically located towards one pole of the embryo: the "embryonic" pole.Thus the blastocyst has a clearly polarised embryonic-abembryonic axis. Over the recent years the progress have been made in address two important and related questions. First, how does the embryo develop its embryonic- abembryonic axis? Second, when do cells first start to differ from each other? With the aim to address both of these question we started by developing means that would allow us to follow undisturbed development in time and space and to identify individual cells of the mouse embryo and follow their fate in natural and also perturbed circumstances.

FIRST DIVISION-INFLUENCE OF CELL SHAPE The fertilised mouse egg appears uniform, yet a product of the previous meiotic division, the second polar body (PB), is an asymmetric feature that conventionally defines the animal pole(5) (Gulyas, 1975; Gardner, 1997). Another asymmetric feature is the fertilization cone, which indicates the sperm entry position (SEP).In addition, upon the egg changes its shape to become flattened so that the SEP will mark the egg's short axis(6) (Gray et al. 2004).We have found that the first cleavage division does not occur at random in respect to these factors, but their "bias" its orientation(7) (Zernicka-Goetz, 2002). In most fertilised the first cleavage is significantly more often along the animal-vegetal axis rather than perpendicular to it. The correlation of the first cleavage with the SEP 18 could be due to the sperm influence on the shape of the egg, as SEP correlates with the egg short axis through which cleavage tends to occur. We found that the cell shape has an over-riding influence on the cleavage, as when we change the egg shape experimentally this led to the egg division through their new short axis. This dominant effect of cell shape might explain some variability in the cleavage orientation between the eggs as the shape of the egg could be influenced by experimental conditions(8) (Plusa et al. 2005).

PATTERN OF CLEAVAGE DIVISIONS AND CELL FATE To understand whether there is any pattern to the development of the early mouse embryo requires non- invasive methods to trace it. This is because the development is regulative and therefore if perturbed, embryo cells will undertake different paths. Thus, only the development of non-invasive labelling and three-dimensional imaging techniques, enabled the origins and fate of individual embryonic cells to be recognised. Such non-invasive tracing indicates that whatever the orientation of the first cleavage, its plane often allows prediction of the orientation of the blastocyst embryonic’abembryonic axis. First, it has ben found that the attached PB not only marks the first cleavage, but later the boundary between embryonic and abembryonic parts of the blastocyst(9) (Gardner, 1997). This led to the proposal that the animal’vegetal axis of the egg is preserved throughout pre-implantation development and correlates with the polarity of the blastocyst. It was subsequently observed that the PB and a bead placed at the SEP together tend to mark the plane between 2-cell blastomeres and, at the blastocyst, the embryonic- abembryonic boundary(10) (Piotrowska & Zernicka-Goetz, 2001). These observations revealed co- relations that would not be expected if patterning of the blastocyst were to develop entirely randomly. In agreement with these findings, it has been shown that 2-cell-stage blastomeres show biased differences in their fate11(Piotrowska et al. 2001; Gardner, 2001; Fujimori et al, 2003). Both 2-cell blastomeres contribute to both ICM and trophectoderm (TE), but one contributes more cells to the embryonic (containing the polar TE) and the other to the abembryonic (containing the mural TE) parts of the blastocyst. These findings are actually consistent with a number of earlier studies demonstrating that one of the 2-cell blastomeres (the earlier to divide) contributes significantly more cells to the ICM, most of which will be in the embryonic part(12). The relationship between the plane separating 2-cell blastomeres and the embryonic-abembryonic boundary of the blastocyst is difficult to quantify and this had led to the variability of reported conclusions and some controversy(13) (Gardner, 2001; Piotrowska et al. 2001; Fujimori et al. 2003; Alarcon & Marikawa, 2003; Chroscicka et al. 2004; Motosugi et al. 2005). Thus to permit quantitative measurement of clone positions, the most recent lineage tracing used a reporter line which enabled the identification of cells in different layers of optically sectioned blastocysts14 (Piotrowska-Nitsche & Zernicka-Goetz, 2005; Piotrowska-Nitsche et al. 2005). This revealed the proportion of embryonic and abembryonic parts occupied by the progeny of specific 2- and 4- cell blastomeres, which confirmed the bias of 2-cell blastomeres to contribute more cells to either the embryonic or abembryonic part. In addition, this demonstrated that this bias depends on the pattern of the second cleavage divisions. THE SECOND CLEAVAGE DIVISIONS AND CELL FATE The second cleavage divisions are asynchronous and their orientation is variable: they occur either approximately along the animal-vegetal axis, meridionally (M), like the first cleavage, or equatorially/obliquely (E)(15). Any combination of the temporal sequence of such divisions is possible, although sequential M and E divisions are most common16 (Gardner, 2002; Piotrowska-Nitsche & Zernicka-Goetz, 2005). We have found that when the earlier of the second cleavages is M and the later E, in the majority of embryos the earlier 2-cell contributes more cells to the embryonic and later to the abemebryonic part. When the earlier second cleavage is E and the later M, the earlier to divide gives rise to either the embryonic or abembryonic part. By contrast, when second cleavage divisions are of similar orientation, the allocation of the blastomeres' progeny appears to be at random. 19 Thus, it appears that whether the cells has divided meridionally rather than equatorially will influence their prospective fate - does it matter for their developmental potential? One way to address the question would be to separate cells and follow their development. Through this route it has been shown that blastomeres of the 2-cell-stage mouse embryo are totipotent: if one is destroyed, the other can support development to term(17) (Tarkowski, 1959). Despite efforts, this has never been achieved for embryos derived from individual 4-cell-stage blastomeres in the mouse embryo. But this can be due to the small size of the resulting embryos rather than differing potency between the 4-cell blastomeres. So are the 4-cell blastomeres identical to each other? To examine this, it is essential to combine each individual 4-cell blastomere with equivalent cells to generate chimeras of single cell type.The common group of embryos in which the earlier second cleavage is M and the later E is an attractive group for such studies as their 4-cell blastomeres have predictable origins and fate within the blastocyst enabling identification of like type-cells from one embryo to another. Combining like cells together to generate chimeras of single cell typesshowed that blastomeres can differ as early as the 4-cell stage. Chimaeras of blastomeres arising from early M divisions (animal’vegetal) developed to term with full success. However, by contrast, development of chimaeras of blastomeres arising from later E divisions ('animal' or 'vegetal') was compromised. Moreover, whereas chimeras constructed from 'animal' blastomeres could develop to term, 'vegetal' chimaeras could not. We think that at least in part this defect might relate to a reduced proliferative ability of blastomeres arising from latere equatorial divisions as they developed to much smaller postimplantation embryos. Thus, my prediction would be that increasing number of blastomeres in "vegetal" chimeras should result in their better development. The above finding was very surprising as blastomeres at the 4-cell-stage were assumed to be identical. However, this new finding is in fact in agreement with the results of earlier studies that showed 4-cell blastomeres can contribute to different cell lineages(18). Indeed Piotrowska-Nitsche and colleagues demonstrated that although the 'vegetal' blastomere's development was compromised when surrounded by like cells, when it was surrounded by blastomeres of other origins it could contribute to all lineages(19).Thus, successful development might depend on the cellular environment in which a cell finds itself.

CONCLUDING REMARKS The patterning of the mouse embryo, as actually with embryos of other species, is an emerging process built on successive asymmetries that gather as the egg develops after fertilisation. But in the mouse, in contrast to many other organisms, cell fate and patterning do not seem to be directed by determinants. Rather, cells 'learn' which fate to adopt from cues they meet on their way. This piecemeal acquisition of information is compatible with a plasticity of the embryo cells to initiate different developmental paths when cell context (neighbours or other elements of the environment) are changed.

1 St Johnston D., Nusslein-Volhard C., The origin of pattern and polarity in the Drosophila embryo, Cell 1992, 68: 201-219; Gurdon J.B., The generation of diversity and pattern in animal development, Cell 1992, 68: 185-99. 2 Tarkowski A.K., Wroblewska J., Development of blastomeres of mouse eggs isolated at the 4 and 8- cell stage, J. Embryol. Exp. Morphol. 1967, 18: 155-180. 3 Johnson M.H., Ziomek C.A., The foundation of two distinct cell lineages within the mouse morula, Cell 1981, 24: 71-80. 4 Zernicka-Goetz M., Developmental cell biology: cleavage pattern and emerging asymmetry of the mouse embryo, Na.t Rev. Mo.l Cell Biol. 2005, 6: 919-928. 20 5 Gulyas B.A., A re-examination of cleavage patterns in eutherian mammalian eggs: rotation of blastomere pairs during second cleavage in the rabbit, J. Exp. Zool. 1975, 193: 235-248; Gardner R.L., The early blastocyst is bilaterally symmetrical and its axis of symmetry is aligned with the animal- vegetal axis of the zygote in the mouse, Development 1997, 124: 289-301. 6 Gray D., First cleavage of the mouse embryos responds to change in egg shape at fertilization, Curr. Biol. 2004, 14: 397-405. 7 Zernicka-Goetz M., Patterning of the embryo: the first spatial decisions in the life of a mouse, Development 2002, 129: 815-829. 8 Plusa B., Hadjantonakis AK., Gray D. et Al., The first cleavage of the mouse zygote predicts the blastocyst axis, Nature 2005, 434: 391-395. 9 Gardner, The early blastocystis..., pp. 289-301. 10 Piotrowska K., Zernicka-Goetz M., Role for sperm in spatial patterning of early mouse embryos, Nature 2002, 409: 517-521. 11 ID., Wianny F., Pedersen R.A. et Al., Blastomeres arising from the first cleavage division have distinguishable fates in normal mouse development, Development 2001, 128: 3739- 3748; Gardner R.L., Specification of embryonic axes begins before cleavage in normal mouse development, Development 2001, 128: 839-847. 12 Hillman N., Sherman M.I., Graham C.F., The effect of spatial arrangement on cell determination during mouse development, J. Embryol. Exp. Morphol. 1972, 28: 263-278; Graham C.F., Deussen Z.A., Features of cell lineage in pre-implantation mouse development, J. Embryol. Exp. Morphol. 1978, 48: 53-72; Surani M.A., Barton S.C., Spatial distribution of blastomeres is dependent on cell division order and interactions in mouse morulae, Dev. Biol. 1984, 102: 335-343. 13 Gardner, Specification of embryonic axes..., pp. 839-847; Piotrowska, Wianny, Pedersen, Blastomeres arising from the..., pp. 3739-3748; Fujimori, Kurotaki, Miyazaki, Analysis of cell lineage in..., pp. 5113-5122; Alarcon V.B., Marikawa Y., Deviation of the blastocyst axis from the first cleavage plane does not affect the quality of mouse postimplantation development, Biol. Reprod 2003, 69: 1208-1212; Chroscicka A., Komorowski S., Maleszewski M., Both blastomeres of the mouse 2-cell embryo contribute to the embryonic portion of the blastocyst, Mol. Reprod. Dev. 2004, 68: 308-312; Motosugi N., Bauer T., Polanski Z. et Al., Polarity of the mouse embryo is established at blastocyst and is not prepatterned, Genes & Development 2005, 19: 1-12. 14 Piotrowska-Nitsche K., Zernicka-Goetz M., Spatial arrangement of individual 4-cell stage blastomeres and the order in which they are generated correlate with blastocyst pattern in the mouse embryo, Mechanisms of Development 2005, 122: 487-500; Piotrowska-Nitsche K., Perea-Gomez A., Haraguchi S. et Al., Four-cell stage mouse blastomeres have different developmental properties, Development 2005, 132: 479-490. 15 Gardner R.L., Experimental analysis of second cleavage in the mouse, Hum. Reprod 2002, 12: 3178-3189. 16 Ibid. pp.3178-3189; Piotrowska-Nitsche, Zernicka-Goetz, Spatial arrangement of individual 4-cell stage blastomeres and..., pp. 487-500. 17 Tarkowski A.K., Experiments on the development of isolated blastomeres of mouse eggs, Nature 1959, 184: 1286-1287. 18 Kelly S.J., Studies of the developmental potential of 4 -and 8-cell stage mouse blastomeres, J. Exp. Zool. 1977, 200: 365-376; ID., Mulnard J.G., Graham C.F., Cell division and cell allocation in early mouse development, J. Embryol. Exp. Morphol. 1978, 48: 37-51; Hillman, Shermann, Graham, The effect of spatial arrangement on..., pp. 263-278. 19 Piotrowska-Nitsche, Perea-Gomez, Haraguchi, Four-cell stage mouse blastomeres..., pp.479-490.

21 R. COLOMBO

The process of fertilization and its stages. From parental gametes to a developing one-cell embryo

Introducing his historical essay on The Interpretation of Development and Heredity in 1930, Edward Stuart Russell reminded the readers that the " problem of development is without question one of the most difficult and intriguing in the whole field of knowledge. That from a minute germ of relatively simple structure there should be gradually built up, by a series of processes beautifully co-ordinated in space and time, the complex organization of the adult is a fact that has never ceased to excite the wonder of mankind. It has provided a constant challenge to the intellect of man, and many and various have been the theories invented to explain it. It ranks as one of the major problems of biology ".1 The " wonder of mankind " for the " beautiful processes " involved in human conception and development is vividly and passionately expressed by the Psalmist: "You created my inmost self, knit me together in my mother's womb. For so many marvels I thank you; a wonder am I".2 Among the scientists who devoted their entire professional life to the investigation of how an embryo is formed and develops into a foetus and an adult there is a common feeling that has been concisely and effectively reported by Lewis Wolpert as follows: " No one who studies development can fail to be filled with a sense of wonder and delight. Development of the embryo is a truly remarkable process. Understanding the process of development in no way removes that sense of wonder ".3 A short review of the biology of human and early embryo development has already been reported in the Proceedings of the Pontifical Academy for Life4 and the general conclusion that was drawn is still consistent with the current scientific evidence on the process of fertilization: "At the fusion of two gametes, a new real human individual initiates its own existence".5 However, more recently, further details on the stages of in vitro mammalian fertilization and the very beginning of embryo development were obtained by a number of observations and experiments of a cytological, genetic and molecular nature. These novel biological data alone are neither supportive nor exclusive of the view of the fertilization process as the beginning of human individual life, a sound concept of biology of mammalian resting on the overall consideration of long-standing evidence as well as recent acquisitions. Nevertheless, most of what has been discovered in the past few years about the structure, function and interaction of gametes, as well as the formation of one-cell embryo and its cleavage, is relevant to a deeper understanding of how a new individual life comes into existence and starts its development. To favour an integration of old and new biological observations on the complex and coordinated process leading to fusion and the start-up of embryo development, the current knowledge of this process and its stages will be considered within an historical perspective. The aim is not to comprehensively review the huge amount of scientific literature on the topic, but to contribute to the advancement of the study of the anthropological and ethical questions concerning the beginning of individual human life by providing an updated short look at some aspects of fertilization and its preludes, with special reference to and man. BRIEF HISTORICAL OVERVIEW OF OBSERVATIONS AND THEORIES ON HUMAN CONCEPTION Antiquity and the Middle Ages Throughout the long history of Western philosophy, biology and medicine, the question of when and how a new human being comes to existence and begins its has always been a matter of deep inquiry and, in some periods, heated controversy too. Well before the discovery of the physiology and genetics of sexual reproduction in animals and , any careful observer of human life could see that a pregnancy originates from an intercourse between a woman and a man in their fertile age, through which the male passes into the womb. During Antiquity, several 22 conjectures were made to explain the link between the presence of seed in the woman's inner genitals and the appearance of a developing embryo. In his Περì ζώων γενέσεως, (384-322 B.C.) supported the view - shared by other Greek writers, notably the author of the Hippocratic Περì φύσιος παιδίου 6 - that the menstrual blood (καταµήνια) is the biological material out of which the embryo's tissues and organs are formed. To this common belief, the Stagyrite added that the male provides the germinal dynamic element (τò 'άρρεν ποιητιχόν) to shape the passive female element (τò θη̃λυ παθητιχόν). Contrary to the Aristotelian notion of male germ only,7 the Hippocratic treatise Περì γονη̃ς holds the " doctrine of the two " (male and female germinal elements),8 whose origin goes back to Alcmaenon of Crotona (born ca. 535 B.C.), Parmenides of Elea (ca. 515-after 540 B.C.), Democritus (late fifth century B.C.),9 and Empedocles of Acragas (ca. 492-432 B.C.).10 The Greek physician and naturalist Claudius Galen (129/130-199/200 A.D.), who practised in Rome under the Emperors Marcus Aurelius and Lucius Commodus, distanced himself from the Aristotelian doctrine by proposing that the female does elaborate her own seed by filtration from the bloodstream within the ovaries (called female ).11 Such seed is then carried through the , which, he correctly noted, lead to the uterus, where intermingling with the male seed occurred. The theory of the two seeds competed for many centuries with the " doctrine of the menstrual blood " but was less successful as the latter in capturing the attention of the philosophers, physicians, and naturalists of the Middle Ages.12 The undisputed authority of Aristotle, both as a philosopher and a scientist, played the principal role in the reception of his theory of generation. Besides this, the biological theory of the male seed as a shaper and organizer and the female blood as a plastic and inchoate material was favoured by its prima facie consistency with the Aristotelian-Scholastic metaphysical theory of substantial form and first matter as the two principles of every existing natural body, the former being the actual and determining principle and the latter representing the potential and determinable principle (hylomorphic theory). 13 The seed and blood theory of conception was well received by both Christians and Muslims also thanks to its apparent concordance with some sentences of the Bible and the Koran, respectively. In the Ancient Testament, the embryo is called the " of the womb " 14 and during the visitation of Mary to Elizabeth the latter exclaims: " Blessed is the fruit of your womb (καρπòς τη̃ς χοιλίας) ".15 Even the example provided by Aristotle to explain the effect of the seed on a woman's blood (" The action of the male in setting the female's secretion in the uterus is similar to that of rennet [puetâ?¦a] upon milk ")16 resembles the words of Job: " Hast thou not poured me out of milk and curdled me like cheese? ".17 The " cheese-analogy " of conception had a great fortune until the Middle Ages: taken to Alexandria, the Aristotelian analogy found its way to the Arabic literature and, through it, re-entered the West.18 Among the Medieval texts, the clotting image can be found in Plate IV of the Wiesbaden Codex B of Liber Scivias by Hildegard of Bingen (1098-1179), who is to have borrowed it from Haly-Habbas' Liber Totius, which appeared as such in Latin in 1523 but actually much earlier, between 1070 and 1085, at Monte Cassino, by the monk and translator of Greek medical texts Constantin the African (ca. 1010-1087), who had entitled it Liber de Humana Natura, giving it out to be his own work. The Koran describes the creation of man by God as follows: "We made the seed [nutfata: (a drop of) seminal fluid] a clot [khalaqna: blood coagulum], then We made the clot a lump of flesh, then We made (in) the lump of flesh bones, the We clothed the bones with flesh, then We caused it to grow into another creation ".19 Again, a reference is made to the mother's blood as the matter from which the is formed. Last but not least, since no evidence was available to ancient and medieval scholars about the existence of eggs in the female reproductive apparatus of mammals, the theory of the two germs as a rational explanation of their sexual reproduction was devoid of half its empirical basis. Indeed Aristotle described the mammalian embryo (kÃ?hma) as " egg-like " ('ωοειδές)20 at the stage when the conceptus begins to be enclosed in a membrane (amnios) resembling that surrounding the amphibian eggs (vitelline envelope). However, according to the Stagyrite, in no way does the " egg-like " embryo 23 originate from an egg formerly present in the woman's body: the mother's contribution to the generative process is exclusively her καταµήνια. The modern concept of eutherian egg - a biological entity produced by the female and capable of developing, as a consequence of a fecundating intercourse, into a new living individual of the same species - will be introduced by William Harvey (1578-1657) in his Exercitationes de generatione animalium. Although Harvey was unable to identify any egg in the dissected body of female deer during the season (probably due to the lack of a suitable optical instrument), he first stated that " all animals were in some sort produced from eggsâ?¦The origins of either [oviparous or viviparous animals] are from an egg or at least something that by analogy is held to be so ".21 Following Aristotle, Thomas Aquinas held that in sexually reproducing animals the efficient cause of generation is the male, while the female is the material cause: " In perfect animals, generated by coitus, the active power (virtus activa) is the of the male (in semine maris), according to the Philosopher in De generatione animalium, but the matter of the fetus (material foetus) is what is provided by the female ".22 According to Aquinas, in generating the offspring the male acts through a medium, his semen, which is the instrumental cause of reproduction. The semen is endowed with a virtus formative that organizes the matter provided by the female in the menstrual blood and remains until the semen has disappeared. In a brief summary of the historical roots of contemporary knowledge about the biology of reproduction, there is no reason to enter into the metaphysical vexata quaestio of the of the human embryo and the succession of the vegetative, sensitive and rational souls according to the writings of the Angelicus.23 However, in view of its implications for the current debate on the biological and anthropological meaning of the earliest biological events in the life of a human individual, it is worthwhile to briefly touch upon the cause of embryo development. For Aquinas, " it is evident that there must be an adequate, ongoing cause of this formative process ".24 At the very beginning, the cause resides in the semen itself: the " animal spirit ", whereby a formative power (virtus formativa) operates to start the conversion of the female's blood into the primordium of the developing embryo, is present in every sperm. The semen's activity allows the appearance of the vegetative soul, followed by the sensitive soul. " And after the sensive soul, by the power of the active principle (virtutem principii activi) in the semen, has been produced in one of the principal parts of the thing generated, the sensitive soul of the offspring begins to work towards the perfection of its own body, by nourishment and growth. The active power (virtus activa) which was in the semen ceases to exist when the semen is dissolved (dissoluto semine) and its spirit vanishes (evanescente spiritu) ".25 Therefore, it is the semen's instrumental causality, imparted to it by the principal cause (the male), what starts up the generative process by acting on the female's blood and preparing it to receive the vegetative and sensitive souls. Finally, when a perfecta dispositio corporis has been reached in the human embryo only, the rational soul, which replaces the sensitive soul and assumes its functions, is immediately created and infused by God. In determining when such a perfecta dispositio is present in the developing embryo, the Aquinas follows Aristotle, according to which the soul is " the first act ('εντελέχεια) of a natural body endowed with organs (σώµατος φυσικοû 'οργανικοû) ".26 In dealing with animal and human physiology, Aristotle emphasizes the importance of senses (an animal must have sensation)27, in particular the sense of touch (without touch it is impossible to have any other sensation, for every body possessing soul has a sense of touch, as has been said)28 as well as movement. According to the Philosopher, the supposed manifestation of movement in the male and female embryo, respectively ca. 6 and 13 weeks after intercourse, represents the first signal of the presence of a sensitive soul.29 To Aquinas, this fulfils the requirement for a body to be properly disposed to receive the rational soul from God.30 The focus on the appearance of senses and their organs, as well as movement, as landmarks of animal development is shared by all pre-modern embryology. This feature of the investigations on generation and intrauterine life is comprehensible on account of the absence of any cellular, genetic or molecular 24 knowledge of the vital functions, the biological structures behind them, and the developmental processes leading to their formation. Therefore, in a correct historical perspective, it is not surprising to read pages such as those mentioned above. It is astonishing, on the other hand, to see the metaphysics of Aristotle and Aquinas sometimes applied today to the question of the anthropological status of the human embryo without a reassessment of its biological basis in the light of contemporary science. Indeed, starting from the middle of the nineteen century, cell theory, cytogenetics, biochemistry, and molecular genetics have added much value to research in the field of reproductive and , and the results of these investigations have something to say as to the role of the sperm in starting up the embryo's development or the intrinsic requirements for a human embryo to become a foetus, a newborn and an adult woman or man. The Seventeenth and Eighteenth centuries A few Renaissance authors anticipated the return to the theory of the two germs, male and female, that would characterized the investigations into sexual generation in modern times; in the seventeenth century the realism of the scientific look at human conception and early development was challenged by the imaginative speculations of a new embryological trend, that of preformist theories. Besides the outstanding contribution of his Quaderni d'Anatomia to the description of foetal development and the foundation of the quantitative approach to ,31 Leonardo da Vinci (1452- 1519) firmly held that the " seed of the female was as potent as that of male in generation ".32 On the opposite side, De Conceptu et Generatione Hominis, by Jacob Rueff (1500-1558), represents the continuation of the Aristotelian-Thomist view of the male seed causing the mother's blood to coagulate in the uterus33 (Fig. 1). The great Flemish anatomist Andreas Vesalius (1514-1564) could have easily disproved this view by his rigorous experimental investigations, but he had no opportunities for dissecting pregnant uteri and his lack of interest in embryology is notorious.34

25

Fig. 1. The Aristotelian theory of the male semen and the female blood coagulum in the uterus as illustrated in RUEFF J., De conceptu et generatione hominis et iis quae circa haec potissimum consyderantur libri , Tiguri: Fraschoverus, 1554. The uterus appears to be filled with the menstrual blood (A-B), upon which the semen progressively acts, building up the vessels and shaping the body's organs (C-F). After approximately forty or ninety days (male and female conceptus, respectively), the fetus appears as formatus (G).

The important contribution of Harvey to the emergence of the concept of in mammals cannot be understated. Nevertheless, in his De Generatione Animalium (Fig. 2)35 there is neither mention of an egg pre-existing to the introduction of the male semen in the female body nor the statement of a direct contact between the semen and something that may resemble an egg, whatever its biological origin may be. According to Harvey, any animal and human being originates from an egg (ex ovo omnia) by a progressive development, and the cause of this process is the sperm. Like Aristotle, Harvey was an upholder of epigenesis, i.e., the gradual and continuous growth and differentiation of the conceptus, which is not present as such before the allows the male semen to enter the female's body. The opposing doctrine of preformation arose almost in Harvey's

26 lifetime. If the actual originator(s) of the preformation theory cannot be named with certainty, a few authors are usually associated with the beginning of this idea. In a letter dated October 31, 1625,36 the Venetian physician Giuseppe degli Aromatari (1586-1660) mentions a work on animal generation he has been preparing for many years but has not found the time to complete. The reference to preformation in this letter is very brief: the author only says that the chick is fashioned in the egg before incubation by the hen, and owes its subsequent growth to the maternal heat and the contained in the egg, while assisted by those " vital principles " derived from the atmosphere. In 1644, Sir Kenelm Digby (1603-1665) refers to a preformation doctrine " held by some ", according to which " the embryo is actually formed in the seed, though in such little parts as it cannot be discerned until each part has enlarged and increased itself by drawing into it from the circumstant bodies more substance of their own nature ".37 Marcello Malpighi (1628-1694), often cited as one of the fathers of the preformation theory, indeed fills a singular position in the history of this doctrine. The Italian physiologist described development as a gradual process but he did not believe the embryo's parts to be formed gradually.

27 Fig.2.Cover of the original Latin edition of HARVEY W., Exercitationes de generatione animalium, London: Pulleyn, 1651. All the parts are formed at once by a kind of " precipitation " after, and as a result of, conception. However, Malpighi explained, these parts are so tiny that they only become perceptible, even under the microscope, when their dimensions have reached a certain extent.38 The contribution of Anthoni Van Leeuwenhoek (1632-1723) to the history of preformism dates from 1677, when he first reported in a letter to Nehemiah Grew,39 secretary of the Royal Society (London), that the semen of a healthy male, collected " immediately after ", contained a huge number of small " animalcules " with " a long tail ", a million of which " would not equal in size a large grain of sand " (Fig. 3A). Over the next few years, the Dutch naturalist devoted himself to the investigation of the nature and characteristics of these " seminal animalcules ". In 1683, Van Leeuwenhoek addressed the importance of spermatozoa in animal reproduction.40 He believed that the sperm impregnates the egg; however, the embryo does not come from the egg, which is concerned only with the nourishment and growth of the conceptus arising ex animalculo. These were the roles which Van Leeuwenhoek supposed the sperm and the egg to play respectively in generation. However, after fifteen years - in a comment on a letter of Dalenpatius that was published simultaneously in Amsterdam, London and Edinburgh and reported on the examination of " the constituents of human semen " by biconvex lenses and the discovery of " certain animalcules " containing a tiny " human body " (homunculus) - Van Leeuwenhoek reproduced the unbelievable drawings of homunculi (Fig. 3B) and stated that he had examined the human seed a hundred times and had never seen anything like the images of Dalenpatius that he considered to be entirely fanciful and imaginary.41 According to Cole, his genuine thought was " that the substance of the human body must be included in the seminal animalcule ", but no " definite human shape will ever be seen in it microscopically ".42 The moderate version of preformism that Van Leeuwenhoek professed is also documented by his criticism of Hartsoeker's seminal homunculus (Fig. 3D): every spermatic animalcule of the ram contains a lamb - he asserted - but the well-known aspect of the latter is not present until it has been nourished and grown in the uterus of the female. Van Leeuwenhoek compared such a development with the life history of a fly, in which the previous forms of the include the adult one, although not in a visible fashion.43

28

Fig. 3. The preformation doctrines (XVII-XVIII centuries). (A) Drawings of spermatic " animalcules ", after VAN LEEUWENHOEK A., The observations of Mr. Antoni Leeuwenhoek on animalcules engendered in the semen, in The Collected Letters of Antoni Van Leeuwenhoek, Amsterdam: Swets and Zeitlinger, 1941: 279. (B) " Homunculi " in the human male semen, after Dalenpatius (drawings by Ibid). (C) Preformation according to the ovist theory: the tiny body of the unborn child is closed into the egg (original illustration by Cook 1925-1926); redrawn by NEEDHAM J., History of Embriology, Cambridge: CambridgeUniversity Press, 1959. (D) Human spermatozoon with included homunculus, after HARTSOEKER N., Essay de dioptrique, Paris: Jean Anisson, 1694. (1729-1799) had a great impact on the debate on reproduction of his time. From accurate microscopic observations performed by himself, the Italian naturalist refuted spermism, considering animalcules as sperm's parasites that had no role in reproduction. However, " it is very natural to believe " - he wrote in 1780 - " that the(se) different orders of foetuses, which appear every year in the ovaries, are not formed successively, but coexist from the beginning with the female [frog],

29 and that they are developed and only then rendered visible ".44 Although Spallanzani was not prone to speculations concerning the presence of preformed human beings in the woman's womb, his undisputed authority indirectly supported those of ovists, some of which believed to see a small but complete body within the human ovule (Fig. 3C). How preformist ideas were influenced by, and exerted an influence on, the pre-existence theory of generation is evident in the words of the French philosopher Nicholas Malebranche (1638-1715), a priest of the Oratory of Cardinal de Bérulle who cultivated a strong interest in the scientific debates of his time: "We see in the germ of a fresh egg, which has not been incubated, an entirely formed . We see frogs in frogs' eggs and we shall see other animals in their germs also, when we have sufficient skill and experience to discover themâ?¦We must suppose that all bodies of men and beasts, which shall be born or produced till the end of the world, were possibly created from the beginning of it,â?¦in other words, that the first were created with all the subsequent individuals of their own species within them ".45 The doctrine of the pre-existence of men in the male gamete was explicitly professed by Nicolas Hartsoeker (1656-1725), the first author to figure an embryo (homunculus) locked up in the head of a spermatozoon46 (Fig. 3D). However, by 1722, Hartsoeker definitely abandoned preformism in any form, and the reason for this change of his mind was the consideration of the regen- eration of animal parts, a phenomenon that he found to be inconsistent with preformation. Indeed, in his reply to Leeuwenhoek's criticism of the homunculus, published posthumously in 1730,47 Hartsoeker made no serious attempts to defend his previous convincement. By the middle of the eighteenth century ovism suffered an eclipse and animalculism, though still in some favour, progressively changed its characteristics. Careful microscopic observations failed to reveal the existence of animalcula or homunculi in the seminal fluid. In the meantime, " epigenesis was slowly but surely gaining ground ".48 However, this theoretical transition from preformism to epigenesis was hampered by some scientists who refused both schools of thought. The Nineteenth century According to Farley,49 in the nineteenth century the debate over the nature of conception and the process of early development was strongly dominated by four influences. In the first few decades, the cultural heritage of pre-existence theories predominated, while toward the middle of the century the birth of the cell theory put the question in a new light; furthermore, the introduction of physical and chemical explanations into biological thought opened a window on the " invisible world " of fertilization; finally, the beginning of the experimental approach to reproduction and embryology by German investigators offered an unprecedented opportunity for verifying old and new hypotheses. In 1803, despite the fact that the epigenetic hypothesis had made important progress, the Scottish naturalist John Graham Dalyell (1775-1851) still expressed his unabated confidence in the existence of the foetus in the egg well before fecundation. This, he said, is " universally known " and rests on " undisputable observations ".50 However, ideas about sexual generation rapidly changed. An example of the " new deal " is provided by the work of Lorenz Oken (1779-1851),51 written when he was a student but not published until 1805. There is no preformation, he says, but every generation begins de novo, and not from an organized stage such as a preformed miniature: nullum vivum ex ovo. Oken opposed preformation in either in the egg or the sperm. When the German scientist Karl Ernst Von Baer (1792-1876) wrote his treatise on embryology (1828),52 ovism, the most long-lived version of preformism, was practically dead and he made its resurrection impossible. " The wealth of careful and sound observations which that great work contains reduced to negligible proportions the rhetorical and argumentative methods of the preformationists, and demonstrated, as such work always must demonstrate, that only by observation and experiment can the biologist hope to advance ".53 The development of the cell theory during the fifties and the sixties of the nineteenth century54 led to the concept of the cell as the fundamental unit of reproduction in all living beings. It was soon recognized that both the ovum and the spermatozoon are single cells, and it was held that development of a new organism would take place through the division and growth of cells. 30 However, at the beginning of modern biology, under the unbalanced influence of the cell theory on all the aspects of life, too much stress was laid on the multiplication of cells during early embryogenesis, so that the individuality of the developing organism was overlooked. As Russell pointed out, it " has been considered that segmentation is essentially a process of duplication and re-duplication of that which is accepted as the fundamental biological unit - the cell - and that differentiation is based upon cell-multiplication ".55 Restatement of " the obvious truth that in spite of cell multiplication the organism is and remains one unity from the earliest to the latest stages of its development "56 is due to the work of August Rauber (1841-1917).57 According to the Bavarian anatomist, the living organism is not constructed like a building, by the adding stone upon stone in an orderly way. On the contrary, its unity and primary plan are there from the very beginning, so that the organism is a whole at any given stage of development and its parts are derived from the whole by self-differentiation: " The fertilized egg is the whole in its youngest state ".58 Rauber too provided a definition of the early stages of development, by which the embryo " divides itself up in different dimensions of space and differentiates itself in orderly fashion chemically and histologically ".59 In the same period, the organismal rather than the cellular approach to understanding the embryo's developmental plan was shared, among others, by Adam Sedgwick (1854-1913)60 and Charles Whitman (1842-1910).61 In his famous lecture on the cell theory, Whitman held that an organism is an organism from the fertilized egg onwards and the continuity of biological organization is the fundamental characteristic of embryo development. However, as he clearly pointed out, this conception of development is not a revival of preformism: " Continuity of organization does not of course mean preformed organs, it means only that a definite structural foundation must be taken as the starting-point of each organism, and that the organism is not multiplied by cell division, but rather continued as an individuality through all stages of transformation ".62 It is surprising how the contemporary debate on the unity and individuality of the developing embryo was so brilliantly anticipated by Whitman! A decade after, one of his pupils at the University of Chicago, Frank Lillie (1870-1947), expressed the same view in an even more incisive passage: " If any radical conclusion from the immense amount of investigation of the elementary phenomena of development be justified, this is; that the cells are subordinated to the organism which produces them and makes them large or small, of a slow or rapid rate of division, causes them to divide, now in this direction, now in that, and in all respects so disposes them that the latent being comes to full expressionâ?¦The organism is primary, not secondary; (â?¦a) property of the whole distinct from the discernible properties of the parts ".63 The availability of the achromatic lens,64 whose images were less clouded by aberrations, played an important role in the understanding of the structure and function of the egg. Using the new lenses, several prominent scientists started investigating the very nature of the mammalian egg and its interaction with the sperm. The prevalent opinion that the ova were fertilized in situ in the ovary was challenged by the Swiss physician Jean-Louis Prévost (1831-1927) and the French scientist Jean-Baptiste Dumas (1800- 1884). In 1824, the two authors gave an excellent account of the development of the Graafian follicle of the and the rabbit, the formation of the corpus luteum, and the early development of the fertilized oocyte.65 They described the rupture of the surface of the ovary and the liberation of the mature oocyte and established the distinction between the " vesicles " in the female gonad (follicles), reported to be seven to eight millimetres in diameter, and the small " " found in the " horns of the uterus " (Fallopian tubes), which " very probably " had been originally lodged in the oviarian " vesicles ". However, it is doubtful whether Prévost and Dumas were the first to observe the mammalian oocyte: the dimensions given by the two investigators (about one millimetre) argue against the possibility that they have really isolated an unfertilized egg. In 1827, Von Baer gave a precise microscopic description of the ovum, first in the dog and then in other mammals.66 But he missed the nucleus, which had been first observed two years before as a " germinal vesicle " in the unfertilized egg of the bird by the Bohemian anatomist Jan Evangelista PurkyneË? (1787-1869), one of the fathers of 31 modern histology.67 This is not surprising, since at Von Baer's times the ovum was still unknown as a cell.68 In the early years of nineteenth century, with the decay of the animalculist theory, spermatozoa were considered to be parasitic organisms specific of the male testis. As Karl Friedrich Burdach (1776-1847) argued in his treatise on physiology, " must be considered the same as entozoa [parasitic worms] as the products of an organic substance which decomposes in the interior of a living organism. Thus, the spermatic animals appear to me to be the entozoans of the semen ".69 By the 1830s, microscopists progressively moved from the parasitic nature of the sperm to the idea that it plays an important role in fertilization. During the following decades, the parasitic theory seems to have collapsed rapidly. This was due both to the decay of the theory of spontaneous generation of parasites and the appearance of the cell theory. Theodor Ludwig Bischoff (1807-1882) was the first to admit that spermatozoa played an essential role in fertilization. The embryo, he argued, develops as a result of an " inner motion " (activation) of some elements that are present in the egg. Such motion would quickly become disorganized and the egg would " dissolve " (degeneration) if a " certain direction and intensity [â?¦is not] given to it, and this is received from the spermatozoa ". This " inner motion " - Bischoff added - is not the " visible motion " of the male germ, but an invisible " molecular motion " within the sperm, of which the " visible motion " is merely a manifestation. It is the " molecular motion " that, " imparted to the atoms of the egg, stimulates the inner motion and its continued regulation, which constitute fertilization ". Therefore, " seed operates by contact, by touching, through a catalytic power ".70 The " contact theory " of fertilization proposed by Bischoff was well received in Germany71 and France.72 While most of its supporters believed that fertilization occurred after mere contact by the sperm with the egg and that no actual entry occurred, Bischoff was less averse to the notion that the sperm penetrates the egg. Indeed, he admitted that the sperm could actually penetrate the egg immediately after this has escaped from the ovary, but, in the same 1847 paper, he added: " I do not wish to claim at all that constituents of the semen penetrate into the interior of the egg and here only exert their influence ".73 As Farley pointed out, at that time " his opposition to penetration was based on his denial of any material role for the sperm ".74 While Bischoff was somewhat vague or ambiguous in his statements on the question of whether the sperm alone or the whole seminal fluid played the vital contact role in fertilization, the British naturalist George Newport (1803- 1854) performed some experiments whose results were " most unfavourable to the belief that the spermatozoa penetrate bodily through the membranes of the ovum ".75 Furthermore, since segmentation (i.e., the first stage of embryo development) occurred soon after the contact of the frog's egg with the sperm, Newport concluded that " the act of impregnationâ?¦must take place or be commenced very rapidly; and, apparently, almost at the instance of contact of the spermatozoon with the coverings of the ovum ".76 However, his most prominent contribution to the understanding of the decisive role of fertilization in sexual reproduction was to recognize that fertilization " entails not only a process by which the egg is excited to develop, but also a process by which characters of the male parent are passed to the offspring ".77 Newport sharply observed that simple contact with the sperm may be sufficient to induce egg activation and division but not enough " to determine the transmission of more or less of the material structural characteristics of the male parent to the offspring ".78 Therefore, it was conceivable that fertilization, besides sperm-egg contact, involved a further step: " The physical entry of seminal material by which male characteristics were somehow passed into the egg. The question of the inheritance of male characters had been of little interest to laboratory-trained investigators and had been glossed over or ignored by them. To a naturalist like Newport, however, such question assumed a fundamental role ".79 In 1852, Henry Nelson (1822-1875) reported that, in the nematode Ascaris mystax, some spermatic particles are visible in the egg after contact with the male gamete has been observed. The author himself stated that " the present investigation appears to be the first in which the fact of the penetration 32 of the spermatozoa into the ovum has been distinctly seen and clearly established " thanks to the nematode's completely transparent egg.80 Surprisingly, despite this conclusion, Nelson then denied any biological role for the sperm in fertilization. His vitalistic ideas (egg activation and division is caused by vitality inherent in it) and propension towards a revival of the ovist theories of pre-existence (life is transmitted from the mother to offspring, pervading and redeveloping itself in each individual member of the species)81 prevented Nelson from recognizing the importance of the sperm's contribution to embryo development. The following year, three memories appeared which describe the entry of the spermatozoon into the eggs of two mussels, a nematode, and the frog. Ferdinand Keber (1816-1871) provided scanty evidence of the sperm passing into the ovum of Unio and Anodonta.82 Georg Meissner (1829- 1905), working at the nematode Mermis albicans, described the of its eggs and the formation of a micropyle through which one or more spermatozoa were supposed to enter the egg;83 however, its report was refuted soon after it was published.84 Newport's contribution85 was of a more robust nature, and the Londoner entomologist is usually credited with the honour of the discovery. He noted the penetration of the of the frog's egg by the sperm and provided the following precise account of his observations. " I have succeededâ?¦in detecting spermatozoa within the vitelline cavity in direct communication with, and penetrating into, the . They were first seen by myself, in company with a friend, on the 25th of March of the present year (1853) within the clear chamber (i.e., the now-called entrance funnel) above the yolk, at about forty minutes after fecondation, when the chamber begins to be formedâ?¦The spermatozoa do not reach the yolk of the frog's egg by any special orifice or canal in the envelopes, but actually pierce the substance of the envelopes at any part with which they may happen to come into contact, as I have constantly observed while watching their entrance. Some time after they have entered the yolk chamber they become disintegrated, and are resolved into elementary granules ".86 In the 1870s it was still generally accepted that fertilization involved the entry and dissolution of one or more sperm cells within an . However, the mode by which the embryo's cells arose from the fertilized egg was a subject of controversy. The role played by the nucleus in the beginning of development was not clear. Some believed that a constriction and splitting of the nucleus into two halves preceded the first cell division, but others - such as the German biologist Eduart Strasburger (1844-1912) - reported that the nucleus disintegrated prior to cell division and that the daughter nuclei appeared de novo.87 In the context of this debate, Oskar Hertwig (1849-1922), trained under Ernest Haeckel (1834-1919) in Jena and then professor of anatomy in Berlin, " posited a morphological theory of fecundation that challenged the generally held physicochemical interpretation ".88 In his two famous papers published in 1876-1877,89 Hertwig claimed that the " cleavage nucleus (i.e., the zygote's nucleus) arises from the conjugation of two different sexual nuclei, a female nucleus which is derived from the germinal vesicle and a male nucleus which is derived from the body of an entering spermatozoon ".90 In the same years, Hermann Fol (1845-1892), who, like Hertwig, was a pupil of Haeckel, investigated fertilization in the and other animals and reported that the germinal vesicle underwent two very rapid divisions. According to his pioneering but accurate observations, only one nucleus, the female , remained within the egg, while the others being expelled. Fol also described the actual penetration of a single sperm into the egg, where - according to the Swiss cytologist - it fused with some protoplasm91 of the female gamete to form the male pronucleus. This pronucleus eventually units with the female one after crossing the fertilized egg.92 The basis of the understanding of pronuclear migration, apposition and fusion was already laid one hundred and thirty years ago. The Twentieth century Notwithstanding the continuous efforts conducted in several laboratories from the second half of the nineteenth century onwards, at the beginning of the 1970s knowledge of the morphological, physiological, and molecular aspects of fertilization in mammals was still fragmentary and rough in 33 comparison with that concerning amphibian and sea-urchin fertilization. " The progress of research was hindered by the internal site of mammalian fertilization, which means that events involved in fertilization of mammalian eggs and early cannot be investigated readily in their natural environment ".93 The advent of mammalian in vitro fertilization (IVF) as a laboratory procedure in veterinary-assisted reproduction of dairy animals first, and then in human clinics, made the interacting gametes and the nonimplanted embryos available for direct investigations. Furthermore, cellular and environmental requirements for a successful IVF and embryo develop- ment forced the biologists to study several aspects of gamete and zygote physiology, pathology, genetics, and biochemistry, as well as the mechanisms underlying the stages of fertilization, in order to improve the chances of yielding a viable embryo. Between 1878 and 1953, numerous attempts were made to have a mammalian oocyte fertilized in vitro. Many reports claimed positive results. However, in the light of current knowledge, most of them do not provide convincing evidence that the fertilization was successful. Most of the pioneers of such techniques used ovarian that were probably not mature enough to be prepared for fertilization.94 To overcome this problem, Rock and Menkin95 cultured human oocytes in vitro before putting them in contact with the spermatozoa but, in retrospect,96 the time allowed for maturation (based on a previous work)97 was insufficient. Furthermore, in place of fertilization, parthenogenetic activation could have occurred in some experiments since it is known that the latter may be induced by cooling the oocytes of some species (for example, rat and hamster oocytes).98 With one exception only,99 none of the investigators involved in the early attempts to fertilize mammalian oocytes in vitro - from Schenk100 to Shettles101 - maintained the physiological temperature (37° C) throughout the experiments to avoid the risk of cooling-induced activation of the female gamete. Last but not least, " it is possible that incubating eggs for prolonged periods of time at less than normal body temperature destroyed their ability to undergo normal fertilization and development ".102 Studies carried out on mammals before 1954 did not demonstrate unequivocally sperm penetration into oocytes, nor have any of them provided clear microphotographic evidence of pronuclei formation and second polar body extrusion.103 The probable reason why most of the early IVF experiments were unsuccessful is the fact that spermatozoa added to the oocyte-containing medium were not capacitated. Soon after the discovery of by Austin104 and Chang,105 several attempts were made to achieve IVF using post-coital spermatozoa recovered from the uterus - i.e., capacitated in vivo - and, within a few years, rabbit oocytes were fertilized in vitro for the first time.106 In 1959, Chang showed that IVF-derived rabbit embryos could develop normally following transfer to foster ,107 and his description of the morphological signs of fertilization (including zygote segmentation) removes any doubt that oocytes could have been fertilized in vivo by attached spermatozoa. During the following years, IVF was extended to a wide variety of mammalian species.108 This marked the beginning of the contemporary season of morphological, ultrastructural, cytogenetic and molecular investigations on fertilization and early embryo development in mammals that has provided and continues to produce a vast amount of data concerning the process of gamete interaction and fusion, and the biological events that start up a new developing organism. Sperm-oocyte fusion, emission of the second polar body, and formation of female and male pronuclei were first studied during IVF in hamsters.109 However, the well-know phenomenon of the " two-cell block " prevented hamster embryos from developing to term.110 IVF of mouse oocytes using spermatozoa capacitated in vitro was described between 1968 and 1971.111 Further pioneering information on IVF in mammals soon came from rat,112 sheep,113 cat,114 guinea pig,115 and dog.116 Bavister, Edwards, and Steptoe first documented human IVF in a convincing way in 1969.117 Evidence reported in their studies includes sperm penetration into the ooplasm, second polar body extrusion, and formation of both pronuclei. Experimentally, fertilization of human oocytes in vitro has become feasible mainly thanks to modifications of the culture protocol used by Yanagimachi and 34 Chang for hamster IVF.118 Some of the human so obtained were able to develop normally to the blastocyst stage in culture.119 However, the first baby generated by IVF was born only in 1978,120 " apparently because the ovarian stimulation regimens may have interfered with establishment of pregnancy or produced incompetent eggs ".121 Advances in non-human primate IVF were more difficult to achieve. After the early reports in the first half of the 1970s,122 unequivocal evidence for IVF in Macacus rhesus and in the chimpanzee was available only in 1983.123 At the time when IVF began to spread rapidly, information on the development of human embryos resulting from natural conceptions was scarce. The first formal description of a human embryo before implantation is ascribed to Hamilton, who reported it in 1949.124 Over fifteen years later, an ultrastructural study of fertilization in humans at the pronuclear stage was performed by Zamboni.125 The fine morphology of 2-, 4-, and 7-cell embryos developed in vivo was also described by Pereda, Croxatto, and Coppo in the next two decades.126 Embryos were recovered by flushing the Fallopian tubes127 and observed under light and electron microscopy. The first investigations of the early events of human sperm-oocyte interactions128 and embryo development129 in vitro were reported between the mid of 1970s and the 1980s. However, since only " a limited number of such embryos can be considered normal if rigid criteria of normalcy are applied ",130 the usefulness of morphological data obtained from these studies is questionable. Sperm penetration through the cumulus oophorous, the outermost investment of the eutherian egg, was investigated, among others, by Austin in 1948131 and Blandau in 1961.132 The role of the acrosomal in cumulus penetration was much debated in the following years and, in 1988, Yanagimachi recognized that " it is somewhat surprising that the exact function of hyaluronidase in fertilization is still the subject of controversy today ".133 Starting from the observation that acrosomal hyaluronidase, at least in some species, is not necessarily involved in facilitating sperm passage through the cumulus, Metz134 and Anand135 provided the first evidence in favour of the notion that surface of spermatozoa also carries a hyaluronidase, which, in part, originates from the male genital tract, and that this surface enzyme, not the acrosomal one, aids in sperm penetration through the cumulus. Once spermatozoa have crossed the cumulus, they must complete the reaction before entering the , the oocyte's innermost investment. This was first noticed by Austin and Bishop in 1958136 and confirmed by several investigators.137 The acrosome-reacted sperm, while passing through the zona, loses all the acrosomal components except for the equatorial segment and the inner acrosomal membrane that covers the anterior part of the head. Evidences on how the spermatozoon crosses the zona were collected through many years of investigation, but no unanimous opinion was reached on a single explanation. Two hypotheses remained: the enzymatic and the mechanical one. Insights into gamete plasma membrane interactions came first from elegant microscopic studies, including light microscopy, scanning and transmission electron microscopy, and video microscopy.138 Modifications of the oocyte plasma membrane (oolemma) surface during maturation of the female gamete were investigated by Eberspaecher and Barros:139 the region overlying the meiotic spindle is free from the microvilli that cover the remaining surface of the oolemma, and sperm-oocyte fusion was shown to occur rarely in this region. Huang and Yanagimachi140 provided evidence that sperm interactions with the oolemma also occur in a spatially defined manner, with the inner acrosomal membrane of the sperm head - which is exposed after the - contacting the oolemma. Following this first approach, the equatorial segment and the posterior head adhere to and then fuse with the oocyte membrane.141 As was shown in the rodent, acrosome-intact sperm can adhere to the oolemma, but only acrosome-reacted head can fuse with it.142 In 1982, investigations on human sperm pointed out that the acrosome reaction is important for the adhesion of the male gamete to the oolemma,143 and four years before Wolf and Armstrong144 reported that the initial attachment of the sperm to this is reversible and requires , although sperm with poor 35 motility can fuse with oocytes.145 The same authors clearly showed that sperm tail movement decreases or stops within few seconds of the fusion.146 The sperm tail is also eventually incorporated into the oocyte's cytoplasm (ooplasm).147 These and other morphological aspects of gamete membrane fusion formed the basis of the knowledge of this fundamental stage of mammalian fertilization that was available two decades ago. At that time, molecular investigations on membrane interactions were started and are still in progress, and results from these studies continuously provide further insights into the complex and marvellous mechanisms of gamete fusion. Up to the 1970s, many studies concerning the process of fertilization by investigators who utilized the light microscope had furnished preliminary but accurate accounts of the events leading up to, and associated with, the formation of the genome of the embryo.148 These investigations provided the necessary background for more detailed studies by employing the greater morphological resolution achieved by the electron microscope and through the following, more recent molecular techniques. Moreover, the early studies indicated that the association of the maternally-and paternally-derived genomes may take essentially two forms. The first one consists in the fusion of the pronuclei together to form a single zygote nucleus. The formation of the zygote nucleus in this way is referred to as " pronuclear fusion " or . Eggs exhibiting such a process are said to have the " type of fertilization ".149 In the second form, both the paternal and the maternal pronuclei give rise to a group of chromosomes ready for the first cell division (zygote segmentation). In this way, there appears to be an intermixing of the maternal and the paternal chromosomes without a fusion of the respective pronuclei. In such cases the nuclear envelopes of both pronuclei disappear prior to the association of the two genomes. In mammals, two sets of chromosomes, one derived from the male pronucleus and the other from the female pronucleus, move together and form a single group that represents the prometaphase stage of the first mitosis. Thus, in this case, a real zygote nucleus (i.e., an interphase- stage nucleus, enclosed in an envelope) does not exist. Eggs demonstrating this series of events, including the human oocyte, are referred to as possessing the "Ascaris type of fertilization ".150 Wilson, who first recognized and classified the two forms of genome union during the last stage of fertilization, indicated that the factors which govern the association of the male and the female pronuclei may be closely but not necessarily correlated with the relationship between the meiotic stage of the egg and the time when it is normally inseminated.151 Fine structural analysis of the various aspects of fertilization in the previously mentioned classes of eggs is a relatively recent field of inquiry, most of the work having been published since 1960.152 This is not because these events were of little interest but, rather, due to the difficulties involved in handling the gametes and embryos for examination. Investigations concerning the development and migration of male and female pronuclei and their subsequent association have been rather minimal in number before the 1980s. The formation of the male pronucleus as a consequence of the breakdown of the sperm's nuclear envelope was first investigated in relation to chromatine dispersion.153 Breakdown of the sperm's nuclear envelope exposes the condensed chromatin to the surrounding ooplasm and appears to allow for its reorganization. It was noted that chromatin dispersion usually begins along the periphery of the sperm's nucleus and produces several circumscribed regions of chromatin conformation, which are less electron-opaque than the condensed form that is present in the nucleus of the male gamete. In the mammals, dispersion usually starts along the periphery of the midregion of the sperm's nucleus and progresses anteriorly and posteriorly.154 In the mouse155 and the hamster,156 the profile of the dispersed chromatin appeared to be ellipsoidal and reminiscent of the original shape of the sperm's nucleus. Development of the male pronuclear envelope was first studied in the sea urchin and in various molluscs and appeared to involve a similar series of events in each case. Fine structural investigations concerning the formation of this lamellar system in the hamster indicated that development of the male pronucleus in this is similar to that observed in invertebrates.157 The appearance of the 36 pronuclear envelope is first noted as an aggregation of vesicles along the periphery of the dispersing vesiculation of the sperm's nuclear envelope and elements of the endoplasmic reticulum, which are maternally derived. This observations supports the concept, still sound, that a deep cytogenetic difference exists between the sperm's nucleus and the male pronucleus and that the latter one is the consequence of a fertilization reaching a stage which already determines the full integration of the two gametes in one new cell. Reviewing this aspect of fertilization as it was known in 1973, Longo stated: " It is unlikely that the male pronuclear envelope originates exclusively from elements derived from the sperm nuclear envelope since: the pronuclear envelope appears to be augmented by the fusion of vesicles during later stages of development, the size of the pronuclear envelope is considerably larger than the sperm nuclear envelope, and redundant sperm nuclear envelope which could delimit the relatively larger male pronucleus has not been observed ".158 Following its formation and up to the time of the pronuclear association, the male pronucleus was shown to continue to undergo morphogenetic changes, which may include its increase in size,159 the progression of chro- matin dispersion,160 and the acquisition of intranuclear structures such as nucleoli161 and annulate lamellae.162 The study of the formation of the sperm aster and the migration of the pronuclei was soon initiated, even before the extensive use of electron microscopy. One of the earliest indications of sperm aster development is the dissociation of from the sperm . The central role of the sperm- contributed centriole in the reconstitution of zygotic centrosome has been established in most mammalian species, including humans.163 In contrast with the strictly maternal centrosomal inheritance in rodents, the association of the centriole with the developing sperm aster as an organization structure in the sea urchin's fertilized eggs was already suggested by Tilney and Goddard in 1970.164 In the late 1960s and early 1970s, the events involved in the association of the male and female pronuclei in an oocyte fertilized at the second methaphase of - as usually happens in mammals and man - have been studied in a few species. Ultrastructural observations of the pronuclei following their migration have been made in the rabbit, the mouse, the monkey, and the human fertilized oocytes.165 After migration, the pronuclei become cen- trally located. In most cases, one of them is proximal to the incorporated sperm's middle piece, and this association suggests that this may be the male pronucleus. Although, in some mammalian zygotes, it is difficult to distinguish the two pronuclei from each other by electron microscopy, Austin stated that they may be identified under the light microscope by their difference in size and shape.166 Following the close apposition of the pronuclei, chromatin begins to condense, as evidenced by the accumulation of electrondense reticular material that is predominantly located along the regions where the pronuclei are in intimate association. Breakdown of the pronuclear envelopes follows chromatin condensation and involves a process of vesiculation that appears to be similar to that observed in the molluscs. During the disruption of the envelopes, the condensing chromosomes become associated with involved in the organization of the . Subsequently, the chromosomes move together and intermix on what becomes the metaphase plate of the first mitotic cleavage. According to the "Ascaris type of fertilization ", a zygote's interphase nucleus was not observed in the rabbit and the mouse, and the parentally-derived genomes - which constitute the one-cell embryo's genome - are enclosed within a single nuclear envelope for the first time at the two-cell stage. As pointed out by Longo in a paper published over thirty years ago, " many texts and reviews present as a " general scheme " of meiosis and fertilization " what " pertains specifically to those eggs which are fertilized at the pronuclear stage, e.g., the sea urchin Arbacia". However, this scheme does not correspond to reality in mammals and man. " Although this situation is a result of an attempt to simplify a complex series of events, it unfortunately distorts the true picture and totally ignores the fact that the egg of most animals are inseminated at the stage of meiosis ".167 This observation is still valid

37 for a number of popular presentations that form the basis of public debates on the beginning of individual human life. PRELUDES TO FERTILIZATION Sexual generation of a new individual requires two highly coordinated biological processes: and fertilization. The first is a necessary prelude to the second, since its products, the germ cells, are essential for the occurrence of fertilization. Therefore, in looking at gamic reproduction, the two processes should be considered ad modum unius. Gametogenesis represents the collective processes of mitosis, meiosis, and cellular that are necessary for the production of mature male and female gametes. is the production of sperm cells in the male testis; is the production of oocytes in the female ovary. The cytogenetic basis of gametogenesis is meiosis,168 a unique form of nuclear division (also called reductional karyokinesis) by which a diploid cell produces genetically distinct haploid gametes. While mitosis (equational karyokinesis) maintains the cell's original level - e.g., the number of forty-six chromosomes in human somatic cells -, meiosis segregates one copy of each homologous into each , thus dividing by two the number of chromosomes that make up the individual diploid karyotype. Two aspects of germ cells make them unique. First, they are the only cells in the body that carry the genome on to the next generation. Second, germ cells only can undergo the reductive meiotic division that plays a fundamental role in generating the correct karyotype in the developing embryo. In the following sections, a few but important features of the processes involved in preparing the male and the female gamete to their eventual fusion during fertilization will be presented and discussed in the light of recent studies on spermatogenesis and oogenesis. Maturation and capacitation of the male gamete Mammalian spermatogenesis is geared towards the efficient production of a large number of sperms from a renewable spermatogonial stem-cell population.169 The entire process takes place within the seminiferous tubules of the testis and is carefully controlled both temporally and spatially. Within the spermatogenetic epithelium of these tubules, the process is sustained by extensive interaction between Sertoli cells170 and germ cells that are subject to endocrine control by the pituitary follicle-stimulating (FSH) and paracrine control by local factors, including the produced by Leydig cells171 under the primary regulation of (LH). Inhibins and activins,172 produced by Sertoli cells, participate in the regulation of the testicular function. Spermatogenesis includes the mitotic division of spermatogonia, followed by the meiotic divisions of the resulting (haploidization of the paternal genome)173 and the transformation of the round so obtained into spermatozoa (spermiogenesis).174 Spermatogonia develop from primordial germ cells (PGCs) that migrate into the undifferentiated gonad early in embryogenesis (3rd to 5th week). During the fetal period of the male's life, spermatogonia enter a dormant phase and their development is arrested. At , they begin to increase in number and spermatogenesis starts. The spermatogonial phase of spermatogenesis involves the proliferation of type-A spermatogonia, whose four increasingly differentiated subtypes (A1 to A4, as they are classified in the rat) are continuously generated from undifferentiated spermatogonia throughout the adult's life. Spermatogonia pass from the type A to the type B morphology and then enter into meiosis I to form diploid preleptotene spermatocytes, which become leptotene, zygotene, pachytene and diplotene primary spermatocytes and, at the end of the first meiotic division, result in haploid secondary spermatocytes. Completion of meiosis II gives rise to the haploid round spermatids. Four spermatids are formed from each primary .175 Finally, spermiogenesis converts spermatids into spermatozoa (Fig. 4) through several morphogenetic steps that include 1) acrosome formation by the coalescence of proacrosomal granules to form a large membrane- bound acrosomal vesicle that moves next to the nucleus and attaches to the nuclear envelope; 2) migration of the centrioles to the 's posterior pole and formation of the flagellum; 3) migration of mitochondria to form a spiral collar around the proximal part of the tail (the so-called middle piece); 4) surrounding of the nucleus by a cylindric band of microtubules (manchette)176 that 38 are associated with the posterior border of the acrosome; 5) condensation of the nuclear chromatine and flatting and elongation of the nucleus; and 6) cytoplasm reduction. In humans, stem-cell proliferation and differentiation, meiosis, generation of haploid germ cells, and morphogenesis of the developing sperm require approximately 2 months, while the spermiogenetic period takes about 5-6 weeks.177

Fig. 4. Structure of the human spermatozoon: (1), head; (2) neck; (3) middle piece, containing the mitochondrial sheath; (4) tail; and (5) end piece. The male gamete's head is partially covered by the acrosome (A); the remaining part is named post-acrosomal region (B). (ROSATI P., DE SIMONE I., GUIDOTTI L. ET AL., Embriologia Generale, Milano: Edi-Ermes, 1993).

In the final stages of spermiogenesis, the profound morphological changes that transform spermatids into spermatozoa result in the loss of all the cytoplasm except for a thin stratum compressed in the perinuclear , a sheat located in the head between the nucleus and the acrosomal and/or the cell membrane. Formation of this theca parallels the extrusion and removal of the cytoplasmic portion

39 (cytoplasmic droplets)178 that is fago- cytated by the Sertoli cells before the sperms are released into the lumen of the seminiferous tubule. The sperm's head has highly specialized structures to interact with the oocyte in the fertilization process. The acrosome is a cap-like structure covering the anterior portion of the sperm nucleus (Fig. 5). The acrosome varies in size and shape from species to species; however, its basic structure is the same in all mammals. It contains a variety of hydrolytic that are released during the " acrosome reaction " and play important roles in the fertilization process, such as the hydrolysis of the matrices when they pass through the cumulus and the zona pellucida. The acrosome originates in the Golgi apparatus and its components are transported from the apparatus to the growing acrosomic structure by the trans-Golgi vesicles, which originate in the stacks during early spermiogenesis.179 Although several proteins localized in the acrosome of the mature human sperm have been identified,180 the organization of these molecules - except for SP-10, proacrosin, acrin1 (MN7) and few others - is not yet well understood. There is growing evidence that acrosomal proteins gradually become compartmentalized during

Fig. 5. Head of the human spermatozoon (section). The inner acrosomal membrane overlies the nucleus, whereas the outer acrosomal membrane apposes the plasma membrane. At the most posterior region covered by the acrosome, the sperm head is referred to as the equatorial segment. (Modified from TALANSKY B.E., Fertilization and early embryonic development in the human, in COHEN J., MALTER H.E., ID. ET AL., Micromanipulation of Human Gametes and Embryos, New York: Raven Press, 1992: 84-112). sperm passage through the . Some of them are further modified during the fertilization process. These findings indicate that acrosomal molecules are not only restricted to a specific region of the acrosome but also undergo ongoing relocation in a stage-specific manner during sperm maturation in the testis and epididymis. Such maturation-associated modifications are considered essential for sperm molecules to reach the correct or final site before fertilization.181

40 During mammalian spermiogenesis, extensive remodeling of chromatin takes place. Nuclear elongation and chromatin condensation occur at the same time of the modifications in the nuclear basic proteins associated with DNA. A number of biochemical events accompany the displacement of histones and the appearance of a set of basic nuclear proteins, such as tH2A, tH2B, H1t, spermatid- specific H2B (ssH2B), haploid germ-cell-specific nuclear protein 1 (Hanp1), testis-specific HMG (tsHMG), histone H1-like protein in spermatids 1 (Hils1), " transition proteins " (TPs) and protamines. Histone synthesis stops during spermiogenesis and histones are replaced by a set of TPs,182 which are subsequently replaced by protamines.183 The whole process results in a greater than six-fold condensation of chromosomes, producing a tightly packaged chromatin structure. Although some aspects of chromatin structure in spermatozoa have been elucidated, the molecular mechanisms that contribute to chromatin restructuring are still under investigation. Most mammals have one protamine form that replaces TPs, but a few species, including humans and mice, have two protamine forms. In mice, gene disruption experiments involving protamine 1 or 2 have shown that both protamines are essential for , and that haploinsufficiency is caused by a in one protamine allele.184 Moreover, when protamine 2 is disrupted, the resultant sperms are unable to generate a developing embryo.185 These results indicate that protamine 2 is required for the process of nuclear compaction during spermiogenesis. Many nuclear proteins are expressed systematically during the nuclear condensation period.186 Almost all of these proteins are derived from histone H1 and undergo complex processes of post-translational modification in mammals. In the human sperm, protamines and histones are present at ca. 85:15 ratio.187 In contrast to its somatic counterpart, the sperm's DNA-protein complex is arrayed into a series of parallel sheets. The stacking of these sheets and their stabilization by disulphide bonds enable them to be assembled into a toroid structure. 188 This condenses the sperm nucleus chromatin so that it becomes at least six times more compact than the somatic nucleus one.189 Nevertheless, some genomic regions still remain in a more relaxed deoxyribonuclease-I-sensitive conformation190 that contains short, histone-enriched segments191 that are bound to the nuclear matrix.192 Studies on nuclear matrix depletion in the mouse have suggested that this structure might provide epigenetic marks that are necessary for early post-fertilization development of the embryo while maintaining the integrity of the paternal genome.193 Maternal chromosomes decondense readily into the female pronucleus following the completion of meiosis II at fertilization. On the contrary, before participating in pronuclear development, paternal chromatin requires an additional processing step, comprising the relief of disulfide bond cross-linking and the replacement of protamines with oocyte-derived histones. It has long been held that the tightly packaged chromatin within sperm nucleus is transcriptionally silent.194 However, in situ hybridization experiments localized both b- and prm2 mRNAs to the head region of normal human spermatozoa,195 suggesting that this RNA is a regular component of the terminally-differentiated male gamete. Further investigations196 " independently confirmed that spermatozoa contain a wealth of both known and unknown protein-encoding and non-coding RNAs. The presence of this suite of RNAs is intriguing when we consider that mature sperm have little if any cytoplasm ".197 Micro-RNAs198 have been identified as well in human sperm.199 At fertilization, all these mRNA are delivered to the ooplasm as a consequence of sperm-oocyte fusion,200 adding a further set of male factors that contributes to zygote formation and development even before the mixing of maternal and paternal chromosomes, and " raising some interesting questions regarding its potential role in early embryonic development ".201 Sperm maturation: acquiring the ability to move and capacitate Testicular spermatozoa are neither motile nor able to undergo the process of capacitation that will enable them to fertilize the oocyte. During their transit from the testis to the eiaculatory duct a complex series of events occurs, called epididymal maturation,202 which allows the spermatozoa to acquire the ability to move and capacitate. These functional changes take place within the epididymis, through which male gametes pass and where they are stored. It takes approximately ten days for human 41 spermatozoa to reach the cauda of the epididymis, the locus of their storage before ejaculation. During this passage, the epididymis provides a specific intraluminar environment where maturation of sperms occurs. To this end, they are bathed in a caput to cauda gradient of ribonucleases,203 glycosidases,204 and proteases, including the 26S proteasome.205 This array of enzymes allows the removal of molecular compo- nents that are extruded from the maturing spermatozoon as unnecessary to (or impeding for) a successful fertilization. Most of mammalian sperms, including the human one, display two types physiological motility: activated motility (also simply referred to as " sperm motility ") and hyperactivated motility.206 The flagellum of an activated sperm generates a symmetrical, lower amplitude waveform that drives the sperm in a relatively straight line. In the hyperactivated sperm, the flagellum's beat becomes asymmetrical and its amplitude increases, resulting in circular or figure- eight trajectories. Both activated and hyperactivated motilities are important for normal fertility: the former is acquired by the sperm during its transit through the epididymis while the latter is developed is association with the process of capacitation.207 Ca2+ is one of the most important ions involved in the regulation of human sperm motility.208 The role of extracellular concentration ([Ca2+]e) in activating spermatozoa has been much debated. While there is no doubt that external calcium is essential for sperm motility, it is the intracellular ion concentration ([Ca2+]i) that must be strictly regulated to allow precise timing for sperm activation. Decreasing levels of [Ca2+]e between the caput and cauda of the epididymis are associated with progressive development of sperm motility and increase in protein tyrosine phosphorylation.209 The molecular mechanisms connected with the detrimental effect of excess [Ca2+]e on sperm motility are still obscure and different hypotheses are under investigation. 210 Several evidences indicate the importance of protein phosphorylation in transducing the stimulatory signals to motility. The adenylate-cyclasecAMP- PKA system has been demonstrated to be involved in tyrosine phos- phorylation of different flagellum's proteins associated with sperm motility,211 and defects in such protein phosphorylation have been described in asthenozoospermic patients, whose mature gametes show reduced motility and hyperactivation.212 There is growing evidence that " epididymal maturation can be considered the quality-control phase that ensures a healthy sperm ".213 Ubiquitin molecular marking of cellular components, including the paternal mitochondria, is essential for a spermatozoon to contribute correctly to fertilization and embryo development.214 Effective removal of paternal mitochondrial structures ensures that exclusive maternal inheritance of these metabolically-essential organelles, a rule also known as maternal mitochondrial homoplasmy.215 This protective mechanism provides a controlled way of removing products of oxidative damage: mitochondria are prone to oxidative damage during sperm's life owing to the limited repair capacity of their genome. Failure of the ubiquitin surveillance compromises embryonic development in humans216 and the first clinical case of heteroplasmy confirms that survival after birth is problematic due to severe exercise intolerance.217 The fusogenic properties of the sperm's plasma membrane that are essential to fertilization require the presence of high concentrations of polyunsaturated fatty acids (PUFA). This predominance of PUFA renders spermatozoa easily susceptible to lipid peroxidation due to attacks from (ROS), a phenomenon known as " oxidative stress ".218 The antioxidant strategies that protect the sperm during its epididymal transit involve a few enzymes, including members of the glutathione peroxidase (GPX) family, catalase, indolamine dioxygenase, and superoxide dismutase.219 Sperm capacitation: getting into the power to fertilize oocytes It has been known for over 50 years that mammalian spermatozoa are unable to fertilize oocytes when released from the male reproductive apparatus. To penetrate the coverings of the oocyte and fuse with it, sperms must be in a specific state of readiness known as capacitation. The term " capacitation " was originally coined to mean " a need for sperms to spend some time in the female tract before they can penetrate the zona " pellucida220 and " the time required for a physiological change in the spermatozoa 42 enabling them to attain fertilizing capacity ".221 With the advent of IVF, biologists started investigating mammalian sperm capacitation outside the female reproductive apparatus and inducing it in the laboratory by gamete manipulation: this " allowed studies of capacitation at the molecular level while obscuring the original definition of this process ".222 Indeed, nowadays the term is sometimes misused. It was long believed that capacitation is a rather static and prolonged property of the sperm, until it was clearly demonstrated in human spermatozoa that " only a small fraction of the sperm population is capacitated at any given time, that the capacitated state is transient (1-4 hours of life span), that it occurs only once in the sperm's lifetime, and that different sperm individuals get to this stage at different time points, resulting in a continuous replacement of capacitated cells within the sperm population ".223 These phenomena raised the intriguing question of why the duration of capacitation in humans is so short and why there are so few spermatozoa capacitated at any given time. Michael Eisenbach and his collaborators224 suggest the hypothesis that the role of these features is to prolong the presence of a number of capacitated sperms in the female genital tract: the continuous replacement of capacitated gametes is a mechanisms to compensate for the lack of a strict time coordination between coitus and by extending the overall fertilizing ability of the deposited sperm population to the few days that follow its penetration of the cervical mucus. Indirect experimental evidence that supports this hypothesis was recently obtained by a comparative study of the time course of in vitro capacitation in rabbit and human spermatozoa: it was more extended in the latter than in the former. Since in the rabbit females ovulation is induced by coitus, while woman's ovulation is periodic (i.e., independent of sexual intercourse), the reported finding " suggests that the capacitated state is programmed to maximize the prospects that an ovulated egg will meet spermatozoa in the best functional state ".225 Capacitation is preceded by the loss or removal of sperm-surface-associated inhibitory factors that originate from the secretions of the male tubules and ducts through which the spermatozoa passed. The removal of such " decapacitation " factors from uncapacitated sperms results in a rapid increase in their fertilizing ability in vitro226 and reintroduction of these factors readily inhibits capacity to fertilize the oocyte.227 The most well-characterized decapacitation factor is DF, a 40 kDa located on the post-acrosomal head region of uncapacitated mouse spermatozoa.228 The identity and molecular mechanisms of action of a number of additional proteins supposed to be involved in blocking sperm capacitation remain largely unknown. Recently, a phosphatidylethanolamine binding protein (PBP) was identified as a novel candidate for a decapacitation factor.229 During the capacitation process, a number of biochemical changes occur on spermatozoa. They include increased adenylyl cyclase activity and elevated levels of cyclic adenosine monophosphate (cAMP); protein tyrosine phosphorylation of a subset of sperm components; increased intracellular pH (pHi); Ca2+ influx; modifications of the sperm plasma membrane proteins, , and lipids; and changing in the lectin-binding patters as a consequence of surface alterations.230 Although cAMP has been known for a long time to play an important role in sperm capacitation, only recently several ligands were discovered that are able to act as " first messengers " when they bind to separate, specific receptors on the sperm's plasma membrane and so affect the production of cAMP.231 Changes in membrane lipids could be related to the unmasking of sperm receptors for zona pellucida proteins that occurs during acquisition of the acrosome reaction competency.232 The role of actin cytoskeleton in mammalian sperm capacitation has been recognized as an important one. Polymerization of globular (G)-actin to filamentous (F)- actin occurs during capacitation and depends on protein kinase A (PKA) acti- vation, protein tyrosine phosphorilation, and phospholipase D activation.233 Actin polymerization is necessary for sperm incorporation into ooplasm234 and for the subsequent decondensation of its nucleus.235 F-actin formation is required for the translocation of phospholipase C from the cytosol to the sperm plasma membrane that occurs during capacitation.236 Maturation and ovulation of the female gamete

43 " The growth of oocytes within ovarian follicles and their development to mature eggs have fascinated biologists for centuries, and scientists have long realized the importance of the 's somatic cells in nurturing oogenesis and delivering the oocyte to the by ovulation ".237 In its development aimed at yielding a mature gamete, competent for fertilization, the mammalian female germ cell goes through several transitions and blocks. These transformations begin with the specification of PGCs in the embryo. In mammals, PGCs appear to be induced de novo from other cells in the early gastrulating embryo.238 The embryological origin of PGCs is extragonadal: to reach the gonadal rudiment, they migrate through various tissues. The mechanisms of PGC migration during are philogenetically highly conserved and involve intrinsic and somatic cues, attraction and repulsion, and amoeboid motility.239 Several factors involved in the molecular mechanisms that lead to the establishment of the have been identified in the PGCs in different species (conserved deter- minants include vasa, tudor, pumilio, nanos, germ cell less, and mago nashi).240 Oct-4, a maternally inherited factor that is essential for the mammalian germline,241 is supposed to act as a transcriptional activator of genes required in maintaining an undifferentiated totipotent state and may repress the transcription of lineage-specific regulatory genes. The number of PGCs increases by mitosis during migration.242 When the PGCs arrive at the developing ovary, if a male sex-determining factor is not is not present in the genome and properly expressed, they are differentiated into oogonia and continue to multiply mitotically until meiosis I begins. From this stage onwards, a new name is assigned to them (primary oocytes) and their number will reduce progressively (ca. 7 millions, between 700000 and 2 millions, and ca. 400000 by five months of , birth, and puberty, respectively). In mammals, female germ line meiosis begins well before birth. However, the process is soon arrested in the diplotene stage of prophase I (P-I) and does not resume until around the time of ovulation during the fertile years of the woman's life. " Germ cell destiny is determined once meiosis is initiated. A meiotic germ cell can either degenerate or complete meiosis but cannot return to the mitotic proliferation ".243 Indeed, in each adult woman, all but a few oocytes (ca. 400-500) will mature to the ovulatory stage and a much more limited number will eventually complete meiosis II when fertilization occurs. In the perinatal ovary, oocytes arrested in the diplotene stage of meiosis I244 and surrounded by a single, squamous layer of somatic cells form a finite population of non-growing primordial follicles. A number of such follicles are recruited and their somatic cells (called granulosa cells) become cuboidal and proliferate to yield a pool of primary follicles committed to the subsequent stages of follicular development throughout the reproductive life span of the woman. From germinal vesicle breakdown to metaphase-II block The oocyte is a highly differentiated cell. It is " specialized to its unique functions and progresses through a series of developmental stages to acquire a fertilization competent phenotype ".245 (Fig. 6) In comparison to mammalian somatic cells, oocytes have a very different cytological architecture. They are the adult female's largest cells (in humans, their in vitro diameter is 160±20 µm, n= 545)246 and contain stockpiles of all the cytoplasmic elements (organelles, macro- and micromolecules) required to support the first rapid embryo segmentations once fertilization occurred. In addition to having vast numbers of organelles typical to eukaryotic cells - such as the endoplasmic reticulum (ER), mitochondria (MTs), and the Golgi apparatus (GA) -, oocytes also possess structures that are not found elsewhere. These include annulate lamellae and cortical granules.247 Furthermore, all animal eggs are

44

Fig. 6. Structure of the human (section). At ovulation, the oocyte, arrested at the metaphase II stage of meiosis, is enclosed within a glycoproteic envelope (zona pellucida) and surrounded by the follicular cells of the corona radiata, which are held together by a matrix. The thin space between the gamete's cell membrane (oolemma) and the zona pellucida is called perivitelline space and accommodates the polar bodies as they are expelled from the oocyte's cytoplasm (ooplasm) at the end of the first and the second meiotic karyokinesis (PB1 and PB2, respectively). (Modified from ROSATI, DE SIMONE, GUIDOTTI, Embiologia Generaleâ?¦). coated by one or more extracellular coverings: mammalian oocytes are surrounded by a thick coat of , called the zona pellucida (ZP),248 and an extracellular matrix, named perivitelline space (PVS).249 The nucleus of oocytes arrested at the beginning of meiosis I is very large and is termed germinal vesicle (GV). The nuclear envelope (NE) of GV contains much more nuclear pore complexes (NPCs) - structures mediating the transport between cytoplasm and nucleus250 - than the nucleus present in a typical , e.g., a fibroblast. In contrast, the size of karyokinetic chromosomes and spindle's microtubule asters is very similar to that of dividing somatic cells. " Although the functional relevance of the enormous GV is unclear, it may be a means of storing spontaneously assembling NE components such as NPCs and lamins over long periods of time ".251 When oocytes exit from the P-I block and re-enter meiosis, the GV breaks down. Soon after the occurrence of this process, resulting from mechanical forces of the cytoskeleton as well as biochemical disassembly of the nuclear envelope protein complexes,252 oocyte's chromosomes progress to the metaphase-I (M-I) plate, rapidly enter anaphase I (A-I) and telophase I (T-I), and then the first polar body (PB1) is extruded. The chromosomes remained within oocyte promptly align again in the second

45 meiotic spindle. Mammalian oocytes arrest at this stage (metaphase II, M-II) and only enter anaphase II (A-II) upon fertilization.253 During meiosis I and II, oocytes are in the karyokinetic phase of cell cycle, characterized by a high maturation-promoting factor (MPF) activity.254 As a consequence of high MPF expression, NE proteins such as lamins and nucleoporines are phosphorylated, preventing lamin polymerization and NPC assembly. However, although MPF activity drops between meiosis I and II, NE does not reform around the chromosomes. As Lénárt and Ellenberg noted, " preventing NE formation is probably an important prerequisite to inhibiting DNA replication in the reducing division ".255 Starting from follicle formation and continuing throughout its development, bidirectional communication between the oocyte and its surrounding granulosa cells is essential for the coordinated maturation of both the gamete and the somatic cells.256 For example, defects in meiotic maturation are documented in mice lacking the junctional protein connexin 37 involved in oocytegranulosa cell interactions.257 Oocytes depend on somatic cells for their growth and development,258 meiosis regulation,259 and modulation of genome transcription.260 Bidirectional signaling is required for the progression of follicle development beyond the primary stage. Specific proteins participating in this regulatory process have been identified and include growth differentiation factor 9 (GDF9, secreted by the oocyte), KIT receptor (oocyte surface) and its ligand KITL (produced by granulosa cells).261 Furthermore, follicles become sensitive to gonadotropins. For example, GDF9 promotes the formation and integrity of the cumulus-oocyte complex by inducing hyaluronan synthase 2, pentraxin 3, and tumor necrosis factor-induced factor 6, and through the suppression of urokinase plasminogen activator.262 From the follicular to the tubal environment Ovulation, triggered by the mid-cycle surge of luteinizing hormone (LH) levels, is the event that marks the transition of the female gamete from the follicular anthrum to the extraovarian environment, normally represented by the endosalpinx lumen, where the oocyte is bathed by the oviductal fluid.263 Just before ovulation, gonadotropins stimulate the granulosa cells to produce and secrete hyaluronic acid (HA), a non-sulfated glycosaminoglycan, that disperses these cells and embeds them in a mucus- like matrix, a process called " cumulus expansion " (or mucification).264 The formation of the cumulus matrix is also controlled by an oocyte-derived factor. Experimental evidences from in vitro studies on cumulus cells strongly argue that GDF9 is the factor that stimulates HA synthesis.265 In a fully expanded cumulus-oocyte complex (COC), HA is present at a concentration of 0.5-1 mg/ml as the predominant matrix component, determining the viscoelastic properties of the complex. Other glycosaminoglycans are also present in the COC matrix, such as chondroitin sulfate, dermatan sulfate and a heparin-like substance. The matrix seems to be anchored to the surface of the cumulus cells by the HA receptor CD44.266 By this time, the oocyte has completed meiosis I, yielding a large secondary oocyte that contains almost all of the cytoplasmic material and a small round structure, the first polar body (PB1), in which half of the mother's homologous chromosomes are packaged. The polar body is localized within the perivitelline space (PVS), i.e., the thin space between the oocyte and the zona pellucida (Figure 6). Meiosis II follows at once but will be arrested in the metaphase and will so remain until fertilization. The spindle apparatus of the second meiotic division appears at the time of ovulation. A second polar body (PB2), containing half of the sister chromatids, will eventually form during fertilization, soon after gamete membrane fusion and the engulfing of the sperm.267 Follicular response to LH surge, culminating in ovulation, is a complex but well-orchestrated process, involving a continuously cross-talking between granulosa cells and the oocyte. LH activates multiple signaling pathways via its specific receptor.268 Furthermore, some signals - for example, Ca2+ and cAMP rising - diffuse into the oocyte through the gap junction network to elicit the gamete's responses.269 Rupture of the Graafian follicle270 occurs within 24-36 hours of LH surge. Locally synthesized prostaglandins appear to be essential to follicular rupture, being primarily responsible for 46 the increase in vascular permeability, which sustains positive intrafollicular pressure.271 The progressive reduction in the tensile strength of the follicular wall results in its complete rupture.272 The flow of fluid and vascular transudate then carries the COC out of the follicle on to the surface of the ovary, where it is picked up by the infundibulum and enters the ampulla of the .273 " The oviduct has been recognized for more than 50 years as a reproductive organ responsible for creating a microenvironment that serves to facilitate gamete functions, fertilization and early embryo development. This evidence has come from numerous species and scientists using a variety of techniques to recover oviduct secretions and subjecting them to functional and compositional analyses ".274 Molecular studies of tubal secretions document that this fluid is biochemically very complex, with proteins as a major component. Although most of these proteins are found in other tissues, a group of oviduct-specific glycoproteins (OSGs or oviductins) has been described in humans275 as well as in other species (mouse, hamster, sheep, cow, pig, horse, and baboon).276 Because OSGs have a unique tubal origin, and their secretion is greatest in the periovulatory period, it has received considerable attention to evaluate their function. The effects of OSG in vitro on the female and male gametes and the embryo have been reviewed in detail elsewhere277 and include maintenance of sperm motility, stimulation of capacitation, increasing of the fertilization rates, and facilitation of the early stages of embryo development. 278 However, the appealing hypothesis that OSGs play a vital role in the promotion of fertilization and preimplantation embryogenesis has been questioned by the observation that OGS null mice have normal fertility.279 Recently, Gabler, Chapmam, and Kilian280 determined that the oviduct fluid contains osteopontin, a glycoprotein found in many tissues and known to be involved in cell adhesion and cell signaling by binding to integrins. Osteopontin is expressed by the oviduct's epithelium and, like OSGs, appears to have multiple beneficial effects on gametes and embryo physiology in vitro. Nevertheless, the null osteopontin mouse is fertile.281 Further studies directed at evaluating the function of specific factors of the tubal environment are in progress and will better define their contributions to the fertilization- and embryo development- promoting action of the oviduct fluid. The picture that is emerging is that endosalpinx secretions collectively provide a failsafe system to ensure fertility: the favorable milieu is not dependent on a single component. THE FERTILIZATION PROCESS The process, its stages and their events: understanding what happens at fertilization Fertilization is neither an instantaneous phenomenon nor a single spatial rearrangement of the elements that make its fulfillment possible. By the contribution of the sperm, fertilization " reorganizes the egg into the zygote through an interacting series of structural and biochemical changes occurring in a distinct temporal and spatial pattern ".282 As most of the physiological occurrences in the life of an organism, fertilization is a complex , i.e., a well-orchestrated series of causes and effects of a cellular, genetic, chemical, and physical nature whose end is to carry out a specific function. In our case, the biological end is the generation of a developmentally totipotent cell (zygote or one-cell embryo) " that is the beginning, or primordium, of a new individual "283 reproduced in a gamic way. Such a geneti- cally and cytologically new cell is the outcome of a great number of coordinated molecular and ultrastructural events that put the interaction of the two gametes forward, and the discreet, successive steps of this progress toward the constitution of the novel biological entity are known as " stages of fertilization ". The distinction of several stages within the fertilization process not only accomplishes the task to organize the single ultrastructural and molecular events into an intelligible pattern that makes sense as the function of fertilization; it also introduces us to a deeper understanding of the cause-effect relationship underlying this fundamental life process. In such heuristic perspective of multistep living phenomena, whose theoretical basis is developed by the philosophy of biology, a special role is ascribed to the identification of the " critical stage "; i.e., the stage where the process will move away from its state of indetermination as to which of two or more 47 possibilities will be actualized or from a condition of reversibility that allows the biological system to stop at equilibrium point or return to its original state. Once the " critical stage " has been entered or passed, the process is pushed forward and - if all the required genetic, molecular and cellular factors are available and no adverse event occurs - it will reach its end in due course. In the following review of the main events that contribute to the beginning and the progression of the stages of fertilization in mammals (with some reference to other animal models where necessary), recent data will be reported and discussed that can contribute to the insight into this fundamental process of the life cycle, the definition of its main cytological, genetic and biochemical features, and the identification of its " critical stage ". According to the recent literature, the events that participate in the process of mammalian fertilization can be grouped into successive stages, the ordinary way by which " two sex cells (gametes) fuse together to create a new individual with genetic potentials derived from both parents ".284 Using several animal models and - more recently - by the study of human IVF too,285 each of these stages has been investigated in some detail and characterized at the ultrastructural, genetic and molecular level, and studies are currently being carried out in many laboratories to shed light on the inmost mechanisms that give rise to and control them. These investigations have resulted in an extensive literature whose contribution to the understanding of the fertilization process is hard to summarize in a limited space. Therefore, a selection has been made of the aspects of contemporary research that further clarify what happens " in the process by which a sperm and an egg unite to form the first cell in the development of a multicellular organism ".286 The reported distinction into stages does not entail the overall unity of the fertilization process that rests on the end toward which all the stages are oriented and for which they provide the full repertoire of structures, functions, and regulations: to " pass the genome from one generation to another and initiate the development of a new organism ".287 Furthermore, since " the fertilization process in mammals consists of numerous events that must take place in a compulsory order to produce a viable zygote ",288 it is such an intrinsic order of causes and effects that allows us to fix the boundaries of the process with respect to its beginning and its end and look for the stage that definitely orients the process to produce its final effect, i.e., " to initiate the embryonic development of a new individual ".289 The crossing the barriers: from gamete contact to perivitelline-space entry The penetration of cumulus oophorus Spermatozoa approach the ovulated expanded cumulus oophorus with the goal to reach the ZP and cross it (Fig. 7). In comparison to the abundant data and considerations on the importance of cumulus- oocyte interaction during oocyte maturation, the role of the cumulus cells in the fertilization process has received less attention. While cumulus-intact oocytes are not required for IVF (indeed, oocytes are routinely " decoronized " before adding the male gametes to the culture medium or injecting the sperm into the oocyte), nevertheless, in some species, cumulus cells definitely improve fertilization rates.290 As clearly

