L'infertilità Maschile Oggi Male Infertility Today

p( ) l’infertilità maschile oggi male gg y y infertility today

editor: Giovanni M. Colpi 4 / 2004 4 l’infertilità maschile oggi male infertility today

editor: Giovanni M. Colpi 4 / 2004 RINGRAZIAMENTI Ringrazio i miei collaboratori Dr. Igor Piacentini e Dr. Giuseppe Piticchio per la revisione dei testi. Giovanni M. Colpi INDICE

HOW FAR ARE ICSI PROCEDURES USING HUMAN SPERMATOZOA GENERATED INTO ANIMAL TESTICLES? MICRO- AND MACRO-CONSEQUENCES OF GERM CELL TRANSPLANTATION TECHNIQUES Sofikitis Nikolaos, Kaponis Apostolos, Giannakopoulos Xenophon, Miyagawa Ikuo 5

AROMATASE INHIBITORS IN MALE INFERTILITY Kaan Aydos, Önder Yaman 41

MAMMALIAN TESTICULAR DESCENT AND MALDESCENT; IMPLICATIONS IN FERTILITY POTENTIAL Charalampos Mamoulakis, Apostolos Kaponis, John Georgiou, Dimitrios Giannakis, Spyros Antypas, Stavros Tsambalas, Xenophon Giannakopoulos, Ikuo Miyagawa and Nikolaos Sofikitis 63

MALE ACCESSORY GLAND INFECTIONS AND INFERTILITY Enzo Vicari, Sandro La Vignera, Alessandro Arancio, Aldo Eugenio Calogero 139

Male infertility today #4 3

HOW FAR ARE ICSI PROCEDURES USING HUMAN SPERMATOZOA GENERATED INTO ANIMAL TESTICLES? MICRO- AND MACRO-CONSEQUENCES OF GERM CELL TRANSPLANTATION TECHNIQUES

Sofikitis Nikolaos1,2, Kaponis Apostolos1, Giannakopoulos Xenophon2, Miyagawa Ikuo1

1Dept. of Urology, Tottori University School of Medicine, Yonago, Japan 2Laboratory for Molecular Urology and Genetics of Human Reproduction, Dept. of Urology, Ioannina University School of Medicine, Greece

Running title: Germ cell transplantation Key words: Spermatogonia/ syngeneic/ testicle/ transplantation/ xenogeneic.

Abstract Contents Results from the transplantation of donor Mechanisms regulating male germ male germ cells into xenogeneic recipient cell meiosis and spermiogenesis. seminiferous tubules indicate that donor The immunological tolerance of the spermatogonia are capable of differentiating testicle. to form spermatozoa morphologically characteristic of the donor species. Germ cell Theses and controversies on syn- transplantation procedures combined with geneic male germ cell transplantation. developments in freezing, culturing, or A. Development and evolution of syngeneic enriching germ cell populations have applica- germ cell transplantation procedures. tions of paramount importance in medicine, basic sciences, and animal reproduction. B. Cryopreservation of male germ cells. Additionally these techniques can serve as C. Germ cell transplantation techniques as an alternative approach for gonadal protec- a powerful tool to reconstitute spermatoge- tion and fertility preservation in patients with nesis in animals with testicular damage and oncological disease. study the mechanisms of genetically This article is a chronological critical caused male infertility. review of the technological advances that fol- D. Culture of spermatogonia cells prior to lowed the initial successful transplantation of transplantation. mouse germ cells into recipient mice. Fur- thermore, the factors responsible for the Xenogeneic transplantation of mam- immunological privilege properties of the tes- malian male germ cells. ticle and the parameters influencing the Transplantation of human male germ potential of mammalian germ cells to under- cells into xenogeneic testicles. go mitosis and meiosis within a xenogeneic testicle are described. Finally the role of Techniques to increase recipient tes- human germ cell transplantation procedures ticular colonization by donor germ cells. in the therapeutic management of non- A. Selection/preparation of donor germ cells. obstructive azoospermia is discussed.

Male infertility today #4 5 B. Preparation of recipient animals. involves mitotic and meiotic division of spermatogonia to produce mature spermatids C. Methods for germ cell transplantation. and spermatozoa. Spermatogenesis occurs Patterns, kinetics, and differentiation within the seminiferous tubules of the testi- of donor germ cells within the recipient cle (Dym, 1983). The spermatogonial type seminiferous tubuli. A stem cells in the testicle can be considered as eternal germ cells because they Potential clinical applications of male are present from the birth to the death and germ cell transplantation techniques. have the capacity to give rise to new sub- A. Preservation of the whole genome of types of spermatogonia and form sperma- patients with oncological disease in recipi- tozoa, as well. The latter cells represent ent animals (using an animal testicle as a highly motile structures that can pass surrogate organ). genetic material from generation to genera- B. Autotransplantation of frozen/thawed tes- tion (McLaren, 1992). Among the type A ticular germ cells in men with oncological spermatogonia there are distinct subpopu- testicular disease (without using a recipient lations, types A1-A4 that have been consid- animal testicle as a surrogate organ). ered the renewing stem cell spermatogonia, and type A0 that have been considered C. Germ cell transplantation techniques for a reserve type of stem cells. The type A4 preservation of endangered species. cells divide to give rise to differentiating D. Survival and differentiation of germ cells spermatogonia as well as to form new type from non-obstructed azoospermic men into cells, the A1 cells to maintain the stem cell recipient human individuals (human to population (Dym & Clermont, 1970). The human transplantation techniques). type A0 cells divide slowly and represent a reservoir to repopulate the testis after an E. The role of human germ cell transplanta- injury as a fine mechanism to maintain nor- tion procedures in the therapeutic manage- mal numbers of types A1-A4 (Clermont, ment of non-obstructive azoospermia 1969). (human to animal transplantation techniques). Numerous factors have been implicated in spermatogonial development. Several F. Assisted reproduction techniques using alternative hormonal mechanisms regulate human haploid cells generated into animal the mitotic and meiotic dynamics of sper- testicles; genetic and immunological risks. matogonia (Meachem et al., 2001). Sertoli cells limit the expansion of the spermatogonial population. Each Sertoli cell supports a MECHANISMS REGULATING MALE defined number of germ cells. Sertoli cells GERM CELL MEIOSIS AND form niches for stem cells. These niches SPERMIOGENESIS allow a certain number of stem cells to Approximately 50% of human infertility reside in or repopulate the seminiferous is attributable to male defects with the clini- tubules. The repopulation pattern of donor cal presentation of abnormal sperm pro- spermatogonia post-transplantation sup- duction, such as oligo-, astheno-, ter- ports the presence of niches of donor germ atospermia, or azoospermia (Sigman et al., cells (Ohta et al., 2000). FSH has an impor- 1997). Spermatogenesis is a process that tant role in the regulation of the kinetics of

6 Male infertility today #4 adult spermatogonia. The mechanism via A1/Bfl-1) and others promoting cell death which FSH mediates its effect is probably (Bax, Bak, Bad, Bim) (Adams & Cory, by acting both as a survival factor and as a 1998). Transgenic loss of the Sertoli cell- mitogen (Meachem et al., 1997). produced glial cell derived neurotrophic fac- Withdrawal of FSH in normal adult rats tor (GDNF) function results in depletion of showed a time-dependent decline in the spermatogonia stem cells reserves, where- number of early germ cell types (Meachem as mice overexpressing GDNF accumulate et al., 1997; Meachem et al., 2001; undifferentiated spermatogonia within the Kangasniemi et al., 1990). Great reduc- seminiferous tubuli (Meng et al., 2000). tions/depletions of gonadotrophins in pri- Recolonization of a recipient testicle by mates (treatment with GnRH-antagonists) transplanted donor germ cells offers an reduced the number of type B spermatogo- excellent approach to understand the nia to less than 10% of controls indicating mechanisms regulating the influence of the that the spermatogonium is a major target testicular microenvironment on spermato- for gonadotrophin stimulation (Zhengwei et gonial development. al., 1998). Treatment with GnRH-antago- Spermiogenesis is a metamorphosis nists provokes a pronounced inhibition of process where no cell division is involved. spermatogonial proliferation indicating that During spermiogenesis alterations can be the first step of mitosis (at the level of A seen in the male gamete nuclear proteins, spermatogonia) is under hormonal influ- the cellular size, the cellular shape, and ence (Schlatt & Weinbauer, 1994). position of pro-acrosomal granules and the Meistrich et al. (1994) provided compelling localization of the centrioles, as well. A fas- evidence that high testicular testosterone cinating cascade of events results in the concentrations have a detrimental effect on formation of sperm tail. Elongation of the spermatogonial development. round spermatid is observed in the steps 7- Stem cell factor (SCF) and its receptor 8 of spermiogenesis in the rat (Sofikitis et c-kit play an important role in spermatogo- al., 1999c). Sofikitis and co-workers (1999c) nial development. Mutation in the gene have shown that a decrease in the intrates- encoding the SCF or the c-kit results in ticular testosterone results in a premature infertility owing to defective migration, prolif- detachment of round spermatids from the eration, and survival of primordial sper- seminiferous tubuli and passage of the matogonia. SCF has been found to act as a round spermatids through the epididymal mitogen (Allard et al., 1996) and survival lumen. The latter spermatids have an factor (Dirami et al., 1999). impaired fertilizing capacity.

Inhibin inhibits the incorporation of triti- ated thymidine into differentiated mouse THE IMMUNOLOGICAL and hamster spermatogonia (van Furth & TOLERANCE OF THE TESTICLE Van Dissel, 1989), whereas, activine stimulates spermatogonial mitosis in vitro The testicle is an immuno-privileged (Mather et al., 1990). Bcl-2 family plays a organ (Selawry and Whittington, 1984; key role in integrating the positive and neg- Selawry et al., 1989; Clouthier et al., 1990; ative signals on spermatogonial survival. Belgrau et al., 1995; Russell and Brinster, There are members of this family that pro- 1996; Clouthier et al., 1996). Selawry et al. mote cell survival (Bcl-2, BcL-xl, Bcl-w, (1985) showed that pancreatic islets of MHC-

Male infertility today #4 7 incompatible donors survived indefinitely in the regulation of immune responses. FasL the abdominal testicles of diabetic BB/W rat protein is expressed by CD4+ T helper cells recipients. Furthermore, it has been shown and by CD8+ cytotoxic T lymphocytes. that the abdominal testicle rather than the tes- Resting T cells normally express low levels of ticle in its original scrotal position is the most Fas. In contrast, during the first hours post- suitable site for extended functional survival of engagement of their antigen receptors, Fas pancreatic islet allo-and xenofrafts (Selawry et expression increases significantly. Post-activa- al., 1989). The colonization of the mouse tes- tion, Fas in T cells becomes functional and ticle by rat germ cells (Clouthier et al., 1996; the T cells undergo apoptosis in response to Russell and Brinster, 1996; among others) crosslinking with FasL (O`Connell et al., and the rat testicle by hamster germ cells 1996). Thus, FasL ligation onto its receptor (Tanaka et al., 1997; Shimamoto et al., 1999; Fas, on sensitized cells, induces programized among others) further confirm the testicular cell death. Griffith et al. (1995) suggested that capacity to inhibit local immune responses. through the expression of FasL, the eye However, the mechanisms responsible for the directly kills activated T cells that might invade unique immunologically privileged status of the globe and destroy the vision. Similarly, the testicle are, as yet, undefined. It has been considering that Sertoli cells constitutively suggested that elevated levels of intratesticu- express FasL transcripts (Belgrau et al., lar testosterone and /or progesterone may 1995), it may be suggested that FasL interact- cause an inhibition of the local immune ing with Fas of activated T cells induces apop- responses (Grossman et al., 1985). However, tosis in the infiltrating T cells and subsequent- Cameron et al. (1990) provided strong evi- ly contributes to the capacity of the testicular dence that Leydig cells and Leydig cell secre- tissue to maintain an immune privilege envi- tory products do not represent a prerequisite ronment. The latter thesis is additionally sup- for the success of transplantation of pancreat- ported by findings in our laboratory showing ic cells into testicles. On the other hand the enhancement of the immune privilege status testicle contains high levels of steroids, mole- of the rat seminiferous tubules during a) intra- cules that are immunosuppressive by nature tubular administration of interferone-γ (known (Russell & Brinster, 1996). The most com- to up-regulate FasL expression) (Shimamoto monly recognized mechanism for the et al., 1999), or b) seminiferous intratubular immunological privilege properties of the testi- transfer of anterior chamber eye cells (known cle is the physical isolation of cell surface anti- to express FasL) from the same recipient ani- gens by the Sertoli cell-barrier. However, mal (Shimamoto et al., 1999). immune privilege in the testicle may not be simply a passive process involving physical barriers (Sertoli cell-barrier) but it may also be THESES AND CONTROVERSIES ON regarded as an active mechanism having the SYNGENEIC MALE GERM CELL capacity to induce cell death (via apoptosis) TRANSPLANTATION TECHNIQUES on potentially dangerous lymphoid and myeloid cells (Belgrau et al., 1995). In fact A. Development and evolution of Belgrau et al. (1995) have shown that expres- syngeneic germ cell transplantation sion of functional FasL by Sertoli cells has a procedures major contribution to the immune privileged nature of the testicle. Fas and its ligand FasL are cell surface molecules known to interact in Brinster & Zimmermann (1994) showed

8 Male infertility today #4 that germ cells isolated from testicles of In another study Brinster & Avarbock donor male mice would repopulate (1994) demonstrated that mouse germ cells immunologically compatible sterile testicles transplanted into the testicles of infertile when injected into the seminiferous tubules mice colonized the recipient seminiferous of these recipient animals. Donor spermato- tubules and initiated donor spermatogene- genesis in recipient testicles showed nor- sis in more than 70% of recipients. The mal morphological features characteristic of most striking result of these experiments the donor species. In that study, germ cells was the production of healthy offspring (by were transplanted into mice carrying the W, mating) from spermatozoa generated within Wv and W44 mutant alleles in the homozy- the recipient testicles by donor germ cells. gous or compound heterozygous conditions Increasing the number of Sertoli cells in the (spermatogenesis does not occur in the donor cell population did not increase the testicles of these mice; very few germ cells efficiency of colonization of the recipient of the most primitive stages can be found in testicles by donor germ cells. Some of the recipients that underwent transplantation the testicles of W/Wv mice). At the end of the experimental period, donor germ cells passed on the LacZ gene to 80% of their were found in 18% of the recipients. In progeny. The initiation and maintenance of additional experiments immunodeficient donor spermatogenesis within the recipient male mice had been treated with 40 mg/kg testicles post-donor germ cell transplanta- of the chemotherapeutic agent busulfan tion seems to be inconsistent with the fact approximately 4 weeks prior to the trans- that the donor spermatogonia have been plantation of donor germ cells. At this dose, injected into the lumen of the seminiferous busulfan destroys almost all endogenous tubules. It appears that donor spermatogo- spermatogonial stem cells, thereby space nia have moved from the recipient adlumi- is created on the basal surface of the nal to the basal compartment of the semi- tubules for the transplanted spermatogonia niferous tubulus, a translocation that does to seed and develop. In addition, the lack of not occur naturally in adult mammalian recipient germ cells facilitates the injection species. The mechanisms regulating the procedure due to a decrease in the resist- translocation of the injected spermatogonia ance to the flow of the donor germ cell sus- from the lumen towards the basal surface pension through the seminiferous tubuli. In of the tubule are still unexplained. On the that study the transferred cells bore the other hand, the success of the experiments of Brinster & Avarbock (1994) and addition- LacZ gene (the expression of this gene is al studies (see Table I) suggest that the detectable histochemically in germ cells recipient Sertoli cell can regulate transloca- using the substrate X-gal). At the end of tion of recipient and donor germ cells a) experimental period among the busulfan- from the basement membrane to the lumen treated recipients 37% were found to be and b) from the lumen to the basement positive for donor germ cells. In tubules col- membrane, respectively. onized by transferred donor cells, the organization of the spermatogenic stages Jiang and Short (1995) transferred pri- within the recipient seminiferous tubules mordial germ cells or gonocytes recovered was normal, characteristic of the donor from male Sprague-Dawley rat fetuses or species and mature donor spermatozoa neonates into the rete testicles of recipient were observed. adult Long Evans rats that had been treat-

Male infertility today #4 9 Table 1: Evolution of male germ cell transplantation techniques.

Author Year Report

First report of successful induction of donor Brinster and Zimmermann 1994 mouse spermatogenesis within a recipient mouse testicle Germ-line transmission of donor mouse Brinster and Avarbock 1994 haplotype in recipient mice Jiang and Short 1995 Rat male germ cell transplantation in recipient rats First report of xenogeneic transplantation of rat Clouthier et al. 1996 germ cells into recipient mice Transplantation of frozen/thawed mouse Avarbock et al. 1996 spermatogonia into recipient mice Ogawa and Brinster 1997 Detailed description of transplantation techniques Germ cell genotype controls cell cycle during Franca et al. 1998 spermatogenesis in the rat Transplantation of hamster germ cells into Ogawa et al. 1999 mouse testicles First successful attempt of germ cell transfer into Schlatt et al. 1999 a primate testicle Pattern and kinetics of mouse spermatogonial Nagano et al. 1999 stem cells into recipient testicles First report of successful production of human Sofikitis et al. 1999 spermatozoa after transplantation of human germ cells into mouse and rat testicles Magnetic cell sorting for enriching viable Schlatt et al. 1999 spermatogonia from rodent and primate testicles Primate spermatogonial stem cells colonize Nagano et al. 2001 mouse testicles Successful hamster seminiferous tubuli Sofikitis et al. 2001 xenogeneic transplantation

10 Male infertility today #4 ed with busulfan. They suggested achieve- colonization was absent. In these regions ment of normal donor spermatogenesis in recipient Sertoli cells were observed that 62% of the recipients. However, due to a were actively phagocytosing donor sperma- lack of a specific marker for the identifica- tozoa. In the latter study, failure of elongation of donor cells it was difficult to con- tion and chromatin condensation of donor clude in that study that the donor cells had spermatids was a major barrier for the com- colonized the seminiferous tubules. In pletion of donor spermatogenesis. The another study, Jiang and Short (1998) pro- authors hypothesized that the failure of vided clear evidence that transplanted pri- achievement of donor spermatogenesis in mordial germ cells could divide within the these areas was due to dysfunction of the recipient seminiferous tubules. The authors testicular somatic cells of the recipient concluded that the pattern of donor cell col- mutant genetic model used rather than due onization and donor spermatogenesis fol- to failure of donor germ cells to develop. lowing transplantation in terms of their spa- Tanemura et al. (1996) transplanted very tial location and connection with the recipi- thin seminiferous tubules of old (33 months ent seminiferous epithelium depend on the of age) BDF1 mice into W/Wv mouse developmental stages of the donor cells at testes. Artificially cryptorchid younger BDF1 transfer. mice were used as controls. Testicular tis- Russell et al. (1996a) described the sue from the above cryptorchid animals ultrastructural features of donor spermato- was transplanted into the right testicles of genesis in immunologically compatible ster- W/Wv mice while testicular tissue from the ile mice post-transplantation of mouse old BDFo mice was transplanted into the spermatogonia. Recipient animals carried left testicle of the same animal. Two weeks mutant alleles and lacked endogenous tes- post-transplantation the most advanced ticular spermatogenesis. Testicular tissue of donor spermatogenic cells on the left side recipient animals was examined by light were spermatocytes, whereas, on the right and electron microscopy 12 to 15 months side both donor spermatocytes and round following donor germ cell transplantation. spermatids were detected. At four weeks Donor mouse germ cells were demonstrat- post-transplantation, elongated spermatids ed to form cellular associations, character- were detected in tubules of the right testi- istic of the donor species, within the recipi- cles but spermatids were still undetectable ent testicle. Donor spermatogonia were in the left testicles. It appears that spermatogenic cells derived from old mice found exclusively in the basal compartment. could not pass through meiosis. The Apparently they had been translocated from authors concluded that either a defective the recipient tubular lumen through the extratubular environment or a defective recipient Sertoli cell junctions eventually to intratubular environment in the donor semi- reside on the basal lamina. In addition, niferous tubuli had caused the arrest of some recipient seminiferous tubules spermatogenesis (of the old mice) in the exposed qualitative and quantitative defects transplanted seminiferous tubules within in donor spermatogenesis. Most of the the recipient left testicles. common abnormalities were seen at the elongation phase of donor spermatogene- Schlatt et al. (1999) injected bull, mon- sis. There were regions in the recipient key, or human germ cells into resected bull, seminiferous tubules where donor germ cell monkey or human testicles, respectively.

Male infertility today #4 11 Introduction of donor germ cells to areas of nance of immature male germ cells during recipient seminiferous tubuli distal to the freezing or cryostorage. Furthermore, in the sites of injection could not be achieved, latter study, the authors proved that post- probably because the endogenous recipient thawing haploid round male germ cells spermatogenesis had not been depleted could fertilize oocytes. and recipient germ cells and seminiferous Avarbock et al. (1996) have demonstrat- tubular fluid impeded the movement of ed that donor germ cells recovered from injected donor cells into the tubules. Distal prepubertal or adult mice have the capacity movement of injected donor cells within the post-freezing/thawing to generate donor recipient seminiferous tubuli could be only spermatogenesis in recipient mouse semi- achieved in immature or partially regressed niferous tubules. Among 30 recipient testi- testes. Rete testis injections were applied cles, 22 (73%) testicles were found to be via ultrasonographically-guided technique positive for donor spermatogenesis, post- in vivo on two cynomolgus monkeys that had been previously treated with a GnRH- transplantation of donor frozen/thawed antagonist. Ultrasonography allowed the germ cells. In the latter study, satisfactory localization of the rete testis and guidance colonization of recipient testicles by donor of the injection needle into the rete testis. frozen/thawed germ cells was found when The authors suggested that this technique propanediol and glycerol had been used as is the most promising one for transplanta- cryoprotective agents. Cryopreservation of tion of germ cells into large testicles. In one spermatogonia cells in men with primary monkey transplanted donor germ cells were testicular damage or men with oncological present in the recipient seminiferous disease offers advantages over cryopreser- tubules four weeks post-transfer. These vation of spermatozoa, since the spermato- cells had the morphological characteristics zoon cannot replicate, and a single sper- of B-spermatogonia and were demonstrat- matozoon carries a defined set of genetic ed as single cells and as small aggregates information. In contrast, cryopreservation of of cells in both the interstitium near the rete spermatogonia allows the whole genetic testis and within the seminiferous tubules. information/potential of an individual to be preserved. Furthermore, an additional number of donor spermatogonia and subse- B. Cryopreservation quently spermatids/spermatozoa can be of male germ cells produced post-transplantation of donor frozen/thawed spermatogonia within the Although advances in cryobiology have recipient testicles (via mitotic divisions and resulted in the development of well defined via meiotic divisions, respectively) since media for cryopreservation of mature sper- subpopulations of the donor spermatogonia matozoa there is inadequate knowledge undergo mitoses and subsequently meios- concerning the constituents of optimal es within the recipient testicles. Frozen chemical media for the maintenance of the male germ lines could be considered as viability and function of immature male biologically immortal. The implications of germ cells. Yamamoto et al., (1999a) in our this technology in basic sciences, medicine, laboratory have shown that the seminal and zoology are tremendous. plasma, milk, and other specific media can serve as cryoprotectants for the mainte-

12 Male infertility today #4 C. Germ cell transplantation This was the first application of the germ techniques as a powerful tool to cell transplantation technique to demon- reconstitute spermatogenesis in strate that a gene knockout that disrupts animals with testicular damage and spermatogenesis has no direct detrimental study the mechanisms of genetically action on the germ cell capacity to prolifer- caused male infertility. ate and differentiate. The authors used coat color differences in offspring to prove that Disruption of spermatogenesis is com- some recipients post-transplantation pro- monly caused by factors affecting either the duced spermatozoa (with fertilizing capaci- germ cells or the Sertoli cells or both of ty) that carried the ERa knockout gene. them. In most cases of male infertility the Both of the estrogen receptors ERa and pathogenesis is not well defined. Recently ERb have been reported in multiple cell several reports have demonstrated the types in the testicle and the epididymis importance of male germ cell transplanta- including germ cells (Hess et al., 1997). tion techniques for the investigation of Studying separately the ERa and ERb mammalian male infertility that occurs due genes indicates a distinct functional role of to a natural gene mutation or a targeted ERa in spermatogenesis (Krege et al., gene deletion. Gene knockout experiments 1998). These experiments have provided often lead to male infertility. In most cases, biological proof that a direct action of estro- the testicular cell types where the disrupted gens on the germ cells is not required for gene is phenotypically important cannot be the completion of spermatogenesis. easily identified. For the characterization of the latter cell types, the transplantation of Johnston et al. (2001) injected testicular germ cells from the evaluated infertile cells from mice with testicular feminization (donor) animals into seminiferous tubuli of pathophysiology into the seminiferous animals with distinct genetic features tubules of azoospermic mice expressing (Ogawa et al., 2000) may offer important functional androgen receptors. Recipient information. Thus, transplantation of germ testicles were analyzed 110 to 200 days fol- cells carrying a disrupted gene into a wild- lowing transplantation. Multiple colonies of type recipient may indicate the testicular complete and qualitatively normal donor- cell types where the disrupted gene is phe- derived spermatogenesis were observed notypically important. within the tubules of the recipient testicles demonstrating that male germ cells do not Mahato et al. (2000) transplanted male require functional androgen receptors and germ cells recovered from testicles of direct stimulation by the testosterone to estrogen receptor-a (ERa) knockout infertile complete spermatogenesis. mice into the seminiferous tubules of germ cell-depleted wild-type mice. The donor Ogawa et al. (2000) transplanted sper- germ cells carrying the knockout mutation matogonia from an infertile mouse strain underwent qualitatively normal mitoses and (Steel [Sl] mouse as donor of germ cells) to meioses within the recipient testicles, and an infertile recipient mouse (dominant white the recipients became fertile. Offspring spotting [W]) to determine if germ cells derived from the recipient mice were from that infertile male mouse were capable proven to have been generated from the of generating spermatogenesis. Post-trans- fertilization of oocytes with spermatozoa plantation the recipient mice were shown to that carried the disrupted gene for the ERa. be fertile. Thus, fertility was restored after

Male infertility today #4 13 transplantation of spermatogonia cells from Transplantation of spermatogonia from an infertile donor into an infertile recipient infertile mice carrying a mutation in the that had a permissive testicular environ- SCF gene into infertile mice with a mutated ment. That study indicates that both: a) c-kit receptor gene leads to qualitatively male germ cells and b) supporting environ- normal spermatogonial repopulation of the ment can retain full functional capability for recipient testicles with developing and dif- a long period of time in the absence of nor- ferentiating donor germ cells due to the mal spermatogenesis. presentation of recipient Sertoli cells producing membrane-bound SCF and donor Another naturally occuring mutation in germ cells expressing the functional c-kit mice that affects spermatogenesis is the receptor. Thus, Ohta et al. (2000) showed jouvenile spermatogonial depletion (jsd) that SCF stimulation is required for differen- mutation. The overall result of this mutation tiation of germ cells but not for spermatogo- is a single wave of spermatogenesis fol- nial stem cell proliferation (mitoses). In lowed by a failure of type A spermatogonial additional experiments, Ohta and co-work- cells to repopulate the testis. When testicu- ers transplanted undifferentiated testicular lar germ cells from jsd animals were inject- germ cells of GFP transgenic mice into ed into W/Wv or busulfan-treated recipients seminiferous tubules of mutant mice with (Boetger-Tong et al., 2000) no donor- male sterility, such as those dysfunctioned derived spermatogenesis was observed in at the Steel (Sl) locus encoding the c-kit lig- the recipients. In contrast, when Boetger- and or at the Dominant white spotting (W) Tong et al. (2000) injected germ cells with- locus encoding the c-kit receptor. In the out jsd mutation into jsd recipients, demon- seminiferous tubules of Sl/Sld or strated that jsd animals could support Sl17H/Sl17H mice, transplanted donor cells donor spermatogenesis for up to seven proliferated and formed colonies of undiffer- months. These data indicate that the jsd entiating spermatogonia but where unable infertile phenotype is due to a defect in the to differentiate further. When these undiffer- germ cells and cannot be attributable to entiated but proliferating spermatogonia functional or anatomical alterations of the were retransplanted into Sl (+) seminiferous testicular somatic cells. Additionally, Ohta et tubules of W-locus-mutant animals, they al. (2001) injected into the seminiferous resumed differentiation. These experiments tubules of B6-jsd/jsd mutant mice donor have indicated very vividly that when germ germ cells derived from the wild type GFP cell transplantation techniques are used to transgenic mouse (B6-+/+GFP). The latter reconstitute spermatogenesis in subjects donor cells were able to undergo complete with testicular damage, stimulation of c-kit spermatogenesis within the recipients indi- receptor by its ligand is necessary for the cating that the jsd mouse possesses opti- achievement of full donor spermatogenesis mal testicular microenvironment for sup- within the recipient testicle. porting germ cell differentiation. The conclusion of these experiments is that gene mutations that lead to germ cell inability to D. Culture of spermatogonia cells undergo meiosis do not preclude the proba- prior to transplantation. bility that the respective testicular biochemical microenvironment allows differentiation of germ cells of a different gonotype. 1. Optimal conditions for culturing donor

14 Male infertility today #4 germ cells pre-transplantation. bovine serum on a feeder layer of STO cells for four months and could still gener- Culture of spermatogonia cells in vitro ate donor spermatogenesis successfully increases their number and allows trans- when transplanted into recipient mice. plantation of a larger number of donor pre- Donor premeiotic germ cells pre-transplan- meiotic germ cells within a recipient testi- tation survived under relatively simple cul- cle. Meiotic differentiation of male germ ture conditions without the addition of cells in culture has been reported by growth factors and/or hormones into the Rassoulzadegan et al. (1993). The authors culture media. In this experiment all the reported that mouse immature germ cells successful donor germ cellular cultures that differentiated up to the spermatid stage in subsequently resulted in spermatogenesis vitro when they had been co-cultured with a following transplantation into recipients had Sertoli cell line. However, in that study, in been supported by STO feeder cells. STO vitro derived spermatids never proceeded feeder cells are known to have a beneficial to form mature spermatozoa. Additionally effect on cultures of embryonic stem cells that in vitro culture system could not be and primordial germ cells (Matsui et al., maintained beyond two weeks. 1992; Resnick et al., 1992). This beneficial Kierszenbaum and co-workers (1994) effect during culture (pre-transplantation) claimed that germ cells could not survive in may be mediated by growth factors and vitro for longer than few weeks. However, cytokines secreted by the feeder layer or by the absence of an assay to evaluate sper- cellular associations between the germ matogonia cell function prevented an accu- cells and the feeder cells (Robertson 1987; rate evaluation of the function of the cells. Smith et al., 1992). Under these conditions The development of the spermatogonial germ cells remained alive in culture up to transplantation technique has provided a 111 days. functional assay to assess the potential for meiosis of any population of spermatogonia It appears that nowadays culture of (Brinster & Zimermann, 1994; Brinster & male germ cells pre-transplantation is pos- Avarbock, 1994). It should be emphasized sible and increasing the number of avail- that the rationale to culture donor germ cel- able for transplantation spermatogonia may lular populations prior to the performance of have a beneficial effect on the transplanta- transplantation techniques is to increase tion outcome. the number of premeiotic spermatogonial cells rather than the number of spermatocytes and spermatids. Among the trans- 2. Human spermatogenesis in vitro planted donor germ cells, the spermatogo- Numerous researchers have aimed to nia are the cells that will move towards the induce human male germ cell meiosis in basement membrane of the recipient vitro. Aslam & Fishel (1998) cultured in vitro tubules and will organize the first colonies cellular suspensions from men with of donor spermatogenesis within the recipi- obstructive and non-obstructive azoosper- ent tubules. mia for 96 hours. Culture medium was a Nagano et al. (1998a) demonstrated modified Eagle`s minimum essential medi- that mouse testicular spermatogonia cells um supplemented with 15 mM HEPES, could be cultured into Dulbecco`s modified pyruvic acid, lactic axid, glutamine, peni- Eagle`s medium containing 10% fetal cillin, streptomycine and 10% fetal bovine

Male infertility today #4 15 serum. Some cells lost gradually their via- reported that application of this method in bility during the first 24 h period. Between patients with spermatogenetic arrest result- 72 and 96 hours of culture all cells lost ed in a birth after fertilization of oocytes eventually their viability. However, during with elongated spermatids obtained by in the culture period 22% of round spermatids vitro meiosis of primary spermatocytes. developed some growth of flagella. Sofikitis Cremades et al. (1999) cultured in vitro and co-workers (1998a and 1998b) in our (co-culture with Vero cells) round sper- laboratory had developed a medium (SOF matids recovered from: a) men with non- medium) for maintenance of the viability obstructive azoospermia with late matura- and function of spermatogonia, spermato- tion arrest and b) one patient with total cytes, and spermatids for relatively long globozoospermia. Mature spermatozoa with periods. With SOF medium it could be pos- normal morphology were detected after five sible to prolong the viability and maintain days of culture in one man with spermato- the function of round and elongating sper- genetic arrest and from the patient with matids in vitro. SOF medium contains lac- total globozoospermia. The authors sug- tate and glucose as energy substrates. It gested that co-culture with Vero cell mono- should be emphasized that the preferable layers could support full maturation of energy substrate for immature germ cells is human round spermatids. In additional the lactate rather than glucose (Sofikitis et studies Cremades et al. (2001) provided al., 1998a). Protection of round spermatids evidence that few round spermatids could against environmental shock and stabiliza- differentiate in vitro up to the elongated tion of the spermatid membrane was spermatid or spermatozoon stage after a achieved by cholesterol that had been seven to 12 day-culture with Vero cells. The added to the SOF medium in a small con- latter spermatids were able to fertilize centration. Vitamins and ferric nitrate had human oocytes and normal embryos were been chosen as components of the SOF generated. medium due to their positive influence in spermatid viability.