48

Fig. 7. The sequence of early stages in . (a) Capacitated spermatozoa penetrate the cumulus oophorus and (b) adhere to the zona pellucida, where the acrosome reaction takes place. One or more acrosome-reacted spermatozoa cross the zona pellucida and (c) enter into the perivitelline space. Adhesion of the sperm head to the oolemma is followed by (d) gamete membrane fusion and the engulfing of the sperm into the ooplasm. (Modified from EVANS J.P., FLORMAN H.M., The state of the union: the cell biology of fertilization, Nature cell Biology 2002, 4(1): S57-S63). pointed out by Olds-Clarke, " it is a mistake to assume that sperm-cumulus cell interactions are superfluous, as sperm penetration of the zona is enhanced significantly if the cumulus cells are left in place ".291 However, the mechanisms by which the cumulus and its extracellular matrix affect sperm interaction with ZP are still not definitively known. They could include chemical signals to sperm signal-transduction pathways possibly altering motility and/or the final steps of capacitation, and cumulus cells may continue to supply the oocyte with useful metabolites even after ovulation. Furthermore, the hyaluronic-rich matrix appears to be continuous with the lining of spaces in the outer ZP and thus could have a beneficial effect as the sperm penetrates it.292 Finally, the physical resistance of the cumulus oophorus is likely to be significant in sperm-ZP interactions.293 To penetrate the cumulus oophorus, sperm use hyperactivated motility294 and a glycosylphosphatidylinositol-anchored surface hyaluronidase, named PH-20.295 Since no proteases have yet been implicated in this penetration, according to some authors sperm hypermotility and the hyaluronidase located on the sperm surface " are necessary, and perhaps sufficient, to digest a path through the extracellular matrix of the cumulus cells ".296 However, mice lacking PH-20 have normal fertility in vivo, although delayed penetration of the cumulus leads to reduced rates of IVF.297 Thus, " there may be additional that compensate for the absence of PH-20 or the importance of enzymatic activities in penetrating the cumulus needs reassessment ".298

49 Sperm-zona pellucida adhesion Sperm-ZP attachment is called " adhesion " rather than " binding " because the first term refers to cell- cell or cell-extracellular-matrix associations, while the second one is more properly reserved for a specific association between two molecules. While one (or perhaps two) of the solubilised ZP glicoproteins bind(s) specifically with various putative " ZP receptors " located in the sperm's cell membrane,299 sperm association with the intact ZP enclosing the oocyte occurs between a cell (male gamete) and an extracellular matrix, and therefore constitutes an adhesion.300 Adhesion of sperm to the ZP occurs in several steps that are not always apparent at the light microscope level of observation. The initial contact between sperm and ZP surface is at the level of the equatorial and/or postacrosomal region of the acrosome-intact male gamete. As documented by video recordings in the hamster, at first the sperm pivots freely around this point of contact, although cumulus cells reduce the degree of movement.301 Drobnis suggested that the cumulus could help orient the sperm head for its penetration of ZP and provide mechanical resistance to direct flagellar forces to the proximal portion of the tail.302 Anyhow, regardless of the eventual role of the cumulus, " it appears that the acrosome-intact sperm is not simply lying in the zona, but actually adhering to it. If there were contact but no adhesion, flagellar movement would move the head off the zona ".303 In the sperm head equatorial region, intermembrane bridges stabilizes the outer acrosomal membrane and the overlying plasma membrane, to which the postacrosomal sheath is closely linked by periodic ridges or projections.304 In mammals (al least in most of them), it is likely that the initial phase of adhesion to ZP occurs only with acrosome-intact sperms.305 Considerable evidence indicates that carbohydrate recognition plays a key role in this interaction. 306 The preferred animal model for investigating the role of carbohydrate recognition in mammalian fertilization is the mouse. Murine sperm initiate fertilization by binding to the ZP, a matrix composed of three major glycoproteins designated mZP1, mZP2, and mZP3.307 mZP3 has been implicated as the component that mediates both initial binding and the induction of the acrosome reaction.308 Its genetic deletion leads to the complete loss of a functional ZP and infertility.309 In mouse, evidence suggests that initial gamete adhesion is mediated by an unusual sperm surface receptor, b1,4-galactosyltransferase-I (GalT) binding to specific oligosaccharide chains on the zona pellucida glycoprotein ZP3.310 Binding of ZP3 oligosaccharide chains induces aggregation of GalT, thus activating, directly or indirectly, acrosomal exocytosis.311 However, more recent studies have questioned whether sperm-egg binding can be solely accounted for by sperm GalT binding to ZP3 oligosaccharides, and suggest that sperm-egg binding is likely mediated by receptors in addition to GalT and ZP3. Gamete recognition appears to be more complex than a single receptorligand interaction, and can be resolved into at least two distinct binding events: a ZP3- and GalT-independent interaction responsible for gamete adhesion, and a ZP3- and GalT-dependent interaction that facilitates acrosomal exocytosis.312 The acrosome reaction Exocytosis of the acrosome (or acrosome reaction, AR), is a morphological alteration that occurs prior to penetration of the ZP. AR " is a fundamental reproductive strategy which is a prerequisite for successful fertilization ".313 As is the case in somatic cell regulated exocytosis, Ca2+ is an essential mediator of the AR.314 However, AR differs from other known exocytotic phenomena in several ways, mainly in that the sperm contains a single secretory vesicle (acrosome) whose release is a singular occurrence. Nonetheless, " it is suspected that sperm uses the same conserved fusion machinery and regulatory components as characterized for other secretory events ".315 Exocytosis is a highly regulated, multistep event including targeting, tethering, priming, docking and fusion of secretory vesicles with the plasma membrane of the cell. There is convincing evidence that AR is completed after the sperm binds to ZP and one or more components of the ZP induce sperm to complete the AR. Currently, it is believed that ZP3, the oocyte's 50 receptor for sperm, is also involved in AR completion.316 ZP3 acts by stimulating the depolarization of sperm (ca. -25 mV) and this effect is specific to this ZP glycoprotein.317 Several steps in ZP3 signal transduction have been recently identified.318 These includes activation of a heterotrimeric GTP-binding protein, transient increase of intracellular pH (pHi), activation of phospholipase C (PLC), and transient elevation of [Ca2+]i through T-type Ca2+ channels. In the following step of the signaling, these initial ZP3-induced events produce additional Ca2+ entry through TRPC family Ca2+-conducting cation channels,319 resulting a sustained elevation of [Ca2+]i to ca. 500 nM that triggers exocytosis. AR occurs some minutes after the start of Ca2+ signal elicited by ZP3. It has been reported that TRPC2 colocalizes with mouse ZP3 sperm binding sites and an antibody directed against the extracellular domains of TRPC2 inhibits the sustained elevation of [Ca2+]i.320 However, TRPC2 may not be the only TRCP involved in AR Ca2+ signaling since TRPC2 null mice are fertile.321 AR is characterized by multiple fusions - at the anterior region of the sperm's head - of the plasma membrane with the outer acrosomal membrane that underlies the first one (Fig. 8). Depolymerization of F-actin enables the two membranes to come into close proximity and fuse.322 As a consequence of these fusion events, numerous -membrane vesicles (composed of plasma and outer acrosomal membrane) are produced and the inner acrosomal membrane (with its associated components) as well as the acrosomal enzymatic contents are exposed to the ZP surface. Lytic enzymes (proteases, glycosidases) are involved in this phenomenon. The proteases could be surface, membrane-anchored enzymes323 or soluble proteases from the acrosomal contents.324 Only acrosome-reacted sperms are found in the PVS and are able to fuse with oolemma.325

Fig. 8. Changes in the morphology of the mammalian sperm head during the acrosome reaction. (A) Intact acrosomal vesicle before the reaction. (B) Fusion of the outer acrosomal membrane and the sperm plasma membrane, leading to the vesiculation of the acrosome. (C) Progressive loss of membranes and acrosomal contents. (D) Completion of the acrosome reaction and exposure of the inner acrosomal membrane. (Modified from YANAGIMACHI R., Mechanism of fertilization in mammals, in MASTROIANNI I., BIGGERS J.D. (eds.), In vitro fertilization and embryo transfer, New York: Plenum Press, 1981: 81-182; TALANSKY, Fertilization and early embryonicâ?¦). Crossing the zona pellucida and entering into the perivitelline space

51 In most mammals, when the sperm begins ZP penetration, the head long axis is usually oblique or even perpendicular to the zona surface.326 Orientation of the sperm head from the parallel geometry (peculiar to adhesion) to the diagonal or orthogonal one (characteristic of penetration) could be determined by the formation of a " shallow depression " or " pit " in the ZP surface around the ghostenclosed acrosomal region of the head. As the depression forms, this region would progressively sink into ZP. In this way, the sperm head's anterior tip points toward the inner ZP surface. Scansion electron microscopy (SEM) studies of hamster and mouse sperm-ZP interaction revealed the presence of a shallow pit around the anterior tip of the ghost-enclosed sperm.327 In considering this question in the light of the information available two decades ago, Hunter328 stressed the need then for a balanced view that would at least invoke a role for both enzymatic and mechanical factors. Yanagimachi329 has detailed much of the evidence for and against the concept of zona lysis. However, sperm lysis of a path through the mammalian ZP is still treated as a fact in most general texts and in many analyses of sperm function. 330 As Austin and Bishop commented in 1957, " a mammalian zona lysine has yet to be demonstrated, but a small hole left in the zona after passage of the spermatozoon testifies to its probable existence ".331 Next year, the same authors noted that " the perforatorium may carry a lysin capable of altering the zona substance in such a way as to permit the spermatozoon to pass through into the perivitelline space ".332 Thus, the belief that spermatozoa utilize acrosomal enzymes to penetrate the much thicker zona pellucida of eutherian mammals was, in a sense, preordained. Since then, a number of hydrolytic and other enzymes have been detected within the mammalian acrosome, 333 and support for the concept of zona lysis has come from (1) the effects of acrosomal extracts or individual enzymes on the integrity of ZP, (2) the robust evidence that an acrosome reaction is required for sperm penetration through the zona, and (3) the studies on the suppression of fertilization in the presence of protease inhibitors. Several lines of evidence now implicitly question the likelihood that penetration of the ZP matrix in eutherian mammals depends upon acrosin and/or other acrosomal hydrolases. Such doubts come from comparative observations of the disposition and behavior of fertilizing spermatozoa and the study of ZP penetration as a function of the architecture of sperm's head and ZP. In view of the evidence now at hand, it can no longer be assumed that sperm penetration of ZP in eutherian mammals depends on the lytic action of acrosomal enzymes. Current data and their interpretations do not prove that lysis has no role in this penetration, but the contrary evidence is too striking to justify continuing supposition that it does. Although eventual solubilisation by acrosome extracts or acrosin has been cited as indirect evidence for lysis as the means of its penetration, the zona shows no response to the acrosomal content released where the sperm head binds and reacts. Even the slower fertilization by acrosin- or galactosyltransferase-deficient mouse spermatozoa334 apparently reflects a delay in development of the acrosome reaction and/or in functional binding, not an inability to penetrate the zona or to undergo fusion with the oolemma and activate the egg. In the case of humans, TEM studies often show the heads of spermatozoa within narrow channels comparable to those generally seen in animal eggs. In human oocytes fixed 48-60 h after , spaces suggestive of local lysis sometimes are present, though with some evidence of degeneration.335 The union of gametes: sperm-oolemma adhesion and cell membrane fusion The adhesion of the acrosome-reacted sperm's head to oolemma The spermatozoon adheres specifically to the oocyte plasma membrane (oolemma) prior to fusing with it.336 Motility of the male gamete is not necessary for adhesion: when injected directly into the PVS, immotile sperms from subjects with primary ciliary dyskinesia are capable to fuse with human oocytes.337 However, only spermatozoa that have undergone the AR are able of binding to and fusing with oolemma using the plasma membrane in the posterior region of the sperm head.338 The strength of sperm to oolemma binding is experimentally well documented by the microscopic observation that some spermatozoa remain adherent to oolemma even after pipette washing of the oocyte from which

52 ZP has been removed: this phenomenon, sometimes called " tenacious adherence ", makes gamete fusion a highly probable event as a follow-up of adhesion.339 For nearly two decades, members of the ADAM protein family340 on the sperm head and integrins on the oolemma have been considered as complementary partners in gamete adhesion and fusion.341 In particular, fertilin α and β and cyritestin on the sperm and integrin α6β1 on the oocyte have been strongly implicated in gamete cell membrane interactions. Fertilin β (ADAM-2) was first implicated because it was identified as the antigen of an antibody, PH-30, that blocked fertilization of guinea pig oocytes.342 Fertilin β (ADAM-1) was isolated and characterized together with fertilin β, since these two proteins form a heterodimer.343 Cyritestin (ADAM-3) was identified in the mouse and monkey by molecular cloning.344 Fertilin α and β as well as cyritestin undergo proteolytic processing between the metalloprotease and disintegrin domains, and this results in the conservation of the disintegrin domain, the cysteine-rich domain, and the EGF-like repeat only on the surface of the mature sperm. Fertilin a is processed intracellularly during spermatogenesis in the testis,345 while fertilin β and cyritestin are cleaved during epididymal maturation. 346 Since they mediate somatic cell adhesion, integrins - as well as other cell adhesion molecules (e.g., cadherins) - have been speculated to mediate sperm-oocyte adhesion. The identification of an integrin ligand-like domain in fertilin α and β and cyritestin347 led to the hypothesis that these sperm ligands could bind to oolemma integrins. Integrin α6β1 primarily recognizes laminins and has been suggested to participate in fertilization. Based on chemical cross- linking experiments with a fertilin β peptide348 and from studies with an anti-α6 monoclonal antibody,349 integrin α6β1 is supposed to be implicated as an oocyte receptor for ADAM-2. However, α6 subunit is not strictly required for fertilization350 and attempts to inhibit the interactions of fertilin β and other ADAM proteins with oolemma, as well as other cell membranes, using the same anti-α6 antibody have been unsuccessful.351 Recently, investigating whether integrins are strictly required for the human spermoocyte adhesion and fusion events, Sengoku352 concluded that two molecular mechanisms are operating in these gamete membrane interactions: only one of them can be inhibited by integrin antibodies and, according to the authors, it does not play an essential role in the human sperm- oolemma adhesion and fusion processes. In the past, a number of functional studies in animals provided evidence that the three ADAM family members above mentioned participate in sperm-oocyte adhesion.353 However, despite this extensive experimental evidence, results from other investigations do not support a decisive role of the sperm's fertilins and the oocyte's integrins in gamete fusion. Males null for fertilin β or cyritestin ([-/-]ADAM-2 or [-/-]ADAM-3 mouse) and females null for integrin α6β1 produce spermatozoa and oocytes, respectively, that are able to adhere to and fuse with each other regularly.354 The reported adverse effects on fertilization of the missing membrane proteins " could be attributed to altered surface organization of mutant sperm and eggs that lack any one of these proteins, such that they may not resemble wild-type gametes ".355 Evidence has been provided that oocyte CD9, a member of the tetraspanin superfamily (TM4SF or tetraspans),356 may play a key role in gamete membrane interactions. CD9 knockout female mouse appear to ovulate regularly and oocyte maturation to M-II stage appears to be normal. However, CD9- lacking oocytes are rarely fertilized.357 The observation that sperm can adhere to oolemma even in the absence of CD9 but do not regularly fuse with it is suggestive of a role for CD9 in sperm-oocyte fusion. Whether CD9 may play a role, direct or indirect (via the tetraspanin web), in sperm-oocyte adhesion or not is a debated question. Experimental data show that ZP-free oocytes treated with anti-CD9 antibodies are characterized by reduced levels of binding of the ADAM sperm ligands.358 This is consistent with " a role for CD9 to strengthen adhesions mediated by these sperm ADAMs rather than the initial molecular interaction of sperm ligand to oocyte receptor ".359 Finally, it is conceivable that CD9 may serve as a receptor for sperm PSG17, a member of the immunoglobulin (Ig) superfamily.360 Recently, a new factor involved in gamete membrane fusion at fertilization has been investigated by Inoue.361 By using a fusion-inhibiting monoclonal antibody and gene cloning, the Japanese scientists 53 identified a mouse sperm fusionrelated antigen and showed that the antigen is a novel protein of the Ig superfamily. They named this protein Izumo and produced a gene-disrupted mouse line. (-/-)Izumo- mice are healthy but males are sterile, since produce normallooking sperm that bound to and penetrated ZP but are unable to fuse with oocytes. The human sperm also contains the Izumo protein. A plausible model of sperm-oocyte adhesion seems to be the mechanism by which leukocytes interact with endothelial cells. In this model, adhesion occurs in a stepwise fashion, starting with loose attachment (called rolling and tethering) and reaching a " firm adhesion " that brings the two membranes into close apposition. In the leukocyte-endothelium system, specific pairs of ligands and receptors mediate each of the subsequent adhesion steps. For example, " rolling " is mediated by selectin-carbohydrate interactions that have rapid kinetics, while " firm adhesion " is supported first by the integrina α4β1-VCAM-1 pair and then by integrin αlβ2/αmβ2-intracellular-adhesion-mol- ecules pairs.362 Sperm-oocyte cell membrane interaction also proceeds step by step, in terms of both spatial arrangements and molecular recognition. Membrane fusion and the engulfing of sperm " Sperm-oocyte fusion is one of the most impressive events in sexual reproduction, and the elucidation of its molecular mechanism has fascinated researchers for a long time ".363 However, because of the limitation of biological materials and difficulties in analyzing protein interactions, many attempts to study membrane fusion at fertilization in mammals failed to reach this goal. Oocytes differ from somatic cells in the structure of their plasma membrane's surface364 that is covered with microvilli with the exception of the region overlying the meiotic spindle. Microvilli surround the sperm head before the sperm-oocyte fusion, and it has been observed that sperm rarely fuse with oocytes at the region lacking microvilli. These observations suggest that the oocyte's microvilli and the sperm head's equatorial segment should be rich in molecules involved in the fusion of the two gametes.365 Ultrastructural investigations indicate that fusion and engulfing of the sperm head involve different regions of the acrosome-reacted spermatozoon and that sperm incorporation by the oocyte is quasi- phagocytic in appearance. 366 Membrane fusion takes place at the equatorial segment of the sperm head, while the rostral portion of the head (i.e., the portion that in acrosomereacted sperms is limited by the inner acrosomal membrane) is incorporated in an oolemma-derived vesicle (Fig. 9). Fifteen years ago, Richard Bronson and his group studied sperm-oocyte interaction at the level of oolemma by

54

Fig. 9. Sperm-oocyte membrane fusion and incorporation of the acrosome-reacted sperm head into an oolemma-derived vesicle in the cortical region of the ooplasm. (A) Fusion occurs between the equatorial segment of the sperm head and oolemma. Inner acrosomal membrane, the limiting membrane of the rostral portion of the acrosome-reacted sperm head, does not participate in the early morphological events of gamete fusion. (B) Oolemma microvilli engulf the sperm head following its adherence and membrane fusion. (C) The rostral portion of the sperm head is completely engulfed in an oolemma-derived vesicle. (Modified from BEDFORD J.M., COOPER G.W., Membrane fusion events in fertilization of eggs, Membrane Surface Reviews 1978, 5: 65-125; BRONSON R., Is the oocyte a non-professional phagocyte?, Update 1998, 4: 763-775. scanning electron microscopy (SEM) using a " stop-fix " technique367 by which a limited number of spermatozoa adhering to and fusing with ZP-free oocytes can be observed serially. SEM microphotographs obtained at the time of sperm entry into the oocyte revealed an apparent elongation of oolemma microvilli over the equatorial segment of sperm head, as these sperms appeared to sink

55 into the cortical ooplasm. While they observed the elongation of microvilli around the individual sperm head, in proximity of other neighboring spermatozoa on the surface of the same oocyte the oolemma did not show the same ultrastructural phenomenon. Sperms adherent to the oolemma where microvilli elongation was not recorded did not enter the ooplasm. " These observations suggest that sperm incorporation by the oocyte involves local gamete membrane interactions on the egg surface and bears certain morphological similarities to events observed during phagocytosis of target particles by macrophages ".368 Although the " phagocytosis analogy " has not been unanimously accepted by the scientific community, it represents a useful working hypothesis that deserves further investigations. Given the presence of phagocytosis-promoting receptors on oocytes, as well as the ultrastructural appearance of the sperm entry of them, it is not unreasonable to propose that phagocytic mechanisms occur during gamete fusion. Functional oolemmal receptors for IgG-FC (FcgR) are present on mammalian oocytes. Both ovulated ZP-free hamster eggs and human preovulatory oocytes obtained from follicular aspirates specifically bind human and murine IgG-Fc peptides.369 Fab preparations of monoclonal antibodies directed against Fcg I, II and III receptors were shown to bind specifically to the oocytes, providing evidence for the presence of these receptors.370 Once gamete membrane fusion takes place, sperm tail beating stops.371 Hence, while motility is essential for penetration of the corona and ZP, it is not required for the incorporation of the sperm's head into the oocyte, as well documented following the microinjection of non-motile spermatozoa from men with dynein arm abnormalities into the PVS.372 via Ca+2 oscillations Sperm fusion with the female gamete immediately triggers a complex series of events, referred to collectively as " oocyte activation " (OA). In mammals, these events lead to exocytosis of the content of cortical granules to block ;373 exit from M-II arrest and completion of meiosis through Ca+2-dependent destruction of cyclin B;374 cytoskeletal remodelling; pronuclear formation, migration and apposition;375 recruitment of maternal mRNAs;376 and initiation of the " mitotic divisions that unveil the complete developmental program "377 (Fig. 10). The signal-transduction pathways that are utilized during OA have been studied for many years. Although ooplasmic free Ca+2 has long been identified as a universal second messenger during OA, the molecular mechanisms by which the fertilizing sperm induces shifts in [Ca+2]i within the oocyte have been systematically investigated only in more recent years. In this section, the current knowledge of signal-transduction mechanisms that are important for OA will be briefly outlined, and some recent findings that shed light on how the sperm triggers these signalling pathways will be mentioned. The earliest detected signalling event during OA is the release of Ca2+ from intracellular stores in the endoplasmic reticulum (ER).378 In M-II mammalian

56

Fig. 10. Central role for the rising of the intracellular Ca2+ concentration in stimulating the major events of mammalian oocyte activation: exocytosis of the content of cortical granules into the perivitelline space, chemical modification of the zona pellucida (" zona hardening "), and block to the entry of further spermatozoa; resumption of female meiosis from the metaphase-II arrest and extrusion of the second polar body; and recruitment of maternal mRNA to initiate protein synthesis. (Modified from ABBOTT A.L., DUCIBELLA T., Calcium and the control of mammalian cortical granule exocytosis, Frontiers in Bioscience 2001, 6: 792-806). oocytes, [Ca+2]i basal levels are ca. 0.1 µM. In fertilized oocytes, Ca2+ release occurs shortly after gamete membrane fusion379 and is manifested as a striking series of [Ca+2]i oscillations of a low frequency (intervals: 6-31 minutes in the mouse; 8-25 minutes in the cow), lasting for a few hours, usually with a decline in amplitude380 (Fig. 11). Calcium levels increase up to ca. 1 µM. Interestingly, in mouse, sperm-initiated [Ca+2]i response ceases in concurrence with the formation of pronuclei,381

57 while in other mammalian species oscillations persist

throughout Fig. 11. Ca2+ oscillations in mouse oocytes after fusion with a spermatozoon during fertilization in vitro, as measured by dual excitation fluorescence photometry. The graph reports the ratio of fluorescence intensity (F340/F380) as a function of time since the male gamete was added. Fluorescence, emitted by the fertilized oocyte in the presence of the fluorescent chelator fura-2 excited by 340 and 380 nm light, reflects the intracellular Ca2+ concentration. Oscillations last for 3-5 hours and cease when the male and female pronuclei are formed. (Modified from MIYAZAKI S., ITO M., Calcium signals for egg activation mammals, Journal of Pharmacological Sciences 2006, 100: 545- 552). the first cell cycle.382 Considering the fundamental role of Ca2+ in OA and the onset of embryo development, it is not surprising that the releasing machinery of this ion within the oocyte undergoes a remarkable reorganization during its maturation. Among other changes, the spatial distribution of ER, the main Ca2+ store of the cell,383 and of the type I inositol 1,4,5-triphosphate (IP3) receptor (IP3R1) changes from the perinuclear region of the GV to distinct focal clusters at the cortex of the M-II oocyte.384 At the same time, the expression of IP3R1 is increased due to the recruitment and translation of IP3R1 mRNA.385 Furthermore, direct modification of IP3R1 and other proteins involved in Ca2+ homeostasis - such as Ca2+ pumps and/or plasma membrane Ca2+ channels - as a consequence of the prevailing kinase environment in the maturing oocyte may also take place. " Collectively, these adaptations are thought to lead to a maximal and spatially organized series of oscillations, the occurrence of which is pivotal for the initiation of normal development ".386 Despite two decades of investigations devoted to elucidating the mechanism by which the sperm initiates the Ca2+ release that is responsible for completing the fertilization process and triggering embryonic development, this biochemical phenomenon continues to draw the attention of many scientists and is far from being adequately explained. Starting from the experimental data available till now, two classes of hypotheses have been put forward.387 The first class suggests that Ca2+ oscillations are due to the sperm binding to an oocyte's plasma membrane receptor that activates a signal-transduction mechanism. The second class attributes the oscillations to the delivery of a soluble sperm factor into the ooplasm as one of the first consequences of sperm-oocyte fusion. The most recent and authoritative reviews on this subject argue in favor of the soluble sperm factor, which is a reflection of a consensus among the vast majority of the specialists in this field.388

58 While the precise mechanism underlying sperm-induced Ca2+ spiking has not yet been made fully clear, it has been established in all animal species stud- ied to date that it involves the activation of the phosphoinositide (PI) pathway. 389 Activation of the PI pathway in oocytes (Fig. 12) results in the production of IP3 and 1,2-diacylglycerol (DAG) via the hydrolysis of phosphatidyl 4,5- bisphosphate (PIP2) by a PI-specific phospholipase C (PLC) isoform.390 Both products of the PI pathway are involved in shaping Ca2+ responses. Increase in the intracellular concentration of IP3 is responsible for mediating Ca2+ release by binding and gating the IP3R1 receptor,391 a tetrameric ligand-gated Ca2+ channel located on the endoplasmic reticulum (ER) membrane, the main Ca2+ store of the cell.392 Production of DAG, either directly393 or indirectly via activation of protein kinase C (PKC), may be involved in the regulation of Ca2+ influx.394 There has been much uncertainty as to the mechanism(s) that triggers activation of the PI pathway and Ca2+ oscillations during mammalian fertilization, and this has led to the proposal of different molecular explanations of this fundamental cellular event. Following the finding that the initial calcium responses proceed unaltered even in the absence of [Ca2+]e, 395 the early hypothesis, referred to as the " conduit hypothesis " (sperm-oocyte fusion would allow extracellular Ca2+ to enter the oocyte), was soon given up. A more robust class of hypotheses, the " receptor hypotheses ", suggests that upon sperm-oocyte membrane contact, receptor-ligand interactions on the surface of the gametes

Fig. 12. Proposed mechanisms of intracellular calcium concentration ([Ca+2]i) increase after sperm- oocyte membrane fusion during mammalian fertilization. Sperms delivers into the ooplasm a soluble substance, most likely a phosphoinositide-specific phospholipase C (PLC_), capable of hydrolyzing phosphatidyl 4,5-bisphosphate (PIP2) into two signaling molecules, inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG). IP3 is involved in intracellular Ca+2 release from the endoplasmic reticulum, the cell Ca+2 store, by binding and gating its receptor IP3R1. DAG may play an integral role in signaling events at the plasma membrane. Together with the rising of [Ca+2]i, DAG may further target and activate protein kinase C (PKC) to the plasma membrane, where PKC may regulate Ca+2 influx to refill the intracellular stores, making possible the persistence of oscillations. The channels and

59 mechanisms responsible for the influx of Ca+2 are unknown, although the transient receptor potential (TRP) channels and the store-operated Ca+2 entry mechanism have been both demonstrated in mammalian oocytes. (Modified from MALCUIT C., KUROKAWA M., FISSORE R.A., Calcium oscillations and mammalian egg activation, Journal of Cellular Physiology 2006, 206: 565-573). relay intracellular signaling events that initiate Ca2+ release in the ooplasm. One of the signaling cascades thought to be engaged by the interaction of gametes is that mediated by protein tyrosine kinases (PTKs). In echinoderm and ascidian fertilization, the Src-family of PTKs (SFKs) may activate one of the PLC isoforms (PLCg)396 thereby triggering Ca2+ release through the production of IP3. In mammals, where the cell-cell recognition process is generally mediated by an integrin/disintegrin interaction involving ADAM proteases and the CD9 tetraspanin fusion protein,397 the PLC is likely to be PLCz.398 Cell-cell recognition is the second species-specific interaction at fertilization. 399 The above- mentioned class of hypotheses postulates that sperm-oocyte receptor interaction includes a signal- transduction component that activates the oocyte. Certainly, integrin receptors can transduce a transmembrane signal to generate a calcium response in general.400 Peptides containing the RDG integrin recognition motif can induce activation of frog and bovine oocytes401 and inhibit sperm binding and fusion.402 However, an earlier report by Abassi and Foltz of an egg membrane receptor for sperm that possessed signal-transduction motifs403 has not yet been confirmed. Nonetheless, " it remains a possibility in mammalian oocytes that integrin signaling may activate tyrosine kinase pathways,404 presumably distinct from the PLCg pathway that has been shown not to operate at fertilization405".406 The second class of hypotheses that account for oocyte's Ca2+ release takes as a premise that such activation signal requires fusion of sperm and oocyte. The distinguishing feature of the " fusion hypotheses " is that activation occurs as a consequence of sperm-oocyte fusion. The idea that the sperm is the vehicle that transmits an activating factor (generically called " sperm factor " [SF]) to the female gamete has been very popular among the investigators,407 mostly because of the observation that the extracts of sperm cytoplasm induces calcium transients when microinjected into oocytes.408 The advent of intracytoplasmic sperm injection (ICSI) as a gamete-micromanipulation technique in IVF409 provided a further evidence that one or more molecules contained in the sperm are responsible for initiating Ca2+ oscillations in mammalian eggs. However, examination of the ICSIinduced [Ca2+]i increase410 revealed that " the events that take place during the interaction of the gamete membranes are pivotal for normal fertilization ".411 The molecular identity of SF has yet to be fully disclosed. There is growing evidence that PLC isoforms expressed in the mammalian spermatozoon412 are winning candidates to be the SF. Initial studies revealed that the sperm-specific PLC, PLCζ,413 exhibits a [Ca2+]i-oscillation-inducing activity very close to that of sperm or extracts of sperm cytoplasm. Injection of recombinant PLCζ or PLCζ complementary RNA (cRNA) in mouse,414 bovine,415 and human oocytes416 evokes sperm-like Ca2+ oscillations. Moreover, as is observed after conventional IVF, PLCζ cRNA-induced oscillations cease at ca. the time of pronuclear appearance.417 To these evidences supporting PLCζ as the SF investigators are looking for, Fujimoto.418 added a further one by localizing the protein to the post- acrosomal region of the mouse sperm's head, which is normally its first part to fuse with the oocyte. Ca2+ release at fertilization is essential for driving the fusion of female and male cell membranes to its regular end, i.e., the beginning of embryo development: " After this plasma membrane interaction occurs, a signal-transduction cascade is initiated that results in the conversion of the oocyte to a diploid embryo capable of forming a new organism ".419 " Egg activation initiates embryonic development "420 and premature termination of [Ca2+]I oscillations prevents the formation of pronuclei,421 the stage of fertilization that precedes and makes possible the appearance of a two-cell embryo some hours after their apposition and the mixing of maternal and paternal chromosomes. The cascade of molecular events that results in Ca2+ release " is responsible for triggering embryonic development ".422 60 Bibliografia [1] Russell E.S., The Interpretation of Development and Heredity: A Study in Biological Method, Oxford: Clarendon Press, 1930: 1. [2] Sal 139 (138), 13-14. [3] Wolpert L., The Triumph of the Embryo, Oxford University Press, New York 1991: 199. [4] Serra A., Colombo R., Identità e statuto dell’embrione umano: il contributo della biologia, in Vial Correa J. de D., Sgreccia E. (a cura di), Identità e statuto dell’embrione umano, Città del Vaticano: Libreria Editrice Vaticana, 1998: 106-158. [5] Ibid., p.146. [6] Cf. Corpus Hippocraticum, Περì φύσιος παιδίου, III (testo curato e tradotto in francese da Joly R., Hippocrate, Paris: Les Belles Lettres, 1970, tomo XI, XVI, 1-2). Come buona parte dei trattati che formano il cosiddetto Corpus Hippocraticum, anche questo fu scritto fra il 430 e il 380 a.C. [7] “ Che la femmina non concorre con liquido seminale alla riproduzione, e tuttavia ci concorre in qualche modo, con il mestruo (καταµηνίων σύστασις), e negli animali non sanguigni, con ciò che è analogo al mestruo, risulta dunque chiaro...Perciò, se vi è il maschio come agente e trasformatore [κινοûν καì ποιοûν], e la femmina come paziente (παθητικόν), la femmina non potrà aggiungere al liquido seminale del maschio liquido seminale, ma materia (‛ύλη)”.Aristotele, Περì ζώων γενέσεως, I, 729a 22-30 (tradotto it. M. Vegetti, Lanza D., Aristotele, vol. V, Riproduzione degli animali, Bari: Editori Laterza, 2001: 187). [8] Cf. Corpus Hippocraticum, Περì γονη̃ς, VI-VII (testo curato e tradotto in francese da Joly, Hippocrate, VI-VII). Per una ricostruzione storica delle teorie contrarie vedi: Boylan M., The Galenic and Hippocratic challenges to Aristotle’s conception theory, Journal of History of Biology 1984, 17: 83-112. [9] Una concisa presentazione delle teorie presocratiche sulla generazione sessuale si trova in Schuhl P.-M., Les premières étapes de la philosophie biologique, Revue de l’Histoire des Sciences 1952, 5: 197-221. [10] Cf. Empedocle, Frammento B 23 (ed. critica di Bollack J., Empédocle, Paris: Editions de Minuit, 1965-1969, tomo I, p.213]. Per una discussione del frammento del filosofo siculo e della sua teoria della generazione vedi: De Ley H.,Empedocle’s sexual theory: a note on fragment B23, Antiquité Classique 1978, 47: 153-165. [11] Cf.. Galenus C., De usu partium corporis humani, XIV, 10.11.14 (Kühn K.G. (a cura di), Claudii Galeni Opera Omnia, vol. IV, Lipsiae: C. Cnoblochii, 1821-1833: 180.193.208). Ulteriori informazioni sulla biologia della riproduzione in Galeno si trovano in Penso G., La médicine romaine: l’art d’Esculape dans la Rome antique, Paris: Roger Dacosta, 1984: 187-190; Preus A., Galen’s criticism of Aristotle’s conception theory, Journal of History of Biology 1977, 10: 65-84; Boylan, The Galenic and Hippocratic challenges...; Id., Galen’s conception theory, Journal of History of Biology 1986, 19: 47- 77. [12] Nella seconda metà del XIX secolo, il grande morfologo Wilhelm His riconobbe l’importanza della disputa secolare fra queste due dottrine per la storia della biologia e ne riassunse lo sviluppo: His W., Die Theorien der geschlechtlichen Zeugung, Archiv für Anthropologie 1870, 4: 197-220; pp.317- 332; 1871, 5: 69-111. [13] Vanni Rovighi S., Elementi di filosofia, Brescia: La Scuola, 1963: 53-71. Vedi anche: Descoqs P., Essai critique sur l’hylémorphism, Paris: Beauchesne, 1924. [14] Gen 30, 2; Dt 7, 13; 28, 4.53; Sal 127(126), 3; 132(131), 11; Mi 6, 7. [15] Lc 1, 42. [16] Aristotele, op. cit., II, 739b 22 (trad.: Vegetti, Lanza, Aristotele..., p.214). [17] Giobbe 10, 10. Cf. Sap 7, 2. [18] Cf. Needham J., A History of Embryology, Cambridge: Cambridge University Press, 1959: 50.83.85. 61 [19] Bonelli L. (a cura di ), Il Corano XXIII, 14, Milano: Hoepli, 1991. [20] Aristotele, op. cit., III, 758 b3 (trad.: Vegetti, Lanza, Aristotele..., p.258). [21] Harveus G., Exercitationes de generatione animalium quibus accedunt quaedam de partu, de membranis ac humoribus uteri & de conceptione, Londini: Octavianum Pulleyn, 1651: 38, b, ex. 63 (traduzione ingl. di Willis R., The Works of William Harvey, London: Sydenham Society, 1847: 460). Come ha osservato E.B. Gasking nel suo ben documentato saggio Investigations into Generation 1651- 1828 (London: Hutchinson, 1967), Harvey “non è affatto chiaro sul significato tecnico che vuole attribuire al termine “ovulo”. Per i biologi moderni, l’ovulo si definisce nel quadro della teoria cellulare, e il termine si riferisce a una precisa struttura morfologica (o citologica). L’ovulo di Harvery ha qualche legame con questa concezione moderna. Harvey lo ritiene prodotto dalla femmina e dotato del potere di svilupparsi dopo il rapporto sessuale, dando luogo al nuovo individuo. E pur negando che il seme entri materialmente in contatto con l’ovulo, insiste che è il seme a conferirgli la capacità di svilupparsi pienamente” (p.28). [22] San Tommaso d’Aquino, Summa Theologiae, I, q. 118, a. 1, ad 4. [23] La questione è presentata e discussa in dettaglio da Pangallo M. nel suo contributo al presente volume: Il pensiero di san Tommaso riguardo all’embrione umano, pp.? [24] Haldane J., Lee P., Aquinas on human ensoulment, abortion and the value of life, Philosophy 2003, 78: 255-278; p.271. [25] San Tommaso d’Aquino, Summa Theologiae, I, q. 118, a. 1, ad 4.; cf. Id., Summa Contra Gentiles, q. 3, a. 11, ad 8. [26] Aristotele, Περì φυχη̃ς, II, 412 b5. [27] Ibid., III, 434 a30. [28] Ibid., III, 435 a14. [29] “ Nel caso della progenie maschile, il primo movimento di solito si registra all’incirca al quarantesimo giorno nel lato destro del grembo, nel caso del sesso femminile, invece, al novantesimo giorno e nel lato sinistro. Tuttavia, non si può assolutamente affermare che questa sia un’accurata esposizione dei fatti, poichè esistono molte eccezioni in cui il movimento si è manifestato sul lato destro sebbene si trattasse di una bambina o sul lato sinistro per un bambino. In breve, questi ed altri fenomeni simili sono solitamente caratterizzati da differenze che possono essere catalogate come differenze di grado. In questo periodo l’embrione comincia a sviluppare le varie parti, essendo stato costituito fino a questo momento di una sostanza simile alla carne senza distinzione di parti”: Aristotele, Περì ζώων µορίων, VII, 3, 583 b 2-10 (traduzione ingl. di DThompson D.W., Historia Animalium, in Smith J.A., Ross W.D. (a cura di), The Works of Aristotle, Oxford: Oxford University Press, 1967: 583). [30] Cf. San Tommaso d’Aquino, In III Sententiarum, dist. III, q. 5, a. 2. [31] Le osservazioni di Leonardo sullo sviluppo del feto umano e sulle dimensioni relative delle sue parti anatomiche sono sorprendentemente in linea con i dati che è oggi possibile ottenere per mezzo delle misurazioni ecografiche. [32] Leonardo da Vinci, Quaderni d’Anatomia. [33] Rueff J., De Conceptu et Generatione hominis et iis quae circa haec potissimum consyderantur libri sex, Tiguri: C. Froschoverus, 1554. [34] Cf. Vesalius A., De Humani Corporis Fabrica libri septem, J. Oporinus, Basilea 1543 [copia fotostatica: Bruxelles: Culture et Civilization, 1964]. [35] In origine, il trattato fu pubblicato in quattro edizioni latine uscite nello stesso anno: Harvey W., Exercitationes de generatione animalium, Ludovicum Elzevirium, Amstelodamium 1651; Id., Ioannem Ravesteynium, Amstelodamium 1651; Id., Joannem Janssonium, Amstelodamium 1651; Id., Octavianum Pulleyn, Londinium 1651. la prima traduzione inglese dell’opera fu pubblicata quattro anni dopo: Harvey W., Anatomical exercitations concerning the generation of living creatures, London: Octavian Pulleyn, 1653. 62 [36] Degli Aromatari G., Epistola ad Bartholomeum Nanti de generatione plantarum ex seminibus (Venetiis, 1625), Philosophical Transactions of the Royal Society 1694, 18: 150-152. [37] Digby K., The nature of Bodies, Paris: Gilles Blaizot, 1644. [38] Malpighi M., De formatione pulli in ovo, Londra: manoscritto, 1772. [39] Van Leeuwenhoek A., The observations of Mr. Antoni Leeuwenhoek on animalcules engendered in the semen, in The Collected Letters of Antoni van Leeuwenhoek, Amsterdam: Swets and Zeitlinger, 1941: 279. La lettera fu pubblicata per la prima volta nel 1679 nelle Philosophical Transactions della Royal Society di Londra. [40] Id., Generation by an animalcule of the male seed. Animals in the seed of a frog, Philosophical Transactions of the Royal Society 1683, 13: 347-355. [41] Id., Concerning the animalcula in semine humano, Philosophical Transactions of the Royal Society 1699, 21: 301-308. In questo articolo Van Leeuwenhoek ha riassunto la lettera di Dalenpatius (pseudonimo di Mr. de Plantade) pubblicata nello stesso anno. I celebri disegni di Dalenpatius, riprodotti da Van Leeuwenhoek, furono quasi certamente un falso; cf. Cole F.J., Early Theories of Sexual Generation, Oxford: Clarendon Press, 1930: 68ss. [42] Ibid., p.56. [43] Van Leeuwenhoek A., Further observations on the animalcula in semine masculino, Philosophical Transactions of the Royal Society 1701, 22: 739-746. [44] Spallanzani L., De la génération de quelques animaux amphibies, in:Oeuvres de M. l'Abbé Spallanzani, Paris: Pierre J. Duplain, 1787: 101. Si tratta della traduzione francese delle Dissertazioni di fisica animale e vegetabile, Società Tipografica, Modena 1780, vol. 2. pt. 1. [45] Malebranche N., De la recherche de la vérité, vol. I, Paris: André Pralard, 1673, cap.6. [46] Cf. Hartsoeker N., Essay de dioptrique, Paris: Jean Anisson, 1694. La prima descrizione degli animalculi nel seme maschile apparsa sulla stampa fu quella dello scienziato olandese Christiaan Huygens, al quale Hartsoeker aveva comunicato la sua fantasiosa ipotesi (Hartsoeker N., Three letters to C. Huygens dated 1678, in Huygens C., Oeuvres complètes, publiées par la Société Hollandaise des Sciences, vol. VIIII, L’Aia: M. Nijhoff, 1888-1950: 96-98). Huygens non menzionò la tesi di Hartsoeker in un primo saggio in cui descriveva gli animalculi dello sperma (Journal des Sçavans 1678, 28: 331-332), ma gli riconobbe il merito di aver formulato l’ipotesi sulla stessa rivista due settimane dopo. [47] Hartsoeker N., Cours de physique... et d’un extrait critique des lettres de M. Leeuwenhoek, L’Aia: Jean Swart, 1730. [48] Cole, Early Theories of..., p.86. [49] Farley J., Gametes & Spores. Ideas about sexual reproduction: 1750-1914, Baltimore-London: The Johns Hopkins University Press, 1982: 35. [50] L’opera principale di Dalyell fu pubblicata a cominciare da due anni dopo la sua morte: Dalyell J.G., The powers of the Creator displayed in the creation, London: John Van Voorst, 1853-1858. [51] Oken L., Die Zeugung, Bamberg, Wirzburg : Joseph Anton Goebhardt, 1805. [52] Von Baer C.E., über Entwickelungsgeschichte der Thiere, Königsberg: Gebrüdern Bornträger, 1828. [53] Cole, Early Theories..., p.127. [54] Nel 1838, Matthias Jakob Schleiden (1804-1881), professore di botanica all’Università di Jena, ipotizzò che “ogni elementro strutturale delle piante si componesse di cellule o di loro prodotti” (Schleiden M.J., Beiträge zur Phytogenesis, Johannes Müllers Archiv für Anatomie, Physiologie und Wissenschaftliche Medizin 1838, 13: 137-176). L’anno seguente, conclusioni analoghe rispetto agli animali furono formulate dallo zoologo Theodor Schwann (1810-1882), allievo del grande anatomista Johannes Peter Müller (1801-1858) presso l’università Humboldt di Berlino. Schwann sostenne che “le parti elementari di tutti i tessuti sono formate da cellule” e che “vi è un unico principio universale dello sviluppo per le componenti elementari degli organismi...e questo principio risiede nella formazione di 63 cellule” (Schwann T., Mikroskopische Untersuchungen über die Übereinstimmung in der Struktur und dem Wachstum der Tiere und Pflanzen, Berlin: Sander’schen Buchhandlung, 1839). Secondo Schleiden, tuttavia, la prima fase della generazione delle cellule era la formazione di un nucleo di “cristallizazione” nella sostanza intracellulare (che chiamò citoblasto), con il successivo ingrandimento progressivo di questo materiale condensato fino a diventare una nuova cellula. Questa teoria –una sorta di variante cellulare della dottrina della generazione spontanea– fu confutata negli anni Cinquanta del XIX secolo da Robert Remak (1815-1865, scopritore dei tre strati germinali dell’embrione iniziale: ectoderma, mesoderma ed endoderma (1842), da Rudolf Karl Virchow (1821-1902), e da Rudolph Albert von Kölliker (1817-1905), i quali dimostrarono che le cellule si formano per suddivisione di cellule preesistenti. Si attribuisce proprio a Virchow il popolare aforisma omnis cellula e cellula, che esprimeva in forma sintetica il consenso sulla cellula come unità biologia della riproduzione oltre che della struttura e della funzione. Nella seconda metà del XIX secolo, Virchow fu il principale sostenitore della teoria cellulare, che contribuì grandemente a diffondere con il suo grande trattato Die Cellularpathologie in ihrer Begründung auf physiologische und pathologische Gewebelehre (Berlin: A. Hirschwald, 1858). Per una storia completa e critica della teoria cellulare e della sua influenza sullo sviluppo del pensiero biologico moderno, vedi: Mayr E., The Growth of the Biological Thought, Cambridge: Belknap, 1982; Harris H., The Birth of the Cell, New Haven: Yale University Press, 1998; Mazzarello P., A unifying concept: the history of cell theory, Nature Cell Biology 1999, 1: E13- E15. [55] Russell, The interpretation of Development..., p.239. [56] Ibid., p.239. [57] Rauber A., Neue Grundlegungen zur Kenntnis der Zelle, Morphologisches Jahrbuch 1883, 8: 233- 338. [58] Ibid., p.251. [59] Ibid., p.332. [60] Sedgwick A., On the inadequacy of the cellular theory of development, Quarterly Journal of Microscopical Science 1895, 37: 87-101. [61] Whitman C.O., The inadequacy of the cell-theory of development, Boston: Wood’s Hell Biological Lectures, 1894: 105-124. [62] Ibid., p.112. [63] Lillie F.R., Observations and experiments concerning the elementary phenomena of embryonic development inChaetopterus, Journal of Experimental Zoology 1906, 3: 153-268; pp.252-253. [64] Alla fine del XVIII secolo, i fabbricanti di microscopi si erano ormai resi conto che le immagini ottenute mediante i loro strumenti erano affette da aberrazioni sferiche e cromatiche. Come osservò George Adams nei suoi Essays on the Microscope (1787), “(A causa della) diversa rifrangibilità dei raggi luminosi, che sovente provoca questa deviazione dalla verità nell’aspetto delle cose, molti hanno immaginato di aver fatto scoperte sorprendenti e come tali le hanno comunicate al mondo, quando in realtà queste erano altrettante illusioni ottiche dovute alla rifrazione disuguale dei raggi” (citato daBradbury S., The Evolution of the Microscope, Oxford: Pergamon Press, 1967: 164). A quel tempo gli stessi fabbricanti erano ormai consapevoli che una delle “illusioni ottiche” –cioè le aberrazioni cromatiche– si poteva ridurre al minimo combinando lenti dotate di proprietà diverse. Ai primi del XIX secolo, “questa combinazione di vetro crown-vetro flint dava immagini soddisfacenti soltanto a basso ingrandimento. Poi però, durante gli anni Venti del secolo, fu possibile ottenere immagini acromatiche ad alta potenza, quando Giovanni Amici e altri riuscirono a produrre lenti combinate dotate di lunghezza focale assai ridotta” (Farley, Gametes & Spores..., pp.35-36). Fra i più eminenti utilizzatori di quei nuovi obiettivi microscopici vi furono le équipe di scienziati che operavano nel laboratorio berlinese di Johannes Müller e in quello di Jan Purkyně a Bratislava. [65] Prévost J.-L., Dumas J.-B.A., De la génération dans les mammifères, et des premiers indices du développement de l’embryon, Annales des Sciences Naturelles 1824, 3: 113-128. 64 [66] Von Baer K.E., De ovi mammalium et hominis genesi: epistolam ad Academiam Imperialem Scientiarum Petropolitanam, Lipsia: L. Vossii, 1827. [67] Purkyně J.E., Symbolae ad ovi avium historiam ante incubationem, Bratislava: Vratislaviae typis universitatis, 1825 (trad. ingl. di Barthelmez G.W., in: Essays in Biology. In honor of H. M. Evans. Written by his friends, Berkeley-Los Angeles: University of California Press, 1943: 53-93). [68] Nel 1839, Theodor Schwann ipotizzò che l’ovulo fosse una cellula, ma senza considerarlo un assunto dimostrato. La questione dell’identità della vescicola osservata da Purkyně nell’uovo di uccello era ancora in dubbio. L’opinione di Schwann era che la vescicola fosse il nucleo e il tuorlo il corpo cellulare. Cf. Schwann, Mikroskopische Untersuchungen... [69] Burdach K.F., Die Physiologie als Erfahrungswissenschaft, Lipsia: L. Voss, 1832: 84. La teoria dei parassiti fu ulteriormente rafforzata quando alcuni microscopisti postularono l’esistenza di “organi” nello spermatozoo. Nel 1939, ad esempio, Gustav Valentin, un professore di fisiologia dell’università di Bonn, riferì della presenza, nello spermatozoo dell’orso, di una bocca, un ano e delle vesciche interne. Un anno dopo, presso la medesima università, Friedrich Gerber descrisse analoghe strutture nello spermatozoo della cavia e descrisse persino la presenza in essa di organi sessuali. Tali speculazioni furono suffragate dal tedesco Christian Ehrenburg e dai francesi Antoine Dugès e Felix Pouchet. Cf. Farley,Gametes & Spores..., p.44. [70] Bischoff T.L.W., Theorie der Befruchtung und über die Rolle, welche die Spermatozoiden dabei spielen, Archiv für Anatomie, Physiologie und wissenschaftliche Medicin 1847, pp.422-440; pp.433- 435. [71] Fra gli scienziati tedeschi che aderirono alla teoria della fecondazione di Bischoff, Rudolf Wagner e Rudolf Leuckart contribuirono grandemente alla sua diffusione nei paesi anglofoni con il loro saggio intitolato Semen, in: Todd R.B. (a cura di), The Cyclopaedia of Anatomy and Physiology, vol.IV, London: Sherwood, Gilbert & Piper, 1836-1859: 472-508. [72] Cf. fra l’altro de Quatrefages J.-L., Recherches expérimentales sur les spermatozoides des hermelles de Tarets, Annales des Sciences Naturelles 1850, 13: 111-125. [73] Bischoff, Die Physiologie als..., p.437. [74] Farley, Gametes & Spores..., p.60. Bischoff riteneva che l’ovulo è un tutto e possiede tutte le parti costituenti(Bischoff, Die Physiologie als..., p.437). Nessuna parte del germe maschile è strettamente necessaria per perfezionare il patrimonio naturale dell’ovulo, “che in genere necessita soltanto di un cambio di movimento per venire eccitato”. Pertanto, secondo lo studioso tedesco, “l’ardua questione dell’ingresso o non ingresso dello sperma e dei suoi costituenti non è affatto essenziale” (Bischoff T.L.W, Entwicklungsgeschicte des Meerschweinchens, Giessen: Ricker, 1852:15). [75] Newport G., On the impregnation of the ovum in the amphibia, Philosophical Transactions of the Royal Society 1851, 141(Pt 11): 169-242; p.221. Dopo aver dimostrato che il carminio non aveva effetti indesiderabili sulla fecondazione, Newport immerse 41 uova di rana in una miscela di liquido seminale dello stesso anfibio e carminio, e osservò che nell’uovo non passava nessun granulo di carminio di dimensioni paragonabili a quelle di uno spermatozoo di rana. [76] Ibid., p.232. [77] Farley, Gametes & Spores..., p.62. [78] Newport, On the impregnation of..., p.242. [79] Farley, Gametes & Spores..., p.62. L’osservazione di Newport circa il possibile ruolo dello sperma nella trasmissione ereditaria di elementi paterni è ancor più notevole se si considera che fu fatta 15 anni prima che Johann Gregor Mendel pubblicasse i risultati dei suoi esperimenti sull’incrocio del Pisum sativum e la loro interpretazione, che collegava i gameti alla trasmissione dei fattori genetici (Mendel G., Versuche über Pflanzen-Hybriden, Verhandlungen des naturforschenden Vereines in Brünn 1866, Bd. IV: Abhandlungen, 3-47). [80] Nelson H., The reproduction of the Ascaris mystax, Philosophical Transactions of the Royal Society 1852, 142: 563-594; p.578. 65 [81] Ibid., p.586. [82] Keber F., De spermatozoorum introitu in ovula. Additamenta ad physiologiam generationis, Königsberg: Bernträger, 1853. [83] Meissner G., Beitrage zur Anatomie and Physiologie von Mermis albicans, Ztschr. Wissensch. Zoo1. 1853, 5: 207-284. [84] Cole, Early Theories..., p.193. [85] Newport, On the Impregnation of the Ovum in the..., 1953, 6: 214-217. [86] Id., On the Impregnation of the..., 1853: 270. [87] Cf. Strasburger E., über Zellbildung und Zelltheilung, Jena: Dabis, 1875: 309. [88] Farley, Gametes & Spores..., p.161. [89] Hertwig O., Beiträge zur Kenntniss der Bildung, Befruchtung und Theilung des thierischen Eies, Gegenbaurs Morphologische Jahrbuch 1876, 1: 347-452; 1877, 3: 1-86. [90] Ibid., 1877, 3: 30. [91] Il termine “protoplasma”, coniato dal botanico tedesco Hugo Von Mohl nel 1846, era usato per indicare la materia vivente presente nella cellula. Al livello più elementare esso si può dividere in citoplasma e nucleoplasma. Questa distinzione rispecchia gran parte delle conoscenze più primitive dell’architettura cellulare che precedettero lo sviluppo di potenti indagini microscopiche (citologia ultrastrutturale) e di analisi molecolari (biochimica cellulare). Il termine “protoplasma” si riferiva alle sostanze organiche e inorganiche che compongono una cellula, dirette in modo misterioso dal nucleo e controllate dalla membrana cellulare. Oggigiorno si sa che il citoplasma è assai complesso sul piano strutturale e che il protoplasma è vivente a causa della complessità degli organelli citoplasmatici e della loro attenta separazione e orchestrazione di molteplici processi biochimici. Nell’ultimo decennio del XIX secolo furono condotti studi pionieristici sulla composizione chimica del protoplasma e furono avanzate varie ipotesi sulla sua natura e origine. Vedi fra l’altro Bütschli O., Investigations on Microscopic Foams and Protoplasm, London: Adam and Charles Black, 894;Delage Y., La structure du protoplasma et les theories sur l'heredite et les grands problemes de la biologie generale, Paris: C. Reinwald, 1895; Cvet M.S., Études de physiologie cellulaire. Contributions à la connaissance des mouvements du protoplasme, des membranes plasmiques et des chloroplasten, Genève: Rey&Malavallon,1896; Fischer A., Fixierung, Färbung und Bau der Protoplasmas, Jena: G. Fischer, 1899; Ghesquier D., La centrifugation et la cellule. La déconstruction du protoplasme entre 1880 et 1910, Revue d'histoire des sciences 2002, 55: 323-377. [92] Fol H., Sur le commencement de l’hénogénie chez divers animaux, Archives de Zoologie Expérimentale et Générale 1877, 6: 145-169. Due anni dopo, Fol discusse più estesamente i medesimi aspetti della fecondazione in: Id., Recherches sur la fécondation et le commencement de l’hénogénie chez divers animaux, Mémoires de la Société de Physique et d'Histoire Naturelle de Genève 1879, 26: 92-397. [93] Bavister B.D., Early history of in vitro fertilization, Reproduction 2002, 124: 181-196; p.182. [94] Schenk S.L., Das Säugetierei Künstlich befruchtet ausserhalb des Muttertieres, Mittheilungen aus dem Embryonischen Institute der Kaiserlich-Königlichen Universität in Wien 1878, 1: 107-184; Pincus G., Enzmann E.V., The comparative behaviour of mammalian eggs in vivo and in vitro, Journal of Experimental Medicine 1935, 62: 665-675;Pincus G., Development of fertilized and artificially activated rabbit eggs, Journal of Experimental Zoology 1939, 82: 65-129. [95] Rock J., Menkin M.F., In vitro fertilization and cleavage of human ovarian eggs, Science 1944, 100: 105-106;Menkin M.F., Rock J., In vitro fertilization and cleavage of human ovarian eggs, American Journal of Obstetrics and Gynecology 1948, 55: 440-452. [96] I tempi di maturazione esatti degli ovociti umani in vitro sono stati descritti per la prima volta da Edwards R.G.,Maturation in vitro of human ovarian eggs, Lancet 1965, II: 926-929. Come osserva correttamente Bavister, “sebbene sia possibile che la maturazione di alcuni ovociti sia giunta a termine

66 entro il periodo durante il quale gli spermatozoi e gli ovuli sono stati incubati insieme, ciò non fornisce ancora la prova che la fecondazione sia stata effettuata in vitro”.(Bavister, Early history of..., p.182) [97] Pincus G., Saunders B., The maturation of human ovarian ova, Anatomical Record 1939, 75: 537- 542. [98] Gli ovociti di ratto usati da J.A. Long (The living eggs of rats and mice with a description of apparatus for obtaining and observing them, Publications in Zoology of the University of California1912, 9: 105-136) sono particolarmente suscettibili di attivazione partenogenetica. Per i dettagli delle condizioni sperimentali, vedi: Austin C.R., Activation of eggs by hypothermia in rats and hamsters, Journal of Experimental Biology 1956, 33: 338-347; Id., Effects of hypothermia and hyperthermia on fertilization in rat eggs, Journal of Experimental Biology 1956, 33: 348-357. [99] Smith A.U., Fertilization in vitro of the mammalian egg, Biochemical Society Simposia 1951, 7: 3-10. [100] Schenk, Das Säugetierei.... [101] Shettles L.B., Observations on human follicular and tubal ova, American Journal of Obstetrics and Gynecology 1953, 66: 235-247. [102] Bavister, Early history..., p.182. [103] Vedi fra gli altri Moricard R., Bossu J., Premières études du passage du spermatozoïde au travers de la membrane pellucide d’ovocytes de lapine fécondes in vitro, Comptes Rendus de l’Academie de Medicine 1949, 33: 659-665;Moricard R., Penetration of the spermatozoon in vitro into the mammalian ovum, Nature 1950, 165: 763;Smith,Fertilization in vitro...; Shettles, Observations on human...Le prove della realizzazione della FIV nei conigli presentate da G. Pincus, E.V. Enzmann (Can mammalian eggs undergo normal development in vitro?, Proceedings of the National Academy of Sciences USA 1934, 20: 121-132) appaiono più convincenti, poiché dopo aver trasferito ovociti che erano stati a contatto con spermatozoi in vitro, hanno ottenuto prole. Sono state avanzate tuttavia delle critiche per il fatto che gli ovociti potrebbero essere stati fecondati dopo il trasferimento all’ovidotto della femmina ricevente. Effettivamente la rimozione di tutti gli spermatozoi aderenti a ovociti appena ovulati e cumulo-intatti mediante procedura di lavaggio è di difficile esecuzione (vedi: Chang M.C., In vitro fertilization of mammalian eggs, Journal of Animal Science 1968, 27(1): 15-22). La stessa obiezione vale per gli esperimenti di Venge O. (Experiments on fertilization of rabbit ova in vitro with subsequent transfer to alien does, in Wolstenholme G.E.W. (a cura di), Mammalian Germ Cells, London: Churchill, 1953: 243-252), che ha riferito della nascita di due cucciolate di conigli: l’intervallo temporale fra il contatto dei gameti e il trasferimento (3h.30’) è considerato insufficiente a escludere la possibilità di fecondazione in vivo (vedi Bavister, Early history of..., p.183). [104] Austin C.R., Observations on the penetration of sperm into the mammalian egg, Australian Journal of Scientific Research 1951, 4: 581-596. [105] Chang M.C., Fertilizing capacity of spermatozoa deposited into the Fallopian tubes, Nature 1951, 168: 697-698. [106] Dauzier L., Thibault C., Wintenberger S., La fecondation in vitro de l’oeuf de la lapine, Comptes Rendus de l’Academie de Science 1954, 238: 844-845; Thibault C., Dauzier L., Wintenberger S., étude cytologique de la fécondation in vitro de l’oeuf de la lapine, Comptes Rendus de la Société de Biologie 1954, 148: 789-790. Questi risultati sono stati confermati da altri studiosi che hanno usato anch’essi spermatozoi capacitati in vivo. [107] Chang M.C., Fertilization of rabbit ova in vitro, Nature 1959, 179: 466-467. Risultati simili a quelli ottenuti da Chang sono stati riferiti fra gli altri da Thibault C., Dauzier L., Analyse des conditions de la fécondation in vitro de l’oeuf de la lapine, Années de la Biologie Animale et de Biochimie et Biophysique 1961, 1: 277-294. [108] Per una disamina critica delle prime tecniche e dei primi risultati della FIV applicata a gameti di mammiferi, vedi:Rogers B.J., Mammalian sperm capacitation and fertilization in vitro: a critique of methodology, Gamete Research 1978, 1: 165-223. 67 [109] Yanagimachi R., Chang M.C., Fertilisation of hamster eggs in vitro, Nature 1963, 200: 281- 282; Id., In vitrofertilisation of golden hamster ova, Journal of Experimental Zoology 1964, 156: 361- 376; Barros C., Austin C.R., and the sperm acrosome reaction in the hamster, Journal of Experimental Zoology 1967, 166: 317-323;Pickworth S., Chang M.C., Fertilisation of Chinese hamster eggs in vitro, Journal of Reproduction and Fertility 1969, 19: 371-374. [110] I primi cuccioli di hamster ottenuti con la FIV sono nati soltanto nel 1992: Barnett D.K., Bavister B.D.,Hypotaurine requirement for in vitro development of golden hamster one-cell embryos into morulae and , and production of term offspring from in vitro fertilized ova, Biology of Reproduction 1992, 47: 297-304. [111] Whittingham D.G., Fertilisation of mouse eggs in vitro, Nature 1968, 220: 592-593; Iwamatsu T., Chang M.C., In vitro fertilization of mouse eggs in the presence of bovine follicular fluid, Nature 1969, 224: 919-920; Id., Further investigation of capacitation of sperm and fertilisation of mouse eggs in vitro, Journal of Experimental Zoology 1970, 175: 271-281; Cross P.C., Brinster R.L., In vitro development of mouse ovocytes, Biology of Reproduction 1970, 3: 298-307; Mukherjee A.B., Cohen M.M., Development of normal mice by in vitro fertilisation, Nature 1970, 228: 472-473;Toyoda Y.M., Yokoyama M., Hoshi T., Studies on the fertilization of mouse eggs in vitro. I. In vitro fertilization of eggs by fresh epididymal sperm, Japanese Journal of Animal Reproduction 1971, 16: 147-151; Id., Studies on the fertilization of mouse eggs in vitro. II. Effects of in vitro preincubation on time of sperm penetration of mouse eggs in vitro, Japanese Journal of Animal Reproduction 1971, 16: 152-157. [112] Toyoda Y., Chang M.C., Sperm penetration of rat eggs in vitro after dissolution of zona pellucida by chymotrypsin, Nature 1968, 220 589-591; Miyamoto H., Chang M.C., In vitro fertilization of rat eggs, Nature 1973, 241: 50-52; Id.,Fertilization of rat eggs in vitro, Biology of Reproduction 1973, 9: 384-393; Toyoda Y., Chang M.C., Fertilization of rat eggs in vitro by epididymal spermatozoa and the development of eggs following transfer, Journal of Reproduction and Fertility 1974, 36: 9- 22; Id., Capacitation of epididymal spermatozoa in a medium with high K-Na ratio and cyclic AMP for the fertilization of rat eggs in vitro, Journal of Reproduction and Fertility 1974, 36: 125-134. [113] Bondioli K.R., Wright R.W. Jr., Influence of culture media on in vitro fertilization of ovine tubal ovocytes, Journal of Animal Science 1980, 51: 660-667. [114] Hamner C.E., Jennings L.L., Sojka N.J., Cat (Felis catus L.) spermatozoa require capacitation, Journal of Reproduction and Fertility 1970, 23: 477-480; Bowen R.A., Fertilization in vitro of feline ova by spermatozoa from the ductus deferens, Biology of Reproduction, 17: 144-147. [115] Yanagimachi R., Fertilization of guinea pig eggs in vitro, Anatomical Record 1972, 174: 9- 19; Rogers B.J, Yanagimachi R., Retardation of guinea pig sperm acrosome reaction by glucose: the possible importance of pyruvate and lactate metabolism in capacitation and the acrosome reaction, Biology of Reproduction 1975, 13: 568-575; Id.,Competitive effect of magnesium on the calcium- dependent acrosome reaction in guinea pig spermatozoa, Biology of Reproduction 1976, 15: 614- 619; Rogers B.J., Chang L., Yanagimachi R., Glucose effect on respiration: possible mechanism for capacitation in guinea pig spermatozoa, Journal of Experimental Zoology 1979, 207: 107-112. [116] Mahi C.A., Yanagimachi R., Maturation and sperm penetration of canine ovarian ovocytes in vitro, Journal of Experimental Zoology 1976, 196: 189-196. [117] Bavister B.D., Edwards R.G., Steptoe P.C., Identification of the midpiece and tail of the spermatozoon during fertilisation of human eggs in vitro, Journal of Reproduction and Fertility 1969, 20: 159-160; Edwards R.G., Bavister B.D., Steptoe P.C., Early stages of fertilisation in vitro of human ovocytes matured in vitro, Nature 1969, 221: 632-635. [118] Yanagimachi, Chang, Fertilisation of hamster....; Id., In vitro fertilisation... [119] Edwards R.G., Steptoe P.C., Purdy J.M., Fertilisation and cleavage in vitro of preovulatory human ovocytes, Nature 1970, 227: 1307-1309; Steptoe P.C., Edwards R.G., Purdy J.M., Human blastocysts grown in culture, Nature 1971, 229: 132-133. 68 [120] Steptoe P.C., Edwards R.G., Birth after the reimplantation of a human embryo, Lancet 1978, II: 366. [121] Bavister, Early history of..., p.184. [122] Gould K.G., Cline E.M., Williams W.L., Observations on the induction of ovulation and fertilization in vitro in the squirrel monkey (Saimiri sciureus), Fertility and Sterility 1973, 24: 260- 268; Kuehl T.J., Dukelow W.R., Fertilization in vitro of Saimiri sciureus follicular ovocytes, Journal of Medical Primatology 1975, 4: 209-216. In entrambi gli studi, la capacità evolutiva degli zigoti generati mediante FIV (o degli ovociti attivati partenogeneticamente?) era scarsa. Ed effettivamente non è stata presentata alcuna prova chiara che la fecondazione fosse avvenuta. [123] Bavister B.D., Boatman D.E., Leibfried M.L. et Al., Fertilization and cleavage of rhesus monkey ovocytes in vitro, Biology of Reproduction 1983, 28: 983-999; Gould K.G., Ovum recovery and in vitro fertilization in the chimpanzee, Fertility and Sterility 1983, 40: 378-383. [124] Hamilton W.J., Early stages of human development, Annals of the Royal College of Surgeons of England 1949, 4: 281-294. [125] Zamboni L., Mishell D.R., Bell J.H.et Al., Fine structure of the human ovum in the pronuclear stage, Journal of Cell Biology 1966, 30: 579-600. [126] Pereda J., Croxatto H.B., Ultrastructure of a seven-cell human embryo, Biology of Reproduction 1978, 18: 481-489; Pereda J., Coppo M., Ultrastructure of a two-cell human embryo, Anatomy and Embryology 1987, 177: 91-96;Pereda J., Ultrastructural observation on early human eggs. Analysis of four concepti, in Motta P.M. (a cura di),Developments in Ultrastructure of Reproduction, New York: Alan R. Liss, 1989: 367-379. [127] Avendaño S., Crosatto H.D., Pereda J.et Al., A seven-cell human egg recovered from the oviduct, Fertility and Sterility 1975, 26: 1167-1172. [128] Soupart P., Strong P.A., Ultrastructural observations on human ovocytes fertilized in vitro, Fertility and Sterility 1974, 25: 11-43; Sathananthan A.H., Chen C., Sperm-ovocyte membrane fusion in the human during monospermic fertilization, Gamete Research 1986, 15: 117-186 [129] Sundström P., Nilsson O., Liedholm P., Cleavage rate and morphology of early human embryos obtained after artificial fertilization and culture, Acta Obstetrica and Gynaecologica Scandinavica 1981, 60: 109-120; Dvorač M., Tesarik J., Pilka L. et Al., Fine structure of human two-cell ova fertilized and cleaved in vitro, Fertility and Sterility 1982, 37: 661-667; Lopata A., Sathananthan A.H., Mc Bain J.C. et Al., The ultrastructure of the preovulatory human egg fertilized in vitro, Fertility and Sterility 1980, 33: 12-20; Van Blerkom J., Henry G.H., Porreco R., Preimplantation human embryonic development from polypronuclear eggs after in vitro fertilization, Fertility and Sterility 1984, 41: 686- 696. [130] Pereda, Ultrastructural observation..., p.367. [131] Austin C.R., Function of hyaluronidase in fertilization, Nature 1948, 162: 63. [132] Blandau R.J., Biology of eggs and implantation, in Young W.C. (a cura di), Sex and Internal Secretions, vol.II, Baltimore: Williams & Wilkins,1961: 797-882. [133] Yanagimachi R., Mammalian fertilization, in Knobil E., Neill J.D. (a cura di), The Physiology of Reproduction, vol.I, New York: Raven Press, 1988: 135-185; p.155. [134] Metz C.B., Seiguer A.C., Castro A.E., Inhibition of the cumulus dispersing and hyaluronidase activities of sperm by heterologous and isologous antisperm antibodies, Proceedings of the Society for Experimental Biology and Medicine 1972, 140: 776-781. [135] Anand S.R., Kaur S.P., Chaundhry P.S., Distribution of β-N-acetylglucosaminidase, hyaluronoglucosaminidase and acrosin in buffalo and goat spermatozoa, Hoppe-Seyler’s Zeitschrift für Physiologische Chemie 1977, 358: 685-688. [136] Austin C.R., Bishop M.W.H., Early reaction of the rodent egg to spermatozoa penetration, Journal of Experimental Biology 1956, 33: 358-365.