Tesarik et al. (1998) demonstrated that XENOGENEIC TRANSPLANTATION FSH stimulates both male germ cell meio- OF MAMMALIAN MALE GERM CELLS sis and spermiogenesis during in vitro cul- Clouthier et al. (1996) transplanted rat ture of human germ cells in GAMETE-100 germ cells into the seminiferous tubules of medium in the presence of Sertoli cells. In immunodeficient mice and generated rat addition, testosterone was found to potenti- spermatogenesis into mouse testicles. In all ate the effect of FSH on meiosis and of 10 recipient mice (in 19 out of 20 testi- spermiogenesis. This effect was probably cles) rat spermatogenesis was achieved. caused by the prevention of apoptosis of Some recipients were found positive for Sertoli cells. Tesarik et al. (1998; 1999) donor spermatozoa within their epididymes. have suggested that human spermatogene- Rusell & Brinster (1996) transplanted rat sis can proceed in vitro with an unusual germ cells into the seminiferous tubules of speed in the presence of high concentra- immunodeficent mice, as well. The success tions of FSH and testosterone. However, of rat to mouse transplants led to a reevalu- the generated gametes are often morpho- ation of the role of the Sertoli cell in terms logically abnormal. Tesarik et al. (1999) of its requirement to support differentiation

16 Male infertility today #4 of germ cells. Rat germ cell associations rat and generated the spermatogenic struc- into the mouse seminiferous tubuli followed tural pattern of the rat. Thus, the length of the characteristic patterns of rat spermato- the spermatogenic cycle of the rat genesis. The recipient Sertoli cells associat- remained the same in the mouse testicle as ed with rat spermatogenesis were identified in the rat testicle indicating that the length ultrastructurally as being of mouse origin. of germ cell cycles was determined by the However, donor spermatogenesis derived germ cells alone and was not influenced by from the xenogeneic germ cells was not Sertoli cells. always qualitatively or quantitatively com- Ogawa et al. (1999a) used the rat as a plete in the recipient animals as donor elon- recipient animal and transplanted rat and gated spermatids were often missing or mouse germ cells into the seminiferous deformed. In recipient tubular regions lack- tubules of rats. They demonstrated that ing donor spermatogenesis, recipient 55% of rat testicles were colonized by Sertoli cells were phagocytosing donor mouse cells. Depletion of recipient endoge- elongated spermatids indicating that some nous spermatogenesis before donor cell recipient Sertoli cells do not support donor transplantation was more difficult in the rat spermatogenesis and develop surface fea- than previously reported for mouse recipi- tures that can recognize donor elongated ents. Transplantation of mouse testicular spermatids and remove them. cells into rat seminiferous tubules was most Franca et al. (1998) transplanted rat successful in cryptorchid recipients pre- germ cells into mouse testicles and the treated with busulfan. Mouse donor cell- mice were killed 12.9-13 days after trans- derived spermatogenesis within the recipi- plantation. The most advanced rat germ cell ent rat testicle was proven. Pretreatment of subtype labeled within the recipient testicle rat recipients with leuprolide had a benefi- was the pachytene spermatocyte of stages cial effect on the colonization of the recipi- VI-VIII of the rat spermatogenic cycle. ent testicle by donor mouse cells. The Recipient animals had been treated with authors suggested that recipient testicular busulfan pre-transplantation. In the recipi- preparation prior to donor spermatogonial ent animals that underwent transplantation cell transplantation is critical and differs of rat germ cells a degree of mouse among species. endogenous spermatogenesis recovered. In our laboratory Tanaka et al. (1997) The most advanced labeled mouse germ and Sofikitis et al. (1998a) transplanted tes- cell types (in recipients killed 12.9-13 days ticular germ cells from cryptorchid ham- post-transplantation) were meiotic cells or sters into the seminiferous tubuli of recipi- young spermatids. The same findings were ent immunodeficient mice, immunodeficient observed if a mixture of rat and mouse rats, and non-immunodeficient Wistar rats. germ cells were transplanted in mice recipi- After mincing the recipient testicular tissue ents. Two separate timing regimens for and epididymal tissue it was found that germ cell development were found in the hamster spermatogonia had differentiated recipient mouse testicle: one of rat duration up to the spermatozoon stage within the and one of mouse duration. Rat donor germ recipient seminiferous tubuli. An interesting cells that were supported by recipient observation was that in some microscopical mouse Sertoli cells always differentiated fields of the droplets prepared from the with a cell cycle timing characteristic of the minced recipient tissue the number of

Male infertility today #4 17 donor spermatozoa was larger than that of achieved hamster spermatogenesis into the recipient spermatozoa. Transmission xenogeneic testicles, as well. The authors electron microscopy revealed hamster demonstrated that hamster spermatogene- round spermatids into recipient seminifer- sis could be produced within immunodefi- ous tubuli. A novel characteristic of those cient mouse recipients. They transplanted studies was that as donors had been used fresh hamster donor testicular cells into the animals with primary testicular damage due seminiferous tubules or the rete testis of to induction of cryptorchidism. We have immunodeficient mice that had been pre- chosen donors with primary testicular dam- treated with busulfan before transplantation. age considering that if the transplantation Among 13 recipient mice, hamster sper- techniques are applied in the future for the matogenesis was identified in the testicles management of non-obstructive azoosper- of all mice. Approximately 6% of the evalu- mia the candidates for such treatments will ated recipient seminiferous tubules demon- be men with primary testicular damage. The strated xenogeneic donor spermatogene- recipients had not been treated with busul- sis. Additionally, frozen/thawed hamster tes- fan. Immature animals with spermatogenic ticular cells post-transplantation were differ- arrest at the spermatogonium or primary entiated within the xenogeneic recipients. spermatocyte stage had been used as However, the outcome of transplantation recipients in all experiments (see next para- techniques using donor frozen/thawed graphs for the advantages and disadvan- germ cells appeared to be inferior to the tages of preparation of recipients with fresh germ cell transplantation techniques. busulfan). The accomplishment of donor Some morphologically abnormal hamster hamster spermatogenesis within a recipient spermatids were recognized into the recipi- Wistar rat testicle indicates that the ent seminiferous tubuli. Specifically, within immunological privilege properties of the the recipient seminiferous tubules hamster testicle of a non-immunodeficient recipient elongated spermatids with abnormal head animal may be occasionally sufficient for shapes were frequently observed. Within the survival of donor xenogeneic cells with- the recipient mouse epididymes the acro- in the recipient testicle. Ooplasmic injec- somes of hamster spermatozoa were often tions of hamster spermatozoa generated absent or poorly formed. It appears that into xenogeneic testicles resulted in fertil- although transplantation techniques can be ization and adequate embryonic and fetal successful in xenogeneic recipients the development up to the delivery of healthy derived haploid cells may have anatomical offspring. The latter animals were proven abnormalities. later to be fertile (Shimamoto et al., 1999; Xenogeneic transplantation of rabbit or first post-transplantation offspring genera- dog testicular germ cells into mouse semi- tion). The second and third post-transplan- niferous tubules (Dobrinski et al., 1999b) tation offspring generations were fertile, as and of porcine, bovine, or equine testicular well. The results of these experiments rep- germ cells into mouse seminiferous tubules resent the first application of assisted (Dobrinski et al., 2000) did not result in the reproductive technology using end haploid production of donor spermatozoa within the products of donor spermatogenesis within a recipient testicles. All the recipient mice xenogeneic recipient testicle. were immunodeficient and had been treat- Ogawa and co-workers (1999b) ed with busulfan approximately four weeks

18 Male infertility today #4 prior to donor germ cell transplantation to to support donor spermatogenesis of ani- deplete endogenous spermatogenesis in mals phylogenetically further removed than the testicles. Post-transplantation donor the hamster. Additionally, since donor sper- rabbit germ cells were present in the testi- matogonia were found in the immunodefi- cles of all recipients and had proliferated to cient recipients, immunological incompati- form chains of cells connected by intercellu- bility might not be responsible for the failure lar bridges or formed more elaborate mesh of achieving donor spermatogonia differen- structures. This pattern of colonization did tiation in the latter experiments. On the not change during the 12 month-observa- other hand, the recipients in the most of the tion period. Dog testicular germ cells (fresh, latter studies were busulfan-treated ani- cryopreserved, or cultured) were found mals. The administration of busulfan may located in pairs, short chains, or small have influenced detrimentally the recipient mesh structures in the recipient seminifer- Sertoli cell secretory function with an over- ous tubules post-transplantation. all result inability of the recipient Sertoli Colonization of recipient seminiferous tubuli cells to support donor germ cell differentia- appeared less extensive than that observed tion (see next paragraphs). In addition, no post-transplantation of rabbit germ cells. efforts were directed to enhance the Porcine donor germ cells formed chains immunological privilege properties of the and networks of round cells connected by recipient testicle (as suggested in a study intercellular bridges but advanced stages of by Shimamoto et al., 1999). Furthermore donor-derived spermatogenesis were not the recipients were not immature in all of observed within the recipient seminiferous the above experiments. Mature animals are tubuli. Transplanted donor bovine testicular considered as less appropriate recipients germ cells initially exposed a similar archi- than immature animals (Shinohara et al., tecture but then developed predominantly 2001). For all the above reasons the into fibrous tissue within the recipient semi- authors’ conclusion concerning the pre- niferous tubules. Few equine donor germ sumed causes of failure to induce donor cells proliferated within the recipient mouse germ cell differentiation in the above experi- testicles. Transplantation techniques of ments cannot be unequivocally adopted. equine germ cells recovered from a scrotal In 1999 Sofikitis et al. (1999a; 1999b) testicle or from a cryptorchid testicle result- reported completion of human meiosis and ed in similar outcome. These results indicat- spermiogenesis within recipient xenogeneic ed that under the experimental conditions testicles. This was the first report of suc- the authors followed, fresh or cryopre- cessful germ cell transplantation procedure served germ cells from a few mammalian with a given long phylogenetic distance species could colonize recipient mouse tes- between donor and recipient. ticles but did not differentiate beyond the stage of spermatogonial expansion. The Nagano et al. (2001) used baboon testi- authors hypothesized that the longer the cles from both prepubertal and postpuber- phylogenetic distance between the donor tal animals and demonstrated that donor and the recipient becomes, the less likely is spermatogonial cells of the baboon, regard- spermatogonial transplantation to result in less of reproductive age, have the capacity complete donor spermatogenesis within the to establish germ cell colonies in recipient recipient testicle. The authors speculated mice. The latter colonies remained within that the mouse Sertoli cell may not be able the recipient seminiferous tubuli for periods

Male infertility today #4 19 of at least six months. The long survival of ent testicular tunica albuginea is easier and baboon germ cells in the mouse testicle more rapid method than microsurgical and the formation of clusters may represent puncture of recipient seminiferous tubuli. (according to the authors’ speculation; Nagano et al., 2001) a process of slow division of undifferentiated spermatogonia fol- TRANSPLANTATION OF HUMAN lowed by apoptosis of early differentiation MALE GERM CELLS INTO stages, as previously described by de Rooij XENOGENEIC TESTICLES et al. (1999) and van Pelt et al. (1990). The Sofikitis et al. (1999a; 1999b) evaluated undifferentiated donor spermatogonia repli- the potential of spermatogonia cells of non- cated but were unable to proceed to differ- obstructed azoospermic men to differenti- entiating spermatogonial stages and finally ate to round spermatids or spermatozoa underwent apoptosis. Recipient animals into the seminiferous tubules of immunode- had been pretreated with busulfan to ficient immature recipient animals. destroy the endogenous spermatogenesis. Recipients were nude rats, which lack B That may have affected recipient Sertoli cell cells, and severe combined immuno-defi- secretory function and donor germ cell dif- cient (SCID) mice, which lack both T and B ferentiation. The maintenance of primitive cells. Recipient animals were immature and baboon germ cell clusters in mouse testi- exposed endogenous spermatogenesis up cles clearly demonstrated that antigens, to the spermatogonium or primary sperma- growth factors, and signaling molecules that tocyte stage at the time of transplantation. interact between baboon germ cells and Human spermatogonia cells had been the mouse seminiferous tubular environ- recovered from 18 non-obstructed ment have been preserved for 100 million azoospermic men with premeiotic sper- years in these widely divergent species (for matogenic arrest who visited for infertility additional information see Kumar et al., evaluation the Department of Urology of 1998). As it is described extensively below Tottori University, Yonago, Japan and the transfer of human germ cells into immunod- M.F.C. Clinic, Yonago, Japan. eficient immature animals resulted in the Spermatogonia cells were transferred completion of human meiosis and spermio- microsurgically into the seminiferous genesis within the recipient testicles. These tubules of ten nude rats and eight SCID latter studies (Sofikitis et al., 1999a; mice (Sofikitis et al., 1999a; Sofikitis et al., Sofikitis et al., 1999b) further support the 1999b). Ten seminiferous tubules per recipi- preservation of interacting signaling mole- ent testicle were microinjected with human cules in the seminiferous tubular microenvi- testicular germ cells plus anterior chamber ronment in widely divergent species. eye cells from the respective recipient. Sofikitis et al. (2001) reported genera- Recipient anterior chamber eye cells (they tion of hamster spermatozoa within xeno- express Fas L) were co-transferred to geneic testicles after hamster testicular tis- enhance the immune privilege status of the sue transplantation (see for details next recipient testicle (Shimamoto et al., 1999). paragraphs). The latter study provided a Previous studies in our laboratory have novel and easy methodology for xenogene- suggested strongly that co-transfer of recip- ic germ cell transplantation since transfer of ient anterior chamber eye cells together donor seminiferous tubules under a recipi- with the donor germ cells within the recipi-

20 Male infertility today #4 ent seminiferous tubulus lumen at the time dures in fragments of recipient seminiferous of transplantation enhancing the immuno- tubuli revealed human spermatogonia in logical privilege properties of the recipient proximity with the recipient animal seminif- testicle, increases the percentage of testic- erous tubulus basement membrane. ular infiltrating recipient lymphocytes that Human primary spermatocytes, round sper- undergo apoptosis with a final overall result matids, and elongating spermatids were a beneficial influence on the survival of the observed, as well in the recipient seminifer- donor germ cells within the recipient semi- ous tubuli via FISH techniques. In a very niferous tubulus and an improvement in the limited number of recipient seminiferous outcome of transplantation technique tubuli recipient spermatogonia and donor (Shimamoto et al., 1999). One hundred spermatogonia could be seen at the basal sixty six to 172 days post-transplantation, compartment of the same seminiferous testicles, epididymes, and vas deferens of tubulus. In addition, recipient sperm heads each recipient animal were evaluated for and human spermatids could be seen in the presence of human spermatozoa. the lumen of the same seminiferous tubu- Transmission electron microscopy and fluo- lus. This mixed donor-recipient spermatoge- rescent in -situ hybridization techniques netic architectural pattern has not been using specific probes against human X-, observed in the rat-mouse transplantation 18-, and Y-chromosomes were also per- model (Russell et al., 1996b). It should be formed in: a) some of the round germ cells emphasized that in contrast to the studies found in the testicles of the recipient ani- by Russell`s group (1996a and 1996b) and mals postmincing and b) some of the recipi- most other researchers’ investigations, the ent seminiferous tubuli. Prior to these recipient animals in our investigation were experiments we confirmed that the probes immature and had a certain degree of used could not react with /label rat or endogenous spermatogenesis (i.e., up to mouse chromosomes. the spermatogonium or primary spermatocyte stage) because busulfan had not been Post-transplantation human sper- administered. This may be the reason for matozoa were observed in minced samples the presence in our study of both donor and from the testicles, epididymes, and vas def- endogenous advanced spermatogenesis in erens of three nude rats and two SCID the same recipient seminiferous tubulus mice. Within the group of the latter five ani- post-transplantation. Furthermore, trans- mals the percentage of motile human sper- mission electron microscopy demonstrated matozoa liberated after recipient tissue human round spermatids (mitochondria are mincing and filtration ranged from 0% to scattered in the cytoplasm) in contact with 19% in testicular samples, 0% to 83% in recipient rat round spermatids (mitochon- the epididymal caudal samples, and 0% to dria are located peripherally only). This find- 88% in the vas deferens samples. ing confirms the presence of mixed human- Fluorescent in-situ hybridization (FISH) animal spermatogenesis in the same semi- techniques in recipient dispersed round niferous tubulus. It appears that human cells from minced testicular tissue demon- germ cells post-transfer into animal seminif- strated human spermatogonia/primary erous tubules can complete meiosis and spermatocytes, human secondary sperma- differentiate up to the spermatozoon stage. tocytes, and human round spermatids (n- Additionally, transmission electron DNA haploid cells). In addition FISH proce- microscopy revealed human spermatids in

Male infertility today #4 21 the lumen of the rat seminiferous tubules. cytes had been collected after mincing and These results have indicated that the filtering (the sediment in the filter was kept mouse or rat Sertoli cell can support and the filtrate was discarded) via a 13 µm- human spermatogenesis. This is an inter- pore size filter testicular biopsy specimens esting finding since there are differences in recovered from non-obstructed azoosper- the duration of spermatogenesis among the mic men with complete spermatogenic mouse, rat, and human species. To explain arrest at the primary spermatocyte stage the development of human spermatogene- (these purification procedures were similar sis within the mouse or rat seminiferous to those we previously published for mouse tubules it may be suggested that the donor spermatogonia/primary spermatocytes germ cells influence the recipient Sertoli purification; Yamamoto et al., 1999b). Prior cells to support/stimulate the survival and to culture, FISH techniques (with probes for differentiation of the donor germ cells. This human chromosomes 18, Y, and X) and suggestion is supported by studies showing confocal scaning laser microscopy (Sofikitis that humoral factors secreted by spermato- et al., 1998a) were applied in subfractions genic cells may affect the viability and pre- of cultured cells to distinguish between: a) sumambly modulate the function of Sertoli human and rat germ cells and b) rat germ cells (Chen et al., 1997). In addition, the cells and rat Sertoli cells nuclei, respective- completion of human meiosis and spermio- ly. Thus the number of each cell type genesis within a recipient testicle suggests preculture was evaluated. Type-2-system that mouse or rat Sertoli cell secretory contained rat round germ cells (60- function influences positively the induction 70x106/ml; prepared as previously of human meiosis. This hypothesis is sup- described for mouse round germ cells; ported by a very recent study in our labora- Yamamoto et al., 1999b) and human sper- tory showing that co-culture in vitro (at 310 matogonia/primary spermatocytes (60- C) of rat Sertoli cells, rat germ cells, and rat 70x106/ml). Type-2-system contained less human spermatogonia/primary spermato- than 100.000 rat Sertoli cell nuclei/ml. In cytes (in the presence of human FSH, rat type-1-system, rat minced testicular materi- FSH, and testosterone) resulted in the al had not been filtered, prior to its treat- completion of the human meiosis (forma- ment with pronase, therefore this culture tion of human round spermatids). In that system was positive for a significant num- study we prepared two in vitro culture sys- ber of rat Sertoli cells (Yamamoto et al., tems. Type-1-system contained rat Sertoli 1999b). In contrast, in type-2-system, the cells (number of nuclei: 2x106/ml), rat rat minced testicular material had been fil- round germ cells (60-70x106/ml) (rat round tered repeatedly (Yamamoto et al., 1999b) germ cells could be separated from rat and the vast majority of rat Sertoli cell elongated spermatids, and spermatozoa by nuclei were removed (they remained in the enzymatic treatment with pronase as we sediment of the respective filters; previously applied for mouse round germ Yamamoto et al., 1999b). Rat FSH had cell purification; Yamamoto et al., 1999b; been added in both culture systems to a enzymatic treatment occasionally decreas- final concentration of 15 ng/ml. Human es the available for culture number of recombinant FSH had been added in both Sertoli cells and human spermatogonia/pri- systems to a final concentration of 50 mary spermatocytes (60-70x106/ml). IU/ml. Water soluble testosterone had been Human spermatogonia/primary spermato- also added in both systems to a final con-

22 Male infertility today #4 centration of 1 µmol/L. Prior to culture in treatment. However, sections from the both type-1-system and the type-2-system recipient testicles examined up to 150 days cellular subfractions were proven by FISH after transplantation showed recipient semi- techniques and confocal scanning laser niferous tubules lined mainly with recipient microscopy to be negative for human round Sertoli cells, whereas, xenogeneic germ spermatids and human Sertoli cell nuclei. cells were not found. The authors speculat- Seventy-two hours post-culture 1.8-4.6 ed that the donor germ cells did not survive (x106/ml) and 0 human round spermatids and colonize the mouse testes because of were found in the type-1-system and type- non-compatible cellular interactions and 2-system, respectively, by FISH techniques immunological rejection resulting from inter- and confocal scanning laser microscopy species differences. However, the conclu- (Yamamoto et al., 2002). Human elongated sions of the study by Reis et al. (2000) can- spermatids or spermatozoa could not be not be unequivocally accepted for the fol- seen. These results represent the findings lowing reasons: a) to identify human germ of 12 culture experiments of type-1-system cells the authors relied on an antibody and type-2-system performed as above against proacrosin which appears only in described. Always human round spermatids late stages (mid-pachytene primary sper- were found post-culture in type-1-systems, matocytes and spermatids) of germ cell dif- whereas, no round spermatids were gener- ferentiation (see comment by Nagano et al., ated in type-2-systems. It appears that ani- 2001); thus, undifferentiated human sper- mal Sertoli cells can stimulate human meio- matogonia and early primary spermato- sis in vitro, in other words, animal Sertoli cytes could not be identified by the authors’ cellular paracrine or endocrine factors can methodology, b) the number of the trans- affect positively human meiosis. planted donor germ cells was low, c) the authors did not use any method to enhance The presence of human spermatozoa the immunological privilage properties of with potential for strong forward progression the recipient testicle as was done by within the epididymal tail and vas deferens Sofikitis et al. (1999a; 1999b) or Shimamoto of one rat post-transplantation (Sofikitis et et al. (1999) d) the recipients used were al., 1999a; 1999b) suggested that the rat neither immature nor newborns (studies by epididymis could induce human sperm mat- Shinohara et al., 2001, support Sofikitis’ uration. Thus, the thesis that epididymal choice to use as host testicles for donor sperm maturation process is strictly a germ cells those of immature animals species-dependent process may need to be rather than mature animals), and e) the reconsidered. recipients used had been treated with In contrast, Reis et al. (2000) failed to busulfan or were aspermatogenic (recipi- establish a complete human spermatogenic ents carried a mutation disrupting sper- line in the testicles of mutant aspermato- matogenesis). Since a) busulfan may have genic (W/Wv) mice and severe combined affected recipient Sertoli cell secretory immunodeficient mice (SCID). The latter function and subsequently may have had a species received germ cells from azoosper- detrimental effect on recipient intratesticular mic men. Spermatogenic cells were profiles of androgen-binding protein impair- obtained from testicular biopsy specimens ing the differentiation of donor germ cells of adult non-obstructed or obstructed (see next paragraphs) and b) Sertoli cells azoospermic men undergoing infertility from aspermatogenic animals may have

Male infertility today #4 23 β α had a secretory dysfunction due to the lack and anti- 1 and anti- 6-integrin antibodies of experience to interact with germ cells (Shinohara et al., 1999). The colonization of (see next paragraphs), the lack of positive recipient testicles by donor germ cells β α findings in the study by Reis et al. (2000) selected with the aid of anti- 1 and anti- 6- may not be due to non-compatible cellular integrin antibodies is significantly enhanced interactions between recipient and human compared with control donor germ cells. cells but it may be attributable at least par- Additionally, donor germ cells selected by α tially to the methods the authors followed anti- 6-integrin antibodies appear to be and the preparation of the recipients pre- twice as effective in colonizing recipient tes- β transplantation. ticles as those selected by anti- 1-integrin antibodies. In contrast, spermatogenic cells selected with an anti-c-kit antibody did not TECHNIQUES TO INCREASE show improved rates of recipient testicular β α RECIPIENT COLONIZATION BY colonization. 1-and 6-integrins could be DONOR CELLS used as markers to identify subpopulations of donor spermatogonial stem cells with a A. Selection/preparation higher potential to colonize recipient testi- of donor germ cells cles. The presence of surface integrins on spermatogonial stem cells suggests that Selecting donor germ cells for transplan- these cells share elements of a common tation techniques those subpopulations of molecular machinery with stem cells in germ cells with specific surface markers other tissues, including hematopoietic, helps to improve recipient colonization effi- intestinal epithelial, and epidermal (Potten ciency. Shinohara et al. (1999) studied the et al., 1992). Shinohara et al. (2000a, known association of stem cells with base- 2000b) examined the colonization ability of ment membranes to identify specific molec- a population of testicular cells enriched in ular markers on the stem cell surface that stem cells. They demonstrated that Sl infer- may influence recipient colonization effi- tile mutant mice can provide an enriched ciency. Selection of mouse testicular cells source of testicular stem cells for transplan- β α with anti- 1 – or anti- 6-integrin antibody tation techniques. In this mutant, the sper- resulted in the isolation of germ cell popula- matogonia stem cells constitute a relatively tions with significantly enhanced ability to high subpopulation of testicular cells (com- colonize recipient testicles. Spermatogonial paratively with the wild type mouse). The cells that preferentially adhered to laminin cryptorchid model provides another rich but not to collagen IV or fibronectin were source of stem cells; one cell in 200 testicu- proven to have the capacity to colonize lar cells is a spermatogonium stem cell. recipient testicles. The ability of fractions of Using germ cell transplantation techniques spermatogonial cells to adhere to laminin as a functional assay to evaluate quantita- suggested that integrins may have a role in tively the presence of spermatogonial stem the identification of the respective fractions cells, it was found that testicular cells recov- β because 1-integrin has the ability to ered from cryptorchid mice were highly α adhere to laminin and 6-integrin forms enriched in stem cells (Shinohara et al., β dimers with 1-integrin (Hynes, 1992). 2000c). It appears that the function of sper- Subpopulations of testicular cells can be matogonial stem cells is not adversely isolated with a system of magnetic beads affected by the elevated temperature of a

24 Male infertility today #4 cryptorchid testicle. Donor testicular germ (Shimamoto et al., 1999). cells from the Sl mutant mouse increased the number of donor germ cell colonies within recipient testicles post-transplanta- B. Preparation of recipient animals tion to a much lower extent than was Tanaka et al. (1997) in our laboratory achieved by spermatogonial cells recovered reported induction of meiosis of hamster from cryptorchid mice. Furthermore, spermatogonia not only within the tubules Shinohara et al., (2000c) employed an in of immunodeficient animals but also within vitro fluorescence-activated cell shorting the tubules of a non-immunodeficient ani- cellular analysis (FACS) in populations of mal. That study was the first that suggested spermatogonia from cryptorchid testicles to that the local immunological privilege prop- develop qualitative and quantitative cellular erties of the testicle of a non-immunodefi- criteria that indicate spermatogonial stem cient animal may be occasionally sufficient cell subpopulations. The most effective to allow survival and differentiation of donor stem cell enrichment strategy was the germ cells. selection of testicular germ cells with α A novel characteristic of three studies proven expression of 6-integrin and low side scatter. This strategy resulted in a 166- carried out in our laboratory (Tanaka et al., fold enrichment in spermatogonial stem 1997; Shimamoto et al., 1999; Sofikitis et cells. al., 1999a) was the presence of a certain degree of endogenous spermatogenesis in Another way to increase the outcome of the recipient animals at the time of trans- transplantation techniques is to enhance plantation. Recipients were immature mice the immunological privilege properties of and rats with arrest at the spermatogonium the recipient testicle by co-transferring or primary spermatocyte stage (in most of recipient anterior chamber eye cells togeth- the cases). Endogenous recipient sper- er with the donor germ cells (Shimamoto et matogenesis had not been destroyed with al., 1999) within the recipient seminiferous toxic agents (i.e., busulfan) prior to trans- tubuli. Co-transplantation of anterior cham- plantation. The presence of a degree of ber eye cells increases the percentage of endogenous spermatogenesis in the recipi- infiltrating leukocytes that undergo apopto- ents at the time of transplantation in our sis post-penetration of a recipient seminifer- experiments may explain the presence of a ous tubule with a beneficial result on the mixed type of donor-recipient spermatogen- survival of the transplanted donor germ esis within the same recipient seminiferous cells and the transplantation technique out- tubule post-transplantation. Brinster & come. Additionally, findings in our laborato- Zimmermann (1994), Brinster & Avarbock ry suggest that infusion of interferone-γ into (1994), Clouthier et al., (1996). Russell et a recipient seminiferous tubule at the time al. (1996), Russell and Brinster (1996), and of donor germ cell transplantation upregu- Nagano et al. (2001) used as recipients ani- lates the FasL expression by the recipient mals with endogenous spermatogenesis Sertoli cells, increases the percentage of absent/disrupted due to genetic/toxic fac- infiltrating leukocytes that undergo apopto- tors, respectively. This may explain the fact sis after the mechanical penetration/injury that in their studies post-transplantation of the seminiferous tubule, and improves recipient seminiferous tubuli showed only the outcome of transplantation techniques donor spermatogenesis or only recipient

Male infertility today #4 25 spermatogenesis. Mixed type of donor and experiments showed that a) busulfan has a recipient spermatogenesis was not long time duration-detrimental effect on observed within the same recipient seminif- Sertoli cell secretory function and subse- erous tubule in the latter studies. Although quently may impair (or decrease) the recipi- the absence of endogenous recipient sper- ent Sertoli cell capacity to support donor matogenesis in the recipients at the time of germ cell differentiation, and b) sterile donor germ cell transplantation may a) aspermatogenic mutant mice have dimin- facilitate the movement of the donor germ ished Sertoli cell secretory function com- cells from the recipient seminiferous tubule pared with SCID mice or nude mice and lumen towards the basement membrane subsequently may support to a less degree and b) be accompanied by a lower recipient donor spermatogenesis. intratubular pressure that decreases the Prolonged administration of GnRH-ago- resistance to the injection of donor germ nists inhibits the secretion of LH and FSH cells, toxic agents administered for disrup- followed by suppression of testosterone tion of endogenous spermatogenesis may production by the testicle and disruption of result in the development of an intratubular spermatogenesis (Hadziselimovic et al., biochemical environment that is not opti- 1987; Ogawa et al., 1989; Meistrich and mal/ideal for colonization/differentiation of Kangasniemi, 1997). Leuprolide reducing donor germ cells. In addition sterile mutant the intratesticular testosterone and subse- animals (used by other authors as recipi- quently disrupting spermatogenesis in the ents) may not be ideal recipients because recipient animals may be used for sup- their Sertoli cells do not have the “experi- pressing endogenous recipient spermato- ence” to support meiosis and spermiogene- genesis in the recipient animals pre-trans- sis and subsequently may not have optimal plantation. Ogawa et al. (1998) and capacity to support the spermatogenesis of Dobrinski et al. (2001) demonstrated that donor germ cells that enter the recipient the efficiency of colonization of recipient seminiferous tubuli non-physiologically. In testicles by donor cells was markedly high- three studies (Tanaka et al., 1997; er after pre-treatment of recipients with Shimamoto et al., 1999; Sofikitis et al., leuprolide. 1999a) in our laboratory most of the recipients were immature animals with arrest at the spermatogonium or primary spermato- C. Methods for cyte stage. The choice of immature animals germ cell transplantation that neither had been treated with busulfan nor were genetically aspermatogenic in the Microsurgical puncture of seminiferous latter three studies is supported by recent tubules was the initial approach to transfer studies in our laboratory showing that donor germ cells into a recipient testicle. androgen-binding protein profiles in testicu- The standard method for donor germ cell lar cytosols are significantly smaller transplantation in our laboratory (Tanaka et (p<0.05) in a) sterile mutant Ww/v mice al., 1997; Sofikitis et al., 1999a; Sofikitis et compared with same age SCID mice or al., 1999d; Shimamoto et al., 1999) is pene- nude mice and b) busulfan treated (4, 8, tration of a seminiferous tubule by two nee- and 12 weeks post-busulfan administration) dles guided by two micromanipulators. nude rats compared with same age nude Several seminiferous tubuli are punctured rats (non-treated with busulfan). The latter in each recipient testicle. Ogawa et al.,

26 Male infertility today #4 (1997) reported two additional methods for without the aid of a micro-manipulator or introducing donor germ cells into recipient pressure injector. It may be considerably seminiferous tubules. First, introduction of more difficult in some species, such as the donor germ cells into the recipient effer- monkey, pig or sheep which have an axial ent ducts and second, introduction of the rete testicle deeply located in the testicle. donor germ cells into the recipient rete It has been suggested (Ogawa et al., testis. Direct injection of donor germ cells 1997) that microinjection of donor germ cell into the recipient seminiferous tubules is suspensions into the recipient seminiferous less invasive and allows entrance of donor tubules, efferent ducts, or rete testicle are germ cells into several tubules at various equally effective in generating donor germ sites on the testicular surface. However, this cell-derived spermatogenesis in recipients. approach is the most time consuming Using a microinjection apparatus, a volume (compared with donor germ cell transfer of 10 to 50-100 µl can be easily injected into the efferent ducts or rete testicle) and into recipient rodent seminiferous tubuli, may be inappropriate and ineffective for which is appropriate for the immature, larger testicles (than the rodent testicle). mature, and post-mature rat and mouse Injection of donor cellular suspensions testicle. However, in animals with larger tes- into the recipient efferent ducts (Ogawa et ticles, larger volumes of donor cell suspen- al., 1997) is a more difficult technique and sions (occasionally larger than 2 ml) are requires careful surgical dissection to needed to fill the recipient testicles. The expose the delicate efferent ducts. Post- development of transplantation techniques transfer of donor germ cells into the recipi- into larger (than rodent) mammalian testi- ent efferent ducts develops excessive intrat- cles is not an easy task. Microinjection into esticular pressure which may result in the seminiferous tubules of calf, bull, mon- intratesticular ischemia with a subsequent key and man is a difficult procedure due to detrimental effect on donor cell viability and a resistant lamina propria and a highly con- on the recipient tubular environment. voluted tubular mass. An alternative tech- However, this technique is faster than semi- nique has been developed by Schlatt et al. niferous tubular injection. An advantage of (1999) for germ cell transfer into the pri- introducing donor cells into an efferent duct mate testicle, using ultrasonographically is that the injecting pipette can be held guided injections into the rete testis. This securely into the duct resulting in little technique allowed an efficient filling of up to donor cell suspension fluid leakage. Thus, 70% of the recipient seminiferous tubules smaller volumes of donor cell suspensions when the secretory activity of the recipient are needed to fill the recipient tubules. testicle had been reduced.