69 [137] Per una panoramica degli studi che sono seguiti negli anni Sessanta, vedi: Bedford J.M., Sperm capacitation and fertilization in mammals, Biology of Reproduction 1970, 2: 128-158. [138] Una rassegna esauriente di questi studi si trova in Yanagimachi R., Sperm-egg fusion, Current Topics in Membrane Transport 1988, 32: 3-43. [139] Eberspaecher U., Barros C., Changes at the hamster ovocyte surface from the germinal vesicle stage to ovulation, Gamete Research 1984, 9: 387-397. [140] Huang T.T.F. Jr., Yanagimachi R., Inner acrosomal membrane of mammalian spermatozoa: its properties and possible functions in fertilization, American Journal of Anatomy 1985, 174: 249-268. [141] Yanagimachi R., Noda Y.D., Physiological changes in the post-nuclear cap region of mammalian spermatozoa: a necessary preliminary to the membrane fusion between sperm and egg cells, Journal of Ultrastructural Research 1970, 31: 486-493. [142] Ibid. Cf. anche Phillips D.M., Yanagimachi R., Difference in tha manner of association of acrosome-intact and acrosome-reacted hamster spermatozoa with egg microvilli as revealed by scanning electron microscopy, Development, Growth and Differentiation 1982, 24: 543-551. [143] Talbot P., Chacon R.S., Ultrastructural observations on binding and membrane fusion between human sperm and zona pellucida-free hamster ovocytes, Fertility and Sterility 1982, 37: 240-248. [144] Wolf D.P., Armstrong P.B., Penetration of the zona-free mouse egg by capacitated epididymal sperm cinematographic observations, Gamete Research 1978, 1: 39-46. [145] Negli anni Novanta del XX secolo, la discinesia flagellare (parametri abnormi di motilità degli spermatozoi) offrirà un modello interessante per studiare il ruolo del movimento degli spermatozoi nei processi di fusione fra spermatozoo e oolemma. Cf. Wolf J.P., Feneux D., Ducot B. et Al., Influence of sperm movement parameters on human sperm-oolemma fusion, Journal of Reproduction and Fertility 1995, 105: 185-192. [146] Wolf, Armstrong, Penetration of the zona-free... [147] Hirao Y., Hiraoka J., Surface architecture of sperm tail entry into the hamster ovocyte, Development, Growth and Differentiation 1987, 29: 123-132. [148] Wilson E.B., The Cell in Development and Heredity, New York: Macmillan, 1925; Rothschild L., The fertilizing spermatozoon, Discovery 1956, 18: 64-65; Austin C.R., Fertilization, Englewood Cliffs: Prentice Hall, 1965; Monroy A.,Chemistry and Physiology of Fertilization, New York: Holt, Rinehart and Winston, 1965. [149] Wilson, The Cell in Development... [150] Ibid. [151] Ibid. Rispetto all’ipotesi di Wilson, gli studiosi hanno suddiviso le classi di uova in quattro tipi, in base al loro stato di meiosi e al momento in cui sono state inseminate. Cf. Austin C.R. , Walton A., Fertilization, in Parkes A.S. (a cura di),Marshall’s Physiology of Reproduction, vol.I, London: Longmans, Green and Co., 1960: 310-416; Dalcq A.M.,Introduction to General Embryology, Oxford: Oxford University Press, 1957. [152] Per una recensione vedi: Austin C.R., Ultrastructure of Fertilization, New York: Holt-Rinehart and Winston, 1968. [153] Longo F.J., Anderson E., The fine structure of pronuclear development and fusion in the sea urchin, , Journal of Cell Biology 1968, 39: 339-368. [154] Bedford J.M., Ultrastructural changes of the sperm head during fertilization in the rabbit, American Journal of Anatomy 1968, 123: 329-358; Id., Sperm capacitation....; Id., The saga of mammalian sperm from ejaculation to syngamy, in Gibian H., Plotz E.J. (a cura di), , New York: Springer Verlag, 1970: 124-182; Pikó L., Gamete structure and sperm entry in mammals, in Metz C.B., Monroy A. (a cura di.), Fertilization: Comparative Morphology, Biochemistry and Immunology, New York: Academic Press, 1969: 325-403; Stefanini M., Oura C., Zamboni L., Ultrastructure of fertilization in the mouse. II. Penetration of sperm into the ovum, Journal of Submicroscopic Cytology 1969, 1: 1-23; Szollosi D., Ris H., Observations on sperm penetration in 70 the rat, Journal of Biophysical and. Biochemical Cytology 1961, 10: 275-283; Yanagimachi R., Noda Y.D., Electron microscope studies of sperm incorporation into the golden hamster egg, American Journal of Anatomy 1970, 128: 429-462; Zamboni L., Fine Morphology of Mammalian Fertilization, New York: Harper and Row, 1971. [155] Stefanini, Oura, Zamboni, Ultrastructure of fertilization....; Zamboni, Morphology of... [156] Yanagimachi R., Noda Y.D., Physiological changes in the postnuclear cap region of mammalian spermatozoa: a necessary preliminary to the membrane fusion between sperm and egg cells, Journal of Ultrastructural Research 1970, 31: 486-494. [157] Yanagimachi, Noda, Electron microscope studies... [158] Longo F.J., Fertilization: a comparative ultrastructural review, Biology of Reproduction 1973, 9: 149-215; pp.163-164. [159] Longo F.J., Anderson E., The effects of nicotine on fertilization in the sea urchin, Arbacia punctulata, Journal of Cell Biology 1968. 46: 308-325; Id., Id., A cytological study of the relation of the to subsequent events of fertilization in urethane-treated eggs of the sea urchin, Arbacia punctulata, Journal of Cell Biology 1970, 47: 646-665. [160] Ibid. [161] Bedford, Ultrastructural changes [...], art. cit.; Yanagimachi, Noda, Electron microscope studies [...], art. cit. [162] Zamboni L., Mastroianni L., Electron microscopic studies on rabbit ova. I. Penetrated tubal ovum, Journal of Ultrastructural Research 1966, 14: 118-132. [163] Fu psotulato 120 anni fa che l’ovocita maturo possedesse tutti gli elementi necessari per lo sviluppo dell’embrione a eccezione di un “centro di divisione” attivo, e sebbene si credesse che gli spermatozoi contenessero un centro siffatto, si pensava che mancassero del substrato protoplasmatico in cui operare (Boveri T., Über die Befruchtung der Eier vonAscaris megalocephala, Sitzungsberichte der Gesellschaft für Morphologie und Physiologie 1887, 3: 71-80). “Sussistono ormai pochi dubbi che negli esseri umani soltanto il gamete maschile possieda un centrosoma attivo” (Palermo G.D., Colombero L.T., Rosenwaks Z., The human sperm centrosome is responsible for normal syngamy and early embryonic development, Reviews of Reproduction 1997, 2: 19-27; p.23). Analisi estensive condotte per mezzo della microscopia elettronica a trasmissione hanno dimostrato la presenza di centrioli negli spermatozoi e negli oviciti fecondati e la loro assenza da ovociti in metafase II; ciò conferma l’ereditarietà paterna del centrosoma nell’uomo (Sathananthan A.H., Kola I., Osborne J. et Al., Centrioles in the beginning of human development, Proceedings of the National Academy of Sciences USA 1991, 88: 4806-4810). Inoltre, la valutazione della distribuzione cromosomica per mezzo dell’ibridazione fluorescente in situ (in inglese FISH, fluorescence in situ hybridization) ha rivelato che il centrosoma dello spermatozoo è l’unica struttura responsabile dell’organizzazione della prima divisione mitotica degli embrioni umani (Palermo G., Munné S., Cohen J., The human zygote inherits its mitotic potential from the male gamete, Human Reproduction 1994, 9: 1220-1225). [164] Tilney L.C, Goddard J., Nucleating sites for the assembly of cytoplasmic microtubules in the ectodermal cells of blastulae of Arbacia punctulata, Journal of Cell Biology 1970, 46: 564-575. [165] Per una rassegna degli studi fino al 1972, vedi: Longo, Fertilization a comparative..., pp.190- 195. [166] Austin C.R., The Mammalian Egg, Oxford: Blackwell, 1961. [167] Longo, Fertilization a comparitive..., p.208. [168] Il termine “meiosi” proviene dal greco µειοũν, che significa “diminuire”, poiché questo tipo di cariocinesi (divisione del nucleo) dà luogo a una diminuzione del numero dei cromosomi della cellula. La meiosi fu descritta per la prima volta nell’uovo di riccio di mare nel 1876 dallo zoologo tedesco Oskar Hertwig (Hertwig O., Beiträge zur Kenntnis der Bildung, Befruchtung und Teilung des tierischen Eies, Morphologische Jahrbücher 1876, 1: 347-434), e poi nuovamente osservata nell’uovo di Ascaris megalocephala nel 1883 dall’embriologo belga Edouard van Beneden (Van Beneden 71 E.,Recherches sur la maturation de l'oeuf et la fécondation, Archives de Biologie 1883, 4: 610-620). Tuttavia l’importanza della meiosi ai fini della riproduzione e dell’ereditarietà fu descritta soltanto nel 1890 da August Weismann, il quale osservò che erano necessarie due divisioni cellulari per trasformare una cellula diploide in 4 cellule aploidi. Nel 1911, il genetista statunitense Thomas Hunt Morgan osservò il fenomeno del crossing-over nelle cellule germinali di e fornì la prima interpretazione genetica della meiosi (Morgan T.H., The origin of nine wing inDrosophila, Science 1911, 33: 496-499). [169] “ Nell’arco della vita di un uomo, il suo testicolo produce fino a 2´1012 spermatozoi”: Grootegoed J.A., The testis: spermatogenesis, in Hillier S.G., Kitchener H.C. e Neilson J.P. (a cura di), Scientific Essentials of , London: W.B. Saunders, 1996: 172-183; p.172. [170] Le cellule di Sertoli sono cellule allungate dei tubuli seminiferi del testicolo, alle quali gli spermatidi si attaccano per nutrirsi durante la spermatogenesi. Un’unica cellula di Sertoli si estende dalla membrana basale al lume del tubulo, benché a livello di microscopia ottica il suo citoplasma sia difficile da distinguere. Le cellule di Sertoli sono caratterizzate dalla presenza di un nucleo vescicolare, ovale, posizionato basalmente che contiene un nucleolo prominente. Queste cellule furono scoperte nel 1865 dal fisiologo italiano Enrico Sertoli, il quale le definì “cellule madri” (Sertoli E., Dell’esistenza di particolari cellule ramificate nei canalicoli seminiferi del testicolo umano, Il Morgagni 1865, 7: 31- 40). [171] Dal nome dello zoologo tedesco Franz Von Leydig, il quale descrisse il tessuto intertubulare testicolare nel 1850 e fu il primo a osservare la scarsità di queste cellule (meno dell’1%) rispetto ad altre cellule del parenchima testicolare (Von Leydig F., Zur Anatomie der männlichen Geschlechtsorgane und Analdrüsen der Säugethiere, Zeitschrift für wissenschaftliche Zoologie 1850; 2:1-57). [172] Deffieux X., Antoine J.M., Inhibines, activines et hormone anti-mullérienne: structure, signalisation, roles et valeur prédictive en médecine de la réproduction, Gynécologie, Obstétrique&Fertilité 2003, 31: 900-911; O'Connor A.E., De Kretser D.M., Inhibins in normal male physiology, Seminars in Reproductive Medicine 2004, 22: 177-185. [173] Champion M.D., Hawley R.S., Playing for half deck: the molecular biology of meiosis, Nature Cell Biology 2002, 4: s50-s56. [174] De Krester D.M., Kerr J.B., The cytology of the testis, in Knobil, Neil, op. cit., pp.1177-1290. [175] Una caratteristica unica della spermatogenesi è il fatto che le cellule germinali maschili in via di sviluppo non completano la citocinesi dopo la cariocinesi (sia mitosi che meiosi), cosicché la cellula figlia resta collegata mediante ponti di citoplasma fino alla fine della differenziazione dello spermatozoo (Dym M., Fawcett D.W., Further observations on the numbers of spermatogonia, spermatocytes, and spermatids connected by intercellular bridges in the mammalian testis, Biology of Reproduction 1971, 4: 195-215). Una spiegazione parziale ma plausibile del fenomeno poggia sul fato che nel maschio la meiosi dà luogo a cellule aploidi contenenti o un cromosoma X o un cromosoma Y: dato che entrambli gli eterosomi possono contenere geni che codificano proteine essenziali alla spermiogenesi e/o alla funzione spermatica, i ponti di citoplasma costituiscono un modo di scambiare prodotti genici fra cellule germinali contenenti X e Y. Si hanno evidenze sperimentali del fatto che gli spermatidi che hanno un citoplasma in comune possono scambiarsi proteine, il che significa che queste cellule sono funzionalmente diploidi (Braun R.E., Beheringer R.R., Peschon J.J. et Al., Genetically haploid spermatids are phenotypically diploid, Nature 1989, 337: 373-376; Caldwell K.A., Handel M.A., Protamine transcript sharing among post-meiotic spermatids, Proceedings of the National Academy of Sciences USA 1991, 88: 2407-2411). [176] La manchette è una struttura transitoria che si sviluppa durante la spermiogenesi. Essa consiste di 3 componenti: un anello perinucleare; un involucro microtubulare inserito nell’anello; placche dense attaccate all’estremità distale dell’involucro. La manchette microtubulare differisce dai singoli 72 microtubuli del citoplasma per la posizione perinucleare che le è specifica; perché rimane strutturalmente stabile per circa 2 settimane; per i suoi tempi organizzativi: infatti comincia a costituirsi quando l’assonema contenente tubulina è già presente. Ulteriori dettagli si trovano in Meistrich M.L., Nuclear morphogenesis during spermiogenesis, in De Kretser D. (a cura di), Molecular Biology of the Male , New York: Academic Press, 1993: 67-97; Tachibana M., Terada Y., Murakawa H. et Al., Dynamic changes in the cytoskeleton during human spermiogenesis, Fertility and Sterility 2005, 84 (2): 1241-1248. [177] Heller C.G. , Clermont Y., Spermatogenesis in man: an estimate of its duration, Science 1963, 140: 184-186;Clermont Y., The cycle of the seminiferous epithelium cycle in man, American Journal of Anatomy 1963, 112: 35-51. [178] Cooper T.G., Yeung, C.H., Fetic S., Sobhani A., Nieschlag E., Cytoplasmic droplets are normal structures of human sperm but are not well preserved by routine procedures for assessing sperm morphology, Human Reproduction 2004, 19: 2283-2288; Cooper T.G., Cytoplasmic droplets: The good, the bad or just confusing?, Human Reproduction 2005, 20: 9-11. 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[194] Hecht N.B., Molecular mechanism of male germ cell differentiation, Bioessays 1998, 20: 555- 561. [195] Wykes S.M., Visscher D.W., Krawetz S.A., Haploid transcripts persist in mature human spermatozoa, Molecular Human Reproduction 1997, 3: 15-19. [196] La complessità di trascrizione degli spermatozoi è stata affrontata per la prima volta analizzando una serie di cloni di cDNa selezionati a caso (Miller D., Briggs D., Snowden H. et Al., A complex population of RNAs exists in human ejaculate spermatozoa: implications for understanding molecular aspects of spermiogenesis, Gene 1999, 237: 385-392). Per un’ampia panoramica delle evidenze attualmente disponibili, vedi: Miller D., Ostermeier G.C., Krawetz S.A., The controversy, potential and roles of spermatozoal RNA, Trends in Molecular Medicine 2005, 11: 156-163. [197] Martins R.P., Krawetz S.A., RNA in human sperm, Asian Journal of Andrology 2005, 7: 115- 120; p.116. [198] I microRNA (miRNA) sono RNA non codificanti lunghi approssimativamente 22 nucleotidi che partecipano alla regolazione dei geni. I miRNAs conferiscono la loro regolazione a livello post- trascrizionale, dove o si scindono o reprimono la traduzione dei mRNA. È stato testato sperimentalmente per la loro funzione un numero relativamente basso di miRNA, e di quelli testati sono state proposte funzioni comprendenti la differenziazione cellulare, la proliferazione, l’apoptosi, le difese antivirali e il cancro. E’ possibile che si scopra che i miRNA svolgono un ruolo praticamente in tutti gli aspetti della regolazione dei geni. Per una panoramica recente dell’identificazione, della struttura e della funzione dei miRNA, vedi: Wang Y., Stricker H.M., Gou D. et Al., MicroRNA: past and present, Frontiers in Bioscience 2007, 12: 2316-2329; Zhang B., Wang Q., Pan X., MicroRNAs and their regulatory roles in animals and plants, Journal of Cell Physiology 2007, 210: 279-289. 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[221] Chang M.C., Fertilizing capacity of spermatozoa deposited into the Fallopian tubes, Nature 1951, 168: 697-698; p.697. Trent’anni dopo la prima definizione di “capacitazione” da parte sua e di

75 Austin, Chang ha riportato l’attenzione sul significato originario del termine: Id., The meaning of sperm capacitation, Journal of Andrology 1984, 5: 45-50. [222] Olds-Clarke P., Unresolved issues in mammalian fertilization, International Review of Cytology 2003, 232: 129-184; p.130. [223] Giojalas L.C., Rovasio R.A., Fabro G. et Al., Timing of sperm capacitation appers to be programmed according to egg availability in the female genital tract, Fertility and Sterility 2004, 82: 247-249; p.247. Queste osservazioni sono in linea con altre precedenti, secondo cui soltanto una piccola percentuale di una popolazione spermatica è un grado di fecondare l’ovocita in vivo: Cohen J., Adeghe A.J.-H., The other spermatozoa: fate and functions, in Mohri H. (a cura di),New Horizons in Sperm Cell Research, Tokyo-New York: Japan Science Press, Gordon and Breach, 1987: 125-134. Per una panoramica esauriente, vedi: Jaiswal B.S., Eisenbach M., Capacitation, in Hardy D.M. 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(a cura di),Introduction to Mammalian Fertilization, Norwell: Kluwer Academic, 2003: 237-256; Breitbart H., Signaling pathways in sperm capacitation and acrosome reaction, Cellular and Molecular Biology 2003, 49: 321-327; Tulsiani D.R.P., Abou-Haila A., Is sperm capacitation analogous to early phases of Ca2+-triggered membrane fusion in somatic cells and viruses?, BioEssays 2004, 26: 281-290; Fraser L.R., Adeoya-Osiguwa S., Baxendale R.W. et Al., First messenger regulation of mammalian sperm function via adenylyl cyclase / cAMP, Journal of Reproduction and Development 2005, 51: 37-46. Per una panormaica aggiornata degli studi sul modo in cui la capacitazione degli spermatozoi è regolata da alcuni “primi messaggeri” fra cui il peptide che promuove la fecondazione, l’adenosina, la calcitonina e l’angiotensina II (che si trovnao tutti nel plasma seminale), vedi: Fraser L.R., Adeoya-Osiguwa S.A., Baxendale R.W. et Al., Regulation of mammalian sperm capacitation by endogenous molecules, Frontiers in Bioscience 2006, 11: 1636-1645. [231] Fraser, Adeoya-Osiguwa, Baxendale, First messenger regulation... [232] Harkema W., Harrison R.A., Miller N.G. et Al., Enhanced binding of zona pellucida proteins to the acrosomal region of the intact boar spermatozoa in response to fertilizing conditions: a flow cytometric study, Biology of Reproduction 1998, 58: 421-430. 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Nella profase I (P-I) avviene la maggior parte degli eventi determinanti che differenziano la meiosi dalla mitosi, fra cui l’appaiamento dei cromosomi omologhi nonché la formazione e risoluzione di double-strand break (DSB) che conduce al crossover o ricombinazione fra cromosomi omologhi. La P-I si può ulteriormente suddividere in 5 sottofasi: leptotene, zigotene, pachitene, diplotene e diacinesi. Durante la P-I è presente una struttura proteinacea, specifica della meiosi, detta “complesso sinaptonemico” (SC). Esso si compone di due elementi laterali, o LE (che nel pachitene si dicono elementi assiali, o AE), i quali formano l’intera lunghezza di ciascuna cromatide sorella, e di un elemento centrale (CE) che “fa da cerniera lampo” fra i due elementi laterali e lega i due cromosomi omologhi in un processo detto “sinapsi”. Le diverse fasi della P-I sono definite dalla formazione del complesso sinaptonemico e si possono seguire mediante immunostaining delle componenti dell’SC, in particolare della proteina degli elementi laterali (proteina 3 del complesso sinaptonemico, o SYCP3) e la proteina dell’elemento centrale (proteina 1 del complesso sinaptonemico, o SYCP1). Vedi ad es. Di Carlo A.D., Travia G., De Felici M., The meiotic specific synaptonemal complex protein SYCP3 is expressed by female and male primordial germ cells of the mouse embryo, International Journal of Developmental Biology 2000, 44: 241-244; Costa Y., Speed R., Ollinger R. et Al., Two novel proteins recruited by synaptonemal complex protein 1 (SYCP1) are at the centre of meiosis, Journal of Cell Science 2005, 118 (Pt 12): 2755-2762. 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79 [272] Zeleznik A.J., Hillier S.G., The ovary: endocrine function, in Hillier, Kitchener, Neilson, op. cit., pp.133-146; pp.140-141. [273] Sebbene le tube di Falloppio abbiano preso il nome dal medico e anatomista italiano Gabriele Falloppio –il primo a descrivere queste strutture nel 1561 (Falloppio G., Observationes anatomicae, Venetiis: M.A. Ulmum, 1561)– Reinier de Graaf fu probabilmente il primo a comprenderne la vera funzione nella riproduzione (De Graaf, De mulierum organis...). [274] Killian G.J., Evidence for the role of oviduct secretions in sperm function, fertilization and embryo development, Animal Reproduction Sciences 2004, 82-83: 141-153; p.141. [275] Verhage H.G., Fazleabas A.T., Donnelly K., The in vitro synthesis and release of proteins by the human oviduct, Endocrinology 1988, 122: 1639-1645. [276] Killian, Evidence for the..., e riferimenti ivi citati. [277] Buhi W.C., Characterization and biological roles of oviduct-specific, oestrogen-dependent glycoprotein, Reproduction 2002, 123: 355-362; Killian G.J., Estrogen-associated glycoproteins in oviduct secretions: structure and evidence for a role in fertilization, in Tulsiani D. (a cura di), Introduction to Mammalian Reproduction, Boston: Kluwer Academic 2002, 187-201. [278] Cf. Killian, Evidence for..., p.148, tavola 4. [279] Araki Y., Nohara M., Yoshida-Komiya H. et Al., Effect of a null mutation of the oviduct-specific glycoprotein gene on mouse fertilization, Biochemical Journal 2003, 374(Pt 2): 551-557. [280] Gabler C., Chapman D.A., Killian G.J., Expression and presence of osteopontin and integrins in the bovine oviduct during the oestrous cycle, Reproduction 2003, 126: 721-729. [281] Rittling S.R., Matsumoto H.N., McKee M.D. et Al., Mice lacking osteopontin show normal development and bone structure but display altered osteoclast formation in vitro, Journal of Bone and Mineral Research 1998, 13: 1101-1111.Come ha osservato Killian con acuto senso critico, “la strategia per valutare la funzione delle singole secrezioni dell’ovidotto in vitro...non deve perdere di vista il fatto che in vivo le singole componenti agiscono di concerto con altre componenti presenti nell’ambiente oviduttale. Ciò spiega forse perché i topi null per proteine oviduttali come l’OSP o l’osteopontina siano fertili” (Killian, Evidence for the..., p.148). [282] Baluch D.P., Koeneman B.A., Hatch K.R. et Al., PKC isotypes in post-activated and fertilized eggs: association with the meiotioc spindle, Developmental Biology 2004, 274: 45-55; p.45. [283] Moore K.L., Essentials of Human Embryology, Toronto: Decker, 1988: 2. [284] Gilbert S.F., Developmental Biology, Sunderland: Sinauer, 2000: 185. [285] Riferendo dati ottenuti tramite osservazioni ed esperimenti sulla fecondazione in vitro e su embrioni unicellulari umani, l’Autore del presente saggio non intende né appoggiare la decisione di effettuare tali osservazioni ed esperimenti, né incoraggiare più l’uso del modello umano di fecondazione e di sviluppo iniziale per indagare la biologia della riproduzione gamica. [286] Mayor S., ACEing GPI release, Nature Structural and Molecular Biology 2005, 12: 107-108; p.107. [287] Primakoff P., Myles D.G., Penetration, adhesion, and fusion in mammalian sperm-egg interaction, Science 2002, 296: 2183-2185; p.2183. [288] Wassarman P.M., Contribution of mouse egg zona pellucida glycoproteins to gamete recognition during fertilization, Journal of Cell Physiology 2005, 204: 388-391; p.388. [289] Larsen W.J., Essentials of Human Embryology, New York: Churchill Livingstone, 1998: 1. [290] Tajik P., Niwa K., Murase T., Effects of different protein supplements in fertilization medium on in vitro penetration of cumulus-intact oocytes matured in culture, Theriogenology 1993, 40: 949- 958; Zhang L., Jiang S., Wozniak P.J. et Al., Cumulus cell function during bovine oocyte maturation, fertilization, and embryo development in vitro, Molecular Reproduction and Development 1995, 40: 338-344; Suzuki K., Eriksson B., Shimuzu H. et Al., Effect of hyaluronan on monospermic penetration of porcine oocytes fertilized in vitro, International Journal of Andrology 2000. 23: 13-21.

80 [291] Olds-Clarke, Unresolved issues in..., Vedi anche Drobnis E.Z., Yudin A.I., Cherr G.N. et Al., Hamster sperm penetration of the zona pellucida: kinematic analysis and mechanical implications, Developmental Biology 1988, 130: 311-323. [292] Yudin A.I., Cherr G.N., Katz D.F., Structure of the cumulus matrix and zona pellucida in the golden hamster: a new view of sperm interaction with oocyte-associated extracellular matrices, Cell and Tissue Research 1988, 251: 555-564. [293] Olds-Clarke, Unresolved issues in..., pp.152-157. [294] Yanagimachi R., Mammalian fertilization, in Knobil, Neill, op. cit., 1988: 135-185; pp.152-154. [295] Lin Y., Mahan K., Lathrop W.F. et Al., A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg, Journal of Cell Biology 1994, 125: 1157-1163. [296] Primakoff, Myles, Penetration, adhesion, and..., p.2183. [297] Baba D., Kashiwabara S., Honda A. et Al., Mouse sperm lacking cell surface hyaluronidase PH- 20 can pass through the layer of cumulus cells and fertilize the egg, Journal of Biological Chemistry 2002, 277: 30310-30314. [298] Dean J., Reassessing the molecular biology of sperm-egg recognition with mouse genetics, BioEssays 2003, 26: 29-38; p.29. See also Talbot P., DiCarlantonio G., Zao P. et Al., Motile cells lacking hyaluronidase can penetrate the hamster oocyte cumulus complex, Developmental Biology 1985, 108: 387-398. [299] Tulsiani D.R.P., Yoshida-Komiua H., Araki Y., Mammalian fertilization: a carbohydrate- mediated event, Biology of Reproduction 1997, 57: 487-494; Bi M., Wassler, Hardy, Sperm adhesion to..., pp.153-180. [300] Thaler C.D., Cardullo R.A., Defining oligosaccharide specificity for initial sperm-zona pellucida adhesion in the mouse, Molecular Reproduction and Development 1996, 45: 535-546; Wassler, Hardy, Sperm adhesion to...; Olds-Clarke, Unresolved issues in..., p.135. [301] Suarez S.S., Katz D.F., Meizel S., Changes in motility that accompany the acrosome reaction in hyperactivated hamster spermatozoa, Gamete Research 1984, 10: 253-265; Katz D.F., Cherr G.N., Lambert H., The evolution of hamster sperm motility during capacitation and interaction with the ovum in vitro, Gamete Research 1986, 14: 333-346. [302] Drobnis, Yudin, Cherr, Hamster sperm penetration.... [303] Olds-Clarke, Unresolved issues in..., p.153. [304] Olson G.E., Noland T.D., Winfrey V.P. et Al., Substructure of the postacrosomal sheath of bovine spernatozoa, Journal of Ultrastructure Research 1983, 85: 204-218. [305] Lee M.A., Storey B., Evidence for plasma membrane impermeability to small ions in acrosome- intact mouse spermatozoa bound to mouse zonae pellucidae, using an aminoacridine fluorescent pH probe: time course of the zona-induced acrosome reaction monitored by both chlortetracycline and pH probe fluorescence, Biology of Reproduction 1985, 33: 235-246. In vitro, tutti gli spermatozoi di topo che aderiscono alla ZP intatta mostrano un pattern B dopo uno staining con clortetraciclina (CTC) (Storey B.T., Lee M.A., Muller C. et Al., Binding of mouse spermatozoa to the zonae pellucidae of mouse eggs in cumulus: evidence that the acrosome remain substantially intact, Biology of Reproduction 1984, 31: 1119-1128), e ciò indica spermatozoi acrosoma-intatti (Saling P.M., Storey B.T., Mouse gamete interactions during fertilization in vitro: chlortetracycline as fluorescent probe for the mouse sperm acrosome reaction, Journal of Cell Biology 1979, 83: 544-555). [306] Dell A., Morris H.R., Easton R.L. et Al., The glycobiology of gametes and fertilization, Biochimica et Biophysica Acta 1999, 1473: 196-205; Tulsiani D.R., Yoshida-Komiya H., Araki Y., Mammalian fertilization: a carbohydrate-mediated event, Biology of Reproduction 1997, 57: 487- 494. [307] Bleil J.D., Wassarman P.M., Structure and function of the zona pellucida: identification and characterization of the proteins of the mouse oocyte's zona pellucida, Developmental Biology 1980, 81 76: 185-202. Gli esperimenti, condotti principalmente sul topo, hanno portato a concludere che la ZP sia composta da tre proteine (ZP1, ZP2 e ZP3). La ZP murina è il modello accettato di struttura della ZP nei vertebrati superiori ed è composta di unità ricorrenti di eterodimeri ZP2-ZP3 organizzati in filamenti incrociati da dimeri ZP1 (Greve J.M., Wassarman P.M., Mouse egg extracellular coat is a matrix of interconnected filaments possessing a structural repeat, Journal of Molecular Biology 1985, 181: 253-264). Studi sui geni che codificano le proteine della ZP hanno condotto alla recente scoperta di un 4° glicoproteina della ZP nell’uomo. Questa non è presente nel topo ma si trova in altre specie di mammiferi (Spargo S.C., Hope R.M., Evolution and nomenclature of the zona pellucida gene family, Biology of Reproduction 2003, 68: 358-362; Lefievre L., Conner S.J., Salpekar A. et Al., Four zona pellucida glycoproteins are expressed in the human, Human Reproduction 2004, 19: 1580-1586). La nuova glicoproteina, detta ZP4, svolge un ruolo strutturale e, insieme alla ZP3, è coinvolta nello sperm binding e nell’induzione della reazione acrosomiale (Yurewicz E.C., Sacco A.G., Gupta S.K. et Al., Hetero-oligomerization-dependent binding of pig oocyte zona pellucida glycoproteins ZPB and ZPC to boar sperm membrane vesicles, Journal of Biological Chemistry 1998, 273: 7488- 7494; Yonezawa N., Fukui N., Kuno M. et Al., Molecular cloning of bovine zona pellucida glycoproteins ZPA and ZPB and analysis for sperm-binding component of zona, European Journal of Biochemistry 2001, 268: 587-594; Chakravarty S., Suraj K., Gupta S.K., Baculovirus-expressed recombinant human zona pellucida glycoprotein-B induces acrosomal exocytosis in capacitated spermatozoa in addition to zona pellucida glycoprotein-C, Molecular Human Reproduction 2005, 11: 365-372; Caballero-Campo P., Chirinos M., Fan X.J. et Al., Biological effects of recombinant human zona pellucida on sperm function, Biology of Reproduction 2006, 74: 760-768). L’identificazione della ZP4 introduce la possibilità che il modello classico di interazione spermatozoi-ZP stabilito nel topo possa non applicarsi alle specie che producono ZP4. [308] Bleil J.D., Wassarman P.M., Mammalian sperm-egg interaction: identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm, Cell 1980, 20: 873-882. [309] Liu C., Litscher E.S., Mortillo S. et Al., Targeted disruption of the mZP3 gene results in production of eggs lacking a zona pellucida and infertility in female mice, Proceedings of the National Academy of Sciences USA 1996, 93: 5431-5436. [310] Wassarman P.M., Jovine L., Litscher E.S., A profile of fertilization in mammals, Nature Cell Biology 2001, 3: 59-64;Rodeheffer C., Shur B.D., Targeted mutations in β1,4-galactosyltransferase I reveal its multiple cellular functions, Biochimica et Biophysica Acta 2002, 1573: 258-270. [311] Macek M.B., Lopez L.C., Shur B.D., Aggregation of β1,4-galactosyltransferase on mouse sperm induces the acrosome reaction, Developmental Biology 1991, 147: 440-444; Gong X., Dubois D.H., Miller D.J. et Al., Activation of a G-protein complex by aggregation of β1,4-galactosyltransferase on the surface of sperm, Science 1995, 269: 1718-1721. [312] Per un’analisi recente delle ipotesi correnti sui meccanismi molecolari alla base del riconoscimento del gamete da parte della zona pellucida, vedi: Shur B.D., Rodeheffer C., Ensslin M.A. et Al., Identification of novel gamete receptors that mediate sperm adhesion to the egg coat, Molecular and Cellular Endocrinology 2006, 250: 137-148. [313] Felix R., Molecular physiology and pathology of Ca2+-conducting channels in the plasma membrane of mammalian sperm, Reproduction 2005, 129: 251-262; p.251. [314] In tutte le specie studiate fino ad oggi, il Ca2+ è assolutamente indispensabile per l’AR fisiologico dello spermatozoo: Florman H.M., Arnoult C., Kazam I.G. et Al., A perspective on the control of mammalian fertilization by oocyte-activated io channels in sperm: a tale of two channels, Biology of Reproduction 1998, 59: 12-16; Publicover S.J., Barratt C.L., Voltage-operated Ca2+ channels and the acrosome reaction: which channels are present and what do they do?, Human Reproduction 1999, 14: 873-879; Darszon A., Beltrán C., Felix R. et Al., Ion transport in sperm signaling, Developmental Biology 2001, 240: 1-14.

82 [315] Tomes C.N., De Blas G.A., Michaut M.A. et Al., α-SNAP and NSF are required in a priming step during the human sperm acrosome reaction, Molecular Human Reproduction 2004, 11: 43-51; p.43. Vedi anche Tomes C.N., Michaut M., De Blas G. et Al., SNARE complex assembly is required for human sperm acrosome reaction, Developmental Biology 2002, 243: 326-338, e i riferimenti ivi citati. [316] Bleil J.D., Wassarman P.M., Mammalian sperm-egg interaction: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein, Developmental Biology 1983, 95: 317-324. [317] Arnoult C., Zeng Y., Florman H.M., ZP3-dependent activation of sperm cation channels regulates acrosomal secretion during mammalian fertilization, Journal of Cell Biology 1996, 134: 637- 645. [318] Per una breve panoramica su questo aspetto dell’AR, vedi Evans J.P., Florman H.M., The state of the union: the cell biology of fertilization, Nature Cell Biology 2002, 4(1), S57-S63; Wassarman P.M., Jovine L., Litscher E.S., A profile of fertilization in mammals, Nature Cell Biology 2002, 4(1), E59- E64. Una descrizione più dettagliata della trasduzione del segnale della ZP3 si può trovare in Florman H.M., Kirkman-Brown J., Brown J.C. et Al., The acrosome reaction: an example of egg-activated signal transduction in sperm, ChemTracts Biochemistry and Molecular Biology 2003, 16: 126-133. [319] La famiglia di proteine TRP (Transient Receptor Potential) sono componenti di canali cationici che vengono attivate in risposta a diversi stimoli, che vanno dal fattore di crescita e stimolazione dei recettori della membrana plasmatica da parte di neurotrasmettitori a una varietà di segnali chimici e sensori. Attualmente, i membri della sottofamiglia TRPC sembrano essere i candidati più forti per i canali di flusso di Ca2+ che vengono attivati in risposta alla stimolazione dei recettori della membrana 2+ plasmatica che dà luogo all’idrolisi di PIP2, alla produzione di IP3 e DAG, e al rilascio di Ca indotto 2+ da IP3 dalla riserva di Ca intracellulare attraverso il recettore dell’IP3. Le interazioni omomeriche o eteromeriche selettive fra monomeri TRPC generano canali distinti che contribuiscono a meccanismi d’ingresso del Ca2+ store-operatedo store-independent (dipendenti o non dipendenti dalle riserve di Ca2+ nel reticolo endoplasmico). Sebbene non sia stato ancora stabilito in modo definitivo quale sia esattamente la funzione fisiologica dei canali TRPC, né come questi siano regolati, è evidente che una varietà di funzioni cellulari è regolata dall’ingresso di Ca2+ attraverso questi canali. Per una rassegna recente della struttura e delle funzioni dei canali TRPC-Ca2+, vedi: Ambudkar I.S., Bandyopadhyay B.C., Liu X. et Al., Functional organization of TRPC- Ca2+ channels and regulation of calcium microdomains, Cell Calcium 2006, 40: 495-504; Minke B., TRP channels and Ca2+ signaling, Cell Calcium 2006, 40: 261-275. [320] Jungnickel M.K., Marrero H, Birnbaumer L. et Al., Trp2 regulates entry of Ca2+ into mouse sperm triggered by egg ZP3, Nature Cell Biology 2001, 3: 499-502. [321] Leypold B.C., Yu C.R., Leinders-Zufall T. et Al., Altered sexual and social behaviors in trp2 mutant mice, Proceedings of the National Academy of Sciences USA 2002, 99: 6376-6381. [322] Breitbart, Cohen, Rubinstein, art. cit. [323] Zhu G.Z, Myles D.G., Primakoff P., Testase 1 (ADAM 24) a plasma membrane-anchored sperm protease implicated in sperm function during epididymal maturation or fertilization, Journal of Cell Science 2001, 114: 1787-1794. [324] Ohmura K., Kohno N., Kobayashi Y. et Al., A homologue of pancreatic trypsin is localized in the acrosome of mammalian sperm and is released during acrosome reaction, Journal of Biological Chemistry 1999, 274: 29426-29432. [325] Talbot P., Dandekar P., Perivitelline space: does it play a role in blocking polyspermy in mammals?, Microscopy Research and Technique 2003, 61: 349-357; Yanagimachi R., Fertilization and development initiation of orthodox and unorthodox ways: from normal fertilization to cloning, Advances in Biophysics 2003, 37: 49-89. [326] Yanagimachi R., Kamiguchi Y., Sugawara S. et Al., Gametes and fertilization in the Chinese hamster, Gamete Research 1983, 8: 97-117. 83 [327] Yanagimachi R., Philips D.M., The status of acrosomal caps of hamster spermatozoa immediately before fertilizationin vivo, Gamete Research 1984, 9: 1-19; Barros C., Vigil P., Herrera E. et Al., Selection of morphologically abnormal sperm by human cervical mucus, Archives of Andrology 1984, 12: 95-107; Jedlicki A., Barros C., Scanning electron microscope study of in vitro pre- penetration gamete interactions, Gamete Research 1985, 11: 121-131; Soldani P., Rosati F., Sperm-egg interaction in the mouse using live and glutaraldehyde-fixed eggs, Gamete Reseach 1987, 18: 225-235. [328] Hunter R.H.F., The Fallopian Tubes, Berlin: Springer Verlag, 1988: 95-96. [329] Yanagimachi R., Mammalian fertilization, in Knobil, Neill, op. cit., pp.135-185; pp.159-162. [330] Vedi, ad es., McLeskey S.B., Dowds C., Carballada R. et Al., Molecules involved in mammalian sperm-egg interaction, International Review of Cytology 1998, 177: 57-113. [331] Austin C.R., Bishop M.W., Fertilization in mammals, Biological Reviews 1957, 32: 296-349. [332] Id., Role of the rodent acrosome and perforatorium in fertilization, Proceedings of the Royal Society 1958, 149: 241-248. [333] Zaneveld L.J.D., De Jonge C.J., Mammalian acrosomal enzymes and the acrosome reaction, in Dunbar B.S., O'Rand M.G. (a cura di), A Comparative Overview of Mammalian Fertilization, New York: Plenum Press, 1991: 63-79. [334] Baba T., Azuma S., Kashiwabara S. et Al., Sperm from mice carrying a targeted mutation of the acrosin gene can penetrate the oocyte zona pellucida and effect fertilization, Journal of Biological Chemistry 1994, 269: 31845-31849;Adham I.M., Nayernia K., Engel W., Spermatozoa lacking acrosin protein show delayed fertilization, Molecular Reproduction and Development 1997, 46: 370-376, Lu Q., Shur B.D., Sperm from β-1,4-galactosyltransferase-null mice are refractory to ZP3-induced acrosome reactions and penetrate the zona pellucida poorly, Development 1997, 124: 4121-4131. [335] Sathananthan A.H., Trounson A.O., Wood C. et Al., Ultrastructural observations on the penetration of human sperm into the zona pellucida of the human egg in vitro, Journal of Andrology 1982, 3: 356-364. [336] Yanagimachi R., Mammalian fertilization, in Knobil, Neill op. cit., pp.135-185; pp.162- 163; Bronson R.A., Bronson S.K., Oula L. Et Al., An investigation of the latency period between sperm oolemmal adhesion and oocyte penetration, Molecular Reproduction and Development 1999, 52: 319-327; Primakoff P., Myles D.G., Gamete fusion in mammals, in Hardy, Sperm adhesion..., pp.303-318; Olds-Clarke, Unresolved issues in..., pp.162-164. [337] Terriou P., Giorgetti C., Hans E. et Al., Subzonal sperm insemination and total or extreme asthenozoospermia: an effective technique for an uncommon cause of , Fertility and Sterility 1993, 60: 1057-1061; Wolf J.P., Feneux D., Escalier D. et Al., Pregnancy after subzonal insemination with spermatozoa lacking outer dynein arms, Journal of Reproduction and Fertility 1993, 97: 487-492. [338] Talbot P., Chacon R.S., Ultrastructural observations on binding and membrane fusion between human sperm and zona pellucida-free hamster oocytes, Fertility and Sterility 1982, 37: 240-248. [339] Redkar A.A., Olds-Clarke P., An improved mouse sperm-oocyte plasmalemma binding assay: studies on characteristics of sperm binding in medium with and without glucose, Journal of Andrology 1999, 20: 500-508; Olds-Clarke, Unresolved issues in..., p.163. Durante studi in vitro sui topi si è osservato che non esiste una corrispondenza “uno a uno” fra il numero degli spermatozoi che aderiscono e il numero degli spermatozoi che si fondono con ovociti da cui è stata rimossa la zona pellucida. Tuttavia, un tasso significativamente basso di “adesione tenace”, resistente al lavaggio è indicativo di scarsa fusione con l’oolemma. [340] ADAM è l’acronimo inglese per “a disintegrin and a metalloprotease”: si tratta di una famiglia di glicoproteine che contengono un dominio disintegrina e metalloproteasi. Struttura e funzioni delle proteine ADAM sono illustrate in: White J.M., ADAMs: modulators of cell-cell and cell-matrix interactions, Current Opinion in Cell Biology 2003, 15: 598-606;Seals D.F., Courtneidge S.A., The ADAMs family of metalloproteases: multidomain proteins with multiple functions, Genes and 84 Development 2003, 17: 7-30; Huovila A.P., Turner A.J., Pelto-Huikko M. et Al., Shedding light on ADAM metalloproteinases, Trends in Biochemical Sciences 2005, 30: 413-422. [341] Per una review di questa evidenza, vedi: Wasserman, Jovine, Litscher, A profile of fertilization...; Evans J.P., The molecular basis of sperm-oocyte membrane interactions during mammalian fertilization, Human Reproduction Update 2002, 8: 297-311; Evans, Florman, The state of the union...; Oh E., Wortzman G.B., Zhu X. et Al., Getting sperm and egg together: the molecules of gamete membrane interactions, ChemTracts Biochemistry and Molecular Biology 2003, 16: 142- 157; White J.M., ADAMs: modulators of cell-cell and cell-matrix interactions, Current Opinion in Cell Biology 2003, 15: 598-606; Civetta A., Positive selection within sperm-egg adhesion domains of fertilin: an ADAM gene with a potential role in fertilization, Molecular Biology and Evolution 2003, 20: 21-29; Kim T., Oh J., Woo J.M. et Al., Expression and Relationship of Male Reproductive ADAMs in Mouse, Biology of Reproduction 2006, 74: 744-750. [342] Primakoff P., Hyatt H., Tredick-Kline J., Identification and purification of a sperm surface protein with a potential role in sperm-egg membrane fusion, Journal of Cell Biology 1987, 104: 141- 149. [343] Ibid.; Waters S.I., White J.M., Biochemical and molecular characterization of bovine fertilin α and β (ADAM1 and ADAM 2): a candidate sperm-egg binding/fusion complex, Biology of Reproduction 1997, 56: 1245-1254. [344] Barker H.L., Perry A.C., Jones R. et Al., Sequence and expression of a monkey testicular transcript encoding tMDC I, a novel member of the metalloproteinase-like, disintegrin-like, cysteine- rich (MDC) protein family, Biochimica et Biophysica Acta 1994, 1218: 429-431; Wolfsberg T.G., Straight P.D., Gerena R.L. et Al., ADAM, a widely distributed and developmentally regulated gene family encoding membrane proteins with a disintegrin and metalloprotease domain, Developmental Biology 1995, 169: 378-383; Heinlein U.A.O., Wallat S., Senftleben A. et Al., Male germ cell- expressed mouse gene TAZ83 encodes a putative, cysteine-rich transmembrane protein (cyritestin) sharing homologies with snake toxins and sperm-egg fusion proteins, Development, Growth and Differentiation 1996, 36: 49-58. [345] Lum L., Blobel C.P., Evidence for distinct serine protease activities with a potential role in processing the sperm protein fertilin, Developmental Biology 1997, 191: 131-145. [346] Linder B., Bammer S., Heinlein U.A., Delayed translation and posttranslational processing of cyritestin, an integral transmembrane protein of the mouse acrosome, Experimental Cell Research 1995, 221: 66-72; Hunnicutt G.R., Koppel D.E., Myles D.G., Analysis of the process of localization of fertilin to the sperm posterior head plasma membrane domain during sperm maturation in the epididymis, Developmental Biology 1997, 191: 146-159; Lum, Blobel, Evidence for distinct... [347] Ibid., Waters, White, Biochemical and molecular... [348] Chen H., Sampson N.S., Mediation of sperm-egg fusion: evidence that mouse egg α6β1 integrin is the receptor for sperm fertilin β, Chemistry and Biology 1999, 6: 1-10. [349] Almeida E.A., Huovila A.P., Sutherland A.E. et Al., Mouse egg integrin α6β1 functions as a sperm receptor, Cell 1995, 81: 1095-1104; Bigler D., Takahashi Y., Chen M.S. et Al., Sequence- specific interaction between the disintegrin domain of mouse ADAM2 (fertilin β) and murine eggs. Role of the α6 integrin subunit, Journal of Biological Chemistry 2000, 275: 11576-11584; Takahashi Y., Bigler D., Ito Y. et Al., Sequence-specific interaction between the disintegrin domain of mouse ADAM3 and murine eggs: role of β1 integrin-associated proteins CD9, CD81, and CD98, Molecular Biology of the Cell 2001, 12: 809-820. [350] Miller B.J., Georges-Labouesse E., Primakoff P. et Al., Normal fertilization occurs with eggs lacking the integrin α6β1 and is CD9-dependent, Journal of Cell Biology 2000, 149: 1289-1295. [351] Evans J.P., Kopf G.S., Schultz R.M., Characterization of the binding of recombinant mouse sperm fertilin beta subunit to mouse eggs: evidence for adhesive activity via an egg β1 integrin- mediated interaction, Developmental Biology 1997, 187: 79-93; Eto K., Huet C., Tarui T. H.Z. et 85 Al., Functional classification of ADAMs based on a conserved motif for binding to integrin α9β1: implications for sperm-egg binding and other cell interactions, Journal of Biological Chemistry 2002, 277: 17804-17810; Zhu X., Evans J.P., Analysis of the roles of RGD-binding integrins, α4/β9 integrins, α6 integrins, and CD9 in the interaction of the fertilin β (ADAM2) disintegrin domain with the mouse egg membrane, Biology of Reproduction 2002, 66: 1193-1202. [352] Sengoku K., Takuma N., Miyamoto T. et Al., Integrins are not involved in the process of human sperm-oolemmal fusion, Human Reproduction 2004, 19: 639-644. [353] Per una panoramica sull’argomento, vedi: Miller D.J., Shi X., Burkin H., Molecular basis of mammalian gamete binding, Recent Progress in Hormone Research 2002, 57: 37-73; Talbot P., Shur B.D., Myles D.G., Cell adhesion and fertilization: steps in oocyte transport, sperm-zona pellucida interactions, and sperm-egg fusion, Biology of Reproduction 2003, 68: 1-9. [354] Shamsadin R., Adham I.M., Nayernia K. et Al., Male mice deficient for germ-cell cyritestin are infertile, Biology of Reproduction 1999, 61: 1445-1451; Cho C., Ge H., Branciforte D. et Al., Analysis of mouse fertilin in wild-type and fertilin β (–/–) sperm: evidence for C-terminal modification, α/β dimerization, and lack of essential role of fertilin α in sperm-egg fusion, Developmental Biology 2000, 222: 289-295; Miller B.J., Georges-Labouesse E., Primakoff P. et Al.,Normal fertilization occurs with eggs lacking the integrin α6β1 and is CD9-dependent, Journal of Cell Biology 2000, 149: 1289- 1295; Nishimura H., Cho C., Branciforte D.R. et Al., Analysis of loss of adhesive function in sperm lacking cyritestin or fertilin β, Developmental Biology 2001, 233: 204-213; He Z.Y., Brakebusch C., Fassler R., Kreidberg J.A. et Al., None of the integrins known to be present on the mouse egg or to be ADAM receptors are essential for sperm-egg binding and fusion, Developmental Biology 2003, 254: 226-237. [355] Wassarman P.M., Jovine L., Qi H. et Al., Recent aspects of mammalian fertilization research, Molecular and Cellular Endocrinology 2005, 234: 95-103; p.101. [356] Le tetraspanine costituiscono una famiglia di circa 30 proteine caratterizzate conservativamente da 4 regionimembrane-spanning (donde il nome) e 2 loop extracellulari (uno grande, uno piccolo), nonché da una serie di altri residui conservati. Le tetraspanine sono espresse principalmente sulla superficie cellulare e ne è stato riferito il coinvolgimento in molti processi fisiologici e patologici, come la fecondazione, la risposta immunitaria, lo sviluppo del sistema nervoso, le metastasi e le malattie infettive (HCV, malaria etc.). La prima tetraspanina fu identificata nel 1981 come un antigene espresso sulle cellule della leucemia linfoblastica acuta e fu denominata CD9. Alcune componenti della famiglia TM4SF potrebbero svolgere un ruolo come “organizzatrici” dei complessi multimolecolari intramembranosi (detti anche laterali ocis) presenti sulla superficie cellulare che associano numerose proteine e definiti “reti di tetraspanine”. Ulteriori informazioni sulle tetraspanine si trovano in: Boucheix C., Rubinstein E., Tetraspanins, Cellular and Molecular Life Sciences 2001, 58: 1189- 1205; Hemler M.E., Tetraspanin functions and associated microdomains, Nature Reviews Molecular and Cell Biology 2005, 6: 801-811. [357] Kaji K., Oda S., Shikano T. et Al., The gamete fusion process is defective in eggs of Cd9- deficient mice, Nature Genetics 2000, 24: 279-282; Le Naour F., Rubinstein E., Jasmin C. et Al., Severely reduced female fertility in CD9-deficient mice, Science 2000, 287: 319-321; Miyado K., Yamada G., Yamada S. et Al., Requirement of CD9 on the egg plasma membrane for fertilization, Science 2000, 287: 321-324. Per una recente panoramica sull’argomento, vedi:Rubinstein E., Ziyyat A., Wolf J.P. et Al., The molecular players of sperm-egg fusion in mammals, Seminars in Cell and Developmental Biology 2006, 17: 254-263. [358] Chen M.S., Tung K.S., Coonrod S.A. et Al., Role of the integrin-associated protein CD9 in binding between sperm ADAM 2 and the egg integrin α6β1: implications for murine fertilization, Proceedings of the National Academy of Sciences USA 1999, 96: 11830-11835; Takahashi Y., Bigler D., Ito Y. et Al., Sequence-specific interaction between the disintegrin domain of mouse ADAM 3 and murine eggs: role of β1 integrin-associated proteins CD9, CD81, and CD98, Molecular Biology of the 86 Cell 2001, 12: 809-820; Wong G.E., Zhu X., Prater C.E. et Al., Analysis of fertilin alpha (ADAM1)- mediated sperm-egg cell adhesion during fertilization and identification of an adhesion-mediating sequence in the disintegrin-like domain, Journal of Biological Chemistry 2001, 276: 24937-24945; Zhu X., Evans J.P., Analysis of the roles of RGD-binding integrins, α4/α9 integrins, α6 integrins, and CD9 in the interaction of the fertilin β (ADAM2) disintegrin domain with the mouse egg membrane, Biology of Reproduction 2002, 66: 1193-1202. [359] Evans, The molecular basis..., p.304. [360] Ellerman D.A., Ha C., Primakoff P. et Al., Direct binding of the ligand PSG17 to CD9 requires a CD9 site essential for sperm-egg fusion, Molecular Biology of the Cell 2003, 14: 5098-5103. [361] Inoue N., Ikawa M., Isotani A. et Al., The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs, Nature 2005, 434: 234-238. [362] Per una descrizione sistematica degli aspetti cellulari e molecolari delle interazioni leucocita- endotelio, vedi: Brown E.J., Adhesive interactions in the immune system, Trends in Cell Biology 1997, 7: 289-295. [363] Kaji K., Kudo A., The mechanism of sperm-oocyte fusion in mammals, Reproduction 2004, 127: 423-429, p.423. [364] Longo F.J., Chen D.Y., Development of surface polarity in mouse eggs, Scanning Electron Microscopy 1984, 2: 703-716. [365] Kaji, Kudo, The mechanism of sperm-oocyte... [366] Thomson R.S., Smith D.S., Zamboni L., Fertilization of mouse ova in vitro, Fertility and Sterility 1974, 25: 211-249;Bedford J.M., Cooper G.W., Membrane fusion events in fertilization of vertebrate eggs, Membrane Surface Reviews 1978, 5: 65-125; Yamagimachi Y., Noda Y.D., Electron microscope studies of sperm incorporation into the golden hamster egg, Journal of Anatomy 1978, 128: 429- 437; Sathananthan A.H., Chen C., Sperm-oocyte function in the human during monospermic fertilization, Gamete Research 1986, 15: 177-186. [367] Bronson R.A., Fusi F.M., Cooper G.W. et Al., Antisperm antibodies induce polyspermy by promoting adherence of human sperm to zona-free hamster eggs, Human Reproduction 1990, 5: 690- 696. [368] Bronson R., Is the oocyte a non-professional phagocyte?, Human Reproduction Update 1998, 4: 763-775. [369] Bronson R.A., Fusi F.M., Fleit H.B., Identification of a oolemmal IgG Fc receptor: its role in promoting binding of antibody-labeled human sperm to zona-free hamster eggs, American Journal of Reproductive Immunology 1990, 23: 876-892. [370] Id., Monoclonal antibodies identify Fcg receptors on unfertilized human oocytes but not spermatozoa, Journal of Reproductive Immunology 1992, 21: 293-307. Inoltre, si è dimostrato che la preincubazione di ovuli di criceto privi di ZP con IgG-Fc umana, per saturare i recettori di Fcg dell’oolemma, abroga la promozione dell’adesione spermatozoo-oolemma da parte di spermatozoi marcati con anticorpi: Bronson R.A., Fusi F.M., Autoimmunity to sperm antigens, Immunology Clinics of North America 1994, 14: 773-786. [371] Yanagimachi R., Sperm-egg fusion, Current Topics in Membrane Transport 1988, 32: 3-43. [372] Wolf, Feneux, Escalier, Pregnancy after subzonal... [373] Cran D.G., Moor R.M., Irvine R.F., Initiation of the cortical reaction in hamster and sheep oocytes in response to inositol trisphosphate, Journal of Cell Science 1988 91(Pt 1): 139-144; Sun Q.- Y., Cellular and molecular mechanisms leading to cortical reaction and polyspermy block in mammalian eggs, Microscopy Research and Technique 2003, 61: 342-348. [374] Hyslop L.A., Nixon V.L., Levasseur M. et Al., Ca2+ promoted cyclin B1 degradation in mouse oocytes requires the establishment of a metaphase arrest, Developmental Biology 2004, 269: 206-219. [375] Jones K.T., Mammalian egg activation: from Ca2+ spiking to cell cycle progression, Reproduction 2005, 130: 813-823. 87 [376] Ozil J.P., Markoulaki S., Toth S. et Al., Egg activation events are regulated by the duration of a 2+ sustained [Ca ]cyt signal in the mouse, Developmental Biology 2005, 282: 39-54; Aoki F., Hara K.T., Schultz R.M., Acquisition of transcriptional competence in the 1-cell mouse embryo: requirement for recruitment of maternal mRNAs, Molecular Reproduction and Development 2003, 64: 270-274. [377] Malcuit, Kurokawa, Fissore, art. cit., p.565. [378] Stricker S.A., Comparative biology of calcium signalling during fertilization and egg activation in animals, Developmental Biology 1999, 211: 57-76; Runft L.L., Jaffe L.A., Mehlmann L.M., Egg activation at fertilization: where it all begins, Developmental Biology 2002, 245: 237-254. [379] Lawrence Y., Whitaker M., Swann K., Sperm-egg fusion is the prelude to the initial Ca2+ increase at fertilization in the mouse, Development 1997, 124: 233-241. [380] Stricker, Comparative biology of calcium.... [381] Jones K.T., Carroll J., Merriman J.A. et Al., Repetitive sperm-induced Ca2+ transients in mouse oocytes are cell cycle dependent, Development 1995, 121: 3259-3266; Marangos P., FitzHarris G., Carroll J., Ca2+ oscillations at fertilization in mammals are regulated by the formation of pronuclei, Development 2003, 130: 1461-1472; Jellerette T., Kurokawa M., Lee B. et Al., Cell cycle-coupled 2+ [Ca ]i oscillations in mouse zygotes and function of the inositol 1,4,5-trisphosphate receptor-1, Developmental Biology 2004, 274: 94-109. [382] Fissore R.A., Dobrinsky J.R., Balise J.J. et Al., Patterns of intracellular Ca2+ concentrations in fertilized bovine eggs, Biology of Reproduction 1992, 47: 960-969; Nakada K., Mizuno J., Shiraishi K. et Al., Initiation, persistence, and cessation of the series of intracellular Ca2+ responses during fertilization of bovine eggs, Journal of Reproduction and Development 1995, 41:77-84. [383] Koch G.L., The endoplasmic reticulum and calcium storage, Bioessays 1990, 12: 527- 531; Berridge M.J., The endoplasmic reticulum: a multifunctional signalling organelle, Cell Calcium 2002, 32: 235-249. [384] Mehlmann L.M., Mikoshiba K., Kline D., Redistribution and increase in cortical inositol 1,4,5- trisphosphate receptors after meiotic maturation of the mouse oocyte, Developmental Biology 1996, 180: 489-498; FitzHarris G., Marangos P., Carroll J., Cell cycle-dependent regulation of structure of endoplasmic reticulum and inositol 1,4,5-trisphosphate-induced Ca2+ release in mouse oocytes and embryos, Molecular Biology of the Cell 2003, 14: 288-301. [385] Miyazaki S., Shirakawa H., Nakada K. et Al., Essential role of the inositol 1,4,5-trisphosphate receptor/Ca2+release channel in Ca2+ waves and Ca2+ oscillations at fertilization of mammalian eggs, Developmental Biology 1993, 158: 62-78; Shiraishi K., Okada A., Shirakawa H. et Al., Developmental changes in the distribution of the endoplasmic reticulum and inositol 1,4,5-trisphosphate receptors and the spatial pattern of Ca2+ release during maturation of hamster oocytes, Developmental Biology 1995, 170: 594-606; Mehlmann, Mikoshiba, Kline, Redistribution and increase...; Xu Z., Williams C.J., Kopf G.S. et Al., Maturation-associated increase in IP3 receptor type 1: role in conferring 2+ increased IP3sensitivity and Ca oscillatory behavior in mouse eggs, Developmental Biology 2003, 254: 163-171. [386] Per una dettagliata raccolta di dati che documentano questa affermazione, v.: Ozil J.P., The parthenogenetic development of rabbit oocytes after repetitive pulsatile electrical stimulation, Development 1990, 109: 117-127; Ozil J.P., Huneau D., Activation of rabbit oocytes: The impact of the Ca2+ signal regime on development. Development 2001, 128: 917-928; Ozil J.P., Markoulaki S., Toth 2+ S. et Al., Egg activation events are regulated by the duration of a sustained [Ca ]cyt signal in the mouse, Developmental Biology 2005, 282: 39-54; Miyazaki S., Shirakawa H., Nakada K. et Al.,Essential role of the inositol 1,4,5-trisphosphate receptor/Ca2+ release channel in Ca2+ waves and Ca2+ oscillations at fertilization of mammalian eggs, Developmental Biology 1993, 158: 62-78; Ducibella T., Huneau D., Angelichio E. et Al., Egg-to-embryo transition is driven by differential responses to Ca2+ oscillation number, Developmental Biology 2002, 250: 280–291. [387] Whitaker M., Swann K. , Lighting the fuse at fertilization, Development 1993, 117: 1-12. 88 [388] Rogers N.T., Hobson E., Pickering S. et Al., Phospholipase Cz causes Ca2+ oscillations and parthenogenetic activation of human oocytes, Reproduction 2004, 128: 697-702; Malcuit C., Kurokawa M., Fissore R.A., Calcium oscillations and mammalian egg activation, Journal of Cellular Physiology 2006, 206: 565-573. [389] Turner P.R., Sheetz M.P., Jaffe L.A., Fertilization increases the polyphosphoinositide content of sea urchin eggs, Nature 1984, 310: 414-415; Stith B.J., Espinoza R., Roberts D. et Al., Sperm increase inositol 1,4,5-trisphosphate mass in Xenopus laevis eggs preinjected with calcium buffers or heparin, Developmental Biology 1994, 165: 206-215. [390] Parrington J., Brind S., De Smedt H. et Al., Expression of inositol 1,4,5-trisphosphate receptors in mouse oocytes and early embryos: the type I isoform is upregulated in oocytes and downregulated after fertilization, Developmental Biology 1998, 203: 451-461; Rongish B.J., Wu W., Kinsey W.H., Fertilization-induced activation of phospholipase C in the sea urchin egg, Developmental Biology 1999, 215: 147-154; Rice A., Parrington J., Jones K.T. et Al., Mammalian sperm contain a 2+ Ca sensitive phospholipase C activity that can generate InsP3 from PIP2 associated with intracellular organelles, Developmental Biology 2000, 227: 125-135. [391] Miyazaki S., Shirakawa H., Nakada K. et Al., Essential role of the inositol 1,4,5-trisphosphate receptor/Ca2+release channel in Ca2+ waves and Ca2+ oscillations at fertilization of mammalian eggs, Developmental Biology 1993, 158: 62-78. [392] Koch G.L., The endoplasmic reticulum and calcium storage, Bioessays 1990, 12: 527- 531; Berridge M.J., The endoplasmic reticulum: a multifunctional signaling organelle, Cell Calcium 2002, 32: 235-249. [393] Bazzi M.D., Nelsestuen G.L., Differences in the effects of phorbol esters and diacylglycerols on protein kinase C, Biochemistry 1989, 28: 9317-9323. [394] Halet G., Tunwell R., Parkinson S.J. et Al., Conventional PKCs regulate the temporal pattern of Ca2+ oscillations at fertilization in mouse eggs, Journal of Cell Biology 2004, 164: 1033-1044. [395] Stricker S.A., Repetitive calcium waves induced by fertilization in the nemertean worm Cerebratulus lacteus, Developmental Biology 1996, 176: 243-263. [396] Carroll D.J., Ramarao C.S., Mehlmann L.M. et Al., Calcium release at fertilization in starfish eggs is mediated by phospholipase Cγ, Journal of Cell Biology 1997, 138: 1303-1311; Giusti A.F., Carroll D.J., Abassi Y.A. et Al.,Requirement of a Src family kinase for initiating calcium release at fertilization in starfish eggs, Journal of Biological Chemistry 1999, 274: 29318-29322; Giusti A.F., Carroll D.J., Abassi Y.A. et Al., Evidence that a starfish egg Src family tyrosine kinase associates with PLC-γ1 SH2 domains at fertilization, Developmental Biology 1999, 208: 189-199; Sato K., Tokmakov A.A., Iwasaki T. , Tyrosine kinase-dependent activation of phospholipase Cγ is required for calcium transient in Xenopus egg fertilization, Developmental Biology 2000, 224: 453-469; Tokmakov A.A., Sato K.I., Iwasaki T. et Al, Src kinase induces calcium release in Xenopus egg extracts via PLCγ and IP3-dependent mechanism, Cell Calcium 2002, 32: 11-20; Sato K., Tokmakov A.A., He C.L. et Al., Reconstitution of Src-dependent phospholipase Cγ phosphorylation and transient calcium release by using membrane rafts and cell-free extracts from Xenopus eggs, Journal of Biological Chemistry 2003, 278: 38413-38420; Runft L.L., Carroll D.J., Gillett J. et Al., Identification of a starfish egg PLC- γ that regulates Ca2+ release at fertilization, Developmental Biology 2004, 269: 220-236. [397] Cho C., Bunch D.O., Faure J.E. et Al., Fertilization defects in sperm from mice lacking fertilin beta, Science 1998, 281: 1857-1859; Eto K., Puzon-McLaughlin W., Sheppard D. et Al., RGD- independent binding of integrin a9b1 to the ADAM-12 and -15 disintegrin domains mediates cell-cell interaction, Journal of Biological Chemistry 2000, 275: 34922-34930; Evans J.P., Kopf G.S., Schultz R.M., Characterization of the binding of recombinant mouse sperm fertilin β subunit to mouse eggs: evidence for adhesive activity via an egg b1 integrin-mediated interaction, Developmental Biology 1997, 187: 79-93; Fenichel P., Durand-Clement M., Role of integrins during fertilization in mammals, Human Reproduction 1998, 13: 31-46; Kaji K., Oda S., Shikano T. et Al., The gamete fusion process is 89 defective in eggs of Cd9-deficient mice, Nature Genetics 2000, 24: 279-282; Miller B.J., Georges- Labouesse E, Primakoff P. et Al., Normal fertilization occurs with eggs lacking the integrin α6β1 and is CD9-dependent, Journal of Cell Biology 2000, 149: 1289-1296; Zhu X., Evans J.P., Analysis of the roles of RGD-binding integrins, a4/α9 integrins, α6 integrins, and CD9 in the interaction of the fertilin β (ADAM2) disintegrin domain with the mouse egg membrane, Biology of Reproduction 2002, 66: 1193- 1202. Un meccanismo di attivazione basato sulle ADAM è stato proposto anche nella rana (Norris J.W., Tomczak M.M., Oliver A.E. et Al., Structural characterization of the ADAM 16 disintegrin loop active site, Biochemistry 2003, 42: 9813-9821). Nel riccio di mare, la reazione acrosomiale smaschera una proteina legante di adesione (adhesion protein binding) , una proteina fortemente idrofoba con un’affinità per i carboidrati (Glabe C.G., Interaction of the sperm adhesive protein, bindin, with phospholipid vesicles. II. Bindin induces the fusion of mixed-phase vesicles that contain phosphatidylcholine and phosphatidylserine in vitro, Journal of Cell Biology 1985, 100: 800-806), il cui recettore è stato identificato (Kamei N., Swanson W.J., Glabe C.G., A rapidly diverging EGF protein regulates species-specific signal transduction in early sea urchin development, Developmental Biology 2000, 225: 267-276). [398] Saunders C.M., Larman M.G., Parrington J. et Al., PLCζ: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo development, Development 2002, 129: 3533-3544. [399] Wassarman P.M., Fertilization in animals, Developmental Genetics 1999, 25: 83-86. [400] Akiyama S.K., Integrins in cell adhesion and signaling, Human Cell 1996, 9: 181-186. [401] Campbell K.D., Reed W.A., White K.L., Ability of integrins to mediate fertilization, intracellular calcium release, and parthenogenetic development in bovine oocytes, Biology of Reproduction 2000, 62: 1702-1709; Iwao Y., Fujimura T., Activation of Xenopus eggs by RGD-containing peptides accompanied by intracellular Ca2+ release, Developmental Biology 1996, 177: 558-567; Viets L.N., Campbell K.D., White K.L., Pathways involved in RGD-mediated calcium transients in mature bovine oocytes, Cloning Stem Cells 2001, 3: 105-113. [402] Campbell K.D., Reed W.A., White K.L., Ability of integrins to mediate fertilization, intracellular calcium release, and parthenogenetic development in bovine oocytes, Biology of Reproduction 2000, 62: 1702–1709. [403] Abassi Y.A., Foltz K.R., Tyrosine phosphorylation of the egg receptor for sperm at fertilization, Developmental Biology 1994, 164: 430-443. [404] Woods M.L., Shimizu Y., Signaling networks regulating b1 integrin-mediated adhesion of T lymphocytes to extracellular matrix, Journal of Leukocyte Biology 69: 874–880, 2001. [405] Mehlmann L.M., Carpenter G., Rhee S.G. et Al., SH2 domain-mediated activation of phospholipase Cγ is not required to initiate Ca2+ release at fertilization of mouse eggs, Developmental Biology 1998, 203: 221-232. [406] Whitaker M., Calcium at fertilization and in early development, Physiological Reviews 2006, 86: 25-88; p.35. [407] Ibid., p.36 e riferimenti ivi citati. [408] Stice S.L., Robl J.M., Activation of mammalian oocytes by a factor obtained from rabbit sperm, Molecular Reproduction and Development 1990, 25: 272-280; Swann K., A cytosolic sperm factor stimulates repetitive calcium increases and mimics fertilization in hamster eggs, Development 1990, 110: 1295-1302; Stricker S.A., Intracellular injections of a soluble sperm factor trigger calcium oscillations and meiotic maturation in unfertilized oocytes of a marine worm, Developmental Biology 1997, 186: 185-201; Wu H., He C.L., Fissore R.A., Injection of a porcine sperm factor triggers calcium oscillations in mouse oocytes and bovine eggs, Molecular Reproduction and Development 1997, 46: 176-189. [409] La microiniezione diretta di uno spermatozoo immobilizzato nell’ooplasma per mezzo di una pipetta sotto osservazione microscopica (Intra Cytoplasmic Sperm Injection o ICSI) è stata messa a punto già nel 1966 per esperimenti su gameti non di mammiferi. A quel tempo la ICSI non veniva 90 effettuata a fini di fecondazione assistita, bensì per dimostrare che la decondensazione del nucleo dello spermatozoo e la formazione del pronucleo maschile non richiedevano una precedente interazione fra lo spermatozoo e le membrane dell’ovocita (Hiramoto Y., Microinjection of the live spermatozoa into sea urchin eggs, Experimental Cell Research 1966, 27: 416-426). Seguirono vari esperimenti, e i miglioramenti della microiniezione ottenuti usando un modello di coniglio hanno dato luogo non soltanto alla formazione del pronucleo a seguito di una fecondazione indotta dalla ICSI, ma anche alla prosecuzione della divisione embrionale e alla nascita di prole viva e normale. La ICSI è stata applicata per la prima volta a ovociti umani nel 1988 (Lanzendorf S.M., Slusser J., Maloney M.K. et Al., A preclinical evaluation of pronuclear formation by microinjection of human spermatozoa into human oocytes, Fertility and Sterility 1988, 49: 835-842). Tali esperimenti hanno dimostrato che gli ovociti umani erano in grado di sopravvivere alla microiniezione e, successivamente, di supportare la formazione di pronuclei maschili e femminili. Nel 1992, Palermo et al. hanno dato notizia delle prime gravidanze umane ottenute in seguito a ICSI (Palermo G., Joris H., Devroey P. et Al., after intracytoplasmic injection of single spermatozoon into an oocyte, Lancet 1992, 340: 17-18). Per una panoramica recente di questa tecnica, delle sue conseguenze e delle sue applicazioni, vedi: Yanagimachi R., Intracytoplasmic injection of spermatozoa and spermatogenic cells: its biology and applications in humans and animals, Reproductive Biomedicine Online 2005, 10: 247-288. [410] Kurokawa e Fissore hanno riferito che, nel topo, le oscillazioni indotte dalla ICSI si verificano con minore frequenza dopo la prima ora e terminano prematuramente (Kurokawa M., Fissore R.A., ICSI-generated mouse zygotes exhibit altered calcium oscillations, inositol 1,4,5-trisphosphate receptor-1 down-regulation, and embryo development, Molecular Human Reproduction 2003, 9: 523- 533). Nel cavallo, inoltre, la ICSI non riesce a indurre regolari oscillazioni del Ca2+ (Bedford S.J., Kurokawa M., Hinrichs K. et Al., Patterns of intracellular calcium oscillations in horse oocytes fertilized by intracytoplasmic sperm injection: possible explanations for the low success of this assisted reproduction technique in the horse, Biology of Reproduction 2004, 70: 936-944). [411] Malcuit, Kurokawa, Fissore, Calcium oscillations and...,p.566. [412] Choi D., Lee E., Hwang S. et Al., The biological significance of phospholipase Cβ1 gene mutation in mouse sperm in the acrosome reaction, fertilization, and embryo development, Journal of Assisted Reproduction and Genetics 2001, 18: 305-310; Fukami K., Nakao K., Inoue T. et Al., Requirement of phospholipase Cδ4 for the zona pellucida-induced acrosome reaction, Science 2001, 292: 920-923; Parrington J., Jones M.L., Tunwell R. et Al., Phospholipase C isoforms in mammalian spermatozoa: potential components of the sperm factor that causes Ca2+ release in eggs, Reproduction 2002, 123: 31-39. [413] Saunders, Larman, Parrington, PLCζ: a sperm-specific trigger... [414] Cox L.J., Larman M.G., Saunders C.M. et Al., Sperm phospholipase Cζ from humans and cynomolgus monkeys triggers Ca2+ oscillations, activation and development of mouse oocytes, Reproduction 2002, 124: 611-623; Fujimoto S., Yoshida N., Fukui T. et Al., Mammalian phospholipase Cζ induces oocyte activation from the sperm perinuclear matrix, Developmental Biology 2004, 274: 370-383; Kouchi Z., Fukami K., Shikano T. et Al., Recombinant phospholipase Cζ has high Ca2+ sensitivity and induces Ca2+ oscillations in mouse eggs, Journal of Biological Chemistry 2004, 279: 10408-10412; Larman M.G., Saunders C.M., Carroll J. et Al., Cell cycle-dependent Ca2+ oscillations in mouse embryos are regulated by nuclear targeting of PLCζ, Journal of Cell Science 2004, 117(Pt. 12): 2513-2521. 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91 [417] Larman, Saunders, Carroll, Cell cycle-dependent Ca2+; Yoda A., Oda S., Shikano T. et Al., Ca2+ oscillation-inducing phospholipase Cζ expressed in mouse eggs is accumulated to the pronucleus during egg activation, Developmental Biology 2004, 268: 245-257. [418] Fujimoto S., Yoshida N., Fukui T. et Al., Mammalian phospholipase Cζ induces oocyte activation from the sperm perinuclear matrix, Developmental Biology 2004, 274: 370-383. [419] Williams C.J., Signalling mechanisms of mammalian oocyte activation, Human Reproduction Update 2002, 4: 313-321; p.313. [420] Evans J.P., Florman H.M., The state of the union: the cell biology of fertilization, Nature Cell Biology 2002, 4(1): S57-S63. [421] Lawrence Y., Ozil J.P., Swann K., The effects of a Ca2+ chelator and heavy-metal-ion chelators upon Ca2+oscillations and activation at fertilization in mouse oocytes suggest a role for repetitive Ca2+ increases, Biochemical Journal 1998, 335: 335-342. [422] Malcuit, Kurokawa, Fissore, Calcium oscillations and...., p.565.

92 GIGLIOLA SICA

THE DEVELOPMENT OF PRE-IMPLANTATION EMBRYO

The expression " pre-implantation embryo " was introduced by Clifford Grobstein in a paper published in Scientific American in 1979.1 It refers to embryo development from zygote to blastocyst stage during the journey through the reproductive tract and includes all the important transitions that occur following fertilization and the beginning of . Human fertilization occurs when a capacitated spermatozoon, with its haploid number of chromosomes, passes through oocyte investments represented by the corona radiata and zona pellucida, and penetrates the oocyte plasma membrane. Capacitation occurs in the female genital tract which primes sperm and increases the probability that it will undergo acrosomal reaction. It consists of the removal of an acrosome-stabilizing factor (a glycoprotein similar to fibronectin) from the outer coat of the sperm.2 Factors released by the corona radiata determine spermatozoon activation, including acrosomal reaction, a change in the tail movement and the capacity of the spermatozoon's membrane of fusing with the oolemma.3 In vivo the whole activation process starts when the spermatozoon is near the oocyte as it can only survive for a very limited time. The acrosomal reaction consists of the fusion of the outer acrosomal membrane and the spermatozoon plasma membrane at several points, forming windows between the acrosoma and the external environment. The spermatozoon nucleus continues to be covered by the inner acrosomal membrane and subacrosomal material. Enzymes are released by the acrosoma that aid the sperm in dispersing the cells of the corona radiata. Among them hyaluronidase (pH-20) digests hyaluronic acid present in the extracellular matrix surrounding the cells and the " corona dispersing enzyme " disrupts cell contacts.4 In the ovarian follicle, the cells which form the corona radiate possess cytoplasmic extensions which reach the oolemma through the zona pellucida. The intercellular contacts develop into intimate and presumably communicative connections. The transport of selected molecules is supported by the presence of gap junctions. When the spermatozoon faces the zona pellucida, it binds to some acidic, highly glycosylated proteins of the zona pellucida, in particular ZP3, which enables it to be recognized. Then, another enzyme (acrosin) is released which helps the spermatozoon to digest its way to the oocyte plasma membrane. The equatorial segment of the sperm head becomes attached to the oolemma which is covered with microvilli. The attachment is due, also in this case, to the existence of a receptorial mechanism.5 On the oocyte surface integrins have been found which are receptors for specific sperm proteins such as members of the ADAM and /or CRISP1 family. 6 However, the molecular basis of this interaction is still poorly understood and experimental evidence does not support a decisive role of these molecules in gamete fusion, with a particular reference to ADAM proteins.7 The nucleus and the centriole of the spermatozoon are incorporated in the oocyte, but recently it has been suggested that some paternal mRNA molecules are involved in the embryonic development.8 The fusion of gametes determines a series of events which mark the existence of a new cell: the zygote. The events are initiated by the hydrolysis of phosphatidylinosytol biphosphate in the oolemma followed by electrical changes and an increase in the level of intracellular calcium.9 Activating factors of paternal origin control intracellular calcium homeostasis, calcium pumps, calcium channels and calcium binding proteins, resetting them in a way that generates and sustains oscillation of free intracellular calcium concentration. This pattern determines the exit of the oocyte from metaphase II arrest and the completion of meiosis. The centriole is responsible for the nucleation of microtubules

93 and function of the mitotic spindle in early human embryos while the centrosome of oocyte origin is inactivated after fertilization.10 The calcium level rise in the oocyte precedes a change in the characteristics of the oolemma and an alteration in the zona pellucida produced by cortical granule exocytosis from the ooplasmic periphery. These granules have an enzymatic content which renders the oolemma impermeable to other sperm and induces a decreased expression of ZP3 in the zona, blocking polyspermy. Sex determination is due to the spermatozoon penetration and, at the same time, a series of biochemical and biophysical processes lead to the formation of pronuclei that include dramatic changes while both the nuclei move to what is described by Stanley Shostak as a " rendezvous with destiny ".11 The male pronucleus, which may be somewhat larger in humans, forms near the site of sperm entry, and the female originates at the ooplasmic pole of the meiotic spindle. The breakdown of the spermatozoon's nuclear envelope (devoid of communication with the cytoplasm) is the first of a series of events in the formation of the male pronucleus. This envelope is substituted by a double membrane in which pores are present and the molecular components of this new envelope derive almost entirely from ooplasm. Sperm chromatin undergo a remodelling; maternal histones (in the place of protamines) and other proteins are incorporated into DNA, which becomes despiralized. Chromatin decondensation is induced by ooplasmic factors produced in the last phases of oogenesis; this event dictates the paternal gene transcription. The oocyte completes its second meiotic division; chromatin becomes decondensed and the mature egg nucleus turns into the female pronucleus.12 Within 3-6 hours of the spermatozoon's penetration in the zygote microtubules are organized to form the aster starting from the centriole. Microtubules also seem to play an important role in the migration of the two pronuclei (which are at a certain distance from each other early on in their formation) towards the centre of the cell: migration lasts 12-16 hours. Pronuclei do not fuse and remain separated by a narrow strip of ooplasm that may contain mitochondria and elements of the smooth endoplasmic reticulum. The breakdown of nuclear membranes of the two pronuclei is followed by the migration of chromosomes to the mitotic spindle. The first cell division occurs 24- 30 hours after fertilization.13 Opinions vary widely as to the importance of the site of sperm entry on polarities in succeeding embryo. Some authors have reported that sperm entry has a significant role in forming the plane of the first cleavage division, others assessed the first cleavage plane in relation to the site of the second polar body. In addition, it has been hypothesized that the whole ooplasm rotates at sperm entry to match the position of the spermatozoon and its microtubular system.14 Two blastomeres, smaller than the oocyte, are formed after the first division. Subsequent mitoses occur with a more rapid rhythm. The result is the formation of the morula composed of loosely aggregated cells. Because growth does not accompany cleavage division, embryonic cells become increasingly small and acquire a nuclear/cytoplasmic ratio characteristic of adult cells. When blastomeres are 8-16, the morula undergoes compaction. This event is associated with profound metabolic and structural changes. From the metabolic point of view there is an increase of RNA and protein synthesis and a change in the ratio between phospholipids and cholesterol. From the structural point of view, the blastomeres remaining in contact with the outside are polarized. Their nucleus migrates to the basal part of the cell while at the apex there is accumulation of actin and clathrin; microvilli protrude from the cell surface. The close contact of cells is due to the expression of adherens junctions which precedes the establishment of tight junctions. Adherens junctions are complex heteromeric structures mainly formed by cadherins, which belong to a superfamily of adhesion proteins. In particular, E-cadherin, the most common member of this family, is present in the oocyte and during early mammalian embryo development; nevertheless, stable E-cadherin mediated contacts do not take place until compaction is initiated.15

94 The membrane proteins of tight junctions forming a permanent seal between cells include occludin and claudin; the cytoplasmic proteins include ZO-1, ZO-2 and cingulin.16 The blastomeres which form the external layer are destined to form the while the cells inside the embryo are destined to form internal cell mass (ICM). These latter show gap junctions which are composed of membrane proteins named connexins. A group of six connexins forms an intracellular channel which serves as communicator for transport of metabolites and molecules regulating cell division.17 The zona pellucida is crucial in determining the differentiation of the two distinct cell populations and, as soon as the differentiation occurs, cell totipotency is lost. Approximately 4 days after fertilization morula is transformed into blastocyst. This transformation is due to the presence at the level of external cells of a NaK-ATPase pump that transports sodium and causes an inward movement of water to generate a large cavity (blastocoele) in which a cluster of non- polar cells situated eccentrically forms the ICM (embryonic pole) where proliferative activity is concentrated. The above mentioned tightly joined and polarized cells of the trophoblast do not contribute any tissue to the fetus but produce and extraembryonic membranes.18 Recent experiments have cast some doubt on the role played by NaK-ATPase in mediating the above events and have suggested that the physiological mediators of fluid movement across the trophoectoderm are aquaporins, small membrane proteins involved in water transport. In addition, Mitogen-Activated Protein Kinases, a family of proteins involved in the control of cell growth and differentiation, seem to influence blastocyst formation. In particular, p38 MAPK has been shown to regulate compaction and preimplantation development.19 During the pre-implantation period embryo is transported through the oviduct into the uterus. On the 7th day after fertilization the blastocyst implants into the uterine mucosa. From the illustration of the different stages of pre-implantation embryo development, it can be clearly seen that relevant morphological changes occur. These changes are associated to metabolic variations in terms of aminoacid uptake, protein synthesis and energetic requirement. The mechanisms by which proliferation and differentiation are controlled are poorly understood, due to the large number of genes and the complexity of the systems involved. Mitotic divisions are subjected to stringent control at what is called the cell cycle check points, which are established at an early stage of pre-implantation development. However, in the human pre-implantation embryo a relatively high frequency of chromosomal abnormalities occur.20 Following fertilization, the most important event is probably embryonic genome activation, wherein transcripts expressed from the zygotic/embryonic genome replace the maternal transcripts that direct early development through mRNA accumulated during the oocyte growth and maturation. This dramatic change drives the transformation of highly differentiated oocyte into totipotent blastomeres of the early cleavage stage pre-implantation embryo. As explained above, protamines are replaced by histones, the methylated haploid parental genomes undergo demethylation following formation of the diploid zygote; maternal control is replaced by zygotic control. Embryonic gene activation is probably a stepwise process. A global transcriptionally repressive state is being formed after a transcriptionally permissive period which may permit the continual expression of genes that are regulated by strong promoters/enhancers. In mice, major gene activation occurs at the late 2-cell stage; in other mammalian species major gene activation seems to occur a few cell cycles later. However, recently a more sensitive assessment of the 1-cell mouse embryo led to the suggestion that zygotic gene activation may begin at that stage and that differences between the transcriptional activity of the male and female pronuclei exist.21 In the human zygote at least seven genes (SRY, ZFY, XIST, HPRT, APRT, DK, and a-globin) are active.22 COX1 gene is expressed during the transition from zygote to 2cell-stage.23 At the pronuclear stage, some apoptosis-related genes (bcl-2, Bax, Bad) are active and continue to be expressed during the whole pre-implantation period. In addition, the expression of the Insulin-like 95 Growth factor gene family begins and homeotic genes such as HOXA4, HOXA7, HOXB4, HOXB5, and HOXC6 start to operate.24 Recently, a study performed on mice established the gene expression profile of more than 20,000 genes from in vitro cultured embryos at different stages (zygote, 2-, 4-, 8- cell embryo, morula and blastocyst) using microarray technique and compared this profile with that of in vivo embryos. At most stages of development there was hardly any significant difference in overall expression patterns. Among the genes examined DNA methyltransferase-1 and cadherin-11 were found to be differently expressed.25 During normal pre-implantation development no spontaneous apoptotic processes occur before embryonic genome activation. Human embryos produced in vitro showed no apoptosis before compaction. All mammalian species show the highest level of apoptotic processes at the blastocyst stage. Apoptotic morphological changes have been observed in 70-80% of all in vivo or in vitro produced blastocysts from mice and humans. Human embryos showed 7-8% of dead cells both in the trophoectoderm and the ICM. Apoptotic processes may have a beneficial effect through the removal of genetically abnormal or mutated cells and a protection function. It is worth mentioning that the quality of the blastocyst predicts its ability to implant and its future growth. When the apoptotic ratio increases, massive cell death disrupts embryo homeostasis and results in growth arrest.26 Growth factors, , amino-acids, carbohydrates and regulate embryo development. Most information concerning these factors is obtained from in vitro studies and may not necessarily identify the actual factors crucial to the embryo in vivo. For example, although glucose is present in oviductal fluid in vitro it can be detrimental to the pre- compacted mouse embryo by causing cleavage arrest or retarding the cleavage rate. Transforming Growth Factor-a (TGF-a), Fibroblast Growth Factor, Platelet-derived Growth Factor, Insulin-like Growth Factor II (IGF-II), Insulin Receptor, IGF-I receptor (IGF-R), Epidermal Growth Factor receptor (EGF-R) have all been detected in the cleavage stage mouse embryo. EGF, TGF-a and EGF-R are particularly associated with the physiology of trophoectoderm, blastocyst expansion and implantation in mice. TGF-a is an embryonic product and may constitute an endogenous regulator. EGF, like insulin, is not produced by the embryos but must be produced by the mother. EGF- Rs were mostly found in the trophoectoderm, while IGF-Rs I are uniformly distributed in blastocyst membranes. A possible growth factor circuitry operates during pre-implantation development in vivo. Interaction exists between pre-implantation embryo itself (autocrine and paracrine) and the maternal environment: trophoectoderm then inner mass are the targets of maternal pancreatic insulin; TGF-a from ICM is directed to trophoectodermal EGF-Rs or endometrial IGF-I to trophoectodermal type I IGF-R, etc. Tumor Necrosis Factor (TNF)-a presents the best evidence of an inhibitory influence on pre- implantation development. This factor and its receptors have been detected in oocytes, oviductal epithelium, uterine and the endometrial glands of several species. TNF-a inhibits cell proliferation and blastocyst formation in vitro. Growth hormone has traditionally been considered a master of postnatal growth, but may participate in a hierarchical circuit, regulating IGF action during the first events of embryonic development.27 These local regulatory systems seem to be orchestrated by the maternal sex steroid profile suggesting that pre-implantation development is maternally modulated. Nevertheless, pre-implantation development is, at least in part, a function of autoregulation as explained before. The early embryo is not a quiescent organism developing in the benign environment of the female reproductive tract with little control of its own destiny.

96 An interesting example of the autoregulation is Paf (1-o-alkyl-2-acetyl-snglycero- 3-phosphocholine), a soluble factor which is synthesized soon after fertilization and persists throughout the implantation phase in all mammalian species studied to date. Released Paf causes a series of alterations in the maternal physiology, including changes in the maternal immune function. Paf also acts in an autocrine fashion acting back on receptors expressed by the early embryo as a survival factor and this is, at least in part, mediated by the mobilization of calcium. In the last stages Paf seems to exert a trophic action, stimulating embryo metabolism, cell cycle progression and embryo viability. Paf is a molecule of extraordinary potency and may also promote embryo migration, modify oviduct function and eventually create beneficial changes in the embryo's milieu.28 In mouse the rate of embryo cleavage is controlled by the Ped (preimplantation embryo development) gene. It seems that this gene has a homolog in humans. Genetic segregation studies and the analysis of congenic mouse strains have mapped the Ped gene to the Q region of the mouse Major Histocompatibility Complex (MHC).29 The Ped gene phenotype of embryos cultured in vitro is maintained which suggests that embryo cleavage is dependent on the genes of the embryo itself.30 Finally, one of the most striking changes which the pre-implantation embryo experiences is in its energy preferences. Embryo culture experiments have shown that the mouse oocyte and zygote absolutely require pyruvate; as such, glucose cannot support embryo development until the 8 cell-stage. After this stage there is a switch to glucose utilization. At the time of compaction there is an increase in energy demand as protein synthesis increases when blastocoele is formed. At the blastocyst stage the embryo displays a considerable capacity for anaerobic glycolisis in view of the anoxic environment it encounters at implantation. The developmental normality and viability of human oocytes and preimplantation embryos is influenced by mitochondrial complement size, copy number, stage specific spatial distribution and level of respiration. Mitochondria participate in the regulation of intracellular calcium homeostasis which has important functional implications. They represent the most abundant organelles in oocytes and only contain a few cristae. This phenotype persists through the cleavage and late morula. Lamellar cristae that completely traverse the mitochondrial matrix are a typical feature of the blastocyst stage. Mitochondrial morphology is seen in the low respiratory activity in the oocyte. During the pre- implantation period the mitochondrial oxidative metabolism is a major contributor of ATP, and in the mouse 80% of ATP derives from the mitochondria. If the ATP supply exceeds cellular demand, it results in a toxic state. In the mouse, after fertilization mitochondria migrate to the perinuclear region and form a condensed aggregate surrounding the opposed pronuclei. A similar transient perinuclear accumulation occurs in the blastomeres during the early cleavage stage. Disproportionate mitochondrial segregation seems to influence embryo viability.31

97

1Grobstein C., External Human Fertilization, Sci Am 1979, 240(6): 57-67. 2Shostak S., The embryo's beginning, in Id., Embryology. An introduction to developmental biology, New York: Harper Collins Publishers, 1991: 231. 3Barbieri M., Carinci P., La fecondazione, in Id., Embriologia, Milano: Casa Editrice Ambrosiana, 2005: 109. 4Primakoff P., Myles D.G., Penetration, adhesion and fusion in mammalian sperm-egg interaction, Science 2002, 296: 2183-2185. 5Shostak, The embryo's beginning..., p. 231. 6Frayne J., Hall L., Mammalian sperm-egg recognition: does fertilin ß have a major role to play?, Bioessays 1999, 21(3): 183-187; Cuasnicù P.S., Ellerman D.A., Cohen D.J. et Al., Molecular mechanisms involved in mammalian gamete fusion, Arch Med Res 2001, 32(6): 614-618. 7Nishimura H., Cho C., Branciforte D.R. et Al., Analysis of loss of adhesive function in sperm lacking cyritestin or fertilin ß, Dev Biol 2001, 233: 204-213; He Z.Y., Brakebusch C., Fassler R. et Al., None of the integrins known to be present on the mouse egg or to be ADAM receptors are essential for sperm-egg binding and fusion, Dev Biol 2003, 254: 226-237. 8Ostermeier G.C., Miller D., Huntriss J.D. et Al., : delivering spermatozoan RNA to the oocyte, Nature 2004, 429: 154. 9Veeck L.L., Zaninovic N., An atlas of human blastocyst, London-New York-Washington: Parthenon Publishing Group, 2003: 20. 10Tesarik J., Paternal effects on cell division in the human pre-implantation embryo, Reprod Biomed Online 2005, 10(3): 370-375. 11Barbieri, Carinci, La fecondazione..., p. 113; Shostak, Reorganizing the zygote..., p. 272. 12Barbieri, Carinci, La fecondazione..., p. 114. 13Veeck, Zaninovic, An atlas of human..., p. 22; Barbieri, Carinci, La prima settimana..., p. 122. 14Edwards R.G., Genetics of polarity in mammalian embryo, Reprod Biomed Online 2005, 11(1): 104- 114. 15Watson A.J., Natale D.R., Barcroft L.C., Molecular regulation of blastocyst formation, Anim Reprod Sci 2004, 82(83): 583-592. 16Fleming T.P., McConnell J., Johnson M.H. et Al., Development of tight junctions de novo in the mouse early embryo: control of assembly of the tight junction-specific protein ZO-1, J Cell Biol 1989, 108(4): 1407-1418. 17Barbieri, Carinci, La prima settimana..., p. 124; Veeck, Zaninovic, An Atlas of human..., p. 62. 18Wiley L.M., in the mouse pre-implantation embryo: NaK-ATPase and the origin of nascent fluid, Dev Biol 1984, 105(2): 330-342. 19Sakkas D., Vassalli J.D., The pre-implantation embryo: development and experimental manipulation, in Geneva Foundation for Medical Education and Research, , 2003 (www.gfmer.ch); Watson, Natale, Barcroft, Molecular regulation of blastocyst..., pp. 583-592. 20Tesarik, Paternal effects on cell..., pp. 370-375. 21Kanka J., Gene expression and chromatin structure in the pre-implantation embryo, Theriogenology 2003, 59(1): 3-19. 22Adjaye J., Bolton V., Monk M., Developmental expression of specific genes detected in high- quality cDNA libraries from single human pre-implantation embyos, Gene 1999, 237(2): 373-383. 23Wang H., Wen Y., Mooney S. et Al., Phospholipase (A2) and cyclooxigenase gene expression in human pre-implantation embryos, J Clin Endocrinol Metab 2002, 87(6): 2629-2634. 24Liu H.C., He Z.Y., Mele C.A. et Al., Expression of apoptosis-related genes in human oocytes and embryos, J Assist Reprod Genet 2000, 17(9): 521-533; Liu H.C., He Z.Y., Tang Y.X. et Al., Simultaneous detection of multiple gene expression in mouse and human individual pre-implantation embryos, Fertil Steril 1997, 67(4): 733-741; Kuliev A., Kukharenko V., Morozov G. et Al., Expression 98 of homeobox-containing genes in human pre-implantation development and in embryos with chromosomal aneuploidies, J Assist Reprod Genet 1996, 13(2): 177-181. 25Wang S., Cowan C.A., Chipperfield H. et Al., Gene expression in the pre-implantation embryo: in vitro developmental changes, Reprod Biomed Online 2005, 10(5): 607-616. 26Fabian D., Koppel J., Maddox-Hyttel P., Apoptotic processes during mammalian pre- implantation development, Theriogenology 2005, 64(2): 221-231. 27Kaye P.L., Pre-implantation growth factor physiology, Rev Reprod 1997, 2(2): 121-127. 28O'Neill C., The role of paf in embryo physiology, Hum Reprod Update 2005, 11(3): 215-228. 29Xu Y., Jin P., Warner C.M., Modulation of pre-implantation embryonic development by antisense oligonucleotides to Major Histocompatibility Complex genes, Biol Reprod 1993, 48: 1042-1046. 30Sakkas, Vassalli, The pre-implantation embryo development and... 31Van Blerkom J., Mitochondria in human oogenesis and pre-implantation embryogenesis: engines of metabolism, ionic regulation and developmental competence, Reproduction 2004, 128(3): 269-280.

* L'autore ringrazia la dr. Anna Giuli e la dr. Fortunata Iacopino per la collaborazione offerta nella raccolta della bibliografia e nella revisione del manoscritto.

99 C. BELLIENI

PRE-IMPLANTATION DIAGNOSIS, PRENATAL DIAGNOSIS

This report will give some brief notions about the latest advances in the field of prenatal diagnosis and pre-implantation diagnosis, and then put forward some evaluations that spring from clinical practice and specialized scientific literature. PRE-IMPLANTATION DIAGNOSIS Pre-implantation genetic diagnosis (PGD) consists of the analysis of single cells (blastomeres) biopsied from embryos three days after fertilization, or from polar bodies taken from oocytes during meiosis. The purpose of these tests is to determine which embryo is free from a disease due to an alternation of a single gene (SGD) or from chromosomal abnormalities.1 The embryos that are determined to be genetically normal with respect to the gene/chromosome in question are then transferred into the mother choosing them over the abnormal ones. Simple Gene Diseases (SGD) The first cases of PGD used the polymerase chain reaction (PCR) to determine the sex of the embryo, thereby allowing the elective transfer of unaffected females in families that are carriers of x-linked diseases.2 These first successes were soon followed by tests based on the PCR for SGDs such as the alpha-1- antitrypsin deficiency and cystic fibrosis, in which a DNA fragment containing the harmful mutation of the disease was amplified and analyzed.3 Now it is possible to analyze more than 40 hereditary diseases in the pre-implantation state.4 To avoid the errors due to amplification failure,5 many PGD tests now use the PCR Multiplex. This technique consists in the simultaneous amplification of various fragments of DNA in a single reaction. The PCR Multiplex has also helped in avoiding another serious problem of the PCR single-cell: namely, the contamination of the reaction with extraneous DNA. Chromosomal analysis In the beginning, chromosomal analysis of human embryos pre-implantation was introduced to identify X and Y-chromosomes in order to determine the sex of the embryo and avoid X-linked diseases. The PCR techniques used initially for this purpose were soon replaced by more valid methods based on fluorescent in situ hybridization (FISH).6 Since then, the indications for the chromosomal test have increased, and the number of chromosomes examined in each cell has also increased significantly. Today chromosomal screening is offered to various groups of patients presumed to be at risk of producing aneuploidy gametes. These include carriers of chromosomal rearrangements such as translocations, 7 previous spontaneous , IVF failures, and older women.8 The identification and elective transfer of chromosomically normal embryos reduces the incidence of children born with trisomy. Moreover, this screening can improve the success of IVF for some patients.9 This occurs because many of the chromosomal imbalances detected in the pre-implantation stage are lethal. The embryos that carry these abnormalities are often morphologically normal in the pre-implantation stage and are consequently transferred to the mother. The cytogenetic investigation of material from spontaneous abortions and cells taken from embryos in the pre-implantation stage indicate that aneuploidy can explain many of the cases in which a developed pregnancy is not achieved.10 For screening the polar body, 11 the major limitation is that it can only be used to discover errors that come from female meiosis, but not all meiotic errors are of maternal origin. NON-INVASIVE PRENATAL DIAGNOSIS: RECENT DEVELOPMENTS Non-invasive prenatal diagnosis techniques exist which are based on the analysis of a series of markers in the mother’s blood (Table 1). Recently a system has been perfected to determine the sex of the unborn child by analyzing the mother’s blood at six weeks of pregnancy. The current recommendations 100 of the National Institute of Clinical Excellence (NICE) are that all pregnant women should be offered a test that will give a minimum rate of accuracy of 60%, and a maximum rate of false positives of 5%. By 2007, these rates should rise to 75% and 3% respectively. The search for fetal red blood cells in the mother’s blood, mesenchymal stem cells and has been used for various prenatal diagnostic tests.12 The main limiting factor appears to be the rarity of these cells in the mother’s bloodstream (enrichment techniques are needed to increase their production). To this picture can also be added the diagnostic possibilities offered by the recent advances in fetal ultrasound.

Table I: Screening test for Down syndrome 11-14 weeks Nucal Translucence (NT) Combined test (NT, hCG, PAPP-A) 14-20 weeks Triple test (hCG, AFP, uE2) Quadri test (hCG, AFP, uE2, inhibin A) 11-14 weeks Integrated test (NT, PAPP-A, inhibin A, hCG, AFT, uE2) Serum-integrated test (PAPP-A, inhibin A, hCG, AFP, uE2)

INVASIVE PRENATAL DIAGNOSIS Both second trimester and chorionic villus sampling (CVS) are consolidated techniques used to obtain genetic information about the fetus. However, there are some other less common techniques, such as cordocentesis, usually performed after 18 weeks of gestation, and early amniocentesis, which can be performed between the 11th and 14th week of gestation. Amniocentesis is considered the ‘gold standard ‘of invasive prenatal diagnosis. It was introduced in 195213 to identify hemolytic diseases in the prenatal stage, and it has been used since the mid-70s to identify genetic diseases. Used initially to obtain fetal cells for the karyotype, second trimester amniocentesis is now used for the molecular and biochemical diagnosis of those fetal disorders in which an analysis of the amniocytes or is informative, as well as to provide very reliable information for defects of the neural tube. The time needed to obtain a karyotype is from 10-14 days. The major disadvantage of second trimester amniocentesis is that the result is usually available only after the 18th week of gestation. CVS and early amniocentesis can be done between the 10th and 14th week and offer an earlier alternative, especially early amniocentesis, but they are not used much because of the risks involved. Although both CVS and amniocentesis are very reliable, the greater rate of mosaicism seen with CVS can result in differences with regard to the positive predictive value (that is, the concordance of a fetal abnormality with an abnormal test result). A collaborative Canadian study has determined that the positive predictive value of amniocentesis is 0,909, while that of CVS is only 0,525.14 Like all the invasive prenatal procedures, both amniocentesis and CVS sometimes lead to pregnancy loss. For second trimester amniocentesis, fetal loss is estimated to be from 0,5-1% above the rate of spontaneous abortion.15 In 2003, the Cochraine library16 analyzed 14 randomized studies to evaluate the risks of the various techniques with respect to fetal death and deformation of the limbs. Various authors maintain that direct visualization of the needle may improve safety.17 One recent randomized study in 2004 observed that early amniocentesis causes a higher rate of and clubfoot than CVS.18 101 Other works confirm the risk for the fetus of invasive prenatal diagnosis.19 RISKS AND CONCERNS REGARDING INVASIVE PRENATAL DIAGNOSIS In the 35 years since Jacobson and Barten reported the first 56 cases of amniocentesis,20 the number of amniocenteses has grown progressively. The International Genetic Laboratory Directory has indicated that 190,000 amniocenteses are performed annually in the USA.21 Recently, efforts have been made to go beyond the need for invasive examinations in pregnancy because of the risk of miscarriage involved. If we consider that in Italy, in 2003, approximately 100,000 amniocenteses were performed, 22 it follows that between 500-1000 normal, wanted pregnancies ended in abortion because of this technique, with the resulting trauma for the women. This is not a negligible fact. We know that this exam is undertaken at times while underestimating its risks. Stranc wrote the following in Lancet23: ‘When CVS or second semester amniocentesis are both indicated, the woman’s preference or the procedure that gives her a greater degree of certainty will be the deciding factorâ?¦Some women want an early prenatal test regardless of the significant rate of risk: some women want an early prenatal test regardless of the possibility of a greater risk of complications connected with the procedure or the relation between the risk from the procedure and the probability of a fetal abnormality. Heckerling and collaborators found that the choice between an early and a late diagnosis was the primary factor in choosing CVS over amniocentesis, and that the miscarriage related to the test, the levels of error in determining the karyotype, with the resulting termination of the pregnancy or maternal morbidity following therapeutic abortion did not significantly influence the choice of the type of test. Similarly, Evans and collaborators reported that older women with a better education, with a lower rate of previous pregnancies and deliveries, were more inclined to choose CVS rather than amniocentesis ‘.24 The psychological risks should not be underestimated of the search for perfection in the unborn child who is entrusted with fulfilling its parents exaggerated expectations: ‘Any unusual fetal exploration, in particular the realization of a karyotype, causes, especially in the mother, a real interruption in her relationship with the child she is bearing, which will only end with a normal result. Parents describe almost all of these suspensions of their project, which is manifested in halting all the material preparations for the birth, but also in a transitory detachment from this child who is suspected of not being supposed to survive, and in the concern to not get uselessly attached. At the slightest abnormality, the suspicion about the quality of the child and the doubt about its present and especially future integrity lead to a reaction of rejection in the parents, a death wish, which is often entirely disproportionate to the real gravity. These sentiments put attachment in grave danger, to the point of a real and proper anticipated mourning, which, if the child survives, will leave an indelible trace. It is easier to correct a diagnosis rather than a psychic representation’.25 The Italian National Bioethics Committee26 also warns about excessive intrusion into the fetal chromosomal patrimony, prefiguring an intrusion into an individual’s privacy,27 especially when diseases that are non lethal and may only appear later are scrutinized: ‘The ability to predict ‘by analyzing the genome in the prenatal stage or the genetic constitution of adult individuals ‘that a subject will become ill with a given disease, or to ascertain that although he has no specific pathologies, he is predisposed to contracting them, can also involve an elevated cost in psychological and social terms. In fact, it is possible to subject an individual to discrimination in various areas of his daily life (at work, as well as by insurance companies and even one’s partner), often only on the basis of a greater probability, but not the certainty that one day he will become sick. Therefore, the need is raised to protect him from a negative use of genetic information that will lead to discriminating and limiting collective behaviors, on any level, of the fundamental rights of the person ‘.28 Lastly, the danger should not be underestimated of abuse in the use of these techniques: ‘They do not know that zero risk does not exist, not even for a 20 year-old, and that in any case there is a 3% possibility that the fetus will have an abnormality. Moreover, invasive techniques are involved that 102 endanger the child’s survival and add almost nothing on the predictive level. At 20 years of age, the risk of having children with a handicap is one in 1,500, whereas the possibility of causing a miscarriage through amniocentesis is one in 200 ‘. Precisely for this reason, the current tendencies are going in the direction of finding alternative techniques to invasive investigation. Moreover, the current approach is to provide more information about the real indications for the various prenatal diagnostic options through an ever more careful use of informed consent. RISKS AND CONCERNS REGARDING PRE-IMPLANTATION DIAGNOSIS The first point to remember is that ‘pre-implantation diagnosis ‘means ‘selection ‘. In other words, it means that we are not before a diagnosis that is made to cure but to eliminate the sick embryos. Today the discussion is not being raised about whether or not it is ethically right to select the embryos, but which ones should be selected. Some argue that it is correct to allow access to pre-implantation diagnosis only for very serious diseases or there will be a risk of falling into procreative consumerism.29 Others explain that in doing this, the dignity of the persons afflicted with these diseases is offended (for example, bifida, mucoviscidosis), and they would feel that their lives are considered ‘unworthy ‘. On the other hand, the supporters of this second theory invoke access to selecting a child only in accordance with the parents’requests.30 In their opinion, even access to should not be prohibited. They explain that what is important is that the unborn child’s sex will be chosen in order to ‘balance ‘the sex of the pre-existing children, but not to choose the sex of the first child. Julian Savulescu writes, ‘couples ought to select the embryos or which are presumed to have the best life on the basis of the genetic information available, including information about non- pathological genes. I also maintain that we should allow selection for the non-pathological genes even if this maintains or increases social inequality. In particular, I will speak about the genes of intelligence and selection on the basis of sex ‘.31 We are thus witnessing a justification of selection even on the basis of the ‘dental abnormalities of the conceived child,32 or ‘if it will be possible to diagnose this in the embryonic stage one day in the future ‘the child’s sexual tendency,33 or even the characteristic of a predisposition for music.34 There is also a tendency to select a child not on the basis of its ‘complete wellbeing ‘, but to impose some characteristics on it that would not seem desirable to most, such as deafness.35 Through pre-implantation diagnosis, by definition embryos are thus eliminated on the basis of genetic characteristics. Then there are also the longterm risks related to the manipulation and removal of 1-2 cells from a group of 8, which are still unknown since there is obviously insufficient follow-up from the viewpoint of time. ONE RISK IN COMMON: THE LOSS OF OUR PRENATAL PRIVACY Recently, there was news about the production of a do-it-yourself kit to determine the unborn child’s sex. The test is possible at five weeks of gestation, precisely in the period when the interruption of pregnancy is allowed in many States. Great protests were voiced, especially by countries like India where the problem of eliminating fetuses of the female sex is very grave. But also in circles sensitive to women’s rights there were complaints about the commercialization of a product with a possible sexist spin-off. So it is more and more possible to act in order to know the ‘secrets ‘of the unborn child, many times not to its advantage. From various parts in scientific literature there is more and more talk about the unborn child’s rights to privacy so as to not suffer from discrimination both before and after birth. In 1989, the World Health Organization (WHO) drew up guidelines for the protection of our prenatal privacy. Every person has a right to not be subjected to discrimination both before birth on the basis of sex, characteristics or future attitudes, and the WHO suggested limiting the possibility for parents to get excessive information about their child: ‘Prenatal diagnosis is only carried out to give parents and doctors information about the health of the fetus. The use of prenatal diagnosis for paternity tests, except in cases of rape or incest, or for selection related to sex, except in cases of disease linked to sex, is not acceptable ‘.36 In 2000 the German geneticist Wolfram Henn wrote: ‘Can there be any doubt that 103 a kit for DNA that promises future parents a good chance of having a tall, slim, brilliant child would be a best seller? I believe that this is incompatible with the principle of nil nocere. Therefore, it is urgent to extend the current prohibition of tests for paternity or sex to any parameter that is not correlated to grave diseases in the unborn child ‘. Today, however, it is being discussed, for instance by David Wasserman of Yale University, whether or not this prenatal selection on the basis of characteristics should only be made for serious diseases (Down Syndrome first of all), or so as to not stigmatize the disabled, if access should be allowed to any possible type of curiosity about the embryo or fetus.37 Dorothy Wertz38 and John Fletcher report: ‘More than a quarter of the geneticists in the Western countries would be ready to perform prenatal diagnosis for the selection on the basis of sex, and 8% state that the patients have a right to any service they can pay for’. In India the problem is so serious that there is a law against selection related to sex, and the State of Maharshtra has passed a law that punishes doctors who take part in these actions.39 Other risks of abuse exist, and the Italian National Bioethics Committee has addressed them (see above). Botkin, a pediatrician and bioethicist from Utah, stresses the following in an article entitled ‘Fetal privacy’: ‘As technology gradually advances, screening will be possible for conditions that do not produce serious defects. Future parents will soon be able to select the most desirable embryo in vitro or terminate the undesirable fetuses until they get the child they desire. The medical profession must assume the responsibility for establishing guidelines for the use of reproductive technology ‘.40 It is obviously right to know before birth about the possibility of curable pathologies, but what can be said about the possibility of revealing a predisposition to the pathologies that are not curable and may appear at an advanced age? Is it a parents’right to know everything about the child when ‘knowing everything ‘is not in the child’s own interest? How can a conflict of interests not be seen because of the possibility that this information may be detrimental to the unborn child? ‘One day in the future, when we will know how to see from the embryo that a child will have such and such characteristicsâ?¦ that it will be male, female, have blue eyes and a bent for mathâ?¦there will be parents who will say: we don’t want him like that. Can you imagine the doctors’embarrassment in 50 years when they are called in for cases like these? ‘.41 The intrusion into prenatal privacy does not only harm the right to privacy because it can lead to terminating the pregnancy; it goes further. Anyone born after prenatal intrusion into his DNA already finds his ‘genetic secrets ‘deciphered from birth. Moreover, he finds himself in a situation in which he knows things that he may have preferred not to know. Privacy regarding information related to the sphere of personal physiology and health can be gotten around if my data, which I would not give to anyone as an adult, is decoded before birth. A FURTHER RISK: THE GENERATION OF SURVIVORS Prenatal-pre-implantation diagnosis raises another question: what is the feeling of children born in the current state of prenatal selective possibilities? Some speak about the current generation as a generation of survivors. Actually, this image may be exaggerated, but everyone born today knows that they were born in a cultural climate where being born is a right of those who conform to their parents’plan. ‘The notion of a wanted child has turned into that of a planned child. A new dichotomous logic has slowly been created: a woman is either pregnant or sterile; a child is either planned and therefore wanted, or not planned and thus undesirable ‘.42 These words say a lot about the cultural climate around conception today: a child is programmed; he is a choice; his birth is postponed more and more; he is an only child or has at most one little brother or sister; he is spoiled so as to free from a sense of guilt, and he is not spoiled with the things he really wants, but with what the wanted in their unfulfilled childhood. Therefore, a child today starts out with the burden of someone who knows that the norm is to conceive in order to satisfy a need, someone who knows that if he was not suitable, he might have been rejected before being born. The fact that his family may not be fully part of this climate and conceived him in an entirely gratuitous way alleviates this burden, but not completely. ‘A child subjected to the desire of others is an omnipotent child on whom it may be difficult to set limits. Before 104 or after him, his parents eliminated one or more children ultimately out of their desire for him so that he could live. So how much must he be worth for whom such a sacrifice was made? ‘.43 Pre-implantation or prenatal diagnoses are thus signs of the fear of an unwanted child, but also signs of the society of the third millennium’s feeling only for those who are ‘in conformity ‘. It is not by chance that conformism is the guideline of the current generation of teenagers that sociologists call ‘echo-boomers ‘, i.e., characterized by living without any real desires, but just reflecting (like an echo) their parents’ideas and aspirations.44 CONCLUSION Lastly, how can the real problem not be mentioned that lies at the basis of all this, of this diagnostic aggressiveness and interference into prenatal life? From what has been reported, I believe that the heart of the question is fear of ‘unexpected ‘life: that is, an ‘undesired ‘child has become ‘undesirable ‘, a real handiphobia45 exists now, a real and proper phobia of the hard, difficult experience of disease and of a sick child, which soon generates escape and repression rather than affection and solidarity. What will be the psychological repercussions on those who come to know that they survived a phratry of embryos? Is it possible to go beyond the consumerist logic that ties birth to the fulfillment of the parents’desires? Is it possible to educate to not be afraid of what ‘has not been planned ‘, knowing that imagined reality is more frightening than real reality? Responding to and overcoming this is an imperative for those who are involved in culture, and for those who are called to protect health.

1Wells D., Delhanty J.D., Pre-implantation genetic diagnosis: applications for molecular medicine, Trends Mol Med 2001, 7: 23-30. 2Handyside A.H., Kontogianni E.H., Hardy K. et Al., Pregnancies from biopsied human pre- implantation embryos sexed by Y-specific DNA amplification, Nature 1990, 344: 768-770. 3Verlinsky Y., Ginsberg N., Lifchez A. et Al., Analysis of the first polar body: preconception genetic diagnosis, Hum Reprod 1990, 5: 826-829; Handyside A.H., Lesko J.G., Tarin J.J. et Al., Birth of a normal girl after in vitro fertilization and pre-implantation diagnostic testing for cystic fibrosis, N Engl J Med 1992, 327: 905-909. 4Verlinsky, Ginsberg, Lifchez, Analysis of the first polar body..., pp. 826-829. 5Delhanty J.D., Griffin D.K., Handyside A.H. Et Al., Detection of aneuploidy and chromosomal mosaicism in human embryos during pre-implantation sex determination by fluorescent in situ hybridisation (FISH), Hum Mol Genet 1993, 2: 1183-1185, Munné S., Weier H.U., Stein J. Et Al., A fast and efficient method for simultaneous X and Y in situ hybridization of human blastomeres, J Assist Reprod Genet 1993, 10: 82-90. 6 Under the abbreviation FISH, a series of techniques are included which make specific traits of the genome fluorescent by using a DNA probe formed by sequences complementary to the ones researched. The probe ‘recognizes ‘these traits and positions itself on the region of the chromosome where they are contained. The technique is used to diagnose different genetic diseases. 7Griffin D.K., Handyside A.H., Harper J.C. et Al., Clinical experience with pre-implantation diagnosis of sex by dual fluorescent in situ hybridization, J Assist Reprod Genet 1994, 11: 132-143; Munné S., Scott R., Sable D. et Al., First pregnancies after preconception diagnosis of translocations of maternal origin, Fertil Steril 1998, 69: 675-681; Iwarsson E., Ahrlund-Richter L., Inzunza J. et Al., Pre- implantation genetic diagnosis of a large pericentric inversion of chromosome 5, Mol Hum Reprod 1998, 4: 719-723; Conn C.M., Cozzi J., Harper J.C. et Al., Pre-implantation genetic diagnosis for couples at high risk of Down syndrome pregnancy owing to parental translocation or mosaicism, J Med Genet 1999, 36: 45-50; Van Assche E., Staessen C., Vegetti W. et Al., Pre-implantation genetic diagnosis and sperm analysis by fluorescence in-situ hybridization for the most common reciprocal 105 translocation, Mol Hum Reprod 1999, 5: 682-690; Munné S., Sandalinas M., Escudero T. et Al., Outcome of pre-implantation genetic diagnosis of translocations, Fertil Steril 2000, 73: 1209-1218. 8Gianaroli L., Magli M.C., Ferraretti A.P. et Al., Pre-implantation genetic diagnosis increases the implantation rate in human in vitro fertilization by avoiding the transfer of chromosomally abnormal embryos, Fertil Steril 1997, 68: 1128-1131; Gianaroli L., Magli M.C., Ferraretti A.P. et Al., Pre- implantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed, Fertil Steril 1999, 72: 837- 844; Munné S., Magli C., Cohen J. et Al., Positive outcome after pre-implantation diagnosis of aneuploidy in human embryos, Hum Reprod 1999, 14: 2191-2199; Kahraman S., Bahce M., Samli H. et Al., Healthy births and ongoing pregnancies obtained by pre-implantation genetic diagnosis in patients with and recurrent implantation failure, Hum Reprod 2000, 15: 2003- 2007. 9Munné S., Wells D., Pre-implantation genetic diagnosis, Curr Opin Obstet Gynecol 2002, 14: 239- 244. 10Hassold T., Chen N., Funkhouser J. et Al., A cytogenetic study of 1000 spontaneous abortions, Ann Hum Genet 1980, 44: 151-178; Sandalinas M., Sadowy S., Alikani M. et Al., 458 Developmental ability of chromosomally abnormal human embryos 459 to develop to the blastocyst stage, Hum Reprod 2001, 16: 1954-1958. 11A polar body is a small cellular structure expelled by an oocyte made up of nuclear material and a small amount of cytoplasm. 12Choolani M., O'Donnell H., Campagnoli C. et Al., Simultaneous fetal cell identification and diagnosis by epsilonglobin chain immunophenotyping and chromosomal fluorescence in situ hybridization, Blood 2001, 98: 554-557; Campagnoli C., Roberts I.A., Kumar S. et Al., Identification of mesenchymal stem/progenitor cells in human first trimester fetal blood, liver, and bone marrow, Blood 2001, 98: 2396-2402. 13Bevis D.C., The antenatal prediction of haemolytic disease of the newborn, Lancet 1952, 1: 395-398. 14Lippman A., Tomkins D.T., Shine J., Canadian Collaborative CVS Amniocentesis Clinical Trial Group, Canadian multicentre randomised clinical trial of villus sampling and amniocentesis: final report, Prenat Diag 1992, 12: 385-467. 15Tabor A., Madsen M., Obel E.B. et Al., Randomised controlled trial of genetic amniocentesis in 4606 low-risk women, Lancet 1986, 1287-1292; Nichhd National Registry for Amniocentesis Study Group, Midtrimester amniocentesis for prenatal diagnosis. Safety and accuracy, Jama 1976, 236: 1471- 1476; Smidt-Jensen S., Permin M., Philip J. et Al., Randomised comparison of amniocentesis and transabdominal and transcervical villus sampling, Lancet 1992, 340: 1237-1244; Bellieni C., Plantulli A., Buonocore G., Amniocentesi: rischi e indicazioni, Biol Ital. 2004, 9: 43-46. 16Alfirevic Z., Sundberg K., Brigham S., Amniocentesis and chorionic villus sampling for prenatal diagnosis, Cochrane Database Syst Rev. 2003, (3): CD003252. 17Carpenter R.J., Hinkley C.M., Carpenter A.F., Midtrimester genetic amniocentesis: use of ultrasound direction vs. blind needle insertion, J Reprod Med 1983, 28: 35-40; Williamson R.A., Varner M.W., Grant S.S., Reduction in amniocentesis risks using a real-time needle guide procedure, Obstet Gynecol 1985, 65: 751-755. 18Seeds J.W., Diagnostic mid trimester amniocentesis: How safe?, Am J Obstet Gynecol. 2004, 191(2): 607-615. 19Sangalli M., Langdana F, Thurlow C., Pregnancy loss rate following routine genetic amniocentesis at Wellington Hospital, N Z Med J. 2004, 117(1191): U818; Id., Pregnancy loss rate following routine genetic amniocentesis at Wellington Hospital, N Z Med J 2004, 117(1191): U818. 20Jacobson C.B., Barter R.H., Intrauterine diagnosis and management of genetic defects, Am J Obstet Gynecol 1967, 99: 796-805.

106 21Rebolloso F., Knutsen T. (a cura di), 1998-1999 International Genetic Laboratory Directory. Association of Genetic Technologists, Lenexa: Kan, 1998. 22De Bac M., ‘In un anno 230 mila esami sui cromosomi e 164 mila sul Dna. Troppi e usati male’, Corriere della Sera (3 novembre 2003); Novelli G., In gravidanza servono sempre? In Italia boom dei testi genetici, Newton (1 dicembre 2003). 23Stranc L.C., Evans J.A., Hamerton J.L., Chorionic villus sampling and amniocentesis for prenatal diagnosis, Lancet 1997, 349: 711-714. 24Heckerling P.S., Verp M.S., Hadro T.A., Preferences of pregnant women for amniocentesis or chorionic villus sampling for : comparison of patients' choices and those of a decision- analytic model, J Clin Epidemiol 1994, 47(11): 1215-1228. 25Vial M., Benoit A., Schneider Z. et Al., Maltraitance du foetus et du nouveau-né, Ann Pédiatr 1996, 43: 446-455. 26Handyside A.H., Lesko J.G., Tarin J.J. et Al., Birth of a normal girl after in vitro fertilization and pre-implantation diagnostic testing for cystic fibrosis, N Engl J Med 1992, 327: 905-909. 27Botkin J.R., Fetal privacy and confidentiality, Hastings Cent Rep. 1995, 25(5): 32-39. 28Comitato Nazionale per la Bioetica, Orientamenti bioetica per i test genetici (19 novembre 1999);http://www.genomica.net/PREDITTIVA/test%20genetici/CNB_test_genetici.pdf. 29Henn W., Consumerism in prenatal diagnosis: a challenge for ethical guidelines, J Med Ethics 2000, 26(6): 444-446. 30Savulescu J., Procreative beneficence: why we should select the best children, Bioethics 2001, 15(5- 6): 413-426. 31Ibid. 32Aldred M., Crawford P.J., Savarirayan R. et Al., It's only teeth are there limits to genetic testing?, Clin Genet. 2003, 63(5): 333-339. 33Dahl E., Ethical issues in new uses of pre-implantation genetic diagnosis: should parents be allowed to use pre-implantation genetic diagnosis to choose the sexual orientation of their children?, Hum Reprod. 2003, 18(7): 1368-1369. 34Robertson J.A., Extending pre-implantation genetic diagnosis: medical and non-medical uses, J Med Ethics 2003, 29(4): 213-216. 35Savulescu J., Deaf lesbians, designer disability and the future of medicine, BMJ 2002, 325: 771-773. 36World Health Organization, Proposed international guidelines on ethical issues in medical genetics and genetic services, Geneva: WHO, 1998. 37Wasserman D., A choice of evils in prenatal testing, FSU Law Review 2003, 30(2). 38Wertz D.C., Fletcher J.C., Ethical and social issues in prenatal sex selection: a survey of geneticists in 37 nations, Social Science and Medicine 1998, 46: 255-273. 39Henn W., Consumerism in prenatal diagnosis: a challenge for ethical guidelines, J Med Ethics 2000, 26(6): 444-446. 40BOTKIN J.R., Fetal privacy and confidentiality, Hastings Cent Rep. 1995, 25(5): 32-39. 41Delefosse S., Fecondation in vitro, Devenir 1999, 11(3): 87-108. 42Chatel M.M., Malaise dans la procréation, Paris: Albin Michel, 1993. 43Bayle B., L'embryon sur le divan. Psychopathologie de la reproduction, Paris: Masson, 2003: 46-47. 44Vatter R.H., Taking stock of America's youth, Stat Bull Metrop Insur Co. 1995, 76(2): 2-9. 45A phobia with regard to handicaps.

107 GIGLIOLA SICA

THE EMBRYO-MATERNAL DIALOGUE AND PREPARATION FOR IMPLANTATION

The establishment of a pregnancy includes a series of events which precede the implantation represented by sperm capacitation, transport of gametes, fertilization, early embryonic development, transport of embryos. All these events occur in the oviduct which provides a variety of molecules, including proteins, creating an adequate environment to sustain oviductalgamete and oviductal-embryo interactions. In fact, communication is not one-way, because the embryo itself produces hormones and other molecules which support the dialogue. The non-ciliated cells of the oviductal epithelium are supposed to provide some proteins (albumin, transferrin, immunoglobulins, transport proteins), cytokines and growth factors in a trasudate deriving from serum and other fluids. In addition, they actively release macromolecules and this biosynthetic activity appears to be estrogen (E)-regulated. One protein synthesized de novo, the E-dependent oviductal secretory glycoprotein (OSP) is conserved in a number of mammalian species. OSP associates with the zona pellucida, perivitelline space, blastomere membranes and pre-implantation embryos. It enhances the binding of spermatozoon to oocyte and penetration; moreover, it markedly influences embryo development. OSP presence is maximal during the time of ovulation, fertilization and early cleavage stages but declines when (P) levels raise. Protease inhibitors which protect the oocyte integrity, embryo and oviductal tissue and promote embryonic development are also secreted. Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) and Plasminogen Activator Inhibitor-1 (PAI-1) belong to this family of proteins. The potential roles of TIMP-1 include improving the embryo cleavage rates, delaying hatching and preventing embryo degradation. PAI-1 is a member of the serpin family of serine protease inhibitors and is the primary inhibitor of the plasminogen activator and tissue-specific plasminogen activator which initiate proteolytic cascades that convert plasminogen to plasmin. PAI-1 controls a series of activities such as remodelling of extra-cellular matrix (ECM), fibrinolysis, cell migration and tumor metastasis.1 Growth factors and cytokines are present in the oviductal fluid and may act both in an autocrine and/or paracrine way to regulate oviductal function and embryonic development. Their relevance is supported by experimental evidence of the enhanced development of in vitro embryos when cultured in the presence of oviductal fluid.2 Moreover, they have been shown to increase blastocyst cell number in culture.3 Growth factors are also produced by endometrial epithelium which contributes to the milieu of the developing embryo. Epidermal Growth Factor (EGF), Transforming Growth Factor (TGF)-a, Insulin- like Growth Factor, Activin, Leukaemia Inhibiting Factor (LIF), Fibroblast Growth Factor seem to play an important role in this context.4 Granulocyte-Macrophage Colony Stimulating Factor is produced by uterine epithelium stimulated by E and interacts with receptors expressed by the embryo. This factor seems to regulate cell number in the internal cell mass (ICM).5 Ovarian hormones control the human , which, through a series of morphological and biochemical processes, prepares the uterus for blastocyst implantation. The endometrium is rendered receptive by these hormones in a limited period of time which is named " window of implantation ". Outside this period, endometrium is no more capable to receive the embryo and may even be hostile to it. Hormonal activity is mediated by a complex pattern of endometrial receptor expression which varies in different cell types and tissue compartments. After the menstrual phase, in the proliferative phase, E regulate cell proliferation which reconstitutes both the glands and the surface epithelium when E receptor (ER) is present in the stroma suggesting 108 that paracrine interaction takes place between the epithelium and the underlying connective tissue. Two types of ER have been found in the endometrium: ER-a, the classical ER mediating the effect of E in the uterus, and ER-b which has been reported to modulate ER-a.6 Proliferation of the connective cells and deposition of the ground substance in the propria also contribute to the growth of the endometrium as a whole. After the proliferative phase, the secretory phase is controlled by P which acts via specific receptors upon glands already developed by the action of E inducing the secretion of glycoproteins useful for embryonic . The glands become highly coiled and cells accumulate glycogen. In this phase, endometrium reaches its maximum thickness as a result of the accumulation of the secretion and edema of the stroma. P also stimulates stromal cells to produce TGF-b which, in turn, suppresses the production of Matrix Metalloproteinase-7 (MMP-7), an enzyme involved in the remodelling of the ECM and effective when hormone levels fall.7 Hormones other than ovarian steroids they take part in the cross-talk between the embryo and the female reproductive tract. During its development, the blastocyst produces Human Chorionic Gonadotrophin (HCG) which alerts the maternal organism of its presence. This hormone interacts with specific receptors located on endometrial cells and expressed at a very high level during the time of the implantation window. In addition, it acts as an autocrine regulator for trophoblast.8 Parathyroid-related Hormone produced by the endometrium has been shown to induce primary and secondary mouse trophoblast differentiation to giant cells in vitro while prolactin, secreted by the decidua, activates receptors on uterine large granulated lymphocytes potentially influencing their role in the post-implantation uterine function.9 It is necessary to remember that after the implantation the endometrial stroma undergoes profound modifications and it is called " the decidua ", a tissue that will be eliminated at the time of parturition. implicates alterations in ECM with a loss of collagen fibers and modifications of the stromal cells. They become enlarged and polygonal with a great amount of endoplasmic reticulum, lisosomes, lipid droplets and glycogen granules. not only provide nutrients to the embryo but represent a sort of barrier which counteracts the invasive action of the trophoblast. Corticotrophin Releasing Hormone (CRH) has been localized in endometrial epithelium, stroma, decidua and trophoblast. It has a potent pro-inflammatory action, inhibits Prostaglandin E2 (PGE2), stimulates Interleukin (IL)- 1 and IL-6 production and participates in the process of decidualization. The blastocyst, in turn, secretes IL-1 and PGE2 which induce CRH expression in the endometrium.10 CRH is involved in the attenuation of maternal immunological attacks to the embryo. It enhances the capability of decidual cells to induce apoptosis of maternal T lymphocytes activated by the presence of the embryo. CRH may inhibit local maternal immune response through stimulation of the expression of pro-apoptotic Fas-ligand peptide in decidual and trophoblastic cells. Fas ligand binds to its receptor Fas and induces the apoptosis of activated cells bearing the Fas receptor on their surface.11 Calcitonin is produced by endometrial gland cells in response to P. It acts via receptors present on the pre-implantation embryo, induces the raise of intracellular calcium levels, activation of adenyl cyclase and integrin expression. It seems to be involved in the passage from the relative quiescence of the early cleavage stages to the enhanced metabolic activity of the blastocyst.12 After hatching, the embryo directs the embryonic pole toward the endometrium which is covered with mucus that will disappear at the time of implantation, a dynamic process that includes: apposition, adhesion and invasion. Apposition involves close proximity between trophoblast and endometrial epithelium. Subsequently, microvilli expressed by trophoblast cell membranes establish contacts with cytoplasmic protrusions exhibited by endometrial cells (uterodomes). These bulbous protrusions, in women, appear about 5-6 days after ovulation and have a half-time of about 48 hours. Finally, the basal 109 membrane is invaded by the trophoblast which penetrates the connective tissue of the endometrial mucosa.13 Integrins, a family of transmembrane receptors that mediate cell adhesion to various types of ligands, including ECM, are involved in the interaction between the embryo and the endometrial surface. During the window of implantation, it has been documented a specific temporal and spatial expression of at least three different integrins (a4b1, a1b1 and avb3), crucial to endometrial receptivity. In particular, the window opens with the appearance of avb3 and closes with the loss of a4b1. On the other hand, the human blastocyst expresses a number of integrin sub-units during the pre-implantation phase. A switch in the integrin repertoire is also observed in the trophoblast.14 Ovarian steroids, cytokines and growth factor regulate integrin expression in both sites, and in particular, P induces the upregulation of some integrins in the endometrium after ovulation. Aberrant expression of integrins is linked to fertility defects.15 The adhesion mediated by integrin receptors requires the presence of ligands such as fibronectin. During the pre-implantation period, fibronectin accumulates in endometrial ECM under the basal membrane, promotes the attachment of trophoblast and modulates protease activity.16 The interaction between trophoblast and endometrium is facilitated by an alteration in the organization of epithelial cells which can undergo apoptosis to support stromal invasion by trophoblast. As far as the epithelial structure is concerned, the apical glycocalix undergoes remodelling. In humans, the mucin MUC-1 disappears from a small area of cells surrounding the attached embryo.17 Moreover, intercellular contact is disrupted; in fact, E-cadherin present at the adherens junctions is degraded and desmosomal proteins are downregulated. Cells become flattened and uterodomes appear.18 The invasive propensity of the trophoblast is supported by the production of MMPs which start to be expressed in the 7day-old embryo. MMPs are induced by IL-1 -a and TNF-a during the first trimester. Leptin and LIF have an opposite effect on MMPs, enhancement and reduction, respectively.19 There is an increasing body of evidence that Leptin, the product of the ob gene, plays an important role in implantation even though this role is not clear. This small peptide is secreted by the adipose tissue, but, at present, it is thought to be involved in the reproductive function, acting at endocrine and paracrine levels. Leptin is present in the endometrium increasing in the and in the blastocyst; moreover, Leptin and Leptin receptors have been found in the implantation sites.20 LIF is a polyfunctional glycoprotein member of the IL-6 family. Its signal is triggered by a high affinity receptor formed by LIF receptor-b and the glycoprotein gp-130 at the level of cell membranes. Both E and HCG stimulate LIF secretion in endometrial epithelial cells and it is likely that LIF promotes decidualization acting in cooperation with other signals coming from the blastocyst. In addition, LIF induces the differentiation of trophoblast in the anchoring phenotype.21 During blastocyst implantation and early pregnancy, a sort of maternal tolerance is observed and pro- and anti-inflammatory cytokines localized in both the human endometrium and in the pre-implantation embryo modulate the immunological processes occurring within the uterus. The IL-1 family, which has probably the widest impact, consists of four related peptides crucial to implantation. These peptides modulate cell proliferation and differentiation, stimulate the production of several other cytokines such as IL-6 and IL-8, TNF-a and prostaglandins, affect integrin and MMP expression. Moreover, they modulate the synthesis of steroids, HCG and CRH in trophoblast and in endometrial cells.22 IL-6 is constitutively expressed by the trophoblast and may modulate its invasive properties by increasing MMP expression, but in the implantation control IL-10, IL-11 and IL-15 are also involved. IL-10 downregulates MMPs and cytotrophoblast invasion in vitro and in vivo. IL-11 is important for the differentiation of endometrial stroma to decidua and IL-15 could act as a mediator of interaction between decidual cells and uterine NK cells which infiltrate the endometrium during the first trimester of pregnancy.23 110 The expression of IL-18 may serve as a marker of endometrial receptivity. Increased expression of this cytokine is deleterious for the onset of pregnancy as a strong stromal anti-IL-18 staining has been found in patients with implantation failure.24 It is worth mentioning that trophoblast cells do not express the Human Leukocyte Antigens (HLAs) apart from HLA-G, a non classical major histocompatibility complex class I molecule. It exerts an immunosuppression inducing apoptosis of activated CD8(+) T cells and down-modulation of CD4 (+) T cell proliferation. HLA-G also regulates cytokine secretion of decidual NK cells. During gestation NK cell numbers decline, and at term, leukocytes are absent.25 Macrophages represent 20-30% of the decidual cells at the site of implantation and persist throughout the pregnancy. They are presumably involved in the adjustment of the feto-maternal immunity.26 It is 20 years since the first report of the release by the human embryo of a soluble factor that causes thrombocyte activation. This factor, named Paf, is also produced by the sperm, the ovary, the endometrium and the fetus. Paf has not only a trophic effect on the embryo itself but is involved in the regulation of maternal immune function. In fact, embryo-derived Paf release determines a modification of T-lymphocyte rosette formation which could represent an evidence of maternal immunosuppression.27 Finally, another important factor which may condition implantation is represented by uterine contractility which is controlled by ovarian hormones. The smooth muscle of the uterus must be relaxed when the trophoblast interacts with the epithelium. P is the most important relaxant factor, promoting local vasodilatation and musculature quiescence by inducing itric oxide. Adequate uterine contractility is also required for embryo transportation along the utero-tubal cavities.28

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1Buhi W.C., Alvarez I.M., Kouba A.J., Secreted proteins of the oviduct, Cells Tissues Organs 2000, 166(2): 165-179. 2Kane M.T., Morgan P.M., Coonan C., Peptide growth factors and pre-implantation development, Hum Reprod Update 1997, 3(2): 137-157; Srivastava M.D., Lippes J., Srivastava B.I.S., Cytokines of the human reproductive tract, Am J Reprod Immunol 1996, 36(3): 157-166. 3Diaz-Cueto L., Gerton G.L., The influence of growth factors on the development of pre- implantation mammalian embryos, Arch Med Res 2001, 32(6): 619-626. 4Buhi, Alvarez, Kouba, Secreted proteins of the..., pp. 165-179. 5Aplin J.D., Kimber S.J., Trophoblast-uterine interactions at implantation, Reprod Biol Endocrinol 2004, 2: 48. 6Ibid.; DUPONT S., KRUST A., GANSMULLER A. ET AL., Effects of single and compound knockouts of E receptors a (ER a) and b (ER b) on mouse reproductive phenotypes, Development 2000, 127(19): 4277-4291. 7Osteen K.G., Rodgers W.H., Gaire M. et Al., Stromal-epithelial interaction mediates steroidal regulation of metalloproteinase expression in human endometrium, Proc Natl Acad Sci 1994, 91(21): 10129-10133. 8Fanchin R., Peltier E., Frydman R. et Al., Human chorionic gonadotropin: does it affect human endometrial morphology in vivo?, Semin Reprod Med 2001, 19(1): 31-35; Yang M., Lei Z.M., Rao C.V., The central role of human chorionic gonadotropin in the formation of human placental syncytium, Endocrinology 2003, 144(3): 1108-1120. 9Aplin, Kimber, Trophoblast-uterine interactions..., p. 48. 10Minas V., Loutradis D., Makrigiannakis A., Factors controlling blastocyst implantation, Reprod Bio Med Online 2005, 10(2): 205-216. 11Makrigiannakis A., Zoumakis E., Kalantaridou S. et Al., Corticotropin-releasing hormone promotes blastocyst implantation and early maternal tolerance, Nat Immunol 2001, 2(11): 1018-1024. 12Aplin, Kimber, Trophoblast-uterine interactions..., p. 48. 13Bentin-Ley U., Sjogren A., Nilsson L. et Al., Presence of uterine pinopodes at the embryo- endometrial interface during human implantation in vitro, Hum Reprod 1999, 14(2): 515-520; Usadi R.S., Murray M.J., Bagnell R.C., Temporal and morphologic characteristics of pinopod expression across the secretory phase of the endometrial cycle in normally cycling women with proven fertility, Fertil Steril 2003, 79(4): 970-974. 14Minas, Loutradis, Makrigiannakis, Factors controlling blastocyst..., pp. 205-216; Sueoka K., Shiokawa S., Miyazaki T. et Al., Integrins and reproductive physiology: expression and modulation in fertilization, embryogenesis, and implantation, Fertil Steril 1997, 67(5): 799-811. 15Minas, Loutradis, Makrigiannakis, Factors controlling blastocyst..., pp. 205-216. 16Wartiovaara J., Leivo I., Vaheri A., Expression of the cell surface-associated glycoprotein, fibronectin, in the early mouse embryo, Dev Biol 1979, 69(1): 247-257; Armant D.R., Bastocysts don't go it alone. Extrinsic signals fine-tune the intrinsic developmental program to trophoblast cells, Dev Biol 2005, 280(2): 260-280. 17Meseguer M., Aplin J.D., Caballero-Campo P. et Al, Human endometrial mucin MUC 1 is up- regulated by progesterone and down-regulated in vitro by the human blastocyst, Biol Reprod 2001, 64(2): 590-601. 18Potter S.W., Gaza G., Morris J.E., Estradiol induces E-cadherin degradation in mouse uterine epithelium during the estrous cycle and early pregnancy, J Cell Physiol 1996, 169(1): 1-14. 19Meisser A., Chardonnens D., Campana A. et Al., Effects of tumor necrosis factor-a, interleukin-1 a, macrophage colony stimulating factor and transforming growth factor ß on trophoblastic matrix metalloproteinases, Mol Hum Reprod 1999, 5(3): 252-260.

112 20Cervero A., Horcajadas J.A., Dominguez F. et Al., Leptin system in embryo development and implantation: a protein in search of a function, Reprod Bio Med Online 2005, 10(2): 217-223. 21Kimber S.J., Leukaemia inhibitory factor in implantation and uterine biology, Reproduction 2005, 130(2): 131-145. 22Minas, Loutradis, Makrigiannakis, Factors controlling blastocyst..., pp. 205-216; Feinberg B.B., Anderson D.J., Steller M.A. et Al., Cytokine regulation of trophoblast steroidogenesis, J Clin Endocrinol Metab 1994, 78(3): 586-591; Yanushpolsky E.H., Ozturk M., Polgar K. et Al., The effects of cytokines on human chorionic gonadotropin (HCG) production by a trophoblast cell line, J Reprod Immunol 1993, 25(3): 235-247. 23Minas, Loutradis, Makrigiannakis, Factors controlling blastocyst..., pp. 205-216; Dunn C.L., Kelly R.W., Critchley H.O., Decidualization of the human endometrial stromal cell: an enigmatic transformation, Reprod Biomed Online 2003, 7(2): 151-161. 24Leede-Bataille N., Olivennes F., Kadoch J. et Al., Detectable levels of interleukin-18 in uterine luminal secretions at oocyte retrieval predict failure of the embryo transfer, Hum Reprod 2004, 19(9): 1968-1973. 25Le Bouteiller P., The role of HLA-G expression in the embryo during implantation, J Gynecol Obstet Biol Reprod 2004, 33 (1 Pt 2): S 9-12. 26Abrahams V.M., Kim Y.M., Straszewski S.L. et Al., Macrophages and apoptotic cell clearance during pregnancy, Am J Reprod Immunol 2004, 51(4): 275-282. 27O' Neill C., The role of paf in embryo physiology, Human Reprod Update 2005, 11(3): 215-228. 28Bulletti C., De Ziegler D., Uterine contractility and embryo implantation, Curr Opin Obstet Gynecol 2005, 17(3): 265-276.

* L'autore ringrazia le dott.sse Anna Giuli e Fortunata Iacopino per la collaborazione offerta nella revisione del manoscritto e nella raccolta della bibliografia.

113 KEVIN T. FITZGERALD

PGD: BIO-MEDICAL INSIGHTS AND ETHICAL CONSIDERATIONS

In April of 1990, Alan H. Handyside and his colleagues published a letter in the journal, Nature.1 The letter was noteworthy in that it reported for the first time established pregnancies that were the result of a process which included pre-implantation genetic diagnosis (PGD) of in vitro fertilized (IVF) embryos before they were transferred to two women. Only female embryos were selected to be transferred to these two women since each had a 50% risk of passing on an mutation that would affect any male offspring. Since testing for the diseases directly was not possible, the researchers tested for the presence of a in the embryos. The researchers then selected only those embryos which had no Y chromosome for transfer because they calculated that any female offspring would not be burdened by the X chromosome mutations if any were inherited since the female offspring would also inherit an X chromosome from their fathers that did not contain the identified mutations. This report of the first successful PGD was the result of research that sought to combine the rapidly expanding field of genetic testing with the expanding practice of IVF. The basic idea behind the development of PGD was to create the ability to test embryos for genetic diseases before they had become implanted in the uterine lining and established a pregnancy. Finding a genetic problem before implantation results in the purported advantage of the termination of an embryo rather than the termination of a pregnancy ‘presuming that other pre-natal testing reveals the genetic problem and the fetus is aborted or dies in utero ‘or the birth of a child with the genetic disease. These purported advantages and goals of employing PGD involve many assumptions, evaluations, and claims that require a thorough and careful analysis as to their accuracy and legitimacy. It is not within the purview of this presentation to present such a thorough analysis. However, certain questions concerning the purported advantages and goals of PGD will be raised as part of this review of PGD technology in order to provide a brief description of PGD technology and some of the ethical issues the technology raises. As stated previously, PGD is a combination of IVF and genetic testing. The advantage of using IVF embryos for testing is their accessibility before a pregnancy is established. These embryos are created in a Petri dish, not inside a woman’s body. The embryos are created using sperm obtained from a donor (who may or may not be the husband of the woman who will receive the transplanted embryos), and eggs obtained by hyperstimulating the ovaries of a woman (who may or may not be the woman who will receive the transplanted embryo) so that many egg follicles might be removed at one time. After fertilization of an egg by sperm, there are both more direct and more indirect methods for testing an embryo using PGD technology. The most common method is the more direct approach, which involves holding the embryo in place as a technician breaches the zona pellucida (a protective layer surrounding the developing embryo) and removes one or two cells from the embryo for testing. This method is done usually around the 6- 8 cell stage of embryonic development, which occurs approximately 3 days after fertilization. Alternatively, the removal of cells from the embryo can be done at the blastocyst stage (an embryo of 100 or more cells at about 5 or 6 days after fertilization) which is technically easier to do and allows for more cells to be removed for analysis. However, blastocyst testing leaves less time for analysis, explanation, and decision-making, before the selected embryos need to be transferred to the uterus. Whichever direct method of obtaining cells for testing from the embryo is used, the object of the testing is to determine which embryos are desirable for transferring to a woman’s uterus.2 Hence, great care needs to be taken not to damage the embryos determined by PGD to be desirable since that would presumably make it less likely that they successfully develop. Obviously the purpose of PGD is undermined if the process itself creates embryos less likely to implant and develop after transfer to a uterus.

114 The more indirect method, used less often than the more direct method, has the advantage of not removing cells from the embryo proper and, hence, incurs less of a chance of damaging the embryo. It also may be the only method available for testing in areas where direct embryo biopsy is prohibited. 3 However, since the embryo is not directly tested the test results are used to infer the maternal contribution to the genetic constitution of the embryo. The cells that are tested are the polar bodies which contain the chromosomes that are discarded as the egg’s complement of chromosomes is reduced to a single copy in order to pair up appropriately with the single copy of chromosomes from the sperm cell during fertilization. The first polar body is created during the meiosis process when the duplicated maternal chromosomes are divided in half from a 4N state (chromosomes having been doubled to four copies to prepare for cell division) to the normal 2N (two copies) diploid state. Using the maternal genetic constitution as a reference, testing this polar body allows one to infer which of the maternal alleles (normally each gene in a human being has two copies, or ‘alleles,’one on each of a pair of chromosomes) have been distributed to the egg. The second polar body results when half of the egg chromosomes are removed so that the haploid (1N) set of maternal chromosomes in the egg can match up with the haploid set of chromosomes from the sperm to produce a normal diploid (2N) embryo. Testing this polar body along with the first polar body allows one to infer the specific maternal allele that has been inherited by the embryo. The obvious limitation of this indirect method is that the paternal alleles inherited by the embryo remain unclear. Another limitation of this method is the risk of polar body degeneration that can result in unsuccessful testing. Regardless of which method is used, the testing methods of the genetic material in the removed cells are the same. If the number or arrangement of the chromosomes in the cells of an embryo is the target of the testing, then the chromosomes in the selected cells are labeled so that their number and composition can be seen by microscopy. If a particular allele or genetic sequence is the target of testing, then the target DNA sequence can be amplified by a process called polymerase chain reaction (PCR). This process allows for enough copies of the DNA sequence to be made so that an accurate reading of the sequence can be done, and any undesired sequences discovered. Of course, the accuracy of the PGD test results depends in part on the level of experience of the people involved in the testing process. Even with an experienced staff, there is a definite risk of PGD testing providing ambiguous or false results. This inaccuracy can be the result of technical errors or be due to some aspect of the embryo itself, such as mosaicism (i.e. the embryo being a mixture of cells with two or more different genetic constitutions).4 A brief internet survey of IVF clinics indicated that a common rate of misdiagnosis cited was around 10%. One fertility center stated the risk of ambiguous or false results at about 5 to 15%.5 In addition to these risks, there are the risks involved in manipulating the embryo in vitro as mentioned above. Though there is not yet sufficient data to ascertain the extent of these risks, especially long term risks after birth, the PGD process of removing one or two cells from an early embryo obviously has the potential to damage the embryo, perhaps even in ways that may not manifest themselves until well after birth. In addition, if PCR is to be used in the process of testing the cell(s) taken from the embryo, the recommendation is for the egg to be fertilized by injecting the sperm directly into the egg (a process called Intracytoplasmic Sperm Injection, or ICSI).6 This procedure carries risks of its own, such as genetic abnormalities or the perpetuation of male infertility factors in offspring.7 In addition to the risks of PGD and ICSI, there are the risks that come from the IVF process itself. There is evidence of a significantly increased risk of multiple births in IVF (obviously dependent upon the number of embryos transferred), as well as an increased risk for low , prematurity, and perinatal mortality in singleton births.8 Therefore, as mentioned above, anything that increases the risk of the IVF/ICSI/PGD process not producing its purported goal of a healthy child or a child with the desired characteristics brings into question the reasons proponents of PGD often use to justify employing the process at all. However, in this consideration of the goals and risks of these processes, it 115 is important to note that even in the European Society of Human Reproduction and Embryology guidelines regarding PGD the concerns for the welfare of any children born via IVF/PGD are only ‘balanced against ‘the couple’s reproductive autonomy and liberty.9 Hence, as we will see below, the welfare of the child to be born via the PGD process is not the only reason, or even necessarily the preeminent reason, for employing PGD. Currently in the United States the cost of PGD is estimated to be between $1500 and $3500.10 This cost is usually not covered by insurance in the United States, and is also in addition to the regular costs of IVF. Still, though the process of PGD is costly and involves risks both determined and undetermined, the number of PGD tests done and the number of babies born after PGD continues to grow (over 1000 born post-PGD testing worldwide).11 This growth in the use of PGD is due, in part, to the rapidly expanding list of diseases or conditions for which one can use PGD to test. One increasingly common reason for PGD testing is to identify embryos with aneuploidy (an abnormal number of chromosomes) or other chromosomal abnormalities. This type of testing is sometimes referred to as ‘low-risk PGD’or ‘preimplantation genetic screening (PGS) ‘.12 Earlier aneuploidy testing was focused primarily on identifying embryos with abnormal chromosome arrangements linked to known diseases such as Down Syndrome (Trisomy 21) and Turner Syndrome (single X chromosome). More recently, research has been focusing on the correlation between embryos with aneuploidy (in all or some of the cells in the embryo) and a reduced rate of pregnancies going to term. This research has evolved rapidly. It began with common PGD testing used to investigate this correlation between poor pregnancy outcomes and embryo aneuploidy.13 Currently this research involves more detailed studies investigating the limitations of PGD testing in establishing this correlation between aneuploidy and birth rates due to the relatively non-uniform and unstable nature of the number of chromosomes found in the different cells of the early embryo.14 Hence, as is often the case in science, further research has revealed that what originally appeared as a relatively simple relationship between an observed condition (aneuploidy in one or two cells of an 8 cell embryo) and a particular outcome (unsuccessful pregnancy), may, in fact, be a much more complicated reality that is more difficult to predict. Such complexity needs to be acknowledged and considered carefully, especially when the reasons often given for employing PGD require high levels of precision and predictability which may not be supported by current data.15 In addition to aneuploidy testing, one can currently find fertility clinics that will test for dozens of genetic diseases and conditions. These genetic conditions range from those with known single mutations (such as sickle-cell anemia and cystic fibrosis) to more complex genetic conditions that will not necessarily result in a disease but only increase the probability of one occurring ‘and some not until much later in life (e.g. Alzheimer disease, Huntington disease, and BRCA-related breast ). In part, it is the desire to not pass on such genetic characteristics to one’s offspring that is driving some of the increased use of PGD. This list of genetic conditions available for testing has the potential to continue its rapid expansion since technically one can test for any genetic sequence once it has been linked to a particular condition or trait of interest. This situation raises the issue of embryo selection based on non-disease related characteristics, which is already present in the case of PGD for sex selection or tissue-typing. In the case of tissue-typing, PGD is used to identify embryos that not only are free of a genetic disease that the parents may harbor, but also are a histocompatibility/human leukocyte antigen (HLA) match for a previous child that has inherited or acquired a disease. Transferring embryos that are histocompatible allows for the possibility of bearing a child who can then be a tissue or organ donor for the sick sibling. This more recent application of PGD raises the issue of embryo selection not primarily for the benefit of the embryos being selected, but for the benefit of another. To this point histocompatibility selection has been done to acquire tissue for a sibling. However, this need not necessarily be the case, though there is a much better chance of a sibling being a HLA match for another person.

116 Currently this tissue-typing issue is focused on the situation of a couple having another child in order to provide tissues or organs for a sick sibling.16 However, human embryonic technology might advance to the point where it would be technically comparable to use the embryos selected to start an line to produce tissue for transplantation instead of transferring the embryos into a uterus. Such a development would remove the matter of the value, quality, and independence of the PGD-selected child’s life from the ethical analysis, and focus the analysis more on the selected embryos and their necessary destruction in the process. Of course, for this technical advance to occur, research would have to be done which would require the destruction of human embryos in order to establish the safety and predictability of the process. As cited in the beginning of this presentation, the earliest use of PGD was to identify and discard male embryos since they had the much greater risk of being born with the genetic diseases being targeted. There is no technical reason that the selection of XX or XY embryos need be limited to reducing the risk of disease. In fact, there is already an extensive debate surrounding the use of PGD for sex selection and ‘family balancing ‘(a version of the sex selection argument that claims additional justification to select the gender of the embryos to be transferred if a family already has more or only children of one gender). This use of PGD for sex selection puts medical issues aside and moves clearly into the realm of individual choice as the basis for embryo selection. If this trend toward using PGD for embryo selection according to a parent’s or customer’s choices persists or increases, proponents of such use of PGD will have to justify the access, costs and risks of the PGD process (not to mention the destruction of human embryos) primarily or exclusively on the basis of individual autonomy and choice. This shift from medical rationales to justifications for using PGD based solely or primarily on individual or group autonomy is already present in the current discussions surrounding PGD. This shift is not surprising since it reflects a similar shift that confronts all of medicine and medical technology. When deciding whether or not it is legitimate to perform a medical or technological intervention on human beings, the medical profession and society must always weigh the benefits to be gained against the harms that will, or might be, incurred. Part of the reason for the current shift away from using medical and therapeutic reasons to justify undergoing some risk, and toward the primacy of individual autonomy as a justification, is a growing confusion as to the meaning of health and therapy. At one end of this issue is the growing acknowledgement among bioethicists that distinctions between therapies and enhancements are not easy, if possible, to determine.17 On the other end, even if some kind of ‘disease ‘or undesirable state is required to be present in order to justify undergoing the risks of a procedure, these parameters are not clear since what is a disease or what is undesirable is very much in the eye of the beholder. For example, at what point do the risks involved in reconstructive surgery outweigh the benefits, especially if the benefits are primarily social acceptance? The current debate over face transplants for severely disfigured individuals is a key example of this tension between undergoing a procedure with high medical risks in order to address what could be primarily a psychological or social harm. In addition, the rapid expansion of genetic and molecular biology information has increased this confusion concerning any definition of health and disease. We already know that alleles which are deleterious under certain conditions can actually be beneficial under other conditions (e.g. sickle-cell anemia and cystic fibrosis). By selecting against certain alleles or genetic conditions will we eventually limit the diversity and robustness of our future generations in order to pursue morphological or behavioural fashions? If so, what will the pressures be on children born that have been selected for some reason ‘including their gender? What if a child selected to be a certain gender decides to change gender either behaviourally or surgically? If autonomy is the ultimate arbiter, do the parents of a child have the right to choose that child’s gender considering the fact that the child might not like the gender that was chosen? Granted parents make many decisions for their children, as they must. However, it is far easier to change a child’s clothes or the instrument that is practiced, if the child does not like them, 117 than it is to alter a physiological characteristic such as gender. Furthermore, will children behave in a ‘healthy ‘manner if they come to know that they have been selected to be resistant to obesity, cancer, etc.? Perhaps such selection would actually result in a more unhealthy population due to a false sense of security. Since we already acknowledge that people can have children for many reasons, including quite selfish reasons, should we exacerbate this situation by allowing PGD to be used to place these selfish desires even more directly upon the child? If individual autonomy becomes the ultimate justification for employing technologies such as PGD, it will become all the more difficult to avoid such undesirable situations. The purpose of this article has been to present a brief review of PGD technology and some of the fundamental ethical issues it raises within the larger arena of medicine and ethics. Judgments reached concerning the legitimate uses of PGD, if any, will both influence and be influenced by the larger ethical issues, such as delineating health and the role of individual autonomy, confronting the medical profession and society. The rapid advance of biological information and medical technology may make these judgments all the more difficult, and yet also all the more important. Considering this importance, one final issue needs to be added to any consideration concerning the use or non-use of PGD: how will our judgments about PGD affect those who are most vulnerable and needy in our societies? Will our judgments create greater divides between those who count and those who don’t count in our societies, or will our judgments about PGD bring those on the margins of society closer to everyone else? On this issue, PGD appears to have an inherent problem ‘any technology that allows some to select against or select out others tends to exacerbate differences and distances among people. Is this what we want?

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1Handyside A.H., Pregnancies from biopsied human pre-implantation embryos sexed by Y-specific DNA amplification, Nature 1990, 344: 768-770. 2For a more detailed explanation of embryo biopsy methods, see: ‘Embryo culture and biopsy ‘section of the European Society of Human Reproduction and Embryology guidelines for PGD found in THORNHILL A., ESHRE PGD CONSORTIUM, Best practice guidelines for clinical pre-implantation genetic diagnosis (PGD) and pre-implantation genetic screening (PGS), Human Reproduction 2005, 20(1): 35-48. 3Ibid., pp. 39-40. 4Baart E.B., Pre-implantation genetic screening reveals a high incidence of aneuploidy and mosaicism in embryos from young women undergoing IVF, Human Reproduction 2005. 5For example, see Advanced Fertility Center of Chicago website (www.advancedfertility.com). 6Thornhill, Eshre PGD Consortium, Best practica guidelines..., p. 40. 7Foresta C., Genetic Abnormalities among Severely Oligospermic Men Who Are Candidates for Intracytoplasmic Sperm Injection, The Journal of Clinical Endocrinology & Metabolism 2005, 90(1): 152-156. 8http://www.dnapolicy.org/tools-content/pdfs/1/64441.pdf. 9Thornhill, Eshre PGD Consortium, Best practica guidelines..., p. 45. 10Ibid. 11www.dnapolicy.org, Reproductive Genetic Testing: Issues and Options for Policymakers, 2004: 23. 12Thornhill, Eshre PGD Consortium, Best practica guidelines..., p. 35. 13Delhanty J.D., Pre-implantation genetics: an explanation for poor human fertility? Annals of Human Genetics 2001, 65: 331-338. 14Baart, Pre-implantation genetic screening reveals a ... 15Shahine L.K., Cedars M.I., Pre-implantation genetic diagnosis does not increase pregnancy rates in patients at risk for aneuploidy, Fertility and Sterility 2006, 85(1): 51-56. 16Thornhill, Ehre PGD Consortium, Best practica guidelines..., p. 38. 17UNITED STATES PRESIDENT’S COUNCIL ON BIOETHICS, Beyond Therapy: and the Pursuit of Happiness, New York: Regan Books-Harper Collins Publishers Inc., 2003: 13-16.

119 MARIE-ODILE RETHORÉ

RENATAL AND PRE-IMPLANTATION DIAGNOSIS FROM THE PARENTS’ VIEWPOINT

The conquests of medicine exist and they are wonderful! Real research goes on and has no intention whatsoever of stopping. It can be carried out perfectly without endangering the life of even one person. A week does not go by without some wonderful discovery…the work is enormous! The difficulties encountered today in carrying this out well did not begin yesterday. When Bourneville asked Public Health for credit to improve the care of the handicapped in his Department at the Salpetriere hospital, he was told: “Idiot children do not deserve such investments. They are absolute nonvalues ”. Despite this, as professor Lejeune liked to say, we will not give up! To give birth to a handicapped child is infinitely painful, and nothing or no one can console that sorrow. Those who are charged with accompanying parents, especially in the beginning, have a heavy responsibility. The child’s future and the couple’s harmony will depend to a great extent on their attitude. These invalidating diseases are dreadful. Research to understand them better and attenuate their consequences must go on. It merits dedicating one’s life to it. As professor Lejeune often said: “We will never give up”. But it should not be done at any price. We have to stop pretending we believe that research is neutral, and that only its applications can be qualified as good or bad. The choices have to be made well before the discovery. The technical means for observing a child during intrauterine life are more and more efficient. Ultrasound or echography (the three-dimensional image of the child in color), the amnioscope, make it possible, when in expert and above all very practiced hands, to “ see ” even the slightest malformations. However, we have to recall at the right and the wrong times that very often one only finds what one is looking for. Some images are difficult to interpret. There are some false negatives that are wrongly reassuring, and some false positives which very understandably create great anguish in the parents’ hearts and even lead to aborting perfectly formed children. The examination of a child’s chromosomes can be made from the seventh week of intrauterine life through the on a biopsy of the placenta, or later at the sixteenth week of life by puncturing the amniotic liquid into which the fetus’ skin peels. The analysis of the karyotype makes it possible to know the child’s sex (which is decisive in the diseases linked to sex), and whether or not a child is affected with a disease through chromosomal abnormality. These diseases are common: one conception in two is affected by them. One of the most frequent diseases full term is Trisomy 21: 1 child in 650,98% of the cases occur in families without particular precedents. Their frequency increases with the mother’s age. They involve more or less severe visceral malformations in the child, but above all a more or less significant delay in psychomotor development from one chromosomal abnormality to another, and for the same abnormality, from one child to another. In the name of the principle of precaution, more and more women undergo amniocenteses. At present, 80,000 examinations are done each year, which represents 11-12% of the women expecting children. The percentage rises to 15% in Paris and its surroundings. At a discussion organized in March 2005 at the Clamart Hospital, professor Frydman, the Head of the Obstetrics Department, said: “ The practice of this examination is constantly growing although it exposes to risks of miscarriage through micro fissuring of the membranes (like the thermal shield of the American shuttle) in a percentage ranging from 0,5-1% of the cases, even 2%, and a risk of premature delivery at 25-26 weeks ”. So today, by wanting to do too much too well, more are caused in women carrying perfectly normal babies in whom no grave abnormalities are detected that would lead to a request for abortion. In this regard it should be noted that at present in Paris, less than 50% of couples ask for 120 abortion in these cases, and they only do so when the chromosomal abnormality involves a mental deficiency. For professor Frydman, this multiplication of amniocenteses due to excessive precaution raises a grave public health problem. He said that it is necessary “ to explain the stakes and the risks of this technique to women because we know how easy it is to upset a pregnant woman by proposing a battery of tests to her in order to, so to speak, reassure her ”. France is the first country in the world for prenatal diagnosis and the detection of fetal malformations through ultrasound, amniocentesis and the dosage of certain “ markers ” present in the mother’s blood in which abnormal concentrations may be associated with diseases or malformations in the child. However, in professor Frydman’s opinion, “ these blood markers researched between the fifteenth and the eighteenth week of pregnancy are far from reliable ”. Since 1996, this examination has been proposed to pregnant women. In 1997, since the test was reimbursed by Social Security at the time, 380,000 samples were taken. This number increased by 20% in 1998, which cost the State 124 million Francs. Today, it is proposed not imposed – to all pregnant women. All the tests performed on the fetus surely create stress and suffering in the fetus, which we are now able to analyzing and measure, and they have consequences for its future. We know now that the composition of the amniotic liquid changes after it is punctured or a blood sample is taken from the fetus. An ultrasound done in that moment shows that the fetus has a withdrawal reaction, just like every child in the world, and its cardiac rhythm accelerates. These observations have led to recommending the use of an analgesic during these tests. In France, anesthesia for the fetus is obligatory if abortion takes places after 24 weeks of intrauterine life. The consequences of maternal anxieties should not be overlooked for the child in utero, but neither should maternal love. Professor Relier, the Head of the Neonatal Medicine Department at the Port- RoyalHospital, strongly emphasized this at a Congress in Italy in 1999. “ It has been demonstrated that like nutritional support or the quality of the exchange between the placenta and the fetus, the mother’s emotion, joy or suffering, has an essential influence. Under this heading, love undoubtedly represents the most appropriate stimulation to the growth and harmonious equilibrium of a quality being ”. Genetic pre-implantation diagnosis is an alternative to prenatal diagnosis. It consists of carrying out a genetic analysis of embryos obtained through in vitro fertilization and the intrauterine transfer of only healthy embryos. For the moment, this test is not authorized by law except for couples that have a particular risk of giving birth to a child affected by a genetic disease recognized as incurable at the time of the diagnosis. Many present this as a means of preventing abortion after prenatal diagnosis, as a lesser evil, in order to lessen the parents’ suffering because an abortion leaves indelible traces in the parents’ heart. It is obvious that if an embryo is eliminated at the four cell stage, then it will not be necessary to kill a fetus, or much less euthanize a child or a sick old person, but this does not seem like a good formula to me! Pre-implantation diagnosis requires in vitro fertilization, which is generally done by injecting a spermatozoid inside each oocyte obtained through ovarian hyper stimulation. When the embryo has six to ten cells, one or two of them are sampled and put in a culture for the requested genetic analysis. The other cells are put in waiting or frozen. According to the results of the cultures, the good embryos are chosen and the bad ones are thrown away. Many questions are raised, first of all regarding the technique used. Can the aggressive act of sampling and removing one or two cells from the embryo at three days of life be detrimental to the further development of the remaining cells that will be implanted in the uterus? Experimental embryology gives a reassuring answer for animals, but do we have a right to experiment on man? In Paris, out of the 872 oocytes collected from January 2000 to July 2001, 731 were fertilized and 127 were implanted. To date, 16 children, including four pairs of , have been born. No diagnostic error has been reported after the control. All of these children will be the subject of special observation for three years.

121 The risks of deviation are easy to imagine aggravated by the absence in our countries of a status of the embryo, which leads to fearing that less protection will be given to the embryo than the fetus. The less traumatic action performed outside of the maternal organism can lead to its trivialization and hence to an extension of the practice towards a kind of predictive medicine with very blurry limits. Requests made out of pure convenience can be feared, such as the choice of sex, or even social pressure or a risk of instrumentalizing the conceived baby not for itself, but to use it as therapy for one of its brothers or sisters. Can what some brutally call babies as medicine be allowed? This was the case for a little born in the United States on August 29,2000, whom his parents called Adam. He was conceived in vitro, then selected from other normal embryos like him, but because of their immunitary incompatibility with the HLA system, they could not be used as donors for a blood cell graft, the only thing that could save the life of the couple’s older daughter. About forty couples in France have requested this kind of manipulation. This procedure is taxing for the mother who has to undergo ovarian hyper stimulations in order to produce several embryos when she is not sterile. After being tested, two embryos will be implanted with an only 12-15% probability of resulting in a pregnancy. Detecting compatibility with the child to be saved is delicate…and what will be done if none of the embryos produced is compatible? What will be done with the other embryos that have not been implanted, even though they are healthy and compatible, if the parents do not want any more children? The forty weeks of this hyper-medicalized pregnancy will certainly seem very long, especially if the sick child’s state worsens. Then won’t there be a temptation to provoke labor, even very prematurely, in order to save the child? How will this “ savior ” child live his situation? What will his attitude be if other grafts prove to be necessary later, and how will he live the death of the older child he was not able to save? There are so many questions that do not have simple answers, but both the medical team and the family concerned must be aware of them. The National Committee of Ethics gave its opinion on this subject in July 2002: “ If medically assisted procreation is no longer aimed at favoring the birth of children for themselves, but to be used to repair another child, we are entering into a humanity that despises itself ”. Some genetic diseases, such as the chorea of Huntington due to a dominant gene located on chromosome 4, only show up later, generally after 50 years of age. When the person who is a carrier of this gene is younger, she is in perfect health and so she may have children, but she has one risk in two of transmitting the bad gene to her child. If she wants to procreate knowing that she has no risk, she can find out if she is a carrier through a genetic test. In fact, most of the people concerned prefer not to know. Do we have the right to announce to a 30 year-old man in perfect health that he has a gene that was detected in his father at 55 years of age which causes dementia? To tell him that there is one risk in two of transmitting the gene to each of his children…that no treatment is known for this disease, and that it inevitably leads to dementia at about 50 years of age…? Everyone has his/her burden to bear throughout life because of his/her history and family, but is it really the doctor’s role to give information in this way that is not always wanted? This has to be asked in order to know how far not to go when medicine is tempted to enter people’s lives forcibly. This implies extreme vigilance in terms of respect for the person and for everyone’s right to accept the test or not as well as each one’s desire to know or not to know. “We can easily imagine what a society would be like in which the search for genetic predispositions would be systematic, or worse, if it would become obligatory…in which the individual would be subordinated to a collective eugenic will ”, warns professor François Gros, Permanent Secretary of the Academy of Sciences. If this 30 year-old man does not want to know – and this is his most absolute right – but he wants to be sure that his child will not have the gene, a prenatal test can be proposed to him that will show whether or not the fetus has received a chromosome 4 from his paternal grandfather. But no attempt will be 122 made to find out if it is the chromosome 4 that brings the bad gene or if it is the other one (1 chance in 2), because if it is announced that it is the “ bad one ”, then the father will know that he is the carrier. But he does not want to know…In this case, the risk is accepted a priori of eliminating a healthy fetus in 50% of the cases. A pre-implantation diagnosis can also be proposed to him so that an embryo will be implanted that is certain to be free from the disease. But what should be done if all the embryos are affected? The failure to implant will suggest to the father that he too is affected…In a situation like this, we can see that the will to not know is not as simple to respect as one might imagine, and so the right to not know cannot be condemned at all and must be respected. You can imagine that in all this, for the families and for the doctors, there is one dreadful temptation. These diseases are grave. Most of them, for the moment, have no therapeutic solution. Everyone advises abortion if any abnormality is detected during intrauterine life. Some even consider it a professional error not to do it. Some families sue because they did not have an abortion when the child comes into the world alive and a carrier of one of these diseases. Many doctors, as if to excuse themselves, propose and even advise abandonment. At present in Paris, one child in four affected with trisomy 21 is abandoned at birth. Some parents resist the temptation with enormous courage that does not take away the suffering. A few days after receiving three young parents who were living through this drama, I received this letter: “ Madame, my heartfelt thanks for your welcome on Tuesday at the hospital. We left you encouraged and recharged. In fact, I think that you have reassured us about the future that is very confusing for us. When this birth was announced, there was a kind of a collapse, then acceptance and lastly fear set in. The panic and fear are starting to fade. Your existence and that of the Lejeune Foundation is reassuring. Thank you again.”. Deliberate abortion is considered an abominable crime by the whole Christian tradition, and John Paul II recalls this in the Gospel of Life: “ The human being is to be respected and treated as a person from the moment of conception… these reasons and others like them, however serious and tragic, can never justify the deliberate killing of an innocent human being…it not infrequently happens that these techniques are used with a eugenic intention which accepts selective abortion in order to prevent the birth of children affected by various types of anomalies. Such an attitude is shameful and utterly reprehensible, since it presumes to measure the value of a human life only within the parameters of normality and physical well-being, thus opening the way to legitimizing and euthanasia as well ” (Evangelium Vitae). Prenatal diagnosis must be proposed and favored if it makes it possible to treat a child from its intrauterine life. At present, this is possible in some but still not very many cases, either through surgery or a medical treatment given to the mother that avoids the occurrence of the malformation. It is our duty as doctors and researchers to do everything possible to favor research in this area. It is a question of life or death for the children affected. If prenatal diagnosis is proposed as a means of selection among children only to choose those whose lives correspond to the required criteria for finding a place in our society, then this would boil down to hiding their human faces definitively with a label that reads, “ condemned to death by the ignorance of medicine ”. It is very difficult to talk to the parents of a child they only know through diagnosis about the affection the child brings who no one has ever seen and cannot be touched and taken into their arms. As soon as possible, I try to help them to look not only at the test results, but also at the person of their child, their little one, quite simply by proposing that they call the child by name…then a great step is taken! The discovery of a grave genetic disease or an incurable malformation during pregnancy is a terrible trial for a couple and for the family as a whole. After the doctor’s announcement, the parents rush to a website where they look up words they do not always understand because they are not doctors, but which sound dreadful to them and condemn their child. 123 For those who reject abortion through the force of their conviction, it is necessary to live from day to day until the delivery, that is, about 20 weeks, and this is terribly long. They are going to meet a lot of well-intentioned people in their family and among their friends who will tell them that their attitude is not reasonable, that they have to think of the future of their other children, and that this handicapped child will be unhappy its whole life, that it will be costly for society, etc. Some of these couples tell me that they change their itinerary to not pass in front of a store with baby clothes where everything seems so happy and serene. When a child’s disease includes the risk of a more or less rapid death after delivery, we naturally speak about welcoming this little one who will not “ go home ”, but also about the way to prepare the other children who are impatiently waiting for their little brother or sister. They should talk to them about the child using his name, and as soon as possible, bring them photos and tell them that he did not suffer and that he fell asleep in his parents’ arms. If the parents are Christians, I help them to prepare their child’s Baptism and Confirmation. I do not know any parents who regretted acting in this way. Despite their suffering, they surprisingly find peace again rather quickly. When I am with these couples that are living through such moments, I feel very poor and have the inner conviction that I am before modern day saints, but holiness does not protect from temptations, and once again we must not forget that a pregnancy is long! The very critical article by Jean-Yves Nau: “ The programmed eradication of Mongolism ” summarizes the situation perfectly. “ Everything is set up so that in the short and mid-term, no more births of children with trisomy 21 will be registered. It should be noted that in so doing, the nature of medical activity changes fundamentally and clearly goes out of the framework of the individual relationship in order to serve de facto a public health policy ”. In this article, Jean-Yves Nau quotes Bernard Andrieu, a philosopher of life sciences: “ In the name of individual freedom, the State sets up a kind of eugenics that does not want to say its name, leaving all the burden of guilt on the individual…Today a woman would have the right…to decide the quality of the unborn child. Is this really the way to avoid eugenic deviation? ”. Eugenics was not born yesterday. Plato gave it a particularly cruel formulation in The Republic: “ It is necessary as often as possible for the élite of men to have dealings with the élite of women and, on the contrary, for outcasts to be with outcasts: the rejects of the former will be raised, but not those of the latter if we want the drive to keep its eminent quality and, when all these dispositions are taken, if we want them to be ignored by everyone except the magistrates ”. In the editorial of the review of the French Society of Genetics (June 1999), Jean Gayon puts the old and the new eugenics in opposition: “A pseudo science puts at the service of those discriminated by class or race a biologically based ideology associated with the worst exactions in the history of the 20th century, a kind of medicine and public health that turns against the sick, the handicapped and the mentally ill. These are the most common representations of eugenics today. The word is now the object of such aversion that the mere qualification of an idea or practice as eugenic is very often equivalent to its condemnation. However, it is said that the eugenic question is returning…We have two reasons for not forgetting this word too quickly. As it regards the past, it reminds us where we absolutely must not return. As it regards in a utopian way a possible future, it enables us to not let ourselves get hypnotized by it in any way, to look it in the face ”. He concludes, “ words are not guilty in themselves; there are only guilty actions ”. To transmit life is to accept the unborn child beforehand with its shadows and lights. It means accepting beforehand to adopt the child each day so that it will become what it really is: namely, this man, this woman, who is irreducible to no other, and after the moment of its conception, who only belongs to itself and its Creator. “Do not be afraid, open the doors wide to Christ ”. This is what John Paul II cried out to the world at the beginning of his Papacy on October 22, 1978. He went on to say: “Today very often man ignores what he has within himself. Very often he is uncertain about the

124 meaning of his life on this earth. He is invaded by doubt that turns into despair. So allow Christ to speak to man ”. If the glory of God is man fully alive, as Saint Irenaeus liked to say, the honor of man is to serve life wherever it is in danger. Today suffering and mutilated man is before science and technology that want to be the master of our destiny before the conscience of the world. John Paul II explained the Church’s position at the closing session of the Meeting of the Pontifical Academy of the Sciences on November 22, 1993: “ The permanent deepening of knowledge about the living person is a good in itself because the search for truth is part of man’s primordial vocation and constitutes the first praise to the One who formed humankind and is at the origin of all things. Science is seductive and fascinating. However, it would not be capable alone of enunciating the ultimate truth and proposing the happiness that man hopes to achieve, nor of dictating the moral criteria to achieve the good. Therefore, it is important to take the measure of the moral problems that lead not to knowledge itself, but to the means of acquiring knowledge and its possible or foreseeable applications. It is not up to the Church to establish the scientific and technical criteria of medical research, but it is up to the Church to recall in the name of its mission the limits within which any process is a good for man ”. He further emphasized: “ The moral criterion of research is always man in his corporal and spiritual essence ”. Most doctors and researchers who let themselves be tempted by these techniques say that they are acting according to their consciences. I am willing to believe this, and in any case, it is not up to me to judge. But to us, as Christian doctors, it is not possible to ask us to have a good conscience. As baptized and confirmed persons, we are asked to enlighten our consciences in the light of prayer, sacramental life, the sacred texts and the Church’s teach- ing which, as Father de Lubac wrote, “ dissipates the darkness where everyone is numb or despairing or pitifully carving out his/her own unending novel as he/she pleases. Without discouraging us from any task, it keeps us from false myths and spares us from the distractions and disgust of all the churches made by human hands. It saves us from ruin in the presence of our God! ”. Consequently, it is up to us to take on our responsibilities, to not be afraid to row against the tide firmly but humbly, knowing that we are sinners and vulnerable and that evil exists, but a Man came on earth to carry this burden on his shoulders. He died for this, but it was to rise again!