Injection of donor cells into the recipient Sofikitis et al. (2001) introduced a novel rete testicle requires less dissection than method for germ cell transplantation: trans- that needed to expose the recipient efferent plantation of donor testicular tissue under ducts and for this reason is a faster the tunica albuginea of xenogeneic ani- approach. However, only one or two rete mals. This method resulted in the comple- testicles penetrations can be performed tion of hamster meiosis and spermiogene- before leakage prevents filling of the sis within hamster seminiferous tubuli under tubules. Rete testicle puncture is the most the rat tunical albuginea (within rat testi- easy technique to transfer donor germ cells cles). It is an easy and efficient method to

Male infertility today #4 27 transfer donor genome within a recipient tion. Four weeks post-transplantation donor animal. In these experiments (Sofikitis et germ cells are divided on the basement al., 2001) a piece of testicular tissue from membrane of the recipient seminiferous cryptorchid hamsters was placed under the tubuli forming a network. Differentiation of tunica albuginea of 13 nude rats. Vascular donor germ cells to more mature germ cell endothelial growth factor was administered stages appears to begin after this initial col- three times a week intrascrotally in the onization and spreading on the recipient recipients rats. Three nude rats, five months seminiferous tubulus basement membrane. later, showed complete hamster spermato- Nagano et al. (1999) observed the pat- genesis into the hamster seminiferous tubu- tern and kinetics of mouse seminiferous li under the rat tunica albuginea. At that tubular colonization by donor cells during a time transmission electron microscopy 4-month-period post-transplantation. The showed full hamster spermatogenesis into recipient testicles were fixed in 4% some transplanted hamster seminiferous paraformaldehyde, washed, and stained tubules, whereas, arrest at the primary with X-gal, as described by Ogawa et al. spermatocyte stage or spermatogonia plus (1997). Using this system it was possible to Sertoli cell-only histology were demonstrat- observe that the recipient seminiferous ed in other transplanted hamster seminifer- tubular colonization process by the donor ous tubules. Vascular endothelial stain cells could be divided into three continuous showed standard or extensive vasculariza- phases. During the initial week transplanted tion in a number of transplanted hamster donor cells were randomly distributed along seminiferous tubules. the recipient tubules and a small number reached the basement membrane. In the period between the first to the fourth week PATTERNS, KINETICS, AND post-transplantation, donor germ cells on DIFFERENTIATION OF DONOR the basement membrane divided and GERM CELLS WITHIN THE formed a monolayer network. Following the RECIPIENT SEMINIFEROUS TUBULI end of the first month, cells in the center of An intriguing question concerning colo- the network differentiated extensively and nization of a recipient seminiferous tubule established a colony of spermatogenesis by donor germ cells is the pattern and tim- which expanded laterally by repeating the ing of donor germ cell divisions post-trans- two previous phases. The number of colo- plantation. Ogawa et al. (1997) and Nagano nized sites in the recipient testicle did not et al. (1998b) used the transgenic mouse change in the period between the first and line B6, 129-TgR (ROSA 26) from the the fourth month. However, the average Jackson Laboratory (ROSA 26) (it was length of donor cellular colonies increased developed using gene trapping methods; it from 0.73 to 5.78 mm between the first and contains the E.coli LacZ [lacZ] structural the fourth month. These experiments have gene) to evaluate the kinetics of donor offered an approach to study in a systemat- germ cell post-transplantation. In the latter ic and quantitative manner the pattern and study it has been found that the donor germ kinetics of the donor germ cell colonization cell suspension remains widely dispersed process. Parreira et al. (1999) evaluated by within the recipient seminiferous tubules morphometric and ultrastructular studies during the first weeks following transplanta- (light and electron microscope) the devel-

28 Male infertility today #4 opment of germ cells transplants. Ten min- tion of donor germ cells in recipient testi- utes post- transplantation donor germ cells cles following the injection of a known num- developed relationships with small recipient ber of donor cells. The relationship between Sertoli cell processes. These relationships the number of transplanted cells and the remained for one week post-transplanta- colonization efficiency was investigated. tion. Clones of donor spermatocytes were The authors found a linear correlation found one month post-transplantation of between the number of injected cells and mouse germ cells into mouse seminiferous the degree of colonization. Transplantation tubules. Up to the end of the second month of 104 donor germ cells per mouse testicle post-transplantation mature donor sperma- only rarely resulted in recipient testicular tozoa were produced and by the end of the colonization whereas after transplantation 5 6 third month an average of 30% of the recip- of 10 and 10 donor germ cells per mouse ient testicles contained donor-derived sper- testicle the extent of donor derived spermatogenesis. The cumulative lateral spread matogenesis in the recipient seminiferous of donor spermatogenesis in a recipient tubuli was directly related to the number of tubule was rapid, moving at approximately transplanted donor cells. It appears that 55-60 µm per day. However, to calculate the approximately 10% of the transplanted length of the colonies of donor cells the spermatogonial cells participate in colony authors used an eyepiece micrometer on a formation in the recipient testicle. Shinohara stereo microscope. This technique should et al. (2001) using this computer-assisted be considered subjective and extremely imaging system technology found a 39-fold labor intensive, thereby limiting its applica- increase in mouse male germ line stem tion for detailed analysis of donor sper- cells during development from birth to the matogenic colonization after transplantation maturity. In addition, they found small differ- (comment by Dobrinski et al., 1999a). ences in the size of the colonies of the donor cells within the recipient testicle Dobrinski et al. (1999a) used a comput- when the donor germ cells had been recov- er-assisted image analysis system that ered from young or adult mice. evaluated quantitatively a wide range of Transplantation of donor germ cells was information available by microscopic obser- more effective when as recipients were vation of specimens. Donor spermatogene- used immature pup testicles (Shinohara et sis in a recipient testicle was identified by al., 2001). This supports the methodology blue staining of donor derived spermato- of the transplantation program in our labo- genic cells expressing the E. coli lacZ struc- ratory employing immature animals exclu- tural gene. Stained seminiferous tubules sively as recipients (Tanaka et al., 1997; containing donor-derived spermatogenesis Shimamoto et al., 1999; Sofikitis et al., were selected for quantitative analysis tak- 1999a; Sofikitis et al., 1999b). The authors ing into consideration their color by color suggested that the microenvironment of the thresholding (range of blue). Colonization pup testicle represents a more hospitable was appreciated as number, area, and biochemical environment for transplantation length of stained tubules. Interactive, opera- of male germ-line stem cells. The coloniza- tor-controlled color selection and sample tion area per donor spermatogonium stem preparation accounted for less than 10% cell was 4.0 times larger in immature pup variability for all collected parameters. This recipient testicles than in adult recipient system quantified the degree of coloniza- testicles.

Male infertility today #4 29 POTENTIAL CLINICAL can be transferred back to patients’ testicle. APPLICATIONS OF MALE GERM Sofikitis et al. (1999b) and Jahnukainen CELL TRANSPLANTATION et al. (2001) suggested that maintenance of TECHNIQUES donor germ cells within a recipient animal A. Preservation of the whole genome may be susceptible to risks concerning of patients with oncological disease transmission of virus from the recipient ani- in recipient animals (using an animal mal to the donor cells. testicle as a surrogate organ) Schlatt et al. (1999) demonstrated a Cryopreservation of spermatogonia (for rapid and effective method for the enrich- transplantation purposes into host animal ment of spermatogonia from testicular cell testicles) from young patients with oncologi- suspensions with a purpose to avoid con- cal disease who are subsequently sched- tamination of donor cell population with uled for chemotherapy treatment is of great malignant cells. Receptors for c-kit are clinical importance. Male germ cells prior to present in the plasma membrane of sper- the performance of chemotherapy can be matogonia. A magnetic cell separation tech- frozen and subsequently post-thawing can nique using c-kit antibodies for detection of be transplanted into immunodeficient ani- spermatogonia allowed fractions of sper- mals to maintain the whole genetic informa- matogonia from rat, hamster and monkey tion of the donor for a certain period. testicles to be purified. This approach could Additionally, xenogenic transplantation of be used to select only desirable cellular patients’ germ cells into an animal testicle populations to transfer into recipients in may increase the number of human sper- order to avoid contamination of the donor matogonia with potential to enter meiosis cellular populations with malignant cells. because subpopulations of the transplanted However, until a) improved methods for human spermatogonia are anticipated to purging testicular cancer cells or b) new undergo mitosis within the animal testicle. techniques for the early detection of con- This is an advantage of combined freezing taminating malignant cells will develop, this and transplantating germ cells from men technique may not be unequivocally with oncological disease over cryopreserva- accepted for routine performance. tion techniques alone because after the former procedures the number of transplanted spermatogonial stem cells will increase B. Autotransplantation of within the recipient testicle allowing later frozen/thawed testicular germ cells in (after the end of chemotherapy) a larger men with oncological testicular number of spermatogonial stem cells to be disease (without using a recipient transplanted back from the surrogate host animal testicle as a surrogate organ) animals to the patients. Thus the recipient Using different techniques frozen/thawed animal testicle can be used to maintain for human testicular germ cells can be transplant- a certain length of time the genome of a ed back to the patient’s testicle without using a patient with oncological disease and after recipient animal testicle as a surrogate organ the end of chemotherapy a larger number as proposed in the previous paragraph. of patients’ spermatogonia cells (compared with the number of spermatogonia that was Eighteen men with unilateral testicular originally transplanted into the recipients) oncological disease underwent unilateral

30 Male infertility today #4 radical orchiectomy in the Department of plantation procedures can be applied for Urology of University of Ioannina. At the preservation of the germ cell line of older time of unilateral orchiectomy germ cells animals unable to breed, economically were isolated from the contralateral healthy valuable animals, and endangered species. testicle. Biopsies from the contralateral tes- Prior to cryopreservation and transplanta- ticle demonstrated absence of neoplasia in tion procedures, a germ cell culture system all patients. Fractions of testicular germ increasing the number of spermatogonia in cells from the healthy testicle were then vitro and will allow freezing of a larger num- frozen. Four out the above 18 patients ber of spermatogonia cells. Then received the same standard chemotherapy frozen/thawed spermatogonia can be trans- protocol (for clinical low-volume stage II ferred to host animal recipients. Thus it is non-seminomatous germ cell tumors) post- possible to maintain and increase further orchiectomy. Six months after the end of the number of donor spermatogonia within the protocol of the administration of the the host animal recipients. Some of the chemotherapeutic drugs (bleomycin, etopo- donor spermatogonia will generate a num- side, cisplatin), all four men were consid- ber of donor spermatozoa within the recipi- ered to be free of oncological disease and ent testicles. These donor spermatozoa can were found to be azoospermic. At that time be recovered and processed for assisted the respective frozen/thawed germ cells reproduction. were transplanted back to the rete testicle of the contralateral to the neoplasia testicle in two patients. Thirteen months post-trans- D. Survival and differentiation of plantation the latter two men showed sperm germ cells from non-obstructed concentration 9x106 /ml, and 4x106 /ml, azoospermic men into recipient respectively, percentage of motile sperma- human individuals (human to human tozoa 13%, and 8%, respectively, and per- transplantation techniques). centage of morphologically normal sperma- Combined secretory dysfunction of tozoa (W.H.O. criteria) equal to 22% and human Sertoli and Leydig cells (for exam- 11%, respectively. In contrast at that time ple due to varicocele) (Sofikitis et al., 1996; the remaining two patients who had Sofikitis et al., 1998a) occasionally results received chemotherapy but did not undergo in azoospermia. Isolation of spermatogonia autotransplantation were still azoospermic. from testicular biopsy material from the lat- It appears that autotransplantation of testic- ter azoospermic men and transplantation ular frozen/thawed germ cells post- into the seminiferous tubuli of men with chemotherapy can result in colonization of proven azoospermia and lack of testicular the human testicle and production of spermatozoa due to Y-chromosome semen samples of sufficient quality for microdeletions may result in production of assisted reproduction technology. donor human spermatozoa by the recipient human testicle. Since as recipient men are chosen individuals with lack of testicular C. Germ cell transplantation spermatozoa and azoospermia due to Y- techniques for preservation of chromosome microdeletions, spermatozoa endangered species. that will appear in the semen post-trans- Cryopreservation and germ cell transplantation are anticipated to be of the donor

Male infertility today #4 31 man origin. An attractive hypothesis is that therapeutic management of the recipient human Sertoli cells and the non-obstructive azoospermia intratubular biochemical environment will (human to animal transplantation support the donor human germ cells to dif- techniques). ferentiate. The above hypothesis is support- A significant percentage of non- ed by experiments in animals demonstrat- obstructed azoospermic men have testicu- ing that intratubular environment from infer- lar foci of active spermatogenesis up to the tile recipients can support differentiation of spermatid or spermatozoon stage (Silbert donor germ cells from infertile subjects et al., 1996; Amer et al., 1997; Silbert et al., (Ogawa et al., 2000). It is also supported by 1997; Antinori et al., 1997; Sofikitis et al., our recent studies showing that there are 1998a; Sofikitis et al., 1998b; Schlegel et no significant differences in androgen-bind- al., 1999). Ooplasmic injections of sperma- ing protein activity in testicular cytosols tozoa (Silbert et al., 1995; Palermo et al., between five azoospermic men with dele- 1998), spermatids (Antinori et al., 1997; tions in the AZFc area of Y-chromosome Amer et al., 1997; Van der zwalmen et al., and 11 obstructed azoospermic men 1997; Sofikitis et al., 1998b) or secondary (androgen-binding protein activity in human spermatocytes (Sofikitis et al., 1998c) testicular cytosols was assayed as we pre- recovered from testicular foci of spermato- viously described; Yamamoto et al., 2002). genesis of non-obstructed azoospermic Thus it appears that the secretory activity of men have resulted in delivery of healthy Sertoli cells of men with Y-chromosome newborns. In contrast, non-obstructed deletions is within “normal” values probably azoospermic men with complete premeiotic indicating that the intratubular environment block in spermatogenesis do not have foci of these men can support donor spermato- of haploid cells in their testicular tissue and genesis. The above described methodology therefore cannot be candidates for assisted may allow azoospermic men without a reproduction programs, nowadays. genetic cause of azoospermia to produce Transplantation of human germ cells into a their own spermatozoa in a recipient host animal testicle may give the opportuni- human testicle with complete arrest in sper- ty to men with premeiotic block (not caused matogenesis due to genetic reasons. by a genetic factor exerting a direct effect GnRH-antagonists may be administered to on germ cells) to produce haploid cells the recipient man prior to transplantation to within a host animal testicle and subse- further disrupt his arrested spermatogene- quently be candidates for ooplasmic injec- sis (i.e., by eliminating any present sper- tions of spermatids or spermatozoa in the matid or spermatocytes and some sper- future. Candidates for such transplantation matogonia). Thus the former men procedures may be non-obstructed (azoospermic men without a genetic cause azoospermic men with premeiotic block due of azoospermia) may be candidates for to acquired testicular damage. assisted reproduction programs. Of course Azoospermic men with an inherent inability several ethical issues should be considered of their germ cells (due to a genetic factor prior to the performance of such tech- affecting directly the germ cells) to undergo niques. meiosis will not have a benefit from such E. The role of human germ cell transplantation procedures. Transplantation transplantation procedures in the techniques into animals represents the only

32 Male infertility today #4 hope for men with incurable complete pre- Considering that the cell nuclear DNA is meiotic block in spermatogenesis due to protected by the nuclear envelop and sur- acquired testicular damage (such as varico- rounded by the respective cell cytoplasm it cele, mumps-orchitis, trauma, and so on) to may be suggested that the nuclear genetic father their own children. material of human spermatids/spermatozoa generated into a recipient testicle has not Considering that testicular foci of been contaminated by the recipient spermatogonia can be found in the vast species-nuclear DNA. However, additional majority of non-obstructed azoospermic studies are necessary to confirm it. men who are negative for haploid cells in Furthermore, the prolonged contact of the therapeutic testicular biopsy material human germ cells with recipient Sertoli (Sofikitis et al., 1998a) it appears that suc- cells /germ cells during human spermato- cessful induction of human meiosis and genesis into the recipient testicle may result spermiogenesis within a recipient animal in binding on the human germ cell cytoplas- testicle has the potential to increase the mic membrane of molecules / antigens that number of non-obstructed azoospermic are characteristic of the recipient species. men who can be candidates for assisted Therefore, subsequent ooplasmic injections reproduction programs in the future. of human spermatozoa or spermatids recovered from animal testicles may lead to the production of human embryos positive F. Assisted reproduction techniques for a recipient antigen molecule that is nor- using human haploid cells generated mally absent in the human species. Finally, into animal testicles; genetic and the presence of a virus within the recipient immunological risks. host testicle may result in infection of the It should be emphasized that application transplanted human germ cells and of the of assisted reproduction procedures using subsequently derived human haploid cells human spermatids or spermatozoa pro- with an overall result transmission of the duced in an animal testicle is susceptible to virus to the human embryonic cells and tis- ethical considerations and genetic, sues after human assisted reproduction immunological, and infection-related risks. techniques.

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Male infertility today #4 37 Shinohara T, Avarbock M, Brinster R. reproductive capacity of the early haploid male Functional analysis of spermatogonial stem gamete. cells in Steel and cryptorchid infertile mouse J Urol 156: 267-270, 1996. models. Sofikitis N, Miyagawa I, Yamamoto Y, Dev Biol 220: 401-411, 2000a. Loutradis D, Mantzavinos T, Tarlatzis V. Shinohara T, Brinster R. Micro- and macro- consequences of ooplasmic Enrichment and transplantation of spermatogo- injections of early haploid male gametes. nial stem cells. Hum Reprod Update 4: 197-212, 1998a. Int J Androl 23(Suppl. 2): 89-91, 2000b. Sofikitis N, Yamamoto Y, Miyagawa I. Shinohara T, Orwig K, Avarbock M, Brin- Ooplasmic elongating spermatid injections for ster RL. the treatment of non-obstructive azoospermia. Spermatogonial stem cell enrichment by multi- Hum Reprod 13: 709-714, 1998b. parameter selection of mouse testis cells. Sofikitis N, Mantzavinos T, Loutradis D, Proc Natl Acad Sci 97: 8346-8351, 2000c. Yamamoto Y, Tarlatzis V, Miyagawa I. Shinohara T, Orwig K, Avarbock M, Brin- Ooplasmic injections of secondary spermato- ster RL. cytes for non-obstructive azoospermia. Remodeling of the postnatal mouse testis is Lancet 351: 1177, 1998c. accompanied by dramatic changes in stem cell Sofikitis N, Mio Y, Yamamoto Y, Miyagawa number and niche accessibility. I. Proc Natl Acad Sci 98: 6186-6191, 2001. Transplantation of human spermatogonia into Sigman M, Lipshultz LI, Howard SS. the seminiferous tubules of animal testicles Evaluation of subfertile male. results in the completion of the human meiosis In `Infertility in the male`, 3rd edition (Editors and the generation of human motile spermato- Lipshultz LI, Howard SS) Mosby, St. Louis, pp. zoa. 173-193, 1997. Presented at The 55th Annual Meeting of the American Society of Reproductive Medicine in Silbert S, van Steirteghem AC, Devroy P. Toronto, Canada, September 25th to 30th,. Sertoli cell only syndrome revisited. Fertil. Steril. (Suppl.), pp. 83-84, 1999. Hum Reprod 10: 1031-1032, 1995. Sofikitis N. Silbert S. Transplantation of human germ cells into Sertoli cell only syndrome. immunosuppressed animal testicles for the Hum Reprod 11: 229-233, 1996. management of non-obstructive azoospermia. Silbert S, Nagy Z, Devroy P, Tournaye H, Presented and published by The American Van Steirteghem AC. Society of Reproductive Medicine, Distribution of spermatogenesis in the testicles Postgraduate Course of ESHRE in Torondo, of azoospermic men: the presence or absence Canada, September 25th to 30th , pp. 41-49, of spermatids in the testes of men with germi- 1999b. nal failure. Sofikitis N, Ono K, Yamamoto Y, Hum Reprod 12: 2422-2428, 1997. Papadopoulos H, Miyagawa I. Smith FF, Tres LL, Kierszenbaum AL. Influence of the male reproductive tract on the Expression of testis specific histone genes dur- reproductive potential of round spermatids ing the development of rat spermatogenic cells abnormally released from the seminiferous in vitro. Develop Dynam 193: 49-57, 1992. epithelium. Hum Reprod 14: 1998-2006, 1999c. Sofikitis N, Miyagawa I, Incze P, Andrighetti S. Sofikitis N, Yamamoto Y, Miyagawa I. Detrimental effect of left varicocele on the Influence of anterior-chamber eye cells, inter-

38 Male infertility today #4 feron-??and caspase-1 inhibitor on the cellular activation still true? immune reactions following transplantation of FEMS Microbiol Immunol 1: 287-292, 1989. spermatogonia. Van Pelt AM & de Rooij D. Hum Reprod 14: 85. Presented at the 15th Synchronization of the seminiferous epithelium Annual Meeting of European Society of Human after vitamin A replacement in vitamine A-defi- Reproduction and Embryology, Tours, France, cient mice. June 26-30, 1999d. Biol Reprod 43: 363-367, 1990. Sofikitis N, Yamamoto Y, Kanakas N, Miyagawa I. Van Schonfeldt V, Krisnamurthy H, Foppi- Two novel methods for germ cell transplanta- ani L, Schlatt S. tion. Magnetic cell sorting as a fast and efficient J Androl 22: 124. Presented at the Annual method of enriching viable spermatogonia from Meeting of the American Society of Andrology, rodent and primate testes. Montreal, Canada, June 15th-19th, 2001. Biol Reprod 61: 582-589, 1999.

Tanaka A, Nagayoshi M, Awata M, Yamamoto Y, Sofikitis N, Miyagawa I. Mawatari Y, Tanaka I, Sofikitis N . Ooplasmic injections of rabbit round spermatid Conclusions from the transplantation of human nuclei or intact round spermatids from fresh, or hamster spermatogonia /primary spermato- cryopreserved and cryostored samples. cytes to rat or mouse testis. Hum Reprod 14: 1506-1515, 1999a. Fertil Steril 68 (Suppl.): S61, 1997. Yamamoto Y, Sofikitis N, Ono K, Kaki T, Tanemura K, Kanai Y, Kanai-Azuma M, Isoyama T, Suzuki N, Miyagawa, I. Kurohmaru M, Kuramoto K, Yazaki K, Postmeiotic modifications of spermatogenic Hayashi Y. cells are accompanied by inhibition of telom- Reinitiation of spermatogonial mitotic differenti- erase activity. ation in inactive old BDF1 mouse seminiferous Urol Res 27: 336-345, 1999b. tubules transplanted into W/W mouse testis. v Yamamoto Y, Sofikitis N, Mio Y, Loutradis Biol Reprod 55: 1237-1242, 1996. D, Kaponis A, Miyagawa I. Tesarik J, Guido M, Mendoza C, Greco E. Morphometric and cytogenetic characteristics Human spermatogenesis in vitro: respective of testicular germ cells and Sertoli cell secreto- effects of follicle stimulating hormone and ry function in men with non-mosaic Klinefelter’s testosterone on meiosis, spermiogenesis, and syndrome. Sertoli cell apoptosis. Hum Reprod 17: 886-896, 2002. J Clin Endocrinol Metab 83: 4467-4473, 1998. Zambrowicz B, Imamoto A, Fiering S, Tesarik J, Bahceci M, Ozcan C, Greco E, Herzenberg LA, Kerr WG, Soriano P. Mendoza C. Disruption of overlapping transcripts in the Restoration of fertility by in vitro spermatogene- ROSA bgeo 26 gene trap strain leads to wide- sis. spread expression of b-galactosidase in mouse Lancet 353: 555-556, 1999. embryos and hematopoietic cells. Van der Zwalmen P, Zech P, Birkenfeld H. Proc Natl Acad Sci 94: 3789-3794, 1997. Intra-cytoplasmic injection of spermatids Zhengwei Y, Wreford NG, Schlatt S, Wein- retrieved from testicular tissue: influence of tes- bauer GF, Nieschlag E, McLaughlan RI. ticular pathology, type of selected spermatids Acute and specific impairment of spermatogo- and oocyte activation. nial development by GnRH antagonist induced Hum Reprod 12: 1203-1213, 1997. gonadotrophin withdrawal in the adult macaque Van Furth R & Van Dissel JT. (Macaca fascicularis). Is the conventional doctrine about macrophage J Reprod Fertil 112: 139-147, 1998.

Male infertility today #4 39

AROMATASE INHIBITORS IN MALE INFERTILITY

Kaan AYDOS Önder YAMAN Department of Urology and Research Center on Infertility, School of Medicine, University of Ankara, Turkey. [email protected]

Since its introduction as an estrogen follicle stimulating hormone (FSH) and suppressor agent, aromatase inhibitors luteinizing hormone (LH). While a wide vari- have extensively been used to treat the ety of effects of FSH on the seminiferous women with breast carcinoma. The employ- epithelium has been described, LH exerts ment of aromatase inhibitors in the treat- its influence on spermatogenesis by stimu- ment of male infertility has been based on lating testosterone secretion from the the theory that increased estrogen produc- Leydig cells (for review see Mak et al., tion or an inappropriate ratio of estrogen to 2000). Although FSH and LH are the key testosterone may have an adverse direct or factors for optimal germ cell survival, their indirect effect on spermatogenesis. In this removal from the circulation either by fashion, aromatase inhibition may improve hypophysectomy or use of gonadotropin- testicular exocrine function by lowering the releasing hormone (GnRH) antagonists serum estradiol-to-testosterone ratio and/or does not prevent entry of stem cells into the increasing FSH. In this article, we first spermatogenic cycle. In fact, the need for describe some of the basic elements of both FSH and testosterone has been hormonal regulation of testicular functions emphasized in the restoration of spermato- and testosterone-estrogen interaction, genesis (Matorras et al., 1997). Receptors which serve as background information for for both hormones can be found on the the ensuing sections. Specific factors affect- Sertoli cells, and testosterone receptors are ing aromatization pathway are described in also found on the peritubular cells detail. Following this, clinical results of aro- (McLachlan et a1., 1994; Mauras et al., matase inhibitors in the treatment of infer- 2000). FSH stimulates the production of an tile men are reviewed. androgen-binding protein (ABP) in the seminiferous epithelium by the Sertoli cell, which acts as a carrier protein for androgen HORMONAL REGULATION within the lumen of the seminiferous OF TESTICULAR FUNCTIONS tubules and within the epididymis. The action of FSH is thus seen as providing a Central regulation and testosterone concentration gradient for testosterone that A large body of evidence available sug- is directed toward the tubular lumen and a gests that for quantitatively normal sper- transport mechanism for androgen to reach matogenesis to occur, the testis requires the developing germ cells and stimulate stimulation by the pituitary gonadotropins- their maturation (Moreno et al., 2000).

Male infertility today #4 41 Thus, the survival of testicular germ cells various studies. When given to embryos of appears to be dependent on gonadotropins emys orbicularis, the nonsteroidal aro- and intratesticular testosterone (Bartke, matase inhibitor letrozole was found to 1995). induce gonads with different degrees of masculinization, from ovary-like to testis- like. There was a strong correlation among Estrogens aromatase activity, gonadal structure, and Mullerian duct status; high levels of aro- Although testosterone is the principal matase were found in ovary-like gonads, sex steroid in testis, the occurrence of however, low levels were found in strongly estrogen synthesis, the existence of abun- masculinized gonads (Richard-Mercier et dance estrogen receptors (ER) in male al., 1995). These results confirm the impor- reproductive tissues (Schleicher et al., tance of estrogens in gonadal differentiation 1984), as well as data from knock out ani- and testicular structure formation as well. mals (Krege et al., 1998) indicate that spermatogenesis regulation is also under the influence of estrogens. Approximately 20% Although it has been known for many of estrogens present in blood are produced years that estrogen administration has by the testis (de Jong FH 1973). Recent deleterious effects on male fertility, data studies have shown that testicular germ from transgenic mice deficient in estrogen cells and sperm, as well as Leydig and receptors or aromatase indicate an essen- Sertoli cells are major cell types that pro- tial physiological role for estrogen in male duce estrogen in the male (Janulis et al., fertility. Data available lead to the interest- 1998). ERalpha and ERbeta genes are ing observation that estrogen receptor func- reported to be coexpressed in germinal and tion is absolutely required for normal sper- nongerminal epithelia of the mature testis matogenesis (Smith et al., 1994) and its of channel catfish and seem to be develop- disruption causes alteration of spermatoge- mentally regulated in spermatocytes. These nesis and infertility (Eddy et al., 1996). observations are, as reportedly, consistent Supportingly, estrogen has been shown to with the concept that estrogens, via interac- regulate the reabsorption of luminal fluid in tion with ERalpha and ERbeta, participate the head of the epididymis and disruption of in the regulation of male gamete develop- this essential function causes sperm to ment and fertility (Bilinska et al., 2000; enter the epididymis diluted, rather than Vecino et al., 2001). Elaborate sperm trans- concentrated, resulting in infertility (Hess et plantation studies have shown that the al., 1997). Additionally, spermatogonial altered sperm function characteristic of the stem cell renewal was promoted by estradi- ERalpha knockout male are the result of ol implantation so that; estrogen is consid- the loss of ERalpha actions in the support- ered as an indispensable “male hormone” ing somatic cells of the testis and epi- in the early spermatogenetic cycle (Miura et didymis rather than in the germ cell (Couse al., 1999). An alternative or complementary et al., 2001). explanation, given the recent identification of estrogen receptors (ERalpha and ERbeta) and aromatase within various cell The implication of estrogens in gonadal types in the testis, is that estrogens also differentiation has further been confirmed in exert paracrine actions within the testis to

42 Male infertility today #4 promote spermatogenesis (Ebling et al., lar testicular level necessary for develop- 2000). ment of spermatogenesis (Carreau et al., 1988). Testosterone secretion in turn regulated by estrogens. Experiments with intact Contrary to the spermatogenesis-regu- and hypophysectomized animals have lating effect of estrogens, elevation of tes- demonstrated that estrogens can inhibit ticular estradiol level has also been report- testosterone secretion by acting directly on ed to be possibly one of the causes of male the testis (for review see Moger, 1980). infertility (Akiyama., 1997). In rats, high doses of estrogen cause tubular damage, disorganization of the cytoarchitecture in Complexity of estrogen-testosterone the seminiferous tubules, vacuolation, interaction has further been proved by a absence of lumen and compartmentaliza- large body of evidences. In cases with ele- tion of spermatogenesis (Leon et al., 1987; vated estradiol-to-testosterone ratio the Gill-Sharma et al., 2001). Likewise, in men suggestion that lowered testosterone is not increase in the rate of aromatase activity a major contributor to impaired spermato- and the concentration of estradiol were genesis was raised based on the investiga- found to correlate with the decreased tion indicating that exogenous testosterone Johnsen’s score count (Ichikawa, 1995). was not capable of restoring declined spermatogonial number in the estradiol-treated rats despite the partial or full restoration of AROMATASE INHIBITION testicular testosterone levels (Meachem et AND TESTICULAR FUNCTIONS al., 1997). However, no evidence was found Intricate relation between estrogens for the involvement of non-androgenic and testosterone Leydig factors in the control of spermatogonial numbers. It is possible that both andro- Normal spermatogenesis depends upon gens and estrogens participate in the regu- the proper interaction of the androgens and lation and maintain of spermatogenesis, estrogens. The data available suggest an and that estrogen deficiency or abundance intricate relation between the testosterone has an impact on the gonad’s output of and estrogen levels in the testis. This rela- sperm. Also, prevention of disruption of tionship may be complex and depend on a spermatogenesis induced by high dose certain ratio between the two hormones. It estrogen, by antiestrogens indicates is well known that estradiol synthesized in crosstalk between androgen and estrogen the testis plays a role in the regulation of receptors in Sertoli cells (Gill-Sharma et al., testicular testosterone production in some 2001). species (Tapanainen et al., 1989). The Leydig cell aromatase activity is, in part, regulated by some certain testicular Aromatase enzyme paracrine factors including estrogens. It is and its functional regulation noteworthy that the Leydig cell testosterone synthesis is highly stimulated by LH and the The biosynthesis of estrogens from addition of seminiferous tubular culture androgens is catalyzed by the terminal medium further increases testosterone out- enzyme aromatase, an enzyme localized in puts which brings about a high intratesticu- the endoplasmic reticulum that consists of

Male infertility today #4 43 two components: a cytochrome P450 (P450 to a lesser extent, the epididymis could aro- Arom, P450 19 product of the CYP19 gene) matize androgens (Pereyra-Martinez et al., and the NADPH cytochrome P450 reduc- 2001). However, in the epididymis a dis- tase. P450 19 is a unique form of the P450 crepancy between the aromatase activity superfamily and also the aromatase has and the mRNA was found pointing out the been supposed to be a good target for the fact that aromatase activity was not strictly development of selective P450 inhibitors regulated at the level of RNA expression (Vanden Bossche et al., 1994). Aromatase and that other mechanisms for this regula- cytochrome P450 is found in gonads as tion should be considered. Physiologic well as adipose tissue and many other tis- studies have shown that aromatization sues (Simpson et al., 1997). Carreau et al. pathway is strikingly dependent on the sur- (2001) have evidenced a positive correla- rounding hormonal milieu in the testis. In tion between a fully developed spermatoge- fact, the Sertoli cell secretes a number of nesis and a strong immunoreactivity for compounds, some of which are postulated both P450arom and ERbeta not only in to serve as chemical signals to the neigh- Sertoli cells but also in pachytene sperma- boring cells (Ritzen, 1983). Interestingly, tocytes and round spermatids. Indeed, aromatase inhibitors consist an example of Leydig cells, Sertoli cells and spermatozoa Sertoli cell secretory products whose pro- have recently been shown to express aro- duction is dependent on the type of sur- matase enzyme as reported in experimen- rounding germ cells. Further, it is known tal studies (Tsai-Morris et al., 1985; Janulis that androgens are the major factor that et al., 1996; Janulis et al., 1998). Human stimulates the production of Sertoli cell spermatozoa, mainly the tail and midpiece inhibin. Sertoli cell aromatase activity that are a potential site of P450-aromatase pro- converts the androgens into the estrogens tein expression and estrogen biosynthesis is also stimulated by FSH (Verhoeven & (Aquila et al., 2002). But the major source Franchimont, 1983). Supportingly, there of circulating estradiol in man is from exist influence of FSH in female gonads for peripheral conversion of androgens to the induction of aromatase enzyme and LH estrogens by the aromatase enzyme in adi- receptors (Erickson et al., 1985). Similarly, pose tissue (Longcope et al., 1978). previous findings demonstrated a direct Nevertheless, the overall data confirm the inhibitory influence of tri-iodothyronine (T3) presence of a functional cytochrome on aromatase activity and estradiol produc- P450arom in the male testis and conse- tion in peripuberal Sertoli cells. In this con- quently, suggest a physiological role for text, a possible role of T3 on the interplay androgens and estrogens in the regulation between testicular steroids and Sertoli cells of spermatogenesis. has been investigated and consequently, thyroid hormone was found to modulate up- regulation of androgen and down-regulation Testicular aromatization has been evalu- of estrogen receptor content in the Sertoli ated extensively (Canick et al., 1979; cells of peripubertal rats, which possibly Dalterio et al., 1983; Ichikawa, 1995). But influences the androgen responsiveness of the mechanism playing role in the regula- the Sertoli cells during spermatogenesis tion of aromatase enzyme activity is more (Panno et al., 1996). complex than known. Indeed, it has been demonstrated that the monkey testis and,

44 Male infertility today #4 These results clearly emphasize the cells (Raeside JI, 1988). Furthermore, importance and complexity of the aromati- transforming growth factor beta 1 (TGF zation pathway interacting with Sertoli cell beta 1) has inhibitory effect on FSH- functions. Otherwise, the role of the aro- induced aromatase activity (Morera et al., matase activity in the peritubular cell-Sertoli 1992). Aromatization is further stimulated cell interactions controlling Sertoli cell func- by activin, alpha-MSH, growth hormone as tion by androgens was further investigated well as FSH but is inhibited by aromatase and testicular peritubular cells were found inhibitor, inhibin, FSHBI (FSH binding to secrete a protein under androgen control inhibitor) (for review see Demoulin & that inhibits induction of aromatase activity Franchimont, 1989; Closset et al., 1991). in Sertoli cells (Verhoeven et al., 1988a). However, the physiologic significance of Coculture with peritubular cells was shown these factors in germ cell-Sertoli cell inter- to increase the sensitivity and/or the action throughout the seminiferous cycle is responsiveness of a number of Sertoli cell unclear. parameters including aromatase activity to androgens. This effect is in part mediated by the secretion of one or more diffusible From the point of the androgen therapy, factors (P-Mod-S) by the peritubular cells aromatase activity is once again important. (Verhoeven et al., 1992). Otherwise, Because testicular androgens may be PModS alone was not found to affect aro- responsible for the decrease in FSH- matase activity at any of the developmental inducible aromatase activity, prolonged stages as examined in rats at various exposure to androgens are expected to stages of pubertal development (Rosselli & suppresses follicle-stimulating hormone- Skinner, 1992). Interestingly, PModS sup- induced aromatase activity in Sertoli cells, press’ the ability of FSH to stimulate aro- as observed in rat trials (Verhoeven et al., matase activity and estrogen production in 1988b). Scaglia & Carrere (1991) demon- midpubertal Sertoli cells. strated that in some oligoasthenoterato- zoospermic or azoospermic patients testicular hormone production is altered. On the other hand, aromatase activity of According to their data, when serum bipha- Leydig cells was also indicated based on sic pattern of hCG-stimulated testosterone the experimental studies demonstrating that was altered, aromatase inhibitors (aminog- Leydig cell tumor had an active lutethimide or testolactone) induced an autonomous aromatase system that is improvement of the acute testosterone accompanied with elevated serum estradiol response only in patients with high or nor- concentrations. So that, although has not mal 17 OH-progesterone response to hCG, been confirmed, in infertile men with Leydig whereas no effects were observed in cell hyperplasia, severely impaired sperm patients with low 17 OH-progesterone production could be resulted from excess response. However, in cases of normal pat- estrogen (Orczyk et al., 1987). Further, the tern of hCG-stimulated testosterone, the effects of fibroblast growth factor (FGF) on aromatase inhibitors induced no changes in testicular aromatase activity was studied the testosterone response. Recently, a previously, and a role has been suggested family with the aromatase excess syndrome for FGF as a paracrine/autocrine agent in is described, in which the condition was the control of estrogen secretion by Leydig inherited in an autosomal dominant man-

Male infertility today #4 45 ner, led to feminizing manifestations in both tumor was shown to be caused by sexes, and was associated with the aber- increased aromatization and in situ estro- rant utilization of a novel transcript of the gen production in Leydig cells of the non- P450arom gene (Stratakis et al., 1998). neoplastic testis and in interstitial or stromal Treatment with an aromatase inhibitor cells of the tumor, as well as increased (testolactone) and a GnRH analog suc- tumor size (Nakazumi et al., 1996). cessfully delayed skeletal and pubertal Increased aromatase activity and impaired development. Afterword, an endocrinopathy androgen receptor functions as well was in men with severe male factor infertility is also encountered in a female patient with identified, that is characterized by a partial androgen insensitivity (Isurugi et al., decreased serum testosterone-to-estradiol 1996). Interestingly, stress seems to exert a ratio (Pavlovich et al., 2001). Another modifying effect upon the aromatase and 5 observation emphasizing this intricate ratio alpha-reductase hypothalamic activity, as is estrogens’ dose-related effects on sper- shown in rats (Tarasenko et al., 1996). matogenesis. Indeed, low doses of estro- Furthermore, excessive or inappropriate gens appear to maintain whilst high doses aromatase expression in adipose fibrob- reversibly disrupt spermatogenesis (Gill- lasts and endometriosis-derived stromal Sharma et al., 2001). Disclosure of these cells, as well as in testicular, hepatic, adre- mechanisms of intratesticular regulation nal and uterine tumors, is associated with permited to discover the aromatase abnormally high circulating estrogen levels inhibitors as a therapeutic agent capable of and/or with increased local estrogen con- correcting certain male factor infertility centrations in these tissues. Elevated estro- cases. Indeed, aromatase inhibition may gen levels will in turn promote the growth of offer potential benefits in the establishment hormone-responsive tissues. Regarding of the estrogen-to-testosterone balance, as these data, a common metabolic abnormal- reported recently (Raman & Schlegel, ity associated with activation of a cyclic 2002). AMP-dependent signaling pathway that gives rise to activation of aromatase in all of the affected tissues is suggested, being a However, aromatization pathway has an common features of excessive aromatase important effect not only in male infertility expression in these disease states (for but also in some other clinical conditions. review see Bulun et al., 1997). Elevated aromatase activation is thought to play a significant role in these diseases. For instance, a study of a large cell calcifying Influence of aromatase inhibition on Sertoli cell tumor of the testis associated regulation of testicular functions with bilateral gynecomastia in an 8-year-old boy postulated that tumoral cells might The theoretic basis of the aromatase stimulate neighboring interstitial cells to dif- inhibition in the treatment of infertile men is ferentiate into Leydig cells and to secrete that increasing the serum testosterone-to- androgens, which in turn might have been estradiol ratio and/or FSH may improve tes- aromatized to estrogens by tumoral cells, ticular exocrine function. Indeed these which results in gynecomastia (Berensztein female hormones or the ratio et al., 1995). Further, impaired spermatoge- androgens/estrogens do play a physiologi- nesis in patients with testicular germ cell cal role (either directly on germ cells or via

46 Male infertility today #4 testicular somatic cells) in the maintenance gonadotropins. In a group of men with a of male gonadal functions and obviously, history of failed fertilization in previous IVF several steps are concerned particularly the attempts, Acosta et al. administered pure spermatid production and the epididymal FSH at least 3 months and observed that sperm maturation (for review see Carreau fertilization rate was significantly improved, et al., 2001). giving 26% term pregnancy rates per transfer (Acosta et al., 1992). Their results emphasized the benefits of systemic FSH One of the proposed effects of aro- administration as an adjunct to assisted matase inhibitors in the treatment of male reproduction in selected cases of severe factor infertility is on FSH secretion. male factor infertility. These results gave Experimental studies showed that there is rise to other studies aimed to stimulate tes- differential regulation of FSH and LH secre- ticular exocrine function by FSH. Although tion in the adult male rat, such that FSH is several studies suggested that patients with more sensitive to the effect of aromatase severe male factor infertility might benefit inhibitor administration (Turner et al., 2000). from FSH in terms of sperm parameters Indeed, treatment with aromatase inhibitors and pregnancy rates (Dirnfeld et al., 2000; resulted in a significant increase in plasma Ashkenazi et al., 1999), other randomized FSH concentrations (Mauras et al., 2000; and placebo-controlled studies did not Trunet et al., 1993). Estrogen is also show any significant improvement of semi- involved in the regulation of FSH secretion nal parameters (Matorras et al., 1997; (Turner et al., 2000). There is a general Kamischke et al., 1998). consensus on the need for FSH in the regulation of spermatogenesis in humans. Various attempts have been made to The mean drawback for hormone thera- induce sperm production in infertile men py in men with idiopathic infertility is with oligozoospermia by administering hMG improper patient selection. In fact, it has or FSH, but the results are still controver- been demonstrated that highly purified FSH sial. Since gonadotropins participate in reg- could be the appropriate treatment for a ulation of normal spermatogenesis and are group of selected patients with oligo- effective in the treatment of hypogonadism zoospermia when FSH plasma level is in (Matsumoto et al., 1986; Mastrogiacomo et the normal range and the testicular tubular al., 1991), they were applied in idiopathic structure is characterized by hyposper- male infertility based on the assumption matogenesis without maturation distur- that increase of gonadotropin concentra- bances (Foresta et al., 2000). Also, the tions may lead to improved function of the presence or absence of testicular FSH seminiferous epithelium with a further stim- receptors is found to be an important pre- ulation of spermatogenesis. The main dictive parameter for the responsiveness to premises of empirical stimulation therapy the FSH treatment (Okuyama et al., 1998). are that there may exist a relative dispro- Further, FSH stimulation in the GnRH test portion between the levels of certain hor- has also been proposed as one of the mones and their target cells, and the methods to select the patients who may radioimmunoassays used to measure benefit from the treatment with pure FSH serum gonadotropins may not accurately (Glander & Kratzsch, 1997). These results reflect levels of biologically active clearly indicate the need of proven criteria

Male infertility today #4 47 being able to discriminate the FSH-respon- increase in FSH concentration. In men der patients from FSH-nonresponder ones. aromatization of testosterone to estrogens was reported to be a prerequisite for the central regulation of gonadotropin secretion Nevertheless, empirical increasement of (Loveland & de Kretser, 1999). In the FSH has not been regarded as useful in the human male, estrogen has dual sites of treatment of all infertile patients since con- negative feedback, acting at the hypothala- ventional semen analysis is a relatively mus to decrease GnRH pulse frequency insufficient tool for the diagnosis of male and at the pituitary to decrease responsive- infertility and determination of the treatment ness to GnRH (Hayes et al., 2000). It has outcome. On the other hand, FSH adminis- been shown that chronic administration of tration has important impacts on the sub- an aromatase inhibitor to intact male dogs cellular organizations of the germ cells, led to an increase in both serum testos- which may not be reflected by standard terone and LH in the presence of complete microscopic semen analyses. Electron inhibition of estrogen production in the microscopic examination has revealed an testes, thus suggesting the aromatizing improved fine architectural pattern, mainly pathway through estrogens being a major involving acrosome, head and chromatin factor in mediating the effects of testos- and middle-piece, in accordance with the terone on the regulation of LH secretion positive changes of functional data (Arnaldi and that the rise in serum testosterone was et al., 2000). Other studies have also a consequence of the elevated serum LH proved the FSH treatment to be effective in (Lunenfeld, 1986). In this fashion, aromati- improving the ultrastructural features of zation of testosterone to estrogen locally semen (Bartoov et al., 1994; Baccetti et al., within the hypothalamus/pituitary gland is 1997). Additionally, FSH administration has responsible for this negative feedback loop been found to prevent apoptosis in a stage- (Turner et al., 2000). specific fashion (Henriksen et al., 1996). Probably, the high efficiency of FSH before IVF in the fertilization rates with no signifi- During the embryonic development, aro- cant changes in seminal parameters may matase inhibitors administered before sexu- be secondary to sperm quality instead of al differentiation of the gonads can induce quantity. sex reversal (Vaillant et al., 2001). In treated females, masculinization of the genital system was characterized by the differenti- In addition to FSH increasing effect of ation of the gonads into a testis or an aromatase inhibitors, lowering of estrogen- ovotestis, and this transdifferentiation to-testosterone ratio is another mechanism occurred all along embryonic and postnatal by which spermatogenesis is expected to development; thus, new testicular be stimulated. Aromatase inhibitors lower cords/tubes were continuously formed while serum estradiol levels in men through the others degenerated. Indeed, studies exam- inhibition of the aromatization of testos- ining the sex-reversing potential of aro- terone to estradiol and androstenedione to matase inhibitors have confirmed the sper- estrone, and does not have any intrinsic matogenesis and spermiogenesis by histo- androgenic or estrogenic activity. Lowering logical examination of the gonads of estrogen, subsequently, induces (Wennstrom et al., 1995). Formation of

48 Male infertility today #4 male-specific structures and regression of porosis, cardiovascular disease, and uro- female primordia are regulated in early genital atrophy). Although in male trials male embryogenesis by testis-determining such inverse effects have not been reported gene (SRY) on the Y chromosome, and even in high doses, further studies are war- directly controls male development through ranted to establish the confident dose- sequence-specific regulation of target response efficacy. genes (Haqq et al., 1993). In mammals, this male-determining gene of the Y chromosome may code for an intrinsic aromatase AROMATASE INHIBITORS IN THE inhibitor. Studies showed the gene’s prod- TREATMENT OF INFERTILE MEN uct had a binding domain that recognizes Clinical trials regulatory elements in the promoter of the aromatase gene. Specifically recognizing Experimental studies suggested that ability of the SRY DNA-binding domain the specific inhibitors of estrogen biosynthesis proximal upstream elements in the promot- might be suitable to some extent for the ers of the sex-specific genes encoding treatment of estrogen-induced impairment P450 aromatase was presented in other of spermato- and spermiogenesis studies (Haqq et al., 1993). (Leschber et al., 1989). In a pioneer study, despite nondetectable amounts of intratesticular estradiol and estrone and higher Consequently, for the cellular physiology amounts of testosterone, androstenedione of sex steroid sensitive cells, the andro- and dihydrotestosterone than testes of con- gen/estrogen ratio may be more important trol dogs were obtained by chronic treat- than only one hormone action per se (for ment of male dogs with an orally active review see Seralini & Mostemi, 2001). This nonsteroidal aromatase inhibitor, no evi- ratio is controlled in vertebrates by aro- dence on any effect of aromatase inhibition matase; its gene expression can be inhibit- upon spermatogenesis was reported ed in different ways, and this is crucial for (Juniewicz et al., 1988). Further, Turner et the treatment of estrogen-dependent dis- al evaluated the effect of anastrozole on eases such as in male infertility for testicular histology and found that the instance. To reach this goal, non-steroidal majority of treated rats had testes with nor- inhibitors have recently been used as first mal spermatogenesis but, occasionally, line agents. So that, testosterone-to-estro- seminiferous tubules showed abnormal loss gen ratio may be a satisfactory selection of germ cells or contained only Sertoli cells criterion, as indicated previously, to predict (Turner et al., 2000). Because no significant the possible outcome of the aromatase inhi- change in the plasma estradiol concentra- bition treatment. Nevertheless, aromatase tions was observed during the treatment inhibitors can modulate the estrogenic bal- period, excess serum estrogens may be ance essential for male gonadal function supposed having significant pathological and available clinical data suggest that they effects on testes. In fact, increase of the may be a useful tool with which to amelio- rate of testicular aromatase activity and the rate the semen parameters. However, com- following elevation of testicular estradiol plete suppression of aromatase enzyme level has been proposed as one of the pos- may have adverse effects, as is evident in sible causes of male infertility (Akiyama, postmenopausal women (increased osteo- 1997), and with anastrozole treatment a

Male infertility today #4 49 decrease in estrogen-to-testosterone ratio be safe and well-tolerated medication and was obtained in association with increased subsequently has been tried in men with semen parameters (Raman & Schlegel, idiopathic infertility. 2002). However, whether the amelioration in spermatogenesis is a result of local suppression of intratesticular estrogen-to- Testolactone has long been used in testosterone ratio or systematic normaliza- advanced female mammary cancer. tion of aromatase activity is a subject of Pharmacological properties were well docu- continued debate. Nevertheless, in these mented in both animals and humans. cases inappropriate ratio of estrogen to Clinical implications of testolactone treat- testosterone is suggested to result in the ment have been evaluated in various condi- pathophysiology of impaired spermatogen- tions related with hormonal disturbances, esis. such as carcinoma of the pancreas (Waddell, 1973), uterine sarcoma (Tseng et al., 1986), pubertal gynaecomastia In several studies, alterations of sper- (Zachmann et al., 1986), benign prostatic matogenesis have been evaluated when hyperplasia (Schweikert et al., 1987), pre- serum estradiol levels were decreased by cocious puberty (Wheeler & Styne, 1990), suppression of aromatase activity and epilepsy (reviewed by Herzog et al., 1991), many suggested a beneficial effect on familial adenomatous polyposis (Tsukada sperm parameters and hormonal profiles in et al., 1992), congenital adrenal hyperpla- men with normal gonadotropins and idio- sia (Laue et al., 1996), and male fertility pathic oligozoospermia. However, inhibitors disturbances as comprehensively docu- of this enzyme with more favorable proper- mented below. It is one of the pioneer aro- ties had been necessary since the most matase inhibitors that has been studied in extensively utilized inhibitor, aminog- men with impaired spermatogenesis. When lutethimide, lacks specificity and causes used 500 mg twice daily for 4 weeks, in frequent side effects. Data available sug- patients with idiopathic oligospermia low- gest that nonsteroidal compounds have ered circulating estradiol levels by about more favorable properties as a specific and 30%, enhanced the secretion of follicle- potent aromatase inhibitor. stimulating hormone, and testosterone (+ 30%), but did not affect serum luteinizing hormone levels (Dony et al., 1985). They are generally described as type 1 inhibitors which are steroidal compounds (testolactone and aminoglutethimide) that Letrozole (CGS 20267) as well, had pre- may be purely competitive inhibitors, or viously been tried in male rats and dose- type 2 inhibitors which are non-steroidal dependent suppression of serum estradiol (anastrozole, letrozole and exemestane) and an increase in serum testosterone and and bind to the haem group of the aro- LH were seen (Bhatnagar et al., 1992). In matase enzyme by co-ordination fashion men, the systemic and subjective tolerabili- through a basic nitrogen atom (for review ty was shown to be good at single doses see Dowsett, 1999). They were originally ranging from 0.02-30 mg (Trunet et al., used in the treatment of women with 1993). Further, when used in boys with con- metastatic breast carcinoma and found to stitutional delay of puberty, letrozole inhibit-

50 Male infertility today #4 ed estrogen synthesis and delayed bone tion (Plourde et al., 1994; Dukes et al., maturation (Wickman & Dunkel, 2001). In 1996). another study, 35 boys who were referred to because of suspicion of delayed puberty were treated by letrozole-plus testosterone Until now, anastrozole has been used in for 5 months, and significant inhibition of male subjects for various experimental and endogenous estrogen synthesis and clinical purposes. Initially, anastrozole was increased LH concentration as well were administered to male canine and when seen (Wickman & Dunkel, 2001). Although given with a 5-alpha-reductase a significant majority of the data about letrozole have increase in both prostate and testicular vol- been obtained from the female studies, ume were observed (Suzuki et al., 1998). there are marked gender differences con- Then, Turner et al. (2000) evaluated the cerning pharmacokinetics, at least as seen effects of anastrozole on male reproductive in rats (Liu et al., 2000). Plasma concentra- function in adult rats. In addition to plasma tion in female rats is about 7.4-fold of that FSH and testosterone concentrations, testis of male rats probably due to estimated ter- weight also increased significantly. As stat- minal phase half-live differences being 10.5 ed previously, histological evaluation of the in males and 40.4 h in females. testes revealed that spermatogenesis was Metabolically, letrozole absorption decreas- grossly normal. The metabolic effects of es slightly under fed conditions, however, in selective estrogen suppression in the male view of the half-life of about 2 d in human using anastrozole, was further investigated this small change in the absorption rate is by Mauras et al. (2002) and 50% decrease not considered to be of clinical relevance in estrogen concentrations with 1 mg daily for treatment with repeated administrations doses was obtained. After 10 days of treat- (Sioufi et al., 1997). ment, there were no significant changes in body composition (body mass index, fat mass, and fat-free mass) or in rates of pro- Anastrozole, which is another potent tein synthesis or degradation, oxidation, and highly selective aromatase inhibitor, muscle strength, calcium kinetics, or bone has also long been employed in the treat- growth factors concentrations. Schnorr et ment of postmenopausal women with al. (2001) used oral anastrozole (loading advanced breast cancer. In experimental dose of 30 mg on the first day, and then 5 studies, anastrozole was found to be selec- mg twice daily) for 5 days in healthy young tive for the aromatase enzyme, having no men; and in summary, anastrozole abol- detectable pharmacologic activity other ished testosterone-dependent inhibition of than aromatase inhibition, and did not inter- GnRH-stimulated LH and FSH release. fere with steroid hormones produced by the Subsequently, Hayes et al. (2002) used 10 adrenal glands. Also in human studies, mg/day anastrozole for 7 days in normal anastrozole had no clinically significant and idiopathic hypogonadotropic hypogo- effects on key enzymes that regulate corti- nadic men and 41% of suppression in sol and aldosterone biosynthesis. 1 to 10 serum estrogen was obtained. However, in mg doses suppress estradiol to the maxi- this study no clinical finding was reported. mum degree measurable. Its absorption is Interestingly, Faglia et al. (2002) treated a rapid and maximum plasma concentrations boy previously operated on for a hamar- occur within 2 hours after oral administra- toma causing precocious puberty and pre-

Male infertility today #4 51 senting with advanced bone maturation and with idiopathic oligozoospermia and report- nearly fused epiphyseal cartilages, with 1 ed a dramatic decrease in the estrogen-to- mg/day anastrozole for 3 yr. Treatment testosterone ratio. Basal and LHRH-stimu- delayed the epiphyseal closure and lated serum gonadotropin levels were unaf- improved the final height. In another study, fected by these changes. Sperm density fifteen men over 65 yr were treated for 9 rose from 10.8 ± 2.5 to 19.8 ± 4.7 x 106/ml weeks with 2.0 mg/day of anastrozole, and and total sperm count from 26.8 ± 6.5 to the rate of bone resorption secondary to 60.6 ± 14.3 x 106. There was no significant estrogen depression was shown to be limit- change in motility. 30% of the wives ed by endogenous estrogen derived from became pregnant. Their data suggested aromatization of testosterone (Taxel et al., that testolactone might be an effective 2001). Further, to assess whether or not treatment for men with idiopathic oligo- anastrozole has any anticoagulant activity, zoospermia and it’s lowering effect of estro- 16 men were given 7 mg of anastrozole gen levels might had been responsible for loading dose on day 1, followed by 1 mg the improvement in spermatogenesis. daily for 10 day. Overall, anastrozole was These ensuring clinical data resulted in the found having no effect on clotting mecha- successful induction of testicular functions nisms or on the pharmacodynamic activity using aromatase inhibitors. of warfarin (Yates et al., 2001). Anastrozole was also tried in men with prostat cancer refractory to androgen-related therapies, Dony et al. (1986) showed a gradually but no patient experienced an objective rise in sperm density from 8.1 ± 1.3 before 6 response or disease stabilization (Santen to 21.3 ± 6.7 X 10 /ml after 6 months of et al., 2001). testolactone treatment, whereas the total sperm count almost threefold increased. Sperm concentrations exceeding 20 X 6 Treatment results in infertile men 10 /ml were achieved in 4 of the 9 patients. Two of these patients’ wives became preg- All the data available have confirmed the nant. The estradiol decrease was accompa- tolerability and safety of aromatase nied by a temporary increase in the levels inhibitors in male gender. Also taking into of FSH, not of LH, but less of testosterone. consideration the role of the estrogens and The testosterone/estradiol ratio almost dou- FSH in the regulation of testicular functions, bled until the end of the treatment period. further attempts were undertaken to treat male factor infertility by this kind of drugs. It was hoped that suppression of estrogen as In a subsequent study, Schill et al. well as increased testosterone would (1987) reported 17% pregnancy rate after induce the spermatogenesis observed treating men with idiopathic oligozoosper- when these patients are treated with an mia for 3 to 6 months with 1 mg testolac- aromatase inhibitor. Although based on tone daily. A significant increase in the seminal parameters there seem to be strik- sperm count, total sperm output and the ing ameliorations, little information is avail- absolute number of motile and progressive- able on pregnancy rates. In a pilot study ly motile spermatozoa was observed. There with the aromatase inhibitor testolactone, was a significant increase of testosterone Vigersky & Glass (1981) treated ten men and FSH after 1 and 3 months of treatment,

52 Male infertility today #4 while the increase in testosterone/estradiol infertile men were found to have testos- ratio was more than twice. terone-to-estradiol ratios in the lowest 20th percentile of those of normal subjects. Aromatase inhibitor testolactone was effec- Maier & Hienert (1988) used 150 mg tive in increasing serum testosterone testolactone/day in combination with 30 mg and/or lowering serum estradiol in all treat- tamoxifen/day and compared the results ed men. Furthermore, oligozoospermic with those of only 30 mg tamoxifen/day. The men with abnormally low ratio had signifi- increase in sperm density was significant in cant increases in sperm concentration and both groups, but no improvement of the motility while on testolactone. Thus, other ejaculate parameters was seen in authors’ data suggested that alteration of either groups. The incidence of gravidity the hormonal milieu in these men with was the same being 15%. This study con- testolactone was responsible for increased firmed that testolactone prevents elevation sperm production. As these results were of estradiol levels during monotherapy with indicative of an endocrinopathy character- tamoxifen. ized by a decreased serum testosterone-to- estradiol ratio, in a further study a group of infertile men with this condition were treat- Itoh et al. (1991) suggested that an aro- ed with two different aromatase inhibitors, matase inhibitor might be effective to male anastrozole and testolactone (Raman & infertile patients with high serum estradiol Schlegel, 2002). Both medications revealed levels. In that study nine male infertile an increase in testosterone-to-estradiol patients were treated with testolactone for 3 ratio in association with increased semen months. Four of them had an increase in parameters. So, the introduction of testos- sperm count (more than 10 x 106/ml rela- terone-to-estradiol ratio monitoring in infer- tive to base line). Serum estradiol levels tile men may be expected to add safety as and estradiol/free testosterone ratio were well as effectiveness for treatment with aro- significantly decreased after treatment. In matase inhibitors in achieving a high preg- particular four patients whose sperm counts nancy rate in their wives. were improved after testolactone treatment had high values of basal serum estradiol levels and estradiol/free testosterone ratio Regarding these promising results, in before treatment, and these values were order to evaluate the role of aromatase inhi- normalized after treatment. Improvement in bition in spermatogenesis and germ cell seminal parameters seemed to be due to maturation, we analyzed the abnormal the testolactone’s aromatase inhibition retention of residual sperm cytoplasm in effect. infertile men before and after anastrozole administration. 64 patients with different degrees of oligoasthenoteratozoospermia In a more recent work, a subset of infer- were included in the study. According to our tile men with severe infertility who had sig- preliminary unpublished data, at the end of nificantly lower testosterone and higher the 3 months, significant increase in serum estradiol than fertile control reference sub- FSH, LH and testosterone levels accompa- jects were identified (Pavlovich et al., nied by significantly suppressed estradiol 2001). According to the results, 71% of were seen (Table 1).

Male infertility today #4 53 Table 1: Serum hormone values before the treatment and at the end of treatment

Before 12 weeks after Mean treatment treatment difference 95% CI

FSH (IU/l) 4.7 ± 0.9 10 ± 3.3 5.3 ± 3 4.5-6.1

LH (IU/l) 3.8 ± 0.6 7.9 ± 2.2 4 ± 2 3.5-4.6

Testosteron 14.2 ± 5.2 25 ± 8 11 ± 6.4 9.4-12.6 (nmol/l)

Oestradiol 59 ± 3 36.7 ± 9 22 ± 8.8 20-24.4 (pmol/l)

p<0.001

Significant improvements of basic sperm consistent with the possibility that aromatase parameters, including concentration, pro- inhibition’s lowering of serum estrogen levels gressive sperm motility and morphology as well as increased FSH, LH and testos- were also found (Table 2). Moreover, the terone, may have been responsible for the mean percentage of sperm with retained improvement in spermatogenesis. cytoplasm, a morphologic characteristic of impaired spermatozoa maturation, was significantly lower after the treatment than Although uncontrolled studies showed a baseline values (Figure 1). positive response to aromatase inhibitors, in a randomized, placebo-controlled double- blind crossover trial sperm output and Although our data do not clearly eluci- semen quality remained unchanged during date the mechanism of this effect, they are either testolactone or placebo treatment,

Table 2. Semen parameters before and at the end of the treatment.