125 W.J. EIJK

THE CRITERIA OF OVERALL INDIVIDUALITY AND THE BIO-ANTHROPOLOGICAL STATUS OF THE EMBRYO BEFORE IMPLANTATION

One of the central subjects in the discussion about whether abortion and experiments on embryos are licit is that of the status of the embryo. The reaching of the status of a human individual or human person means that the embryo has a connected moral status, a dignity from which come the rights attributed to all human beings. Before reaching this status it is said to be licit to abort the embryo or to use it as material for experiments, thereby eliminating it. The difficulty is that many divergent opinions exist regarding the moment when one should attribute a moral status to the embryo. Thus indicators as to when an embryo becomes a real human being or a human person are variously identified as conception, nidation (after about two weeks), the beginning of cerebral activity, the ability to feel pain, having life outside the womb (in general from the twenty- fourth week of pregnancy),1 birth, or a stage of development after birth. In recent years there has been an increasingly widespread tendency to indicate a precise moment when an embryo is said to become a human individual and thus reference is made to a gradual progressive humanization or a gradual growth in the dignity of the embryo. This means that a duty is said to exist to protect the embryo that is proportionate to its level of development. This was, amongst other things, the argument used by the Dutch government to legalize experiments with human embryos in 2002 ‘the early embryo has a dignity of such a character that the advance of medicine can compensate the loss of embryos for research.2 Apart from conception, the moments listed above concede few possibilities of attributing to the embryo before the seventh day after fertilization the dignity of a human individual. The moments listed above, however, are conclusions that draw their validity from criteria employed to assess the status of the embryo. The criterion used depends upon the vision of man that is adopted as a point of departure. It is therefore necessary to make a net distinction between: 1) the moment when one attributes to the embryo the status of a human individual with all connected rights; 2) the criterion used to assess the status of the embryo; and 3) the anthropology that is the foundation of this criterion. The great variety of moments that are seen as the beginning of the existence of the human individual makes the discussion about the status of the embryo rather difficult. When it is not clear which criterion is used and which anthropology is taken as a point of departure, the discussion, inevitably, becomes marooned. For this reason, we must first of all consider the various criteria employed, first and foremost in relation to the relevant biological data. The distinction between extrinsic and intrinsic criteria helps us to clarify the terms of the question. The extrinsic criteria are those that do not derive from the embryo as such but from external factors. Both in the past and still today the following extrinsic criteria are to be encountered in the discussion about the status of the human embryo: human relationships: the embryo becomes a human individual when it establishes relationships with other human beings; positive law: the embryo becomes an individual when it is recognized as such by positive law; and the decision to give to an embryo that has been created through in vitro fertilization the possibility of further development. The intrinsic criteria, on the other hand, refer to certain characteristics of the embryo itself, namely: independence from the body of the mother: the embryo becomes a human individual when it is no longer part of the organism of the mother; human biological nature: the embryo is a human individual because of the simple fact that it is in biological terms a human being; individuality: the embryo becomes a human individual only when it can no longer divide itself thereby giving life to a or uniting itself to another embryo; being a person: the embryo becomes a human individual with all connected rights when it becomes a human person; and intrinsic finality: the embryo, even though it is 126 not yet a human individual, must be respected as such because of its intrinsic finality, that is to say because of the fact that it will become a human individual. Only after establishing the criterion to be employed can one indicate the moment in the development of the embryo when the embryo receives the moral status of being human: conception, the beginning of cerebral activity, the moment of birth or the moment when society recognizes it as being a subject of rights. One should bear in mind that the criteria that have been listed above do not exclude each other. Certain currents of thought apply some of them contemporaneously. To achieve a detailed assessment, however, it is necessary to analyse them separately.

EXTRINSIC CRITERIA Human relationships: the embryo becomes a human individual when it establishes relationships with other human individuals In the philosophy of the twentieth century, and especially in structuralism, there exists a strong tendency to look for the specific character of man not in what he is but in his relationships. Some thinkers even see the relationships of man as the only specific characteristic that distinguishes man from other living beings. In the view of the French moralists Ribes, Pohier and Roqueplo, the embryo reaches a fully personal and human status only by having human relationships. The example is that of an embryo that was beforehand wanted by its parents and in a certain sense by society as well. When there is not this intentionality on the part of the parents to conceive the child, and they have also tried to prevent conception, the embryo is said not to have a specifically human status.3 For this reason, the stages of the biological development of the embryo, in this vision, have no relevance. Obviously, there are important objections to this approach. It implies that one could deny all respect even to an unwanted new born child, with the possibility of eliminating him or her. Indeed, one could not indicate a precise moment when the embryo begins to acquire a human status. In this way one could also deny the status of being human to certain adults. What should we thus think of the elderly Indian woman of Calcutta who was dumped in a garbage heap in a sack of plastic by her daughter? Did she for this reason cease to be a human person? And did she become a human person again when she was brought by the sisters of Mother Teresa to one of their homes to be looked after with love? On the basis of its obvious shallowness and the extreme conclusions to which it leads, the reduction of the human being to pure relationality does not find many adherents. Others are of the view that the status of a human being and the personality of the individual emerge at the moment of nidation: nidation, because it implies the beginning of a close relationship with the mother, makes clear transcendence towards the other, which is considered essential for the human person. The human body, in fact, is the foundation and the real symbol of this transcendence towards the other.4 On the basis of this, Böckle and others, during the 1960s and 1970s, justified the use of interceptives and the day after pill: the fact that before nidation the embryo, not having human relationships, is not yet to be considered a human individual makes possible a comparative assessment of the values involved: the value of the embryo who is not yet a human person, on the one hand, and the welfare of the mother in situations of emergency, on the other. This implies that the use of the day after pill is acceptable in the case of rape and the use of the coil is also acceptable when there are grave reasons for , such as the need to prevent a pregnancy, or demographic reasons.5 However, it is not correct to assign too much importance to the moment of nidation, as though an existential relationship did not exist between the mother and the embryo before this event. Such a relationship is already created with the fusion of the spermatozoon and the ovule as an outcome of the sexual relationship of the parents. In addition, even before nidation the embryo receives necessary nutriments and oxygen for its growth from its mother. Thus nidation is not the beginning of a transcendental relationship with the mother that is said to characterize the embryo as a truly human individual. 127 The embryo becomes a human individual when it is recognized as such by positive law It appears evident that positive law guarantees and protects the objective rights of every human individual. In our pluralistic society the only practical solution to the controversy about the status of the human embryo in the opinion of many is that the status of the embryo should be defined through democratic consensus. Thus whether the embryo merits respect is said to depend exclusively on what has been established in this field by law. In the majority of countries procured abortion is allowed within the limit of a certain period of time and on certain conditions. In some countries experiments with human embryos before the time when they are implanted in the womb in natural conditions, that is to say up to fourteen days after conception, have been legalized (for example in England and the Netherlands). In our society many people do not dwell upon the question of the objective status of the embryo but adapt to the positive law in force. Hubert Marktl, formerly head of the Max-Plank- Gesellschaft, who presents as alternative the ideas that a human being is a purely biological fact or a concept that is recognized from a cultural point of view, refers to an act of recognition by which a living being during its development becomes a human being in the full sense.6

Civil law In a democracy, compromise is often inevitable and in many cases acceptable. However, the truth, including that about the status of the embryo, cannot be established by means of a statistical inquiry. It would be extremely dangerous for a society to determine what status should be attributed to human persons or to certain stages of development through the establishment of a consensus. Even if in a nation, in line with a law accepted by the majority, expulsions were to take place, we would not conclude that the members of persecuted ethnic minorities were not persons with a moral status and connected rights. Thus also the objection to that effect that unborn children, unwanted children or handicapped children have a low quality of life or will constitute a grave burden for their parents is not an objective reason to refuse then a moral status acknowledged by the law. Even people who request asylum in another country in which they have sought refuge are not always well accepted, nor can they expect an easy future. Despite this fact, even if forced to flee from their countries, they remain persons with the right to be aided and helped by the countries that receive them. In this area one may notice that a doctrine such as the doctrine of the Church, which is based upon objective reality, is neither authoritarian nor intolerant. Ethical relativism, which is held by many to be an essential precondition for democracy, on the other hand, is. According to the Encyclical Evangelium Vitae: ‘It is precisely the issue of respect for life which shows what misunderstandings as contradictions, accompanied by terrible practical consequences, are concealed in this position. It is true that history has known cases where crimes have been committed in the name of truth. But equally grave crimes and radical denials of freedom have also been committed and are still being committed in the name of ethical relativism. When a parliamentary or social majority decrees that it is legal, at least under certain conditions, to kill unborn human life, is it not really making a tyrannical decision with regard to the weakest and most defenceless of human beings? ‘(n. 70). Ethical relativism is not only a threat to the life of the weakest human beings, especially the unborn, who have no opportunity to vote, but also to democracy itself. Democracy is not an end in itself, but, like every other form of government, a means by which to assure the common good. Obviously, the common good, which involves all the conditions that are necessary for each individual member of society to be able to fulfil his or her destiny, requires respect for life, which is a fundamental good. Although freedom is a higher good than that of physical life, a human being cannot exercise his or her own freedom without being alive. Life is, therefore, a fundamental good as regards freedom. The non- recognition of life as a fundamental good constitutes a grave threat to freedom and democracy: ‘The value of democracy stands or falls with the values which it embodies and promotes. Of course, values such as the dignity of every human person, respect for inviolable and inalienable human rights, and the 128 adoption of the common good as the end and the criterion regulating political lifeâ?¦If as a result of the tragic obscuring of the collective conscience, an attitude of scepticism were to succeed in bringing into question even the fundamental principles of the moral law, the democratic system itself would be shaken in its foundations, and would be reduced to a mere mechanism for regulating different and opposing interests on a purely empirical basis ‘(Ibid.).

Positive divine law Amongst those that invoke Holy Scripture there are some who base the moral status of the embryo on a law held to be revealed, which in turn is based upon a specific translation of Genesis 9,6, that is to say: ‘Whoever sheds the blood of man in man, his blood will be shed ‘,7 instead of the usual translation ‘whoever sheds the blood of man, by man shall his blood be shed ‘(my italics).8 Genesis 9,6, translated in the first way, implies a prohibition on procured abortion. In combining a literal exegesis of isolated Biblical texts with a biological criterion, as some groups of Orthodox Jews do, there is an affirmation of the licit character of scientific research with embryos created by means of in vitro fertilization but not transferred into the maternal womb, in order to develop therapies for illnesses which as yet cannot be cured by man. The translation the blood of man in man requires, in fact, only respect for the intrauterine embryo and not for the extrauterine embryo. In this way, the creation of human embryos for the purposes of research and therapeutic cloning is also assessed as being admissible. Does Genesis 9, 6 justify therapeutic cloning? The usual translation ‘whoever sheds the blood of man, by man shall his blood be shed ‘implies a general prohibition on the killing of human beings. The by man in Hebrew is ba-’a-da-m. The prefix be’can have two meanings: in, as a preposition to indicate a location, or by in a causal sense. Which is the more correct translation?9 First of all it is necessary to observe that the original text allows, in principle, both translations.10 The translation by man in the context of Genesis 9,6 is, however, more evident and is used by practically all the versions of Holy Scripture. Two arguments in favour of this interpretation are the following: the phrase ba-’a-da-m is found nineteen times and nowhere does it mean in man11; secondly, Genesis 9,6 is constructed in the original Jewish text with a chiasm, a rhetorical device that involves the crossed arrangement of two words that are connected, like a mirror image: whoever sheds the blood of a man; by man shall his blood be shed. The words blood and man are used in an upside down way. This implies that the second part of the word man does not apply to the relative, as appears in the translation blood of man in man, but to the principal sentence by man shall his blood be shed. A second point, which is more important, concerns the use of Holy Scripture in theology in general and in moral theology and bioethics in particular. Even if the translation man in man, and thus the unborn human being in the maternal womb, were correct, Genesis 9,6 would not anyway constitute a justification for the killing and the exploitation of human embryos outside the womb: Genesis 9,6 would then say explicitly that it is not licit to kill human embryos outside the maternal womb. But the fact that this is not explicitly said does not mean that it is licit to kill embryos outside the maternal womb. Such a conclusion is a logical error: one cannot draw a positive conclusion from a negative premise. However much the human authors of the Bible were inspired by the Holy Spirit, the Bible is not a source for the natural sciences. For the authors of the Bible the possibility did not exist of creating embryos and of saving their lives outside the maternal womb. In Revelation it was not realized that biotechnology would arrive at the point of generating humans outside the womb, in a laboratory. To make a comparison: the principles of the social doctrine of the Church are formulated on the basis of the social questions and issues of the nineteenth century which were characterized by industrialization and the emergence of the proletariat. These principles without doubt have a Biblical foundation but, as they are, they were not formulated in Holy Scripture. Biblical texts cannot be interpreted on their own ‘they must be interpreted in relation to the whole context of the Bible. Holy Scripture does not give a univocal answer to the question of what the status 129 of the human embryo is.12 Given that within the Bib le we do not find explanatory criteria, supplementary criteria are required for a sound interpretation. In the the most important criteria are offered by Tradition and by the Documents of the Magisterium. The possibility of development: the embryo is to be considered a human individual if the possibility of further development exists An embryo conceived through in vitro fertilization which is not then implanted in the womb but which remains in the laboratory will live for at the most nine or ten days given current technical possibilities. Only if the embryo is transferred into the womb will it have a possibility of developing. The decision not to implant it has important consequences for the status of the embryo, as Tauer observes: ‘The question of normal conditions for a zygote in a test tube, if one does not intend to proceed with the transfer of the embryo and its implantation, raises doubts. If the normal conditions of a zygote in a laboratory are essentially the same as the oocyte before fertilization, something that appears to be true, then the zygote will never develop as a person. Thus it would be better to classify it as a possible person, a person who could become such only on certain conditions that are possible from a causal point of view (and deliberately chosen) ‘.13 If the embryo were destined to be transferred into the uterus it would have a higher status. This means that it should be classified as a potential person because it has a real possibility of developing. It would then have a value that is greater than a purely instrumental value.14 After concluding that a human embryo in a test tube, given that it is not able to feel and to act and is not conscious, has a weak moral status,15 Meyer and Nelson conclude that the status of the embryo is determined by the gametes from which it comes, that is to say from its genetic parents. These last have the exclusive right to decide whether the embryos are to be used for the procreation of their own children, of the children of other people, for research, or whether they must be simply thrown away. The use of embryos created in a laboratory for more than fourteen days must be avoided because some people consider this moment as constituting the morally significant beginning of the individuation of the embryo.16 The status of the embryo, understood in this way, is made to depend on the choices of others, first and foremost the researcher and the parents. One could argue that this choice can be made only during the stage prior to the implantation of the embryo and that in this case the intrinsic possibilities of the embryo are taken into consideration. However, an extrinsic criterion, that is to say the arbitrary decision taken by others, conditions judgement on the question as to whether the embryo has the same status as a gamete or whether it has a higher status. The intrinsic possibilities are thus decidedly denied.

Assessment The extrinsic criteria are not suitable for indicating the moral status of an embryo because they are secondary to what an embryo is. Only on the basis of intrinsic criteria can one have an objective judgement on the respect due to an embryo. Apart from this and apart from the criticisms expressed above, there is yet another fundamental objection: in the extrinsic criteria biological factors either have no role or have only a marginal role. However, this is inadmissible given that a human being is a substantial unity with a spiritual and material dimension. The material aspect is an intrinsic dimension of a human being and thus it is impossible both to identify a human being with this dimension and to conceive of a human being in a way that leaves aside his or her physical/biological dimension and does not attribute to that dimension an intrinsic role.

130 INTRINSIC CRITERIA From what has been said above it may be understood that one can employ intrinsic criteria alone to establish a definition of an embryo as a human individual and that these criteria must take into consideration the biological data as well.

The independence of the body from the mother: the embryo becomes a human individual when it is no longer a part of the organism of the mother At the end of the 1960s and during the 1970s feminist groups upheld the right to procured abortion with the motivation that the unborn child forms a part of a woman’s body and thus a woman should be able do with it as she so pleases ‘we are the governors of our tummies. The idea that the unborn child forms a part of the body of the mother is in conformity with an interpretation of the position of Roman law in relation to the status of the foetus which arose after the first translation of Digesta into German in 1831 and which was followed by nearly all of the commentators.17 I am referring here to a statement made by Ulpian (a Roman jurist who died in 228 AD): partus, antequam edatur, mulieris portio est vel viscerum.18 Given that in the translations the terms portio and viscera are understood in a physical sense, the general view emerged that Ulpian saw the unborn child as a part of the body of the mother (mulieris portio) or of her organs (viscerum) until the time of birth. Employing subtle arguments and on the basis of a precise reading of the context and the status of the unborn child in Roman law in general, Waldstein concludes that muilieris portio means ‘the interest of the mother ‘and that viscera should be translate as ‘the most beloved of someone ‘, own child or ‘part of the family of someone ‘. This implies that before birth the child belongs to the sphere of interest of the mother and that before birth the father has almost no right over the child.19 The discussion about the status of a pre-implantation embryo concerns, first and foremost, the ethical problems of in vitro experimentation on an embryo that has been created by artificial fertilization and thus is not a part of its mother. The biological data provided by modern science, however, have made clear that the embryo, beginning with conception, has its own existence. It relies on its mother for food, liquids and the expulsion of organic matter. However, its development and its growth as an individual are guided from conception onwards by its own genome, which is different from the genome of its mother. For this reason, on the basis of contemporary genetic knowledge, one can in no way argue that an unborn child is a part of the body of the mother. On the basis of this argument it is not admissible for the mother to claim the right to dispose of the life of the embryo. Human biological nature: the embryo is a human individual because of the simple fact that it is biologically a human being The Wilkes based their rejection of procured abortion on the fact that human life, from a biological point of view, begins with conception. For them, theology and philosophy were of no use in solving the question of the status of the embryo given that in both disciplines there exist many divergent opinions on the subject.20 The biological definition of the beginning of life, that is to say conception, which cannot be called into doubt by anyone, was, in the view of the Wilkes, thus the most solid criterion by which to attribute a moral status to the human embryo starting with conception. This conclusion, however much it may be held to be interesting by the pro-life movements, meets certain objections that are insuperable. It does not take into account the fact, for example, that, as will be seen below, many modern ethicists make a distinction between human beings in a strictly biological sense and human persons. The embryological and biological facts, in themselves, interpreted in various ways in the various visions of man, cannot provide a definitive answer about the status of the embryo. According to certain visions of man it is to be excluded that the embryo is a human being from conception. In addition, a purely biological definition would lead to a biologistic and materialistic conception of man which holds that man cannot have an intrinsic dignity but at the very most an instrumental value. An appeal to the mere biological presence of a human being, leaving aside other aspects such as the spiritual dimension and its intrinsic finality, is insufficient, as we will see below. 131 Individuality: the embryo becomes a human individual only from the moment at which it cannot divide itself and thus give life to a twin or unite itself to another embryo In England in 1990, on the Recommendation of the Warnock Commission, a law was passed that allowed experiments on in vitro embryos on certain conditions until the fourteenth day after conception. In its report, which was published in 1984, this Commission concluded that the early embryo, because it still had the possibility of dividing, could not be considered as being an individual being and thus could not be considered as being a human individual either.21 Even some Catholic ethicists have adopted this approach.22 The moralist Häring has stated that: ‘The greatest objection to the theory of animation at the moment of fertilization is raised by the phenomenon of identical twins ‘.23 The Warnock Commission held that the beginning of the individuality of the embryo was the moment of the formation of the and assumed that before this (but not afterwards) the embryo is able to divide into two individuals that are genetically identical. The primitive streak is the oblong concentration of cells at each end of the embryonic disk which emerges on the fourteenth or fifteenth day after conception. It is a manifestation of the anteroposterior axis of the embryo and appears in the place where the nerve tube will develop after a short period of time, and from which the brain and the spine will form. In this place a number of strata of differentiated cells form after their migration. At the most two primitive streaks can form in the but because of the differentiation that has just begun this will not lead to the division of the embryo. This period of two weeks coincides roughly with the period before the implantation of the embryo in the mucous of the embryo, which is completed between the eleventh and thirteenth day after conception. In relation to this period, reference is often made to a pre-embryo, a term that suggests that the embryo is not yet a human individual and thus does not deserve to be respected as such.24 This line of reasoning has its origin in the presumption that the embryo is not an individual as long as the possibility of scission exists and thus cannot even be considered a person because a person is the most complete individual being. A first problem is whether it is really certain that monozygotic twinning is possible until the moment of the formation of the primitive streak. In the textbooks on embryology on the whole three stages are indicated where twinning can occur: in relation to the stages of the formation of the extra-embryo membranes, that is to say of the trophoblast, of the amnion, and of the chorion.25 When twinning takes place before the formation of the trophoblast, which takes place on the fifth day, each twin has its own amnion and its own chorion (and thus its own placenta). It is thought that this is the cause of twinning in more or less one-third of cases. After the formation of the trophoblast but before the formation of the amnion, which takes place on the ninth day, the twinning involves a division of the internal cell mass where two amnions and a chorion shared by both the embryos (and one placenta) come to be formed. This is said to the be the cause of twinning in more or less two thirds of cases. Very rarely a division takes place after the formation of the amnion, that is to say within the embryonic disc, finally before the appearance of the primitive streak. In this case the twins come to have a single amnion and single chorion (and a single placenta). It should be observed that this analysis is based upon a morphological theory based upon a deduction from the arrangement of the placenta and the membranes of the foetus visible during the foetal stage and during birth. The examples of embryos to support this theory are rare.26 Whatever the case, there is no direct observation of twinning through the division of the embryonic disc. There is also an absence of images of the early stages of this kind of twinning with normal embryos. In 1955 Corner showed a photograph of two monster embryos (a part of the Carnegie collection) of an age of 28 to 30 days to be found together within a single chorion which was presented as ‘absolute proof that twinning can take place after the amniotic cavity has been formed ‘.27 However, Corner himself states that it is

132 not possibly to exclude totally that the amniotic membranes and the chorion-membranes can fuse with each other, thereby producing pseudo-mono-amniotic or pseudo-mono-chorianic twins.28 Contemporary textbooks of embryology also state that the idea of twinning based upon the formation of the various different membranes remains a theory. Rahilly and Müller refer to the ‘presumed way in which monozygotic twins develop in the human being ‘.29 One should ask whether it is justifiable to base a conclusion with very major conclusions for the respect due to the embryo before implantation, that is to say that experiments with embryos are legitimate up to fourteen days after fertilization, on a supposition for which there is no scientific evidence. The fundamental question is: does the possibility of the separation of the embryo really exclude its individual being and thus its being a person? There is another interpretation that is possible, that is to say that man is able to procreate in an asexual way until the formation of the primitive streak. When I, when digging in the garden, cut a worm in two, both the parts of the worm carry on in their own way in an apparently undisturbed fashion. It appears a rather unattractive thought that something of the same kind can happen in man, but who can prove that the contrary is the case? That asexual procreation is possible in man as well would appear to be demonstrated, or so assert Ashley and O’Rourke in the third edition of their textbook Health Care Ethics, by scientists who will be able in time to clone human adults, who are without doubt seen as persons, through nuclear transplant. 30 A further argument that is said to exclude that the embryo during the first stages of its development is an individual focuses on the possibility of the recombination of embryos. In experiments with animals it has been demon- strated that it is possible to combine two31 or at the most three embryos32 into a single embryo that contains genetically different cells which come from the original embryos. The discovery at the end of the 1960s of the existence of men with cells with a twofold chromosome X and cells with one chromosome X and one chromosome Y is seen as an indication that recombination also takes place in human embryos.33 From a scientific point of view this phenomenon, however, could also be explained by a colonization of cells from a dizygotic twin or from the mother in the body of the individual involved during intrauterine development. However, even if such individuals were really the result of a recombination of two embryos, this phenomenon would not constitute proof that the embryo during its first stages is not an individual. One could also well object that in the case of the recombination of the two embryos the body of one is absorbed by the other, which manages to conserve its individuality, and thus that the first embryo cease to exist as an individual and dies from a metaphysical point of view. Many people see proof of the individuality of the early embryo in the fact that the composition of the genetic material of the chromosomes is established at the moment of fertilization. Others object that the development programme that the chromosomes contain is not immediately active after conception. At the outset the energy in the embryo is provided by the Altmann’s granules, which come from the mother. Thus the development of the very first stages of the embryo is not guided by the DNA of the zygote but by the DNA of the Altmann’s granules which come from the mother, from the messenger RNA and from the proteins that were present in the spermatozoon and the ovule.34 This, too, is not in itself a valid reason for doubting the individuality of the embryo. The development programme in the chromosomes, although it becomes active immediately or after only a few days, is established from conception onwards and will guide or regulate the successive development of the embryo if factors of disturbance do not intervene. The results of the most recent research indicate, however, that the DNA of the embryo begins to guide its development practically at the zygote stage, when a first gene that is responsible for gonadic differentiation is already active.35 The criterion for the individuality of the embryo is often likened to the criterion which observes that the embryo will become a person. This point will be discussed later in this paper. This implies that the embryo cannot be considered a person until it is an individual, an assumption that in itself is obviously

133 right. Thus it is that Ford identifies the formation of the primitive streak with the moment of animation.36 The person being: the embryo becomes a human individual when it becomes a person The question whether the embryo is a person or not seems to be a clear and simple one. If it is a person, it deserves respect as such. If it were only a preembryo or a potential person, it would have less rights. However, the moment when the embryo becomes a person is very much debated. It depends first of all on the vision of man that one takes as a point of departure. In addition, even when there is a single vision of man there can be different ideas about the moment when the human embryo has to be seen as a person.

The criterion of animation Until the recent past in the Catholic world the discussion about the moment when the human embryo becomes a person was connected with the moment of animation. For that matter, the traditional Christian vision of man, too, defending both direct animation and indirect or delayed animation, did not provide a definitive answer to the question as to when the embryo becomes a person. The theory of direct animation which implies that the embryo is animated by a human soul from conception onwards has its origin in the writings of Hippocrates. In the view of Hippocrates the embryo was born from the sperm of the father which coagulated in the womb. The blood that was there, not secreted during pregnancy as it is during , was used by the embryo to nourish itself.37 The embryo was a human individual from the very beginning and thus had a human soul. The opposing theory was that of indirect or delayed animation espoused by Aristotle. In his thought the body of the embryo arose from the menstrual blood retained in the womb during pregnancy. This blood, understood as the material cause of the embryo, in Aristotle’s view, was coagulated by the sperm as an efficient and formal cause, like milk under the influence of fig juice or the curd of cheese.38 Thus the blood was transformed into the body of the embryo. Through the sperm the menstrual blood received a vegetative soul: thus the physical part, the body, comes from the woman and the soul from the man.39 In this way, at the end of the first week, the blood became a living being, comparable to a plant. The vegetative soul was replaced a little time afterwards by a sensitive soul, and this was borne out by the formation of sense organs. This soul was in turn replaced by a rational soul which came from outside and had to have a divine origin.40 The rational soul could not be present from the outset because its activity required a certain level of development of the organs, especially the sense organs: ‘The soul is, therefore, the first act (perfection) of a body that has a life potentially. The body is such when it possesses organsâ?¦If we want to mention something common to every soul, it is that the soul is the first act of natural bodies that possess organs ‘.41 On the basis of his observations of aborted embryos, Aristotle concluded that the male embryo was animated by the rational principle of life on the fortieth day and the female embryo on the eightieth day.42 The choice between the theory of indirect animation or the theory of direct animation was clearly determined by a difference at the level of the vision of the development of the embryo. Differently from Hippocrates, Aristotle, thinking that the body of the embryo arose from the menstrual blood, could not assume that the embryo was animated starting with conception. His belief that only an organic body could be animated made it unthinkable, in fact, that an amorphous piece of blood contained a human soul as a principle of life. Here Thomas Aquinas followed Aristotle, although not without modifications and additions to Aristotle’s thesis.43 Until the last century there were still Thomists who supported the theory of delayed animation on the basis of the requisite of what they called a sufficient arrangement of matter to be animated by a rational soul, as is stated in number 15 of the twenty-four Thomist theses published by the Sacred Congregation of Studies on 27 July 1914: ‘On the contrary, the human soul exists on its own and when it can be infused in a subject sufficiently disposed, it is created by God, and by its nature it is incorruptible and immortal ‘.44

134 In 1827 Karl-Ernst Von Baer discovered the ovule in mammals and in man and also the mechanism of fertilization, as a result of which it was definitively proved that the human body does not begin as a coagulate of blood but as a fertilized ovule. For the majority of theologians this was the reason to believe that animation took place at the moment of conception and not later.45 However in order to defend procured abortion, theologians, moralists and ethicists took up the theory of delayed animation again from the 1960s onwards.46 And given that today researchers have to deal with a fertilized ovule, with an embryo brought about by in vitro fertilization, the theory of delayed animation also acts to justify experiments on embryos. To support this theory, on the one hand reliance was placed on an Aristotelian argument according to which animation requires a certain development of the sensorial system: ‘The minimum that we should suppose before admitting the presence of a human soul is the availability of these organs: the senses, the nervous system, the brain and especially the cortex. Given that these organs are not yet mature during the very first stages of pregnancy, I think it is certain that a human person only exists after a few weeks ‘.47 On the other hand, theologians and ethicists often referred ‘and they still refer ‘to certain scientific discoveries in the field of embryology that were made last century: 1) the spontaneous loss of fertilized ovules to a notable degree; 2) the formation of monozygotic twins; and 3) the possibility of recombining two or three embryos into a single individual. This paper has already discussed the last two phenomena, but not the first. On the basis of experimental observations, in the 1920s and 1930s Needham postulated that up to 50% of fertilized ovules were lost spontaneously. For many people this makes it improbable that the fertilized ovule is already animated.48 This, in fact, would mean that one half of human persons with a soul created directly by God are lost during the first weeks to the first months of pregnancy. For that matter, this objection is not new but goes back to Augustine49 and to Anselm.50 For them it was unthinkable that a conceived human at the very first stages of development was already animated because this would mean that such individuals had the possibility of being reconciled with God through baptism. This argument as such, however, is not necessarily in contrast with direct animation. The high mortality rate of children, which until the nineteenth century was around 50%, does not constitute an argument by which to call into question the fact that they are persons.51 From the moment when it is animated the embryo becomes a person and thus attains the highest level of the human individual. The division of the embryo into twins, which is possible until the formation of the primitive streak at the fourteenth or fifteenth day after conception would, according to the Warnock Report, as we have seen, prove that the embryo during the first stages is not an individual and thus not even a person. The moral philosopher Norman Ford concludes, therefore, that animation can only take place after the formation of the primitive streak.52 We have observed, however, that the possibility of twinning, or of recombination with other embryos, does not exclude the early embryo being a human individual.

The criterion of the manifestation of activity that is specifically human In contemporary secular bioethics, discussion about the status of the embryo is shaped above all else by the anthropology of identity theory. This theory, which originated in Australia and which is accepted by many ethicists in the Anglo-Saxon world and ‘albeit unconsciously ‘by many medical doctors as well, is characterized by a strong dualism which separates the biological nature of man and the specific functions that render him a person. That which is specifically human is psychological consciousness, the rational faculty and the capacity for social communication. It is clear that in this vision the embryo could never be a person before a certain development of its nervous system. Tauer thinks that when the nervous system has developed to the point of registering certain experiences that come from the environment the embryo has matured a mental personality which draws the embryo near to being a person in the strict sense. These experiences can be unconscious but as we know from 135 psychoanalysis they can already lead to the formation of memories that act subsequently on the consciousness. On the basis of this, Tauer thinks that there are sufficient reasons for attributing to the embryo in the seventh week not only a moral value but also the beginning of being a person in a morally significant sense.53 Others, such as McMahan, believe, instead, that the embryo becomes a human being at a subsequent moment: ‘I believe that the most credible view is that we are embodied mindsâ?¦I began to exist when the brain of this body ‘my body ‘acquired for the first time the ability to have awareness ‘.54 This implies that the human being begins his or her existence between the twentyeighth and the thirtieth week. Engelhardt, on the other hand, in order to be able to speak about a per son requires the actual presence of self-awareness, a manifest rational activity and a manifest capacity for social communication. Given that such functions are probably present only a notable time after birth, the unborn and the newly born ‘including mentally handicapped people who have never had a rational capacity ‘are said not to be human persons to the full with the accompanying connected moral status. Before being persons they are said to be only human beings in the biological sense.55 This demonstrates all the more the urgent need for careful anthropological reflection on the human biological nature of the early embryo. This vision has various practical consequences for other fields of medical ethics as well. If applied strictly, a patient in a permanent vegetative state could no longer be seen as a person. And some have suggested that he or she could thus be seen as a donor of organs.56 A fundamental objection to identity theory is that it encounters difficulty in explaining the human person as a unity. The human being is considered in antithesis to the human person, like biological nature and spiritual nature, that is to say the rational capacity.

The intrinsic finality: the embryo, even were it not yet a human individual, must be respected as such because of its intrinsic capacity to become that individual Assuming that the early embryo is not a human individual, would it not be obvious to conclude that its elimination by means of procured abortion and its use in research or in therapeutic cloning are licit, specifically because of the fact that they do not involve the killing of a human individual? McMahan’s view is that this would not amount to the killing of one of us but only the prevention of their existence.57 As an argument in favour of the licit character of procured abortion or experiments with embryos, reference is made to the fact that the Christian tradition preferred the theory of delayed animation until the nineteenth century.58 This raises the question why Christian theologians, although accepting this theory, unanimously rejected abortion ‘until the second half of the century ‘even when it took place before the assumed moment of animation. Here the famous text of Tertullian is indicative: ‘Given that killing is always forbidden, the destruction of a foetus during the period in which the blood is transformed into a human being is also illicit. The prevention of birth is the same as early killing; it makes no difference whether one kills life already born or interrupts life already on its way to birth and being developed: he who will be a man is already a man, just as the fruit is already in the seed ‘.59 By the transformation of blood Tertullian was alluding to conception as understood by Aristotle, in whose view the blood that was in the womb was not expelled during pregnancy, as was the case in normal periods of menstruation, but remained there and was transformed within the body into an embryo under the influence of the active force of the male semen. When this process was still under way, Tertullian affirmed, there was something in the womb that should be respected as a human individual, at least because it would become a human individual. As an argument to strengthen this thesis it was added that every fruit is already virtually present in the seed. The fundamental argument in this text is that the process of the development of the embryo takes place in a way that has a purpose. In the conceived human being, and above all in the semen, there is the intrinsic finality of becoming a human individual. From this springs the need for respect. In his commentary on the Gospel According to St. Luke, Ambrose says that ‘to check your levity you 136 recognize the hands of your author who forms a man in the womb. He is working and you violate with your lasciviousness the secret of the sacred womb? ‘.60 Here one is not dealing with abortion. Ambrose seems to state that unchecked sexual passions lead to sterility. Whatever the case, he teaches us that the formation of the embryo in the plan of the creative action of God is a process with a finality. We can find the same thought in St. Augustine: ‘And yet in all men who are born ill, God, in forming the body, in giving them life and nourishing them, does that which is good ‘.61 He is not thinking of a direct intervention on the part of God upon the biological development of the embryo but of a transcendental causality that includes the direct biological causes.62 The same finality linked to the doctrine of the creation is evident in the way in which St. Thomas Aquinas describes the origin of man.63 The prevention of procreation has been seen by Christian theologians as a rejection of the fulfilment of a purpose of marriage which is established in the order of the Creation. And it was on the basis of this thought that the Fathers of the Church and medieval theologians placed on the same level the use of means to bring about sterilization (contraceptives), the killing of a (both an animated and not yet animated) foetus, and infanticide: ‘However they betray themselves when they reach the point of exposing their own children born against their will. They hate raising and keeping near to themselves those children that they feared to generate. When, therefore, dark inequity becomes cruel towards their own children, generated against their wishes, a clear inequity is brought to light and a secrete turpitude is bared by a manifest cruelty. At times, this voluptuous cruelty, or, if one wants, this cruel voluptuousness, is pushed to the point of obtaining contraceptive substances and in the case of failure to the killing in some way in the womb of the conceived foetuses and their expulsion, with the desire that their own child perishes before living or, when it is already living in the womb, that it is killed before being born. There can be no doubt: if both of them are of the same stamp, they are not spouses; and if they behave like that from the outset they do not unite in marriage but in lustfulness. If then it is not both of them who behave like this, I would venture to say that either she in a certain sense is the prostitute of the husband or he is the adulterer of the wife ‘.64 Although in the view of these theologians abortion before animation could not be held to be murder, nonetheless they refer to an illicit intervention because this violates the intrinsic finality of the embryo to reach the moment of animation. At the most in certain circumstances the abortion of the foetus, seen as being inanimate, is assessed in a less severe way65 or, in a case where the life of the mother is in danger, it is explicitly allowed.66 The return of the theory of delayed animation amongst Christian theologians with the passing of the centuries does not in any way, however, support the conclusion which holds that on the basis of Christian tradition abortion or the elimination of embryos for research purposes is legitimate. This tradition also attributed to the inanimate embryo a moral status and a connected dignity because of its intrinsic finality. According to contemporary biology, this is to be found in the development programme that is carried out under the guidance of the chromosomes, whose composition is established since conception. If one wanted to use one element from tradition, why are the other elements of tradition neglected, elements that are compatible with the data of contemporary embryology?

Assessment Which intrinsic criterion and which basic anthropology should we take as a point of departure in considering the embryo during the first week after conception? From what has been observed hitherto in this paper it emerges that the embryo during the first seven days of its life is a being with its own life that is separate from the life of the mother; a human being from a biological point of view; an individual and a being with an intrinsic finality. However, can we also conclude that the embryo before implantation is a human individual or a human person? In his assessment of the status of the embryo in his Encyclical Evangelium Vitae John Paul II, avoiding declaring expressly that the moment of animation coincides with conception, refers to the 137 conclusions of modern biological science with a rhetorical question: ‘Even if the presence of a spiritual soul cannot be ascertained by empirical data, the results themselves of research on the human embryo provide a valuable indication for discerning by the use of reason a personal presence at the moment of the first appearance of human life: how could a human individual not be a human person? ‘(Donum Vitae,67 n. 1; Evangelium Vitae, n. 60). Taking as a point of departure contemporary knowledge of embryology and above all of modern genetics, how can one not identify, by the use of reason, the early embryo with the human individual or with the human person?

THE IDENTIFICATION OF THE EARLY EMBRYO WITH THE HUMAN INDIVIDUAL OR THE HUMAN PERSON A solely materialistic explanation, such as that which typifies identity theory, on the specific functions of the human mind, is insufficient. The process of thinking, which is developed with abstract ideas, although dependent upon sensorial information, is in the final analysis an immaterial function. The same may be said of freedom: material processes, like chemical processes, which unfold according to a pre-determined model, do not explain freedom. Without a spiritual dimension, human freedom would not exist. Both man’s capacity for reason and his freedom pre-suppose that in him there is a spiritual principle of life. To be a human individual or a human person, the embryo must have both a spiritual dimension and a physical dimension. However, the presence of a spiritual dimension cannot be demonstrated through the method of research of the positive sciences. In an empirical way the spiritual dimension is ascertained only in the actualized capacity to perform functions that have in the final analysis their origin in the spirit of man. In the embryo before implantation and after conception, manifest signs of a spiritual dimension are lacking. The process of thinking and the process of willing are functions in which both the spiritual dimension and the corporeal dimension of man have their own role, but in an integrated way. The content of rational consciousness is the symbols that derive from the sensorial experience of the environment and a person’s own body. The fact that this content is absent in the early embryo because of the fact that the sensorial organs are not sufficiently developed does not in itself exclude the possibility that the capacity to think and to will are already present in potential terms, a potential that will be gradually actualized in a way that is proportionate to the development of the senses. Indeed, I will attempt to demonstrate that it is difficult to think that the spiritual dimension is not present from the moment when the embryo manifests itself as a human being in a biological sense, that is to say from conception. To return to the embryo before implantation, we must ask ourselves the following question: can we identify a being whose human biological nature alone is observed as a human individual or human person or not? According to the passage from the Encyclical Evangelium Vitae quoted above, the contemporary knowledge of embryology and of genetics can provide a ‘valuable indication for discerning by the use of reason a personal presence at the moment of the first appearance of human life ‘(Evangelium Vitae, n. 60). How can these sciences be useful in discovering a personal presence in the human embryo from conception? Whatever the case, the contemporary knowledge of embryology contradicts the classic notion in opposition to direct animation (and thus humanization), a notion according to which the human embryo is said to begin its development as a coagulate of blood, that is to say as a non-living being, and thus a being that is not animated. Contemporary embryology confirms the view which holds that the human embryo, from conception, is a biologically human living being. To this should be added the fact that from conception the development of the embryo takes place in an autonomous, co-ordinated, continuous and gradual way.68 There are no caesuras in the successive process of development as there could be if there intervened during the course of the development of the embryo another mechanism of co-ordination or integration of the life of the embryo, to be interpreted as a moment when the embryo truly becomes a human individual by receiving a spiritual dimension. 138 Genetics has discovered the mechanism of this development of the embryo: beginning with conception the embryo is guided by the genome, the conception constitutes the result of the fusion of the chromosomes of the ovule with the chromosomes of the spermatozoon. Knowing that the genome is the most important foundation of the biological identity of a human being, we can ask ourselves what indications genetics can provide, in addition to the indications provided by embryology, to hold that the human embryo is a human person from fertilization. To answer this question it is necessary to know what the ontological identity of the human person is. The ontological identity of man, on the basis of the classic definition of the person of Boethius naturae rationalis individual substantia,69 which was taken up by St. Thomas Aquinas,70 implies in a certain sense a dual identity, that is to say a generic identity, rational nature, and a numerical identity, given that generic nature does not exist in itself but in relation to an individual substance.71 In reading in a newspaper of the death of ten people because of a train crash I come to know about the generic character, the specific identity, of the victims, that is to say that they are human beings. In fact, however, they are individuals who, in having their own numeric identity, actualize this generic identity in a different way. In essential terms, the numeric identity means that this concrete individual with his or her own characteristics is a human person, independently of the state of his or her development, of his or her physical perfection, of his or her success or his or her defects. The person is the most complete individual being: individual character, therefore, belongs essentially to the person. This means that the numeric identity is an intrinsic dimension of the human person. The question is: what role does the genome, which is present from conception, play in the definition of the generic identity and the numeric identity of the human individual? The genome is the deepest biological foundation of the body. Given that a human being is substantial unity of spirit and body, the genome has an intrinsic role as regards the generic identity. Given that the intrinsic identity, too, is an intrinsic aspect of the human person, an equally fascinating subject is what the role of the genome is as regards the numeric identity. One aspect of the numeric identity is that intellectual capacity can differ notably between the various individuals of the human species. What explains this difference? Should one suppose that the various human individuals do not have the same spiritual dimension? Taking as a premise that the human spirit (soul) is the substantial (or better subsistent) form of the human individual,72 a human spirit that differed amongst the members of the human species would have as its consequence that these members would not have the same generic identity. Whatever the case, the capacity to think employing abstract concepts and the capacity to act freely are not in themselves different from human being to human being. The difference in intellectual capacity is explained with reference to the differences in the neuronal networks of the brain as a result of which the capacity to compute sensorial data can vary notably. In the final analysis the difference lies not in the capacity to think employing abstract concepts in itself but in the disposition of the material dimension of the human individual. The material dimension, therefore, is of determining importance for the numeric identity.73 Now, we know that the neuronal networks are present in a virtual sense in the genome (even though other environmental factors also probably have their role in the anatomical and functional development of the brain). The DNA contains the biological basis of all the features that characterize the human being from conception until death. Given that the numeric identity as a material basic dimension and as an intrinsic dimensions constitutes ‘together with the generic identity of man as a being formed of spirit and body ‘the ontological identity of a human being, it is difficult to think that the formal principle, the spiritual dimension, is not yet present at the moment of conception, whereas there is, obviously, the material dimension.74 This argument also provides an answer to identity theory which refers to a human person only in the presence of a manifest rational consciousness. In the emergence of the numeric identity the development of rational consciousness plays a special role although not an isolated one. In this the spiritual dimension and the material dimension have an intrinsic role given that the intellectual capacity is realized through an integrated function of the capacity to think employ- ing 139 abstract concepts, on the one hand, and of the capacity of the neuronal networks of the brain to compute sensorial data, on the other. For this reason it is difficult to think that the spiritual dimension is not present in a state that is so fundamental for the development of the numeric identity of the human individual. One understands that during the first week after conception the embryo does not yet possess these neuronal networks given that the nervous system begins its development from the twenty-first day onwards. However, all the neuronal structures are already present in a virtual sense in the DNA from conception, including their contribution in a biological sense to the numeric identity of the human individual. An objection of Lanza and Donceel to this argument is that this implies a coincidence of a formal causality with an efficient causality of the spiritual dimension: the spiritual dimension, if present since conception, would be both the formal cause and the efficient cause of the human body.75 The formal cause cannot be the efficient cause of the generation of the thing of which it is the substantial form. Here it is useful to distinguish between generation and growth.76 Development subsequent to the moment of the beginning of existence is different from generation it is growth. Growth is a process of a living being that has already been generated. The spiritual dimension, once the human body has been formed, is the moving principle, that is to say the efficient cause, of life. It is the root of all the processes of life, including the process of growth of the embryo. We have seen, amongst other things, that Lanza and Donceel think that a certain development of the neuronal structures and of the brain is necessary to reach a disposition on the part of the embryo of such a kind that the spiritual dimension can inform it with its existence. However, the early brain, although it demonstrates at a macroscopic level and perhaps also at a microscopic level a certain resemblance with the adult brain and has some functional activities, still has to undergo a large number of macroscopic, microscopic and biochemical changes to make possible the complex electrochemical processes that are required for the thinking process. Even after the seventh weeks, the moment starting with which, in the view of Tauer, the embryo, after engaging in experiences of the environment which can influence successive consciousness, acquires a psychic personality, the brain has still not yet completed its development. The fact that experi- ences during that stage are still ones of which the embryo is unaware does not exclude a contemporary spiritual state. Moreover a human being who certainly has spiritual dimension, given the manifest rational consciousness, can have experiences of which he or she is unaware which influence the development of his or her identity or whose meaning in relation to the creation of identity emerge only subsequently. In the light of current medical knowledge it is rather risky, therefore, to argue that a certain level of the development of the brain or the neuronal structures is necessary in order to be able to state that a human being has a spiritual dimension.

CONCLUSION In the assessment of the various and different criteria that are employed to assess the status of the human embryo it is of essential relevance that this embryo has a biologically human status from conception. The embryo is a living being whose development, guided by the genome that is present and active from conception onwards, takes place in an autonomous, co-ordinated, continuous and gradual way. An indirect humanization is hardly compatible with the fact that the specific identity of a human being is intrinsically made up both of the spiritual dimension and the material dimension, above all given that the biological foundation of the physical dimension, the DNA, is present from conception onwards. In addition, the DNA is the biological basis of the numeric identity, which is also an intrinsic element of the human individual. The fact that the numeric identity constitutes, together with the generic identity, the ontological identity of the human individual is at times difficult to reconcile with the thesis of indirect or gradual humanization. Although it is impossible to demonstrate empirically a personal presence from conception onwards, philosophical reflection on the bio-anthropological status of the human embryo indicates an incongruence of indirect or gradual humanization with the vision of the human individual as a substantial unity of spirit and body. 140

1 This is what the American Nobel prize winner, Harold Varmus, asserts. In his view one cannot provide an answer to the question as to the moment when a human being begins to exist. He agrees with the idea proposed by those who see the embryo as a human from the moment the neurons begin to develop, the blood begins to circulate, and the embryo can then survive outside the womb. ‘One can state that there is full individuality only after birth ‘, see the interview with him in: VARMUSS H., Ich sehe eine moralische Pflicht zum Embryoverbrauch, Frankfurter Allgemeine Zeitung 2001, p. 43. 2 KAMER T., Vergaderjaar 2000-2001, 27(5): 4-6. This assessment of the status of the embryo is also very widespread in the Protestant world, see: HONECKER M., Divergenzen in der evangelischen Ethik beim Untergang mit Embryonen, Zeitschrift für Medizinische Ethik 2003, 49(2): 123-136, especially p. 127. 3 CENTRE CATHOLIQUE DES MÃ?DECINS FRANÃ?AIS, Avortement et respect de la vie humaine, Paris: Editions du Seuil, 1972: 93-104; pp. 174-184; pp. 194-204. 4 BÃ?CKLE F., Um den Beginn des Lebens, Arzt und Christ 1968, 14: 70; SPORKEN P., Voorlopige diagnose. Inleiding tot een medische ethiek, Utrecht: Ambo, 1969: 68-69 (Sporken also uses other arguments to indicate that nidation is the initial moment of human life: the possibility of the division of embryos which then gives rise to twins; and the large loss of embryos prior to nidation ‘these are subjects to which I will return later in this paper. In his book: ID., Ethiek en Gezondheidszorg, Baarn: Ambo, 1977: 118 he moderates this idea and argues that nidation is a first, albeit fundamental, step, in the gradual process of the hominisation of the embryo side by side with the stage of the differentiation of the neurons of the brain. 5 BÃ?CKLE, Um den Beginn des Lebensâ?¦; SPORKEN, Voorlopige diagnose. Inleidingâ?¦, pp. 94- 97; ID., Ethiek en Gezondheidszorgâ?¦, pp. 154-158. 6 MARKTL H., Von Caesar lernen heiÃ?t forschen lernen, Frankfurter Allgemeine Zeitung 2001, p. 52. 7 The Dutch Rabbi Evers, for example, is convinced that the human embryo, on the basis of this particular translation of the text of Genesis 9,6, when outside the womb does not deserve to be protected: ‘Analyzing the text one reads only whoever sheds the blood of man in man, his blood will be shed. The obligation to protect life is subordinated to staying in the maternal womb ‘. See EVERS R., EVERS A.P., Bijbel positief over klonen van embryo’s, Trouw 2004, p. 14. The authors see the embryo before forty days after conception as inanimate life. In vitro embryos, because they cannot go on living without artificial aid, are thus said not to be living humans. As regards the experimental use of in vitro embryos being inanimate is not, however, important ‘also embryos within the maternal womb are not yet animated. What is important is the translation of Genesis 9,6 in which the authors see a law that attributes the right to protection only to the embryo that is in the maternal womb. 8 Taken from Bibbia di Gerusalemme, Bologna: Edizioni Dehoniane, 1996. 9 EIJK W.J., Therapeutisch kloneren nog problematischer dan reproductief kloneren: een bijdrage vanuit katholiek-bijbels perpectief, Pro Vita Humanae 2005, 12(2): 47-53. 10 CONNERY J., Abortion: the Development of the Roman Catholic Perspective, Chicago: Loyola University Press, 1977: 13. 11 Gen 6: 3; 9: 6; Ex 8: 13, 14; 9: 10; 13: 2; Num 8: 17; 17, 15; 31: 11, 26; Lev. 24: 20; 2 Sam 23: 3; Jer. 32: 20; 9: 15; Mic 7: 2; Ps 68, 18; 78, 60; 118, 8; Qo 2: 24. On this point I have consulted J. Liesen, professor of Exegesis at the Higher Seminary of Rolduc and member of the International Theological Commission who bases his reply on EBEN-SHOSHAN A., Qonqordantsia chadasha, Jerusalem: Kiryat-Sefer, 1986. 12 EIJK W.J., Embryo en christelijke mensvisie: wanneer wordt het embryo een menselijke persoon?, Pro Vita Humana 1994, 1(3): 107-116.

141 13 TAUER C.A., Personhood and Human Embryos and Fetuses, The Journal of Medicine and Philosophy 1985, 10: 264. 14 Ibid., pp. 263-264. 15 This conclusion is based upon the seven intrinsic and relational criteria advanced by Warren to indicate the moral status of every being: the living being should not be killed without good reason; a feeling being should not be treated cruelly; moral agents have full and equal rights to life and freedom; human beings who can feel but not act have the same moral rights as those human beings who act morally; ecologically important (living or non-living) entities have a stronger moral status than the status they would have if they were independent of the eco-system; animals that are a part of human community have a stronger moral status than the status they would have on their own; and within the framework of the first six criteria moral agents must respect the recognition of moral status by others (transitivity of respect), see: WARREN M.A., Moral Status: Obligations to Persons and other Living Things, Oxford: University Press, 1997: 148-177. 16 MEYER M.J., NELSON L.J., Respecting what we Destroy. Reflections on Human Embryo Research, The Hastings Center Report 2001, 31(1): 16-23. 17 CONNERY, Abortion: the development of theâ?¦, pp. 22-23. 18 ULPIAN, Digesta, 25, 4, 1, 1. 19 WALDSTEIN W., Ulpian and the Legal Position of the Unborn Child in Roman Law, communication during the Plenary Session of the Pontifical Academy for Life, 2006. 20 WILKE J.,WILKE B., Abortion: Questions and Answers, Cincinnati: Hayes Publishing Company, 1988: 5-6. 21 The Warnock Report, nn. 11.5 and 11.22, in WARNOCK M., A Question of Life. The Warnock Report on Human Fertilization and Embryology, Oxford: Basil Blackwell, 1985: 59; p. 66. 22 FORD N.M., When Did I Begin?, Cambridge: University Press, 1988; SHANNON T.A., WOLTER A.B., Reflections on the Moral Status of the Pre-Embryo, Theological Studies 1990, 51: 612-614; CAHILL L.S., The Embryo and the Fetus: New Moral Contexts, Theological Studies 1993, 54: 127- 130. 23 HÃ?RING B., Medical Ethics, Middlegreen: St. Paul Publications, 1991: 73. 24 Some are of the opinion that the term pre-embryo does not suggest this because according to classic embryology one can speak of an embryo only after its implantation in the mucous of the womb. Before that moment one should speak about blastogenesis and subsequently of the genesis of the embryo. See: DE WERT G.M.W.R., GERAEDTS J.P.M., IVF, pre-embryo research en ethick, Metamedica 1988, 67: 106-133, especially the footnote on page 1. However, the term pre-embryo was never used in classic embryology and has been introduced recently. 25 RAHILLY R.O., MÃ?LLER F., Developmental Stages in Human Embryos, Washington: Carnegie Institute of Washington, 1987: 13-26, in particular figure 5.2 on p. 26; GILBERT S.F., Developmental Biology, Sunderland: Sinauer Associates, 2000: 362-363; CARLSON B.M., Human Embryology and Developmental Biology, Philadelphia: Mosby, 2004: 53. 26 CORNER G.W., The Observed Embryology of Human Single-Ovum Twins and other Multiple Births, Am. J. Obstet. Gynecol 1995, 70: 933-51, in particular p. 934 (I am grateful to prof. Gonzalo Herranz for pointing out to me during the Meeting the lack of evidence for this theory). 27 Ibid., p. 943. 28 Ibid., pp. 946-947. 29 RAHILLY, MÃ?LLER, Developmental Stages in Humanâ?¦, p. 26; CARLSON, Human Embryology andâ?¦, p. 53. 30 ASHLEY B.M., O’ROURKE K.D., Health Care Ethics. A Theological Analysis, St. Louis: The Catholic Health Association of the United States, 1989: 212. 31 MINTZ B., Genetic Mosaicism in Adult Mice of Quadriparental Lineage, Science 1965, 148: 1232- 1233. 142 32 MARKERT C.L., PETTERS R.M., Manufactured Hexaparental Mice Show that Adults are Derived from their Embryonic Cells, Science 1978, 202: 56-58. 33 HELLEGERS A., Fetal Development, Theological Studies 1970, 31: 5; DE LA CHAPELLE A., SCHRÃ?EDER J., Early Fusion of Two Human Emrbyos?, Annals of Human Genetics 1974, 38: 63- 75; MAYR W.R., PAUSCH V., SCHNEDL W., Human Chimaera Detectable Only by Investigation of her Progeny, Nature 1979, 277: 210-211. 34 SHANNON, WOLTER, Reflections on the Moral Status ofâ?¦, p. 608. 35 PERGAMENT E., FIDDLER M., CHO N. ET AL., and Pre-Implantation Growth, Human Reproduction 1994, 9: 1730-1732; FIDDLER M., ABDEL-RAHMAN B., RAPPOLEE D.A. ET AL., Expression of SRY Transcripts in Pre-Implantation Human Embryos, American Journal of Medical Genetics 1995, 55: 80-84. 36 FORD, When Did I Begin?â?¦, pp. 170-177. 37 HIPPOCRATES, Du foetus de sept mois, vol. VIII, Paris: Littré, 1851: 492. 38 ARISTOTLE, The Generation of Animals, Cambridge: Harvard University Press, 1979: 95-111. 39 Ibid., II, IV, 738 b, pp. 184-185. 40 Ibid., II, III, 736 b, pp. 170-171. 41 ID., De Anima, II, I, 412 a. 27 ‘412 b 1, and 4-6, in SIWEK P., Aristotelis de anima, vol. II, Rome: Pontificia Università Gregoriana, 1954: 92-93. 42 ID., De animalibus historiae, VII, III, in ID., Opera omnia, vol. III, Paris, 1927: 137-138. 43 Thomas discusses this topic in several of his works: ST. THOMAS AQUINAS, Scriptum super libros sententiarum Petri Lombardi, 2, d. 18, q. 2, a. 3, Paris: Mandonnet, 1929; ID., De potentia, q. 3, ad 9, in ID., Quaestiones disputatae, vol. II, Turin-Rome: Marietti, 1965: ID., Summa Contra Gentiles, 2, 87-89, Romae: Apud Sedem Commissionis Leoninae, 1934; ID., Summa Theologica, 1, q. 76, a. 3, ad 3, and 1, q. 118, a. 2, ad 2; ID., De spiritualibus creaturis, a. 3, ad 12, in ID., Quaestiones disputatae, vol. II; ID., De anima, a. 11, inIbid. 44 SACRA STUDIORUM CONGREGATIO, Theses quaedam, in doctrina Sancti Thomae Aquinatis contentae, et a philosophiae magistris propositae, adprobantur, XV, AAS 1914, 6: 385: ‘Contra, per se subsistit anima humana, quae, cum subiecto sufficienter disposito potest infundi, a Deo creatur, et sua natura incorruptibilis est atque immortalis ‘, DENZIGER HEINRICH, Enchiridion Simbolorum, n. 3615; cf. HERING H.-M., De tempore animationis foetus humani, Angelicum 1951, 28: 18-29; LANZA A., La questione del momento in cui l’anima razionale è infusa nel corpo, Roma: Istituto Grafico Tiberino, 1939. 45 GURY J.P., Compendium theologiae moralis, Rome-Tourin, 1866: 431; GENICOT E., SALSMANS I., Institutiones theologiae moralis, vol. I, Leuven/Brussel, 1931: 375; PRÃ?MMER D.M., Manuale theologiae moralis, vol. II, Barcelona: Herder, 1945: 38. 46 DONCEEL J.F., Immediate Animation and Delayed Hominization, Theological Studies 1970, 31: 76-105. 47 Ibid., p. 101. 48 RAHNER K., The Problem of Genetic Manipulation, in Theological Investigations, vol. IX, London: Darton-Todd, 1981: 226, footnote on page 2; DONCEEL, Immediate Animation andâ?¦, pp. 99- 100; DIAMOND J.J., Abortion, Animation, and Biological Hominization, Theological Studies 1975, 36: 312-313. 49 See the footnote n. 64. 50 ST. ANSELM, De conceptu virginali, 7 (PL 158, 440): ‘Quod autem mox ab ipsa conceptione rationalem animam habeat, nullus humanus suscipit sensus. Sequitur enim ut quoties susceptum semen humanum, etiam ab ipso momento susceptionis perit antequam perveniat ad humanam figuram; toties damnetur in illo anima humana; quoniam non reconciliatur per Christum: quod est nimis absurdum ‘. 51 FORD, When Did I Begin?â?¦, pp. 180-181. 52 Ibid., pp. 170-177. 143 53 TAUER C.A., Personhood and Human Embryo and Fetuses, pp. 253-266. 54 MCMAHAN J., Cloning, Killing, and Identity, Journal of Medical Ethics 1999, 25(2): 77-86, quotation from p. 83. 55 ENGELHARDT H.T., Viability and the Use of the Fetus, in BONDENSON W.B., ID., (eds.), Abortion and the Status of the Fetus, Dordrecht: D. Reidel, 1983: 184-191; ENGELHARDT H.T., The Foundations of Bioethics, New York-Oxford: Oxford University Press, 1996: 135-140. 56 TRUOG R.D., FLETCHER J.C., Brain Death and the Anencephalic Newborn, Bioethics 1990, 4: 199-215. 57 MCMAHAN, Cloning, Killing, and Identityâ?¦, p. 83. 58 DUNSTAN G.R., The Human Embryo in the Western Moral Tradition, in DUNSTAN G.R., SELLER M.J. (eds.), The Status of the Human Embryo. Perspectives from Moral Tradition, London: King Edward’s Hospital Fund for London, 1950: 55. 59 TERTULLIAN, Apologeticus adversus gentes pro christianis, c. IX (PL 1, 319-320): ‘Nobis vero, homicidio semel interdicto, etiam conceptum utero, dum adhuc sanguis in hominem delibatur, dissolvere non licet. Homicidii festinatio est prohibere nasci; nec refert natam quis eripiat animam, an nascentem disturbet: homo est, et qui est futurus; etiam fructus omnis jam in semine est ‘. 60 ST. AMBROSE, Expositio Evangelii secundum Lucam, l. I, 44 (PL 15, 1632): ‘Ad cohibendam petulantiam tuam, manus quasdam tui auctoris in utero hominem formantis advertis. Ille operatur, et tu sacri uteri secretum incestas libidine? ‘An indication of this thought is already to be found in the first Christian texts, for example in the Letter of Barnabus (between the first and second centuries) XX, 2, in which the author says that those who follows the way of the shadows are, amongst other things killers of children, destroyers of the plasma created by God (PG 1, 1230; here quoted from: QUACQUARELLI A., I Padri Apostolici, Roma: Città Nuova Editrice, 1986: 213; DIDACHÃ? (+ 100 d.C) V, 2: on the way of death there walk amongst others killers of children, destroyers of the creatures of God (PG I Padri Apostolici, p. 33). 61 ST. AUGUSTINE, Sermo CLVI, c. II (PL 38, 851): ‘Et tamen in omnibus qui nascuntur infirmis Deus quod bonum est operatur, formando corpus, vivificando corpus, alimenta praebendo ‘. Cf. ID., Contra Julianum Pelagianum, l. V, 34 (PL 44, 804): ‘Ut autem concipiatur fetus atque nascatur, divini est operis, non humani ‘. 62 ID., De anima et ejus origine, l. I, c. XVI (PL 44, 488-489). 63 ST. THOMAS AQUINAS, Scriptum super libros sententiarum Petri Lombardi, 2, d. 18, q. 2, a. 3; ID., De potentia, q. 3, ad 9; ID., Summa contra gentiles, 2, 87-89; ID., Summa Theologica, 1, q. 76, a. 3, ad 3, en 1, q. 118, a. 2, ad 2; ID., De spiritualibus creaturis, a. 3, ad 12; ID., De anima, a. 11. 64 ST. AUGUSTINE, De nuptiis et concupiscentia, l. I, c. XV (PL 44, 423-424): ‘Produntur autem quando eo usque progrediuntur, ut exponant filios, qui nascunutur invitis. Oderunt enim nutrire vel habere, quos gignere metuebant. Itaque cum in suos saevit, quos nolens genuit tenebrosa iniquitas, clara iniquitate in lucem promitur, et occulta turpitudo manifesta crudelitate convincitur. Aliquando eo usque pervenit haec libidinosa crudelitas, vel libido crudelis, ut etiam sterilitatis velenam procuret; et si nihil valuerit, conceptos fetus aliquo modo intra viscera exstinguat ac fundat, volendo suam prolem prius interire quam vivere; aut si in utero jam vivebat, occidi antequam nasci. Prorsus si ambo tales sunt, conjuges non sunt: et si ab initio tales fuerunt, non sibi per connubium, sed per struprum potius convenerunt. Si autem non ambo sunt tales, audeo dicere, aut illa est quodammodo meretrix mariti, aut ille adulter uxoris ‘; LOMBARDUS P., Sententiae, l. IV, d. 31, c. 3-4; ST. THOMAS AQUINAS, Scriptum super libros sententiarum Petri Lombardi, IV, d. 31. 65 CONNERY J., Abortion: the Development of the Roman Catholic Perspective, Chicago: Loyola University Press, 1977: 142-148. 66 GRISEZ G., Abortion: the Myths, the Realities, and the Arguments, New York: Corpus Books, 1970: 165-184.

144 67 CONGREGATION FOR THE DOCTRINE OF THE FAITH, Donum Vitae, I, 1, AAS 1988, 80, pp. 78-79. 68 SERRA A., COLOMBO R., Identità e statuto dell’embrione umano: il contributo della biologia, in AA.VV., Identità e statuto dell’embrione umano, Città del Vaticano: Libreria Editrice Vaticana, 1998: 143-146. 69 BOETHIUS, Contra Eutychen et Nestorium, 3, in RAPISARDA E. (ed.), Opusculi Teologici, Catania: Centro Studi sull’Antico Cristianesimo, 1960: 52: ‘Quocirca si persona in solis substantiis est atque in his rationabilibus substantiaque omnis natura est nec in universalibus sed in individuis constat, reperta personae est definitio: naturae rationabilis individua substantia ‘. 70 ST. THOMAS AQUINAS, Summa Theologica, 1, q. 29, a. 1; ID., De Potentia, q. 9, a. 2; ID., I Sententiarum, d. 25, q. 1, a. 1; cf. LOBATO A., La persona, Rome: Pontificia Universitas S. Thoma, 1973: 231. 71 FETZ R.L., Personbegriff and Identitätstheorie, Freiburger Zeitschrift für Philosophie und Theologie 1988, 35: 69-106. 72 This Aristotelian-Thomist thesis was taken up by the doctrine of the Catholic Church during the Council of Vienna of 1312 (DENZINGER HEINRICH, Enchiridion Simbolorum, n. 902), during the Lateran Council of 1512-1517 (Ibid., n. 1440) and in the Encyclical Veritatis Splendor (n. 48ss). 73 In relation to this whole question Thomas Aquinas has fascinating and inspired ideas. For him, the subsistent form is the same for all human persons. Indeed, beings with a different form belong to different species. However, although it is true that all men have the same form, how then can the differences between them be explained? This question is a fascinating one and concerns above all else the evident difference between human beings in their capacity to understand things, a capacity that is directly linked to the spiritual form of man. The answer of Thomas Aquinas is that the difference in form between men can only be accidental: ‘There is a dual formal variety. There is the variety of the form in itself as regards its essential contents; and such diversity leads to a variety of species: however, there is also a variety of the form not in itself, but in an accidental way, which derives from the variety of matter, in the sense that a better arranged matter will participate in the form in a more worthy way; and such variety does not cause a distinction between species and this is the variety of souls ‘(ST. THOMAS AQUINAS, Scriptum super libros sententiarum, II, d. 32, q. 2, a. 3, ad 1). This provides the basis for his explanation of the differences between individuals within the same species, that is to say the numeric distinction of individuals: ‘The difference of form that comes solely the different arrangement of matter does not make a diversity according to the species but only according to the number. Indeed, there are different forms of different individuals, diversified according to matter ‘(ID., Summa Theologica, 1, q. 85, a. 8, ad 3: ‘â?¦Differentiae formae quae non provenit nisi ex diversa dispositione materiae, non facit diversitatem secundum speciem, sed solum secundum numerum; sunt enim diversorum individuorum diversae formae, secundum materiam diversificatae’). Thus it is also explained why a man can understand himself better than someone else, even though they have the same spiritual form: intellectual capacity also depends on the disposition of the lower faculties which the intellect needs for its activity, that is to say the imagination, the cognitive faculty and the sense memory (Ibid., 1, q. 85, a. 7 in c.). 74 Many people, hearing the word identity, spontaneously think of identity cards or police records. This meaning of identity involves data such as skin colour, hair colour, eye colour, height and weight, physical size and possible mental characteristics. Sociology makes a distinction between natural identity and conventional identity. By natural identity is meant the ability of a person to say I, to see all things in relationship to himself or herself and to enter into conversation with other people. Conventional identity or role identity is the result of the social integration of a human individual which makes him or her a member of a community. A third concept of identity could be added to these two, that is to say the concept of the identity of the autonomous self, which is derived from the Kantian conception of an autonomous person: this concerns the identity of the person who manages to subject 145 himself or herself freely to the laws and the general conventions of society (see: FETZ, Personbegriff and Identitätstheorieâ?¦). All the various kinds of identity hitherto listed which we think of spontaneously or which are employed by sociology do not apply to the embryo before implantation. For this reason the early embryo, because it cannot appeal to a feeling of solidarity between individuals or to society with other human beings in general is in a situation that in a certain sense is disadvantaged. The foundation of solidarity, in fact, is that one recognizes in one’s neighbour something of oneself. However, all these types of identity cannot be seen as being totally independent of what the embryo is: they do not concern, in fact, the deepest level of the identity of the human person. The physical and mental characteristics of police records, the natural and social identity of the description provided by sociology, and also the identity of the autonomous self, which must grow in all of us, can change and indeed they do change during life. These types of identity are accidental. It is necessary to find the ontological identity of the person who is the subject of all these changes. The concept of identity of the social sciences and the ontological concept of Boethius complete each other. The human individual is perhaps able to develop various social identities but only thanks to his or her generic identity and numeric identity, which also provide the limits to the possibilities of developing a social identity. In limiting oneself to social identity and forgetting about ontological identity one runs the risk of attributing being a person only to those people who are able to achieve a social identity in line with a certain standard. 75 LANZA, La questione del momento in cui l’animaâ?¦, pp. 230-231; DONCEEL, Immediate Animation andâ?¦, p. 101. 76 CHOLLET A., Animation, in Dictionnaire de Théologie Catholique, vol. I, Paris, 1923: 1314.

146 MARIO PANGALLO

THE PHILOSOPHY OF SAINT THOMAS ON THE HUMAN EMBRYO

HUMAN LIFE IN CLASSICAL AND CHRISTIAN PHILOSOPHY PRIOR TO ST. THOMAS The aim of this presentation is to illustrate the position of Saint Thomas on the human embryo, highlighting those aspects which are more important from a theoretical point of view, in order to define the identity of the embryo and its status from a perspective that is not only biological but also ontological. As an introduction to the subject, I consider it appropriate to refer to the course of history prior to St. Thomas, that is, to the school of thought of the so-called ancient classical and Christian times on the concept of human life, which Aquinas and (other) medieval philosophers drew on. On this point, it is easy to establish that the philosophical discourse on human life in general, and on human life at its origin, in ancient Greek and Hellenistic times, cannot exclude reference to the soul (the psyche). In fact, it is known that the psyche is the vital principle of every living body: to be alive means being an animated body. The problem of the soul, that is, of life, is the fundamental problem of the so-called Greek ‘naturalist ‘philosophers. Moreover, while the soul appears to be a mere physical reality, subtle and penetrating, as reflection upon it develops, especially thanks to the Pythagoreans, the soul assumes the dimension of a spiritual reality; according to Plato, the human soul possesses something that is divine, something which is also essential to the world of Ideas. In a sense, the soul is a synthesis of finite and infinite in the way in which it communicates energy to the finite human body which allows it to exist and to act in a manner that is open to that which transcends what is finite. Platonic terminology and later, neo-platonic terminology, include the notion of ‘logos ‘in their discussion on the human soul; man is seen as a ‘micro-cosmos ‘, the living (being) in whom the ancient saying ‘all in all ‘manifests itself and makes itself understood, capable of grasping the soul of the whole world, that is, the logos which is immanent in Nature, fount of life and order. Plato maintains that the human soul is a substance in itself. It is a principle of movement. It is rational, spiritual and immortal, that is, inseparable from the body. These principal constitutive notes of Plato on the human soul are acknowledged by Aristotle, albeit with decisive modifications, which can be understood right from the consideration of human life at its origin, of embryonic life. According to Aristotle, the discussion on life is certainly a discussion on the soul, but also a discussion on the generation and corruption (degeneration) of living forms. Aristotle holds that the soul is a substance, insofar as it is a form or first act of a natural body, which has the potential of life and is provided with organs (De Anima, I, 1, 412, 19-21; 28- 30; 412 b, 11-12). The Aristotelian concept of the soul as a form and principle of the body’s organization, quite rightly, comes close to the notion of a ‘genetic code ‘or ‘genetic programme ‘inscribed on our chromosomes. Such a comparison, on one hand, makes sense; on the other hand, it is inappropriate. In any case, it is clear that the soul is intimately united to the body in generation according to the Stagyrite: what we now call the human embryo is such from the beginning, that is, from the moment of the fertilization of the female ovum by the male sperm, a synthesis of soul and body. As a matter of fact, it is a ‘body ‘, that is, secondary matter, organized matter, inasmuch as it is informed by the soul. The soul, being a form, is the cause by which the body possesses the properties which characterize it; that is, it is living, perceiving, thinking. With regard to its principle of living, Aristotle refers to the soul as ‘nutritive ‘(or, more generally, vegetative); as regards its principle of perception, he speaks of the soul as ‘sensitive ‘; with regard to it being a form of the properly human activities involved in thinking, he speaks of the soul as rational or intellectual. The inferior soul is a condition of there being one that is superior, and the former contains the potential of becoming the latter, thus being able to perform the functions of the latter. This is illustrated by Aristotle with a geometric example, which we will see later being adopted by St. Thomas, of a pentagon in which a square is drawn, in which, in turn, a triangle is drawn (De Anima, II, 3, 414 b 29-32). The doctrine of supplantation or succession of souls in human embryos allows Aristotle to amalgamate the philosophical aspect of the consideration of the soul with the 147 empiric/scientific aspect of looking at human life. The teleology innate in the inferior form, which guides embryonic development to superior functions, is the guarantee of unity of a living being in the different phases of the embryo’s development, contrary to the Platonic trichotomy; the soul is essentially the enteléchia of the body, the beginning which contains within itself the end (telos) for which the body exists, develops and acts. In this way, the Platonic doctrine of the soul as a principle of movement (that is, of any change or transformation) of the body is assimilated by Aristotle in the field of his hylomorphic interpretation of the phenomenon of generation and corruption of living beings, without the possibility of giving rise to dualistic or extrinsicist misunderstandings, which had been caused by the Orphic and Pythagorean traditions. The relationship of succession, which links souls to each other in life at its origin, ultimately means that the true, real and unique soul of a man is the intellectual soul, and, to be more precise, the active or productive intellect (nous poietikòs). In fact, only the active intellect is an act by essence, being capable at all times of operating; the active intellect, writes Aristotle, is ‘separated ‘(choristòs; De Anima, III, 4, 429 b 5) and comes to us ‘from outside ‘and thus contains something divine within itself, and is immortal. Aristotle writes: ‘It was a good idea to call the soul the place of forms, though this description holds only for the intellective soul, and even this regards the forms only potentially, not actuallyâ?¦After strong stimulation of a sense we are less able to exercise it than before, as e.g. in the case of a loud sound we cannot hear easily immediately after, or in the case of a bright colour or a powerful odour we cannot see or smell, but in the case of the mind, a thought about an object that is highly intelligible renders it more and not less able afterwards to think of objects that are less intelligible: the reason is that while the faculty of sensation is dependent upon the body, mind is separable from it. Once the mind has become each set of its possible objectsâ?¦its condition is still one of potentiality, but in a different sense from the potentiality which preceded the acquisition of knowledge by learning or discovery: the mind too is then able to think itself ‘.1 As we can see, Aristotle emphasizes at the end of the excerpt that the highest moment of the intellective or spiritual life of man is self-awareness. Further on, Aristotle returns to two aspects of intellective life, passive and active, stating, ‘and in fact mind as we have described it, is what it is by virtue of becoming all things, while there is another (mind) which is what it is by virtue of making all things: this is a sort of positive state like light; for in a sense light makes potential colours into actual colours. Mind, in this sense, is separable, impassive, unmixed, since its essence is activity, for the active factor is always superior to what is passive, the originating force to the matter which it forms ‘.2 Thus, it seems clear to me that the humanity of man can be identified precisely with the presence of a vital principle that is per se incorporate, being united substantially to the body, which is the rational soul, capable of carrying out the duties of the sensitive and vegetative soul. In this sense, it can be said that the intellect is the form of forms (eidos eidòn; De Anima, III, 8, 432 a 2). St. Thomas interprets Aristotle as being in favour of the unity and uniqueness of the substantial form, entering in conflict with the numerous advocates of the plurality of souls of man in the XIII century. The soul is substance, but it is not complete substance: without the body provided with organs, the functions of which the soul is the origin or active potential cannot be performed. In this sense, according to Aristotle, the soul that is vegetative and sensitive is united to the organs, without which it cannot operate, but its essence is different compared to that of the organs, as there is a difference between an organ and the function for which an organ is in the body (De Anima, II, 12, 424 a 25-30). From this it becomes clear why, as stated above, an excess of tangible qualities destroy the sensors, but they do not suppress the soul (if not per accidens and in some cases). By means of the relationship between anterior and posterior, and though differentiating between the three souls of man, Aristotle emphasizes without ambiguity that they form a unity, a unique soul, which is the formal, locomotive and final cause of the body and thus the primary condition of the end-purpose of a living person. Furthermore, as evident from reading the 2nd book of De Anima, especially the first three chapters, 148 Aristotle proposes a hierarchical order of vital and mental functions, when dealing with the relationship of succession which connects the three souls and different faculties. These functions are manifested gradually in human development, and rational function is highest: this is the last with respect to the others, and therefore represents the end to which the others are orientated, and their fulfilment. Since Aristotle, as I have already mentioned, seeks to unite the ontological aspect to the biological aspect in a harmonious way, it appears convenient to him to sustain, together with certain scientists of his era, that the fulfilment of the human vital principle, that is, the spiritual soul, is last not only in a teleological sense, but also in a chronological sense, managing to inform the human body only after a certain degree of development of its embryonic life, that is to say more or less after about 40 days of life for a male foetus and after about 80-90 days for a female foetus. As opposed to the Empedoclesian and Democritean schools of thought, and to other authors for whom male and female are combined in embryonic development, according to Aristotle and many other ancient philosophers, the active principle of generation is only the father, the donor of semen; the mother contributes to generation by providing the matter, (katamenia, Latin menstrual), and is therefore completely passive. Later, Galeno tries to unite the two lines of thought by attributing a certain activity in generation also to the mother, through her own semen, although having a subordinate role compared to that of the male semen. In conclusion, Aristotle, in the scientific field, opts in favour of epigenesis, in compatibility with the theories described above, that is, the theory which supports the idea of a gradual appearance of the differentiation of organs in embryonic development, as opposed to Preformism. For example, he writes in Generation of Animals: ‘An animal does not become at the same time an animal and a man or a horse or any other particular animal. For the end is developed lastâ?¦For at first all such embryos seem to live the life of a plant. And it is clear that we must be guided by this in speaking of the sensitive and the rational soul. For all three kinds of soul, not only the nutritive, must be possessed potentially before they are possessed in actuality ‘.3 It is interesting to note, when reading the same text, why, according to Aristotle, more souls cannot pre-exist from the moment of fertilization: ‘Now that is impossible for them all to pre-exist is clear from this consideration. Plainly those principles whose activity is bodily cannot exist without a body, e.g. walking cannot exist without feet. For neither is it possible for them to enter by themselves, being inseparable from a body, nor yet in a bodyâ?¦It remains, then, for the reason alone so to enter and alone to be divine, for no bodily activity has any connection with the activity of reason ‘.4 It seems to me that it can be said, in the light of this affirmation, that in Aristotle the succession of the three souls in the embryo does not imply that the intellective soul is the result of the evolution of the two previous souls: it represents an advance in quality, caused by the immateriality of the intellect, which has divine origins. For this reason, St. Thomas does not see an opposition in Aristotelian physiology and anthropology to Christian Creationism, according to which the spiritual soul is created directly by God and infused in the human embryo that is adequately predisposed. When does this infusion animae rationalis occur? And how and when does one have that perfecta disposition corporis presupposed by the creative act? Before examining and evaluating the answer of St. Thomas, I will mention the principal positions prior to the Angelic Doctor, which can be found in the Fathers of the Church and in Scholastics. In Patristics, four lines of thought can be distinguished as regards the modality of the transmission of the soul: 1) the theory of pre-existence of souls which is represented for the most part by Origen; 2) the theory of Traducianism, according to which the human soul is materially transmitted by the parents, represented for the most part by Tertullian; 3) the theory of Generationism, which conceives the tradux (shoot) as being a spiritual form transmitted by the parents and thus, with regard to the problem of transmission of the soul, can be assimilated by Traducianism; 4) the theory of Creationism, by which the human spiritual soul is created directly by God and infused in the body of the one who has been conceived. The Creationist theory clearly prevailed in the Patristic era; Origenism was condemned, and Traducianism and Generationism were also finally rejected by the Church, despite the variants of St. 149 Augustine, who, eventually confessed his own inability to resolve the problem adequately (De natura et origine animae, I, 15, 25). It is known that the problem of the modality by which the soul is transmitted was connected to the theological question of the modality by which original sin is transmitted to a conceived being. As for the time of the moment of infusion of the intellective soul, in Patristics, already two opposing theories could be outlined which reappeared in the Medieval era: the theory of belated animation, which more or less follows Aristotle and which I have already explained above, and the theory of immediate or simultaneous animation, which means that the intellective soul is already present in the conceived being from the moment of fertilization. St. Augustine, rejecting both the Platonic trichotomy and the Aristotelian distinction between three souls (and thus the theory of their succession in the human embryo), held that only a single soul existed in man, simple and immortal, the origin which gave not only vegetative and sensitive life to the organism, but its own ‘personal ‘existence: the human soul is present in a body which is still unformed, although it is not yet capable of operating in a specifically human sense. In Oriental Patristics, the Cappadocian Fathers declared themselves as being in favour of immediate animation: St. Basil is favourable towards simultaneous transmission, especially for ethical matters; what has been conceived is a man from the moment of fertilization, and to kill it is equivalent to homicide, quite apart from the distinction between a formed and unformed foetus. Comparing the embryo to a seed of grain in which the essence of grain already exists, St. Gregory Nyssan sustains in De hominis opificio (chap. 29), that the rational soul is present from the beginning in a conceived being, it guides its development and is indeed the origin of its differentiation into various organs; just like the development of the seed, the development of the human embryo is not due to a faculty or formative strength that is foreign to the rational soul, but innate within it, and infused by the same in the corporeal substrate of the conceived being. There is no shortage of voices in favour of belated animation among the Greek Fathers, such as Theodore of Cyre, for example, who defends belated animation in his work Therapy of Pagan Diseases with scriptural as well as philosophical arguments. St. Maximus the Confessor declares himself decisively in favour of immediate animation in his work Ambigua: it is absurd to think of a stage in which the embryo generated by humans can be a plant or an animal. It should be noted that, from a metaphysical point of view, the question of the animation of the embryo in Christian philosophy is related to the question of the embryo’s being a person, although this does not appear clearly from the start. In fact, taking into consideration the renowned definition of a human person divulgated by Boethius, to be a person means existing with a rational nature; therefore, the moment at which a body is animated by a rational soul, it exists with a rational nature, and is thus a human person. It should also be noted that the question in Christian philosophy is additionally conditioned by the Christological question: the Word became incarnate at the same moment in which the body of Jesus was conceived, and must be considered to be completely man from the beginning. Thus the theory of belated animation had to include an exception for Christ, whereas, for the supporters of immediate animation, the Mystery of the Incarnation of the Word created no philosophical problems. For a series of reasons, which I do not consider necessary to treat in this location, in High Medieval times and in the Scholasticism of the XI-XIII centuries, the theory of belated animation became prevalent. The latter may be attributed, in varying degrees, to authors such as St. Anselm, William of Champeaux, Hugo of St. Victor, William of Conches (according to whom the rational soul is created by God after the fifth week of embryonic life), Peter Lombardi (cf. 2 Sent., dist. 18, 8; 3 Sent., dist. 2, 2), William of Auvergne, John of La Rochelle, St. Bonaventure (according to whom the rational soul is composed of a form and an incorruptible matter), and St. Albert the Great. In the XIII century, the question became intertwined with the question of unity or plurality of the substantial forms in man, but not by means of a direct connection: in fact, the supporters of belated animation belonged as much to the group of supporters of unity of the substantial form (the Aristotelian theory) as to the group of supporters of a plurality of substantial forms in man (the neo-platonic theory). Among the few texts which sustain immediate animation, De motu cordis by Alfred of Sareshel, an author of the beginning 150 of the XIII century, is worth a mention: as the embryo is living, it is given life by the rational soul, which is peculiar and specific to humans, otherwise the embryo would not live a human life, it would not be a human embryo, which is unconceivable. However, the position of Alfred is rather confused and his influence is limited. Many Medieval authors, in treating embryonic life, put forward the idea, taken from Aristotle, that a vis formativa exists, innate in the human embryo, and is transmitted to it by the paternal semen. As a result of the latter, it develops in a human way although not yet informed by the specifically human soul (that is, the intellective soul). This idea, as we shall see, was entirely accepted by St. Thomas Aquinas.