12 weeks Before after Mean treatment treatment difference 95% CI Ejaculate volume (ml) 3.1 ± 0.7 3.2 ± 0.8 0.1 ± 0.5 0.2-5.7 Sperm concentration 26 ± 7.5 36 ± 10 9.9 ± 7.4 8.0-11.7 (106/ml) Sperm motility (%) 29 ± 6.9 37 ± 10 8.7 ± 7.3 6.8-10.5 (grade a + b) Normal forms (%) 18 ± 4.9 33 ± 7.4 15 ± 6.8 13.4-16.8 Spermatozoa with RC* (%) 14 ± 3.6 6.6 ± 4.6 7.3 ± 4.1 6.3-8.3 *RC = retained cytoplasm. p<0.001

54 Male infertility today #4 Figure 1. Graphical representation of baseline and at the end of treatment percentage of spermatozoa with retained cytoplasm.

20 18 16 14 12 10 8 6 % spermatozoa with con RC* 4 2 0 Baseline 3 months after treatment and no pregnancy occurred during the 16- In conclusion, most clinical trials with month study (Clark & Sherins, 1989). aromatase inhibitors have in fact resulted in Contrary to the usual reports, however, a tendency to improved seminal parame- serum estradiol and testosterone levels dur- ters. It is thought that this improvement is ing testolactone exposure did not change dependent on suppression of estrogen-to- from basal and placebo values. testosterone ratio, with an associated Nevertheless, these data have been inter- increase of FSH. However, placebo-con- preted to indicate that chronic administration trolled, double-blind, randomized additional of testolactone fails to maintain aromatase studies are needed to determine if there is inhibition and is not efficacious in the treat- a subpopulation of infertile men who will ment of idiopathic oligozoospermic infertility. benefit from aromatase inhibitors.

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60 Male infertility today #4 ment of hypophyseal gonadotropik function in 8, 2001. male rats Fiziol Zh Im I M Sechenova 82: 39- Verhoeven G & Cailleau J. 45, 1996. Testicular peritubular cells secrete a protein Taxel P, Kennedy DG, Fall PM, Willard AK, under androgen control that inhibits induction of Clive JM, Raisz LG. aromatase activity in Sertoli cells. Endocrinolo- The effect of aromatase inhibition on sex gy 123: 2100-10, 1988a. steroids, gonadotropins, and markers of bone Verhoeven G & Cailleau J. turnover in older men. J Clin Endocrinol Metab Prolonged exposure to androgens suppresses 86: 2869-74, 2001. follicle-stimulating hormone-induced aromatase Trunet PF, Mueller P, Bhatnagar AS, Dick- activity in rat Sertoli cell cultures. Mol Cell es I, Monnet G, White G. Endocrinol 57: 61-7, 1988b. Open dose-finding study of a new potent and Verhoeven G & Franchimont P. selective nonsteroidal aromatase inhibitor, CGS Regulation of inhibin secretion by Sertoli cell- 20 267, in healthy male subjects. J Clin enriched cultures. Acta Endocrinol (Copenh) Endocrinol Metab 77: 319-23, 1993. 102: 136-43, 1983. Tsai-Morris CH, Aquilano DR, Dufau ML. Verhoeven G, Swinnen K, Cailleau J, Cellular localization of rat testicular aromatase Deboel L, Rombauts L, Heyns W. activity during development. Endocrinology The role of cell-cell interactions in androgen 116: 38-46, 1985. action. J Steroid Biochem Mol Biol 41: 487-94, Tseng L, Tseng JK, Mann WJ, Chumas 1992. JC, Stone ML, Mazella J, Sun B, Amalfitano Vigersky RA & Glass AR. TG, Wallach RC. Effects of delta 1-testolactone on the pituitary- Endocrine aspects of human uterine sarcoma: testicular axis in oligospermic men. J Clin a preliminary study. Am J Obstet Gynecol 155: Endocrinol Metab 52: 897-902, 1981. 95-101, 1986. Waddell WR. Tsukada K, Church JM, Jagelman DG, Chemotherapy for carcinoma of the pancreas. Fazio VW, McGannon E, George CR, Surgery 74: 420-9, 1973. Schroeder T, Lavery I, Oakley J. Noncytotoxic drug therapy for intra-abdominal Wennstrom KL & Crews D. desmoid tumor in patients with familial adeno- Making males from females: the effects of aro- matous polyposis. Dis Colon Rectum 35: 29-33, matase inhibitors on a parthenogenetic species 1992. of whiptail lizard. Gen Comp Endocrinol 99: 316-22, 1995. Turner KJ, Morley M, Atanassova N, Swanston ID, Sharpe RM. Wheeler MD & Styne DM. Effect of chronic administration of an aro- Diagnosis and management of precocious matase inhibitor to adult male rats on pituitary puberty. Pediatr Clin North Am 37: 1255-71, and testicular function and fertility. J Endocrinol 1990. 164: 225-38, 2000. Wickman S & Dunkel L. Vanden Bossche HV, Moereels H, Koy- Inhibition of P450 aromatase enhances mans LM. gonadotropin secretion in early and midpuber- Aromatase inhibitors—mechanisms for non- tal boys: evidence for a pituitary site of action of steroidal inhibitors. Breast Cancer Res Treat 30: endogenous E. J Clin Endocrinol Metab 86: 43-55, 1994. 4887-94, 2001. Vecino P, Uranga JA, Arechaga J. Wickman S, Sipila I, Ankarberg-Lindgren Suppression of spermatogenesis for cell trans- C, Norjavaara E, Dunkel L. plantation in adult mice. Protoplasma 217:191- A specific aromatase inhibitor and potential

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62 Male infertility today #4 MAMMALIAN TESTICULAR DESCENT AND MALDESCENT; IMPLICATIONS IN FERTILITY POTENTIAL

Charalampos Mamoulakis, Apostolos Kaponis, John Georgiou, Dimitrios Giannakis Spyros Antypas, Stavros Tsambalas, Xenophon Giannakopoulos, Ikuo Miyagawa and Nikolaos Sofikitis

Laboratory of Molecular Urology and Genetics of Human Reproduction, Department of Urology, Ioannina University, Ioannina, Greece

Running head: Undescended testis in mammals Key words: Testicular maldescent, spermatogenesis, hormones, mammals

ABSTRACT (TMD) are controversial. However, there is a general agreement on the seasonality of Mammalian male sex determination is TMD at least in the northern hemisphere. an active process involving complex inter- Epididymal malformations, impaired testicu- actions among several genes. SRY and lar histology due to intrinsic testicular SOX9 are both responsible for testis forma- defects, mild hypogonadal state or tion by initializing and maintaining respec- increased germ cell apoptotic rate mainly tively Sertoli cell differentiation. Male sexual due to abnormal testicular temperature may differentiation is governed by testicular hor- account for impaired fertility in TMD. The mones. Testicular descent (TD) and scrotal theory of an intrinsic testicular pathologic evolution occurs exclusively but not univer- process might plausibly explain the associ- sally in mammals. Although still debatable, ation with testicular cancer. Management of this evolutionarily costly process aims at human TMD aims to preserve future fertility. least to secure lower than core body testic- Surgery is currently the only effective ular temperatures essential for viable measure to bring the testis into the scro- sperm production and storage. TD in scrotal tum. It should take place before the first mammals is a multistaged process involv- year. Hormonal treatment may serve as an ing interplay of several anatomical struc- ancillary treatment to stimulate germ cell tures and hormonal factors. The gubernac- maturation and improve long term results. ulum seems to posess a key role, especially during transabdominal TD (TTD). PHYSIOLOGY OF MAMMALIAN SEX Androgens and Mullerian inhibiting sub- DETERMINATION AND SEXUAL stance have a rather limited, if any, role dur- DIFFERENTIATION IN THE MALE ing TTD. Leydig cell-derived insulin-like 3 During mammalian embryogenesis each hormone acting directly upon the gubernac- individual, regardless of sex chromosome ulum and homeobox genes represent good genotype, has the potential to develop both candidate controllers of TTD. Inguinoscrotal male and female reproductive systems. In TD is mediated by androgens possibly act- order a normal male or female development ing indirectly upon the gubernaculum, in to occur, a selective mechanism is required conjunction with mechanical (abdominal to ensure the development of only one of pressure) factors. Reports on secular the two genital systems while the other trends of human testicular maldescent must regress. Development of the male

Male infertility today #4 63 phenotype is dependent by the presence or either side of the embryo filling much of the absence of the Y chromosome inherited by coelomic or body cavity. During develop- the father at fertilization (genetic sex estab- ment it forms into three structures which lishment of the mammalian embryo at the develop anteroposteriorly along the time of fusion of the gametes) and can be Wolffian duct: pronephros, mesonephros divided into two distinct stages: sex deter- and metanephros. The pronephros is ves- mination and sexual differentiation. The tigial in mammals but mesonephros can sex-determining process is set in motion serve as a primitive kidney during embryo- only during the period of organogenesis genesis in some species. The definitive kid- when the gonads develop within the devel- ney is the product of the interaction oping urogenital system resulting in either between the metanephric mesenchyme at ovary or testis formation, whereas sexual the posterior end of the urogenital system differentiation (controlled by the presence and the ureteric bud, which grows out of the or absence of hormones produced by the Wolffian duct. The Mullerian duct originates gonads during fetal development) is the within each mesonephros by vagination of subsequent process that ultimately results the coelomic epithelium. It runs parallel to in either the female or the male phenotype. the Wolffian duct but turns toward the mid- line at the posterior end of the THE GENITAL SYSTEM DURING THE mesonephros and fuses with the compan- PRE-SEX DETERMINATION PERIOD ion duct. The Wolffian duct produces Prior to sex determination the mam- branching tubules, which extend through malian gonadal anlage is formed as a bipo- the mesonephros towards the coelomic tential primordium (derived from the inter- epithelium. Cells in the coelomic epithelium mediate mesoderm) with the capacity to dif- proliferate, delaminate and join the gonadal ferentiate into either testes or ovaries cell population, accumulating on the sur- depending on the presence of the Sry gene face of the mesonephros. During the bipo- (sex-determining region on Y; proximal to tential stage (4th-6th gestational week in the pseudoautosomal region on the short human (GW)), primordial germ cells arm). Knockout experiments in mice have derived from the extraembryonic mesoderm implicated five genes in the formation or at the base of the allantois (caudal wall of survival of the gonadal primordium: Wt1, the embryonic yolk sac) (4th GW) migrate Sf1, Lhx1 (Lim1), Lhx9, and Emx2 via the hindgut and mesonephros and pop- (Koopman, 2001). During the initial stage of ulate the gonads (5th-6th GW) which arise sexual development (indifferent or bipoten- as a thickening of the ventrolateral surface tial stage), genetically XX and XY embryos of each mesonephros and are first visible at both develop two pairs of genital ducts, ~10 days post-coitum in the mouse (dpc). starting with the male or mesonephric or This process causes mesonephros to bulge Wolffian ducts (which have the potential to forming the genital ridges composed there- differentiate into the vas deferens, epid- fore of somatic cells derived from edymides, seminal vesicles and ejaculatory mesonephros and the primordial germ cells ducts), followed by the female or para- that have migrated and populated the indif- mesonephric or Mullerian ducts (which ferent gonad (Ginsburg et al., 1990). Germ have the potential to differentiate into the cells differentiate into gonocytes upon oviducts, uterus, and upper vagina). entering the testicular cords (see below) to The intermediate mesoderm is found on become fetal spermatogonia by 15th GW.

64 Male infertility today #4 MOLECULAR BASIS OF MALE SEX in experiments with transgenic mice DETERMINING PROCESS (Koopman et al., 1991; Vidal et al., 2001) and in mutational analysis of mice and The gonad initially develops in a non- humans with sex reversal (Foster et al., specific manner, being morphologically 1994; Gubbay et al., 1992; Hawkins et al., identical up to ~12 dpc, and the 7th GW in 1992; Wagner et al., 1994). Almost all SRY XX or XY mouse and human embryos mutations responsible for 46, XY sex rever- respectively. In normal XY embryos, sal cluster in the HMG domain (Harley et expression of the Y chromosome gene Sry al., 1992) which represents therefore the in somatic cells (pre-Sertoli cells) (Hacker most critical part of the SRY protein. Thus, et al., 1995; Jeske et al., 1995) of the geni- SRY and SOX9 proteins are both neces- tal ridge at the time of male sex determina- sary and sufficient for male sex determination initiates the differentiation of Sertoli tion in humans and mice. However, their cells (Lovell Badge et al., 2002) and deter- requirement and functions might not be mines the fate of the indifferent gonad to conserved in all mammalian species testis rather than ovary (Gubbay et al., (Bowles et al., 2001; Nagai, 2001). All 1990; Sinclair et al., 1990; Palmer & species of both subclasses of the Burgoyne, 1991). The human SRY gene Mammalian class (Simpson’s classification) encodes a DNA-binding motif, referred to (Novacek, 1992) possess SRY gene as the high mobility group (HMG), which is homologs (Foster et al., 1992; Gubbay et relatively conserved among different mam- al., 1990; Sinclair et al., 1990): Prototheria malian species unlike the rest of the protein (Monotremata: duck-billed platypus and (Tucker & Lundrigan, 1993; Whitfield et al., spiny ant-eater or echidna), and Theria 1993). More than 100 genes have been (Metatheria: Marsupialia: opossums, kanga- identified containing this domain referred to roos etc and Eutheria or placental mam- as an (HMG) box, and those with the most mals). However, SRY gene homologs are extensive homology (>60%) to the SRYH- absent from two vole species and the spiny MG box gene are collectively termed SOX rat (Just et al., 1995; Soullier et al., 1998). (SRY-type HMG box) genes. The transient Therefore, SRY is not probably the original nature of Sry expression in the genital ridge mammal sex-determining gene that defined of the mouse (~1.5 days) suggests that Sry the Y chromosome and can be replaced as acts as a switch toward Sertoli cell fate a trigger and get lost in the future, as have rather than involved in the maintenance of many other Y-borne genes in recent evolu- cell identity or function. Therefore, Sry must tionary history (Marshall Graves, 2002). in some way activate other genes that are Although the SRY and SOX9 genes involved in defining and maintaining Sertoli appear to be primarily responsible for male cell identity. The autosomal gene Sox9 is a sex determination, the regulation of their strong candidate for this type of down- expression as well as their mode of function stream gene because it is upregulated in still remain elusive. The expression of SRY Sertoli cells just after Sry expression (De in the gonad is followed by complex interac- Santa Barbara et al., 2000) (~11.5 dpc) and tions involving activation and repression of persists throughout life (Kent et al., 1996; other male-specific genes, cell migration Morais da Silva et al., 1996). The impor- and cell proliferation in the developing male tance of both SRY and SOX9 proteins in gonad (Clarkson & Harley, 2002). It has sex determination has been substantiated been proposed (Hammes et al., 2001) that

Male infertility today #4 65 the Wt1 gene must have a role in the acti- lar changes in the developing male gonad vation or transcript stability of Sry (+KTS through the activation of the intracellular isoforms of Wt1 gene; specification of male signaling molecule fibroblast growth factor 9 gonad formation). (-KTS) isoforms of Wt1 (Fgf9) (Colvin et al., 2001). On the other gene are also required for the development hand, SOX9 appears to be primarily neces- of both the male and female gonad but they sary for the activation of AMH/MIS gene in are not essential for sex determination conjunction with other genes such as SF1 (Clarkson & Harley, 2002). The (+KTS) and and Wt1 (-KTS), GATA4 and HSP70 (–KTS) isoforms arise from an alternative (Arango et al., 1999; Clarkson & Harley, splicing event that incorporates or omits 2002; Swain & Lovell Badge, 1999). respectively three amino acids-Lys, Thr, STRUCTURAL ORGANIZATION OF Ser-between zink fingers 3 and 4 (Hastie, THE TESTIS, HORMONE 2001; Little et al., 1999). It has been sug- PRODUCTION, AND MALE SEXUAL gested that SRY acting as a transcription DIFFERENTIATION factor antagonizes the function of repres- sors of male development (anti-testis Once Sertoli cell differentiation is trig- genes) such as WNT4 and DAX1 (Jordan gered by SRY the fate of the gonad has et al., 2001; Swain et al., 1998). WNT4 is been decided, a number of genes associat- known to be involved in gonad differentia- ed with these cells, such as the Desert tion, however, it remains unclear which hedgehog (Dhh) and the Mis, become acti- gene(s) is directly downstream of its signal. vated (11.5 and 12 dpc respectivelly) It is likely that DAX1 gene transcription is (Bitgood et al., 1996; Munsterberg & Lovell upregulated by WNT4 (Suzuki et al., 2002). Badge, 1991) and a number of events fol- DAX1 (an orphan nuclear receptor) may low leading to the structural/functional antagonize the function of SRY by several organization of the testis and finally the ways (Clarkson & Harley, 2002): indirect sexual differentiation (Swain & Lovell inhibition of SRY action by binding to anoth- Badge, 1999). The testis starts organizing er orphan nuclear receptor, SF1 (Ito et al., into two distinct compartments: the testicu- 1997), which is involved in the activation of lar cords (progenitors of the seminiferous male-specific genes such as the anti- tubules) and the interstitial region. Initially, Mullerian hormone gene/Mulerian inhibiting Sertoli cells form clusters around germ substance gene (AMH/MIS) (Arango et al., cells and produce, as yet, unknown 1999; de Santa Barbara et al., 1998), direct signal(s) that trigger the movement of cells competition with SRY for similar DNA struc- from the mesonephros to the gonad which tures involved in the regulation of male-spe- give rise to the endothelial and peritubular cific genes expression (inhibition of SRY- myoid cells that surround these clusters induced SOX9 activation), antagonization of (cords formation) (Buehr et al., 1993; WT1 (+KTS) function. Sry is required for Martineau et al., 1997; Merchant Larios et proliferation of the coelomic epithelium al., 1993). The peritubular myoid cells (Sertoli precursors) (Schmahl et al., 2000) deposit a basal lamina in collaboration with and migration of cells from the Sertoli cells, sequestering germ cells inside mesonephros into the gonad (peritubular testis cords and arrest them in mitosis pos- myoid and vascular cells) (Capel et al., sibly via a DHH-induced signal (Bitgood et 1999), two processes that are crucial for al., 1996). As a result germ cells entry into testis development. SRY might direct cellu- meiosis (believed to initiate the ovarian

66 Male infertility today #4 pathway and block testis formation) is differentiation upon a program that is inher- opposed (Hung Chang Yao et al., 2002). ently female. The androgen producing Leydig cells are excluded from the cords and remain in the 2. TESTICULAR DESCENT AMONG interstitial region in close proximity to arteri- DIFFERENT MAMMALIAN SPECIES: al and lymphatic vasculature. They start EVOLUTIONARY IMPLICATIONS producing testosterone once the testicular Testicular descent (TD) from the initial cords have been formatted (~12.5-13 dpc, position on the urogenital ridge (which in th during the 8 GW). It is suggested that most species is revealed in the adult by the testosterone production is repressed by the position of the ovary) occurs exclusively in presence of Wnt4 which is downregulated mammals (Theria). However, the degree of by Sry and steroidogenesis is allowed to TD is highly variable, and abdominal testes proceed (Vainio et al., 1999). are found in a wide variety and large num- Jost’s pioneering experiments investi- ber of taxa. Furthermore, genera within gated the influence of fetal hormones dur- mammalian families often show variations ing development (Jost, 1947; Jost, 1953). in testes position (Carrick & Setchell, 1977; He was the first to suggest a model accord- Harrison & Lewis, 1986; Kinzey, 1971). Six ing to which, two fetal testicular hormones mammalian orders contain species with regulate sexual differentiation in eutherian internal testes as well as species with mammals, one that stimulates Wolfian duct external testes (Marsupialia: kangaroo etc, differentiation and masculinization of the Chiroptera: bats, Rodentia: rats, mice, por- external genitalia and one that inhibits the cupines, prairie dog, chinchilla etc, development of the Mullerian ducts. Carnivora, Perissodactyla (odd-toed ungu- Subsequently, testosterone (converted by lates): horses, rhinos etc and Artiodactyla 5a-reductase in target tissues to DHT) (even-toed ungulates): pigs, hippos, induces differentiation of the Wolffian ducts camels, deer, cows etc) (Kinzey, 1971; into vas deferens, epidedymides, seminal Pfeifer, 1956; Weir, 1974). Testes position in vesicles and ejaculatory ducts, while MIS mammals can be generally classified (detectable at Sertoli cells by the 7th GW) is secreted by Sertoli cells after the 8th GW (Williams & Hutson, 1991a) as trans- and actively causes regression of the abdominal and inguino-scrotal. Each cate- Mullerian ducts, most likely by interactions gory can be schematically further divided with membrane-bound serine/threonine into three groups respectively: embryonic kinase receptors on mesenchymal cells (no descent from the ancestral and embry- surrounding the Mullerian ducts (Baarends onic position; (position 1), intermediate et al., 1994; di Clemente et al., 1994; Lee & (partially descended, intra-abdominal posi- Donahoe, 1993). The indifferent gonads of tion; position 2), internal inguinal (position XX individuals will differentiate into ovaries 3) and emergent (some part protrudes that do not produce MIS during fetal devel- through the inguinal ring; position 4), opment, thereby creating permissive envi- beyond the inguinal ring but not within a ronment for Mullerian duct differentiation. true scrotum (position 5), scrotal (into a true The lack of testosterone results in the pas- scrotum; position 6). Non-mammalian sive regression of the Wolffian duct system. species such as fish, birds, turtles, croco- Thus, MIS and testosterone produced by diles have testes located in position 1 the fetal testes impose a male pattern of (Freeman, 1990).

Male infertility today #4 67 MAMMALIAN SPECIES WITH TRUE Catacea had scrotal testes during an earlier INTRA-ABDOMINAL TESTES: epoch, but this feature has been lost to a PRIMARY AND SECONDARY variable extent in subsequent millennia TESTICONDA (Meek, 1918) to protect them from getting too cold, as even a brief period of extreme There is a number of mammalian testicular cooling can render an animal per- species whose testes do not descend at all manently sterile (Young et al., 1988). It has like non-mammalian vertebrates (position been suggested that the absence of the 1). These animals are called primary testi- pelvic girdle together with the development conda and the phenomenon is called testi- of structures in and beyond the caudal condia (Van der Schoot, 1996). Prototheria abdominal region, particularily the caudal (Monotremata: the duck-billed platypus and hypaxial musculature, precludes the out- the spiny ant-eater or echidna) (Grant, growth into caudal direction of hollow 1984) as well as some Eutheria organs (such as the processus vaginalis) (Hyracoidea: the rock hyrax (Procavia and from the abdominal cavity (Van der Schoot, Heterohyrax) (Glover & Sale, 1968; Van der 1995). Schoot, 1996), a small herbivore living in rocky outcrops throughout much of Africa; MAMMALIAN SPECIES WITH Proboscidea: both extant elephant species PARTIALLY DESCENDED TESTES (the Asiatic (Elephas maximus) (Short, The testes of the prairie dog (Rodentia: 1972) and the African elephant (Loxodonta Cynomys) remain in a similar position as Africana)) (Gaeth et al., 1999; Short et al., those of the narwhale (Anthony, 1953). This 1967) as well as Sirenia: the sea cow is the case also as far as most of Dugong dugon (Marsh et al., 1984)) are Insectivora are concerned (Erinaceidae: classified to primary testiconda. hedgehog, Talpidae: mole, Soricidae: Aquatic mammals (Catacea: porpoise, shrew, and Macroscelididae: elephant dolphin, whale), have testis which are par- shrew) with the difference that these ascro- tially descended within the abdomen (posi- tal mammalian species demonstrate sea- tion 2) (Meek, 1918). However, testicular sonal changes in the position and the size position varies in different families. The of the gonad (enlargement and protrusion testes of the common porpoise (Phocaena of the testes during spring (breeding sea- phocaena) are latero-caudal to the kidneys son) into sacs near the base of the tail) (Ping, 1926). In the fin whale (Balaenopter (Marshall, 1911; Pearson, 1944). The testes physalus) the testes lie slightly below the of armadillios and sloths (Edentata) lie kidney and a genital cord tethers the cranial between the bladder and the rectum portion of the vas deferens to the inguinal (Wislocki, 1928). region (Ommaney, 1932). The narwhale Chinchilla (Rodentia) is a representative (Monodon monoceros) has its testes in a mammalian species in which TD arrests pelvic position adjacent to the bladder neck just outside the inguinal region (position 4). just inside the inguinal region (position 3) Instead of a true scrotum, these animals (Meek, 1918). Testicondia in Catacea is present thin-walled diverticulae cauda-later- considered secondary as they are judged, al to the base of the penis. Although testes evolutionarily, the descendants of terrestrial are inside the inguinal apertures, the cau- mammals (ungulates) with TD (Van der dal epididymis protrudes into the diverticu- Schoot, 1995). It has been speculated that lum (Roos & Schackelford, 1955). The

68 Male infertility today #4 southern elephant seal (Carnivora: except in the adult during the breeding sea- Pinnipedia: Mirounga leonine Linnaeus) is a son, when they are pendulous because of typical example of a mammal with testes in scrotal relaxation (Erickson et al., 1968). position 5 (extra-abdominal testes in sepa- Similarly, the woodchuck (Rodentia: rate inguinal pouches rather than a single Marmota Monax), has pouches beside the scrotum, no dartos present, rudimentary tail into which the testes descend seasonal- cremaster muscle) (Bryden, 1967). ly (Rasmussen, 1917), while the testes of Aplodontia rufa (Rodentia) are in a semi- MAMMALIAN SPECIES WITH scrotal position during the breeding season DESCENDED TESTES INTO A TRUE (Pfeifer 1956). In young squirrels (Rodentia: SCROTUM Squiridae), the testes are either scrotal or Even in cases where TD within a true inguinal but after sexual maturity they are scrotum is present (position 6); the exact never more than temporarily retracted site and degree of development of the scro- (Mossman, et al., 1932). In all living pri- tal sac may vary widely among mammalian mates the testes descend into the scrotal species. The hyaena’s testes (Carnivora) for sac and do not re-enter the abdomen example usually complete their descent into (Martin, 1969). It is worth noting however, two posterior shallow scrotal pouches but that in loris and potto (Lemuroidea) the may occasionally be concealed subcouta- testes descend seasonally into the scrotum neously in the perineum (Mathews, 1941). (Rau & Hiriyannaiya, 1930). The testes of On the other hand, the kangaroo (subclass Rhesus macaque monkey descend to the Theria: infraclass Metatheria: order scrotum before birth like in human but then Marsupialia) as well as most of the marsu- re-ascend to the inguinal canal after birth to pials (e.g. the opossum (Didelphis)) has re-enter the scrotum finally at puberty permanently descended testes within a (Miller, 1918). large pendulous pre-penile scrotum EVOLUTIONARY HYPOTHESES ON (Chase, 1939; Renfree, 1992) (in contrast TESTICULAR DESCENT AND to most other mammals with permanent TD SCROTAL FORMATION IN that develop post-penile scrota) (Le Gros MAMMALIAN SPECIES Clark, 1962). The testicular position relative to the scrotum may also vary with (breed- The reason for the developmental fate ing) season and/or sexual maturity. Such and ultimate position of the testis and epi- varieties are observed in some Carnivora, didymis has been the subject of consider- Rodentia and only in three species of the able contention among developmental biol- Primate order (see below) (Miller, 1918; ogists, mammalogists, evolutionary biolo- Rau & Hiriyannaiya, 1930). The fox, ferret, gists and reproductive physiologists. TD is weasel, stoat, cat, and dog (Carnivora), as developmentally, physiologically and evolu- well as the mouse and rat (Rodentia: tionarily costly process (Moore & Persaud, Muridae) possess a global scrotum 1993). Therefore its benefits must be great between the inguinal region and the anus to outweigh the cost. What the benefits of and exhibit permanent TD, irrespective of TD may be is poorly understood and the the breading season (Hill, 1939; Sisson, question only partially overlaps that of the 1940). On the contrary, in the bear adaptive value and evolution of the scro- (Carnivora) the testes are scrotal from tum, since the development of the latter is infancy and are held close to the body not a prerequisite for the former. Several

Male infertility today #4 69 hypotheses have been put forward to malian species, as well as for its failure to explain the evolution of the scrotum and TD. explain the observed variability in the The most important of these are: a) the degree of TD (Werdelin & Nilsonne, 1999). hypothesis of temperature dependency of It is well known that special mechanisms spermatogenesis (Moore, 1926); b) the exist for cooling the testis (venous plexuses mutation rates hypothesis (Short, 1997);c) carrying cool blood to the arteries supplying the display hypothesis (Portman, 1952);d) the testis: countercurrent heat exchangers) the cold storage hypothesis (Bedford, in a number of ascrotal taxa (southern ele- 1977); and e) the training hypothesis phant seal like other true seals (Blix et al., (Freeman, 1990). 1983; Rommel et al., 1995), Cataceans The oldest and classical hypothesis to (Pabst et al., 1995; Rommel et al., 1992;)) explain TD and scrotal evolution originated while others have core body temperatures with Moore (1926) states that testes (possibly evolved to accommodate ascrotal descend into a highly specialized low-tem- condition) that obviate the need for such perature environment (the scrotum) mechanisms (hedgehog, (Carrick & because such an environment is necessary Setchell, 1977) primary testiconda such as for viable sperm production. It is well known Monotremata (Macdonald, 1984)). that in many mammals normal spermatoge- Hyracoidea and Proboscidea (elephants) nesis and epididymal storage require below on the other hand, represent an exception core body temperatures (Carrick & Setchell, to a rather distinct pattern of primary testi- 1977, Cowles, 1958; Cowles, 1965; conda (characterized by low body tempera- Jameson, 1988; Setchell, 1998; ture) (Hanks, 1977). Hyraxes are known to VanDemark & Free, 1970; Waites, 1970). have poor internal thermoregulation, con- Spermatogenesis can be hampered at tem- trolling their body temperature to a large peratures between 35-38°C (Cowles, 1958; extent by behavioral means. (Macdonald, VanDemark & Free, 1970), while abdominal 1984) This together with their small size, temperatures can detrimentally affect long- testes placed close to the dorsal body wall term spermatozoa storage of many species and their seasonal reproductive activity (Bedford, 1977). Most mammals have (Glover & Sale, 1968) would make it rela- acquired a large number of sophisticated tively easy for them to regulate behaviorally anatomical and physiological adaptations of their testicular temperature to the proper the scrotum (Table 1) (Bedford, 1977; one for spermatogenesis. The elephants Freeman, 1990; Hutson & Beasley, 1992; have a testicular temperature equal to their Jameson, 1988; Kinzey, 1971; Setchell, core body temperature (>36°C) (Carrick & 1978; Waites & Moule, 1961; Williams & Setchell, 1977) and their testicular veins Hutson 1991a) many of which are also ran straight into the posterior vena cava present in the human, to keep the epi- with no sign of a pampiniform plexus that didymis and testis cool (~3-5°C below core could cool the testis (Gaeth et al., 1999). body temperature). This hypothesis has These animals are regarded as “immune” to been seriously criticized from a number of high temperature effects on sperm viability authors (Bedford, 1977; Bedford, 1978; (Hanks, 1977). However, a temperature Carrick & Setchell, 1977; Freeman, 1990;) range of 36-38°C is not so high to cause merely on the basis of the relationship aspermia, though temperatures above 38°C between core body temperature vs. testicu- do seem to have this effect (Waites, 1970), lar temperature in scrotal vs. ascrotal mam- and the fact that elephants belong to pri-

70 Male infertility today #4 Table 1. Scrotum as a low-temperature environment: Thermoregulation at testicular/epididymal level

Anatomical characteristic Physiological role Pendulous scrotum Physical separation from the body core (e.g. horse, bull, howler monkey) Obliteration of processus vaginalis Keeps testis outside abdominal cavity (e.g. chmpanzee, gorilla, capuchin, spider monkey, human) Thin and pigmented scin Convection/radiation of heat Large surface area Convection/radiation of heat Absence of subcoutaneous fat Convection of heat Total absence of hair fur Heat loss (over caudal epididymis) (e.g. rat)/or at least at the distal end Cremaster muscle Controling of testicular dependency in response to external temperature Dartos muscle Controling of scrotal dependency in response to external temperature Scrotal veins located against the skin Convection of heat Pampiniform plexus formed by testicular Counter-current cooling of incoming blood veins around internal spermatic artery within the spermatic cord Testicular artery (sometimes) winds around Heat loss before breaking up to supply the testis (up to three times in rams) the testis Fat pad between testis and epididymis Insulation of caudal epididymis from the testis Fat pad in inguinal canal (e.g. rat) Testicular insulation from abdominal cavity mary testiconda is therefore not by itself nation for the evolution of the scrotum and sufficient to reject the temperature hypothe- TD is not the disruption of spermatogenesis sis for TD and evolution of the scrotum per se, but the need to keep the mutation (Werdelin & Nilsonne, 1999). The hypothe- rate in the male germ line under control. In sis is further supported by the phylogenetic humans it is known that mutation rates are approach (Werdelin & Nilsonne, 1999) higher in males than in females (Shimmin which provides strong evidence towards the et al., 1993; Shimmin et al., 1994). This, selective passage from the presence of a coupled with the fact that elevated tempera- scrotum to the absence of one. Thus, the tures have long been known to be associat- variation in testicular position could be seen ed with increased mutation rates, suggests as the result of the developmental and that cooling the testes will assist in keeping physiological difficulties encountered during male germ line mutation rates at acceptable the evolutionary pressure towards doing levels (Cowles, 1965; Ehrenberg et al., away with the scrotum and descensus. 1957). A modification of this hypothesis has The “display” hypothesis has been sug- recently been proposed by Short (1997). gested by Portman (1952). It is based on According to this theory, the causal expla- the assumption that the testes descend to