THE POSITION OF ST. THOMAS WITH REGARD TO THE HUMAN EMBRYO St. Thomas adheres to the Aristotelian doctrine of the succession of souls in the human embryo and holds that the rational soul is infused after about 40 days from the moment of fertilization. According to the Angelic Doctor, the male semen derives from the surplus of food (and not from the substance of the generator) and possesses a virtus formativa, which is added to the vital spirit (spiritus vitae) of the sperm and, helped by a triple form of heat (elementary heat, heat of the soul and heat of heaven), it guides human embryonic development.5 The doctrine of the succession of souls is consistent with the Platonic Dionysian principle of contiguity of the grades of perfection, according to which the supreme point of the inferior ontological grade touches the lowest point of the grade immediately above it and to which it is teleologically orientated. 6 Before entering into an analysis of the principal texts of Thomistic embryology, it is appropriate to recreate, in an approximate way, the general psychological-metaphysical context in which such texts were inserted. As for Aristotle, so too for St. Thomas, the human soul performs a triple function, vegetative, sensitive and intellective, so that the sensitive and vegetative functions are contained in their intellective counterpart as a triangle and square are contained in a pentagon.7 Nevertheless, the succession of souls in the human embryo is not to be interpreted as a progressive realization of an inferior form, as if the vegetative soul potentially contained the sensitive soul, and the latter contained the rational soul: it concerns rather a substitution of the inferior form when the superior form arises.8 The rational soul is the only substantial form of the human body (a theory that was notably opposed in the XIII century by the Augustinists and by many exponents of the Franciscan school, and condemned in Paris in 1277), and is the first act of the human body that is duly organized and predisposed to receive it. The union between the human body and intellective soul is immediate, and on the basis of that union, the human embryo can be called a person.9 The nature of the intellective soul is spiritual and immortal, having been created directly and immediately by God, and infused when the body is perfectly predisposed.10 Moreover, one must put the embryology of St. Thomas in the context of a sort of physics and metaphysics in which forma dat esse materiae, a principle which, in my opinion, has two important meanings for St. Thomas: the form constitutes the formal being, that is, the form is the constitutive element of every real essence, insofar as it is an act of the matter for all that regards the corporeal substances: in this sense, one can also say forma causat esse materiae; the form is principium essendi, in the sense of being a subject which renders actual what is a potential substance and enables it to receive its being; in this second sense, the form can be referred to as complementum substantiae, insofar as it unifies, in the substantial act, the dynamic reality of the act of being, both at a categorical and transcendental level.11 Therefore, the rational soul communicates the act of being to the human body, and thus makes of it a spiritual and immortal being similar to God, having come out, so to speak, directly and immediately from Divine creative virtue.12 Having made these preliminary statements, we may proceed to analyze the principal Thomistic texts which concern the human embryo. From the time of the Commentary on the Sentences of Peter Lombardi (the Magister, who, as I have already mentioned, was also in favour of belated animation), St. Thomas, baccalaureus sententiarius, affirms that a conceived being achieves the reception of a soul only after a certain degree of bodily development. In the Commentary on the 2nd book of the Sentences, dist. 18, q. 2, a. 3, one asks oneself 151 ‘if the sensitive soul is ex traduce’. After having exposed and criticized the theories of Plato, Themistius and Avicenna, St. Thomas considers the position of Aristotle to be more reasonable: ‘But the position of Aristotle is much more reasonable. Indeed, nothing begins nor is made or generated if not according to the way in which it has its being. And therefore, we consent to the fact that the sensitive and vegetative souls are ex traduce. Now, the modality of this transmission is the following. In order for every univocal and close agent to induce its species in a patient, and food ‘inasmuch as it is a patient and altered ‘is transformed into nutrition for the body (because it nurtures in the measure in which it is potentially flesh ‘as Aristotle says), it is eventually necessary for (the food) to receive the species and virtue of the . Therefore, before its final assimilation ‘when carried out in a partly determined act, such as flesh or bone’, it contains within it the virtue of the species in a manner which is not determined to become this or that. In fact, the determination towards this or that is in accordance with the virtue specific to the determined part. Therefore, as the semen is the residue of the last food which is very near to the final conversion, it contains the whole within it potentially, and not some part of it in actuality. In truth, before (the semen) is released by means of the action of vegetative virtue ‘separated from the remaining part of its gender’, it contains within it this indistinct potency ‘just as the form of the whole is not in a part of it if not potentially so ‘. On the other hand, when the semen becomes separated, it is rendered actual having a potency or formâ?¦Now, while the potentiality present in female menstruation is a passive potentiality ‘as pieces of wood and stones are potentially houses, the potential present in the male seed is an active potential ‘that is to say, just as the form of the house in the mind of the architect is potentially a house. Consequently, Aristotleâ?¦compares the potential present in the male semen to art, and Avicenna and Averroes call it formative virtue. This formative virtue, as regards its way of working, is halfway between the intellect and the other forces of the soul. In fact, the other forces avail of determined organs in their operations; whereas the intellect uses none of these; as regards the formative virtue mentioned above, it avails of something corporeal in its operation, which does not yet have a determined species. In truth, the subject and organ of such formative virtue is the vital spirit enclosed within the seed so that, as it contains such a spirit, the semen becomes frothy; and this is the cause of its whitenessâ?¦It should also be said that a triple heat becomes joined to this corporeal spirit ‘that is, elementary heat, which is the instrument that frees and utilizes and activates that spirit; the heat of the soul, which is life-giving; the heat of heaven, by virtue of which the spirit moves to a determined species. And by the virtue of this triple heat, the formative virtue converts the matter prepared by the woman into the substance of the limbs, according to the way in which the transformation of the body is to be in its growth-as Aristotle says. And while its production advances towards the perfection of the organs, the soul begins to be ever more in act in the semen while first, it had been so in potential, so that the conceived being participates in the work of the nutritive life from the start, and so it is referred to as living its plant life; and so on, until it reaches full likeness to its generator ‘.13 It is clear that full likeness to one’s generator is achieved only when the conceived being is informed by the intellective soul. The text in which St. Thomas treats the ontological and biological state of the embryo to quite a large degree is found in Quaestiones disputatae de Potentia, q. IIII (De creatione quae est primus effectus divinae potentiae), in article 9 (Utrum anima rationalis educator in esse per creationem, vel per seminis traductionem). In Corpus articuli, before manifesting his own line of thought, St. Thomas recalls some of the opinions of ancient Classical and Christian times. The first theory is that of Traducianism, immediately after that of the pre-existence of souls with respect to bodies and, finally, the theory of immediate animation is mentioned. ‘With regard to this question, in ancient times various authors maintained different positions. In fact, some affirmed that the soul of the child is reproduced by the soul of the parent, as the body is reproduced by the body. On the other hand, others affirmed that all souls are created one by one, but they maintained that, at the beginning, they had all been created together outside the bodies 152 and that they united themselves to the bodies gen- erated, either through the motion of their own will, according to some, or following the order and through the action of God, according to others. Alternatively, others affirmed that souls are infused into the bodies in the same moment in which they are created. For some time, all these opinions were maintained, and there remained doubt as to which one of them was true, as can be seen in the case of Augustine. Subsequently, however, the first two were condemned by the judgement of the Church, and the third was approved ‘.14 Having made these preliminary considerations, St. Thomas answers the question of the article, stating that the rational soul cannot be transmitted with semen: ‘Then looking more closely at the situation, the opinion ‘which is now under discussion ‘has been rightly condemned, that which maintains that the rational soul is reproduced with the semen. And this can now be seen on the basis of three arguments ‘. The first argument is based on the subsistent being of the rational soul. As the rational soul, in contrast with the vegetative and sensitive soul, possesses a subsistent being, it comes to being in a different way from that of the other forms: indeed, whereas the other forms are generated by a matter that is susceptible to receiving different forms, the rational soul, being immaterial in itself, cannot be generated from a substratum material, but comes to being from nothing, and is thus due to a divine creative act, given that being made from nothing means precisely, to be created. Conversely, those who maintain the contrary should admit that the rational soul is not immaterial and thus is not subsistent and is not immortal.15 The second argument is similar to the first: as the rational soul is an entirely spiritual form, in no way can it be reproduced through the generation of the body nor by means of some innate capacity in the semen.16 In the third argument, St. Thomas, recalling from Aristotle the fact that the rational soul is capable of operations separate from the body in virtue of its own subsistent being, affirms that it cannot be informed by the potency of matter.17 After having answered the question of the article, St. Thomas proceeds to respond to the objections. Among these, the 9th objection is of particular interest to us: ‘The embryo, before being brought to completion by the rational soul, manifests a certain activity in its soul, for it grows, nurtures itself and has sensations. However, there is no activity in the soul if there is no life. Therefore, it is alive. On the other hand, the vital principle of the body is the soul. Therefore it has got a soul. However, it cannot be said that another soul is subsequently added, for in this case there would be two souls in a single body. Thus the same soul which was generated from the beginning in the semen is the rational soul ‘.18 In answering to this objection, the Angelic Doctor outlines his embryology, clearly rejecting not only the theory by which the rational soul is transmitted with the semen but also the theory of immediate animation. In this case also, before answering, St. Thomas puts forward some contrasting opinions. The first opinion is attributed to Gregory of Nyssan: ‘Some people established a parallel in the human generative process between the development of the rational soul and the development of man’s body. They affirm that, as the human body is virtually present in the semen, without possessing in actuality the completeness of the human body, which consists in the differentiation into organs, but attains such completeness little by little due to the capacity of the semen, thus at the beginning of (the process of) generation, the soul is in the semen, which has, by some virtuality, all the wholeness which will then appear in the complete man, though not having it in actuality, given that the activities of the soul are not apparent, but it is acquired little by little with the passing of time in such a way that at first, the activities of the vegetative soul appear in the soul, after which appear those of the sensitive soul, and finally, those of the rational soul. Gregory of Nyssan comes close to this opinion in the book that he wrote on man ‘.19 The first part of the answer of St. Thomas on this opinion is of interest to us, in which the Angelic Doctor, following Aristotle, maintains that it is not possible to ascertain that the rational soul is present in the semen right from the beginning, as the embryo has not yet carried out any activity, due to the insufficiency of its organs: ‘Indeed, it is said in the 2nd book of De Anima that the potential of life which is in a physical organic body, of which the soul is the act, is not what it is without the soul, like the seed and the fruit. From this one can understand that the semen is in potential 153 with respect to the soul in such a way that it is without a soul. Given that the semen is not yet assimilated by the limbs in the final assimilation (in fact, in this case its discharge would be a sort of corruption), but is the residue of the final digestive process, as said in the 15th book of De Animalibus, it was not yet in the body of the man who generates a complete existent being with its soul. For this reason, the soul can only be in it at the beginning of its division. However, supposing that the soul divides with it, this cannot be said of the rational soul, which, not being an act of some part of the body, cannot divide itself if the body divides itself ‘.20 A second opinion considers the succession of souls in the human embryo to occur in such a way that, one would have three souls differing in their essence at the end of embryonic development: a theory that is thus very close to those who support the plurality of substantial forms in man. St. Thomas answers: ‘It is impossible for there to be more than one substantial form of a unique and identical thing. Indeed, as the substantial form makes being not only from a certain point of view, but absolutely, and places a certain thing in the category of the substance, if the first form does this, the second which is added to it, on finding the substrate already constituted in the substantial being, is added to it in an accidental way. Thus it follows that the sensitive soul and rational soul in man are united to the body in an accidental way. And it cannot be said that the vegetative soul which, in a plant, is a substantial form, is not a substantial form in man, but a disposition to the form, because what belongs to the category of a substance cannot be an accident of anything, as said in the 1st book of Physics’.21 A third opinion holds that the rational soul of man derives partly from the outside, as regards the intellective-spiritual nature, partly from the inside, as regards the vegetative and sensitive nature. St. Thomas retraces this opinion to that by which the substantial form is brought into act in successive moments, a hypothesis that is to be absolutely excluded for the Angelic Doctor for, as has already been said, according to him (and to Aristotle himself) it is unacceptable to think that a subsistent spiritual form can, partially or totally, derive from another precedent form informed from a material substrate: no evolutionary process can produce a spiritual entity.22 This is followed by the confutation of two other opinions, by which the human embryo is supposed not to have a soul until it is fully developed as a result of the infusion of the rational soul. St. Thomas answers that, right from the beginning of the separation, there is no soul in the semen, but rather the capacity of the soul (virtus animae). This capacity then acts, disposing the matter towards reception of the soul; then the vegetative soul and sensitive soul are formed, as shown by the fact that, at a certain stage of its development, the human embryo manifests organized vital functions and, subsequently, also its own sensitivity. Aquinus observes that the vital activities of the human embryo, such as nutrition and growth, cannot depend on the soul of the mother, and neither can its sensitivity: in fact, one is dealing with ‘immanent operations ‘, (a technical term of Aristotelian-Thomistic physics), characteristics of a living being, which, precisely because they are immanent, cannot depend on a principle external to the embryo.23 In order to clarify the question further, St. Thomas introduces an explanation of the difference that exists between the generation of a human embryo (and that of an animal embryo) and the generation of the elements, such as water or air. According to St. Thomas, the generation of the elements is simple, whereas in the generation of an animal embryo, different substantial forms appear, for which the generative process contains in itself several generations and corruptions, and it cannot be accepted that a unique and identical substantial form are brought into act gradually, as he had already demonstrated above.24 Thus one arrives at the conclusion of the answer to the 10th objection, which summarizes well the principal embryological theory of St. Thomas: ‘Therefore, in this way, as a result of a formative capacity which is in the semen right from the beginning, once the form of the sperm is removed, another form is introduced, and once this is removed, a soul that is both sensitive and vegetative is introduced, and once the latter is removed, not as a result of this capacity but from the Creator, the soul that is jointly rational, sensitive and vegetative is introduced. On the basis of this explanation, the answer is thus that the embryo, before it has the rational soul, is alive and has a soul, and once the latter is removed, the rational soul is introduced. And therefore, one does not have the 154 consequence of there being two souls in the same body, or that of the soul being transmitted with the semen ‘.25 The text is a synthesis of the Thomistic doctrine of belated animation, which the Angelic Doctor proposes more or less in this form in other works of his, with different variations of no theoretic relevance. Let us see the other principal Thomistic texts on the subject.26 In Summa Theologiae, I, q. 118, in dealing with human generation as regards the soul (De traductione hominis ex homine quantum ed animam), St. Thomas dedicates the first two articles to our theme. In the first article, it is asked whether the sensitive soul is transmitted to the human embryo through the male semen. St. Thomas answers that the soul of the generated embryo is caused by the (sensitive) soul of the generator, in the sense that it derives from the action of the vital strength contained within the semen of the gen- erator. In the answers to the objections, we find the description of the activities of the male semen after coitus, of which it has been spoken previously: in addition, it can be noted that St. Thomas believes he can reconstruct the processes of formation of the vegetative and sensitive souls of the embryo by means of an examination of the transformations undergone by the matter of the embryo as a result of the action of the sperm in the female uterus.27 In the second article, it is asked whether the intellective soul is caused by the semen. We already know that the answer of the Angelic Doctor is in the negative: ‘Since it is an immaterial substance it cannot be caused through generation, but only through creation by God ‘.28 At the end of the answer to the 2nd objection, we find a proclamation of the theory of belated animation of the human embryo: ‘We must therefore say that since the generation of one thing is the corruption of another, it follows of necessity that both in men and in other animals, when a more perfect form supervenes the previous form is corrupted: yet so that the supervening form contains the perfection of the previous form, and something in addition. It is in this way that through many generations and corruptions we arrive at the ultimate substantial form, both in man and other animals. This indeed is apparent to the senses in animals generated from putrefaction. We conclude therefore that the intellectual soul is created by God at the end of human generation, and this soul is at the same time sensitive and nutritive, the pre-existing forms being corrupted ‘.29 In Summa Contra Gentiles, St. Thomas dedicates chapters 86 and 87 of the 2nd book to the origin of the human soul, proposing the same theories as examined above, that is, that the human soul is not transmitted with semen (Ibid., chap. 86) and that the human soul is produced by God through creation (Ibid., chap. 87); then, in chap. 88, he puts forward the arguments of those who affirm that the human soul is produced from semen, and in chap. 89, he confutes the same arguments one by one. The theory of belated animation is held by St. Thomas indirectly when, in chap. 86, he states: ‘The vegetative and sensitive souls are generated by the generation of the body, and date their existence from the transmission of the male semen, but not the intellectual soul ‘(which thus supervenes in a subsequent moment).30 The Angelic Doctor expresses himself even more explicitly in chap. 89: ‘The fact that the conceived being is first an animal before it is a man does not demonstrate that the rational soul is transmitted by semen. In fact, the sensitive soul, whereby the human foetus was an animal, does not last, but its place is taken by a soul that is both sensitive and intellective, for which (the embryo) becomes both an animal and a man ‘.31 As for the rest, the chapters of Summa Contra Gentiles mentioned above present the same doctrines that are found in De Potentia, with some of the arguments set out differently. In Compendium Theologiae, chap. 93, St. Thomas speaks of the rational soul as the ultima et perfectissima forma of the human embryo created by God, supremum agens; Aquinus writes: ‘It is evident that the rational soul is the ultimate and most perfect form which the matter of generable and corruptible bodies can acquire. Therefore, the natural causative agents conveniently produce in the inferior bodies the dispositions and the preceding forms; while the supreme causative agent, that is, God, causes the ultimate form, which is the rational soul ‘.32 155 The context is always that of the defence of the creation of the spiritual soul by God, against Traducianism (de productionae animae rationalis, quod non sit ex traductione). The same position is found in Quaestiones quodlibetales (q. 5, a. 1), where it is reaffirmed that the doctrine of creation of the intellective soul can be demonstrated from the subsistent being of the spiritual human soul, which cannot be produced ‘per actionem compositorum ‘but is ‘ab extrinseco per creationem ‘.33 In his Commentary on the Letter of St. Paul to the Romans, St. Thomas refers again to the idea that a capacity exists in semen to render the body in a disposition to receive the soul, but without containing the soul in any way.34 I believe it is futile to talk at length, quoting and analyzing the other Thomistic texts which bring up again the same concepts about human embryonic life and the development of the foetus, and do not draw away from the theory of belated animation. A special place is occupied, in our problem, by the Christological texts of St. Thomas, when they deal with the Incarnation of the Word and the conception of Christ. Let us consider the principal text of Summa Theologiae, III, q. 33, a. 2 St. Thomas raises the following problem: ‘Whether the body of Christ received the soul in the first instant of its conception ‘(utrum corpus Christi fuerit animatum in primo instanti conceptionis). In the preceding article, the first (Summa Theologiae, III, q. 33, a. 1), Aquinas had stated that the Body of Christ was formed perfectly right from the first moment of conception: ‘In the first instant in which the various parts of the matter were united together in the place of generation, Christ’s body was both perfectly formed and assumed by the Word ‘.35 The reasons for this are twofold: 1) he who formed the body of Christ in the womb of the Virgin Mary was the Holy Spirit, Whose power is infinite and thus capable of form- ing in an instant what natural causes form in a gradual and progressive way; 2) he who was to assume that body was the Person of the Son; and thus it was unbecoming that He should take to Himself a body that was not perfectly formed.36 From these preliminary statements, it is clear that, in the following article, St. Thomas could no longer hold the theory of belated animation in the case of the body of Christ; he had to admit a perfecta dispotitio corporis of the embryo in primo instanti conceptionis and thus instantaneous animation and the immediate taking up of the human body on the part of the Word in the same instant of conception. Therefore, St. Thomas writes: ‘For the conception to be attributed to the very Son of God, as we confess in the Creed, when we say, who was conceived by the Holy Ghost, we must needs say that the body itself, in being conceived, was assumed by the Word of God. Now it has been shown above that the Word of God assumed the body by means of the soul, and the soul by means of the spirit, i.e. the intellect. Wherefore in the first instant of its conception Christ’s body must needs have been animated by the rational soul ‘.37 In article 3 of the same question, the Angelic Doctor affirms with decision the instantaneity of the assumption of human nature on the part of the Word, with exquisitely theological reasoning: ‘If Christ’s flesh had been conceived before being assumed by the Word, it would have had at some time an hypostasis other than that of the Word of God. And this is against the very nature of the Incarnation, which we hold to consist in this, that the Word of God was united to human nature and to all its parts in the unity of hypostasis: nor was it becoming that the Word of God should, by assum- ing human nature, destroy a pre-existing hypostasis of human nature or of any part thereof. It is consequently contrary to faith to assert that Christ’s flesh was first of all conceived and afterwards assumed by the Word of God ‘.38 However, according to St. Thomas, the case of the body of Christ remains an exception; in fact, in the same Christological context, he reaffirms that immediate animation of the human embryo right from the first instant of conception is valid only for the conception of Christ and not for other men. Therefore, St. Thomas replies to the objection of those who called upon the authority of Aristotle (in the generation of man, one is first living, then an animal and finally a man; thus Christ could not receive the soul in the first instant of conception): ‘What the Philosopher says is true in the generation of other men, because the body is successively formed and disposed for the soul: whence, first, as being 156 imperfectly disposed, it receives an imperfect soul; and afterwards, when it is perfectly disposed, it receives a perfect soul. But Christ’s body, on account of the infinite power of the agent, was perfectly disposed instantaneously. Wherefore, at once and in the first instant it received a perfect form, that is, the rational soul ‘.39 It may be observed that, ipso facto, St. Thomas, in accepting immediate animation in the single case of the conception of Christ, at least accepts that the theory of immediate animation is not metaphysically absurd, which, in my opinion, permits us to lead the question back to the area of physical doctrines, certainly connected to the metaphysical principles, but especially conditioned by the scientific knowledge of the era. It seems to me that the analysis of the principal texts of embryology in the works of St. Thomas, particularly in those where the theory of belated animation is held, allow a synthesis to be made on the subject. It emerges clearly from an analysis of the texts that St. Thomas follows the doctrine of belated animation not only due to the authority of Aristotle and the wide spread which this doctrine had in the XIII century, but above all, for reasons of a physical-metaphysical nature. Among these reasons, the main ones are the following: a perfecta disposition corporis is necessary in order that the rational soul can be infused in the embryo. In fact, the intellective soul can become the substantial form of the body only when the latter has acquired those organs which render it an adequate instrument of human intelligence in its specific operations. In other words, the rational soul is created and infused by God only when the embryo has received an adequate organization. The generation of animals, in contrast with other natural processes, is achieved as a result of a sequence of different forms: in man, this sequence of different forms culminates in the infusion of the rational soul, which substitutes the previous form (the sensitive soul) and takes over its functions. In this sense, the specifically human substantial form starts its being in the matter only in the final instant of the process of alteration of the matter itself; thus an inchoatio formae substantialis is to be excluded. The rational soul cannot be potentially contained in the human embryo from the first instant of conception, and then come into actuality when embryonic development is concluded; in fact, the substantial form does not accept in its being a development from potency to act, that is, the motion, because motion is proper to the quality. The male semen is not of the substance of the generator but derives from the surplus of food, as already said; the female ovum is considered only unformed matter; thus, in the first instant of conception, there is not yet an individual human with its own identity, because the embryo does not immediately have its soul, not even vegetative, but possesses it only after various processes of transformation of the subiectum. Having made this clear, one may ask if these reasons determine whether to adhere to the position of St. Thomas, or whether it is not more reasonable to adhere to immediate animation; in this second case, one may also consider whether immediate animation contradicts the fundamental principles of Thomistic metaphysics or rather, is compatible with them.

A THEORETIC REFLECTION ON THE POSITION OF ST. THOMAS To propose a theoretic reflection on the embryology of St. Thomas is not exactly the specific aim of the present discussion. Therefore, I will confine myself to the essential aspects of my elaboration on the problem of animation of the human embryo, which favours immediate animation, although adhering firmly to Aristotelian-Thomistic metaphysics. With regard to the human embryo in primo instanti conceptionis, genetics has reached a conclusion that was completely unknown in the era of St. Thomas: the human embryo, right from the beginning of its existence, is a living human being, a ‘third ‘party, from a genetic point of view, with respect to the father and mother. We have seen that St. Thomas agreed that the human embryo was living from the beginning, but, unaware of the laws of genetics, did not consider it necessary to accept the immediate presence of a vegetative soul, for he did not consider the embryo to be a human being from the beginning, with its own identity distinct from that of his parents. Furthermore, as the infusion of the 157 rational soul, according to St. Thomas, occurs only when the material disposing causality becomes adequate with respect to the spiritual form, the question can be narrowed down to establish whether the substrate material is adequately disposed from the moment of conception, or rather, after a certain time following conception. In my opinion, it may be affirmed that an adequate dispositio corporis is ontologically achieved from the moment of conception. In fact, the body is already programmed, from the time of the first phase of fertilization, in its general plan of development, from its genetic composition, albeit with the contribution of other factors: therefore, I fail to see why the act of being cannot be imparted to it immediately, with the infusion of the substantial form which is the rational soul and, at the same time, the sensitive and vegetative souls, according to the principle by which a form contains and can perform the functions of the inferior forms, as St. Thomas himself says. The need to accept a sort of metamorphosis in the embryo is not evident, that is, that it is firstly a plant, then an animal and finally, a man; this would compromise the unity and identity of the act of being in a conceived being, as Paolo Zacchia, the archiater of Innocent X, had already observed in the 2nd volume of his Medico-Legal Questions (published posthumously in 1661), although containing arguments that were rather confused and not well organized from a philosophical point of view. In answering the principal arguments in favour of belated animation, summarized above in § 2, it is necessary to presume that an important distinction exists between the actus essendi, that is the act of the substantial form, and existentia, intended as factualness, as the fact of existing physically in the world, a distinction which seems to me to be deep-rooted in the same texts of Aquinas as opposed to the extrinsicism of Avicenna’s ontology. On the level of existence, or rather of the material actualization of a being in its programming, there is undoubtedly a structuring in strata. However, on the level of being, I consider the vegetative, sensitive and rational souls to be transmitted to the human embryo in a single act which brings it into being; this implies simultaneity on an ontological level, and differentiation on an existential level. Due to the inadequate scientific knowledge of those times, St. Thomas did not distinguish these levels in the question on animation, because it did not appear necessary to him to do so: however, it seems to me that it has already been demonstrated that the notion of a Thomistic actus essendi differs from the notion of esse existentiae of a certain current of thought in Scholasticism of a formalistic basis, and from the concept of existence of Existentialism of the XX Century.40 While St.Thomas maintains that succession of different forms occurs in the embryo, I believe that the occurrence of a succession of manifestations and progressive exercise of different functions of which the rational soul is capable, should be confirmed: first, the vegetative function, then the sensitive, and finally the intellective function. This does not mean nor assert the presence of an inchoatio formae, and neither that of the becoming of the substantial form: in fact, as regards the precise act of being, the rational soul is already entirely in act from the beginning of conception; conversely, as regards the manifestations of its active potencies, progression is needed: St. Thomas himself supported the real distinction between the essence of the soul and its operative faculties and thus, its own operations. The human embryo is therefore a person from the moment of conception, although it has not yet manifested existentially all that being a person entails, and will not necessarily succeed in doing so. In fact, it may occur that the embryo dies in the initial phases of its development, or that it becomes a monstrous foetus, e.g. severely brain-damaged. Such human ‘monsters ‘are not subspecies of men ‘subhuman ‘from an ontological point of view; they are only so from an empirical point of view, because an adequate solid existential development is excluded for them on the basis of accidental reasons. In contrast, the Thomistic supporters of belated animation consider human monstrous foetuses to belong to the ‘reductive ‘human species.41 However, those who deny the dignity of a person to human monsters must therefore fail to consider the terminally ill who are affected by pathologies such as Alzheimer’s disease as being fully people, as

158 their life is reduced to a vegetative state, with severe damage to some parts of the specific instrumental organ of human rational knowledge. The supporters of belated animation take recourse to the objection of monozygotic twins. This is the objection: if the human embryo possessed a spiritual soul from the moment of conception, its subdivision during the first two weeks of life would not be possible: because it should be a human individual and the human individual is indivisible; because the spiritual soul should also divide itself, which is never divisible nec per se nec per accidens. I reply: it is certainly true that, in the case of monozygotic twins, neither the spiritual soul nor the human individual is subdivided, as such. Subdivision, not per se but per accidens, occurs in the substrate material, giving rise in this way to a new embryonic reality which, in the very instant of the subdivision, is informed by another spiritual soul created immediately by God. Monozygotic twins thus maintain a great resemblance as regards the substrate material, but each one possesses, in the first act, their own human, numerical and personal identity, being unique and unrepeatable. From what has been said, it emerges that the theory of immediate animation does not necessarily presuppose preformism, as perhaps, in the first half of the XX century, some Thomists, who were favourable to simultaneous animation, led people to believe, against their will.42 In fact, the theory of immediate animation does not imply that all of the organic differentiations are already contained in the embryo from the first instant of conception; it implies, rather, that there exists in the human embryo, from the first instant of conception, a disposing causality of the substrate material that is capable of guiding the gradual appearance of the differentiations according to the programme of the human species; the fact that this process can be interrupted or altered, causing degeneration into human monsters, depends on accidental causes, which can be genetic or derive from external agents (e.g. a pharmacological therapy). Therefore, it appears to me that the theory of instantaneous animation is also compatible with that of epigenesis. In conclusion, the theory of immediate animation, although not being in agreement with the embryology of St. Thomas, is not in contradiction with the fundamental principles of Thomistic metaphysics.

CONCLUSIVE ETHICAL CONSIDERATIONS The supporters of belated animation cannot accept that the embryo is a person from the moment of conception, and therefore, if they are consistent, cannot accept the existence of the conceived being’s rights in the first two weeks of its life. To be consistent, they must consider abortion performed before the infusion of the rational soul to be a seriously illicit act of violence, but which cannot be classified as a crime against a human person, rather a grave attempt on life according to the order established by nature and, furthermore, a crime against the family, that can be associated with artificial con- traceptive practices. Real and true homicide would occur only when abortion is carried out after the infusion of the rational soul in the embryo.43 Nevertheless, the ancient advocates of belated animation acknowledge that, from a moral and juridical point of view, even abortion carried out in the initial phases of the life of the embryo can be considered equivalent to that performed in subsequent phases, by a practical principle of prudence: as it regards human life, neither probabilism nor even probabiliorism can be applied, rather a mitigated tutiorism must be applied: that is, in doubt, the human embryo must not be killed, even if the more probable hypothesis is in favour of its not being a person.44 This explains why most moralists and canonists of the first half of the XX century, even if they were Thomists, did not fully accept the theory of belated animation. However, some recent supporters of belated animation, or authors who, in any case, deny the rights of the conceived being in the first two weeks of life, insist on accepting the liceity or, at least, the lack of gravity, in abortion during the first phases of the life of the embryo; these people call the embryo a ‘pre-embryo ‘in the first phases.

159 I will decline from quoting names and studies, as it is not the polemic aspect which is of interest here; rather, it is important to me to emphasize that the theory of belated animation inevitably provokes a tension between practice and theory in the field of a Christian viewpoint; the latter does not represent an argument of a determining nature, but it is, at least, always a substantial indication to the contrary. The theory of immediate animation, however, does not consider the moment in which abortion is performed to be important for the purposes of an ethical evaluation: the conceived being has a right to life and its killing is a crime against the human person. The abolition of a distinction between an animated foetus and an unanimated foetus, sanctioned by Blessed Pius IX in the Constitution Apostolicae Sedis of 1869, has an eminently practical value for the supporters of belated animation, as it implies the same moral evaluation and same sanction for the killing of the embryo in whatsoever moment of his life; on the other hand, for the supporters of immediate animation, the abolition of the distinction between an animated foetus and an unanimated foetus is above all, the logical consequence of a theoretical position that is favourable to the embryo’s being a person from the first instant of conception.45 Even now the Catholic Church, using prudence, does not pronounce herself in a definitive way on a question that is per se of a supremely philosophical nature. Nonetheless, it appears to me that the direction taken by the Magisterium is that of the theory of immediate animation. In fact, in the Instruction of the Congregation for the Doctrine of the Faith Donum Vitae, of 1987, an important statement of the Declaration on Procured Abortion of 1974 is recalled and elaborated by the same Congregation: ‘From the time that the ovum is fertilized, a new life is begun which is neither that of the father nor of the mother; it is rather the life of a new human beingâ?¦Right from fertilization is begun the adventure of a human lifeâ?¦this teaching remains valid and is further confirmed, if confirmation were needed, by recent findings of human biological science which recognize that in the zygote resulting from fertilization the biological identity of a new human individual is already constituted. Certainly no experimental datum can be in itself sufficient to bring us to the recognition of a spiritual soul; nevertheless, the conclusions of science regarding the human embryo provide a valuable indication for discerning by the use of reason a personal presence at the moment of this first appearance of a human life: how could a human individual not be a human person? The Magisterium has not expressly committed itself to an affirmation of a philosophical nature, but it constantly reaffirms the moral condemnation of any kind of procured abortion. This teaching has not been changed and is unchangeable. Thus the fruit of human generation, from the first moment of its existence, that is to say from the moment the zygote has formed, demands the unconditional respect that is morally due to the human being in his bodily and spiritual totality. The human being is to be respected and treated as a person from the moment of conception; and therefore from that same moment his rights as a person must be recognized, among which in the first place is the inviolable right of every innocent human being to life ‘.46 Not only abortions, but also all operations which manipulate the human embryo to the point of damaging the same, freezing included, which take place in the various techniques of artificial fertilization, are gravely contrary to the dignity of man.

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1 ARISTOTLE, De Anima, 4, 429 b 1-10. 2 Ibid., III, 5, 430 a 15-20. 3 ARISTOTLE, De Anima, II (B), 3, 736 b, 1-4; 12-16. 4 Ibid., II (B), 3, 736 b, 21-29. 5 Ad Tertium Dicendum quod illa vis activa quae est in semine, ex anima generantis derivata, est quasi quaedam motio ipsius animae generantis: nec est anima, aut pars animae, nisi in virtute; sicut in serra vel securi non est forma lecti, sed motio quaedam ad talem formam. Et ideo non oportet quod ista vis activa habeat aliquod organum in actu; sed fundatur in ipso spiritu incluso in semine, quod est spumosum, ut attestaur eius albedo. In quo etiam spiritu est quidam calor ex virtute caelestium corporum, quorum etiam virtute agentia inferiora agunt ad speciem, ut supra (q. 115, a. 3, ad 2) dictum est. Et quia in huiusmodi spiritu concurrit virtus animae cum virtute caelesti, dicitur quod homo generat hominem, et sol (ARISTOTLE, 2 Physic., c. 2, lect. 4). Calidum autem elementare se habet instrumentaliter ad virtutem animae, sicut etiam ad virtutem nutritivam, ut dicitur in 2, ID., De Anima, c.4, lect. 8, 9; ST. THOMAS AQUINAS, Summa Theologiae, I, q. 118, a. 1, ad 3). On the belief of St. Thomas that semen is the residue of the final digestion, cf. ID., De Potentia, q. 3, a.9, ad 9 (b), where the Angelic Doctor makes reference to Generation of the Animals by ARISTOTLE (I, 18, 725 a. 11-13; 724 b. 23-726 a. 28). 6 ST. THOMAS AQUINAS, De Veritate, q. 16, a. 1; ID., De substantiis separatis, 8, at the end. 7 ‘Similiter dicendum esset quod vegetativum et sensitivum sunt in intellectivo, ut trigonum et tetragonum in pentagonoâ?¦Unde anima intellectiva, quamvis sit ab exteriori agente, habet tamen virtutes quas habent anima vegetativa et sensitiva, quae sunt ab interioribus agentibus ‘(ST. THOMAS AQUINAS, De Unitate Intellectus contra Averroistas, Città di Castello, 1886: 469). Manifestum est autem quod primum quo corpus vivit, est anima. Et cum vita manifestetur secundum diversas operationes in diversis gradibus viventium, id quo primo operamur unumquodque horum operum vitae, est anima: anima enim est primum quo nutrimur, et sentimus, et movemur secundum locum; et similiter quo primo intelligimus. Hoc ergo principium quo primo intelligimus, sive dicatur intellectus sive anima intellectiva, est forma corporis. ‘Et haec est demonstratio Aristotelis in De Anima. (ID., Summa Theologiae, I, q. 76, a. 1). 8 ID., De Potentia, q. 3, a. 9, to 9 (c). 9 ‘Et ideo sicut corpus habet esse per animam, sicut per formam, ita et unitur animae immediate, in quantum est forma corporis ‘; ID., In Lib. Anima, II, lect. I, Taurini: Ed. Pirotta, 1925: 234; ID., Summa Theologiae, I, q. 76. 10 ‘In generatione aliorum hominum locum habet quod dicit Philosophus propter hoc quod successive corpus formatur et disponitur ad animam: unde primo, tamquam imperfecte dispositum, recepit animam imperfectam et postmodum, quando perfecte est dispositum, recipit animam perfectam ‘(ST. THOMAS AQUINAS, Summa Theologiae, III, q. 33, a. 2, ad 3). Among the many Thomistic texts in which the spirituality and immortality of the human soul is held, cf. Ibid., I, q. 75, aa. 5-6. 11 For a wider knowledge on this subject, I take the liberty of recommending referral to my book: PANGALLO M., Il principio di causalità nella metafisica di S. Tommaso, Città del Vaticano: LEV, 1991: 53-64. 12 ‘Ad Quintum Dicendum quod anima illud esse in quo ipsa subsistit, communicat materiae corporali, ex qua et anima intellectiva fit unum, ita quod illud esse quod est totius compositi, est etiam ipsius animae. Quod non accidit in aliis formis, quae non sunt subsistentes. Et propter hoc anima humana remanet in suo esse, destructo corpore: non autem aliae formae ‘(ST. THOMAS AQUINAS, Summa Theologiae, I, q. 76, a. 1, ad 5). 13 Sed positio Aristotelis multo rationabilior est: quia nihi incipit vel fit vel generatur nisi secundum modum quo esse habet: et ideo concedimus animam sensibilem et vegetabilem ex traduce esse. Modus autem traductionis talis est; cum enim omne agens univocum et proximum inducat speciem suam in 161 patiente, et cibus, secundum quod est patiens et alteratum, in nutrimentum corporis cedat (quia nutrit secundum quod est potentia caro, ut in 1 Gener., text. 39 et in De Anima a text. 45 usque ad 50 dicitur), oportet quod in fine speciem et virtutem nutrimenti recipiat. Ante ergo ultimam assimilationem, quando efficitur actu pars determinata, ut caro vel os, est in eo virtus speciei indeterminate ad hoc vel illud: quia determinata ad hoc vel illud est secundum propriam virtutem determinatae partis: et ideo cum semen sit residuum ultimi cibi propinquissimi ad ultimam conversionem, est in eo potentia totum et non actu aliqua pars: ante vero quam resolvatur per actum virtutis generativae separatum a reliquo sui generis, est in eo potentia illa indistinta sicut forma totius non est in parte nisi in potentia: quando autem separatur, efficitur actu habens talem potentiam vel formamâ?¦Haec autem potentia non est passiva in semine maris sicut dicimus ligna et lapides esse in potentia domus (sic enim est potentia in mestruo mulieris), sed est potentia activa, sicut dicimus formam domus in mente artificis esse potentia domum; unde arti comparat eam Philosophus in 17 de Animalibus (De generat. animal, 2, 20); et hanc potentiam Avicenna et Commentator in 7 Metaphysic. vocant virtutem formativam: quae quidam virtus quantum ad modum operandi media est inter intellectum et alias vires animae. Aliae enim vires utuntur in suis operationibus determinatis organis: intellectus autem nullo: haec autem utitur aliquo corporali in sua operatione quod nondum habet determinatam speciem. Subiectum autem et organum huius virtutis est spiritus vitalis inclusus in semine; unde ad continendum huiusmodi spiritum semen est spumosum, et haec est causa albendinis eiusâ?¦Illi autem corporali spiritui coniungitur triplex calor: scilicet calor elementaris, qui est sicut instrumentum resolvens et consumens et huiusmodi operans; et calor animae, qui est vivificans; et calor caeli cuius virtute movet ad speciem determinatam: et virtute huius triplicis caloris, virtus formativa convertit materiam a muliere praeparatam in substantiam membrorum per modum quo est transmutatio corporis in augmento ut 15 (De generat. animal, 2, 1 et 3) de Animalibus dicitur; et secundum quod proceditur in perfectione organorum, secundum hoc anima incipit magis ac magis actu esse in semine, quae prius erat in potentia: ita quod conceptum primo participat opera vitae nutritivae, et tunc dicitur vivere vita plantae; et sic deinceps, donec perveniat ad completam similitudinem generatis ‘. 14 Respondeo. Dicendum quod, circa hanc quaestionem antiquitus diversa dicebantur a diversis. Quidam namque dicebant, animam filii ex parentis anima propagari, sicut et corpus propagatur ex corpore. Alii vero dicebant, omnes animas seorsum creari; sed ponebant a principio eas extra corpora fuisse creatas simul, et post modum corporibus seminatis coniungebantur, vel proprio motu voluntatis, secundum quosdam, vel Deo mandante et faciente, secundum alios. Alii vero dicebant, animas simul cum creantur, corporibus infundi. Quae quidem opiniones, quamvis aliquo tempore sustinerentur, et quae earum esset verior in dubium verteretur, ut patet ex Augustino (in X super Genes. ad litter., cap. XXI et XXII), et in libris quos scribit de origine animae; tamen primae duae postmodum iudicio Ecclesiae sunt damnatae, et tertia approbata. 15 Diligenter autem consideranti apparet rationabiliter illam opinionem esse damnatam quae ponebat animam rationalem cum semine propagari, de qua nunc est quaestio. Et hoc tribus rationibus potest videri ad praesens: Prima est, quia rationalis anima in hoc a ceteris formis differt, quod aliis formis non competit esse in quo ipsae subsistant, sed quo eis res formatae subsistant; anima vero rationalis sic habet esse ut in eo subsistens; et hoc declarat diversus modus agendi. Cum enim agere non possit nisi quod est, unumquodue hoc modo se habet ad operandum vel agendum, quomodo se habet ad esse; unde, cum in operatione aliarum formarum necesse sit comunicare corpus, non autem in operatione rationalis animae, quae est intelligere et velle; necesse est ipsi rationali animae esse attribui quasi rei subsistenti, non autem aliis formis. Et ex hoc est quod inter formas, sola rationalis anima a corpore separatur. Ex hoc ergo patet quod anima rationalis exit in esse, non sicut formae aliae, quibus proprie non convenit fieri, sed dicuntur fieri facto quodam. Sed res quae fit, proprie et per se fit. Quod autem fit, fit vel ex materia vel ex nihilio. Quod vero ex materria fit, necesse est fieri ex materia contrarietati subiecta. Generationes enim ex contraris sunt, secundum Philosophum (lib. I De Generatione animalium, capit. XVIII): unde cun anima vel omnino materiam non habeat, vel ad minus non habeat 162 materiam contrarietati subiectam, non potest fieri ex aliquo. Unde restat quod exeat in esse per creationem, quasi ex nihilo facta. Ponere autem quod per generationem corporis fiat, est ponere ipsam non esse subsistentem, et per consequens cum corpore corrumpi. 16 Secunda ratio est, quia impossibile est actionem corporeae virtutis ad hoc elevari quod virtutem penitus spiritualem et incorpoream causare possit; nihil enim agit ultra suam speciem; immo agens oportet esse praestantius patiente, secundum Augustinum (XII super Genesim ad litteram, capit. XVI). Generatio autem hominis fit per virtutem generativam, quae organum habet corporale; virtus etiam quae est in semine, non agit nisi mediante calore, ut dicitur in XVI de Animalibus (lib. II De Gen. animal., cap. III); unde, cum anima rationalis sit forma penitus spiritualis, non dependens a corpore nec communicans corpori in operatione, nullo modo per generationem corporis potest propagari, nec produci in esse per aliquam virtutem quae sit in semine. 17 Tertia ratio est, quia, omnis forma quae exit in esse per generationem, vel per virtutem naturae, educitur de potentia materiae, ut probatur in VII Metaph. Anima vero rationalis non potest educi de potentia materiae. Formae enim quarum operationes non sunt cum corpore, non possunt de materia corporali educi. Unde relinquitur quod anima rationalis non propagetur per virtutem generantis; et haec est ratio Aristotelis. 18 Praeterea, embrio antequam anima rationali perficiatur, habet aliquam operationem animae; quia augetur et nutritur et sentit. Sed operatio animae non est sine vita. Ergo vivit. Vitae vero corporis principium est anima. Ergo habet animam. Sed non potest dici quod adveniat ei alia anima; quia tunc in uno corpore essent duae animae. Ergo ipsa anima quae prius erat in semine propagata, est anima rationalis. 19 Quidam namque assimilaverunt in generatione humana progressum animae rationalis progressui corporis umani, dicentes, quod sicut corpus humanum in semine est virtualiter, non tamen habens actu humani corporis perfectionem, quae distinctione organorum consistit, sed paulatim per virtutem seminis ad perfectionem huiusmodi pervenitur; ita in principio generationis est ibi anima, virtute quadam habens omnem perfectionem quae postea apparet in homine completo, non tamen eam habens actu, cum non apparent animae actiones, sed processu temporis paulatim eam acquirit; ita quod primo appareant in ea actiones animae vegetabilis, et postmodum animae sensibilis, et tandem animae rationalis. Et hanc opinionem tangit Gregorius Nyssenus in lib. quem fecit de homine. 20 Dicitur enim in II De Anima, quod potentia vitae est in corpore physico organico, cuius actus est anima, non est abiiciens animam, sicut semen et fructus; ex quo datur intelligi, quod semen est ista in potentia ad animam quod anima caret. Secundo, quia cum semen nondum sit ultima assimilatione membris assimilatum (sic enim eius resolutio esset corruptio quaedam) sed sit superfluitas ultimatae digestionis, ut dicitur XV De Animalibus, nondum fuit in corpore generantis existens anima perfectum; unde non potest esse quod in principio suae decisionis sit in eo anima. Tertio, quia dato quod cum eo decideretur anima, non tamen potest hod dici de anima rationali; quae cum non sit actus alicuius partis corporis, non potest deciso corpore decidi. The principal Aristotelian texts to which St. Thomas refers are: De Anima, II, 1, 412 b. 25; De Animalibus I, 18, 725 a. 11-13; 724 b. 23-726 a. 28; I, 19, 726 a. 28-b1. 21 Et iterum impossibile est unius et eiusdem rei esse plures formas substantiales nam cum forma substantialis faciat esse non solum secundum quid, sed simpliciter, et constituat hoc aliquid in genere substantiae, si prima forma hoc facit, secunda adveniens, inveniens subiectum iam in esse substantiali constitutum, accidentaliter ei adveniet; et sic sequeretur quod anima sensibilis et rationalis in homine corpori accidentaliter uniantur. Nec potest dici quod anima vegetabilis quae in planta est forma substantialis, in homine non sit forma substantialis, sed dispositio ad formam: quia quod est de genere substantiae nullius accidens esse potest, ut dicitur in I Physic. 22 Sed hoc nullo modo potest stare: quia vel hoc ita intelligitur quod natura intellectualis sit alia anima a vegetabilis et sensibili, et sic redit in idem cum secunda opinione: vel intelligitur ita quod ex istis tribus naturis constituatur substantia animae in qua natura intellectualis erit ut formae, et natura sensibilis et vegetabilis erit ut materiale. Ex quo sequitur quod cum natura sensibilis et vegetabilis sint 163 corruptibiles, utpote de materia eductae, substantia animae humanae nonpossit esse perpetua. Sequitur idem etiam inconveniens quod inductum est contra primam, scilicet quod forma substantialis successive educatur in actum. 23 Alii vero dicunt, quod embrio non habet animam, quousque perficiatur anima rationali: operationes autem vitae quae in eo apparent sunt ex anima matris. Sed hoc non potest esse: nam in hoc viventia a non viventibus differunt, quia viventia movent se ipsa secundum operationes vitae, quod de non viventibus dici non potest; unde non potest esse quod nutriri et augeri, quae sunt propriae operationes viventis, sint in embrione a principio exstrinseco, scilicet ab anima matris. Et praetera virtus nutritiva matris assimilaret cibum corpori matris, et non corpori embrionis; cum nutritiva deserviat individuo sicut generativa speciei. Et iterum sentire non posset esse in embrione ex anima matrisâ?¦Et ideo aliter est dicendum, quod in semine a principio suae decisionis non est anima, sed virtus animae; quae fundatur in spiritu qui in semine continetur quod de natura sui spumosum est, et consequens corporalis spiritus contentivum. Ista autem virtus agit disponendo materiam et formando ad susceptionem animae. 24 In generatione autem animalis apparent diversae formae substantiales, cum primo appareat sperma, et postea sanguis, et sic deinceps quousque sit forma hominis vel animalis. Et sic oportet quod huiusmondi generatio non sit simplex, sed continens in se plures generationes et corruptiones. Non enim potest esse quod una et eadem forma substantialis gradatim educatur in actum, ut ostensum est. 25 Sic ergo per virtutem formativam quae a principio est in semine, abiecta forma spermatis, inducitur alia forma; qua abiecta, iterum inducatur alia: et sic primo inducatur anima vegetabilis; deinde ea abiecta, inducatur anima sensibilis et vegetabilis simul; qua abiecta inducator non per virtutem praedictam sed a creante, anima quae simul est rationalis sensibilis et vegetabilis. Et sic dicendum est secundum hanc opinionem quod embrio antequam habeat anima rationalem, vivit et habet animam, qua abiecta, inducitur anima rationalis. Et sic non sequitur duas animas esse in eodem corpore, nec animam rationalem traduci cum semine. 26 I am leaving aside the question of the chronology of the writings of Aquinas, given that there is not a significant evolution in his ideas, as regards the human embryo, and I am keeping to the theoretical matter of the various texts. 27 Ad Quartum Dicendum quod in animalibus perfectis, quae generantur ex coitu, virtus activa est in semine maris, secundum Philosophum in libro 2 De Generat. Animal. (cc. 3, 4); materia autem foetus est illud quod ministratur a femina. In qua quidem materia statim a principio est anima vegetabilis, non quidem secundum actum secundum, sed secundum actum primum, sicut anima sensitiva est in dormientibus. Cum autem incipit attrahere alimentum, tunc iam actu operatur. Huiusmodi igitur materia transmutatur a virtute quae est in semine maris, quousque perducatur in actum animae sensitivae: non ita quod ipsamet vis quae erat in semine, fiat anima sensitiva; quia sic idem esset generans et generatum; et hoc magis esset simile nutritioni et augmento, quam generationi, ut Philosophus dicit (1 De Gen., et Cor., c. 5, lect. 14). Postquam autem per virtutem principii activi quod erat in semine, producta est anima sensitiva in generato quantum ad aliquam partem eius principalem, tunc iam illa anima sensitiva prolis incipit operari ad complementum proprii corporis, per modum nutritionis et augmenti. Virtus autem activa quae erat in semine, esse desinit, dissoluto semine, et evanescente spiritu qui inerat. Nec hoc est inconveniens: quia vis ista non est principale agens, sed instrumentale: molto autem instrumenti cessat, effectu iam producto in esse. (ST. THOMAS AQUINAS, Summa Theologiae, I, q. 118, a. 1, ad 4). 28 Similiter etiam anima intellectiva, cum habeat operationem sine corpore, est subsistens, ut supra habitum est: et ita sibi debetur esse et fieri. Et cum sit immaterialis substantia, non potest causari per generationem, sed solum per creationem a Deo (Ibid., I, q. 118, a. 2). 29 Et ideo dicendum est quod, cum generatio unius semper sit corruptio alterius, necesse est dicere quod tam in homine quam in animalibus aliis, quando perfectior forma advenit, fit corruptio prioris: ita tamen quod sequens forma habet quidquid habebat prima, et adhuc amplius. Et sic per multas generationes et corruptiones pervenitur ad ultimam formam substantialem, tam in homine quam in aliis 164 animalibus. Et hoc ad sensum apparet in animalibus ex putrefatctione generantis. Sic igitur dicendum est quod anima intellectiva creatur a Deo in fine generationis humanae, quae simul est et sensitiva et nutritiva, corruptiis formis praeexistentibus. (Ibid, I, q. 118, a. 2 ad 2). 30 Igitur anima nutritiva et sensitiva esse incipiunt per seminis traductionem, non autem intellectiva. (ST. THOMAS AQUINAS, Summa Contra Gentiles, II, c. 86). 31 ‘Conceptum prius esse animal quam hominem, non ostendit rationalem animam cum semine propagari, Nam anima sensitiva per quam animal erat, non manet, sed ei succedit anima quae est simul sensitiva et intellectiva, ex qua est animal et homo simul ‘(Ibid., II, c. 89). 32 Manifestum est autem quod anima rationalis est ultima et perfectissima forma quam potest consequi materia generabilium et corruptibilium. Convenienter igitur naturalia agentia in inferiora causant praecedentes dispositiones et formas; supremum vero agens, scilicet Deus, causat ultimam formam, quae est anima rationalis (ID., Compendium Theologiae, c. 93). 33 Anima autem vegetabilis et sensibilis non sunt formae subsistentes, alias remanerent post corpora; unde oportet quod fiant a generante per actionem compositorum, sicut et ceterae formae materiales. Sola autem anima intellectiva, quae habet esse subsistens, cum maneat post corpus, est ab extrinseco per creationem. Si autem sensibile et vegetabile et intellectivum in homine in diversis substantiis animae radicantur, tunc et vegetabilis et sensibilis hominis in generante erit (ST. THOMAS AQUINAS, Quaestiones Quodlibetales, q. 5, a. 1). 34 ‘Licet in semine non sit anima, est tamen in semine virtus dispositiva corporis ad animae receptionem, quae cum corpori infunditur, etiam ei suo modo conformatur, eo quod omne receptum est in recipiente per modum recipientis (ID., In Epist. Pauli ad Romanos Expositio, cap. V, lect. III). 35 ‘Et ideo in primo instanti quo materia adunata pervenit ad locum generationis, fuit perfecte formatum corpus Christi et assumptum ‘(ID., Summa Theologiae, III, q. 33, a. 1). 36 Sed ipsa formatio corporis, in qua principaliter ratio conceptionis consistit, fuit in instanti, duplici ratione. Primo quidem, propter virtutem agentis infinitam, scilicet Spiritus Sancti, per quem corpus Christi est formatum, ut supra (q. 32, a. 1) dictum est. Tanto enim aliquod agens citius potest materiam disponere, quanto fuerit maioris virtutis. Unde agens infinitae virtutis potest in instanti materiam disponere ad debitam formam. Secondo, ex parte personae Filii, cuius corpus formabatur. Non enim erat congruum ut corpus humanum assumeret nisi formatum. Si autem ante formationem perfectam aliquod tempus conceptionis praecessisset, non posset tota conceptio attribui filio Dei, quae non attribuitur ei nisi ratione assumptionis (Ibid.). 37 Ad hoc quod conceptio ipsi Filio Dei attribuatur, ut in Symbolo (Apostolorum) confitemur, dicentes, ‘qui conceptus est de Spiritu Sancto ‘; necesse est dicere quod ipsum corpus, dum conciperetur, esset a Verbo Dei assumptum. Ostensum est autem supra (q. 6, aa. 1, 2) quod Verbum Dei assumpsit corpus mediante anima, et animam mediante spiritu, idest intellectu. Unde oportuit quod in primo instanti conceptionis corpus Christi esset animatum anima rationali. (Ibid., III, q. 33, a. 2). 38 Si autem caro Christi fuisset concepta antequam susciperetur a Verbo, habuisset aliquando aliquam hypostasim praeter hypostasim Verbi Dei. Quod est contra rationem incarnationis, secundum quam ponimus Verbum Dei esse unitum humanae naturae, et omnibus partibus eius, in unitate hypostasis: nec fuit conveniens quod hypostasim praexistentem humanae naturae, vel alicuius partis eius, Verbum Dei sua assumptione destrueret. Et ideo contra fidem est dicere quod caro Christi prius fuerit concepta, et postmodum assumpta a Verbo Dei (ST. THOMAS AQUINAS, Summa Theologiae, III, q. 33, a. 3). 39 In generatione aliorum hominum locum habet quod dicit Philosophus, propter hoc quod successive corpus formatur et disponitur ad animam: unde primo, tanquam imperfecte dispositum, recipit animam imperfectam; et postmodum, quando perfecte est dispositum, recipit animam pefectam. Sed corpus Christi, propter infinitam virtutem agentis, fuit perfecte dispositum in instanti. Unde statim in primo instanti recepit formam perfectam, idest animam rationalem (Ibid., III, q. 33, a. 2, ad 3). 40 I refer in particular to the studies, already well known, of Etienne Gilson and Cornelio Fabro, who, although coming from different hermeneutic perspectives, converge in emphasizing the originality of 165 the metaphysical notion of esse ut actus in St. Thomas, which cannot be reduced to the notion of existentia. The same real distinction between essentia and esse in a finite being cannot be interpreted simply as a distinction between essentia and exsistentia. Cf. FABRO C., Partecipazione e causalità , Torino: SEI, 1961; ID., Dall’essere all’esistente, Brescia: Morcelliana, 1957; ID., Esegesi tomistica, Roma: PUL, 1969. 41 On this point it is worth noting the affirmation of the eminent moralist Lanza, supporter of the theory of belated animation, in his historical-systematic essay: LANZA A., La questione del momento in cui l’anima razionale è infusa nel corpo, Roma, 1939: ‘It is certain that the human monster, no matter how malformed and far from the perfection of a normal individual it is, belongs to the reductive human species (p. 279). This opinion reflects a conviction which was then widespread among supporters of belated animation, even after the study of Lanza. For a concise exposition of the reasons in favour of belated animation, see: HERING H., De tempore animationis foetus umani, vol. XXVIII, Angelicum 1951, 28: 18-29; HUDECZEK M., De tempore animationis foetus umani secundum embryologiam modernam, vol. XXIV, Angelicum 1952, 29: 162-181. 42 GREDT J., Elementa philosophiae aristotelico-thomisticae, vol. I, Freiburg: Herder, 1937: 347-351; Ibid., pp. 421-422. Gredt wrote the following: ‘Adnotatio circa Aristotelis veterumque scholasticorum doctrinam de generatione. Veteres, cum cellulas germinales ignorarent, semina neque organisata neque viventia putabant ‘(Ibid., pp. 350-351); ‘attamen cum recentibus optime dicitur: statim ab initio, coniunctis cellulis germinalibus, haberi organisationem specialem et dispositiones proximas ad animae intellectivae infusionem ‘(Ibid., p. 422). The formulation mentioned above is accused of preformism by Hudeczeck: ‘Et si respondetur, quod materia haec sit semper a principio disposita ad animam humanam suscipiendam quia ex ea nonnisi homo generari potest, caute hoc accipiendumâ?¦Quodsi aliqui hoc est similia affirmant, dicendum est eorum scientiam, in certo sensu preformisticam, novo progressu Biologiae iam superatam esse ‘(Ibid., p. 177). 43 Lanza writes the following: ‘Considering things from an abstract point of viewâ?¦the evaluation must be different, both moral and juridical of the crime of abortion before and after infusion of the rational soul: after animation, it is a matter of true violation of the right which the person has to life; whereas before (animation), in absence of the rational soul, one cannot speak about a similar violation; but it is a question of violence, sempre illecita (always illicit), inflicted on the order established by nature, which, from a moral point of view, may be traced back to being on the same level as contraceptive practices and may be considered a crime against the family ‘; LANZA, La questione del momento in cuiâ?¦, p. 297). 44 In fact, Lanza himself specifies the following: ‘However, this does not prevent us also, at the current point in the debate, from considering as obligatory, in a practical sense, the regulations dictated on the subject by the ecclesiastic legislation and inspired by those wise criteria of practical prudence which have always guided the discipline of the Church ‘(Ibid.). 45 The Pontiff Sixtus V, already in the Constitution Ad effraenatam of 1588, although maintaining the theory of there being a distinction between an animated and an unanimated foetus, inflicted the same punishments foreseen for voluntary homicide also for those who procured abortion, in whatsoever moment of gestation. The severity of the Constitution of Sixtus V was mitigated by Gregory XIV in 1591. As regards the reflections of the Apostolicae Sedis of Pius IX in the moral-theological debate at the end of the eighteenth century on craniotomy and on abortion in the first phases of the life of the embryo, I take the liberty of recommending referral to my own work: PANGALLO M., La craniotomia nella Summula Theologiae Moralis del Card. Giuseppe D’Annibale, Divinitas 1986, 2: 167-174. 46 CONGREGATION FOR THE DOCTRINE OF THE FAITH, Donum Vitae (February 22, 1987), Part I, n. 1.

166 P. IDE

IS THE HUMAN EMBRYO A PERSON? STATUS QUESTIONIS AND DETERMINATION

This brief intervention intends to clarify the following question: is the human embryo a person?1 By embryo I mean a human being after its conception, or, in other words, after fertilization. Moreover, a zygote is readily called a ‘one-cell embryo ‘today. Later I will define what I mean by the name person. To respond to the question raised, I will only refer to data from the sciences and philosophy. I will thus leave aside the properly theological approach and all the more willingly because the Catholic Church invites us to do this: ‘How could a human individual not be a human person? The Magisterium has not expressly committed itself to an affirmation of a philosophical nature ‘.2 Lastly, in order to clarify the subject as much as possible, it will be presented in the rigorous form of a quaestio disputata.

STATUS QUESTIONIS The negative response At first glance, it seems that the answer is negative. In this perspective, a zygote has to pass through a certain number of stages before it can be qualified as fully or really human.3 According to the criteria of humanization chosen, an embryo becomes a full-fledged subject: when the two genetic patrimonies of paternal and maternal origin recombine in the new genome of the embryo, or between 21-22 hours after fertilization;4 after implantation;5 on the fourteenth day6 when all totipotentiality has disappeared and the primitive line emerges;7 when the unity of the human organism becomes irrevocably signified by the emergence of an organization and the impossibility for homozygote twins to form between the 14thand the 18th day;8 when the principal organs come into being (the eighth week);9 when some electro-encephalographic activity is detected;10 when the brain becomes capable of certain functions such as sensation, memory or learning (twentieth week);11 ‘at the stage of maturation that allows autonomous life ‘;12 that is, at the sixth or seventh month,13 when self- awareness sets in for which a date much later after birth cannot be fixed;14 the criterion of parents’recognition which is even more variable if it requires knowledge about the existence of the zygote and so is necessarily after its emergence.15 Lastly, there are those who affirm the continuity between gametes and zygote and thus make human individuality refer back to them.16 Logic teaches us that the crux of a demonstration lies in its middle term, not its conclusion. Given that in the name of the same argument, sometimes two different dates are put forward, I will distribute the demonstrations in a way that is not contingent on this calendar of access to humanization ‘of which the multiform distribution shows that it is somewhat uncertain ‘but on arguments. I will isolate six of these arguments, of which the first five are the most common ones. I will develop the sixth because it is still somewhat present among Catholics, and this will enable me to enter into the debate with the Thomist philosophers. The possibility of homozygote twins A person is an individual and so etymologically he is an indivisible being. Now, an embryo is divisible, at least up to fifteen days, as the existence of homozygote twins shows: that is, twins that come from the same initial cell. So an embryo is not a human person. One cannot speak about a human person until the possibility of the emergence of real twins has passed. ‘If we consider that a fertilized ovum is a person ‘, wrote Xavier Thévenot: ‘How then can it be accounted for philosophically and theologically that in the case of a split into twins (which is possible up to 14 days after conception) one person can become two persons? How can a reality whose individuality is not even certain be considered a person? ‘.17

The presence of totipotent cells 167

A complete living individual is a differentiated organism made up of many organs ordained to one or more functions. Until the eight cell stage,18 the embryo is made up of totipotent cells: that is, cells capable of becoming any organ. Therefore, in the beginning, we are not in the presence of a fully constituted individual, a fortiori a human person.

The need for a conscience A person is an individual capable of suffering, relations, reason, freedom, etc.19 To exercise one of these abilities supposes the presence of a brain. But the central nervous system does not begin to be formed until six to eight weeks after the fusion of the gametes. So as long as it is not endowed with a brain, an embryo is not a person.20 The argument can be refined based on Paul Ricoeur’s position in his principal book: ‘Soi-meme comme une autre’. The hermeneutics of self are caught up in a dialectic of sameness and ipseity that is resolved in the concept of narrative identity. In other words, the self has access to its ipseity through narration. In fact, the constitution of self, grasping its permanence, is declined in two versions: identity as sameness (or identity-idem) to which responds a permanence of the substratum, and identity as ipseity (or identity-ipse) to which narrative permanence responds. The first permanence introduces some unsolvable apories.21 So the question can only be answered ‘yes ‘by referring to another kind of permanence that is introduced by the word through which the subject enters into self-possession and understanding of self.22 Quite obviously an embryo has an identity-idem; it does not have an identity- ipse. Its self cannot be narrated. Consequently, the embryo cannot be human from the outset.

The need for recognition by others The I is only awakened by a You that recognizes it, in this case, the parents. This recognition of the new life has nothing spontaneous about it: it implies a desire for a child and a parental project. Therefore, an embryo only becomes a person in certain conditions defined by its parents. ‘An embryo becomes a person under two conditions ‘, explains professor Rene Frydman. ‘It must have the physiological abilities to develop and it must be accepted. It only belongs to the desire of the people who have elaborated it in thought and in practice. A woman decides in conscience whether or not she wants to bear it. A destiny cannot be found for embryos against the parents’willâ?¦What is important are democracy and dignity ‘.23

The frequency of spontaneous abortions The spontaneous abortions that occur during the first days of ontogenesis are numerous.24 What is natural comes about more often than what is not. Isn’t the extreme regularity of physical laws a criterion of their naturalness? Consequently, it would be against nature for a zygote to be a human person. Based on the figure of half the non-implanted fertilized cells, the theologian Karl Rahner asks: ‘How can it be thought that 50% of human beingsâ?¦never get beyond a priori this first stage of human existence? ‘.25

The need for an organized body I will present this argument in detail because of the fact that it is common among philosophers of a Thomist school ‘at least since the 70s.26 The apory can be formulated based on the Aristotelian definition of the vital principle. The soul is the first act of an organized body. Now, a zygote and an embryo in their first stages do not present sufficient organization to perform an operation preparing for intellective life. So an embryo is not a suitable subject for a human soul.27 Benedicte Mathonat refines the argument. The starting point is once again the Aristotelian definition of the soul: ‘The body, the subject of the soulâ?¦is organized’. To be organized is to be a principle of operation: ‘The organization in question isâ?¦that which manifests the body’s power to operate ‘. ‘The 168 body, the subject of the soul, is thus one that is capable of carrying out the operations of human life ‘. The operation proper to human life is intellectual operation. But ‘the human intellect only has knowledge based on the senses ‘. So, ‘the human soul has for its own subject a sensible body ‘. But the nervous system is the organic principle of sensibility.28 Therefore, to be informed by a human soul, an embryo must have a nervous system. It is quite obvious that this is lacking at the starting point.29 The objection will be made, however, that far from trying to find a way to get around the magisterial teaching, Aline Lizotte30 defends herself expressly for not presenting a doctrine coherent in any way with the Magisterium, especially with Donum Vitae. The exhortation requires that God’s creative presence be discerned from conception. Doesn’t mediatist infusion reject this? The response to this objection is another original contribution of the article. Inspired by Aristotle, Aline Lizotte distinguishes two modes of presence of the soul in the embryo: according to formal causality and according to final causality. The soul is present in the body according to the first modality (formal) when it is united to it as a potential act that receives it, like the form to the matter that is proportioned to it, in view of constituting a being that is substantially one, with an intrinsic unity that rejects all dualism. The soul is present in the body according to the second modality (final) when it guides the entire becoming as an attractive and actualizing end. The finality, in the full Aristotelian meaning, is surely a term, a result, and even the effect par excellence, but it is much more than this: in fact, before being an effect, the end is a cause. A cause only acts through its reality; it only gets its efficacy from its effectiveness. So it is endowed with a kind of realism which, for lack of comprehension, a considerable number of current concepts have passably exhausted. In other words, the presence by way of finality is a very real presence. It can be said that the end is present intentionally if intentional is not opposed to real.

THE POSITIVE RESPONSE However, some facts put the thesis of a differed personalization up for question.

The precocity of intrauterine life Today physiology and psychology take into consideration more and more the life of the little man even in intrauterine life.31 From the dawn of the ‘self ‘, it appears that the fetus is already capable of feeling, sensing, imagining and memorizing, as established by one of the speakers at the colloquium, prof. Bellieni.32 An ever-growing body of facts also suggests recognizing the occurrence of psychic traumas in a period that is closer and closer to conception. 33 Doctor Benoit Bayle, a practicing hospital psychiatrist in the Henri Ey Hospital Center in Chartres, believes that some phenomena of embryonic life seriously hamper the development of the future child or adult’s personality. Those who speak about subjective experience indicate, even in words, the existence of a subject that lives the experience. This is why dr. Bayle speaks about psycho genome and conceptional scene.34

The trauma of abortion Today the traumas connected with voluntary (and even spontaneous) interruptions of pregnancy cannot be denied. In one remarkable recent work on abortion, the sociologist Luc Boltanski recalls this and, at the same time, shows the many strategies set up by society to close its eyes to the humanity of the embryo in order to eliminate it better.35

The wound of exclusion We are very sensitive to any form of exclusion, and this is good, especially of the most vulnerable persons. Now, no one is more vulnerable and more innocent than a new being in its mother’s womb.36 ‘The only way to be fair with life is to respect the smallest of the living ‘.37 It could thus be said that the good health of a society is judged by its ability to welcome this eminently fragile being which is the human embryo. Moreover, there is a striking parallelism between the current reasoning to justify the 169 use of embryos in the eyes of politicians and the people in the name of their presumed lack of human nature, and the reasoning used five hundred years ago with regard to the nature of the Indians of Latin America. Both are vulnerable beings which our desire for power runs the risk of eradicating. King Charles V and his Council were shocked to learn about the inhuman treatment inflicted on the Indians. But, to put it briefly, since he needed money to wage his wars, he did not totally reform the ‘Indian ‘laws. In the same way, many political or medical personalities think that the embryo constitutes the first stage of a human person, but they hide behind the potential profits from research for the sick, the need to keep pharmaceutical laboratories in their country, etc.38

RESPONSE We are sorely lacking a philosophy of nature adapted to the newness not only of the cosmological vision brought by the many scientific discoveries over the past century, but also to the demands for ethical regulation with regard to technology. I would like to propose four arguments that will be presented in a progressive and apparently exclusive way. In reality, they seem complementary to me, and I hope that the limits of one will be compensated by the limits of the other. I will introduce them with a brief, particularly epistemological introduction.

Preliminary conditions The strictly philosophical reasoning that I am going to develop implies two preliminary conditions. The first is of a scientific order. I will presuppose the many developments that have been made, with their known authority, by professors Colombo, Sica and Bellieni, regarding fertilization (thus the emergence of a zygote or single cell embryo) and the first stages of embryonic life. The second preliminary condition is of an epistemological order.39 This has to do with the link between philosophy and science. To state it simply, we are before two equally partial and thus biased positions. The first position of continuity considers that there is a passage with no break from scientific facts to philosophy: for our subject, that is, to the question of the identity of the embryo. But this position, which is basically scientist, leads to the worse consequences. It underlies most of the mediatist positions. It denies any differences in views.40 Following Emile Boutroux, Maurice Blondel makes a strong critique of scientism. The sciences join calculation and experience in their method, experimentation and argumentation, empiricism and formalization, but how can the success of this wedding be explained? Science uses this felicitous harmony: moreover, it lives it daily, but it does not take it into consideration.41 This wedding shows spirit: ‘Our sciences do not bring or make the world and man. It is man who makes science, who dominates it always, just as he dominates, even without but better with this universe in which he seems immersed, but which he always infinitely surpasses with just one surge of his thought or one act of his freedom. It is not the world that questions and commands us, nor does it produce science; it is we who make science and throughout the world ask ourselves about another mystery different from the one the scientists ponder ‘.42 So the spirit is ‘that power without bounds in this infirmity without remedy ‘, which are the sciences.43 The second position, which is the complete opposite, considers that the two types of disciplines are in discontinuity. An epistemological position of this kind supports, for example, the reasoning of some Thomist philosophers. 44 To state it briefly, philosophy is to the sciences what universal affirmations based on common sense are to more particular affirmations based on hypotheses, even paradigmatic categories. The degree of certitude is a func- tion of universality and foundation on very common facts.45 But a position of this kind leads to a dualism that makes the fields of learning watertight and is refuted in practice. So we cannot do without an epistemological reflection and an attempt to articulate the two fields of knowledge, while respecting both their autonomy and their interaction and, we dare say, their hierarchy.46 On this subject, it could be enlightening to refer to Maurice Blondel again ‘precisely, the 170 distinction between the two types of thought, noetic and pneumatic.47 It is obvious, writes , ‘that the philosopher of nature who studies, from his viewpoint, the origin and genesis of the living, must have first hand information at his disposal about the results and the development of scientific research. This is a necessary condition for the validity of his reflection. He must prove to be ready to make revisions because the philosophy of nature, like science, is measured against the duly certified facts ‘. While confirming that science enriches philosophy from within and not laterally through the great amount of facts it brings, he adds: ‘This, however, does not mean renouncing hastily, and without making a serious confrontation with the scientific facts, some intuitions of a philosophical nature which are knowledge acquired from the heritage of Aristotle and here of Saint Thomas in particular ‘.48

THE PERSON AS AN INDIVIDUAL

Presentation To speak about a person is to speak about an individual being of the human species, and individuality is characterized by this dual note: uniqueness (divisum ab alio) and indivisibility (indivisum in se). Embryology and genetics show us that from conception, the embryo constitutes: a being with a specific nature, in this case a human nature, different from any other animal and living species, for example, from that of the pongids (chimpanzee, gorilla, orangutan); and an individual being, both unique and distinct from any other49 ‘individual and original, in its genome and its phenotypic expression. This is all the more true of the embryo which, in its development, manifests, on the one hand, its indivisibility more (when the possibility of real twins disappears), and, on the other, gains in originality (by making unique relations with the environment that shape its morphology or re-echo on the genome; and even more by appropriating the biological datum one day through its freedom and thus in a relationship).50 So it must be firmly asserted that the human zygote is an individual. And this individuality must be qualified as human because the genetic characteristics are specific before they are individual. Lastly, it must be said that this individuality is personal. The argumentation is based implicitly on totality and continuity. In the name of totality: ‘Since the human person is a whole ‘body, spirit, soul ‘, as a nurse writes, ‘it would seem logical to think that this developing human life already has that indivisible wholeâ?¦Isn’t an embryo also apparently different from a newborn just as a newborn is different from a forty year-old man? And yet it is and always will be the same person, genetically defined forever, until his biological death ‘.51 In the name of continuity: ‘Today, if the embryo is sacred ‘, says the gynecologist Jacques Milliez, ‘this is from the formation of the zygote. There is in fact an absolute continuum of phenomena from fertilization which creates no more doubt for scientists ‘.52

Limits Is it sufficient to show that a zygote is a human individual in order to conclude that it is a human person? The assertion that from fertilization until death, without interruption, the embryo is fully endowed with individuality, in order to conclude with its spiritual nature, raises some questions.53 In fact, this argumentation is supported by a philosophical presupposition that could be described as Parmenidian. It stresses continuity, even sameness. It overlooks the emergence of real new elements, and these are rightly indicated by the supporters of a differed personalization: uterine implantation and exchanges with the mother, indivisibility, the neural plaque, the organs, especially the brain, the conscience, the parental project, recognition by others. In what name are these novelties devalued, or even denied? Moreover, this continuity holds for a genome, and in what name should a genome be given value against the phenotypic and epigenetic determinations? Furthermore, these new features are only potentially present. In this way we come to the difficulties of defining an embryo as a potential person. Lastly, the continuity in question only results from what is observable. The considerable break 171 implied by generation or corruption defies the solely empirical viewpoint: this is true of death. It is also true, on the other hand, about the emergence of life. Consequently, the failure to observe a break in development does not mean at all that a substantial becoming has not come about, in this case the irruption of an intellective soul. Deep down, we find the dilemma again that set Parmenides of Elea against Heraclites of Ephesus, at least in the way it was systematized by Aristotle.54 Its solution, in any case, needs to be questioned. Doesn’t the conception of otherness and history underlying the criterion of continuity result from a problem of identity-idem, to use Ricoeur’s words? In addition, let us take up the traditional definition of person proposed by Boethius, which has profoundly marked the West. While it was made for the needs of Christology, its perspective is strictly rational: ‘The person, he says, is the individual substance of rational nature ‘.55 The above approach stresses individuality directly, but it only shows rationality indirectly, based on continuity. Consequently, it cannot be said that the zygote is a person. Lastly, it can be asked if the argument is not too remote. In fact, it is based on the notion of individuality, which is metaphysical, when the question refers to the field proper to the philosophy of nature and even bio-philosophy. A reasoning is conclusive if it gives its own reason, which is proximate.56 Above, while the neo-Aristotelians and neo-Thomists favorable to a mediatist personalization affirm very strongly the human individuality of the embryo and its continuity after conception, they oblige us to refine our approach to the human being. In dialogue with them I will now proceed. The person as an organized body animated by a spirit Based on and postulating the Aristotelian definition of the soul as the life principle, the next two paragraphs will seem to many researchers and even non-scientists to reawaken in a naive way an issue from another era, or even the polemics caused by vitalism, which the advances in the biological sciences, especially genetics and molecular biology, seemed to have definitively averted. However, I do not believe that this approach is useless, even if it requires a significant up and a patient captatio benevolentiae. First, it is still used by the Thomist (and the Aristotelian) supporters of the mediatist position.57 Next, it makes it possible to respond to the last objection (the proportion of the argumentation). Moreover, the paradox of life,58 the apories of biological reductionism,59 the significant multiplicity of explicative models of life,60 the ‘ontological complexity ‘61 of a ‘ world ‘62 show indirectly that the theme in question cannot be declared obsolete so readily, or at least that a place is left for approaches other than the ones proposed today. Lastly, on the purely philosophical level, the sole cosmological perspective continues to be insufficient. This is why we will frame it with two other approaches: the first is more metaphysical and the second more phenomenological.

Statement As we know, Aristotle distinguished living beings from inert beings, the former having a life principle called the soul. In a properly classic definition, Aristotle defined it as the first act of an organized body.63 This definition asserts two things in particular: on the one hand, the psuchè is an act, a form; on the other, it exercises its action on a fit subject: namely, a body that is sufficiently differentiated and functional to be a principle of the operations proper to the living being, in other words, an organized body or organism. Obviously, the fertilized cell has a real structural organization, with different levels of integration from the nucleotide to the cell in its totality, passing through the gene, the genome in its totality. Consequently, the one-cell embryo is the subject of a human, spiritual soul. Let us develop this argument. What is an organized body? To explain the meaning of the term organ, it seems best to me to refer to the detailed explanation that Thomas gives of the term organized when he comments on Aristotle’s definition of the soul in his Peri psuchès:64 ‘Next Aristotle comes to this part of the definition that deals with the subject of the soul. 172 Just as he said that the soul is the act of a physical body that has potential life, he says again that this describes any organized body. He calls an organized body one that has a diversity of organs. This diversity of organs is necessary to the body, the subject of life, because of the diversity of operations of the soul (Diversitas autem organorum necessaria est in corpore suscipiente vitam propter diversas operationes animae). In fact, since the soul is the most perfect form among the forms of corporal beings, it is the principle of different operations. It thus requires a diversity of organs for its perfectibility. On the other hand, the forms of inanimate things, because of their imperfection, are principles of few operations; hence they do not require this diversity of organs in keeping with their perfection ‘.65 An organ is thus defined as a material principle of operation, here the operational principle of the living being. Organ often evokes a structure first of all. However, the term has a meaning that is not only static but also dynamic. The Greek etymology, organon, means ‘instrument ‘. For Aristotle, an organ is a physical instrument moved by the principal cause, which is the soul in view of its finality, the vital operation, what presentday biology calls function. And if a living body is necessarily an organized body, the reason is as follows: to be organized is to be endowed with many organs. Contrary to an inert being, a living being must carry out a great number of operations, starting with the fundamental operations of life like assimilation. But, as we have just said, an organ is the material instrument, the principle of operation. This is why an animated body is an organized body. Furthermore, three degrees of life can be distinguished: vegetative, sensible and intellective.66 The preceding principles make it possible to specify the nature of the organized body that is needed according to the type of living being. A living body will be informed by a vegetative soul (in other words, it will be a vegetable) only if it is suited to vegetative operations and hence endowed with organs that carry out these actions: namely, for Aristotle, nutrition, growth and generation. In the same way, an animated body will only be able to be actualized by a sensitive soul (characteristic of an animal) if it has the degree of organization sufficient to carry out the operations proper to animal life: that is, sensation in the least, and the first and fundamental sensation is touch. Lastly, a spiritual soul that carries out the operations that do not depend on matter does not have an organ of its own. On the other hand, it requires a perfection of sensible life and of its organs sufficient to prepare the acts of intelligence and will adequately. This is why it is impossible to think without the presence of a brain even though it is not the organ of thought. As we can see, the realism of Aristotelian hylemorphism calls for more than individuation in order to speak about a living being. This individuality of the living body is a necessary but insufficient condition to speak about an animated being. The soul is the principle of being but also of operation. The body united to it, according to its own mode as the material cause, is also informed by the soul and the instrument directed by it, and so it is equipped for this end: in a word, it is organized. Is the zygote an organized body? So the question raised is made clear: does the fertilized cell have the organic perfection, that is, the organization, which enables it to be the (material) principle of operation of a spiritual soul? No, answer the disciples of Saint Thomas favorable to mediatist animation (and with them a considerable number of supporters of other philosophical or implicitly Aristotelian tendencies). In fact, these operations cannot be carried out without the presence of organs, especially the central nervous system and above all the brain. This is why a zygote without these organs is still not fit enough to be informed by a human soul. On my part, I believe that it is necessary to answer yes in view of the knowledge acquired from the biological sciences and interpreted philosophically. This is for two reasons: 1) a structural reason (affecting being), 2) and a functional reason (concerning acting). 1) Some speak about organization, some differentiated complexity: in other words, system. As we have seen, in the example of any cell, a zygote has a structure of an unprecedented complexity and organization that defies any current attempt to establish a model. This specialized structuring holds 173 both for the genome present in the nucleus and for the cytoplasm, the structure of which has been revealed by tomography through electronic cryomicroscopy (with high spatial resolution).67 Far from being encumbered chaos, this complex ordering is structured. Doesn’t it respond to the need for organization of the subject of the human soul? Moreover, our morphological knowledge about the cell taken separately shows us an organization that is much more complete and complex than what the ancients knew about the human body in its totality, but they did not refuse to make the body the matter of the rational soul. Moreover, Thomas Aquinas saw in this harmonious complexity the specificity of the human body and the reason for which it is the proportioned subject of the spiritual soul: ‘It was necessary for the body to which the intellective soul is united to be a mixed body and, among all the others, the one that received the most balanced composition (aequalitatem complexionis) ‘.68 So the argumentation still holds today and the zygote can receive this spiritual soul. Above all, it is recalled that in the end Aristotle stated that the embryo is only ‘an assembling of undifferentiated flesh ‘.69 In fact, ‘the first combination of a female and a male is called an embryo ‘.70 To the point that it can be called a larva: ‘In one sense, it seems that almost all beings generate a product that starts out as a larva: the embryo in its most imperfect form is in fact something like a larva, and among the whole of viviparous and oviparous beings whose eggs are complete, the embryo is indistinct at first and then it develops ‘.71 Based on the experience of the emergence of the first movement, as well as on the observation of aborted embryos, Aristotle, followed by Thomas, thought that ‘conception (that is, the presence of a human soul) was acquired at forty days for a male embryo and at ninety days for a female embryo, but not without great variations among individuals. 72 While what was known at the time about the organic structuring of a forty or ninety day embryo was very real,73 it was extremely rudimentary and much sketchier than what the least of our cells reveals. And yet, it was enough for the Stagirite. Who would dare to deny that a one-cell animal (protozoan or protophyte) is living if Aristotle’s ever- valid distribution is used, and if it performs the three operations of nutrition, growth and reproduction? Quite obviously, does a protozoan or a protophyte have any visible or even differ- entiable organs? So an organized body is not identified with a body endowed with visible and isolated, identifiable organs. This is not to say that Aristotle’s definition is wrong: an organic body continues to be the necessary subject for life; an organ continues to be the principle of vital operation, as we shall say again. On the other hand, the adjective ‘organic ‘and the term ‘organ ‘can no longer be tied only to morphologically identifiable reality, as the philosopher of Stagira did. And what is true of vegetative operation is also true of the sensitive act: animal life begins and exists before the eyes can perceive it. This no longer refers to an organism in the sense of differentiated entities. But in this way we have already anticipated the second point. 2) An organ is defined by its structure, but even more by its function, its finality: namely, to be a principle of action. Are the operations that authorize the structure of a fertilized egg proper to man? Can a human zygote be the principle of operations proper to a rational living being? It could be claimed that structure (morphology) is to functioning (physiology) what the first act is to the second act. One is as one acts, according to the scholastic axiom already enunciated. In reality, an argument of this kind is too universal to come to a conclusion: it is borrowed from metaphysics and is not proportioned to our question, which refers to the philosophy of nature. However, it should not be discarded: if Aristotle and Thomas thought that the slightly differentiated structure of a forty day old embryo was a principle of operation sufficient to be the subject of a human soul, what would they say today in view of the extraordinarily complex structures of the cell? In fact, the operations of the zygote are of a complexity commensurate with its structure. To the most complex genome of the living world, which is human DNA, corresponds the most sophisticated activity of the so-called biological universe.

174 First, the biological sciences teach us that the zygote is intensely active from the beginning. It performs the metabolic functions characteristic of all living beings. So it is in the least the subject of a vegetative soul. Next, until we are more fully informed, we unfortunately do not have studies regarding the sensitive operations of the zygote (human or animal). The only observation, for example, of elementary tactile sensation would oblige us to conclude, in a definitive way, that this living being is informed in the least by a sensitive soul, or to take up the more precise formulation of the Contra Gentiles again, that it lives an animal life. The fact remains, however, that a zygote has everything a protozoal has and noth- ing of a protophyte.74 Now ‘and this fact is worth stressing ‘protozoans are proof of sensibility, in this case, tactile knowledge, and, for some, of mobility. Finally, is it necessary to take the decisive step and say that, even without neurons and a brain, the union of the human ovum and spermatozoid is fit to receive a spiritual principle? How can it be asserted that the zygote performs acts of intelligence and will? But this is not required either by Thomas or by his disciples who support mediatist animation (if not, a child at birth would still not be a human being!). It could be answered that the sensitive operations are acts of the body-soul combination, while the operations of the spirit call for being free from all material causality, all corporeity. Consequently, it is possible to assert the presence of a concomitant spiritual soul. But it is possible to say more, and it should be said on behalf of the human soul that although it is spiritual, it remains the act of the body. The unity of the person requires a human body to be fit to be informed by a spiritual soul. So it must differ from the solely animal organism that reduces a solely sensible soul to the act. In fact, the organization of the zygote is the principle of sensible operations that prepare the spiritual acts. In reality, the genome of the fertilized egg, from the fusion of the gametes, contains all the information within itself: that is, the plan of organization, in view of building the organism, in particular the prefrontal cortex which, through the mediation of the internal senses, prepares for the operations of the spirit. Moreover, from being passively stored, this information unfolds, from fertilization, in immediate and tireless activity. As François Jacob wrote thirty years ago: ‘Each egg thus contains in the chromosomes received from its parents, its whole future, and the stages of its development, the form and the properties of the being that will emerge from it. The organism thus becomes the realization of a program prescribed through heredity. For the sake of a Psychè the translation of a message has been substituted ‘.75 It is not by chance that the great book of the Nobel Prize for medicine describes the history of biology as an marvelous plunge into the more and more microscopic structures of the living. This is undoubtedly a consequence of the reductionist method, but it can also be interpreted as a tribute to the miniaturized determination of the living, and more precisely, the differentiated hierarchy of the degrees of determination present in the animated being. The preceding facts suggest stating clearly that from fertilization an embryo is a person. Not a potential person, as the Consultative Committee of Ethics says, but a person with a potential. This assertion is based on the fully human character of the body. The human zygote is the organized body suitable to receive a spiritual and immortal soul as its first act.

Objection The following objection has been made to this demonstration: the above argument is based on the initial activity of the zygote. Some animal species are endowed with a genome that is almost as complex and active. Consequently, from the beginning, a human zygote does not present any specificity with regard to the fertilized animal egg. Why then would it be informed by a spiritual soul? ‘The extraordinary activity of which the embryo is capable from its very first stages, from fertilization ‘, writes Georges Cottier, ‘does not yet constitute proof of the presence of a spiritual soul. In fact, a similar active power is found in the embryos of other animal species ‘.76 Another difficulty could be added: our argument is based even more on the complex organization of the genome. The human zygote shares this structural complexity in particular with the chimpanzee since 98.4% of the genes are 175 common between the human species and that of these pongids. This can be stated differently: everything in the complete human body signifies its humanity. Aristotle already showed this with great accuracy of observation, from the fine grain of man’s skin to his vertical stance, passing through his articulated voice and the opening of his mouth.77 Now, in reverse, nothing in the zygote or in its genome manifests this difference.

Response These arguments are all the more impressive because they are given in figures and seem to lead to the following comparison: man differs from an animal by just a little more than one percent. ‘The major error (often made) ‘, retorts the biologist Jean-Didier Vincent, ‘consists in saying that we are 99% comparable to chimpanzees ‘.78 First of all, the quantity is not negligible. The is made up of 3 billion nucleotidic bases. According to the latest estimates, the difference is 1.2% and so there are less than 40 million nucleotides of difference between man and chimpanzees. Second, the quantity does not tell us everything. The order must also be taken into consideration. The direct analysis of portions of the genome shows that there are selective mutations within genes (one nucleotide being replaced by another),79 insertions or disappearances of short sequences of DNA,80 duplications of fragments of genes,81 and rearrangements of entire pieces of chromosomes.82 Put end to end, all these elements make the genetic distance between man and the chimpanzee rise to 5%. Consequently, Svante Paabo, the Director of the Department of Genetics at the Max-Planck Institute of the Anthropology of Evolution in Leipzig, states: ‘This small 1.2% (difference between the DNA sequences of man and the chimpanzee) can mean a lot, especially if the different rearrangements, duplications and deletions observed are added to the 40 million selective mutations ‘.83 Along the same line of ideas, while the genes resemble one another very much, the karyotypes differ: man’s includes 46 chromosomes, that of the chimpanzee 48, and that of certain species of monkeys up to 70. The differences in the architecture of the chromosome bring a difference in the expression of the genes and so also of the regulator genes on which many genes depend. Furthermore, proximity and genetic similarity are not troubling unless they are interpreted in a naively linear and analytical way. Now genetics is oriented more and more towards not a linear, but rather a combinatorial, even a ‘systemic ‘understanding of the genome.84 ‘The combining effect of the genes explains how small genetic differences can have considerable consequences on beings ‘, as the geneticist Axel Kahn notes.85 The problem raised by the continuity between human and animal genomes is also considerably intriguing to experts in neurogenesis in particular. They in fact come up against one troubling paradox.86 To state it simply, from a worm to a mouse the number of genes increases from six to eight times. On the other hand, from a mouse to Man, the number stays approximately constant, that is, around thirty thousand genes. An even stranger fact is that judging from the available observations, they seem to be relatively similar. However, it is obvious that these two organisms have profoundly different brains: 87 from a mouse to a man, the surface and the layers of the prefrontal cortex (or the front part of the brain) have grown considerably, going from a few per cent to almost 30%.88 Therefore, apart from the philosophical question regarding the moment of the zygote’s animation, this non-linearity raises an apory that requires a solution on a strictly scientific level. In this case, two explanations seem to be proposed at present: the first is of a combinatorial order: it seems that a great reserve exists in the 30,000 genes; the second, which takes time into consideration, is of a sequential order: the genes are not expressed only once but many times over the course of development. Although spectacular in its results, the difference can be very economical in its means: that of the critical role played by certain genes in the separation of the species. For example, one gene codes the enzyme called CMAH. The function of this enzyme is to help to add a particular sugar, sialic acid, to the surface of the cells. In humans, different from primates, a mutation has affected this gene. 176 Consequently, the CMAH cannot be coded. A study coor- dinated by Ajit Varki from the University of California in San Diego, has shown that this inactivation of the gene undoubtedly took place a little before the spectacular growth of the brain in hominids, or even two million years ago. This concordance in time makes it possible to suggest that the mutation of the gene of CMAH may have played a role in the increase in brain volume. 89 Be that as it may, this hypothesis shows that a small cause (genetic) is capable of generating great effects (phenotypic).90 Finally, it is not pointless to recall the epistemological need for a correct distinction between scientific and philosophical discourses: to conclude from the slight genetic distance between man and a chimpanzee (a scientific fact) the non-specificity of man (a philosophical statement) is a sophism. This is how one article dedicated to this subject concludes: ‘It is necessaryâ?¦to mistrust pseudo- philosophical conclusions that would emanate, according to some authors, from the works of biologistsâ?¦By deducing from them that man is nothing, an accident, is not ‘a consequence of scientific facts. In the same way, the unique character of a human being is not put up for question by the small genetic distance that separates him from a monkey. The value attributed to scientific information does not result from science ‘.91

Limits The preceding argumentation, however pertinent, has some limits. In addition to the difficulty related to the current lack of observation of a properly sensitive operation of the zygote (the organ in its agere), the difficulty remains of the subject’s disposition (the organ in its esse). The objection about the great similarity of genomes between a pongid and man, although not conclusive, shows the difficulty of discerning, in the individuated and active organization of the one-cell embryo, the body prepared to receive the act of the spiritual soul. In fact, the distance is always open between the initial potential and the final act and, according to the thesis supported, the supporters of the immediatist or the mediatist position will always work to stress, for the former, the organization that is already there, and for the latter, the actuality that is still missing. This apory may come from an overly unilateral emphasis on the psuchè as the form.

THE PERSON AS A BODY TO BE ORGANIZED BY A SPIRIT

Presentation Until now, we have considered the soul as a formal cause: in other words, as the act of the human body. Now, the principle of the living being also performs an efficient function,92 and this is in the extension of the form, as Michel Bastit emphasizes in more general terms: ‘The efficient cause is distinguished from the formal cause in that it determines when it can act or when it acts, but it is confused with it in the sense that the efficiency is only the external extension of the causality of the formal cause already active in the efficient cause ‘.93 The question of the animation of the zygote can be taken up contingent not on the dispositions of the subject but of the motor of the embryo’s development. In other words, all argumentation has been based until now on the being of the zygote, but it has not taken into consideration its becoming and cause. In my opinion, this argument of a dynamic order confirms in a decisive way the argumentation taken from the soul as a form of the body. Based on this new perspective, the discussion between immediatists and mediatists can be formulated in another way: the process that leads from the zygote to the formed human being, endowed with visible organs, is, for the former, an accidental becoming, precisely what Aristotle calls a quantitative movement of growth, and, for the latter, a substantial becoming, precisely what Aristotle calls the generation of a new substantial form, in this case a human from, accompanied by the corruption of a form that is substantial and sensitive (or even a dual process of generation-corruption in Aristotle and Thomas). All becoming requires a cause. In the case of the substantial becoming of the living being, this cause must be a univocal agent. What could it be? Stated more simply, any becoming requires a 177 proportioned cause. Anthropogenesis is a becoming. What can the motor cause be that actualizes the subject? The embarrassment is great for Thomas Aquinas, who follows Aristotle very faithfully. It is worthwhile to follow the ever rigorous formulation that Aquinas gives to this by asking if the seed can cause the animal soul: ‘Any being that generates will generate a being similar to itself. So the being generated must be in act in the cause of its generation. The sensitive soul is not in act in the seed, neither it, nor a part of it: because no part of the sensitive soul is there if not in a certain part of the body. In the seed there is no particle (particular) of the body because no particle of the body comes from the seed or the virtue of the seed. Consequently, the sensitive soul is not caused by the seed ‘.94 A simple reading of the objection shows how in the knowledge of the era, the seed cannot contain a particle of the body. It will be necessary to go back to this need for a total, absolute epigenesist, without any pre-formation. In refusing to accept any creation of the sensitive soul,95 and not being able to base himself on a capacity organically present in the seed, Thomas thus has to refer to a vis activa, an energy or active power that will only be partially within the seed.96 This causality is threefold: the causality inside the seed, its formative power lies in the foamy character of the sperm,97 ‘as its whiteness manifests ‘(albedo). Thomas also speaks about a ‘spirit ‘(intellectus or spiritus) present in the seed,98 but this term should not be misleading. Averroes explains that if the Philosopher uses the term spirit to designate the virtue present in the seed, this should be understood in a figurative sense: what is proper to the spirit is to operate through an organ; and the seed is rightly devoid of this.99 But this internal causality acts in fact by delegation: ‘The active virtue that is in the seed, derived from the very soul of the generator, is a kind of motion (motio) of the generator’s own soul ‘. More precisely, the seed is an instrument agent moved by a principal agent which is the soul of the generator, in this case, the father. The formative power of the soul, ‘because it is based like its own subject on the spirit that the seed is fit to contain, because it is something foamy, it brings about the formation of the body in that it acts through the power of the father’s soul, to whom generation is attributed as its principal agent, and through that of the soul of what is conceived, even once the soul is in him ‘.100 Lastly, Thomas adds a third cause, following Aristotle: the sun. In fact, we have seen that through its heat the seed can generate. All heat comes from the heavenly bodies as its first cause. Hence Aristotle’s famous formula: ‘What generates man is man plus the sun ‘.101 Therefore, there is a triple cause concurring in the emergence of the sensible soul and these causes are conjugated according to the dual hierarchy of instrumental cause-principal cause, univocal cause- equivocal cause: a) the instrumental agent, which is the seed; b) the principal agent, which is the father’s generative soul and moves as the univocal cause; c) the equivocal agent, which is the sun.102 So it is understandable that since the vis formativa is only instrumental, it asks for all its energy from much more powerful causes: the soul of the begetter and the celestial bodies. But what the seed gains in power it loses in proximity and immanence. Can philosophy stay with a vision of this kind if it allows itself to be enlightened by the new understanding of the beginning of the animated being offered by the biological sciences? By now, St. Thomas’mysterious vis formativa has been identified with the amazing capacity present in the zygote attested to by biology. This is certain in the chromosomes, but it has also been perceived better and better in the histones, the cytoplasm and so in the whole zygote as it enters into progressive interaction with the environment. While Aristotle and later Saint Thomas needed ‘the vertu of the human act of generation ‘, to use Maritain’s words, the biological sciences have made immanent the process of development. The philosophy of the living being cannot ignore this greater contribution, which cannot be deduced from common notions and is absolutely certain, regardless of what may come from different knowledge: every animated being is a cell or composed of cells. Every cell contains a genome that brings the building program of the whole organism and the ordering of the agents to carry it out. Therefore, we know that however little it may be, the zygote contains in its heart, that is, in the nucleus, the whole of what it will become. This new knowledge has to be assessed, which is probably 178 the most decisive information from the biological sciences and assuredly the most undreamed of information for the ancients and those of the middle ages. In philosophical terms, the biological sciences have made the ontogenesis of the living being totally immanent. Aristotle understood that the originality of the living being consisted in its self-motion; but this was intended for his otherwise complete being (the Stagirite makes growth one of the three operations of the living being), at least for his being that already had visible functional organs. But embryology and genetics make this autonomy and thus the immanent ability of self-organization go back to the origin and so self-motion includes the whole of becoming. Therefore, it is no longer necessary to have recourse to these ‘crutches ‘, these transitive, exterior principles that are the generative act and the sky. The vis informativa has been identified: it is made up by the genome. Any explanation provided by Aristotle and Thomas that took away from the zygote’s own spontaneity and immanent activity must now be given back to it. It can be said, moreover, that today the process has been explained of the univocal cause exercised by the living being: the efficient and material mechanism ‘which, for Aristotle, resulted from the observation that like generates like ‘has been clarified by genetics. Parental action has no need to continue in the seed through a mechanism that deprives the zygote of its efficacy.103 As long as the presence of the genome and its transmission through the gametes was unknown (and unknowable without instrumental mediation), the hypothesis of a continued action of the generative act, albeit transitive and at a distance, proved to be coherent. The knowledge acquired from biology has made this activity obsolete: the parents’efficient action stops on the threshold of fertilization. In the same way, a zygote is no longer an instrument moved by the principal agent, which is the parental soul. The most important fact, writes Elio Sgreccia regarding the zygote and the emergence of a new human being, ‘is that this new program is not inert, nor is it executed with the help of the maternal physiological organs that use the program in the same way that an architect uses the project as a passive model; this has to do with a new project that builds itself and of which it is the principal craftsman. Although the information systems of maternal origin that had brought the ovum to maturation remain active for a certain time, from the first moment of fertilization, the control systems of the zygote enter into action and take control completely even well before implantation ‘.104 To state this in another style ‘which does not fail to be symbolic: in giving ‘in abandoning to the gametes the secret of life, the parents offered all the essentials to the new being and granted it, in complete confidence, the greatest possible autonomy. By detaching themselves now from any direct and essential participation in the biogenesis, the parents offer the newly conceived, on the part of the mother, a warm and intimate reception that is enveloping and nourishing, and, on the father’s side, in a more mediatist way, the external but very real protection that is still a form of love. To go back to a more ontological style, in the debated issue De anima, Thomas takes up again the Aristotelian definition of the soul: the soul is actus corporis organici physici and adds quia anima facit ipsum esse corpus organicum: 105 ‘The soul is the act of a physical, organized body because the soul makes this body organized ‘. So in this way the soul is not only the term, but also the principle of organization. In other words, the soul as the formal cause has for its subject the organized body; but as the efficient cause, it has for its matter the body to be organized.

Limits This response is not sufficient. We have seen that the classic concept of instrument does not take into sufficient consideration the specificity of the genome’s action. Moreover, some, not without apprehension, will see the manes of Hans Driesch’s vitalism rise up again in this approach and they will fear a circular logic.106 Furthermore, the chromosomal DNA plays an essential role in the development of the human being from conception. But once again it is necessary to situate its role: it is the privileged instrument of development but it is not development. It is the material support for information; it is not genetic information (systematized in the famous code that is thus falsely iden- tified with DNA) or much less the information that is the soul, the principle of being and operation. 179 Also, the above argumentation implicitly identifies the genome and the zygote and isolates it abstractly from the cytoplasm. The fertilized cell constitutes a whole and it is the totality that is living. Just to give a very recent example, an Anglo-American team has supposedly just demonstrated that the spermatozoid, far from giving the oocyte only its chromosomes, also transfers a series of DNA messengers, six of which have been identified.107 Lastly, genome and phenotype (this notion itself has taken on many meanings, according to the level of organization considered: cellular, tissue, organic, individual) can be considered in a relation of potentiality to actuality. But they also constitute two levels of organization and thus of actuation. Now, this difference is never thought of as such. Doesn’t the observation of this unimaginable thing call for a specific reflection, or doesn’t it even manifest an intrinsic limit to the application of the pair act- potential in understanding the structure of the living being, and so isn’t this conducive to a new study? A last approach, which is not exclusive but complementary to the preceding ones, will hopefully make it possible to revoke these apories.

THE PERSON AS AN ORGANIZING CENTER

Presentation Let us make a backward zoom and consider not only the relation between the genotype and the phenotype, but also the relation of the zygote in its totality to the multicell embryo that it will become, until it becomes a complete adult body. For the supporters of mediatist information, only a body endowed with functional macroscopic organs is fit to receive a spiritual form. Here a dual problem, exegetic and doctrinal, is raised. First the exegetic problem: the question raised is double. Aristotle requires a living, animated body to be organic, that is, organized. For the Greek philosopher, this epithet is identified in the presence of organs, macroscopic organs that are sensibly identifiable and individually isolable. This dual identification was understood from the time of the Greeks down to the Middle Ages and still in the Renaissance. But the knowledge acquired from the different biological sciences compels an in-depth revision from at least two viewpoints: on the one hand, the organic structure as physiological activity begins well before the eyes can perceive it. We have dealt with this point above. On the other hand, the functional activities of the living being are not only related to the organs but to ubiquitous systems (think of the immune, nervous, hormonal, vascular systems). Therefore, it is ambiguous to continue to identify organic with endowed with organs at least as long as organ is too strictly defined as an isolated and macroscopic principle of action. Moreover, it has been demonstrated that organic can be taken in another sense that is no longer passive but active: organizer. The body would then be both already organized and still to be organized. Therefore, the soul is no longer present at the end of the process (as the formal cause), but also, in a necessary way, at the beginning (as the efficient cause of operations but also of growth). Next the doctrinal problem: to consider the relation existing between the zygote and the embryo, reference can be made first to the resources of traditional metaphysics. Based on factual information from common sense or science, the supporters of immediate animation and differed animation will interpret them in their own way, giving value to one aspect or another: a) in terms of intrinsic cause, the immediatists will stress actuality more and the mediatists potentiality; b) in terms of efficient cause, the former will refer to the notion of immanent becoming and principal cause, and the latter to transitive becoming (doing) and the ability to produce organs. Without denying the validity of the preceding categories and their pertinence in clarifying the question of the identity of the zygote, we could ask if it might not be useful to have recourse to phenomenology.108 I do not mean post-metaphysical phenomenology but phenomenology closely joined to metaphysics.109 To state it in an overly brief, even caricatural way, phenome- nology in this sense constitutes one approach to physical phenomena (a fortiori of the reflexive subject), considered in 180 their immanence, given to themselves, in order to explore their resources and see how they exceed themselves, leading to a finished intrinsic foundation that is not reduced at all to a mechanical cause or even an opening where transcendence is played out.110 Since a development of this kind is still largely unexplored, it does not or would not involve a kind of ‘metaphysics to the second power ‘, as Blondel says, but a ‘phenomenology to the second power ‘. In the framework of this phenomenology to the second power (and here of the philosophy of nature), I would refer to the distinction between essence and emergence as elaborated by Hans Urs Von Balthasar,111 who far from leaving it in a solely phenomenal perspective, imbues it with metaphysics.112 And I would put forth the following hypothesis: the genome is to the phenotype and the zygote is to the formed organism what essence is to appearance. 113 Let us develop this dual point: the genome constitutes the essence, in the phenomenological meaning of the term, of the living being. In fact, the phe- notypic, morphological organization expresses what is hidden but actually present. Now, the difference, which is not static, between the essence and the more dynamic appearance, between the secret, even mysterious essence and the figure (Gestalt),114 unveiled in the manifestation, pertain to the essence of a one, same event of development.115 Moreover, the objections have rightly stressed the active role of the genome: it constitutes an agent which, like an architect, directs the material transformations and enables the molecules present in the cytoplasm to get organized into a structured and functional totality. Lastly, the genome constitutes an active center. As the Bioethical Center of the University of Milan notes with regard to the zygote: ‘The biological center or coordinating structure (the italics are mine) of this new unit are the new genome (italics in the text) with which the one-cell embryo is endowed ‘.116 It will be noted that the text speaks about a ‘center ‘and not a coordinated but ‘coordinating ‘center, thus stressing the efficient role. Furthermore, in the cells, the macromolecule of DNA is situated in a place called the ‘nucleus ‘. In the beginning (1530), this term designated the hard part of a fruit, then later the compact part in the center of a natural or artificial element, and afterwards it progressively expanded to the whole area of the natural sciences (astronomy, geology, medicine, biology, electricity, physics, meteorology), human sciences (psychoanalysis, linguistics), and even to technologies (in the fifteenth century the central axe of a staircase; in the seventeenth century, the full part of a spring).117 Lastly, in a figurative sense, nucleus designates ‘a small group of persons ‘‘that has given rise to a larger group ‘(1794) or carries out ‘an activity in a hostile environment ‘(1844). By analogical extension, this nucleus means the center, secret place and active source, attesting to the ubiquitous presence of that figurative logic whereby the essence of a being is revealed through the mediation of its appearance. What is true of the relations between genotype and phenotype is also true of relations between the zygote and the embryo in ontogenesis. The zygote appears to be the active center from which everything comes, with order and efficacy. The interior-exterior distinction ceases to be just symbolic or spatial and becomes ontological. Angelo Serra also comes to this conclusion: ‘The newly conceivedâ?¦is a totally human, developing individual which, in an autonomous way, moment after moment, and with no discontinuity, builds its own form by carrying out, through an intrinsic activity, a plan projected and programmed in its own genome ‘.118 In a view of this kind, the totality is sealed (in the first act, not in potentiality) in this unique fragment, which is the heart, the center. This constitutes at the same time the hidden essence, the source of every manifestation and the cause of all dynamism. But it is the soul that moves the body. Consequently, a phenomenological perspective concludes with the presence, both motor and manifestative, of the intellective soul at the heart of the zygote.119

Confirmations This hypothesis finds eloquent confirmation in scientific, especially embryological data. In fact, the departure point of this discipline is the following problem: ‘Every one of us began his/her life in the 181 form of a cell, the egg ‘, writes Nicole Le Douarin. ‘In this case, for the human species, a small corpuscle of living matter 100 µm in diameter. When we linger over this idea, it arouses incredulity and questions ‘the Greeks would have said, admiration and astonishment. From this comes the fontal question of embryology: ‘How can it be that from this one, isolated cell the constituents of the body of an adult spring up, made up of many billions of harmoniously ordained cells to form organs as different and complex as the brain, the members, the eyes or the face? ‘120 How can it be stated better that the zygote is the foundational cell, the heart from which the whole living being comes? The biology of development has progressively shown that life has come to the cells not in a kind of continuous growth, but starting from organizational centers, sources of induction, that spring up in a discreet way and act only once. The first of these was discovered by the biologist Hans Spemann121 (who took up the experiments on regulation done by Hans Driesch). But experimental studies have established that Spemann’s organizer is preceded upstream by an even more primordial center, the one discovered by the Dutch embryologist Pieter D. Nieuwkoop that now bears his name.122 Furthermore, ‘it has now been established that Nieuwkoop’s center is put in place at fertilization .123 This hypothesis is not as far from Aristotle’s vision as it may seem. In fact, for the Stagirite, the heart is a principle of the living being. It is first chronologically: ‘The heart is the first part that is differentiated and it exists in act ‘124 and ontologically. 125 While the first principle (like the seed) contains the whole living being only in potentiality in the embryo, where in a certain way all the organs are found in potentiality ‘,126 this principle is actualized, and, for this reason, individualized in this very first organ which is the heart; moreover, it contains the principle of growth.127 So for Aristotle the heart is the equivalent of what will later be among embryologists the principle of organization. Rodolphe Kempf also comes to this conclusion in an article comparing Spemann to Aristotle on this point. He states that it is authorized to see in Spemann’s organizer center the contemporary analogy of the heart in Aristotle’s theory. As he explains: ‘The philosopher sees his major ideas confirmed by present- day science: animal morphogenesis uses a center because an animal is fundamentally a centered individual ‘.128 This organizer center is the instrumental cause, under the motion of the spiritual soul, of the construction and manifestation of the totality. Therefore, a dual phenomenal stratification should be affirmed (and distinguished). According to the first visible reality (here the living organized body, but already the structure of the zygote), expresses, reveals an invisible reality (here the spirit that animates). This more classic perspective129 is not the one developed here, even if it is implicitly present. The second stratification is internal to the body: the somatic structure itself verifies this demonstrative logic and, once again, on different levels: the nucleus unfolds in the organization of the cell; the initial and initiating cell, the zygote, is the center and source of the whole architecture of the body. Lastly, when the organism has already unfolded and opened up, it gives itself some partial centers of organization (Nieuwkoop’s). What is real thus seems to obey a logic of phenomenal construction and this follows a process of stacking into Russian dolls, which the fractals attempt to systematize. Be that as it may, a living being is constituted from a nodal or nuclear or cordial reality130 that presents the quadruple characteristic of being geographic because it is central, historic because it is original, dynamic because it is fontal, and phenomenologicalontological become it is the mysterious essence that is expressed without ever being exhausted, in the organic totality.131 Now we have just seen that meta-physical phenomenology makes the interior-exterior/essence-figure distinction significant. It thus gives value to the morphological capacity of the embryo and makes the membrane much more than an exterior frontier: it is the exterior manifestation that a living being gives itself based on its innermost depths.132

SOME RESPONSES TO THE ARGUMENTS

Homozygote twins

182 The argument taken from homozygote twins confuses individuality and indivisibility: an individual is characterized by its individuality (in act), not by its indivisibility (in potentiality). Moreover, the emergence of monozygote twins is depicted as a split of the embryo in two. Now, more often than not it involves the detachment of one blastomere from many others. So it is not one embryo that becomes two, but one that comes away from the other. If we absolutely want an image, we might refer to the rich biblical symbol of Eve’s birth from a part of Adam (the side, a metaphor of the heart, is a significant fragment of the totality). Vincent Bourguet makes this correction: ‘Rather than saying that one becomes two, it should be said that the initial zygote remains after the split into one of the two twins ‘.133 Furthermore, two realities are misleading. The first is the term twin itself. It implies the perfect equality or even convertibility between the two embryos, while it has more to do with a process of filiation (asexual but not parthenogenetic). This term, which came from the morphological resemblance, erroneously ebbed back to the origin. The second reality consists in the small time period of time between the emergence of the first and the second twins, which tends to be overlooked compared to the scale of a lifetime. And yet, this difference is considerable if it is considered not on the chronological but on the ontological level: one of the beings is not the origin, but at the origin ‘corporal ‘of the other. One indication points this out: if it takes several days to go from one cell to 64 cells, imagine what it would be in an adultâ?¦ Lastly, Philippe Caspar has demonstrated, in my opinion in a definitive way, one of the principle historical origins of the conceptual confusion between individuality and indivisibility: Leibniz’s metaphysics of the monad. Just to take one example, there is a striking connection, which is not only conceptual but verbal, between Leibniz’s theory and that of Jean-François Malherbe and Edouard Boné: ‘By individual I mean a being that destroys its own separation and whose fusion with another being is impossible ‘.134 In fact, Leibniz blocked the concepts of monadic individual and indivisibility: according to the principle of indiscernibles, two strictly identical individuals could not exist. Now, two homozygote twins are genetically identical so they cannot exist: one substance cannot be divided into two, nor can one be made out of two ‘.135

Totipotent cells Totipotentiality can be interpreted in two opposite ways: as an undifferentiated indetermination, as the objection does, or as a reserve put at the service of the good of the embryo. This second line of interpretation is what proves to be true, as shown by the famous embryologist A. Pedersen: the development process requires the embryo, at early stages, to be able to accept modifications in the program and impose them on the cells that are already differentiated in the interest of the whole that is being formed.136 Moreover, the recent discovery of the presence of pluripotent cells in the adult (what are called stem cells) is in favor of the second hypothesis. They serve to repair the sick organs. For example, a Swedish team has shown that neural stem cells were able to form normal hearts.137 In the light of this objection, which is almost as recurrent as that of the homozygote twins, I wonder if we should not ask ourselves in a more general way and from a metaphysical viewpoint about the concept of potentiality and actuality inferred by the new information introduced by the biological sciences. In a word, wouldn’t it be necessary to go from the act conceived of as a reduction in potentiality, to the act in the sense of a power over the potentiality or a power beneath it? Therefore, the indetermination of the totipotent cells constitutes a potentiality over which the soul can exercise its empire, its act: the act is not an imperfection waiting for determination, but a perfection, a source of determination, a power beneath it.138

The embryo as a being with relations and speech The argument comes close to the sophism of the red fish: remove the water from the container and the fish will die; put the water back, but it will not necessarily come back to life. So the brain is the 183 condition of thought, not its cause. As the great biologist Pierre-Paul Grassé also noted: ‘It was pre- sumed that a fÅ?tus was not a man until it acquired its nervous system. It is none of this. This acquisition, which is made at the very beginning of embryonic life, does not change the nature of the being that is already endowed with all the potentialities of its species. It does not add anything to it, not even conscience, which will only appear after birth ‘.139 More fundamentally, those who object base themselves on the non-significant character of human life; they have postulated that the only source of meaning is the spirit.140 A concept of this kind dates back to the divorce between nature and spirit characteristic of the Galilean-Cartesian heritage. Furthermore, those who object reduce zygote-mother communication too much to an exchange of nourishment and information. The environment and the container are not extrinsic to the content, in this case the living being, except in a geometric reduction of the place to space. The mother gives more to the zygote than nourishment and information: the warm, loving protection of her body, the protective secret hiding place, the communication of her intimate emotions, starting with her joyful acceptance of motherhood, etc. Far from being just physical and physiological, this primordial envelopment plays a sensible and even a really spiritual function since it concerns both the body and the three degrees of life.141 Without going into a detailed evaluation of the approach proposed by Ricoeur to ipseity, we could ask if in wanting to exorcize the ontologizing or even biologizing risks of the identity-idem, he does not fall into the symmetric error of devaluating corporal continuity too much, the spiritual origin of every human being, and of not seeing how much narration presupposes and appropriates these primordial facts? Behind this error, moreover, there is the post-Cartesian dichotomy of nature and freedom, and a desperate lack of metaphysical reflection.142

Recognition by others It is essential to the humanization of the new being for it to be recognized by its parents, especially its mother. When a baby is not spoken to or given any sign of love, an affective deprivation is produced that can lead to death.143 The objection thus indicates a truth that will never be stressed enough and which can be summarized in a drastic formula: only some children are adopted; all children should be adopted through an act of parental recognition.144 This recognition, however, supposes meaning; it does not constitute it. It is not because the parents accept the embryo that it is a man, but because it is a man they raise the question of accepting it. This is what the founder of maternology states in another way: ‘A child does not come from thought unless it abandons itself to the flesh, if it becomes flesh. Any mental or genetic manipulation turns against the flesh; any opening of the spirit to the flesh leads it back to the source where life is made. And life comes back to the flesh. Life does not come out of a laboratory; there would only be some life, not a living being. Life comes back to the flesh even though it has become spirit ‘and the spirit plunges back into its substance which is not thought, but the sensation from which it can think, its immersion in the dream that makes the flesh possible, the union of the flesh ‘.145 Lastly, the objection affirms in practice what it denies in theory. No coherent biologist constructs his object or decides about its identity through an act of speech. In his biological practice, René Frydman knows that the human gametes and zygotes present under the eye of his microscope obey specific laws and that if he transgresses them, he will not observe anything and, apart from any ethical evaluation, he will not achieve any fertilization in vitro. The number of spontaneous abortions The argument is erroneous from a scientific viewpoint. The number of spontaneous abortions has been over-evaluated for a long time (like many other authors, Patrick Verspieren speaks about half, without citing his sources). Out of 198 conceptions diagnosed through tests based on HCG, A.J. Wilcox and his team count 155 clinically identifiable pregnancies and 136 brought to term.146 Moreover, we know today that spontaneous miscarriages are not random, arbitrary events. Most early miscarriages are 184 linked to nonviable abnormalities. So the embryo aborted by nature constitutes an organism that is insufficiently fit or even resistant to animation. A , for example, makes it unfit for this human identity. It is well-known, says Anne MacLaren, ‘that in our (human) species there is a very high incidence of chromosomal abnormality ‘, much more than for any other known animal species, and we do not know why.147 The error is also philosophical. Let us concede that spontaneous abortions are frequent, or even that the causes may not be pathological. However, does their frequency make them natural? This is a property or even an accident, not the essence of the naturalness of a process. A process is considered natural because it is aimed at an accomplishment: the acquisition of a determination, what Aristotle would call a form.148 Spontaneous abortion is by definition the disappearance of a being, thus the privation of a determination. Moreover, it constitutes a failure. The failure occurs by accident: by definition, this is outside the intentional order. But nature is the principle of movement by itself and not by accident.149 Consequently, in all rigor it cannot be stated that a spontaneous abortion is natural.150 Of course, the current use of the term natural has gradually identified it with spontaneous, in which case it would be redundant, pleonastic to describe a spontaneous abortion as natural. But in its full, rigorous meaning, natural means an innermost logic present in physical beings, and the commonly known polysemy of this epithet risks leading to a dangerous shift in meaning. Moreover, this presumably natural behaviour can be used even less as a basis for imitating it.

The need for an organized body This argument has been answered at length in the body of the text, so I will not go back to it. I would only add one psychological observation: isn’t the blockage between organization and the presence of identifiable organs another offense to the intelligence through methodological monism, that is, through exclusive polarization on one type of approach to what is real? In fact, a zygote can be considered from the genotypic or phenotypic viewpoint regarding its final macroscopic morphology (I did not say completed) or regarding its initial microscopic morphology (I did not say sketched out). It is significant that Aline Lizotte does not adopt or take up the genetic viewpoint, and that at least as an objection, she does not tackle the question of the status of the genome’s organization (on the dual structural and operative plan).151 Vince Bourguet ‘like Philippe Caspar ‘has rightly criticized the undue privilege given to morphological and anatomical criteria. Although he was not able to distinguish the dual morphological (being) and physiological (acting) aspect present in the terms organization and organ and the importance Aristotle gave to the latter, in the name of his definition of nature as the internal principle of operation, Vincent Bourguet rightly stresses the confusion between human and adult: ‘An individual can belong to the human species without having any morphological property of an adult’. This ‘is the case of the zygoteâ?¦it is not a potential human being, it is at present a human being with the potential to be an adult ‘. Overturning the classic interpretation, the author feels that it is necessary to let oneself be instructed by the case of the living human being as to what individuality is rather than dumping our more general concept of individuality on it: ‘The study of man teaches us that he exists first in the form of one, then of several cells closed in a membraneâ?¦, this seems to us to be the teaching of biology concerning manâ?¦A human being goes through the different orders of reality, from elementary to complex, and to conscience, and it is this ascending trajectory he goes through that defines his individuality ‘.152 To cure the intelligence of this unilateralism or, in any case, to favor a dialogue between the two perspectives, isn’t it necessary to regain the ability to wonder before the prodigious organization present in the embryo? The philosopher Nicolas Malebranche, in assiduously cultivating the sciences, felt this when he observed the formation of the chicken in the egg: ‘Yes, Ari, the egg is the work of an infinite intelligence ‘.153 The process of embryonic development, says Lewis Wolpert, ‘is one of the most exciting problems of modern biology ‘.154 Moreover, the advances in the biological sciences 185 over the past one hundred fifty years have forbidden us to disconnect our structural and physiological view of the living being from its historical approach (which has nothing historicist about it). History teaches us the decisive importance of the beginning. In the same way, the sciences of the living lead us to wonder at these beginnings that are not only very rich in promise but in actual content: ‘The unity of the origin of all the cells of a metazoan has more importance than their current multiplicity ‘, said Georges Canguilhem. ‘The origin of the metazoan is the egg; that unique initial cell that partitioned and divided itself, not some pre-existing cells that would be grouped together ‘.155 Doesn’t the fact that a living being is not built through agglomeration but interior growth signify the unfathomable riches of the origin?

CONCLUSION In every era, man has been impressed by the extraordinary development that takes place between fertilization and birth. Today, however, the sciences do not allow us to see this only as a passage from potentiality to actuality. From the starting point, a zygote is already an extraordinarily organized and active being, in other words, a being in act. It is not a potential person, but a person with a potential. Some years ago, a President of the French Republic stated that the twentieth century was the century of biology.156 We hope that the 21st century will be the century of a philosophy of biology capable of accounting for it in a satisfying way. As a Christian, I will add one word: from the creation of the world, the only real and absolute newness ‘I am speaking about newness in being not in acting ‘is the human being. Ever since God, through his creative Word, made the universe from nothing, but even more from his love, he did not intervene directly, immediately, to create a new being before the emergence of the first man. And he acts, he moves, if I may say this, through every embryo he inserts into the fabric of the universe and humanity. This says something about its importance and its value.

1 For details about the arguments and references, allow me to refer to: IDE P., Le zygote est-il une personne humaine?, Paris: Téqui, 2004. 2 CONGREGATION FOR THE DOCTRINE OF THE FAITH, Donum Vitae (February 22, 1987), I, 1, AAS 1988, 80: 79. As we know, this essential passage is quoted by JOHN PAUL II, Evangelium Vitae (March 25, 1995), n. 60. 3 JONES H.W., Ethics of in vitro fertilization: 1984, inIn vitro fertilization and embryo transfer, Annals of the New York Academy of Sciences 1985, 442: 577-582. In this article, the author presents the main arguments. 4 This theory of the so-called karyogamie is found in DAWSON K., Fertilization and Moral Status: a Scientific Perspective; SINGER P., Embryo Experimentation, Cambridge: Cambridge University Press, 1990: 43-52. 5 ETHICAL ADVISORY BOARD (DHEW), HEW Support of Research Involving Human in vitro Fertilization and Embryo-Transfer, Washington: US Government Printing Office, 1979. And for the DHEW, uterine implantation is completed on the fourteenth day. 6 The famous Rapport Warnock (WARNOCK COMMITTEE, Report of Inquiry into Human Fertilization and Embryology, London: Her Majesty’s Stationery Office, 1984) allows using the human embryo for experimental purposes until this date, but without calling for any precise arguments. However, the argument of the primitive line is found in one member of the Warnock Committee, dr. Anne McLaren, which will be spoken about later. 7 The argument based on the formation of the linea primitiva is found in the Waller Commission in Australia: during the course of this formation, she states: ‘The differentiation of the embryo becomes evident ‘(Committee to Consider the Social, Ethical and Legal Issues arising from in vitro fertilization, chairman: WALLER L., Report of the embryos produced by in vitro fertilization, Melbourne, 1984). 186 ‘The emergence of the primitive line is the sign that an embryo in the proper sense and a human individual has been formed and begun to exist. To speak before this stage about the presence of a real human being in an ontological sense would not have meaning ‘(FORD N.M., When did I Begin? Conception of the Human Individual in History, Philosophy and Science, Cambridge: Cambridge University Press, 1988: 168). 8 This is the opinion defended by the Dominican Father PASTRANA G., Personhood and the beginning of human life, The Thomist 1977, 41: 247-294. 9 Starting from this stage, one no longer speaks about an embryo but a fetus. However, I do not make this distinction, just as I do not distinguish between embryo and pre-embryo for analogous reasons, as I will explain. 10 GOLDENRING J.M., The Brain-Life Theory: towards a Consistent Biological Definition of Humanness, Journal of Medical Ethics 1985, 11: 198-204. ‘Human life can be seen as in a continuous spectrum from the beginning of cerebral life in the uterus (eighth week of gestation) to cerebral death. In any event, tissues and systems of organs can be present, but without the presence of a functional human brain they cannot constitute a human being, at least in the medical meaning of the term ‘. 11 KOREIN J., Ontogenesis of the Fetal Nervous System: the Onset of Brain Life, Transplantation Proceedings 1990, 3: 82. 12 COMITÃ? CONSULTATIF NATIONAL D’Ã?THIQUE FRANÃ?AIS POUR LES SCIENCES DE LA VIE ET DE LA SANTÃ? (now abbreviated as CCNE), Avis sur les prélèvements de tissus d’embryons ou de fÅ?tus humains morts à des fins thérapeutiques, diagnostiques et scientifiques (May 22, 1984), reproduced in CCNE, Avis de recherches sur l’embryon, Arles: Actes Sud/Inserm, 1987: 11-31, here p. 13. Before, according to the formula that has become famous, ‘the embryo or fetus must be recognized as a potential human person ‘(Ibid. p. 15. The emphasis is mine). 13 According to the exegesis of BOURGUET V., L’être en gestation. Réflexions éthiques sur l’embryon humain, Paris: Presses de la Renaissance, 1999: 167. Regarding the critique of this conception, cf. pp. 167-195. 14 This, for example, is the position of Hans Tristam Engelhardt that requires the presence of rational activity implying self-awareness and an ability to communicate (ENGELHARDT H.T., BONDENSON W.B. (eds.), Viability and the Use of the Fetus, Philosophy and Medicine 1983, 13: 184-191). 15 Cf. the fourth argument below. 16 MORI M., Aborto e morale, Milano, 1996. 17 THÃ?VENOT X., Le statut de l’embryon, Projet. Vers la procréatique 1985, 195: 45-56. 18 I refer to the presentation made in this same book by professor SICA G., The development of pre- implantation embryo. To be more precise, a distinction must be made between the totipotentiality of blastomeres considered separately ‘according to the observations, it disappears after the eight cell stage ‘and the totipotentiality of the blastocyst, thus of the embryo considered in its totality which, on the other hand, remains for fifteen days (hence the late emergence of twins). 19 As we can see, this argument can take on many forms, according to the criterion put forward. This is how someone like Peter Singer can stress the ability to suffer (SINGER P., La libération animale, Paris: Grasset, 1993: 37) and draw ethical conclusions from it (ID., Pratical Ethics, Cambridge: Cambridge University Press, 1993). 20 ‘From an ontological viewpoint, the embryo before five months is the body of a being deserted by conscience, reason, intelligence, memory, the ability to have relationships with self, others and the world that acknowledge an undeniable relation. As far as humanity is concerned, both come more from nothingness than from being, they float blindly through life but not yet or no longer in the human ‘(ONFRAY M., Féeries anatomiques. Généalogie du corps faustien, Paris: Grasset, 2004). 21 RICOEUR P., Soi-même comme un autre, Paris: Seuil, 1990; ID., Cinquième étude, L’identité personnelle et l’identité narrative. 22 ID., Sixième étude, Le soi et l’identité narrative. 187 23 ‘L’embryon n’est pas un hors la loi ‘, L’Express of 19 November 1992. Cf. FRYDMAN R., Dieu, la médecine et l’embryon, Paris: Odile Jacob, 1997: 79-107. 24 Anne Fagot-Largeault and Geneviève Delaisi de Parseval speak about ‘two out of three fertilized eggs that die over the cause of the first six weeks ‘, Qu’est-ce qu’un embryon? Panorama des positions philosophiques actuelles, Esprit 1989, p. 95. 25 ‘Ã? propos du problème de la manipulation génétique ‘: RAHNER K., Ecrits théologiques, tome XII, Problèmes moraux et sciences humaines, Paris: DDB-Mame, 1970: 80. 26 Precisely after the well-known article by DONCEEL J.F., Immediate Animation and Delayed Hominization, Theological Studies 1970, 31: 76-110. 27 This is the constant position of Lizotte: ‘It appears difficult to state that the embryo in the first days, when it is still only a blastomere or morula, can have the presence of a human soul whose principal function is to be intellective and voluntary. It does not seem to have, and far from it, the sufficient organization of matter to act as an instrumental and organic support for these operations of intellective life. A brain is needed to think! ‘(LIZZOTTE A., Réflexions philosophiques sur l’âme et la personne de l’embryon, Anthropotes 1987, III(2): 155-195, here p. 156). ‘So a zygote does not have the potentiality for the operation of sensible life since it still does not have neuronal organization, and this privation is an important obstacle that makes the operation impossible ‘(Ibid., p. 186). ‘Matter is not simply potential form, in the first act (entéléchéia), it is potential operation (énergéia). If the soul must be the first act of this triple causality (formal, efficient and final), not only is its presence necessary to make the matter living: the matter must be able to perform the operation for which it is animated. As Aristotle teaches, the faculties or powers through which the soul carries out its operation cannot exist previous to the body, the instrument of this operation. As to intelligence, it does not perform its act through an organ. The fact remains, however, that it needs the activity of the senses in order to act and, as Aquinas would add, the senses must have reached their maximum perfection in order to serve the rational soul. If the soul is the formal cause, it must be the first act of a body with potential life; a body that has sufficient organization to be immediately capable of the operation ‘(Ibid., pp. 179-180). Italics in the next. The author quotes ST. THOMAS D’AQUINAS, Q.D. De Anima, q. un., a. 8; ID., In Aristotelis Librum De Anima Commentarium, L. II, l. 2, n. 240; cf. KREIT J., Bref résumé de la foi chrétienne. Compendium theologiae, Paris: Nouvelles Editions Latines, 1985: XVI. 28 MATHONAT B., Le début de la vie humaine chez Saint Thomas, Cahiers de la Faculté Libre de Philosophie Comparée 2000, 59: 79-114; pp. 104-107. This argument agrees with the one developed by Shea, whereby cerebral activity makes it possible for embryonic life to go from the ‘cellular level ‘, which is fragmented, to the ‘holistic or unified level ‘. In fact, the brain makes possible ‘the unification of the different organs and different tissues into a unique human individual ‘. (SHEA M.C., Embryonic Life and Human Life, Journal of Medical Ethics 1985, 11: 205-209. Now, a human being is necessarily unified. So we have to wait for the emergence of cerebral activity to be in the presence of a really human embryo. 29 The author proceeds, but with prudence and only in a note, to specify a time range for the moment when the human soul is infused: ‘Between the third and fourth week, which is between the 21st and 28th day ‘(Ibid., note 67: 107). No precise argument is given. In any event, both in Benedicte Mathonat and in Aline Lizotte, the only scientific facts mentioned pertain to the area of embryology, hence the visible morphology of the organs. 30 MATHONAT, Le début de la vie humaine chezâ?¦, pp. 110-112. 31 Only on the somatic level, for a long time the newborn was ‘the business of obstetricians, and mothers. A pediatrician only intervened later ‘, states the founder of neonatology, RELIER J.P., L’aimer avant qu’il naisse. Le lien mère-enfant avant la naissance, Paris: Robert Laffont, 1993: 19.

188 32 BELLIENI C.V., L’alba dell’io. Dolore, desideri, sogno, memoria del feto, Firenze: Società Editrice Fiorentina, 2004. The title itself refers to a great classic about fetal and neonatal perceptions: HERBINET E., BUSNEL M.C., L’aube des sens, Paris: Stock, 1981. 33 ASTELLI-HIDALGO N., Sauver ce qui était perdu et le fruit de tes entrailles. La guérison des blessures reçues dans le sein maternel, Paris-Fribourg, 1993. Cf. the clarifications by HENNAUX J.- M., La guérison des souvenirs et des blessures reçues dans le sein maternel, Nouvelle revue théologique 1997, 119(1): 65-84. 34 BAYLE B., Introduction à l’étude de la scène conceptionnelle contemporaine, Université de Marne-la-Vallée, 1997; ID., Embryon sur le divan. Psychopathologie de la conception humaine, Paris: Masson, 2003; ID., L’enfant à naître. Conception, grossesse et gestation psychique, Paris: Ã?rès, 2005. 35 BOLTANSKI L., La condition fÅ?tale. Une sociologie de l’engendrement et de l’avortement, Paris: Gallimard, 2004. 36 COLOMBO R., La vulnerabilità nella ricerca biomedica. Il caso dell’embrione umano, in SGRECCIA E., VIAL CORREA J. DE D. (eds.), Etica della ricerca biomedica per una visione cristiana, Vatican City: Libreria Editrice Vaticana, 2004: 217-244. 37 HENNAUX J.-M., Le droit de l’homme à la vie de la conception à la naissance, Bruxelles: IET, 1993: 28. 38 I owe this vivid analogy to Benoit Laplaize and would like to thank him here for it. 39 Cf. for details, IDE, Le zygote est-il une personneâ?¦, chap. 4. 40 Cf. on this subject the useful clarifications made by Paul Ricoeur, in the dialogue, although it did not take place, with Jean-Pierre Changeux (RICOEUR P., Ce qui nous fait penser. La nature et la règle, Paris: Odile Jacob, 1998). 41 BLONDEL M., L’action, vol. II, L’action humaine et les conditions de son aboutissement, Paris: P.U.F., 1963: 104-123; ID., L’action. Essai d’une critique de la vie et d’une science de la pratique, Paris: Alcan, 1893: 51-86. 42 Ibid., p. 122. 43 Ibid., p. 451. 44 Cf. for example DE KONINCK C., Les sciences expérimentales sont-elles distinctes de la philosophie de la nature?, Culture 1941, II: 4. Developed by BOYANCÃ? M., Le savant et le philosophe. Notes sur la connaissance commune, Actualité de la philosophie, Paris: Nouvelles Editions Latines, 1989: 61-80. 45 ARISTOTLE, Physiques, L. I, ch. 1, 184 a 16-b 14, Paris: Les Belles Lettres, 1990: 29-30. We recall that for Martin Heidegger, chapter 1 of book 1 of Physics constitutes ‘the classic introduction to philosophy ‘; even today it makes entire libraries of philosophical works superfluous. Anyone who has understood that chapter can risk taking the first step along the road of thought ‘(ID., Le principe de raison, Paris: Gallimard, 1962: 153). Cf. the commentary on the prologue made by St. Thomas Aquinas to Aristotle, ST. THOMAS AQUINAS, Physiques d’Aristote, L. I, l. 1, n. 6 à 11, Turin: Marietti, 1965: 4 à 6. Cf. the remarkable article synthesizing: ID., Summa Theologiae, Ia, q. 85, a. 3. 46 Cf. IDE, Le zygote est-il une personneâ?¦, pp. 80-95. 47 BLONDEL M., La pensée, vol. II, Les responsabilités de la pensée et la possibilité de son achèvement, Paris: Alcan, 1934. 48 COTTIER G., L’embryon humain et l’âme spirituelle, Nova et Vetera 2001, LXXVI(4): 35-51, here p. 48 and p. 49. 49 BERNIER R., L’ontogenèse de l’individu: ses aspects scientifiques et philosophiques, Archives de philosophie 1991, 51(1): 33; p. 38; HUARTE J., L’individualité de l’embryon humain, Bioéthique 1991, II(5): 248. Different biochemical studies have shown that the number of differences between proteins belonging to two individuals of a same vertebrate species, and here the human species, rises to 6,700. So the genetic diversity is identical. Consequently, each stem cell during the course of meiosis 189 can produce 26,700 different gametes, or 102,017: that is, 1 followed by 2,017 zeros! Now, 6 billion (6 x 109) human beings live on earth, and it is estimated that 80 billion preceded them after the emergence of homo sapiens. Furthermore, astrophysics estimate at approximately 1080 the number of stable particles composing the existing universe. Consequently, not only is the probability that two genetically identical human individuals will exist so low that it is practically impossible, but there is also not enough matter in the cosmos to make the total number of different possible sex cells. 50 IDE P., La nature humaine, fondement de la morale, Proceedings of the Bioethical Colloquium, Paris: Ã?d. de l’Emmanuel, 2004: 79-155, here pp. 142-153. 51 BOURGOIS Ã?., La bioéthique pour tous, Paris: Le Sarment, 2001: 191. 52 MILLIEZ J., Le quotidien du médecin, 1999: 29. 53 The answer is surely affirmative to the question raised negatively ‘and thus closed a priori ‘by the Instruction Donum Vitae mentioned at the beginning of this article: ‘How could a human individual not be a human person? ‘(I, 5), but through a mediation that is not proposed by a text that does not claim to be philosophical. 54 Moreover, these are not the only presuppositions of the discussion that dates back to pre- Socratic themes. So it could be asked if the value given to one date or another ‘I am thinking of the seven days of the pre-embryo, the fourteen days of the Warnock Report, the twelve weeks of the French law ‘does not result from a privilege granted to numeric harmony and so a certain pythagorism? 55 BOETHIUS, De duabus naturis, PL 64, 1343. Cf. the most recent translation: ID., Contre Eutychès et Nestorius, III, 1, inTraités théologiques, Paris: GF Flammarion, 1999: 75; NÃ?DONCELLE M., Les variations de Boèce sur la personne, Intersubjectivité et ontologie. Le défi personnaliste, Paris: Béatrice- Nauwelaerts, 1974: 235 à 271 (cf. especially the synthetic table from p. 267 to p. 270); cf. ST. THOMAS AQUINAS, Summa Theologiae, Ia, q. 29, a. 1. It is known, for example, that De Saint-Victor R., criticized Boethius’definition and chose another definition of the person (DE SAINT-VICTOR R., De Trinitate, L. IV, 21-23, Paris: Le Cerf, 1959: 278-285). Let it be said in passing that in light of Boethius’definition, I do not understand how Benedicte Mathonat both denies that the zygote is a person and asserts that it is ‘an individual with a reasonable nature ‘(MATHONAT R., Le zygote, de la puissance à l’acte. Clarification de termes, Cahiers de la Faculté Libre de Philosophie Comparée 2002, 62: 65-97, note 51: 85). Cf. the very similar approaches of the manual by Armand Cuvillier: a person is the ‘form that psychic life takes in the normal man and which supposes: 1) individuality; 2) conscience; 3) a function of synthesis that establishes a unity and continuity in mental life ‘, CUVILLIER A., Nouveau vocabulaire philosophique, Paris: Armand Collin, 1956: 138; and from the classic dictionary by André Lalande: a person is ‘an individual being in that he has the characteristics that allow him to take part in the intellectual and moral society of minds: self- awareness, reason ‘, LALANDE A., Vocabulaire technique et critique de la philosophie, Paris: PUF, 1962: 759. 56 BOETHIUS, Organon, vol. IV, Seconds Analytiques, Paris: Vrin. 57 To the above names could be added: HUBERT B., Le statut de l’embryon humain: une relecture d’Aristote, Nova et Vetera 2001, LXXVI(4): 53-81. In a more nuanced way: COTTIER, L’embryon humain et l’âmeâ?¦ In the opposite sense, but always based on the Aristotelian definition: SERANI MERLO A., L’embryon humain, sa vie et son âme. Une perspective biophilosophique, Nova et Vetera 2004, LXXIX(1): 89-103; ANTONIOTTI L.-M., La vérité de la personne humaine. Animation différée ou animation immédiate, Revue Thomiste 2003, CIII: 547-576. 58 KAPLAN F., Le paradoxe de la vie. La biologie entre Dieu et Darwin, Paris: La Découverte, 1995, especially chapters 1 and 7. 59 Cf. on this subject, the fascinating developments of DUCHESNEAU F., Philosophie de la biologie, Paris: PUF, 1997. Reductionism is problematic in all the questions at the heart of biology and biophilosophy: the species (chap. 1), teleology (chap. 2), the two approaches, to genetics, Mendel’s and

190 the molecular approach (chap. 3), the two structures, syntactic and semantic, of the biological theories (chap. 4), and the synthetic theory of evolution (chap. 5). 60 FOX KELLER E., Expliquer la vie. Modèles, métaphores et machines en biologie du développement, Paris: Gallimard, 2004. 61 ‘The disunity of the sciences is not only an unfortunate consequence of the limits of our calculation abilities and our cognitive faculties, but it reflects exactly the ontological complexity underlying the world ‘(DUPRÃ? J., The Disorder of Things: Metaphysical Foundations of the Disunity of Science, Cambridge: Harvard University Press, 1993: 7). 62 CARTWRIGHT N., The Dappled World: a Study of the Boundaries of Science, Cambridge: Cambridge University Press, 1999: 1. 63 Precisely, ‘the soul is the first act (entéléchie) of a natural body with potential life ‘and he specifies, ‘that is, an organized body ‘(ARISTOTLE, De l’âme, L. II, ch. 1, 412 a., 26-28, Paris: Vrin, 972: 68). For a pedagogical approach, cf. IDE P., Le corps à cÅ?ur. Essai sur le corps, Versailles: Saint- Paul, 1996, part II, chap. 5. 64 Here I will not go into the criticism contrary to the classic concept of organ. Doesn’t the much more unitary view of organism suggest replacing it with system? Moreover, this kind of perspective obviously supports the immediatist thesis even more. In fact, the genome and the one-cell embryo constitute systems that are highly sophisticated as well as functional. 65 PIROTTA A.M. (ed.), In Aristotelis Librum De Anima Commentarium, L. 2, l. 1, n. 230, Turin: Marietti, 1959: 61 (cf. the translation is slightly different given by VERNIER J.-M., Commentaire du traité de l’Ã?me d’Aristote, Paris: Vrin, 1999: 131); ARISTOTLE, De l’âme, L. II, ch. 1, 412 a. 28-30, 68. The continuation of the commentary on the same passage is not without interest because it illustrates the principles raised in n. 230 commenting on Aristotle’s elliptical letter (Ibid., b1-b3): ‘However, among the souls, the souls of plants are the most imperfect ones; this is why, in plants, the diversity of the organs is more rudimentary than in animals. So to show that any body receiving life is an organized body, he takes plants as an argument in which there is a lesser diversity of organs. This is what he says in these words: the parts of plants themselves are different organs. But ‘the parts of the plants are of an extreme simplicity ‘(ID., Parties des animaux, L. II, ch. 10, 655 b. 32s.). That is to say, they are very similar. The diversity found in the parts of animals is not found in plants. The foot of an animal, for example, is made up of different parts, like the flesh, the nerves, the bones and other things. But the organic parts of plants are not made up of such diverse parts. ‘Aristotle shows that the parts of plants are organic by showing that different parts are ordained to perform different operations. So the leaf protects the pericarp, the part where the fruit is born. But the pericarp in turn protects the fruit. The roots are for plants what a mouth is for animals because both capture food, like the roots in plants and the mouth in animals ‘(Ibid., nn. 231-232). Regarding this difference between plants and animals, cf. the interesting convergence with what is stated by HEGEL G.W.F., Philosophie de la nature, in l’Encyclopédie des Sciences philosophiques (especially §§ 343-345). 66 Aristotle distinguishes five types of faculties (ARISTOTLE, De l’âme, L. II, ch. 3, 414 a. 31-32), of which the first refers to vegetative life, the next three (appetitive, sensitive and locomotive) to sensible life, and the third (dianoetic) to intellective life. 67 MEDALIA O., Macromolecular Architecture in Eukaryotic Cells Visualized by Cryoelectron Tomography, Science 2002, 298: 1209; PLITZKO J., In Vivo Veritas: Electron Cryotomography of Cells, Trends of Biotechnology 2002, 20(8): 40. 68 ST. THOMAS AQUINAS, Summa Theologiae, Ia, q. 76, a. 5. In the same way: ‘The greater the diversity, the more perfect the soul ‘(ID., De Spiritualibus creaturis, q. un., a. 4); IDE, Le corps à cÅ?urâ?¦, pp. 134-138. 69ARISTOTLE, Histoire des animaux, vol. II, L. VII, ch. 3, 583 b. 10, Paris: Vrin, 1957: 469.

191 70 ID., De la génération des animaux, L. I, ch. 20, 728 b 34; Ibid., ch. 18, 724 b. 18, Paris: Les Belles Lettres, 1961: 38; Ibid., ch. 18, 724b. 18. 71 Ibid., L. III, ch. 9, 758 a. 32-36. 72 ID., Histoire des animaux, L. VII, ch. 3, 583 a. 35-b. 28. 73 Aristotle says that a male embryo aborted on the fortieth day is the size of a giant , but ‘the members are quite visible as well as all the organs including the penis and the eyes, which, as in the other animals, are considerably large ‘(Ibid., 583 b. 17-19). 74 For example, it is heterotrophic and has no skeletal wall. For Robert Gorenflot and Monique Guern, there are six differences (cf. Table in Organisation et biologie des Thallophytes, Paris: Doin Ã?d., 1989: 2). A seventh can be added: undetermined growth in the vegetable in contrast with a given period of growth, from the beginning of existence, in the animal. Cf. also GENEVÃ?S L., Biologie végétale. Thallophytes et microorganismes, Paris: Dunod, 1990: 1-2. 75 JACOB F., La logique du vivant. Une histoire de l’hérédité, Paris: Gallimard, 1970: 10. The relation still needs to be understood between the chromosomes and the future of the subject (including inside its body) in a non-deterministic sense. 76 COTTIER, L’embryon humain et l’âmeâ?¦, p. 49. 77 Cf. for example, the interesting developments of SOUCHARD B., Aristote, de la physique à la métaphysique. Réceptivité et causalité, Dijon: Ã?d. Universitaires de Dijon, 2003: 62-73. He shows in this passage that the most metaphysical approaches, such as the one leading to the distinction between potentiality and actuality, are rooted in the somatic-psychic structure of man. 78 VINCENT J.-D., FERRY L., Qu’est-ce que l’homme? Sur les fondamentaux de la biologie et de la philosophie, Paris: Odile Jacob, 2000: 183. 79 PENNISI E., Comparative Biology Joins the Molecular Age, Science 2002, 298: 719. 80 BRITTEN R., Divergence between samples of Chimpazee and human DNA sequences in 5% counting indels, Proc. Natl. Acad. Sci. USA 2002, 99: 13633. 81 EICHLER E.E., Recent duplication, domain accretion and the dynamic mutation of the human genome, Trends in Genetics 2001, 17(11): 661. 82 NAVARRO A., BARTON N.H., Chromosomal Speciaton and Molecular Divergence. Accelerated Evolution in Rearranged Chromosomes, Science 2003, 300: 321. 83 PAABO S., La Recherche 2004, 377: 73-76, here p. 74. 84 FOX KELLER E., Génome, postgénome. Quel avenir pour la biologie?, La Recherche 2004, 376: 30-37, here pp. 31-33. Cf. also ROSSIER J., La complexité après le séquençage, Pour la science 2003, 314: 92-96. 85 Communication to the United Nations World Conference: KAHN A., Génome, biologie et racisme (Durban, September 2001), summarized in Le Monde (5 September 2001). 86 The relative number of genes constitutes one of the two paradoxes noted by Jean-Pierre Changeux, the second paradox is the variability (CHANGEUX J.P., Un modèle neurocognitif d’acquisition des connaissances, in ID., La vérité dans les sciences, Paris: Odile Jacob, 2003: 61-79). 87 PROCHIANTZ A., Le développement et l’évolution du système nerveux, in MICHAUD Y. (ed.), Qu’est-ce que la vie?, Paris: Odile Jacob, 2000: 302-310. 88 FUSTER J., The Prefrontal Cortex, New York: Raven Press, 1997. 89 CHOU H.-H., HAYAKAWA T., DIAZ S. ET AL., Inactivation of CMP-N acetylneuraminic acid hydroxylase occured prior to brain expansion during human evolution, Proc. Natl. Acad. Sci USA 2002, 99: 11736. 90 This suggests, in another context, the butterfly effect developed by the fractal theory. 91 SCHALCHLI L., MORANGE M., Ces gènes qui font l’homme, La Recherche 2003, 12: 30-33, here p. 33. 92 ‘The soul is the cause and principle of the living body ‘(ARISTOTLE, De l’âme, L. II, ch. 4, 415 b. 7). 192 93 BASTIT M., Les quatre causes de l’être selon la philosophie première d’Aristote, Louvain-La- Neuve: Peeters, 2002: 308. Cf. all of the remarkable chapters 7 to 9 on form, efficiency and end. Along the same order of ideas, cf. ROMEYER DHERBEY G., Les choses mêmes. La pensée du réel chez Aristote, Lausanne: L’âge d’homme, 1983: 208-217. 94 ST. THOMAS AQUINAS, Summa Theologiae, Ia, q. 118, a. 1, 3rd argument. 95 ID., Q. D. De Potentia, q. 3, a. 9. 96 The following quotations without references are taken from the response of: ID., Summa Theologiae, Ia, q. 118, a. 1, ad 3um. 97 ‘In the sperm there is always that which makes the seeds fruitful, that is, what is called warmth. Now this warmth is neither from fire nor a substance of this kind, but the gas stored in the sperm and in the foam, and the nature inherent in this gas which is analogous to the astral element ‘(ARISTOTLE, De la génération des animaux, L. II, ch. 3, 736 b. 35-737 a. 1). 98 Moreover, Aquinas compares the seed to art, which, as a virtue of the practical intellect, is in relation to the spirit (ST. THOMAS AQUINAS, In Aristotelis Libros Metaphysicorum Commentarium, L. VII, l. 6, n. 1398 et l. 8, n. 1456. 99 ID., Q. D. De Pot., q. 3, a. 9, ad 28um. 100 ID., Somme contre les Gentils, L. II, ch. 89, I, § 7, Paris: GF Flammarion, 1999: 371. 101 ARISTOTLE, Physique, L. 2, ch. 2, 194 b. 13. 102 Lastly, for the case of the spiritual soul, a fourth cause should be added that is not named with the three preceding ones: when the matter is sufficiently ready, the ‘divine ‘part (to use Aristotle’s words), that is, the Spirit, will be infused. Now, matter is not able to reduce a spiritual principle that transcends it. Since it can only be infused, created, Thomas says that the intervention will be needed of the primary Cause which is God. 103 A mechanism that does not fail to recall the Aristotelian explanation of the movement of projectionâ?¦ 104 SGRECCIA E., Manuel de bioéthique. Les fondements de l’éthique biomédicale, Montréal: Wilson & Lafleur Itée, 1999: 458-459. 105 ST. THOMAS AQUINAS, Q. D. De Anima, q. un., a. 1, ad 15um. 106 However, it is recalled that Jacques Maritain (in the preface of DRIESCH H., La philosophie de l’organisme, Paris: Marcel Rivière, 1921) tried to clarify things by distinguishing the concept elaborated by Driesch of entéléchie (or vital factor E) from the Aristotelian concept of soul. 107 These are the results reached by: OSTERMEIER G.C., MILLER D., HUNTRIS J.D., Reproductive biology: delivering spermatozoan RNA to the oocyte, Nature 2004, 429(6988): 154; cf. Le Monde, (Friday 14 May 2004), p. 26. 108 If the philosophical context was not so different, Hegel’s distinction between in itself and for itself could have been helpful, which the German philosopher applies to the embryo. For example: ‘If the embryo is indeed in itself a man, it is not for itself. Man is for himself only as a cultivated reason that has made itself what it is in itself ‘(HEGEL G.W.F., Phénoménologie de l’Esprit, vol. II, Paris: Aubier-Montaigne, 1966: 55). 109 It is understandably out of the question to develop this point here. I refer to the works of TOURPE E., Donation et consentement. Une introduction méthodologique à la métaphysique, Bruxelles: Lessius, 2000; second part: L’objection phénoméniste à la métaphysique, pp. 115-172; GABELLIERI E., Saint Thomas: une ontologie sans phénoménologie?, Revue thomiste 1995, XCV(1): 150-192, but also to other Catholic phenomenologists that grant a more or less great place to metaphysics (Jean-Yves Lacoste, Jean-Louis Chrétien, etc.). The first two follow the tradition both of Thomas and of Blondel and Balthasar. 110 Blondel tries to join the two dimensions: phenomenal and ontological. On the one hand, ‘a real definition (of being) implies not only the descriptive, but also the genetic and explicative process of everything it isâ?¦So a semantic of being could not be raised initially; it first involves making an 193 inventory of the realities we call beings ‘. (BLONDEL M., L’être et les êtres, Paris: Alcan, 1935: 366- 367). At the same time, it is necessary to rise up ‘against a phenomenology that would presume to be sufficient for the science of being and replace the integration of the phenomena with the profound reality of beings ‘(Ibidâ?¦, pp. 374-375). 111 The Swiss theologian developed this distinction from a strictly philosophical perspective in a major work: VON BALTHASAR H.U., La Théologique, vol. I, La vérité du monde, Namur: Culture et Vérité, 1994. 112 ‘Balthasar’s approach ‘, writes André-Marie Ponnou-Delaffon, ‘reconciles phenomenology and metaphysics. If ‘a real phenomenologyâ?¦is denied access to the self of the being without the mediation of its appearance ‘, it can no longer ‘neglect the self of the being to be reduced to its appearance ‘(BARBARIN P., Théologie et sainteté, Paris: Le Cerf, 1999: 127). Allow me to refer to the analysis I propose of this properly metaphysical dimension in IDE P., Ã?tre et mystère. La philosophie de Hans Urs Von Balthasar, Namur: Culture et vérité, 1995, especially chap. 1 (for the analysis) and 4 (for the defense of his originality that cannot be reduced to the categories of classic metaphysics, for example, substance-accident). It would be even more erroneous to believe that ‘phenomenology ‘is identified with ‘descriptive ‘. 113 Let us discard one objection from the start: it may seem strange or even out of place to introduce a metaphysical distinction of this kind into the biological area. Balthasar never hid the fact that he owed it to Goethe who had conceived of this intuition based on his observation of plants. (cf. GOETHE W., La métamorphose des plantes, Paris: Ã?d. Triades, 1975: 21992; IDE, Ã?tre et mystèreâ?¦, pp. 177- 180). On this subject (an understanding of nature based on the pair interiority-appearance) the Naturphilosophie (Friedrich Schelling, Franz Von Baader, Hans André) contains a rich perspective that is still underexploited by the philosophy of nature. 114 An approach of this kind, moreover, is not so strange if we could believe in Aristotle’s reflection, especially when he reflects on the limit and form proper to the living being: ‘For all the beings whose constitution is natural, there is a limit and a proportion in size as in growth ‘(ARISTOTLE, De l’âme, L. II, ch. 4, 416 a 16-17; ID., De la génération des animaux, L. II, ch. 6, 745 a. 5). 115 The affirmation by Serra and Colombo is similar: ‘Individuality belongs to the dynamic, diachronic (phenotype) form of an organism and not to its genome which is conservative (genotype, the genetic content of the information in its cells) ‘. They go on to say: ‘The individuality of every organism is based on the uniqueness of its life cycle and not on the uniqueness of its genome ‘. SERRA, COLOMBO, Identità e statuto dell’embrione umanoâ?¦, p. 119. 116 CENTRO DI BIOETICA, Identità e statuto dell’embrione humano, Medicina e Morale 1989, 4: 665-666. 117 Regarding these different meanings cf.: REY A., Dictionnaire historique de la langue française, vol. II, Paris: Le Robert, 1998: 2403. 118 SERRA A., Il neoconcepito alla luce degli attuali sviluppi della genetica umana, Medicina e Morale 1974, 3: 333-366. The italics are mine. 119 It is significant that in one article of this volume dedicated particularly to St. Thomas’theory of the human embryo (PANGALLO M., The philosophy of Saint Thomas on the human embryo, pp. 209- 239), Msgr. Pangallo uses the dual vocabulary of phenomenology and classic ontology: ‘I believe that the succession of progressive manifestations and explications of the different functions of which the rational soul is capable should be affirmed ‘. And further on: ‘The human embryo is thus a person from conception, even if it has still not manifested existentially everything implied by the personal being ‘. Moreover, doesn’t the evocative distinction inspired by Cornelo Fabrio, which he proposes between actus essendis and existentia, result from phenomenology imbued with metaphysics? 120 LE DOUARIN, Des chimères, des clones et desâ?¦, p. 15.

194 121 Cf. the article princeps that earned him the Nobel Prize 11 years later: ‘Ã?ber die Determination der ersten Organanlagen des Amphibienembryonem ‘, SPAEMANN M., Wilhelms Roux’Archiv für Entwicklungmechanik der Organismen, 1918, 43: 448-555. 122 ‘The Formation of the in Urodelean Amphibians. Induction by the ‘, Wilhelms Roux’Archiv für Entwicklungmechanik der Organismen, 1969, 162: 341-373. 123 LE DOUARIN, Des chimères, des clones et desâ?¦, p. 159. Regarding this item, cf. the detailed developments in part 2, chap. 1 and 2. 124 ID., De la génération des animaux, L. II, ch. 4, 740 a. 3-4; ID., Les parties des animaux, L. III, ch. 4, 666 a. 20. 125 Cf. ARISTOTLE, De la jeunesse et de la vieillesse, ch. 3, 469 a. 5-6; ID., De la génération des animaux, L. II, ch. 6, 743 b. 25. Hence the comparison of ID., Des parties des animaux, L. III, ch. 7, 670 a 23-26. To go into detail, cf. BYL S., Note sur la place du cÅ?ur et la valorisation de la mesotes dans la biologie d’Aristote, L’Antiquité classique 1968, 37: 467-476. 126 ARISTOTLE, De la génération des animaux, L. II, ch. 4, 740 a. 1-2. 127 Ibid., L. II, ch. 1, 735 a. 15-17. 128 KEMPF R., Le principe du vivant dans l’embryon d’Aristote et le centre organisateur du développement dans l’embryologie expérimentale, Nova et Vetera 2003, LXXVIII(3): 79-100, here p. 95. 129 Allow me to refer to two personal developments that are more philosophical (IDE, Le corps à cÅ?urâ?¦, L. II, ch. 6) and theological (ID., Don et théologie du corps dans les catéchèses de Jean-Paul II sur l’amour dans le plan divin, in Jean-Paul II face à la question de l’homme, Proceedings of the 6th International Colloquium of the Guilé Foundation, Zurich: Guilé Foundation Press, 2004: 159-209. 130 Regarding the heart in this radical sense, but applied to man, cf. the brilliant developments of SIEWERTH G., L’homme et son corps, Paris: Plon, 1957, especially the decisive and rich passage from pp. 123-124. 131 For Bénédicte Mathonat, the relationship I establish between genome and macroscopic organization is of the order of immanent action, of acting. Quite to the contrary, in her opinion, this relationship is of the order of transitive activity (doing, art). In fact, it consists of transforming one matter in view of a purpose, and this is indeed how the genome operates: ‘The activity of the genome of the zygote achieves the macroscopic organization of the human body ‘(MATHONAT, Le zygote, de la puissance à l’acteâ?¦, p. 81). The author of the movement has the ability to produce the form; this still has to be introduced into the matter. So at the starting point, the form is not actually present. In other words, in the order of doing, the principle and the term of action differ like potentiality and actuality. To take up the classic image: the construction of a house or even the ability to build it is not the constructed house. The genome is the architect and the phenotype is the completed house. So the genotype is to the phenotype what the architect is to his work. Consequently, the difference between genome and organization is not that of the first act and the second act, but that of potentiality and actuality: ‘What is first in actuality on the level of the genotype is the ability to produce organs and nothing else ‘(Ibid., p. 83): that is, not the actual existence of the organs at all. The effort should be welcomed to consider, based on philosophical categories, the distinction between genotype and phenotype. Having said this, this critique calls for at least two remarks. Unquestionably, the genome (and the zygote itself) plays an efficient role in the construction of the embryo, but isn’t the relation one of transitivity and thus of exteriority? In fact, the nucleus is internal to the zygote and this is the internal source based on which the whole living being grows and sculpts a form (in a figurative sense). Now, growth is an immanent becoming. Moreover, from another viewpoint that is far from descriptive, which presents an ontological value, the embryo is built based on a source, an essence. The metaphor of the construction must be spun out. If the house, as the final cause, only exists potentially in the materials, it exists in actuality as the exemplary cause (that is, the formal separated cause) in the spirit 195 of the architect who must also be in actuality in order to build the house. What will be the efficient cause that is so perfect it will be able to organize the matter and in this way model the complete living being? The intellective soul is the principal cause and the genome will be the joint privileged instrument. 132 This conception is not without a profound relation with the theory developed by ANZIEU D., Le Moi-peau, Paris: Dunod, 1985; ID., Une peau pour les pensées, Montréal: Guenoud, 1988; ID., La fonction contenante de la peau, du moi et de la pensée, in ID., Les contenants de pensée, Paris: Ã?d. Anzieu, Paris: Dunod, 1993; ID., Le penser, du Moi-peau au Moi pensant, Paris: Dunod, 1994. It should be noted that although a psychoanalyst, Anzieu established a foundational relation between the psychological and the biological: ‘Any psychic activity is supported by a biological function. The self- skin gets its support from the different functions of the skin ‘(ANZIEU, Le Moipeauâ?¦, p. 39). 133 BOURGUET, L’être en gestationâ?¦, p. 115. 134 MALHERBE J.F., BONÃ? E., Engendrés par la science, Paris: Le Cerf, 1985: 138. Italics in the text. An encounter that is all the more fascinating than in note 49 following on from this text, Malherbe and Boné cite the scholastic saying about the two aspects of unity pointed out above, which show to what extent Leibniz’s conception of individuality has intoxicated and deformed the scholastic conception of the unum. Regarding the position of the work by Ibid.; cf. CHAPELLE A., FAVRAUX P., Bioéthique et foi chrétienne. Ã? propos d’un livre récent, Nouvelle Revue Théologique 1986, 108: 249-267; cf. also the report by ETIENNE J., Revue Théologique de Louvain 1986, 17: 453-456. 135 LE ROY G. (ed.), Discours de métaphysique et Correspondance avec Arnauld, Paris: Vrin, 1970: 44. 136 PEDERSEN A., Potency, Lineage and Allocation in Preimplantation Mouse Embryo, in ROSSANT J., ID., (eds.), Experimental Approach to Mammalian Embryonic Development, Cambridge: Cambridge University Press, 1986: 3-33. 137 CLARKE D.L., JOHANSSON C.B., FRIZEN J. ET AL., Generalized Potential of Adult Neural Stem Cells, Science 2000, 288: 1660-1663. Is it true that the capacity for differentiation is much greater in vivo than in vitro? 138 While this hypothesis is entirely different from that of a phenomenology of nature (2. d), it is in line with its extension: it attempts to understand the difference that exists between the different levels of actuality that are affirmed in the living being. 139 GRASSÃ? P.P., L’homme en accusation. De la biologie à la politique, Paris: Albin Michel, 1980: 288. 140 This is the case, for example, of the Australian bioethicist Peter Singer: ‘We have rejected the traditional principle of the sanctity of human life because this principle gives an enormous significance to something ‘the biological species ‘that really no longer has any intrinsic moral significance ‘(SINGER P., KUHSE H., Should the Baby Live? Problem of Handicapped Infants, Oxford: University Press, 1985: 129). The author considers the infanticide of handicapped newborn babies legitimate and entirely logical. 141 Regarding this primary spiritual and loving relationship between the mother and child, cf. the very remarkable work by SIEWERTH G., Aux sources de l’amour. Métaphysique de l’enfance, Parole et silence 2001. The author treats intrauterine life very little (p. 45ss.), but it could be useful to extend his reflections on life right after birth to the period that precedes it. 142 This is finally sketched out by the tenth study of Soi-même comme un autre, but it stops on the threshold of the act without ever taking up the question of being. Last, in Michel Onfray’s charge, which is argued very little, we note an amusing lapsus: deserted means left. Does this mean, then, that all the capacities denied by the author existed before five months?! It should be noted that rather then being provocative, the development of the essay is marked by an underlying resentment of Christianity, while the preface is marked by an amazingly delicate tone. Onfray refers there with great sensitivity to 196 the cancer suffered by his companion. Here his heart is speaking; there it is the intellectual who is both hurtful and hurting. 143 This is what was established by John Bowlby, the founder of the theory of attachment (cf. the excellent synthesis done by PIERREHUMBERT P., Le premier lien. Théorie de l’attachement, Paris: Odile Jacob, 2003). 144 This is what psychological and social constructivism rightly stresses (IDE, La nature humaine, fondement de laâ?¦, pp. 89-92). 145 DELASSUS J.-M., PAPILLAUD K., Clone ou enfant?, Paris: Dunod, 2003: 163-164. In the same issue of the Nouvel Observateur in which Rene Frydman makes his intervention, Tony Anatrella responds to the researcher through a vicarious interviewer: ‘To make the existence of the embryo only depend on an external, subjective viewpoint (the parental project) opens up to all kinds of arbitrariness. In one case, by showing the ultrasound scan, it is affirmed that someone is there. In the other, he does not exist in the absence of a project concerning him. But is the notion of project so clear? I received a young, 20 year-old woman for a consultation who was a few weeks pregnant and in conflict with her mother who was encouraging her to have an abortion in these terms. The daughter answered her in this way: if you oblige me to lose it, you kill me at the same time ‘. (Le Nouvel Observateur 1997, 1711: 14-15, here p. 15). 146 WILCOX A.J., WEINBERG C.R., O’CONNOR J.F. ET AL., Incidence of Early Loss of Pregnancy, New England Journal of Medicine 1988, 319: 189-194. Cf. below for other references. 147 MCLAREN A., Genetics of the Embryo, Campus Verlag, 1990: 42-53, here p. 43; BRAUNSTEIN G.D., KAROW W.G., GENTRY W.D. ET AL., Subclinical Spontaneous Abortion, Obstetrics and Gynecology 1977, 50(1): 41-44; EDMONDS D.K., LINDSAY K.S., MILLER J.F. ET AL., Early Embryonic Mortality in Women, Fertility and Sterility 1982, 38(4): 447-453. 148 Regarding the meaning of the term natural, allow me to refer to the developments of the article already cited: La nature humaine, fondement de la morale, pp. 108-142. 149 This whole argumentation is based on the Aristotelian conception of nature (ARISTOTLE, Physique, L. II, ch. 1, 192 b. 21-23). Our criticism of the naturalness of abortion is close to the critique the Philosopher makes of the presumably antifinalist argument taken from the existence of a monster. In fact, a monster constitutes an accident, a failure, a shortcoming not in the order of the efficient cause, but the final cause. In fact, if a motor did not exist, the defect could not exist. If there was no biosynthesis of the proteins, the genotypic abnormality would not be expressed. Consequently, a failure fails the purpose, and so it concerns the finality. Therefore, far from contradicting the existence of the finality as is often believed, a monster confirms it (Ibid., L. II, ch. 8, 199 a. 33-b. 13; cf. the enlightening commentary by ST. THOMAS AQUINAS, In octo libros Physicorum Aristotelis expositio, L. II, l. 14, nn. 263-266: 129-130). 150 Much less can an action be based on this observation. We know that this argument is used sometimes to legitimize early provoked abortions. Wouldn’t this be falling into the same rut as the naturalist ethics that have nothing to do with what the Church calls natural morality? 151 Along the same order of ideas, the need for differentiated, visible organs is presented as an obvious thing by Mathonat. The principle is that the body must ‘be organized, that is, provided with instruments of operations ‘. Now, the conclusion that necessarily results from this is as follows: ‘The organization characterizing the subject of the human soul must be that of an organic differentiation that makes possible in particular the powers of sensible operations; hence a body endowed with a nervous system in the least ‘(MATHONAT, Le zygote, de la puissance à l’acteâ?¦, p. 71). It is stated elsewhere: ‘With the genome, everything is possible, but nothing is achieved yet with regard to the organization needed so that the organic dispositions will exist necessary for the operative principles of the human beingâ?¦A macroscopic kind of organization is necessary for the sensible capabilities of the human being to exist ‘(pp. 84- 85). So in this way these declarations never define what they mean by organ, macroscopic, precise corporal order, etc. Next, what becomes of this in the case of a protozoan or a metazoan made 197 up by a very small number of cells? They do not include macroscopic organs; and yet, they are animals that perform sensitive operations. So it is advisable to revisit the definition of the term organized in the Aristotelian definition of the soul. Lastly, an Aristotelian mediatist cannot be disinterested, by right or in fact, in the moment when spiritual animation is a reality. The fact that one is unable to determine the precise instant, like the supporter of immediate animation, does not keep one from asking about the approximate time range of its infusion. Now, on behalf of the principle invoked of a proportion between visible and functional organs and the presence of the intellective soul, the creation of the latter must be delayed considerably. The spirit cannot even perform its elementary acts without the presence of a brain that is already well developed or even organized. The neuronal cells do not stop multiplying until the end of the sixteenth week, but everything remains to be done: the future architecture, the figure, is barely sketched. From this viewpoint, the position of Jacques Maritain, who delays this animation to the seventh month, is coherent. But does this Aristotelian mediatist follow him? Benedicte Mathonat makes ‘the specific macroscopic organization of the human body ‘(p. 86) the adequate subject of the human soul. But an organization of this kind is far from being precocious, especially if the phenomena of neoteny are taken into consideration. 152 BOURGUET, L’être en gestationâ?¦, pp. 84-86. Italics in the text. Cf. also pp. 137-142. 153 MALEBRANCHE N., Entretiens sur la métaphysique et la religion, in Oeuvres, Paris: Gallimard, 1992: 880. 154 WOLPERT L., The Triumph of the Embryo, Oxford: Oxford University Press, 1991. 155 CANGUILHEM G., La connaissance de la vie, Paris: Vrin, 1971, quoted by SAULNIER C., Biologie et philosophie. Essai sur l’individualité biologique, Montpellier: Presses de l’Université de Montpellier, 1955: 119. 156 Comments reported by MAYR E., Qu’est-ce que la biologie?, Paris: Fayard, 1998: 7.