Male infertility today #4 71 an external position (scrotum) because of didymis but sometimes the seminiferous their importance in social competition, vesicles or vas deferens) always precedes either as a character in female choice of the testes in descent, descending to the mates or as a signal of dominance in male- body wall or to a scrotal-like out-pocketing male competition. In some taxa the scrotum in most species with abdominal testes is brightly or at least distinctively colored, (even in non-mammalians e.g. in passerine which serves to call attention to the sexual birds sperm is stored in a cloacal protruber- organs in the taxa involved. This is the case ance lower-than-body core temperature by in primates for example, where the pres- several degrees (Wolfson, 1954) (Bedford, ence of a pigmented scrotum is associated 1978; Glover, 1973; Glover & Sale, 1968; with polygynous or promiscuous breeding Harrison, 1969). However, the main flaw of systems characterized by intense male- this hypothesis is its incompatibility with the male competition (Freeman, 1990). fact that in a few ascrotal taxa, the epi- Although there is a number of observations didymis lays subcoutaneously or intra- consistent with this hypothesis, its failure to abdominally (e.g. hyraxes (Glover & Sale, explain the variable location of the testes 1968), elephant shrew (Woodal, 1995)). among ascrotal species combined with the From the phylogenetic point of view (see fact that pigmented scrota are not always above) (Werdelin & Nilsonne, 1999) readily presented (nocturnal animals, furred Bedford’s hypothesis cannot be justified pigmented scrota) by the relatively small although it explains why the epididymis percentage of genera-owners, weakens its remains in a distal position in some taxa, explanatory power (Freeman, 1990). It is while the testes have migrated out of the more possible that scrotum has evolved for scrotum during the process of scrotal loss. some other function first and subsequently The “training” hypothesis was formulated has been retained and furthermore, co- by Zahavi (Freeman, 1990). It represents a opted as a signaling device by some radical departure from the other hypothe- species (most notably primates) due to ses presented above since it views the their particular sexual behavior systems scrotum as a purposely hostile environment (Harrison & Lewis, 1986). In this way, social for sperm rather than an optimized one. The behavior can act as a constraint against the main idea is that, TD into a scrotum occurs adaptive pressure towards loss of the scro- because spermatozoa must get “trained” for tum rather than an evolutionary mechanism their future endeavor in the physiologically towards scrotal developmetal (Werdelin & rigorous female environment. The physio- Nilsonne, 1999). logical basis for this hypothesis is that the The “cold storage” hypothesis originated degree of TD is inversely correlated to the with Bedford (1977). It was suggested that testicular blood flow (Setchell, 1978; Waites TD into a scrotum (whose primary function & Sethell, 1969) directly correlated to is to store sperm in a cool environment) is a hemoglobin affinity for oxygen (under lower secondary phenomenon, subsequent to the scrotal temperatures) (Eckert & Randall, epididymal descent (evolutionarily “prime 1983) and therefore directly correlated to mover” in scrotal evolution), serving merely the degree of oxygen and pH stress on as a mechanism for providing structural maturing sperm. Direct consequence of support for the epididymides in some spermatogenesis under episodes of physio- species. It was pointed out that the sperm logical oxygen and pH stress, is an storage organ in mammals (usually the epi- increase in size, number and efficiency of

72 Male infertility today #4 maturing spermatocyte mitochondria in a from the Latin word meaning helm or rud- way that parallels the response of striated der) for the unique structure attached to the muscle cells during anaerobic training. If testis (caudal ligament), which he believed mitochondria are trained more efficiently in to be responsible for steering the male descended testes and perform better dur- gonad into the scrotum. Since Hunter’s first ing active sperm transport in the female description, the gubernaculum has been reproductive tract (better swimming and/or described by many workers and numerous longer survival of spermatozoa), TD will theories have been proposed up to date to enable a male to produce a physiologically- explain the cause and mechanism of TD superior ejaculate which would then have (Heyns & Hutson, 1995; Williams & Hutson, an advantage in the between-ejaculate 1991b). Among these theories, the role of competition characterizing many mam- the gubernaculum has indisputably occu- malian mating systems (Ginsberg & Huck, pied a central place. Although it is not uni- 1989). The main strength of this hypothesis versally accepted that the gubernaculum is that it can account for the entire range of “holds the key to the mystery of “descent”, variation in testicular position among mam- the development of this structure is unique malia by providing an explanation for the to the male fetus and offers the most obvi- strategies behind both scrotal and ascrotal ous explanation of why the fetal testis testes: scrotal testes produce fewer sperm descends while the ovary does not. of higher quality, while ascrotal testes pro- Furthermore, there is strong evidence duce more sperm of lower quality. Zahavi’s derived from the mammalian evolutionary hypothesis has been criticized on the base history suggesting that the gubernaculum that the blood supply to the testes could be has an essential role on TD. Reptiles as modified without resorting to an elaborate well as primary testiconda mammals such and costly complex such as the TD and as hyraxes (Van der Schoot, 1996) and ele- scrotal development (Werdelin & Nilsonne, phants (Gaeth et al., 1999) show either no 1999). sign of a gubernaculum at any stage of their development, or at least partial (only 3. PHYSIOLOGY OF TESTICULAR gubernacular cord) gubernacular develop- DESCENT AND PATHOPHYSIOLOGY ment (e.g. Hyrax capensis) (Van der OF TESTICULAR MALDESCENT IN Schoot, 1996). On the other hand second- SCROTAL MAMMALIA ary testiconda such as Catacea (e.g. the In ancient times investigators new from common dolphin (Delphinus delphis), the animal studies that testes descend from the Black sea harbour porpoise (Phocoena abdominal cavity to the scrotum but it was phocoena), the gray whale (Eschrichtius first observed in the 17th century that the robustus), the sperm whale (Physeter same process occurs in the human fetus as catodon)) present gubernaculae primordia well (Weil, 1885). The study of TD really ever since the time of oncet of their sexual originated with John Hunter (1786), an differentiation (Van der Schoot, 1995).In English anatomist who published the first theses mammals, gubernaculuae develop important and highly accurate description further into large masses of dense connec- of TD in human. In 1762 and 1786, he tive tissue in the ventral-caudal abdominal described the fetal testis and epididymis in region at the site of the insertion of the the abdomen and he was the first to intro- mesonephric inguinal ligament. This is duce the term “gubernaculum” (derived strong evidence that Catacea are the

Male infertility today #4 73 descendants of terrestrial mammals (ungu- monkey is not complete until puberty late) with TD (see above). (Kinzey, 1971). The exact etiology of testic- Despite the extensive work on TD during ular maldescent (TMD) is not known but the last centuries, the matter still remains normal hypothalamo-pituitary-gonadal axis controversial since no unified theory does is usually a prerequisite for normal TD. exist as yet. This is probably due to the Abnormal sexual differentiation is associat- incorrect or incomplete observations many ed with TMD. Even though hormones of these theories are based on, the kind of cause TD, hormonal deficiency does not material used (e.g. histo-anatomical studies appear to be a common cause of TMD. on autopsy material cannot determine the Where true hormonal deficiencies are rec- forces responsible for descent) as well as ognizable (i.e. abnormalities of the hypo- (mainly) the type of animal model used and thalamic-pituitary axis) the testes are unde- the indiscriminate extrapolation of findings scended. However, TMD in terms of defined among different spiecies (Heyns & Hutson, hormonal syndromes comprise a very small 1995). In fact there is no entirely satisfacto- subpopulation of the total number of chil- ry animal model for TD. Therefore, confu- dren with undescended testes (UT), since sion has been produced by comparing TD the majority of boys with UT show no of different species due to the marked mor- endocrine abnormalities after birth. phological and topographical differences EMBRYOLOGY OF TESTICULAR among the structures involved (Wensing, DESCENT 1986). Although it has been noticed as long as 1856 that TD should be studied primarily Multi-staged TD was proposed first by in the human in order to avoid false analo- Gier & Marion (1969) as initial nephric dis- gies drawn from animals (Cleland, 1856), placement by degeneration of the the difficulties and limitations of research mesonephros at 7th-8th GW, transabdomi- on fetal processes in human or even in nal passage of the testis from the large mammals (which serve as much bet- metanephros to the inguinal ring by 21st ter models for the study of TD), account for GW and finally inguinal transit of the testis the ample use of Glires (especially from the peritoneal cavity along the proces- Rodentia: e.g. rat, mice but also sus vaginalis at 28 GW. The fact that the Lagomorpha: e.g rabbit) as experimental first stage of TD proposed by this model models to study TD (Heyns & Hutson, has been completed very early during 1995; Wensing, 1986). embryonic development precludes a major TD is a profound example of sexual role of this step in sexual dimorphism dimorphism. It is a complex process that (Hutson et al., 1997). More recently, a appears to be multi-staged involving an biphasic model, with two morphologically interplay of different anatomical structures and hormonally distinct phases, has been and hormonal factors. TD has been accept- suggested to explain TD in normal males ed as being under hormonal control ever (Hutson, 1985). According to this hypothe- since Engle (1932) showed that pregnancy sis the first (transabdominal) phase of TD urine or anterior pituitary extracts caused (TTD), comprising the relative movement of premature TD in prepubertal monkeys. the testis (compared with the ovary) from its However the relation of this effect to the initial posterior abdominal position (adja- normal prenatal descent in man has cent to the kidney by the 8th GW) to the remained unclear, since descent in the internal inguinal ring, is regulated by a non-

74 Male infertility today #4 androgenic hormone/factor and a possible TTD takes place between 15.5-17.5 dpc in role of MIS was suggested (see below). The the mouse (Hadziselimovic et al., 1980) second (inguinoscrotal) phase of TD (ISTD) and between 16-20 dpc in the rat (Van der consists of the androgen-dependent Schoot, 1993a). ISTD needs development descent of the testis from the internal of the processus vaginalis, prior dilation of inguinal ring to the scrotum. This phase the canal by the bulb and some abdominal begins at the 26th GW (Backhouse, 1982; pressure to force the testis through the Heyns, 1987). Between 26th-28th GW, the canal (Hutson et al., 1997). Distal attach- testis descends rapidly through the inguinal ment of the gubernaculum has been experi- canal and then moves slowly towards the mentally shown to be important in the nor- scrotum reaching it at the 35th-40th GW mal development of the processus vaginalis (Backhouse, 1982). In the human, migra- (Clarnette, et al., 1996). During the 8th GW, tion of the gubernaculum and the testis the inguinal canal begins development as a occur simultaneously while in rodents, caudal envagination of the abdominal wall migration of the gubernaculum precedes that forms in conjunction with caudal elon- TD (inguinoscrotal gubernacular migration: gation of the processus vaginalis (Schneck rat at 3-5 days post-natally, mouse at ~7 & Bellinger, 2002). The processus vaginalis days post-natally; ISTD: rat at 3-4 weeks develops as a herniation of peritoneum at post-natally, mouse at ~2 weeks post-natal- the deep internal inguinal ring and anterior ly) (Hadziselimovic et al., 1979; Wensing, aspect of the gubernaculum. This peritoneal 1986). diverticulum later elongates within the Between 10th-15th GW (TTD), the cra- gubernaculum to form an annular cavity, nial suspensory ligament (CSL) that holds dividing the gubernaculum into a central the urogenital tract near the developing mesenchymal column and an outer parietal diaphragm regresses (see below) while, layer. Following complete descent of the simultaneously (8th-15thGW), the guber- testis, the central gubernacular column naculum (that has appeared by the 7th GW involutes by dissolution of the extra-cellular (Schneck & Bellinger, 2002) as a conden- matrix. The residual tissue of the column sation of mesenchymal tissue within the forms the fibrous attachment of the testis to sub-ferous fascia on either side of the ver- the scrotum. The cremaster muscle devel- tebral column that extends from the gonad ops within the outer parietal layer of the to the fascia between the developing exter- gubernaculum forming a bilaminar sac in nal and internal oblique muscles) enlarges rodents, and a strip of muscle in ungulates caudally (see below) (Hutson et al., 1997). and primates (Wensing et al., 1980). The Gubernacular caudal (bulb) enlargement attachment of the cremasteric muscle to keeps the testis anchored close to the the inguinal abdominal wall varies from future inguinal region via traction applied species to species (Wensing, 1986). Its through the gubernacular cord which short- own nerve supplies the gubernaculum: the ens as it becomes incorporated into the genital branch of the genitofemoral nerve bulb (Wensing, 1968; Wensing, 1973a). The (GFN) (Hutson, 1970; Tayakkanonta, 1963). net effect of these processes is the tether- Careful dissection of the distal gubernacu- ing of the testis near the groin and permis- lar end in humans and pigs shows that the sion of its relative descent while the kidney lower end of the gubernaculum is free with migrates cranially during enlargement of no firm attachment to the scrotum the abdominal cavity (Hutson et al., 1997). (Backhouse, 1982; Heyns, 1982). This is

Male infertility today #4 75 strong evidence against the concept of the disputed in the dog (Kersten et al., 1996). gubernaculum pulling the testis down to the There is a critical period (~17-18 post- scrotum, since it is not anchored inferiorly. conceptional day) during which CSL devel- The force of movement may come from the opment can be abolished by androgens in inta-abdominal pressure (Elder et al., 1982; the male rat (Van der Schoot P & Emmen, Frey et al., 1983; Frey & Rajfer, 1984; Gier 1996). Since the rat testis starts to produce & Marion, 1969; Hadziselimovic et al., testosterone on 15.5-16.5 post-conception- 1979; Heyns & Hutson, 1995; Quinlan et al day (Warren et al., 1975), this androgen al., 1988) transmitted directly or indirectly to effect is an early phenomenon during male the testis via the lumen of the processus sex differentiation, with an onset well before vaginalis and the gubernacular cord, fetal testosterone production reaches its respectively (Hutson et al., 1995). maximum on 18.5 post-conceptional day (Habert & Picon, 1984). Furthermore, TRANS-ABDOMINAL PHASE OF androgen receptor (AR) is expressed in the TESTICULAR DESCENT primordial cells of the CSL of both males 1. Regression of the cranial and females, during this critical period (ini- suspensory ligament under the tially hormone-independent expression of influence of androgens AR) (Emmen et al., 1998). However after The CSL (derived from mesonephric 18 post-conceptional day AR is expressed mesenchyma) is a muscular cord-like intensively only in the male CSL primordi- structure, which borders the cranial part of um despite the loss of its androgen respon- the mesonephric mesentery, attaching the siveness. Thus the mesenchymal cells of ovary and genital duct to the cranio-lateral the CSL can be considered as a direct tar- surface of the dorsal abdominal wall, near get for fetal testicular androgens which the ventral aspect of the last rib. The pri- have a strict time-specific morphogenic mordium of the CSL is present in both effect upon CSL. The relative absence of sexes of, among other mammalian smooth muscle cells in the CSL of male species, the rat, pig, dog, cattle and fetuses, as compared to the female, indi- human (Kersten et al., 1996; Van der cates that androgens suppress smooth Schoot & Emmen, 1996). The prevention muscle cell differentiation in the CSL by of outgrowth of the fetal CSL in male possibly initiating a complex interaction rodents is an androgen-dependent among macromolecules (collagen, process (prenatal exposure of females to fibronectin, laminin) of the extra-cellular androgens prevents development of the matrix and peptide growth factors (e.g. CSL, whereas males prenatally exposed TGF?1) (Emmen et al., 1998). This critical to anti-androgens show CSL development period seems to correlate with the precise in a female-like fashion) (Barthold et al., time of androgen action on TD (Husmann & 1994; Van der Schoot & Elger, 1992) as McPhaul, 1991a; Spencer, er al., 1991). well as in human (patients with complete Whether or not CSL regression is the or partial androgen insensitivity syndrome key factor in TD is disputed. CSL regression (CAIS/PAIS) preserve CSL), pig and cattle is probably a prerequisite of TD (Emmen, et (Clarnette et al., 1997; Van der Schoot & al., 1998). However, it has been shown that Emmen, 1996). However, androgen regression of CSL alone is insufficient to dependency of CSL regression has been cause gonadal descent (46,XX individuals

76 Male infertility today #4 exposed prenatally to androgens retained (Van der Schoot, 1993b) demonstrated the their ovaries in a normal position) (Scott, participation of a fetal testicular factor in 1987). TD does not seem to be interrupted gubernacular development and gonadal in cases with CSL retention in testicular descent. However, it is less clear which tes- feminized mouse (Tfm) (testes descended ticular hormone/factor induces gubernacular at the internal inguinal ring) and human outgrowth and regulates TTD. The testicular with CAIS or PAIS (testes descended at the hormone/factor promoting TTD via out- internal inguinal ring or beyond) (Hutson, growth of the gubernaculum has remained 1986). However, bilateral intra-abdominal unknown for decades. It received names testes associated with bilaterally retained such as “factor X”, (Habenicht & Neumann, CSL have been described in the dog 1983) and “third hormone” (Van der Schoot (Kersten et al., 1996). Furthermore, the et al., 1995). testes of testicular feminization rat are intra- 2.1. Evidence against androgenic abdominal, inguinal and scrotal in 20%, control of the trans-abdominal phase of 67% and 13% respectively, suggesting that testicular descent CSL retention possibly has a role in TTD of There is strong evidence that both GSR the rat. Most authors generally agree that and TTD are not under androgenic control in CSL has a limited role in at least some a great number of mammalian species. species (Barthold et al., 1994; Husmann & Although AR-positive cells have been identi- Levy, 1995; Hutson et al., 1995). fied in the gubernacular mesenchymal core 2. The “gubernacular swelling both in the rat (Husmann & McPhaul, 1991b) reaction” and the pig (Heyns & Pape, 1991), their Caudal enlargement of the gubernacu- maximum expression in the rat is seen lum during relative TTD is known as the (Husmann & McPhaul, 1991b) after GSR “gubernacular swelling reaction” (GSR) and becomes almost complete in this animal and it is caused by rapid cell proliferation and an both the receptor binding affinity and capaci- increase in glycosaminoglycans and ty are lower than in known androgen-sensi- hyalouronic acid (Backhouse, 1982; tive target tissues (e.g. prostate) in the pig Backhouse & Butler, 1960; Heyns et al., (Heyns & Pape, 1991). Clinical examples 1986; Heyns et al., 1990). The hydrophilic supporting this theory include patients with nature of hyaluronic acid makes the end of CAIS/PAIS who have bilateral UT. In the the gubernaculum bulky and gelatinous sim- majority of these cases testes are palpable ilar to the Wharton’s jelly found in the umbili- bilaterally in the labioscrotal folds or inguinal cal cord. GSR has been linked closely to regions, respectively (Ahmed et al., 2000; trans-abdominal migration of the testis Hutson, 1986). In addition there is a great (Wensing, 1973a). The (hormonal) control number of experimental studies supporting of GSR (and therefore TTD) remains contro- this hypothesis. In the fetal pig, raccoon, dog versial. Development of a male-like guber- and mouse with testicular feminization syn- naculum (and partial ovarian descent) in drome, GSR and TTD remain normal bovine freemartin (a female fetus exposed (Fentener van Vlissingen et al., 1984; to the blood of a male twin by chorioallanto- Hutson, 1986; Wensing et al., 1975). ic anastomosis) (Lillie, 1917; Van der Schoot Furthermore, GSR and TTD is not impaired et al., 1995) and in female rabbit fetuses after exposure of monkeys, rats, mice and that had been grafted with a fetal testis, rabbits prenatally or dogs postnatally to

Male infertility today #4 77 cryproterone (Elger et al., 1977; Hutson & suppression of a possible MIS effect on the Beasley, 1988; Wensing, 1973b;) or rats gubernaculum (Hutson, 1987; Hutson & (prenatally) to flutamide (Shono et al., 1994). Donahoe, 1986; Hutson & Watts, 1990). In addition, injection of androgens into the MIS has been further investigated as a fac- rat or pig fails to stimulate GSR in fetal tor mediating TTD, because in patients with females (Wensing, 1973a), while androgen persistent Mullerian duct syndrome replacemet after orchidectomy does not pre- (PMDS), which is a familial autosomal vent gubernacular atrophy in the fetal dog recessive disorder due to mutations in the (Baumans, et al., 1982; Baumans, et al., MIS and/or MIS II receptor genes (Imbeaud 1983). However, in vitro studies of androgen et al., 1996), both testes are located in the effect upon gubernacular cell proliferation ovarian position in ~60-70% of cases conclude to inconsistent results (Emmen et (Hutson et al., 1987) and the gubernaculum al., 2000a; Fentener van Vlissingen et al., is feminine-like i.e. thin and elongated 1988; Kubota et al., 2002; Visser & Heyns, (Hutson et al., 1994). However, there is 1995). This is probably due to anatomical/ strong evidence against a significant role of structural differences of gubernaculae MIS in GSR and TTD. Normal TD occurs in among species (e.g porcine gubernaculum fetal rabbits immunized against bovine MIS proper consist of mesenchymal cells only, in despite partial retention of the Mullerian contrast to the gubernaculum of the rat that ducts (Tran et al., 1986). In addition, semi- possesses a muscular outer layer-anatomi- purified bovine MIS failed to induce in vitro cal equivalent of the porcine cremaster mus- DNA synthesis and cell division of cultured cle) (Wensing, 1986). fibroblasts from fetal porcine gubernaculums (Fentener van Vlissingen et al., 1988), 2.2. Evidence against a significant role while MIS appears to have a minimal prolif- of the Mullerian inhibiting substance erative activity on rat gubernacular cells in A biphasic model of TD was proposed vitro due to a week expression of MIS II by Hutson (1985), suggesting that a non- receptors (Kubota et al., 2002) (culturing rat androgenic hormone, possibly MIS, regu- gubernaculae in the presence of testes lates GSR and TTD. The conception is from MIS mutant mice do not decrease based on experiments showing that exoge- thymidine uptake compared with the effect nous estrogens in the fetal mouse cause of control or MIS+/- testes (Emmen et al., retention of the Müllerian ducts 2000a)). Furthermore, mice homozygous (Hadziselimovic, 1983), inhibition of GSR for targeted mutations in the MIS or MIS II (Raynaud, 1958; Shono et al., 1996), and receptor genes present with normally complete TTD blockage (Hutson, 1987) descended testes and normal GSR, while which is not reversed by simultaneous ovarian descent is not observed in trans- exogenous exposure to androgens (Hutson genic femal mice overexpressing MIS & Watts, 1990). These results led to the (Bartlett et al., 2002; Behringer et al., 1994; hypothesis that estrogens have a primary Mishina et al., 1996). However, it should be effect of inhibiting Mullerian duct regres- noted that male homozygous Tfm/MIS dou- sion, as well as a second primary effect of ble mutant mice presents as pseudermaph- inhibiting GSR and block TTD not by sup- rodite with severely impaired TTD although pressing androgen secretion as it had been the Tfm and MIS mutants taken individually postulated before (Hadziselimovic et al., do not demonstrate impaired TTD 1979; Hadziselimovic et al., 1980) but via (Behringer et al., 1994). This fact may imply

78 Male infertility today #4 that there is an as yet unknown mechanism with “descendin”/ ”gubernaculotropin”-like via which androgen may regulate MIS activ- activity has been identified and probably ity on the gubernaculum (Catlin et al., represents the long sought-after factor 1993) or vice versa to govern TTD. A further “descendin” or “gubernaculotropin”. The argument against the proposed role of MIS Insl3 gene product, also known as Leydig on GSR and TTD is that, nearly all patients cell insuline-like (Ley l-L) hormone or relax- with inta-abdominal UT do not have persist- in-like factor (RLF), is a member of the ent Mullerian ducts, while the intra-abdomi- insulin peptide hormone superfamily nal location of the testes in PMDS has (Adham et al., 1993; Pusch et al., 1996; been suggested to be due to the anatomic Zimmermann et al., 1997) which includes at connection of the testes with the Mullerian least eight members (insulin, insulin-like ducts (anatomic blockade by the persistent growth factor-I and II, relaxin, early placen- ducts) rather than due to the absence of ta insulin-like peptide (Insl4 or EPIL) GSR (Guerrier et al., 1989; Hutson, 1986; (Chassin et al., 1995), insulin-5 (Insl5 or Josso et al., 1997). RIF-2) (Conklin et al., 1999; Hsu, 1999), and insulin-6 (or RIF-1; higly expressed in 2.3. The role of Insulin-like 3 the spermatocytes and round spermatids of In an in vitro study on the proliferation of rat and human) (Hsu, 1999; Kasik et al., fetal pig gubernaculum cells (Fentener van 2000; Lok et al., 2000)) implicated in the Vlissingen et al., 1988), it was found that regulation of diverse biological functions the extract from fetal porcine testis during including cell growth, cell differentiation and the first phase of TD contains a low molec- energy balance. The mature hormone, like ular weight (<3.5kD) fraction to which insulin, is composed of an A and a B chain gubernaculum cells are responsive. It was linked by disulfide bonds (Adham et al., suggested that this bioactive fraction proba- 1993). The Insl3 gene is expressed in a dif- bly contains the factor(s) that initiate TD. A ferentiation and sexual dimorphic pattern number of polypeptide growth factors (epi- (Adham et al., 1993; Ivell & Bathgate, dermal growth factor (EGF), insulin, fibrob- 2002). Maximal expression is observed at last growth factor, platelet-derived growth both fetal and mature adult-type Leydig factor and transforming growth factor β) and cells (which replace fetal Leydig cells testicular hormones tested (androgens, between post-natal days 5-15) whereas it is MIS (see above), inhibin), showed only only weakly expressed in prepubertal minor stimulatory activity on gubernacular immature Leydig cells and the ovary (with a cells and were therefore excluded as puta- sole exception so far known to be the rumi- tive regulators of TTD. This unknown factor nant ovary that exhibits a very high expres- received the name “descendin” and later it sion) (Ivell et al., 1997; Zimmermann, et al., was proposed (Visser & Heyns, 1995) that 1977). Insl3 transcripts are found in the it corresponds to a novel hormonal peptide developing testis of all mammalian species of molecular weight ~26 kD secreted by the so far examined (Adham et al., 2000) but fetal testis cells that was named “gubernac- not in the gubernacular bulb or in other ulotropin”. neighbouring tissues (Nef & Parada, 1999). Recently, based on gene knock-out They are first detected at 13.5 dpc in mice studies (Kubota et al., 2001; Nef & Leydig cells (exclusive source in the testis) Parada, 1999; Zimmermann et al., 1999), a (Ivell et al., 1997), remain constant through new hormone named insulin-like 3 (Insl3) the 3rd postnatal week, when increases are

Male infertility today #4 79 observed coincident with the first wave of of knock-out adult mice (Nef & Parada, spermatogenesis and germ cell maturation 1999; Zimmermann et al., 1999). It has and reach their highest level in adult testis been recently shown that intra-scrotal (Adham et al., 2000). In contrast, in orchiopexy may rescue fertility in this model females, transcripts are first detected at (Nguyen et al., 2002). Thus infertily of post-natal day 6 in theca cells of small Insl3-/- mouse model may be attributed to antral follicles (expression is correlated with the icreased environmental temperature the selection of the follicles to become pre- resulting from TMD (Nef & Parada, 1999; ovulatory (Irving Rodgers et al., 2002) and Zimmermann et al., 1999). Heterozygous in a lesser degree in the luteal cells of the (Insl3+/-) male mice are fertile and may ovary (Zimmermann et al., 1997). present with partial (either uni- or bilateral) It has been shown that homozygous UT with a gubernaculum that appears to be (Insl3-/-) male mice are viable but sterile, differentiated but not embedded in the exhibiting bilateral UT located high in the abdominal wall causing a delay in TD dur- abdomen due to developmental abnormali- ing adulthood (dosage sensitivity of Insl3 ties of the gubernaculum. The gubernacu- for TD) (Nef & Parada, 1999). In contrast, lum resembles that of the wild type females there is a mildly altered phenotype in and is characterized by the appearance of homozygous females, with impaired fertility a flat thin bulb, elongated cord, absence of due to disturbed cycle length (prolongation) normal structural organization into outer and increased ovarian apoptosis, particu- (myoblasts) and inner (mesenchymal) lay- larly in follicles and corpoa lutea (Spanel ers, presence of some muscular develop- Borowski et al., 2001). Based on these ment but absence of mesenchymal core results Insl3 seems to play an essential role (Kubota et al., 2001; Nef & Parada, 1999; in TTD, it does not seem to be essential for Zimmermann et al., 1999;). Apart from the spermatogenesis but its potential role(s) in gubernaculum and testicular position, the male and female fertility remains to be fur- remainder of the genital tract (including ther clarified. regression of CSL) as well as sexual In vitro studies of the effect of Insl3 and behaviour and serum testosterone levels androgens upon rat gubernacular prolifera- appears normal. These results indicate that tion activity, suggest that both are required Insl3 stimulates the outgrowth and differen- for the induction of gubernacular develop- tiation of the gubernacular primordium in an ment and differentiation (Emmen et al., androgen-independent way in male mice, 2000a, Kubota et al., 2002). However, there while androgen-mediated regression of the is strong evidence that in vivo Insl3-mediat- CSL occurs independently from Insl3 (Nef ed activity upon the gubernaculum is & Parada, 1999; Zimmermann et al., 1999). androgen-independent (Adham et al., Although Insl3 expression increases dra- 2002). This conclusion is supported by matically during puberty (Zimmermann et results with transgenic mice overexpressing al., 1997), which suggests an additional active Insl3 in the pancreas during prenatal role for this hormone in male fertility, the and postnatal development of females and inta-abdominal testes in the newborn Insl3 males. Expression of the transgenic allele knock-out mouse are histologically normal. in the Insl3-deficient mice rescues TMD in However, if left in this position until adult- male mutants. On the other hand, all trans- hood, histology deteriorates to the Sertoli- genic females display bilateral inguinal her- cell-only state leading to infertility in 100% nias and descended ovaries over the blader

80 Male infertility today #4 (attached to the abdominal wall via the well- of the Brca2 gene (corresponding to the developed CSL and the gubernaculums) previously characterized human 13q12-13 due to Insl3-induced gubernacular develop- region) (Couch et al., 1996) affecting sever- ment (shortenin of gubernacular cord, al genes. Direct sequencing of the affected enlargement of gubernacular bulb and dif- region led to the identification of a new ferentiation of the bulb into mesenchyme in gene named Great (G protein-coupled the center and myoblasts in circumferential receptor affecting testis descent) (Overbeek layers). Administration of DHT during pre- et al., 2001), mutations of which have natal development suppresses the forma- recently been proposed to be responsible tion of the CSL allowing the ovaries further for the phenotype in crsp mice (Gorlov et descend into the processus vaginalis. al., 2002). Great gene is composed of 18 A critical aspect concerning Insl3 func- exons (Overbeek et al., 2001) and encodes tion and the underlying molecular mecha- a novel G protein-coupled receptor (GPCR) nisms governing gubernacular differentia- named mouse GREAT (mGREAT) (Gorlov tion awaits identification of a physiological et al., 2002). In particular, mGREAT is an receptor. Recently, a transgenic insertion orphan leucine-rich repeat-containing mutant mouse strain called crsp (cryp- GPCR (LGR) that belongs to the same sub- torchidism with white spotting) that exhibits group of LGRs with a Drosophila a phenotype strikingly similar to Insl3 melanogaster LGR (DmLGR3), a snail LGR mutants has been generated (Overbeek et (Lymnacea stagnalis, LsLGR), and two al., 2001). Homozygous mutants human LGRs (LGR7 and LGR8) (Hsu et (crsp/crsp) of both sexes exhibit a al., 2002). Expression of Great is restricted decreased early postnatal viability, lower to the testis, brain, and skeletal muscles, body weight and white spotting (WS) visible with the highest level of expression in the at post-natal days 3-5 as a variable sized gubernaculum. LGR8 has been proven to white streak on the back in the midtrunk be the human ortholog of mGREAT with region plus a white belly patch. crsp/crsp 82% overall identity at nucleotide and males are completely sterile, presenting amino acid level and same exon-intron with small bilateral UT located in a high structure (Gorlov, et al., 2002; Hsu et al., intra-abdominal position with an extended 2002). It has been recently shown that gubernaculum. However, the rest of the INSL3 specifically activates GREAT/LGR8 genital system appears normal and periph- receptors through the adenylate cyclase eral blood testosterone levels within normal pathway in the gubernaculum leading to range. In contrast, crsp/+ males as well as gubernacular cell proliferation (Hsu et al., crsp/crsp and crsp/+ females share a wild 2002; Kumagai et al., 2002). type phenotype. Surgical scrotal descent of Two points of interest concerning crsp the homozygous mutants’ testes reveals mutation are also worth-noting. First, UT presence of sperm in the epididymis, indi- has been reported in some patients with cating that crsp is, like Insl3, not essential Rieger syndrome type II (hereditary glauco- for spermatogenesis. Complete arrest of ma). A gene responsible for this syndrome spermatogenesis is attributed to high tem- has been mapped close to BRCA2 (Phillips perature environment of the intra-abdomi- et al., 1996). The relevance of this gene to nal testes. The crsp transgenic integration the crsp mutation remains to be elucidated. is accompanied by a ~550 kb deletion in Second, there may be a relationship the distal part of chromosome 5 upstream between crsp-induced WS phenotype and

Male infertility today #4 81 inactivation of a gene responsible for UT. Prenatal exposure to estrogens, such as Melanoblasts and somatic nerves are 17α- and 17β-estradiol, estriol, DES, derived from the neural crest during devel- inhibits Insl3 gene transcription in embryon- opment, while neural crest derivatives are ic Leydig cells, thus providing an explana- possibly implicated in TD (UT in human is tion for the lack of GSR and UT (Emmen et often associated with neural tube defects) al., 2000b; Nef et al., 2000). Evidently, (Hutson & Beasley, 1988). Furthermore, it abnormally high levels of estrogenic mole- has been suggested that proper innervation cules can overcome the normal fetal buffer- of the cremaster muscle and gubernaculum ing mechanisms (serum α-fetoprotein that are essential for normal TD (Hutson et al., binds efficiously estrogen compounds (Keel 1997). Additionally, there is at least one & Abney, 1984; Nunez et al., 1974) and more example of association among UT, estrogen sulfotransferase in Leydig cells, WS and behavioural abnormalities (sug- which facilitates the catabolism of estro- gesting abnormalities of the neural system) gens (Hobkirk & Glasier, 1992; Song et al., described in foxes homozygous for a semi- 1995)) that control the concentration of free dominant mutation in the gene encoding estradiol (either produced in situ by Sertoli the STAR protein (Belyaev et al., 1981). cells (Dorrington & Armstrong, 1975; Taken all these together, it could be Pomerantz, 1980) or concentrated by the assumed that crsp mutation may in some testis (Kelch et al., 1972) (from exogenous way disturb proper functioning of embryonic sources) in Leydig cells. Estrogen inhibitory neural crest cells resulting both in UT and effect must be mediated via indirect estro- WS. gen-receptor (ER) transcriptional control The observation that in utero exposure (Insl3 proximal promoter does not reveal to exogenous estrogens in rodents and sequences obviously related to estrogen- humans (Gill et al., 1979; Greene et al., response elements) (Zimmermann et al., 1942; Hutson et al., 1987; Raynaud et al., 1998) of Insl3 regulatory genes possibly 1958; Shono et al., 1996) as well as the (Majdic, et al., 1997) other than SF1 absence of Insl3 hormone in mice (Kubota (Emmen et al., 2000b; Nef et al., 2000). et al., 2001; Nef & Parada, 1999; SF1 is a direct mediator of Insl3 gene tran- Zimmermann et al., 1999) both induce bilat- scription in Leydig cells (Zimmermann et eral inta-abdominal UT in newborn males, al., 1998). Intrestingly, Insl3 expression has triggered investigation for a possible reappears between post-natal days 0-7 relationship between these two hormones. suggesting that Insl3 gene transcription is It has been suggested that UT induced by permanently blocked in the fetal-type the nonsteroidal synthetic estrogen diethyl- Leydig cell population. Exposure of adult stilbestrol (DES) is due to failure of guber- males to estrogens does not block Insl3 nacular development (Raynaud et al., 1958; gene expression (Nef et al., 2000). This Shono et al., 1996), but the exact mecha- indicates that Insl3 gene regulation in fetal nism is poorly understood. It has been and adult population of Leydig cells is dif- demonstrated that estrogens inhibit fetal ferent. In fetal Leydig cells, Insl3 expression Leydig cell development (Hadziselimovic et is estrogen-sensitive and gonadotropin- al., 1980) in a fetal pituitary gland- and independent, whereas in adult Leydig cells, androgen-independent way (El Gehani et the expression is estrogen-insensitive and al., 1998; Grocock et al., 1988; Hutson, gonadotropin-dependent (Balvers, et al., 1987; O Shaughnessy et al., 1998). 1998).

82 Male infertility today #4 Human INSL3 gene maps to chromo- were first discovered within the homeotic some 19p13-p12, and consist of two exons: complex (HomC) of Drosophila (Akam, exon 1 encodes for a signal peptide (that is 1989). They encode transcription factors all present in the preproprotein), the B chain of which share a helix-turn-helix DNA-bind- and the first eight aminoacids of the C-pep- ing motif, the homeodomain (Kissinger et tide; exon 2 encodes for the rest of the C- al., 1990). In mammalian genomes, four peptide and the A chain (Burkhardt et al., Hox complexes designated HoxA, B, C, D 1994). Insl3 appears to be necessary for and a total of 39 genes are found, located the development of the mesenchymal com- on four different chromosomes (Branford et ponent of the gubernaculum (the predomi- al., 2000). These genes display remarkable nat muco-polysacharide structure in conservation throughout evolution with human, dog, or pig gubernaculum in con- nearly the same sequence in mice as in trast to the gubernaculum of rodents which humans (Duboule & Dolle, 1989). Thus, is mainly composed of cremasteric muscle murine Hox a, b, c, d (chromosome 6, 11, with a mesenchymal core) (Bartlett et al., 15, 2, respectively) correspond to human 2002; Donaldson et al., 1996). The Insl3+/- HoxA, B, C, D (chromosome 7, 17, 12, 2, male mouse phenotype appears to resem- respectively) (Kolon et al., 1999). They have ble many cases of human TMD, where par- a key role in the morphogenesis of seg- tial TD at birth often is self-corrected (Nef & mental structures along the primary antero- Parada, 1999). However, only two studies posterior body axis, such as branchial arch- so far (Marin et al., 2001a; Tomboc, et al., es, vertebrae, cranial nerves and ganglia 2000) have implicated INSL3 gene muta- (Botas, 1993; Krumlauf, 1994). They are tions (3 in total) in human TTD while the expressed in many cell types acting as majority of studies have failed to indicate master regulatory switches or transcription such an association (Baker et al., 2002; factors that specify axial identity. In addition, Koskimies et al., 2000; Krausz et al., 2000; they control the growth and differentiation of Lim et al., 2001; Marin et al., 2001b; cells (Botas, 1993). The single Drosophila Takahashi et al., 2001). Furthermore, a mis- Abdominal B (AbdB) gene at the 5’ end of sens mutation affecting the structure of the HomC (which specifies the identity of LGR8 has been recently reported among the Drosophila posterior segments) is rep- 60 affected individuals (Gorlov et al., 2002). resented by 5 paralogous groups within Although these results point to the notion mammalian Hox clusters (16 genes in total) (Krumlauf, 1994). These AbdB-related that mutations involving the human INSL3 genes constitute a dinstinct subfamily of and/or the LGR8 genes are not common HOX genes that exhibit posterior domains causes of UT, it is difficult to interprete their of expression (Krumlauf, 1994; Izpisua phenotypic effects and there is a great Belmonte et al., 1991), including the devel- need for further functional studies in order oping genitourinary system in vertebrates to clarify their potential influence on TD. The (Celniker & Lewis, 1987; Dolle et al., 1991). exact signifficane of this hormone in the Specifically the AbdB-related Hoxa-10 gene pathophysiology of TD awaits generally fur- is expressed in posterior domains of the ther testing. developing mouse embryo, including inter- 3. The role of Homeobox genes mediate mesoderm, which gives rise to the Other genes possibly implicated in TTD gonads and genitourinary tract (Benson et include homeobox (Hox) genes. Hox genes al., 1995).

Male infertility today #4 83 Recent studies of murine knockout mod- fifth lumbar nerves are transformed to first els of Hoxa-10 (Satokata et al., 1995; Rijli to fourth lumbar nerve identity) (Rijli et al., et al., 1995) have demonstrated male 1995; Satokata et al., 1995), it has been mutants which are fully viable, but present speculated that TMD in this model might be with uni- or bilateral UT (small testes vary- due to incorrect specification of GFN ing from perinephric to lower abdominal motoneurons and/or abnormal target tissue space in location) as well as developmental innervation (Rijli et al., 1995). Mutations of abnormalities of the gubernaculum and Hoxa-11, also expressed in para- GFN with resultant abnormal spermatogen- mesonephric mesenchyme, affect similarily esis and sterility. Anterior homeotic trans- the male reproductive tract development. formations of the male reproductive tract Direct comparison of gubernacular defects with the proximal ductus deferens trans- (abnormally thin, elongated with apparent formed into the epididymes have also been failure of formation of the cord and muscu- reported (Benson et al., 1996). Male lar bulb) do not reveal any overt differences homozygotes are normally virilized, in con- between Hoxa-10-/- and Hoxa-11-/- trast to testicular feminization (Lyon & (Branford et al., 2000). Similarily Hoxa- Hawkes, 1970) and GnRH-deficient 10+/-/Hoxa-11+/- transheterozygotes dis- (Radovick et al., 1991) models of TMD. play analogous male reproductive tract Therefore, hormonal derangements appear defects. However, Hoxa-10-/+ and Hoxa-11- unlikely to cause TMD in Hoxa10-deficient /+ male mice (Branford et al., 2000), pres- mice (Satokata et al., 1995; Rijli et al., ent with normal TD and gubernacular 1995). In situ hybridization at 15.5 dpc indi- development. Thus, Hoxa-10 and 11 appear cates that both the gubernacular cord and to be functionally redundant in the develop- bulb are sites of strong Hoxa10 expression ment of the male reproductive system, such in the male mouse embryo and this expres- that the quantitative loss of any of the two sion continues postnatally (Satokata et al., Hoxa-10/Hoxa-11 alleles results in similarly 1995). Furthermore, analysis of the guber- severe abnormalities (Branford et al., naculum in Hoxa10-deficient males reveals 2000). that shortening of the cord and outgrowth of HOXA10 is located at the human chro- the bulb have failed to occur (Satokata et mosome 7 and consists of 2 exons. An al., 1995). Cremasteric myocytes, although increased amount of genetic alterations present, are disorganized and reduced in (point mutations and polymorphisms) locat- number (Satokata et al., 1995). ed in exon 1 of this gene have been identi- Furthermore, the processus vaginalis, the fied in some boys with UT compared to nor- inguinal canal and the scrotal sac are either mal controls (Kolon et al., 1999). This was missing or partially formed (Rijli et al., most evident in familial cases of UT. 1995). Since the above findings are com- However, functional analysis of the muta- bined with a morphological (anterior) trans- tions has not yet been performed and spe- formation of vertebrae (anterior homeotic cific associations between mutations and transformation of L1 vertebra to T13, caus- TTD remain to be studied. Further exami- ing an extra T13 vertebra) and spinal nation of the paralogous genes of HoxA10 nerves (i.e. the last thoracic nerve is trans- (HoxB10, C10, and D10) and its ortholo- formed to an intercostal nerve identity, the gous genes (HoxA9 and A11) will also help first lumbar nerve is transformed to a last elucidate the role of these regulatory genes thoracic nerve identity, and the second to in TTD.

84 Male infertility today #4 INGUINOSCROTAL PHASE OF et al., 1992) has been demonstrated, TESTICULAR DESCENT although this is disputed by some authors in favor of involution and eversion of the 1. The role of abdominal pressure and gubernacular cone (Elder et al., 1982; gubernaculum Hadziselimovic et al., 1979; Radhakrishnan The second phase of TD, ISTD, is cur- & Donahoe, 1981). Recently it has been rently thought to be regulated by a combi- suggested that the gubernacular bulb nation of mechanical and hormonal factors. actively proliferates after birth in rodents It is hypothesized that the abdominal pres- and possibly differentiates into new cremas- sure caused by the growth of the viscera ter muscle cells (Hrabovszky, et al., 2002) (Frey et al., 1983; Frey & Rajfer, 1984; while in human (Costa et al., 2002), guber- Quinlan et al., 1988), combined with puta- nacular connective tissue undergoes such tive gubernacular morphological and com- an extensive remodeling during TD that positional changes contribute to the testes finally gubernacullum becomes an essen- push down through the inguinal canal tially fibrous structure rich in collagen and (Hutson et al., 1997). Conditions that result elastic fibers. Such changes decrease its in UT hypothetically associated with size and, thus contribute to other forces decreased intra-abdominal pressure that cause the testis move toward the scro- include prune-belly syndrome, cloacal tum. Due to lack of smooth muscle cells, exstrophy, omphalocele, gastroschisis, and and the small amount of striated muscle a number of syndromes that include both cells, active contraction of the human UT and congenital abdominal wall muscular gubernaculum is less likely to be an impor- defects or agenesis (Kaplan et al., 1986; tant factor in ISTD. Koivusalo et al., 1998). Abdominal pressure probably has an ancillary role in TTD, but 2. The inguinoscrotal phase of thereafter it plays a more significant role testicular descent is androgen- during ISTD (Attah & Hutson, 1993; dependent Quinlan et al., 1988). Androgens may also It is generally agreed that ISTD is andro- play a role together with abdominal pres- gen-dependent (Hadziselimovic, 1983; sure at this stage of development to deliver Hutson & Beasley, 1992; Schneck & the testis into the scrotum (Frey & Rajfer, Bellinger, 2002). Migration of the gubernac- 1984; Frey et al., 1983). Frey et al. (1983) ulum beyond the inguinal region is absent demonstrated experimentally that in the in gonadotropin-deficient animals (Grocock presence of androgen a silicone prosthesis et al., 1988) as well as in both animals with descends from the abdominal cavity into testicular feminization and humans with the scrotum, but this occurs less frequently complete androgen resistance (Hutson, when androgen is removed. 1986). Furthermore, about 50% of animals The role of the gubernaculum in ISTD treated prenatally with the anti-androgen remains controversial. Regression of the flutamide also have deraranged gubernacu- bulky bulb, which is particularly prominent lar migration and delayed regression (Goh in the pig, was thought to be sufficient to et al., 1994a; Husmann & McPhaul, 1991a; allow the testis to reach the scrotum Husmann & McPhaul, 1992; McMahon et (Wensing, 1968; Wensing, 1973a; Wensing, al., 1995; Spencer et al., 1991) Regression 1986). A significant active migratory phase of the gubernacular bulb appears andro- in human (Heyns, 1987) and rodents (Fallat gen-dependent since in the human with

Male infertility today #4 85 complete androgen resistance the guber- reported to be 20% the amount measured naculum remains enlarged, with failure of in the urogenital sinus (George & Peterson, extra-cellular matrix resorption (Hutson & 1988). Donahoe, 1986; Radhakrishan & Donahoe, 2.1. The genitofemoral nerve hypothesis 1981). Quite recently a new hypothesis has The way (direct or indirect) of andro- been generated according to which, androgenic action on the gubernaculum remains gens act primarily on the GFN, rather than controversial. It seems quite possible that directly on the gubernaculum. As a result, androgens act idirectly on the gubernacu- the GFN releases a neurotransmitter (pro- lum to mediate ISTD. AR localization stud- posed to be the calcitonin gene-related ies in humans have demonstrated the pres- peptide (CGRP)) that acts as a second ence of the receptor in the cremaster mus- messenger of androgenic stimulation on cle but not in the gubernaculums (Johansen & Klein, 1993). However, these the gubernaculum to mediate ISTD (GFN studies were performed on postnatal tis- hypothesis) (Hutson & Beasley, 1987). The sues only, and it is not presently known origin of the GFN hypothesis lays on the whether the AR is expressed in the guber- experiments of Lewis (1948) who transect- naculum or cremaster muscle at the time of ed the GFN in newborn rats and prevented human ISTD. Genetic alterations (mutations by this means TD, while testing the “traction or polymorphic CAG trinucleotide repeat theory”. At that time the cremaster muscle expansions of AR exon 1 which are directly was being considered as a source of mus- associated with underviriliztion) appear to cular traction to pull the testis into the scro- be an unlikely cause of isolated UT in tum. The idea was that since the cremaster human (Lim et al., 2001b; Sasagawa et al., muscle is supplied by the GFN, transection 2000; Suzuki et al., 2001; Wiener et al., of the nerve would paralyze the muscle and 1998). Specific ARs have been found in cul- prevent TD. Many workers were critical to tured porcine gubernacular fibroblasts this experiment because they believed that (Oprins et al., 1988), but the total AR con- contraction of the cremaster would pull the centration as well as both receptor binding testis up to the groin, rather down to the affinity and capacity have been reported to scrotum. Nevertheless, it was intriguing that be significantly lower than in recognized denervation blocks a process that is under androgen target organs such as the apparent androgenic control, leading to the prostate and urethra, and comparable to speculation that androgens act via the that of male or female striated muscle nerve (Hutson & Beasley, 1987). (Heyns & Pape, 1991; Heyns et al., 1988). Androgens should lead to prenatal GFN 5α-reductase activity has been reported to modification (Hutson & Beasley, 1987), as remain constant throughout gestation in the even in species with postnatal gubernacular gubernaculum of the pig, at lower levels migration, the nervous system would than those seen in prostate or urethra require earlier modification. Studies with (Heyns et al., 1993). The AR level of antiandrogens confirm that ISTD, although expression at the mesenchymal core of rat occurring postnatally in rodents, can only gubernaculum declines during ISTD be blocked by prenatal treatment (Goh et (Husmann & McPhaul, 1991b). The total al., 1994a; Husmann & McPhaul, 1991a; amount of specific androgen bound in the Husmann & McPhaul, 1992; Shono, et al., neonatal rat gubernaculum has been 1994; Spencer et al., 1991).

86 Male infertility today #4 The GFN hypothesis is consistent with or after selective sensory denervation with the results of distal transection of the capsaicin (which depletes CGRP from the gubernaculum in neonatal rats, which pre- nerves) gubernacular contractility, in vents gubernacular postnatal migration and response to exogenous CGRP in organ cul- ISTD while proximal transection which pre- tures, is restored due to an increase in the serves gubernacular nerve supply (the gubernacular receptor content (Hrabovszky GFN supplies the gubernaculum from its & Hutson, 1999; Terada et al. 1995). posterio-caudal surface (Tayakkanonta, A role of the GFN and its neurotrans- 1963; Larkins & Hutson, 1991)) fails to mitter CGRP in TD has been well estab- block ISTD (Beasley & Hutson, 1988; Bergh lished (Beasley & Hutson, 1987; Beasley, et al., 1978). Furthermore, this hypothesis & Hutson 1988; Bergh et al., 1978; Goh predicts that neuronal anomalies affecting et al., 1993; Hrabovszky & Hutson, 1999; the GFN nucleus should be associated with Lewis, 1948; Momose et al., 1992; Park UT. This is in fact the case for children with & Hutson, 1991; Shono et al., 1995; spina bifida, particularly when the Terada et al., 1994; Terada et al., 1995; myelomeningocele is in the high lumbar Yamanaka et al., 1993). However, the region (above L4; the origin of GFN) exact mechanism by which circulating (Hutson et al., 1988). A similar result can androgens act on the GFN is not yet be obtained by neonatal rat spinal cord known. Intrauterine flutamide exposure transection at the level of low thoracic-high during gestational days 16-19 leads to lumbar region leading to a 75% incidence UT in rats at a frequency of ~50% of UT (Hutson et al., 1988). (Husmann & McPhaul, 1991a; Spencer There is plenty of evidence supporting et al., 1991). The exact mechanism is the role of CGRP in stimulating the devel- uncertain, but it has been suggested that oping gubernaculum. In organ culture, flutamide prevents circulating androgens neonatal rodent guberaculae show a dose- from masculinizing the GFN during this dependent response to exogenous CGRP critical phase of development (Hutson & by rhythmic contractions (Park & Hutson, Beasley, 1987). A specific search for AR 1991; Momose et al., 1992). Prior transec- in the fetal rat lumbar spinal cord shows tion of the GFN results in the gubernaculum androgenic binding as early as 15 days becoming more sensitive to CGRP (Shono of gestation (Cain et al., 1994a), which is et al., 1995), which is consistent with up- consistent with androgens stimulating regulation of CGRP receptors (Terada et differentiation of the GFN and its nucle- al., 1995). CGRP receptors are most abun- us. Furthermore, a significant decrease dant during the period of gubernacular in the neural diameter and the number of migration to the scrotum (Yamanaka, et al., myelinated axons within GFN ipsilateral 1993). Prenatal flutamide treatment to UT has been reported in flutamide- increases the density of CGRP receptors in treated rats (Husmann et al., 1994). the gubernaculums (Terada et al., 1994).In Previous studies focused on the motor mutant trans-scrotal rats (TS; congenitally nucleus of the GFN (L1-L2 in the spinal TMD from unknown causes-natural model cord) as the primary target organ of circu- of UT), CGRP receptors are diminished lating androgens (Barthold et al., 1994; and the gubernaculum is resistant to Barthold et al., 1996; Goh et al., 1994a; exogenous CGRP (Goh et al., 1993; Terada Goh et al., 1994b; Larkins, 1991). The GFN et al. 1995). However, after GFN transection motor nucleus is sexually dimorphic in

Male infertility today #4 87 rodents, but antiandrogen treatement causing on the sensory nucleus of GFN instead es just minor decrease in its cell content of the motor nucleus as previously thought (Barthold et al., 1996; Goh et al., 1994b). (Hrabovszky et al., 2000). Despite the The sexual dimorphism of cell number in extensive work, the GFN hypothesis is still the newborn rat is lost after flutamide treat- questionable since CGRP has been shown ment but due to an increase of the number to fail to induce murine TD (Houle & Gagne, of cell bodies in females and not due to a 1995). Additionally, CGRP is a neuromus- significant loss of cell bodies in males as cular neurotransmitter that has been expected (Goh et al., 1994a). Furthermore, demonstrated to act on the muscular com- it has been claimed that androgens have no ponent of the rodent’s developing guber- effect on the GFN mortor nucleus since naculum, which is primarily cremasteric both in rats (Barthold et al., 1994) and mice muscle (Husmann & Levy, 1995). Human (Larkins et al., 1991) with testicular femi- gubernaculum consists mostly of nization, its size is found to be similar to mucopolysacharides and does not contain that of normal animals while rat GFN almost any muscular component (target of motoneurons virtually lack CGRP CGRP action) (Costa et al., 2002; Heyns et immunoreactivity (Barthold et al., 1994; al., 1990). This fact most likely excludes Newton et al., 1990; Schwindt et al., 1999). CGRP from playing a significant role in Dense CGRP immunoreactive varicose human TD (Husmann & Levy, 1995; fibres surrounding GFN motoneuron cells in Schneck & Bellinger, 2002). male rats which are absent in females have Alternatively, Levy and Husman (1995) been described (Newton et al., 1990), and have proposed an important role for EGF in recently it has been shown that the rat sen- the mediation of androgen action. sory nucleus of the GFN (L1-L2 dorsal root Supraphysiological doses of EGF can ganglia) is also sexually dimorphic reduce the incidence of flutamide-induced (Hrabovszky et al., 2000). In addition, it has TMD in rodents, and vice versa, decreased been reported that flutamide decreases the EGF concentrations in the serum of dams number of cells both in male and female rat result in a significant incidence of UT and GFN sensory nucleous, but a significant epididymal abnormalities in the offspring. decrease of the CGRP immunoreactive EGF stimulates gonadotropin sectretion by cells is detected only in males (Hrabovszky the placenta, and it has been proposed that et al., 2000). Furthermore, it has been EGF effects may reside mainly in the regu- shown that in TS rats the sensory nucleus lation of placental function (Levy & of the GFN ipsilateral to an UT contains Husmann, 1995). However, there are no significantly more CGRP immunoreactive data up to now about the role of EGF in the cells compared to the contralateral normally descent of human testis. descended testis (Hrabovszky et al., 2001). It has been hypothesized that TMD in this 4. EPIDEMIOLOGY OF TESTICULAR strain may be due to a primary defect of MALDESCENT CGRP receptors and the increased number INCIDENCE OF TESTICULAR of CGRP immunoreactive cells represent MALDESCENT an insufficient compensatory mechanism. Based on these results, the GFN hypothe- Isolated UT represents the most com- sis has been recently modified to include a mon congenital abnormality of the male possible role of circulating androgens act- genitalia of human and other mammalian

88 Male infertility today #4 species (e.g. in the dog the incidence is horse (Wintzer, 1986) that exhibit more estimated ~1.2-5% (Boothe, 1993)) and commonly abdominal and incomplete generally one of the most common human abdominal TD (testicle in the abdominal congenital malformations at birth, affecting cavity-tail of the epidymis and vas deferens more than 3.5% of male newborns into the inguinal cannal) with a ratio abdom- (Berkowitz et al., 1993; Ghirri et al., 2002; inal/inguinal ~2:1. Racial as well as ethnic James & Jean, 1998; John Radcliffe differences (Heinonen et al., 1997; Hospital Cryptorchidism Study Group, Berkowitz & Lapinski, 1996) have been 1992; Scorer, 1964; Thong et al., 1998). reported, although there are studies recog- Unilateral UT is more common than bilater- nizing no such differencies (Berkowitz, et al in mammals (the incidence of bilateral al., 1993). In horses UT is more commonly UT is estimated ~1.6-1.9% of cases in seen in ponies (Wintzer, 1986), while in the human (Schneck & Bellinger, 2002), ~10- dog an increased incidence (~2.7 times 16% of cases in horses (Wintzer, 1986) and greater risk) is observed in small breeds ~20.2% of cases in dogs (Johnston, et al., (<9.1Kg, e.g. Chihuahua, Yorkshire terrier 2001)). In most cases in human, testes e.t.c) (Boothe, 1993; Johnston, et al., have descended at or below the internal 2001). There is strong evidence that the inguinal ring (intra-abdominal UT comprises incidence rate is much higher (up to 10 a small group of ~5-10%) (Hutson et al., times) in premature infants (<37 GW, birth 1997). This is the case also in the dog that weight < 2500 g) than in full-term babies exhilit intra-abdominal UT in ~18% of cases (Berkowitz et al., 1993; Ghirri et al., 2002; (Boothe, 1993; Johnston, et al., 2001) James & Jean, 1998; John Radcliffe (Figures 1, 2) in contrast for example to the Hospital Cryptorchidism Study Group,

Figure 1. Undescended tesis. Edwards’ model applied on data from Greek population (1991-1998) indicates that the maximum incidence rate is observed during spring (peak in March) and the minimum incidence rate is observed during autumn (trough in September): x2EDWARDS value at 2df is statistically significant (p<0.001)k

Male infertility today #4 89 Figure 2. Undescended tesis. Adjusted frequencies fitted to the simple harmonic curve given by Edwards’ test. Data are satisfactorily described by the simple harmonic curve since the two curves do not differ significantly (x2 goodness of fit test value at 11 df is not statistically significant; p>0.2)

1992; Scorer, 1964; Thong et al., 1998). Hispanic ethnicity, family history of UT, con- Since the testes normally descend during sumption of cola-containing drinks during late pregnancy, TMD can be considered as the pregnancy, low birthweight, and a normal phenomenon in premature chil- preterm delivery (Berkowitz & Lapinski, dren and spontaneous descend may be 1996). expected. The completion of TD usually SEASONAL TRENDS IN THE occurs during the first weeks after birth INCIDENCE OF TESTICULAR (approximately 70%-77% of UT will sponta- MALDESCENT neously descend early, usually by 3 months of age) so the incidence of UT by the end of Temporal analysis (investigation of sec- infancy declines to approximately 0.7-1% ular and cyclic trends) has been widely and remains constant thereafter (James & employed as a means to elucidate the etiol- Jean, 1998; Schneck & Bellinger, 2002;). ogy of diseases. The investigation of sea- Factors that predict complete early postna- sonality (the most important type of cyclic tal TD include bilateral anomaly, normal variations) is especially used for the basic scrotum anatomy and non-ectopic position, etiological description of congenital abnor- in contrast to the presense of a small or malities. Temporal analyses suggest that poorly rugated scrotum and hypospadias there is an increasing secular trend in the (John Radcliffe Hospital Cryptorchidism incidence of UT during the last few decades Study Group, 1992). On the other hand fac- in some countries (Berkowitz et al., 1993; tors that may indicate “late descenders” John Radcliffe Hospital Cryptorchidism (after 1 year of life) include black or Study Group, 1992; Scorer, 1964), however

90 Male infertility today #4 there are still contrary reports (Ghirri et al., al., 1989; Jackson & Swerdlow, 1986). The 2002; Kaleva, et al., 2001a). Furthermore detection of such a pattern in a country large regional differences among related such as Greece with almost no significant countries have been reported (e.g. in differences in daylight length among sea- Finland the incidence is clearly lower than sons is strong evidence against the theory in Denmark) (Kaleva, et al., 2001a; Kaleva, of light. Furthermore, we have showed that et al., 2001b). More epidemiological studies the maternal peripheral serum human using standardized diagnostic criteria and chorionic gonadotropin (β-hCG) levels at examination techniques are required in the 26th GW (beginning of the ISTD) are order to obtain a more precise estimation of significantly lower during winter compared the secular and regional trends in the inci- to summer (Mamoulakis et al., 2002a). dence of UT, and detect possible environ- Considering that increased incidence of UT mental effects (Toppari et al., 2001). On the was observed during March or May, it other hand, there seems to be a general appears that the beginning of the ISTD in agreement on the seasonality of UT with December-January or February-March peri- spring and autumn being the seasons of od respectively, is accompanied by an significantly highest (peak in March) and increased incidence of UT. Thus low envi- lowest (trough in September) incidence ronmental temperatures at the start of ISTD respectively (Berkowitz et al., 1995; Czeizel may be related with an increased probabili- et al., 1981; Hjertkvis et al., 1989; Jackson ty of failure of the testis to start or complete & Swerdlow, 1986). Most researcher have ISTD. Since ISTD is androgen-dependent, it attempted to attribute the above phenome- appears that low environmental tempera- non to analogous seasonal variations of tures at the 26th GW are accompanied by a sex hormones. It has been suggested that decrease in maternal β-hCG profiles and variations in daylight length influencing the detrimentally affect the embryonic Leydig maternal pineal gland function may alter cell secretory function and the embryonic the secretion of melatonin, which in turn androgen production. It is known that may influence maternal pituitary and ovari- human embryonic testicular androgen pro- an hormonal activity and maternal hormon- duction in the late gestational period is al balances causing alterations in fetal tes- influenced not only by the embryonic ticular function. The latter could be respon- luteinizing hormone (LH) but also by the sible for the maldescent (theory of light). maternal hCG which is more potent as However, by strictly applying statistical gonadotrophin than the LH (Clements et tools especially designed for the investiga- al., 1976). The decreased embryonic tion of seasonality in congenital malforma- Leydig cell secretory function may finally tions with respect to their recommended result in an increased incidence of limitations, we have recently showed partial/total failure of ISTD and subsequent- (Mamoulakis et al., 2001; Mamoulakis et ly development of UT. The above hypothe- al., 2002a) that the monthly incidence of sis is supported by the decreased inci- births of children with isolated UT in Greece dence of UT in September. Our results follows a documented cyclic pattern of sim- have shown clearly that the occurrence of ple harmonic type (peak in March/May, the 26th GW in June-July period is accom- trough in September) similar to that panied by a decreased incidence of UT. We observed in other studies in Northern have suggested that relatively high environ- Europe (Czeizel et al., 1981; Hjertkvis et mental temperatures at the 26th GW do not