198 A. GIL LOPES

THE PRE-IMPLANTATION EMBRYO BETWEEN BIOLOGY AND PHILOSOPHY: THE INDIVIDUAL BEING

To properly address the questions related to the pre-implantation embryo it is necessary to analyze, from the current scientific point of view, the different possibilities for the generation of a human embryo. First of all, as it happens in the natural way, the embryo is generated by the fecundation process that follows the sexual act and allows through the union of both, the feminine and the masculine gametes, the formation of a new and unique zygote. This embryo results from an interpersonal relation, ideally based in the responsible love that unites husband and wife. Some decades ago, the methods developed for animal reproduction were introduced under the scope of the ‘medically assisted reproduction ‘for humans. This technology made it possible to generate a human embryo without the sexual act but with the concourse of the gametes. Few decades ago, other techniques were introduced in order to promote the fecundation in laboratory tubes, out of the human body. The zygotes and pre-implantation embryos obtained in this way may be preserved at low temperatures for long periods of time before adequate transfer to human uterus, what would allow its development. Up to this point of the scientific and technological progress, both the genetic contribution of father and mother are necessary to originate a new embryo. Nowadays these methods are currently used for human procreation. In several countries the legislation allows the production of large number of pre- implantation embryos for this specific purpose, originating large number of frozen embryos without any chance of proper development. In Brazil, for instance, after three years of preservation, with the parents consent, it is legally allowed to use them for scientific research. Starting few years ago, the cloning methods were developed and made it possible to obtain a zygote without the concourse of both gametes. We may expect that in few years it will be possible to stimulate a somatic cell by appropriate biochemical factors in order to signal all the events involved in the processes that would originate an embryo by the proper sequence of expression and suppression of the different genes involved in the embryogenesis. On the other hand, it is expected that differentiation and trans-differentiation processes will be elucidated and as a consequence, culture of specific cell types will be obtained from adult somatic cells. I would like to stress the fact that the actual interest of most studies on the pre-implantation embryo is threefold: 1) studies conducted in order to establish appropriate methods to select in vitro generated embryos for artificial or, as usually called, medically assisted reproduction; 2) studies based on the use of pre-implantation in order to obtain stem cells for scientific or therapeutic use; 3) studies related to human cloning for scientific or therapeutic use of stem cells. This means that, in this context, the pre-implantation embryos will not survive, representing one face of the culture of death. At this point it is necessary to consider that from the biological point of view, life does not starts at each conception since the vital phenomenon is maintained through the reproduction and it is neither extinct nor re-established, but follows from generation to generation. It is just the life of a new being that begins. Moreover, each genome contains all the information that confers not only all the characteristics of its species, but determines the uniqueness of the being. Even if the clones produced in the laboratory without the concourse of the masculine and feminine gametes may have not a father and a mother, they are human and have the humanity as parents. From the human genome only human beings can be generated. The human embryo is one in the sense of unique, concrete, complete, particular, substantial, separate and total. 199 Although some elements are not present in act in the zygote, they are potentially present. Since the first moment the zygote is a human person. In other words, he/she has the essence that will not be other one, but only the one of a human being that is since the beginning, and this is his/her identity. What will be transformed in act is not his /her essence of a human being, but the accidents that confer him/her the proper characteristics of each stage of the development. The zygote presents three fundamental features: the ability to sense characteristics of the environment through different sensors (receptors), the ability to integrate these different signals or biological messages and integrate them, the ability to produce proper responses in order to adapt itself to the environment. Actually, since the very first moment the zygote has a real dialogue with the biological environment. This makes it possible the articulation between genomic and phonotypical characteristics. The zygote has not only its individuality and identity but interacts with the environment and is able to adjust to it and autocorrect itself. In other words, the zygote is able to recognize the self and the non self and establish a real biochemical and physical dialogue with the environment. The language evolution assessed as an interaction of one person with his/her environment can be analyzed from the zygote formation to the last phase of his/her vital cycle. As a zygote, his/her language is based on the basic mechanisms of cell signaling. The developing processes that lead to a progressive structural complexity give the necessary basis to respond in a proportional way, in a more complex way, to the environment by means of a language that acquires new organizational dimensions. To ascribe to the zygote the extra uterine language is senseless since changes in the environment implies in changes in the form of communication. The communication established in the extra uterine life is an evolution of the condition found in the human zygote. In other words, no other species develops an intellective language with the same human characteristics only based in the exposition to a proper and new environment. Under this point of view the language development that supports the relational dimension is a continuous process. A proper image of the wholeness and oneness of the pre-implantation embryo life is may be seen in the analogy with a musical score, an orchestra and a music. Actually, when a zygote is formed, not only the complete score (sheet music) is present in the genome, as well as all the biochemical instruments will be sequentially synthesized and introduced in order to perform the cellular multiplication and differentiation, structural organization and temporal development of a completely new and unique being. Not only are intrinsically defined the human characteristics as well as the time span that denotes the cycle of life both of the individual cells and the whole organism. As occurs to all living species, each phase of human life is regularly and uniformly determined, even the total length of human life has a natural limit. In the same sense that the first sounds are part inalienable of a music that once initiated will reach its fullness only at the last note, when a new zygote is formed, actually, a cascade of biological processes starts and promotes sequentially all the events of life that will end only at the death of this new being. At the first sounds of Salve Regina, Veni Creator, the 9th Symphony or any other musical composition, we do recognize their identity, even if they reach their completeness only in the final note of the last beat (compass). Actually there is a continuum without break that gives not only its identity but also its oneness, unity, wholeness and fullness. At any stage of the development, since the formation of the zygote, we face the same and unique new being.

200 IGNACIO CARRASCO DE PAULA

THE EMBRYO BEFORE IMPLANTATION: BETWEEN NATURE AND THE PERSON

The ethical argument about the embryo during the pre-implantation stage in general, and, at a concrete level, the discussion about the possibility of using it as a subject for experimental research or as a possible donor of tissues and/or cells, is strictly dependent upon the ontological problem, that is to say the identification of the true nature of identity of the embryo. Indeed, between the ethical question (how one should behave towards a subject) and the ontological question (who or what that subject is) there is a solid relationship given that respect due to an entity must be commensurate with its value or preciousness. By value we here mean not the possible price established by the rules of the market but the objective good quality that springs from the perfection of the being. In this sense we believe that living beings rightly deserve a greater respect than inanimate things and above all that rational beings deserve far greater regard than that due to irrational beings. Indeed, we have coined a special word to indicate the specific value or preciousness of man ‘dignity. For this reason, choosing the crux of the ontological question should be a decisive step in providing a decisive answer to the ethical question regarding the human embryo. Indeed, if one takes into account that persona significat id quod est perfectissimum in tota natura,1 and if one managed to demonstrate that the embryo before pre- implantation is really a person ‘a demonstration that I believe plausible ‘then there would be imposed in an apodictic way the duty to unconditionally respect its life and its integrity. All of this justifies the enormous interest amongst scholars in the question of the identity of the embryo. However, the moral question does not only depend upon ontology. For example, the Instruction Donum Vitae, an Document of the Magisterium of the Catholic Church which is important, among other things, because it explicitly applies the personalist norm to the human embryo from conception onwards, that is to say the duty to treat it with the regard due to a person, does not even attempt to base this ethical conclusion on the personal identity of the embryo,2 but rather upon three other arguments3: a biological argument: the data provided by embryology and genetics authorize the thesis that we are in the presence of an individual human being during the first stages of his or her development4; a biographical argument: it is evident that to destroy an embryo means to impede the birth of a human being; an ethical argument: a general principle of morality establishes that it is never licit to act with a doubting conscience: given that a doubt continues as to whether the embryo is really a person or not, it is necessary to respect it as such otherwise one accepts the risk of committing a murder.5

HUMAN NATURE AND THE PERSON After making this necessary premise, I would like to make clear that the reflections that I will propose here are not to be located in the context of the question of the ontological identity of the embryo but rather in that connected with the question of the respect due to the embryo. At the origin of these reflections is an experience that was gone through on a number of occasions last year in Italy during the debates that preceded the referendum on the law governing the procedures of medically assisted procreation. Indeed, in these circumstances it occurred that when a discussion centred around the basic features of the question nearly always two postulates were taken a priori for granted, but which in reality may not be taken for granted: the first postulate: those who declare that they are in favour of the application of the personalist norm have the obligation to demonstrate unequivocally that the embryo is a person. The second postulate: to demonstrate that the embryo is a person is also necessary in order to establish whether it possesses or does not possess a real human nature, because it is believed that the embryo either is a person or is not human.6 It is specifically in this perspective that I would like to draw attention to the theses that I would now like to advance, theses that are to be located at the opposing pole of the two postulates that have just 201 been cited, namely: the first thesis: the personalist norm is pertinent to the human embryo independently, as well, of the metaphysical problem of its personal identity. The second thesis: one cannot exclude, at least at a theoretical level, the existence of entities that belong to the human species, that is to say that are in possession of a real, albeit imperfect, human nature, which have not yet achieved the status of a person. These two theses have different epistemological values. Whereas the first obeys the logic of the practical intellect, of ethics, the second falls to the full under the logical rules of the speculative intellect and thus creates a rigorously metaphysical subject that we could summarize in the following way: person is the way of being that is specific to human nature; human nature can be a person both in actu and in potentia; until it passes from the potential to the act, thereby achieving the completion of personal being, this nature is human but imperfect; however, one should speak about true human nature because what has not been possessed in a potential way cannot become actual. In this sense, for example, could be interpreted the famous statement by Tertullian: ‘A man who will be a man is already a man ‘(Apologeticum, IX, 8).

THE MORAL QUESTION It would be extremely interesting to dwell upon the metaphysical dimensions of what has just been outlined. However, given that at this Round Table I have been asked to pay attention above all else to the ethical aspects, I will here propose to examine on what theoretical bases scholars of great authority, indeed scholars representative of the best Catholic moral tradition such as St. Thomas Aquinas or St. Alphonse of Liguori, on the one hand concede that the embryo during the first stages of its development cannot be considered a person, and, on the other, in practice, have a similar approach to the personalist principle, that is to say that from the moment of fertilization the embryo deserves the unconditional respect due to a human person. On this point I believe that it is indispensable to examine not only the arguments adopted in favour of this thesis but also the concepts that make up their theoretical foundation and that allow them to implement a practice that is equivalent to the personalist norm.7

ST. ALPHONSE OF LIGUORI, A CRIME AGAINST LIFE The position adopted by St. Alphonse was the common view of Catholic moral tradition until the beginning of the twentieth century. Post-Trent morality, more than illustrating the positive duty to welcome and respect unborn life, preferred to emphasize the gravity of an infraction of this duty. Now, it cannot be doubted that for Liguori every attack on the life of a newly conceived human is always a grave matter; however, only in the foetus which is already infused with a spiritual soul ‘and thus a person ‘can one speak about murder; otherwise, this is a crime against generation, that is to say a sin to be located at the level of contraception.8 For St. Alphonse, therefore, the ontological question played a secondary role, to the point that he does not even invoke the principle of doubt, which for that matter is often used in other parts of his Theologia moralis. For him the incontrovertible evidence that, for example, abortion involves the interruption of the growth of a human life, which, following logic, is a very grave fact, although when perpetrated in the first weeks of pregnancy does not constitute true murder, was sufficient. As a result, in the approach of St. Alphonse the pre-implantation human embryo in no case could be used for research purposes or as a possible donor of biological material. One could only take into consideration the possibility of using the remains of an embryo once death has been ascertained, and this in specific conditions, such as, for example, that scandal is avoided and any causal relationship between the death and the possible use of the cadaver is excluded. But here we enter a set of issues that are completely marginal to the subject of this paper.

202

ST. THOMAS AQUINAS The position of Thomas Aquinas is well-known. He was, moreover, cited on a number of times in the debates in Italy about the law on medically assisted procreation. In continuity with Aristotle,9 and sharing with him the metaphysical concepts of matter and form and the erroneous biology of the time, Aquinas argued that the embryo came to possess a rational soul only a few weeks after fertilization. Now, without a spiritual soul there is no person, without a spirit there cannot be an individual substantial subject of a rational nature. In this light one can deduce: either that Aquinas was a ‘liberal ‘ante litteram who believed that the embryo during the first stages of its development is without dignity or that he thought that it was not indispensable to recognize the presence of a spiritual soul in the embryo for there to be an obligation to respect it unconditionally. To express the point in another way: to the ontological question: during the first fifteen days is the human embryo a person? Aquinas would certainly have answered: it is not, not even during the subsequent fifteen days or at least ‘according to the authority of Aristotle ‘it does not appear to be so10; to the ethical question can we therefore manipulate it, exploit it or eliminate it for proportionate reasons? Aquinas would have equally certainly replied: absolutely not, there are no proportionate reasons that can justify such a thing, the deliberate killing of an innocent human being is not ethically allowed, even before that human being has received a spiritual soul. In order to assess the real weight of these statements it is necessary to emphasise that St. Thomas never posed these two questions to himself in an explicit way and that he was not even directly interested in the question of the status of the human embryo. The majority of the texts that may have some significance here are in a particular theological context the Christological context (Jesus as perfectus homo from the moment of conception) or the eschatological context (whether unborn children will have or not have the benefit of resurrection). With respect to conclusions at an ethical level, the evident corporeal continuity between an embryo and an adult man ‘we have all been embryos ‘is sufficient to deduce that the embryo really shares in the nature that makes human every man who has reached the full of his being. The human embryo, in fact, before receiving the spiritual human is always human, that is to say a living being that belongs to the human species, and possesses a life that is truly human although it is imperfect and destined to a give way to a life that is human in the full sense ‘personal life.11 It is clear that this concept of imperfect human nature as opposed to perfect human nature creates a number of difficulties for the modern mentality, above all because the mod- ern mentality has distanced the metaphysical notion of nature and tends to read every relationship between the perfect and the imperfect in an exclusively dialectic and evolutionistic key. For St. Thomas Aquinas, however, things stood differently, firstly because the very concept of life allowed gradations, 12 and secondly because the relationship between perfect and imperfect was seen in the perspective of the key concept of participation,13 according to which omne imperfectum est quaedam participatio perfecti.14 Participation does not belong to metaphoric or symbolic language but indicates a way of being real, a condition or identity that is really possessed, although not in a full but in an incomplete or partial way. For this reason, to understand the embryo as someone who participates from the moment of conception in the personal being of man authorizes us to argue that such a subject must be respected as a person, that is to say in an unconditional way.

203

1 ST. THOMAS AQUINAS, Summa Theologiae, 1 29 3. 2 A thesis, for that matter, that is held to be very probable. See CONGREGATION FOR THE DOCTRINE OF THE FAITH, Donum Vitae (22.2.1987), I, 1. 3 Naturally, the Instruction presents above all arguments ex auctoritattibus theologicis, in particular from Holy Scripture and Tradition. 4 The Document refers to the ‘recent findings of human biological science which recognize that in the zygote resulting from fertilization the biological identity of a new human individual is already constituted ‘(CONGREGATION FOR THE DOCTRINE OF THE FAITH, Donum Vitae, I, 1). 5 This is a principle proposed by the Declaration on Procured Abortion (18.11. 1974, n. 13), even if a doubt existed concerning whether the fruit of conception is already a human person, it is objectively a grave sin to dare to risk murder. 6 Perhaps this second postulate has been influenced by an erroneous interpretation of a clearly rhetorical question to be found in Donum Vitae (I, 1): ‘How could a human individual not be a human person? ‘. 7 This question is not applicable to the position of the Magisterium because its doctrine is valid for what it actually teaches (for example, the embryo should be respected unconditionally) and not because of the ‘reasons ‘that are provided to support such teaching: these can change or even be absent. 8 See ST. ALPHONSE OF LIGUORI, Theologia moralis, l. 3, t. 4, c. 1, d. 4, n. 394. 9 ARISTOTLE, De Generatione animalium, II, (B) 1, 731 b. ‘6, 745 b. 10 The presence of the spiritual soul, according to the erroneous biology of the time, required a body omnia membra distincta even if still very small: ‘In quantitateâ?¦sicut magna formica’(ST. THOMAS AQUINAS, In III Sent., 3 5 2 ad 3). It is advisable to recall that these interpretations have been superseded by contemporary biological knowledge. 11 ‘Embrio antequam habeat animam rationalem non est ens perfectum, sed in via ad perfectionem’(ID., De Potentia, 3, 9 ad 10). 12 ‘In generatione animalis et hominis in quibus est forma perfectissima, sunt plurimae formae et generationes intermediae, et per consequens corruptiones, quia generatio unius est corruptio alterius. Anima igitur vegetabilis, quae primo inest, cum embryo vivit vita plantae, corrumpitur, et succedit anima perfectior, quae est nutritiva et sensitiva simul, et tunc embryo vivit vita animalis; hac autem corrupta, succedit anima rationalis ab extrinseco immissa’(ST. THOMAS AQUINAS, Contra Gentiles, 2, 89 11). 13 The concept of participation should be considered here in the qualitative sense of partialiter esse and not in the quantitative sense of partem capere. Cf. FABRO C., La nozione metafisica di partecipazione, Torino, 1969. 14 ST. THOMAS AQUINAS, Summa Theologiae, I, 93, 2 ad 1.

204 ROBERT SPAEMANN

WHEN DOES THE HUMAN BEING TO BE A PERSON?

Person is not a sortal term, not the term for any species, sort, or group that would subsume individuals of a certain class of objects and thus identify them as examples of this sort. In order to know that someone is someone, and not something, a person and not a thing, we must already have identified him or her as the living being of a certain kind; e.g. as a human being. Neither does person refer to a genus, like mammal, under which we could subsume a variety of species. As Thomas Aquinas puts it, person is rather a nomen dignitatis. To call someone a person means to acknowledge for this someone a certain status, indeed the status of being one’s own goal. The concept of being one’s own goal is familiar from Kant’s categorical imperative: ‘Act in such a way that you never use humanity in your own person or in the person of another merely as means but at the same time also as end ‘.1 This imperative does not prohibit every instrumentalization. We could not exist without constantly utilizing one another as means for our ends. But such instrumentalization must be in principle one of mutuality. We may not reduce persons to the status of mere means for our ends. From this follows e.g. the prohibition of slavery. The reason for this prohibition is that the human being as rational and therefore free is in a position to set his or her own goals and also for reasons of insight and justice to relinquish these goals. For this reason the human being has a rightful claim to be subjected to merely to those consequences of actions that can be justified in regards to him or her. As free beings, persons are subjects with rightful claims to justification. I mentioned that the notion of the person as being one’s own goal is familiar to us from Immanuel Kant; in fact, it is of course older than that. Thomas Aquinas put it succinctly: homo naturaliter est liber et propter seipsum existens.2 Unlike other living beings, free beings are characterized by their possessing a different relation to their essence, their nature and how they are qualitatively. They are not mere exemplars or instances of their particular kind, into which an external observer might place them, but they stand in a subjective relation to their nature and how they are qualitatively. One could put it this way: they not only are their nature; they have their nature. But, unlike Sartre’s claim, they are not subjectivities without an essence, that first provide themselves with how they are. They already possess how they are, but they are not simply identical with it. Their being is having a nature. As both Ovid and the fairy tales of various peoples bear witness, human beings have always dreamt of metamorphosis, in which human beings are transformed into other beings and yet, most interestingly, still remain themselves in the process. Again: in distinction to all things and living beings, persons relate to what they are. Who they are is not completely identical with what they are. In a famous essay, Harry Frankfurt noted that human beings do not merely wish what they wish. They can wish to have other wishes.3 Think, for example, of drug addicts. They can be discontent with their own appearance and character. Frankfurt speaks of secondary volitions. Persons can have secondary volitions. For this reason, persons can also make promises, i.e. they can determine their future volition by granting to others a claim upon it. For this reason, too, persons can also be forgiven. One need not nail them down to be forever precisely the ones who have done this or that. We can and must allow others to reject their deeds. Unlike the ancient concept of person, our concept of person has been shaped by Christian theology. In the doctrine of the Trinity, we think of God as an essence for which it is characteristic to be had in a threefold manner. In Christ we think of a person who has two natures; but in such a way, that this one person is defined through having the divine nature. And it is this divine person who assumes the second, human nature. The doctrines of Trinity and Christ have prepared the structural insight into the essence of personality.

205 Considering what we have said until now, it might seem as if the conclusion would follow that the recognition of human beings as persons should depend upon the actual presence of those characteristics that define personality. It might seem to be implied that only those beings are to be considered persons which actually have something like self-consciousness, a presently conscious relationship to oneself and one’s life. Such a claim has in fact been made repeatedly in the debates of recent decades. While denying the person- hood of embryos, young children, the severely, mentally handicapped, and the aged senile, the demand has been articulated to replace the concept of human dignity with that of personal dignity in the constitutions of European countries and in the documents of the United Nations. This line of thinking is not without some support in the European tradition. True, it is clearly opposed by the great revolutionary, Kant, but it can claim some support in the view of Thomas Aquinas that all human beings apart from Jesus Christ have in the first cycles of their embryonal existence an animal soul before this is replaced through a direct act of divine creation by a human and thus personal soul. For scientific reasons, this view no longer has many defenders. By contrast, John Locke’s understanding of person has become increasingly dominant. Limiting his ontological commitment strictly to the content of interior or exterior empirical perception, Locke excluded all the results of ontological transcendence as well as all the results of transcendental reflection. For him, personhood is therefore not a way of being, which comes to be knowable by certain states of consciousness, but rather personhood is nothing more than that very state of consciousness itself: the state of a subjectivity experiencing itself as identical over the extended flow of time. Since empiricism deals only with the states of exterior or interior experience, but not with anything like the bearer of such states, thus there are for this brand of empiricism no unconscious or sleeping persons. David Hume took the next step and simply denied personhood altogether. To be precise, as he thought, there is no experience stretching across time. There is admittedly recollection, but as a present experience recollection happens here and now. Recollection is not the presence of the past but the presence of a current picture of what we now take for the past. This is also why recollection can deceive us. Thus, he concludes, there are only momentarily present experiences, nothing like an identity stretching across time to which the personal pronoun I refers. D. Parfit’s book: Reasons and Persons, continues along this line from Locke. Parfit denies a continuity of the person into and beyond sleep. Those who are asleep are not then persons, and those who awake are no longer the same persons as before falling asleep. Each phase of sleep puts an end to personal existence. Whoever wakes up inherits from the one who passed away in sleep certain contents of memory based on the physiological identity of the human animal and its brain. Interestingly, Parfit provides here a new account of the obligations we bear towards ourselves, obligations otherwise accounted for only by religious arguments. The obligation that I take care of my health is an obligation towards a being distinct from myself, a kind of progeny. Clearly, personhood is separated here from human existence. There are human beings as living organisms, and there are the personal states of many ‘yet in no way of all ‘of these human beings. The being of the person does not begin with her existence as a living human organism, but rather with the gradual awakening of certain conscious states. The extent to which this understanding has quietly spread can be seen in an explanation offered by no less a figure than the head of the German Bishops’Conference in the context of the debate about the so-called brain-death criterion for organ transplantation: it could well be, he suggested, that the death of the person precedes the death of the human being. I would like to argue against this view by defending the thesis that personhood is not a property but rather the being of the person; and that therefore the person does not begin later than the existence of a new human life no longer identical with the parental organism. Persons are not a natural species that we could identify by description. No one can prescribe for us when we should or should not use the term person. We are dealing here with a question that is not primarily theoretical, but practical and ethical. To name someone a someone and not a something is an 206 act of recognition, of acknowledgement, to which no one can be forced; and yet this decision is also not arbitrary. Joined to our use of the word person, the act of acknowledging someone as someone and not as something has an immanent logic to it. The unjustified limitation of the circle of those who are so acknowledged changes the nature of such recognition even towards those who are still acknowledged as persons. The unjustified dating of the beginning of this acknowledgement at the beginning of life leads to an unjustified termination in the final stage of life. A person is someone and not something. There is no continuous or gradual transition from something to someone. It would be incorrect to say: ‘Someone is something with these and those qualities ‘. Someone is not something. In order to say what we mean by someone, we are forced into a tautology: we call someone, who has such and such qualities. But this is also incorrect. We consider some beings, namely human beings, as someone, even when they do not possess these qualities de facto. We could well characterize our own position with a sentence from Wiggins in Sameness and Substance: ‘A person is any animal, the physical make-up of whose species constitutes the species’typical members thinking intelligent beings, with reason and reflection, and typically enables them to consider themselves as themselves, the same thinking beings, in different times and places ‘.4 The actual presence of the typical traits of persons is not in fact the condition of personhood. This is clear in the common use of the personal pronouns I and you. We all say: ‘I was born in such a such a year and place ‘; and: ‘I was conceived in the city such and such ‘, although the being that was conceived and born could not yet say I. The personal pronoun, I, does not refer to the Ego. The Ego in this sense is the invention of philosophers. The personal pronoun, I, refers rather to a living being that only sometime later began to say I. The identity of this living being is independent of that about which it has actual memory. Thanks as well as accusations can be directed towards someone for deeds that he or she had long forgotten. A mother, of course, says to her child: ‘When I was pregnant with you or when I was giving birth to you; not when I carried within me an organism, from which you then later came to be ‘. All the attempts to isolate personhood from the vitality and existence of the human organism are counter-intuitive; they are incompatible with the language commonly used by normal human beings. This normality is also the condition for human beings to develop the properties characteristic of persons. No mother has the feeling that she needs to talk at a thing and by talking long enough to condition this thing, a something, until it, too, finally begins to speak. For this reason, too, children do not first learn from computers how to speak. Rather, in her interaction with the baby, the mother retraces the child’s niveau in order to relate to the child as a human being to a human being. She speaks not just about, but to the child, says you to the child; she treats the child as a small person. And only because the child has been treated as a person, will the child ever become that which he or she was from the beginning and as which that child was considered from the beginning. Whoever severs the personhood of human beings from their being alive severs as well the bonds of interpersonality, within which persons can first become what they are. Persons are found only in the plural. Even the use of the word person for God makes full sense only in the context of the doctrine of the Trinity. A further argument against binding personhood to the actual presence of certain properties is this: to place such conditions is to transform the act of acknowledgement into an act of co-opting or drafting. This would mean subjecting those who arrive later to the arbitrary demands of those who already mutually acknowledge one another. It is the latter who determine the properties on the basis of which someone may be co-opted or voted into the community of persons. The arbitrariness of such demands is reflected in the extreme diversity among scientists as to the beginning of personal rights. Some want to let the protection of life begin in the third month of pregnancy; others, only from birth on; still others, only six weeks after birth. It is only logical consistency when Peter Singer denies that children under two years have something like a right to life. Once we abandon the sole criterion of belonging to the species homo sapiens and originating from other members of this species, then it is merely a question of power, which human beings are granted personal rights and which ones are not. It belongs, 207 however, to the very dignity of the person that she assume her rightful place within the community of persons not as a co-opted or voted in by others but as a native member. Each and every human being belongs to the community of persons by virtue of the fact that he or she belongs to the family of human beings, i.e. is related to other human beings. Evolutionary biology, represented for example by Ernst Mayr, has given up the attempt to define the human species as a class to which its members belong on the basis of similarity, as is the practice in classifying inorganic things. The concept of population has replaced the concept of class. An animal belongs to a population by genealogical relation, that is, through shared descent and through sexual interaction. In the case of human beings, family relations are more than simply a matter of biology; they are also personal relations. Father and mother, son and daughter, brother and sister, grandfather and grandmother, grandson and granddaughter, male and female cousins, uncle and aunt, brother-in-law and sister-in-law are certain places within an interpersonal structure. Whoever fills such a place, holds this place from the very beginning of his or her biological existence and retains it for the rest of their lives and beyond. This is something quite different from almost all animals. An embryo is the child of his or her parents from the first moment of existence. As a member of the human community he or she is a member of the community of persons, and as a member of the community of persons he or she is person, quite independently of any properties. It is said of Peter Singer that in quite an impressive way he takes diligent care of his mother, who suffers from Alzheimer’s. When asked once in an interview how this can be reconciled with his view that Alzheimer’s extinguishes the personality, he is said to have replied: ‘But that is my mother. And that is exactly the point: the mother remains his mother and the son remains her son. Quite independently of whether it is realized subjectively by both persons, the relationship is a personal one. For this reason, the mother remains a person as long as she remains alive; just as the son is son since he began to live. Were biological relations not something personal as well, how could it make sense that illegitimate and adopted children, at the latest during their puberty, develop the desire to come to know their biological fathers or parents? They consider their relation to an unknown relative as part of their personal identity. It might well be noted here that something analogous holds true for the sexual relationship between man and woman: this relationship, too, is never merely biological. Where it is reduced to this alone, it is always a deprivation. The intelligent dimension of the perversity of the Marquis de Sade is that this is desired precisely as deprivation and humiliation. But the converse here is also true: what is personal for human beings always has its biological dimension. It is no accident that eternal life is portrayed as the cena vitae aeternae, a community at the table of food and drink. In the faith of the Church, the virginity of the mother of Jesus is not exclusively spiritual; and conversely, what is spiritual finds its expression in the biological fact that Jesus was conceived without the physical cooperation of a man. The question as to the temporal beginning of human personhood is in a certain, real sense unanswerable. Personhood is something supra-temporal. By personhood human beings participate in the mundus intelligibilis. Personhood means that the human being is a being capable of truth; and truth is supra-temporal. We are here today together in Rome: that is something that was always true and will remain true forever. Inasmuch as personhood is a participation of supra-temporality, every attempt to pinpoint its temporal beginnings will have something futile about it. Similarly it will be impossible to pinpoint the exact moment of death; looking back, we can say that this person is no longer alive. Conversely, as soon as we are dealing with a human being, we always have to say: this is a person. This is exactly what Kant had in view, when he wrote: ‘Just as what is conceived is a person, and just as it is impossible to develop a notion of conceiving by means of physical operations any being endowed with freedom, so, too, is it in a practical sense an entirely correct and even necessary idea to consider the act of conception as the very act by which we have placed a person in the world ‘.5

208 One could put it like this: the identification of the coming-to-be of the person with human conception is the consequence of the more fundamental impossibility of pinpointing the beginning of the person in time. Those who suggest a later point in time claim to know more than they can. This holds true for Thomas Aquinas as well. Thomas accepted this identification only for the humanity of Jesus Christ. For all other men he supposes that the spiritual or personal soul is created directly by God only on the fortieth day after conception, when the new soul replaces the animal soul that had previously existed. The background for this view is to be found in Aristotle’s thyrathen, the ancient doctrine that spirit, as quite other than a part of the soul, cannot be conceived with the soul, but must enter the human being from outside.6 This doctrine was based on the insight that spirit cannot be defined as a biological function but must be understood as participation of trans-temporal reality. In opposition to Aristotle, Thomas taught that spirit is part of the soul, but precisely in the sense that the soul defines itself through its spirituality. For this reason he denies that a soul of this kind comes to be by conceiving, but rather only by an immediate act of creation without the secondary causality of the parents. This doctrine can no longer be held, once we know that the development of the human being, directed by our genetic program, occurs with strict continuity and that the inherited features provide even the spirituality of the human being their individual contours. This in turn renders the assumption implausible that the origin of the personal soul is not due to conception; for this assumption stands and falls with the assumption of successive animation. If the spiritual soul takes possession of the human being only at some later point, then human parents conceive only an animal, a living being, just like all other animals. But when there is no such animal pre-phase, then what do they conceive? A non-living organism? But we would call this conception. When, however, the human being lives from the very start by a spiritual soul, then it cannot be that the animation has nothing to do with the conception by the parents. This is surely the sense of the passage in Pope John Paul II’s Encyclical, Evangelium Vitae, when he says that the creation of the human soul by God has been inscribed into the act of human conception.7 This is no obiter dictum, but a challenge for theological anthropology to think through again the connection between conception and the tyrathen of the creation of the spiritual soul. In conclusion I want to consider briefly the question of the beginning of human life, which we must identify with the beginning of human personhood. This question as to the beginning of human life cannot be answered without the participation of the biological sciences. But please allow the philosopher here three observations. It cannot be that we talk of human life but really understand by this expression something other than the life of a human being. As Aristotle8 and then St. Thomas put it, vivere viventibus est esse. There is no anonymous human life. Wherever there is embryonal life, it is either the life of the mother or the life of a new human being. Science is agreed that the life of a new DNA structure is not the life of the mother. It is thus the existence of a new human being distinct from the mother. It cannot be that we admit that the life and so the personhood of this new human being only begins once a certain degree of independence from the life of the mother has been obtained: so e.g. with nidation, since it is said that only then is an independent development possible. The latter claim is true, but what then develops independently already exists before it finds its appropriate place in the uterus. And of course even then it doesn’t develop independently, but it needs constant transfer aid from the maternal organism; of which it continues to have need even after the birth, insofar as independence implies freedom from the need for help from others. Independence in this sense is achieved only several years after birth; and total independence is something we never achieve. Human existence is always dependent on a certain degree of solidarity. Was is demanded of Cain is that he know where his brother is. ‘Am I my brother’s keeper? ‘is the answer of a murderer. But what about identical twins? I said: human life is always the life of a human being, a particular human being. But must we say: until we can decide regarding embryonal life whether we are dealing with one, two, or three human beings, we cannot yet be dealing with personal life at all? This does not 209 seem to me a necessary conclusion. First of all: when later identical twins reach the age of reason, they will say, just as other human beings do, that they were conceived at a such and such time. They will refer to the beginning of the human life that was theirs. When in this context they say we instead of I, the personal pronoun we is not meant any less personally than I. We always means a multiplicity of persons, not an anonymous, subject-less life. When we as Christians understand our personhood as our being spoken to by God, then this means with regard to twins: God always saw from the beginning in the life that began there the two or three persons whose common beginning we are dealing with. Equally possible is the opinion of the embryologist Blechschmidt, who assumed that, with a zygote that will later divide, we are dealing at first with one person, from whom then a second originates, as with Eva from the side of Adam. We begin to enter here a realm of speculation, but not an unjustifiable one. Even the opposite assumption that we first have an anonymous life that then divides into two persons is speculation, but one that stands in contradiction to fundamental ontological insights. It suffices that there be alternative speculative accounts of a kind that is compatible with such insights. If someone wants to prescribe for these questions radically empirical abstinence, they should at least be consistent, as consistent as David Hume, who, in confessing that he did not know what to make of the concept of person also admitted not belonging to such human beings who are happy to have an I.

1 KANT E., Fondazione della metafisica dei costumi, in Scritti morali, Torino: UTET, 1970: 88. 2 ST. THOMAS AQUINAS, Summa Theologiae, IIa IIae, 9.64, a. 2, ad 3. 3 FRANKFURT H., Freedom of the will and the concept of a person, The Journal of Philosophy 1971, 68: 5-20. 4 WIGGINS D., Sameness and substance, Cambridge: Harward University Press, 1980: 188. 5 KANT I., Metaphysik der Sitten, A 114, 1797. 6 ARISTOTLE, De generatione animalium, II, 3, 736b 27-28. 7 JOHN PAUL II, Evangelium Vitae, n. 43. 8 ARISTOTLE, De anima, II, 4, 415b 13.

210 JEAN-MARIE LE MÉNÉ

WHY IS IT A DUTY TO PROTECT BY LAW THE PRE-IMPLANTATION EMBRYO?

The place of this communication in the program (the word of the end?), the field of expertise that it mobilizes (the law) and the imperative formulation that it takes (a duty) will contribute to give a particular tone to my intervention. When the practitioner of the law is consulted, he must give a fast answer. He is not interrogated to tell if the pre-implantation embryo has or has not to be protected. This point is given for granted. He is asked what are the mainsprings of this protection, presented as a duty. The answers of the jurist will therefore be simple: if there is an injustice, it is first necessary to stop it; then one has to fight the deep causes of this injustice.

TO PUT AN END TO THE GENOCIDE OF THE PRE-IMPLANTATION EMBRYO What the jurist observes is that the pre-implantation embryo1 is the object of a culpability and of a condemnation so general and radical that one can, without excess of language, speak of genocide. His ability to be created and then destroyed serves for his definition. Before even to be, he is useful and used. Rejected for what he is, he is coveted for what he has. Such a total availability of a being is without an equivalent in history.

TODAY, THE GENOCIDE OF 75% OF THE PRE-IMPLANTATION EMBRYOS An one-sided indictment since the beginning of the extracorporeal fertilization In the different reports, nationals as well as internationals,2 which aim at providing norms, the presentation of the pre-implantation embryo is done with a constant effort to demonstrate that it cannot belong to the human species. When the very early embryo is evoked in a text, this is to overwhelm him with an indictment. The main arguments of these charges are the following: first, he is guilty not to be an individual. How to protect a being who has the audacity to be able to become several, insofar as the embryo, in this early stage, can be multiplicated or divided? Then, he is guilty not to resemble us, the adults. The morphological argument used as a criterion of humanity ‘strange as it is indeed for a legal procedure ‘leads to watch for the first revealing trace of a recognizable human shape: it is the famous primitive streak of the 14th day that founds the utilitarist fiction of the preembryo. The pre-implantation embryo is once more guilty not to present the biologic properties that characterize the human being already born: absence of immunological properties, absence of sensitivity to teratogenic agents, sexual indifferentiation. Finally, he is guilty of nomadism, as he has no attach, no permanent dwelling, being not yet implanted in the uterus, and this makes him a being that would have no relationship, and therefore one who doesn’t have its place in the society. And, above all, he is guilty of not being aware of what he is going to become, guilty of an uncertain future, of unsure prospects. The nature allowing the spontaneous abortion of a certain number of fertilized ovocytes, on what basis would be the scientists prevented from merely anticipating this natural selection? In some cases, cannot the embryo evolve into a tumor? ‘From then on, how to consider the embryo of the first day as a human person? ‘, wrote M. Etienne Beaulieu, ex-president of the FrenchAcademy of Sciences.3 These reproaches addressed to the pre-implantation embryo present a point in common. The embryo is guilty to be what he is, that is guilty of being apparently elusive, unsteady and mysterious; up to the point of generating incomprehension, irrational reactions and denial of humanity. Guilty in 75% of the cases

211 The pre-implantation4 embryo is, in practice, submitted to the double constraint of desire and of quality which draw a simple frame of evaluation. In the first place, the pre-implantation embryos are either desired ‘one will say that they come up within the setting of a parental project ‘or not desired if they are not or are no more the object of such parental project. In the second place, the embryos can be either healthy, or unhealthy. The crossing of these basic alternatives ends up in four scenarios and only four: embryos desired and healthy, embryos desired and unhealthy, embryos non desired and healthy, embryos non desired and unhealthy. Embryos desired Embryos non desired Healthyi embryos Suspended sentence Guilty Unhealthyi embryos Guilty Guilty

A priori, only the desired and healthy embryo merits to be protected because he is the only one innocent. The desired but unhealthy embryo is guilty to be unhealthy and will therefore be eliminated. The proof of it is that most of the rich countries have developed systems of pre-implantation diagnosis of the unhealthy embryos in order to give birth only to healthy children. It goes without saying that if these embryos were not guilty, the society would not eliminate them, but would take care of them and would even undertake some research in order to heal them. Of course, the non desired and unhealthy embryo is two times guilty. It is therefore two times sentenced to death, due to the lack of desire from the part of his parents to have him living and to the lack of a sufficient quality from his part. As for the non desired but healthy embryo, he is also guilty. Guilty of the offense of hindering the solidarity between generations. Guilty to deprive the community of the gift of its precious stem cells (ES). Indeed, the right of the embryo to live is at a lower level than the right of the sick adults to be treated with the stem cells of a healthy but non desired embryo. ‘In these conditions, to refuse to use the embryo, is to deny medical care ‘, one can sometimes hear. ‘The right to life of the embryo that would deprive the patients of the benefit of cellular therapy would be a crime against humanity ‘, some are also telling. If one makes the account, in 75% of the cases the pre-implantation embryos are actually guilty, in the legal sense of the term, insofar as one can inflict them the capital punishment. The three quarters of these embryos contribute indeed either to make sick the society, when they are themselves sick, or not to heal it when they do not deliver themselves willingly to the science. In what regards the desired and healthy embryos, that constitute the remaining quarter, they are not definitely safe and sound. The respect that is granted to them is not a uniform protection from fertilization to birth, but a gradual, progressive protection, adapted to each moment of their development. It is necessary to keep in mind that the fact for the would-be child to be desired is revocable ad nutum, at all times and that his apparently good health may weaken. Let’s say that the desired and healthy embryo is in a situation of reprieveâ?¦

TOMORROW, THE GENOCIDE OF 100% OF THE CLONED EMBRYOS It seems that the observers, Christian observers in particular, have not taken an exact measure of the gravity of this culpability of the pre-implantation embryo that has become a given notion, widely admitted. This has led to the withdrawal of the principle of the unavailability of the human being: the embryo can be sorted out, frozen, appropriated, submitted to experimentation, given, destroyed, etcâ?¦ This notion is the antechamber for the authorization of human cloning. The human cloning, consequence of the availability of the embryo

212 In 1998, some American teams have, for the first time, isolated and put into culture stem cells extracted from human embryos. Since then, various therapeutic perspectives have open up very quickly ‘the so called cellular therapies ‘starting from the creation of cell lines derived from the ES cells. The therapeutic character of these researches is, in fact, questionable especially since that, in 1991, a French team showed that there was in the blood of the adults a mesenchymal stem cell of a multipotent nature.5 From that time on, there has been, therefore, an opened ethical alternative to this cellular therapy with ES cells that require the lives of human embryos. But it should be observed that, in the various reports to Parliaments on bioethic’s matters,6 after having recommended the use of the surplus embryos, the experts immediately expressed some reserves concerning the immunocompatibility problem that would come from the injection of ES cells in an adult organism. They recommended therefore systematically to move toward the solution of the nuclear transfer, which means to authorize human cloning. The ideological link between the use of the pre-implantation embryo and the practice of cloning is blatant. Here is the proof of it. The different reports have all made comments on the possibility of the immune rejection of the ES cells retrieved from surplus embryos, whereas the difficulty is not there. These embryonic cells, precisely, are not rejected by the recipient organism. They develop into it. The question that arises, on the contrary, is the one of the control and of the mastery of this development that tends to take place in an anarchical way (tumors). In other words, the argument presented to support the shift from the use of surplus embryos to the practice of human cloning is not scientifically established. But it is efficient: cloning has thus become an unavoidable technical consequence of the availability of the human embryo. The human cloning, justification of the availability of the embryo Not only does the transgression of using the human embryo leads logically to cloning, but that initial transgression also justifies, a posteriori, human cloning. Human cloning is considered by some as an instrumentation that concerns cellular artifacts produced without fertilization, and not human embryos. The biologist Henri Atlan,7 for example, doesn’t see why it should be necessary to consider, from an ethical point of view, a clone as a human embryo, just because it can become one. ‘It is a question of embryo’s potentiality, that is a potentiality of potentiality! From that point of view, if one considers that an embryo is a person, or a potential person since fertilization, it would be a lot more coherent to oppose the use of the surplus embryos ‘that are true embryos produced in vitro by fertilization ‘than to oppose the use of cells produced by transfer of somatic nuclei in ovocytes ‘, explains Atlan. The perversity of the reasoning reaches there its utmost. The embryo is not respectable as long as the important matter is to campaign actively for using it. Then, once victory would have been obtained, the embryo would become again respectable in order to justify the shift to human cloning, considered, all things done, as a minor transgression. The argument, there again, is efficient. In France, when the Minister of Public Health, Jean-François Mattei, brought to vote the bioethics’law of August 4, 2004, including both the use of surplus embryos for research and the prohibition of human cloning, his adversaries didn’t fail to show him the incoherence and the fragility of his position. Why not allow human cloning if the human embryo is not worth anything anymore, except for those who try to slow down the process? Effectively, the dike of the respect of the pre-implantation embryo having given way, the practical authorization of human cloning is only a simple matter of political calendar. But, no mistake about that, we are shifting paradigm. In the spiral of transgression, the availability of the embryo doesn’t indicate the crossing of one supplementary degree, but a change in the nature of the transgression. If abortion is still a departure from the interdiction to kill, the survival of the surplus embryo (and of the cloned embryo) is already a departure from the obligation to kill.

213 This hold over the human being, total and totalitarian, feeds itself from the effects of a disloyal and permanent trial in which there are no more judges, and no more defenders. Because ‘what can do the law if the delirium is today in science? ‘, wrote psychoanalyst Monette Vacquin.8

TO PUT AN END TO THE UNFAIR PROCEEDINGS AGAINST THE PRE-IMPLANTATION EMBRYO The failing of the judges It is not the absolute legal emptiness that one sometimes imagines when one hears recriminations against the absence of a status for the embryo. There are effectively norms within the law, especially of penal nature, that integrate the recognition of the human being since his conception, without always drawing all consequences from this. Let’s mention for example the International Covenant on Civil and Political Rights (19 Decembre 1966), entered in force March 23, 1976, and that stipulates that capital punishment cannot be pronounced against pregnant women (article 6, 5). This text constitutes an explicit recognition of the autonomy of the life of the fetus or even of the pre-implantation embryo. Paradoxically, the laws legalizing abortion (at least the French law) often recognize, in a first article, the humanity of the embryo or of the fetus ‘since the beginning of his life ‘, before allowing, in a second article, some breaches in this principle. These are besides the best proofs of the humanity of the embryo or of the fetus. Because, for a law to allow to kill, it has to start from the assumption that there is matter for doing it. To suppress a being who would not be human would not be to kill. And there would be therefore no need for a law. In several countries (of which France until 2001), the law punishes the involuntary homicide of the child to be born. In such dispositions, there is also the affirmation that the earliest human life ‘even before the implantation stage ‘should be respected. And of course, even though it has never had a direct impact in criminal matters because of the fiction of birth that it postulates, the maxim infans conceptus pro nato habetur ‘the conceived child will be held for born every time that his interest is involved ‘has been the object of a large application in law. As prof. Jérôme Lejeune observed it, ‘the legal wisdom had not waited for molecular biology to affirm that the child is a heir, before even to be born ‘.9 Yet, imperceptibly, in spite of the criminal law and of this maxim that are without any ambiguity regarding the respect of the human being, the uninterrupted practice of abortion, and then the practice of the free disposition of the pre-implantation embryo, toppled over this jurisprudence. It is somehow as if the judges reasoned that way: ‘Since abortion is permitted, therefore the embryo is nothing ‘. Thus, they invented false legal debates around the notions of person and human being for concealing the emptiness of their judgments. The false legal debates around the notions of person and of human being The unfair proceedings that is conducted against the pre-implantation embryo rests on the idea that this one would not have the legal status of a person. But it is time to put a term to this legal swindle in not making the law tell what it doesn’t tell. Indeed, the legal person of the civil law doesn’t have anything to do with the legal person of the criminal right. Let’s listen to M. Jerry Sainte Rose, magistrate, prosecuting attorney in the Court of Cassation, in France: ‘The personality which, in the sense it has in the civil law, is acquired at birth according to the dominant doctrine, is an abstract construction destined to facilitate the trade between men. It has been extended to the legal entity of a personâ?¦The human person (instead), which is nowhere defined, is protected in her integrity and in her dignity by the criminal law, whether she is titular of rights, whether she is alive or no, whether she is viable or no ‘. And the high French magistrate shows that the civil death, that consisted once in stripping some convicted of all his rights, civil, civic and political, never meant that one could jeopardize his life. ‘The (legal) personality is not therefore a condition of the human’s protection by the law ‘. He also recalls 214 that an attempt of murder can be committed on a cadaver. ‘Thus, the child to be born would not benefit of any protection from the criminal law whereas a person already dead is protected by that law? ‘. Even more, the French jurisprudence having recognized that the child coming from a rape could ask for the repair of this act, ‘the quality of victim has been recognized therefore to someone who was not even conceived at the time of the facts ‘. Is it reasonable to refuse it to the pre-implantation embryo? ‘We have no choice but to accept that it is with the physical person, with the being of flesh and blood that the criminal law is interested in what regards violences, even the lightest ones. The position taken by the Court of Cassation that subordinates the respect of life to the person’s legal status makes a cheap deal of the autonomy of the repressive right ‘, concludes M. Saint Rose.10 One another takeover by force from the jurisprudence drove the French Constitutional Council to consider, in a decision of July 27, 1994, that the pre-implantation embryo didn’t belong anymore to humanity. Not only would he not be a person, but he would not be either human! The matter for the Constitutional Council was to declare that the first bioethics laws of 1994 which, in particular, allowed the FIVET and the DPI, were conform to the French Constitution. In its answer to the submission of the case by the parliamentarians, the Council estimated that ‘the principle of the respect of every human being since the beginning of his life was not applicable (to the in vitro embryos) ‘.11 Let’s give to prof. Bernard Edelman the care to comment this text: ‘These different preambles, it is necessary to say it with the whole brutality that is needed, are clearly inadmissible. Against the legislator’s will, the Constitutional Council has created an under category of human beings, resuscitating that way the slavery in a new form. Firstly, if respect is the distinctive sign of every human being ‘as dignity is the sign of the person ‘a human being who doesn’t deserve respect anymore is thrown out of humanity. He is an outcast. Secondly, if he is an outcast, no legal obstacle opposes its use, except if he is granted guarantees, like an animal or a biotope. Thirdly, a human being thrown out of humanity deserves the name of slave. What is indeed a slave, if not a human being reduced to a mere biological existence, in such a way that he is, indifferently, a living instrument or a pure material submitted to the will of others? ‘.12

The true finalities of the law At this point, it is urgent to recall the finalities of the law. Far from the legal positivism that sees in the law only the addition of existing rules, the law is not the reflection of a policy, it doesn’t dictate the rules of human conduct, it is not an answer to the aspirations of the individuals or to the demands of the pressure groups. It is not the slave of the authorities, nor of the economy. Fundamentally, the law is at the service of the judge who makes judgments to give back to each one what it is his by right, according to its nature. To take the expression of Saint Thomas: suum cuique tribuere. ’Having before him litigants who quarrel for a good or a dignity, a public office or the repair of a damage, the judge has for profession to define the part that comes back to each one, to assign what is its own to each one. The legislators and the jurists are the auxiliaires of the judge. They aim at what is just, which means the best sharing out; all efforts for reducing this relational end to simple, particular ends, are as many failures. They are busy looking for the best proportion between people and things in a social group ‘.13 As for the law, let’s continue with Saint Thomas who gives the following definition of it: ‘Lex is quaedam rationis ordinatio ad bonum commune ab eo qui curam communitatis habet promulgata’’the law is a prescription of reason with a view to the common good promulgated by the one who has in charge the community ‘. And it is Pope Jean-Paul II, in his last book, who had given the best commentary on it: ‘As a prescription of reason, the law leans on the truth of the being: God’s truth, man’s truth, the truth of the reality created itself in its whole. This truth is the basis of the natural law. The legislator adds to it the act of promulgation. It is what took place on the Sinai for God’s law, it is what takes place in the parliaments for the different forms of legislative interventions ‘.14

215 One understands, from then on, that a law that would not be a prescription of reason and/or that would not be established with a view to the common good, would be unjust: lex injusta non est lex. To disobey an unjust law can become therefore a prescription of the charity.

THE TREASON OF THE DEFENDERS The treachery of the proceedings against the pre-implantation embryos is due, finally ‘and especially! ‘to the treason of their defenders. These, not only affect to ignore the human nature of the embryo, but also ignore actually the finalities of the law as we just reminded them, up to the point to permit themselves some real semantic and legal deliriums. The attempts to rename or to disqualify the embryo The first treason consists, for not being able to change the human nature of the embryo, to want to change his name. In October 1999, prof. Jean-François Mattei, geneticist, future minister of Public Health in France, in an interview granted to the magazine Science et Vie, proposed ‘in order to avoid having to speak of embryo’s manipulation ‘to rename the living structure. ‘Would then have a right to this inviolable status of human embryo only that being which is coming from the fertilization of two sexual cells and which thrusts toward life through his successful nidation in the maternal uterus ‘.

Living structure coming from cloning Living structure coming from sexual fertilization In vitro ? ? Nidation missed in the maternal uterus ? ? Successful nidation in the maternal uterus ? Embryo

As one can note from this table, the formulation is generous in the extent of the exclusion to which it leads. It allows indeed ‘with one writing ‘to cross out of humanity: all the embryos coming from human cloning, comprising those who would arrive to birth, all the non implanted embryos, including those coming from a sexual fertilization. They have to be estimated in fonction of the desire of the parents and of the quality of their project. The second treason consists in suggesting to reconsider the summa division of the Roman law that distinguishes people and things. Another Frenchman, prof. Claude Sureau, gynecologist, ex-president of the National Academy of Medicine, member of the CCNE, made himself the apostle of this legal revisionism. In the newspaper La Croix of February 17, 2000, he declared: ‘A priori, one has to respect the embryo constituted of a few cells, but if some strong scientific arguments plead in favor of his use, one cannot reject them ‘. ‘Obviously, there is not a unique answer to the question of knowing if the embryo is a person or a thing. The embryo, then the fetus, represents an evolutive entity. It is necessary to get out of the Roman law that only distinguishes people and things and to create a law adapted to medicine, especially to the medicine of procreation ‘.15 Or else: ‘By what strange intellectual aberration, by what surprising submissiveness to a binary doctrine supposedly inherited from the Roman law, the jurists persist in refusing to see the evidence: the reality of a specific status (legal, philosophical, ontologic and, of course, medical) of the antenatal being, neither a thing, since he can become a man, nor a person although he is very near from it, with a status that evolves along pregnancy, deserving the respect and the attention that we grant to our patients, but of which we must, sometimes, accept the transgression ‘.16 Finally, would not the status that one grants to the human being inform more on the one who proposes it that on the one who receives it? The pre-implantation embryo needs above all defenders 216 The pre-implantation embryo has no need for a status that would come as a derogation from common law. He especially doesn’t need to have a specific law or distinctive laws designed for him. It would be even very dangerous to accept, on this topic, the principle of a discussion that could only lead to the elaboration of an infra-human status.17 The pre-implantation embryo pre-exists to the law. One does not have to wonder whether the penal law protects the embryo or not. From the moment it punishes, by definition, homicide, murder and assassination, the law protects naturally the life of the human being, including when this human being is at the stage of the preimplantation embryo. It is because his natural defenders betrayed him that the embryo finds himself today aside from an application of the law in his favor. But the legal basic instruments of this application does exist. Of course, one can and one has to improve them.18 But it is above all indicated to make use of them. The pre-implantation embryo doesn’t need therefore anything else than defenders. How should they act? In two directions: the reason and the passions. To paraphrase Pascal, it is a question of both increasing the reason and decreasing the passions. The radical novelty, and the difficulty, in regard to the question of abortion as it arose in the years 70, stays in the diffusion of a generalized ignorance concerning the embryo. In other words, 30 years ago, it was only a question of decreasing the passions because everybody was aware of what was abortion. Unfortunately we know what has been the result of this. Today, it is necessary first to remind the intelligence what is the pre-implantation embryo before urging to the unconditional respect of his life. This is to tell how tough is the task. Christians cannot at all free themselves from a personal involvement in this task. They are those who should be the natural defenders of the embryo. I already wrote, and I keep it, that, if it is not necessary to be a Christian to defend life, on the other hand it is necessary to defend life to be a Christian. Then, it belongs to the entire Church, clerics and laymen, at all levels, to be convinced about that, which is far from being the case. For this purpose, it would appear to me indispensable: to create, in every diocese, a strategic cell specialized in the respect for life, distinct from the pastoral care for the family, composed of experts convinced of the humanity and of the personhood of the embryo, and charged with the implementation of an active resistance to the programmed genocide of the pre- implantation embryo, antechamber of human cloning; to impose on all those who have a function of teaching or a pastoral assignment in the Church, and this down to the level of the parish, the duty to express themselves systematically before every electoral consultation, and at least once a year, in order to recall: that to vote in favor of a candidate whose convictions are not respectful of the embryo constitutes a complicity with the homicide of this embryo and therefore a serious lapse in charity; that the Christian politicians should not be content with not doing but that they have also the ardent obligation of doing positive and innovating propositions to protect the embryo. To restore a communication that brings back explicitly the name of the human embryo among the elements of the current language, avoiding all abusive semantic distinction between embryo and pre- implantation embryo, between reproductive cloning and therapeutic cloning, etc. In the formations, the teachings, the spirituality, the pastoral cares, the apostolates, the caritative movements, the embryo must appear as an other fully-fledged human being. The less Christians speak about the human embryo, the more they abandon him, the more his adversaries seize him, exploit him and destroy him. Let’s not leave them either destroying the very name of the embryo. If today the world lacks the words to express man in his fullness, should not the Christians tell him these words?

217

1 I consider here the expression ‘pre-implantation embryo ‘in its more general meaning, as the embryo which is not yet implanted in the uterus, either because he is migrating toward it, or because he is in vitro. 2 See in particular the Palacios Report of the Commission of Science and Technology of the Parliamentary Assembly of the Council of Europe (Document 5943 of September 1988). 3 BEAULIEU E., Le défi bioéthique, Autrement 1991, p. 120. 4 A pre-implantation embryo having survived to pre-implantation diagnosis can fall victim of prenatal diagnosis. 5 Team of prof. Gérard Milhaud, member of the French National Academy of Medicine and of the Scientific Committee of the Foundation Jérôme Lejeune. 6 For example, the Report n. 3208 to the french National Assembly regarding the revision of the laws on bioethics, 2001. 7 ATLAN H., Le clonage humain en arguments, Genève: Georg Editeur, 2005. 8 VACQUIN M., Le magasin des enfants, Folio, 1994. 9 LEJEUNE J., L’enceinte concentrationnaire, Paris: Le Serment Fayard, 1990. 10 SAINTE ROSE J., La protection pénale de l’enfant à naître, 2004. 11 In a judgement of the administrative Court of Paris, on July 5, 2005, it has been answered to the Fondation Jérôme Lejeune that, because the ES cells cannot be considered as embryos, their importation for research do not damage the respect due to the embryoâ?¦(as if the importation of elephants’tusks did not damage the elephants!). 12 One another author, the biologist Jacques Testard, has qualified of praetorian this decision of the Constitutional Council. 13 VILLEY M., Philosophie du Droit, Dalloz, 1986. 14 JOHN PAUL II, Mémoire et Identité, Flammarion, 2005 (The Pope develops there, in particular, the counterexample of the Reichstag delegating full powers to Hitler and the example of the Parliaments that authorize abortion). 15 Bulletin de l’Odre des Médecins, January 2001. 16 La Revue du Praticien, Gynécologie et Obstétrique 2004, p. 87. 17 SUREAU C., Son nom est personne, Albin Michel, 2005. In this book, the author argue explicitly in favor of a law that would grant the embryo the status of animal. 18 For example, the private bill n. 309 presented to the french Senate by prof. Jérôme Lejeune in 1990: LEJEUNE, L’enceinte concentrationnaireâ?

218 P. WOJCIECH GIERTYCH

BEGOTTEN, NOT MADE

In no way does the traditional subordination of sciences suggest a lack of confidence in the specific competence of each field of enquiry. While respecting the achievements of every science and its methods, it perceives a different, higher angle from which the studied object may be viewed. The capacity to step back and raise a deeper question is a sign of wisdom, in particular if in searching for an answer the penetrating curiosity of the mind and its capacity to arrive at truth on various levels of enquiry is respected. Recent developments in the biological and medical sciences have brought to light a wealth of information about the earliest stages of human life. The human embryo from the moment of its coming into existence through fecundation, given the natural surroundings of the mother’s body, even in its pre-implantation stage, manifests an inherent capacity for development and growth, leading to the birth of a child. The biological sciences explain with great detail this natural process. The medical sciences have the capacity to diagnose potential illnesses and in some cases to undertake therapeutic action even at the earliest embryonic stage of human existence. This rapidly expanding knowledge of prenatal human life is the substratum for further enquiry on the level of higher sciences. Philosophy in its wonder on the human being, his nature, personhood and dignity cannot fail to take into account this newly available knowledge. The classical portrayal of the most perfect being in nature, that is the human person, which defines it as an individual substance of the rational nature, finds elements of confirmation in the uniqueness and otherness of the human embryo in respect to its parents, from the very moment of conception, as is known now through contemporary biological knowledge. Philosophy with its rational reflection, when it steps back outside the accumulated biological data and raises questions about human nature, begins with wonder and attempts then to clearly define the ontological status of the human person. While biology supplies information about human bodily processes, it cannot inform about the genesis of the spiritual soul, the source of the supreme dignity of the human person. Rational enquiry however, following the hylemorphic intuitions of Aristotle and Aquinas (even while rejecting their conclusions about retarded animation, based on their limited biological knowledge) can arrive at the conclusion about the necessity of the existence of a unifying principle, called the soul, that sparks off the first act of the living human organism, which has in itself the potentiality to grow from the stage of the zygote to embryo, foetus, child and finally adult and that holds together the biological material of the body in view of the whole and its finality. The non- attribution of any specific organ for the source of life, against the Cartesian approach, is again confirmed by biological knowledge, which observes the dynamism of life leading to human adulthood, right from the moment of fecundation, even before any specific organs develop. This anthropological perception becomes then the most serious grounds for an ethical support of the dignity and rights of the embryo and for the rejection of invasive procedures that encroach upon these rights and distort the biological and ethical environment in which human life develops. Philosophy however, is not the supreme source of cognition that is available to us. The revealed Word of God, transmitted in the Church, is the source of an even higher realm of knowledge, that of theology. In setting together the disclosed truths about the human being created by God so as to enter into a childlike relationship with Him with the truths about the supreme human being, Jesus Christ, the Word of God, made man, theological reasoning sheds further light on the mystery of man, his nature, supernatural finality and dignity. A theological regard at the humanity and extreme fragility of the minute embryo, aware of the magnitude of divine love, only increases the wonder that biology and philosophy can offer, and grants further insights that can direct in faith an approach, that will be truly respectful of the perceived mystery. The first temptation noted in Holy Scripture is one of spiritual pride: ‘You will be like gods ‘(Gn 3, 6). Scientific technologies, with their reductionist approach, treating the human being not as a gift of God 219 and as a person with its inherent dignity, but as an object that can be manipulated at will propose a eugenic approach, in which a future child can be ‘customized ‘at will. Instead of being begotten, not made, received from God, the future child is proposed to be constructed so that it will have a chosen sexual identity, immunity from sicknesses and programmed potential characteristics, with the assumption that those children that have been conceived and do not fit the expected model can be discarded or used as an available source of ‘biological material ‘, (and therefore logically also, those that at a later stage in life will turn out to be different from the expected model may be discarded also). The technical possibilities that biological sciences are expanding, while offering new and unimagined perspectives of healing the sick, easily also generate a proud, demonic derision of the mystery of human generation, with the expectation that scientific inventiveness will soon surpass anything that the Creator of human nature has designed. The aspiration for such intellectual and technical excellence that will be able to replace the logic of God-given natural processes with haphazard, rootless and directionless ingenuity, divorced from any respect for the personhood of the most fragile of human beings is frightening. Respect for the pain of parents, unable to conceive a child (either through no fault of their own, or as a result of earlier contraceptive poisoning) cannot justify the application of techniques, which infringe upon the rights of the future child. A child cannot be produced at any price, when that price means the destruction of sibling embryos or the deprivation of any truly natural parents. A child is not an object; it is a person that in its growth and development needs to be nurtured by parents with whom it has a natural, biological and psychological bond. Again, the purely biological nurturing of the child by the mother, even at the embryo stage is clearly described by science today. As the pregnancy proceeds the biological nurturing grows into a psychological and spiritual bond. The right to possess a child, in or outside marriage, which claims the use of techniques now made possible for this end, is an unjustified elevation of having a child above that of being a parent. The extent of this mental distortion and its future implications are hard to imagine. God always forgives the penitent and will forgive also those who have sinned with the pride of ‘child manufacture ‘. Children, brought into existence through such techniques, if spiritual graces will be given them, may also in the future forgive those who have profoundly wounded them. But nature will never forgive. The introduction of manipulative techniques that do not respect the inherent structure and finality of nature will in time bring about, unknown to us as yet, nefarious consequences. The Catholic faith teaches us to venerate Mary, the Virgin Mother of Jesus. In her maternal love she exercised, as all true mothers do, a certain power and responsibility over her infant Child. She decided for her Child, about his food, clothing, shelter and education. In her virginal love, that retained its primacy within her maternal heart, she persevered in the loving fascination and openness towards the mystery of God as it unfolded itself in her life. Both as Virgin and as Mother, she loved the God made man, that was conceived in her body and to whom she transmitted life. Even though, the vocation of Mary is unique, the quality of her love can serve as a model for the purification of the hearts of all mothers, who need to retain within their love for their child that initial fascination, joy and openness to the mystery of the new life that has a being, identity, personhood and future of its own. The love of parents should not and cannot be tainted by temptations to customize a child at whim. It should primarily be an acceptance of the new individual that has been conceived in its own personal dignity before parental hope can try to direct the child to some distant future, accepting always that parental dreams will not have the last word. The temptation to manipulate the child even in its earliest stage of development is primarily a male temptation that does not take into account the mystery of conception as it takes place within the body, but also within the psyche and the spirit of the mother. Just as male sexual organs are outside the body and the conception of a child takes place outside the father’s body, so also the transmission of life takes place outside the male psyche and spirit. In some sense, for the male psyche it is not so essential whether the fecundation takes place within the woman’s body or in a ‘test tube ‘. It happens outside his body and mind. For the mother, the conception of a child that takes place within her, initiates not only a physical and biological process, but also a psychological and 220 spiritual encounter with the hidden child, in some sense akin to the spiritual encounter with the Blessed Trinity that indwells the soul of all baptized Christians. The mentality of contraception, abortion and of encroaching upon prenatal privacy not only wounds the woman’s body; it distorts her capacity to receive the child as a free gift of God (singing a joyful Magnificat) and her eucharistic capacity to give generously (whispering to her child: ‘Take this and eat of it. This is my body. This is my blood that I give to you! ‘). The study of the biological, medical, philosophical and juridical issues that arise with newly available scientific knowledge about the human embryo in the phase of its implantation, allows for a clearer perception not only of the physical, but also of the moral object of any diagnostic or therapeutic (or even worse: experimental, commercial or eugenic) intervention upon the embryo. As such it enables an unambiguous moral evaluation of the new ethical challenges that humanity is facing with the advance of biological technology. It may be useful however, to remind in this context that ‘activity is morally good when it attests to and expresses: the voluntary ordering of the person to his ultimate end and the conformity of a concrete action with the human good as it is acknowledged in its truth by reason ‘(John Paul II, Veritatis Splendor, n. 72). A precise grasping of the dignity and personhood of the human embryo grounded in biological knowledge, philosophical reasoning and theological insight can allow for the acknowledgement of the truth about the human embryo and the truth about any envisaged action of which the fragile embryo would be the object. A full understanding however, of the moral challenges that potential parents, pregnant women and their husbands and medical staff involved in prenatal science face, needs to focus not only on the external act itself, which is the moral object of any envisaged action, but also on the interior act of the agent, which can be open to the spiritual transformation of grace, allowing the infused virtues of faith, hope and divine charity to intervene, thereby excluding any proud replacement of God, any despair in the face of unwanted foreknowledge or any egoistic possessiveness of the projected ‘ideal ‘child. Pastoral ministry among all those, who in any way are drawn into the realm of prenatal life, requires a deeper initiation into the life of God, the Source of all life.

221 P. SERGEJ FILIMONOV

SI PUÒ CONSIDERARE L'EMBRIONE COME PERSONA?

In ambito scientifico dobbiamo sempre essere pronti ad accettare che le nostre conoscenze siano imperfette e a volte erronee. Secondo il noto fisico Albert Einstein dobbiamo essere in grado di rinunciare a stereotipi e nozioni usuali. Ciò corrisponde all'essenza del metodo teologico apofatico che spesso viene applicato nella tradizione della Chiesa orientale. Esistono "due metodi principali in teologia: il metodo positivo catafatico (teologia affermativa) che per via di affermazione porta a una certa conoscenza di Dio rivelato nella creazione, e quello negativo (teologia negativa) che per via di negazione porta alla cognizione perfetta relativamente a ciò che è al di là dell'esistente.1 Prendendo in esame la complessa questione della personalità dell'embrione solo con il metodo catafatico, si potrebbe ottenere un risultato erroneo dovuto alla nostra ignoranza sulle questioni riguardanti l'anima dell'embrione. Quando gli scienziati e i filosofi moderni cercano di affermare che l'essere umano nella fase preembrionale non è persona, dobbiamo ricorrere attivamente all'argomento apofatico: nessuno scienziato potrebbe provare che l'embrione non è persona dal momento della fecondazione. Secondo il parere della maggioranza dei Santi Padri la persona è composta da anima e corpo. Essi esistono nella persona non uniti e non separati. S. Gregorio di Nissa dice che l'anima non sta nel corpo come in un recipiente o un sacco, ma piuttosto il corpo sta nell'anima, non è il corpo che possiede l'anima ma è l'anima che possiede il corpo e non esiste neanche una parte consacrata della stessa in cui non esista interamente".2 Quando parliamo del futuro di una persona ne parliamo dal punto di vista della Provvidenza divina. Perché da centinaia e migliaia di spermatozoi e ovocellule è avvenuta la fusione proprio di questi? Non lo sappiamo. Non possiamo negare che il Signore Onnisciente abbia previsto anche queste migliaia di eventuali vite non realizzate che sarebbero state persone diverse. Ai Suoi occhi sono già state persone anche se gli zigoti non si sono formati ed i bambini non sono nati. Il motivo per cui il Signore dal non essere ha chiamato alla vita proprio questa persona, è un mistero Divino. Si può pensare che il Signore non sapesse della futura nascita di Isacco da Abramo e Sara quando erano giovani e appena sposati? Ma il Signore ha scelto Isacco invece di altri che non sono nati. Potremmo dire: la formazione dello zigote è il risultato della reciproca fusione di spermatozoo ed ovocellula secondo le leggi biologiche conosciute da noi o è il risultato del funzionamento delle energie divine inconcepibili per la nostra percezione? I Santi Padri sono del parere che il corpo e l'anima vengano creati contemporaneamente. Il Reverendo Padre Anastasio il Sinaita scrive: "Come non c'era corpo prima dell'anima, così non c'era neanche l'anima prima del corpo".3 E Giovanni Damasceno sottolinea: "Il corpo e l'anima sono stati creati contemporaneamente e non prima l'uno e poi l'altra".4 Il tentativo di definire con metodi scientifici il momento a partire dal quale l'embrione diventa persona mi sembra erroneo, irrealizzabile e sacrilego. Nei programmi divini è già una persona. "L'embrione acquisisce l'anima durante il concepimento". L'anima viene creata al momento del concepimento e "in seguito l'anima continua ad agire durante tutto il tempo in cui è viva la carne; perché con la crescita del corpo anche l'anima dimostra la sua attività".5 In questa prospettiva possiamo dire che la persona diventa personalità nel tempo, man mano che l'anima agisce. Sarebbe più giusto trattare il non nato come il nato, senza cercare di definire il momento della personificazione. Quando il Signore dice che ha tessuto la persona nel seno (Sal 138, 13-16), chi potrebbe spiegare che cosa si intenda col concetto del tessere e in quale momento della tessitura l'embrione diventi persona? Se il Signore avesse voluto, ce lo avrebbe rivelato. E se non lo ha fatto, vuol dire che dobbiamo trattare il non nato come nato, indipendentemente dalla fase di sviluppo dell'embrione. L'embrione è un essere umano? Senza dubbio. Però, l'embrione è persona? Nelle opere dei Santi Padri non troviamo l'affermazione diretta che l'embrione sia persona. Non possiamo negare che l'embrione sia un essere umano, ma non possiamo affermare che sia una persona in tutta la sua pienezza così come ce l'immaginiamo. All'embrione è 222 piuttosto applicabile il concetto di persona non realizzata o potenziale. Quando un giardiniere vuol piantare fiori, prende i semi di quei fiori di cui ha bisogno. Non prende un seme di papavero se vuol far crescere un garofano e non prende un seme di garofano se vuol piantare una rosa. Il seme di un fiore, pur non essendo piantato nel terreno, ci fa venire in mente il fiore stesso. Il seme di un fiore ha delle qualità, la potenzialità di un fiore non realizzato. Anche l'embrione, allo stesso modo, ha tutte le qualità e potenzialità della persona non ancora nata. L'immagine divina nella persona è l'immagine perfetta: secondo S. Gregorio di Nissa è anche un'immagine inconoscibile visto che, pur riflettendo la pienezza del suo Archetipo, deve anche possedere la Sua inconoscibilità. Perciò non possiamo definire in che consista l'immagine divina nella persona umana anche se molti Santi Padri hanno espresso opinioni a riguardo. "S. Gregorio di Nissa vede la caratteristica di una persona creata secondo l'immagine del Signore prima di tutto nel fatto che "la persona è libera dalla necessità e non è subbordinata al dominio della natura, ma può autodeterminarsi liberamente". Essendo creata secondo l'immagine del Signore la persona è una creatura personificata. È una personalità che non deve essere definita dalla sua natura, ma può definire la natura cercando di diventare simile al suo divino Archetipo".6 In modo apofatico non possiamo negare che l'embrione avente la natura umana non sia persona. Ma allo stesso tempo non possiede ancora appieno la libertà di autodeterminarsi. "Le persone umane hanno in comune la stessa natura. Nella persona la differenza della natura e della personalità non è meno percepibile che la differenza della natura nelle tre persone divine. Prima di tutto dobbiamo renderci conto che non conosciamo l'essere persona, l'ipostasi umana nella sua vera manifestazione libera da tutte le sostanze estranee. Quello che di solito chiamiamo personalità indica piuttosto gli individui...Però in un certo senso l'individuo e la personalità hanno significati opposti; l'individuo vuol dire un certo insieme di personalità ed elementi che fanno parte della natura comune, mentre la personalità è ciò che distingue dalla natura. Quando vogliamo definire, caratterizzare qualche personalità, cerchiamo di trovare le caratteristiche individuali, le particolarità del carattere che s'incontrano anche in altri individui e non possono essere del tutto personali in quanto fanno parte della natura comune. E in fin dei conti comprendiamo che quello che per noi è più tipico della persona, quello che la rende se stessa è indefinibile perché nella sua natura non c'è niente di ciò che si riferisce alla personalità sempre unica, incomparabile ed eccellente.7 "S. Gregorio di Nissa insegna che l'essere persona è la liberazione dalle leggi di necessità, libertà dalla subordinazione al dominio della natura, possibilità di autodefinirsi liberamente. La maggior parte delle volte la persona agisce secondo i propri impulsi naturali; è determinata dal suo temperamento, dal suo carattere, dalla sua ereditarietà, dall'ambiente cosmico o socio-psicologico ed anche dalla propria storicità".8 "L'essere personale è l'apice della creazione perché attraverso la volontà e la carità può assimilarsi a Dio. Creando la personalità, l'onnipotenza divina realizza un certo intervento radicale, qualcosa di assolutamente nuovo: Dio crea esseri che come Lui possono decidere e scegliere".9 Per quanto riguarda l'embrione o il preembrione (accettando che il nuovo essere umano dallo stadio di zigote è già persona) è difficile parlare di un essere già in grado di decidere e scegliere o dimostrare carattere. Nell'interpretazione teologica un nuovo essere umano dai primi momenti della sua esistenza è già persona; nel senso culturale, sociale, psicologico e storico tale essere non si è ancora manifestato come persona. Ciò significa, accettando che l'embrione sia una persona e non possa non esserlo e tenendo in considerazione tutti gli aspetti di cui sopra, che bisogna intendere l'embrione come personalità dinamicamente e autonomamente sviluppantesi, persona potenziale, non realizzata ma che sta realizzandosi, non definita ma che sta definendosi. Il criterio di realizzazione e definizione si identificherà con la sua capacità di realizzare la scelta cosciente e libera: essere con il Signore o respingerlo. Il noto bioeticista e teologo ortodosso protopresbiterio dr. John Breck a tal proposito afferma: "La questione principale è legata all'individualizzazione - il processo che permette di parlare dell'esistenza dell'individualità sviluppante o ontologica cioè dell'individuo umano diverso da altre 223 persone e di conseguenza di personalità come tale. Attualmente embriologi ed eticisti, in base alle loro conclusioni, si sono divisi in due gruppi. Secondo alcuni, la totipotenza e la bassa differenzialità di blastomeri, ossia di cellule embrionali iniziali, con l'alta percentuale di rigetto naturale di ovociti fecondati testimoniano chiaramentel'animazione ritardata e la caratterizzazione dell'embrione nella fase del preimpianto come preembrione. Per altri, invece, l'individualità genetica (esistente dal momento della singamia) e la continuità del processo di sviluppo testimoniano in modo convincente l'animazione immediata. Vuol dire che già dal momento della fecondazione siamo in presenza dell'individuo umano qualificato come persona e i cui diritti vanno difesi per legge. Questi argomenti sollevano problemi morali molto seri. Se l'animazione, ossia l'individualizzazione, ha luogo solo dal momento in cui appare la stria primitiva durante l'impianto, i diritti umani dell'embrione non sembrano essere indiscutibili. I sostenitori di tale opinione in maggioranza sono d'accordo sulla necessità di tutelare il preembrione, come individuo umano potenziale, contro manipolazioni arbitrarie. D'altra parte, pratiche come l'utilizzo del preembrione a fini di ricerca, la fecondazione artificiale e la contraccezione vengono ritenute completamente ammissibili. Allo stesso modo si ammette anche l'interruzione della gravidanza che risulti da violenza o incesto e difendono l'aborto "secondo la volontà della donna". Ma se si accetta come assolutamente certo il fatto che la differenziazione cominci dal momento della fecondazione e l'animazione avviene simultaneamente alla singamia, tutte le conclusioni relative alla finestra di due settimane vanno riviste. In tal caso la totipotenza risulta essere una chimera, cioè di un sogno ibrido che prende per vero ciò che si desidera, la scienza diviene riduttiva e la fecondazione in vitro e tutti gli altri esperimenti con materiale embrionale rappresentano azioni che calpestano i diritti della persona. Indipendentemente dal modo in cui è stato concepito l'embrione, l'interruzione volontaria della gravidanza in qualsiasi fase, diventerà un atto omicida moralmente inaccettabile poiché annienta la persona che si sta sviluppando. Quale è la posizione della Chiesa ortodossa? L'antropologia ortodossa con la sua tesi più importante sulla sacralità e santità della vita umana richiede che la vita umana sia riconosciuta come tale dal momento del concepimento. Ma mentre l'embriologia moderna rivela un'incertezza su tale affermazione, il rifiuto dell'aborto in qualsiasi fase della gravidanza (in base alla seconda regola di Basilio il Grande, non c'è differenza tra il feto formato e non ancora formato) avvicina la posizione della nostra Chiesa al giudizio cattolico sull'animazione immediata e non ritardata. Allo stesso tempo la Chiesa ortodossa polemizza con la dottrina cattolica dell'animazione nella misura in cui quest'ultima si basa sulla tradizione aristotelica e tomista (Libro delle Regole, L. 13). La stessa terminologia se prendiamo ad esempio le espressioni animazione o infusione nel corpo dell'anima immateriale dotata di ragione, principio dell'individualità immateriale o originalità viene percepita nella mentalità ortodossa come dualistica, ereditata dall'origenismo. Per i Padri orientali (come pure per la tradizione biblica) l'anima è la sede della personalità (Gn 1, 26-27). Nel senso stretto della parola si dovrebbe dire io sono anima e non io ho l'anima. Quindi sembra che la radice delle antiche discussioni sull'animazione ritardata o immediata stia in quella antropologia che anima il corpo materiale con l'anima razionale, creata separatamente e infusa nel corpo al momento della fecondazione, dell'impianto o in un'altra fase dello sviluppo. Gli adepti della teoria dell'animazione ritardata si basano, in genere, su due fatti biologici: la scissione simultanea dei gemelli monozigoti e le "perdite", cioè il rigetto spontaneo di ovociti fecondati prima dell'impianto: "La morte prematura di embrioni causata dalle perdite naturali ci presenta una difficoltà logica simile alla morte prematura di bambini in seguito a malattie o incidenti. Quest'ultimo caso, che ci colpisce per la sua tragicità, si spiega con la perdita del legame affettivo con il bambino che ha lasciato questo mondo e che viene vissuto con molta sofferenza dai genitori e da tutti quelli affettivamente coinvolti. È ovvio che tra la madre e l'embrione tale legame non esiste. Ma ciò non cancella il valorepersonale dell'embrione poiché dal punto di vista ortodosso tale valore sorge non dall'infusione dell'anima, ma dal rapporto del Signore con la Sua creatura. Quindi il valore personale dipende non dall'anima infusa e neppure dai rapporti coscienti dell'individuo con altri 224 individui, ma dall'amore divino che abbraccia le creature fatte a Sua immagine dal momento del concepimento fino al passaggio alla vita nuova. Ecco perché i malati in coma o nello stato vegetativo persistente rimangono persone nel senso pieno della parola, concepite e confermate come tali dal Signore stesso".10Relativamente alla divisione gemellare si possono proporre le considerazioni seguenti. È noto che i blastomeri individuali possiedono una certa totipotenza anche prima della gastrulazione e dell'isolamento. Una o più cellule della morula possono essere separate dalla massa principale e, visto che le cellule separate hanno tutta l'informazione genetica dello zigote iniziale, è possibile il loro sviluppo in un gemello monocoriale. Tuttavia bisogna utilizare il termine totipotenza con molta prudenza. In realtà i gemelli con lo stesso materiale genetico sono identici non nel senso letterale della parola. Anche se ambedue si sviluppono dallo stesso genoma, in virtù della metilazione e dell'azione del citoplasma dell'ovocita (acido ribonucleico materno) sono differenziati geneticamente, poiché (qui seguiamo Geffreys e Suraney) i blastomeri stessi si differenziano a cominciare dal livello di due cellule con le prime manifestazioni del genoma. Così, nel periodo del preimpianto, le cellule possono sdoppiarsi e in casi rari possono riunirsi di nuovo. La domanda "in questa fase c'è un'anima o ce ne sono due?" non ha più ragion d'essere se si considera ogni unità come anima esistente e non come avente un'anima, perché intendiamo per anima la capicità, data dal Signore, che attualizza l'esistenza individuale personale. Nel caso della riunificazione l'esistenza personale è espressa non da due, ma da una esistenza individuale. Quindi, gemelli monocoriali non possono essere simili, la loro identità è di carattere genetico, ma è limitata dalla metilazione in modo che ogni unità si sviluppi in una personalità diversa e irripetibile. Così, il fenomeno della totipotenza, ammissibile con certe riserve, non ci impedisce di vedere nell'embrione l'esistenza umana personalizzata e individualizzata. E nonostante il fatto che la formazione della stria primitiva indichi la fine della totipotenza e della possibilità di divisione gemellare, la differenziazione cellulare inizia non in questo momento, ma quasi subito dopo la fecondazione, cioè alla fase di due cellule. Sebbene il trofoblasto sia un segno visibile del sorgere della vita, il programma iniziale che determina lo sviluppo successivo degli organi vitali, funziona già, in attesa di manifestarsi in un dato momento del ciclo vitale (a tal proposito il professor Germin Grisset nota: "Il fatto che la maggior parte dell'energia dello sviluppo fin dall'inizio venga utilizzata per la formazione del trofoblasto, non vuol dire che non ci sia lo sviluppo dell'embrione. Poiché i materiali ausiliari scartati durante la nascita precedentemente erano organi vitali dell'individuo non nato e parte integrante della sua personalità non meno che il nostro cuore, polmoni, reni e stomaco parte integrante di noi stessi. È naturale che, durante lo sviluppo, appaiano prima gli organi che sono più importanti per le prime fasi della vita"). In condizioni normali, lo sviluppo della persona è una catena ininterrotta di cambiamenti: formazione dello zigote, impianto, formazione del sistema nervoso, nascita. Ad eccezione del primissimo momento inziale, in questo processo non esiste altro momento in cui si possa dire che "la vita umana comincia qui e adesso". Ragionando in questo modo, inevitabilmente ci troviamo di fronte al problema della valutazione morale di procedure come la fecondazione in vitro e l'aborto (su indicazioni mediche o desiderio della donna). Relativamente all'idea dellafinestra di due settimane per giustificare la fecondazione extracorporea, l'interruzione della gravidanza indesiderata o per ottenere il permesso alla sperimentazione con materiale embrionale, le recenti scoperte embriologiche dimostrano che talefinestra non esiste. Se è vero che la differenziazione cellulare avviene già a livello di due cellule, allora la definizione della forma iniziale dell'esistenza come preembrionale, nel migliore dei casi è un errore, nei peggiori casi è una bugia cosciente. Tale differenziazione - indicata chiaramente dall'impossibilità di formazione di chimere dopo la divisione cellulare di quarto livello, condizionata dal fenomeno di metilazione e anche dalla natura della divisione (a condizione di una corretta comprensione) - conferma la convinzione della Chiesa che la vita umana inizia dalla fecondazione, dalla singamia. 225 Ma anche se gli embriologi riuscissero a provare indiscutibilmente che tale differenziazione non esiste (in altre parole, che la metilazione non ha niente a che fare con la persona e il preembrione non è nient'altro che una massa di cellule non differenziate), "ciò non influenzerebbe la convinzione ortodossa che la vita umana inizi dal concepimento, ossia dalla fecondazione".11 Nel prossimo futuro le cosidette forme preembrionali potranno essere chiamate in un altro modo o in maniera più precisa, potranno apparire preembrioni ed analoghi, ma questo fatto non cambia la sostanza, al contrario dimostra imperfezione, mutabilità e relativismo delle nozioni scientifiche. "E la cosa sta non nell'inerzia del conservatorismo, ma in quel fatto incontestabile che l'anima umana, la forza di animazione donata dal Signore, è presente sin dall'inizio, nel momento in cui i pronuclei della sperma e dell'ovulo si fondono formando lo zigote, base della persona nuova e irripetibile. L'inizio della vita stabilisce tutto lo sviluppo successivo e in condizioni normali porta alla nascita del bambino. Dunque, non è moralmente ammissibile nessun argomento a favore dell'aborto, della fecondazione artificiale o della sperimentazione su materiale embrionale. Queste possibilità non possono fare a meno del confronto con questà verità assoluta".12 Perciò, in conclusione, rispondendo alla domanda se l'embrione sia persona dal momento del concepimento, dal punto di vista della teologia ortodossa si può rispondere positivamente, ma con la riserva che il nuovo essere umano è una persona potenziale che si rivela, si realizza e si sviluppa dinamicamente.

1Rasskasovsky S., Il prete. Le fondamenta della dottrina religiosa ortodossa, San Pietroburgo: RHGI, 1993: 53-56. 2PG. T. 45. Col. 217; Migne J.-P., Patrologiae cursus completus, series Graeca, Paris. 3PG. T. 89. Col. 724; Migne, Patrologiae cursus... 4Giovanni Damasceno, Il santo. Esposizione esatta della fede ortodossa, Mosca, 1992: 79-80. 5Vlakhos I., Il Metropolita. Psicoterapia ortodossa. Laura di Santa Trinità e di S. Sergio, 2004: 107. 6Lossky V., Saggio sulla teologia mistica della Chiesa orientale, Mosca, 1991: 91 7Ibid. 8Ibid., pp. 92-93. 9Ibid., pp. 93-98. 10Lossky, Saggio della teologia mistica della Chiesa..., pp. 93-98. 11Breck J., The Protopresbyter. The Sacred Gift of Life, Moscow: Palomnik, 2004: 194-200. 12Ibid.

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