Male infertility today #4 91 influence detrimentally (in terms of TD) the trough (diminished androgen production by hormonal profiles/balances of the mother or the fetal testis) in winter may be speculat- fetus with an overall result a minimal distur- ed. Under these settings it could be hypoth- bance on the beginning/completion of the esized that the coincidence of the 8th or ISTD. Such a suggestion could explain the 26th GW with the winter period could results on the seasonal variations of UT not increase the risk for the phenotypical only in Greece but also in Northern Europe expression of hypospadias or UT, respec- (Hungary, Grand Britain, and Sweden) tively. β-hCG is a possible example of such (Czeizel et al., 1981; Hjertkvis et al., 1989; an androgen stimulator since it shows sea- Jackson & Swerdlow, 1986). In contrast, the sonal variation both in early (Kiely et al., theory of light cannot explain the seasonali- 1995) and late (Mamoulakis et al., 2002a) ty of births of children with UT in Greece pregnancy with lower levels in winter. Since and the similar seasonal variations in the no significant differences in daylight length northern and southern part of Sweden that are found among seasons in Greece, the exist despite the large differences in day- detection of such paterns (Figures 1-4) is light hours between the north and south strong evidence against the theory of light. Sweden (Hjertkvis et al., 1989). Seasonal alterations of weather tempera- In another study (Mamoulakis et al ture may be given as a possible example of 2002b), based on 1178 cases of isolated an alternative cause contributing via the UT and 542 cases of simple hypospadias above described mechanism to the season- we have evaluated the epidemiological rela- al variations of UT and hypospadias tionship, in terms of seasonality by month observed. of birth, between these two usually associ- Recently, based on experimental, epi- ated congenital malformations (Hjertkvis et demiological and clinical evidences, a new al., 1989; John Radcliffe Hospital concept has been proposed according to Cryptorchidism Study Group, 1992; which poor semen quality, testicular cancer, Weidner, et al., 1999) (Table 2) We have UT and hypospadias are symptoms of one shown that the monthly incidence of underlying entity, the testicular dysgenesis hypospadiac births in Greece follows a doc- syndrome (TDS), which may be increasing- umented cyclic pattern of simple harmonic ly common due to adverse environmental type with autumn being the season of sta- influences probably upon a susceptible tistical predominance (peak in October) and genetic background (Skakkebaek et al., spring the season of lowest incidence 2001). TDS is suggested to be the result of (trough in April). Considering that increased disruption of embryonal programming and incidence of hypospadiac births is observed gonadal development during fetal life. We during October, it appears that the begin- may suggest that at least some cases of ning (8th GW) of the crucial period of nor- TMD could be attributed to the relatively mal differentiation of human male external lower levels of placental β-hCG during win- genitalia (8th-16th GW) takes place in late ter months that acting upon a testis already winter. Since both crucial embryonic peri- being dysgenetic by it nature, (with a bor- ods for the final TD and the differentiation- derline Leydig cell function) possibly result development of the male urethra are con- in disanalogous lower levels of androgens sidered to be androgen-dependent, the produced. If these relatively lower placental existence of an androgen stimulator that fol- β-hCG levels are observed early (8th—9th) lows a cyclic pattern of variation with a or late in gestation (26th GW) the manifes-

92 Male infertility today #4 ) Ei frequencies 1 mula ni' = c ni ™mi / mula is divided (i.e. k = 12). ') (E i )(n i )(n Ei frequencies frequencies frequencies 1 ') (E i Undescended testis Hypospadias )(n i )(n i (m live births frequencies frequencies Undescended testis and hypospadias: Data from Greek population (1991-1998) ) Expected frequencies for Edwards' test are proportional Edwards' to [1 + _ Cos (_i-_max)] (see Figures 1-4 and ref.) Expected frequencies for details) 334 for Table 2. Table Ei Adjusted frequencies are used in order to adjust Edwards' test for a variable population at risk. a variable test for Adjusted frequencies are used in order to adjust Edwards' the for are calculated by They k mi where c is a scale factor such that ™ni' = ™ni and k is the number of the sectors in which the given time span (one year) time span (one year) of the sectors in which given such that ™ni' = ™ni and k is the number k mi where c is a scale factor JanuaryFebruaryMarchAprilMay 33346June 31423 39001July August 33851September 36346 33338October 36526 98 36261 105 34060November 105 35167December 106 37144TOTAL 80 102.8 103 116.9 961 94.2 112 86 109.5 34712 96 104.3 77.0 110.1 103 108.1 421175 91.9 92.3 108.0 112.9 88.3 95.5 111.9 88 83.2 97.0 107.1 84.4 1178 46 43 89.2 100.0 46 44 88.7 86.1 1178 24 48.5 50 48.1 33 49 41.5 96.5 45.7 46 47 1178 46,4 25.3 42,6 66 48.4 32.0 47.1 43,7 39,4 47.5 47.0 37,6 48 51,1 62.5 542 37,9 52,8 52,4 40,1 48.6 47,8 542 50,1 542 (E Month of birth male Total Observed Adjusted Expected Observed Adjusted Expected

Male infertility today #4 93 Figure 3. Hypospadias. Edwards’ model applied on data from Greek population (1991- 1998) indicates that the maximum incidence rate is observed during autumn (peak in October) and the minimum incidence rate is observed during spring (trough in April): x2EDWARDS value at 2df is statistically significant (p<0.01)

Figure 4. Hypospadias. Adjusted frequencies fitted to the simple harmonic curve given by Edwards’ test. Data are satisfactorily described by the simple harmonic curve since the two curves do not differ significantly (x2 goodness of fit test value at 11 df is not statistically significant; p>0.2)

94 Male infertility today #4 tations may be hypospadias or UT respec- Jones et al., 1998), maternal smoking tively. The presence of TDS manifestations (Biggs et al., 2002), analgetics consump- may vary with the severity of underlying tion during pregnancy (Berkowitz & testicular dysgenesis. Lapinski, 1996), maternal blood group (A Rh+ and B Rh+) (Ghirri et al 2002), nulipar- RISK FACTORS OF TESTICULAR ity (Biggs et al., 2002; Hjertkvist et al., MALDESCENT 1989; Mayr et al., 1999), maternal obesity Establishing epidemiological factors that (Berkowitz & Lapinski, 1996) and, family affect the risk of UT becomes more compli- history of UT (Berkowitz & Lapinski, 1996). cated due to the complex interactions It has been reported that the occurrence of among anatomy, heredity, hormonal milieu, UT in families is 1.5-4% among the fathers, and socioeconomic or environmental condi- 6.2 among the brothers, and 0.67 among tions and there is not always a uniform first-degree male relatives of index patients agreement among the authors. with UT, supporting a multifactorial pattern Nevertheless, the risk factors for the pres- of inheritance (Czeizel et al., 1981). ence of an UT at birth and by the 1st year of age need to be considered. As described 5. CONSEQUENCES OF above, UT is more common in premature UNDESCENDED TESTIS: IMPAIRED and low birthweight male newborns. FERTILITY & TESTICULAR CANCER However, after adjusting for birthweight it Although UT is occasionally considered has been shown that gestational age is not as a minor congenital abnormality, its close an independent risk factor for UT. On the association with subfertility or infertility other hand, the effect of birthweight is not (Chilvers & Pike, 1989; Hadziselimovic, influenced by adjusting for gestational age. 1984; Leissner et al., 1999; Rozanski & Therefore, low birthweight is the principal Bloom) and the fact that it represents a determinant of UT at birth and at 1 year of well-established risk factor for testicular age, independent of the length of gestation cancer (Chilvers et al., 1986; Depue 1983; (Hjertkvist et al., 1989; Jones et al., 1998; Henderson et al., 1979; Krabbe et al., 1979; Mayr et al., 1999; Weidner et al., 1999). Morrison, 1976; Moss et al., 1986; Pottern Other risk factors that have been reported et al., 1985; Schottenfeld et al., 1980) indi- include: congenital malformations malfor- cate its clinical and epidemiological impor- mations (Biggs et al., 2002; Hjertkvist et al., tance. 1989; John Radcliffe Hospital Cryptorchidism Study Group, 1992; A. IMPAIRED FERTILITY IN PATIENTS Weidner et al., 1999) (mainly hypospadias, WITH A HISTORY OF TESTICULAR other genitourinary and digestive complica- MALDESCENT tions, dislocation/subluxation of the hip), complicated pregnancy (Biggs et al., 2002; 1. Epidemiology of impaired fertility Hjertkvist et al., 1989; Jones et al., 1998; Uni- or bilateral UT is a common etiolog- Mayr et al., 1999) (toxemia of pregnancy, ic factor of azoospermia (David et al., oligohydramnio, pregnancy induced hyper- 1979). The incidence of azoospermia is tension, pre-eclampsia, hyperemesis), estimated around 0.4-0.5% in the general cesarean section (Hjertkvist et al., 1989), population (Hadziselimovic & Herzog, twin gestation (Berkowitz et al., 1993), 2001). However, about 89% of untreated breech presentation (Biggs et al., 2002; patients with bilateral TMD develop

Male infertility today #4 95 azoospermia (almost all men are infer- with controls (Coughlin et al., 1998; Lee et tile)(Hadziselimovic, 2002; Hadziselimovic al., 1998). & Herzog, 2001; Weidner, 2002). Among 2. Pathogenesis of impaired fertiity men who have untreated unilateral UT, a The pathogenesis of reduced fertility percentage of 50-70% are azoospermic or seen in patients with UT has not been fully oligozoospermic; (Weidner, 2002). clarified and the exact cellular and molecu- However, the incidence of azoospermia in lar mechanisms involved remain still elu- unilateral UT has been reported to be sive. The following alterations have been around 13% regardless the patient is treat- discussed aetiologically: epididymal mal- ed or untreated (Hadziselimovic, 2002).It formations, decreased number of tubules has been postulated that paternity would be containing spermatogonia, decreased a better index for verification of fertility than number of spermatogonia per tubule, mild sperm count (Lee, 1993) since it is known concomitant hypogonadal situation, gener- that men with subnormal sperm counts can ation of sperm antibodies, contralateral father children (Barfield et al., 1979). testicular damage, increased apoptotic Fertility data among men attempting pater- rate in germ cells, microdeletions in the nity indicate that this index is significantly euchromatic region of the Y chromosome compromised in men with previous bilateral long arm (Yq11). UT (61.5-65.3%) but not (as initially thought) in men with previous unilateral UT 2.1. Epididymal malformations (89.7%) compared with the general popula- Epididymal anomalies are associated tion (93.2-94.6%) (Lee et al., 1996; Lee et with TMD in 36-43% of cases (Gill et al., al., 1997; Lee & Coughlin, 2001; Lee & 1989; Koff & Scaletscky, 1990; Mollaeian et Coughlin, 2002). Furthermore, men with a al., 1994). They can be classified into two single testis regardless of the origin of the groups: anomalies of ductal fusion and loss (unilaterally maldescended testis not anomalies of ductal suspension. Anomalies found at surgery or found to be atrophic at of ductal fusion usually accompany intra- orchiopexy and was either retained or abdominal and higher inguinal location of orchiectomized) have similar paternity rates the testes (Gill et al., 1989; Mollaeian et al., compared to either men with corrected UT 1994). They are consisted of loss of conti- or controls (Lee & Coughlin, 2002). There is nuity between testis and the epididymis or evidence of subfertility requiring a longer an absent or attenuated segment of the exposure to regular intercourse without epididymis or vas deferens. The most com- contraception (>12months) only in men with mon anomaly of ductal fusion is a flimsy previous bilateral UT (Lee et al., 1996; Lee attachment of the head of the epididymis to et al., 1997). Neither preoperative testicular the testis (50%), whereas complete separa- location Lee et al., 2000) nor small testicu- tion or atresia of the epididymis and atresia lar size at orchidopexy (Lee et al., 2001) in of the vas deference occurs in 5-21%. men with previous unilateral UT appear to Anomalies of ductal suspension occure be a major determinant of fertility according more frequently in the more distal testes to paternity, sperm counts or hormone lev- (Gill et al., 1989; Mollaeian et al., 1994). els. Higher FSH levels, lower sperm counts, They are classified as an angulated epi- and parenchymal suture placement at didymis (separated from the testis) by a orchidopexy correlate inversely, both in wide mesentery (80%), a partially separat- men with previous unilateral UT compared ed epididymis with an elongated tail associ-

96 Male infertility today #4 ated with a long loop vas deferens (12%) matogonia into primary spermatocytes), and a normally attached epididymis with an prophase of the first meiotic division is elongated tail associated with a long loop accompanied by increases in the total num- vas deference (8%). Such anomalies may ber of both germ cells and Ad spermatogo- coexist with an excellent testicular histology nia (Hedinger, 1982; Huff et al., 1989; Huff and may possibly explain the decreased et al., 1993; Nistal & Paniagua, 1984; fertility of some patients with TMD. Schindler et al., 1987;Scorer & Farrington, 1971). 2.2. Impaired testicular histology The histologic hallmarks associated with Impaired fertility has been linked to the UT are evident between 1st and 2nd year reduced number of testicular germ cells of life and include interstitial (peritubular) because patients with the lowest total germ fibrosis (Elder, 1988), hypoplasia of Leydig cell counts have the poorest spermiograms cells (the earliest postnatal histologic in adulthood (Hadziselimovic et al., 1987a). abnormality; evident from the first month of The balance between germ cell prolifera- life) (Huff et al., 1991), reduced numbers of tion, differentiation and apoptosis (see Leydig cells (Hadziselimovic et al., 1986; below) is critical to the maintenance of nor- Huff et al., 1989; Huff et al., 1993), age- mal spermatogenesis. Disruption of the fine independent/testicular location-age regulation of either of these processes may dependent Sertoli cell special degeneration lead to infertility. Normally, testicular germ pattern (dilated elements of rough endo- cells mature from the primitive gonocytes plasmic reticulum, vacuolization of the cyto- th (fetal stem cell pool) that appear at 8 GW plasm, mitochondria with poorly preserved (Holstein et al., 1971) through a series of cristae, increase in electron density of the steps, including the appearance of fetal matrix, elongation of the nuclei, irregulari- th spermatogonia at 15 GW (Holstein et al., ties of the nuclear membrane) (Rune et al., 1971) and adult dark spermatogonia (Ad; 1992), delayed disappearance of gono- adult stem cell pool (Clermont, 1966)) at 3 cytes, delayed apearance of Ad spermato- months of age (Seguchi & Hadziselimovic, gonia, failure of primary spermatocytes to 1974) to primary spermatocytes (the first develop and reduced total germ cell counts meiotic form) by 3-4 years (Seguchi & (Huff et al., 1989; Huff et al., 1991; Huff et Hadziselimovic, 1974). Therefore, during al., 1993; Huff et al., 2001). Reduced total the prepubertal period, two major matura- germ cell counts have been associated with tional steps occur within the testis: a) at 2-3 defects in the two normal spermatic matu- months of age the adult stem cell pool is rational steps during the prepubertal period established and replaces the fetal stem cell (Huff et al., 1989; Huff et al., 1991; Huff et pool; disappearance of gonocytes, appear- al., 1993; Huff et al., 2001), as well as to ance of Ad spermatogonia, and dramatic the enhanced Sertoli cell special degenera- reduction in the total number of germ cells tion pattern (Rune et al., 1992). Males with per tubule (Hadziselimovic et al., 1986; Huff UT are born with germ cells, although the et al., 1989; Huff et al., 1991; Huff et al., number of these cells may be reduced 1993) and b) at 4-5 years of age an (Cortes, 1999; Hadziselimovic, 1977; increase in germ cell maturation, and prolif- Hadziselimovic et al., 1987; Hedinger, eration as well as the onset of meiosis is 1994). No reduction in the mean number of observed; transient appearance of primary germ cells is detected during the first 7 spermatocytes (transformation of Ad sper- months of age (Huff et al., 1991). The

Male infertility today #4 97 abnormal persistence of the untransformed (FSH), LH, testosterone levels, and a mini- gonocytes to Ad spermatogonia results in a peak of testicular weight and volume total germ cell count that is similar to nor- (Cassorla et al., 1981; Forest et al., 1974; mal controls until about the 7th month, Siebert, et al., 1982). These findings sug- when secondary degeneration of untrans- gest that a surge in luteinizing hormone formed gonocytes leads to a decrease in releasing hormone (LHRH) causes LH the total germ cell count. The persistence of release. The latter stimulates Leydig cell high total germ cell counts in the first year production of testosterone which triggers of life is the basis for the traditional concept the maturation of germ cells. The cause of that UT is histologically normal until the normal onset of meiosis at 4-5 years of age second year of life (Huff et al., 2001). is the subject of continued study. An However, a reduction in the number of sper- increase in the levels of LH in urine matogonia has been reported to start as (Hadziselimovic et al., 1986) and increased early as 6 months of age in intra-abdominal prominence of juvenile Leydig cells testes (Hadziselimovic et al., 1987b). It has (Hadziselimovic, 1977) simultaneous with also been shown (based on the evaluation the appearance of primary spermatocytes of biopsies using the spermatic index) that in normal boys suggest that the onset of both intra-abdominal and intra-canalicular meiosis may also be stimulated by a minor testes appear to have the same fertility increase in hypothalamic-pituitary activity. potential (Hadziselimovic et al., 1987a; In UT, Leydig cell hypoplasia is one of McAleer et al., 1995; Wilkerson et al., 2001) the prominent early in life findings indicating exhibiting a sharp decline in the spermatic a possible defective gonadotropin secretion index during the first 1.8 years, which (Hadziselimovic et al., 1986; Hadziselimovic reaches a critically low value as early as 8- & Herzog, 1976; Huff et al., 1991). 9 months of age (Wilkerson et al., 2001). Furthermore, it has been reported that the normal surge in LH and testosterone at 2-3 2.2.1. Hypothalamic-pituitary axis months is blunted in boys with UT (Gendrel function et al., 1980). These findings suggest that in Several observations favor the hormonal infants with UT Leydig cells are understimu- (testosterone)-dependent explanation for lated by a defective hypothalamic-pituitary germ cell differentiation and maturation dur- axis and, therefore cannot produce a surge ing testicular development. Patients with in testosterone of sufficient magnitude to CAIS present with defects in the two post- trigger normal germ cell maturation and the natal steps of spermatogenesis (Sertoli formation of an adequate adult stem cell cell-only syndrome and absence of Ad pool. The cause of the defective onset of spermatogonia by the 1st year of life, meiosis in patients with UT is also a subject absence of primary spermatocytes and of debate. Low levels of urinary LH and Leydig cell hyperplasia) (Hadziselimovic, et FSH (Hadziselimovic, 1987), and impaired al., 1999) indicating that both the transfor- LH and FSH responses to stimulation with mation of gonocytes into Ad spermatogonia gonadotropin releasing hormone (GnRH) and the development of primary spermato- (Canlorbe et al., 1974; Forest, 1979). cytes are androgen-dependent steps. However, it should be noted that there are Furthermore, in normal male, the first matu- also contrary reports suggesting a normal rational step is accompanied by a transient gonadotropin secretion in infants with UT surge in serum follicle stimulating hormone (Bartolini et al., 1979; Cacciari et al., 1976;

98 Male infertility today #4 DeMunick Keizer-Schrama & Hazebrock, 1993). Apoptosis is a distinctive form of cell 1986). The fact that similar (but less severe, death that is defined by characteristic mor- of later onset, and less progressive) phological and biochemical events that changes are also seen in the contralateral result in the efficient elimination of cells descended testis (CDT) in cases of unilat- from a tissue without eliciting an inflamma- eral TMD, possibly supports the theory of tory response (Kerr et al., 1972; Wyllie et hypogonadotropic hypogonadism as a al., 1980). The hallmark of apoptosis is the cause of the increased incidence of infertili- rapid internucleosomal fragmentation of cell ty seen in males with unilateral UT DNA by nuclear endonuclease into multi- (Clermont, 1966; Hadziselimovic et al., mers of 180-200bp (Schwartz & Osborne, 1986; Huff et al., 1991; Huff et al., 1993; 1993; Schwartzman & Cidlowski, 1993). Huff et al., 2001, McAleer et al., 1995). Observations on the cellular morphological However, as we have recently shown (Ono changes include condensation and mar- & Sofikitis, 1997), a thermal injury on the gination of nuclear chromatin, cytoplasmic CDT in cases of unilateral UT could also condensation, budding of the cell mem- explain the impaired function of the scrotal brane, cellular shrinkage and fragmentation testis and the impaired fertility observed in into membrane bound apoptotic bodies such cases (see below). Nevertheless, it which are rapidly phagocytosed by should be noted that the development of macrophages or other neighbouring cells the CDT in unilateral UT remains controver- (Kerr et al., 1972; Majno & Joris, 1995). sial (Leissner et al., 1999) since there are Apoptosis may be caused by noxious also reports suggesting a primary (intrinsic) agents but, unlike necrosis, it may also abnormality in both testes even when only occur spontaneously or after a physiologic one is descended (Skakkebaek et al., stimulus such as a surge or withdrawal of a 2001), while others claim that the CDT is hormone (Tapanainen et al., 1993). not affected in unilateral TMD (Kirby et al., Deletion of a subset of testicular germ cells 1985; Mancini et al., 1965). by apoptosis occurs normally in the adult mammalian testis (Allan et al., 1992; 2.2.2. The role of apoptosis Bartke, 1995; Billig et al., 1995) as well as It has been recently suggested that the in the prepubertal human testis (Heiskanen degenerative process of testicular germ et al., 1996). It has been recently recog- cells in UT is associated with increased nized that apoptosis serves as an important apoptotic DNA fragmentation of these cells physiologic mechanism to limit the germ (Guo et al., 2001; Heiskanen et al., 1996; cell population to numbers which the Sertoli Henriksen et al., 1995a; Jara et al., 2002; cells can support (Allan et al., 1992; Bartke, Nandi et al., 1999; Ogi et al., 1998; Ohta et 1995; Billig et al., 1995; Sinha Hikim et al., al., 1996; Shikone et al., 1994; Socher et 1995). In addition to physiologic germ cell al., 1997; Tomomasa et al., 2002; Wang et apoptosis, massive increases in germ cell al., 1998; Watts et al., 2000; Yin et al., apoptosis are observed after various testic- 1997; Yin et al., 1998). Sertoli and Leydig ular injuries, including toxicant exposure cells do not show apoptosis in UT (Ohta et (Blanchard et al., 1996; Brinkworth et al., al., 1996). The normal cycle of a cell 1995; Lee et al., 1997; Lee et al., 1999; includes growth, differentiation and death. Richburg & Boekelheide, 1996), radiation Death may occur by two different mecha- (Hasegawa et al., 1998; Meistrich, 1993), nisms: necrosis and apoptosis (Buja et al., treatment with chemotherapeutic com-

Male infertility today #4 99 pounds (Meistrich, 1993), alterations of hor- tion or orchiopexy. It has been suggested monal support (Billig et al., 1995; that TMD may elicit an autoimmune Henriksen et al., 1995b; Sinha Hikim et al., response against sperm antigens before 1995; Tapanainen et al., 1993; Troiano et and during puberty possibly due to impaire- al., 1994; Woolveridge et al., 1999) and ment of the blood-testis barrier by the ele- raised testicular temperature (Lue et al., vated temperature (Sinisi et al., 1998). 1999; Yin et al., 1997; Yin et al., 1998), indi- However, in a longitudinal case control cating that a specific pathway is activated study including 61 pubertal boys with UT corrected surgically at a prepubertal age, when testicular environment cannot support we have failed to detected an autoimmune spermatogenesis. response against sperm surface antigens The reason for the increased incidence (Mamoulakis et al., preliminary experi- of apoptosis in testicular germ cells in ments), while antisperm surface antibodies terms of UT remains unclear. Two putative have also not been detected in prepubertal testicular injuring factors have been sug- boys with unilateral UT (Mirilas & De gested to trigger apoptosis (Wang et al., Almeida, 1999). The similar effect on both 1998), acting either concurrently or inde- testes has been taken as an evidence to pendently, via molecular mechanisms exclude thermal injury as the sole cause of which still have not been fully clarified; defective germ cell proliferation and favor defective hormonal support due to impaired the theory of the deficient hypothalamic- hypothalamic-pituitary-gonadal axis and pituitary-gonadal axis (Hadziselimovic, hyperthermia due to abnormal testicular 1977) (see above). We have recently location (see above). There is a controversy reported an increase in contralateral testic- regarding the level of apoptosis in the ular temperature associated with increased (CDT) in cases of unilateral TMD. It has blood flow in CDT (Ono & Sofikitis, 1977). been reported that the CDT exhibits similar- This novel mechanism could explain the ly enhanced level of apoptosis (Wang et al., detrimental effects of UT to its descended 1998), but there are also contrary reports partner (Ono & Sofikitis, 1977). suggesting either no apoptotic elevation in Briefly, three distinct systems have been the CDT (Ohta et al., 1996; Watts et al., so far implicated in the regulation of the 2000; Yin et al., 2002) or even a significant- apoptotic signal pathway in the testis in ly increased labeling of apoptotic cells per order to secure normal spermatogenesis. tubule in the CDT compared with its malde- These include the p53 protein, the Fas- scended partner (Heiskanen et al., 1996). system and the Bcl-2 family of proteins Since a direct thermal injury on the CDT is (Richburg, 2000). Abnormal deviation of not obvious, it has been claimed that these systems due to abnormal hormonal increased apoptosis in the CDT might be state and/or elevated testicular temperature mediated by an immune reaction (autoanti- may play a role in the increased testicular body response against the development of apoptosis identified in the gonads of testicular components) induced indirectly by patients with TMD leading to their impaired the thermal injuring effect on the malde- fertility. scended testis (Rapaport et al., 1969). High The tumor suppressor phosphoprotein titers of antisperm surface antibodies have p53 is a widely described regulator of both been recently reported in a number of chil- cell proliferation (Tsukuda et al., 1993) and dren with TMD independent of testis loca- intra-cellular apoptotic-signaling pathway

100 Male infertility today #4 (Skeikh & Fornace, 2000). It has been spermatocytes securing the avoidance of implicated to both spontaneous and injury- damaged DNA replication transmission (Mu induced apoptosis in mitotically active sper- et al., 2000). matogonia and possibly in meiotic and The Fas-signaling pathway is composed postmeiotic testicular germ cells as well of the interacting proteins Fas (CD95/APO- (Beumer et al., 1998; Hasegawa et al., 1) and Fas ligand (FasL, CD95L/APO-1L) 1998; Yin et al., 1998). It has been pro- (Nagata & Goldstein, et al., 1995). Fas is a posed that p53 is the main inducer of heat- type I transmembrane protein belonging to enhanced UT-related testicular germ cell the tumor necrosis factor/nerve growth fac- apoptotis in mice (Socher et al., 1997). tor superfamily, that contains a “death However, the p53-dependent pathway is domain” and is capable of initiating apopto- responsible only for the initial (first) phase sis when stimulated by receptor cross-link- (androgen-independent) (Ohta et al., 1996) ing or binding to its ligand FasL (a tumor of apoptosis enhanced by heat and is necrosis factor-related type II transmem- Fas/FasL-independent (Yin et al., 2002), brane protein) (Nagata & Goldstein, et al., while subsequent apoptosis involves a p53- 1995). The FasL-Fas interaction triggers the independent pathway (Yin et al., 1998) death of cells expressing Fas (Nagata & mediated via the Fas/FasL system (Yin et Goldstein, et al., 1995). The Fas expression al., 2002). (see below). Early reports have is universal but particularly is seen in thy- suggested that p53-mediated apoptosis mus, spleen and nonlymphoid tissues, could be a a protective mechanism in the while FasL expression is generally more human and other species for the avoidance restricted to lymphoid organs and interest- of propagation of unrepairable DNA dam- ingly, it is abundant in the testis, with a sug- age induced by high temperatures (Yin et gested localization to Sertoli cells (French al., 1998). Another point of interest is that et al., 1996). Besides the role of Fas-sys- p53 does not seem to play a role in the tem in maintaining the immune privilege apoptosis observed in the epididymis of the status of the testis (Bellgrau et al., 1995), cryptoepididymis mouse model (Jara et al., the identification of both FasL (constituitive- 2002). The exact molecular mechanism by ly expressed in Sertoli cells) and Fas (in which p53 mediates high temperature- select types of germ cells) in rodent and induced germ cell apoptosis in UT is not human testis implicates this system in the clear. It has been shown that the testicular regulation of spontaneous germ cell apop- orphan receptor-2 (TR2) expressed in tosis and implies a key role of the Sertoli pachytene primary spermatocytes (Lee et cell as a paracrine regulator of spermatoge- al., 1996) is repressed via a p53-dependent nesis (Lee et al., 1997; Pentikainen et al., signaling pathway in the Rhesus monkey 1999). The Fas-signaling system is also (Mu et al., 2000). The TR2 is a master reg- implicated in the apoptosis induced after ulator that controls many signaling path- testicular injury (exposure to toxicants, radi- ways, and it is likely that it plays very impor- ation, hyperthermia) (Lee et al., 1997; Lee tant role in the process of spermatogenesis et al., 1999). Especially after testicular (Mu et al., 2000). It has been hypothesized hyperthermia (in contrast to toxicant expo- that higher testicular temperatures may sure), there is a selective increased expres- increase p53 with a subsequent repress of sion of Fas, while FasL is not upregulated TR2 which in turn negatively influences (Lee et al., 1999). This implies that hyper- spermatogenesis on the level of primary thermia selectively targets the germ cells;

Male infertility today #4 101 the supporting capacity of Sertoli cells is cells in the majority of the tubules leading not altered (Sertoli cells are not obviously to infertility (Furuchi et al., 1996). affected/FasL is not upregulated) but the Transgenic mice overexpressing Bcl-2 or affected germ cells are eliminated by the Bcl-xL are also infertile and display a disor- up-regulation of Fas (Lee et al., 1999). ganization of the seminiferous epithelium Recently it has been shown that the Fas (Rodriguez et al. 1997). Intrestingly, both pathway is responsible for the second (p53- Bax and Bcl-2 have been involved in the independent) phase of apoptosis in TMD testicular germ cells apoptosis induced by (Yin et al., 2002). There is a controversy hyperthermia (Yamamoto et al., 2000) or regarding the role of androgens on Fas-sig- androgen withdrawal (Woolveridge et al. naling pathway, since both up-regulation 1999). In addition, it has been reported that (Nandi et al., 1999) and down-regulation these proteins may play an important role in (Woolveridge et al., 1999) of testicular Fas the germ cell apoptosis observed in experi- gene expression in correlation to germ cells mentally-induced unilateral UT (Xu et al., apoptosis after testosterone withdrawal has 2000) and cryptoepididymis mouse models been reported. Apart from the two appar- (Jara et al., 2002). ently interactive but independent apoptotic pathways responsive to heat stress in UT 2.2.3. Microdeletions of the Y (p-53- and Fas-signaling pathways), there chromosome and testicular maldescent appears to be additional yet unknown The role of the Y chromosome in sex sequential apoptotic patways (Yin et al., determination is well established (see 2002). above). However it has been shown that the The Bcl-2 family of proteins is a widely control of spermatogenesis is another Y recognized group of apoptotic regulators chromosome-closely related function (Adams & Cory, 1998; Korsmeyer, 1999). (Tiepolo & Zuffardi, 1976). Tiepolo and This family consists of both antiapoptotic Zuffardi (1976) were the first to hypothesize (e.g. Bcl-2, Bcl-xL, Bcl-w) and proapoptotic a correlation between Y chromosome dele- (e.g. Bax) members (Gross et al., 1999). tions and male infertility (Tiepolo & Zuffardi, The ratio of the above molecules provides 1976). These authors examined the kary- information on the susceptibility of cells to a otype of 1170 men and observed large stimulus inducing death (Oltvai et al., deletions including the entire heterochro- 1993). There is evidence that Bcl-2 family matic region of the Y chromosome long arm members and their regulated expression (Yq12) and an undefined amount of the are critical for functional spermatogenesis. adjacent Yq11 part in six azoospermic Bax-deficient male mice exhibit an inappro- males. They suggested that the deletions priate accumulation of premeiotic germ were the cause of the azoospermia and it cells as well as accelerated apoptosis of was postulated that a genetic factor located mature germ cells that enter meiosis lead- in Yq11 was important for male germ cell ing to complete cessation of sperm produc- development. This gene cluster was defined tion and infertility (Knudson et al., 1995). as “azoospermia factor” (AZF). During the Bcl-w-deficient mice are also infertile (Print subsequent years, many genes have been et al., 1998). Transgenic mice misexpress- mapped on the Y chromosome, while the ing Bcl-2 in spermatogonia display an accu- development of sequence tagged sites mulation of spermatogonia prior to puberty (STS)- and yeast artificial chromosome but during adulthood exhibit loss of germ clones (YAC)-based mapping revealed the

102 Male infertility today #4 genetic complexity of the AZF locus. These play a role in the production of factors stim- analyses permited the detection of intersti- ulating/impeding TD, we conducted a tial submicroscopic deletions not visible at screening of more than 180 children with all cytogenetic level (microdeletions). They are possible phenotypical expressions of uni- or detectable only by STS-polymerase chain bilateral TMD (anorchia/total atrophia, intra- reaction (PCR) or Southern hybridization. abdominal location, inguinal/upper scrotal Three nonoverlapping regions (spermato- location, retractile testis and ectopia). genesis loci) may be deleted in infertile Genomic DNA was extracted from peripher- men (AZFα, AZFb and AZFc) (Vogt et al. al blood lymphocytes using a salting out 1996) containing genes that are expressed technique and amplified by routine PCR. in the testis and are therefore considered The set of STS primers and the conditions candidate genes for the AZF phenotype of amplification were chosen according to (AZFα: USP9Y, DBY; AZFb: RBMY; and current laboratory guidelines for molecular AZFc: DAZ) (Foresta et al., 2001a). It is cur- diagnosis of Yq11 microdeletions (Simoni et rently clear that Yq11 microdeletions repre- al., 1999). PCR products were separated sent the most frequent genetic etiology of and visualized under UV light on 2% severe testiculopathy with an overall inci- agarose gels using ethidium bromide. In dence ~8% in infertile patients (Foresta et none of these patients did we detect Yq11 al., 2001b). microdeletions (Mamoulakis et al., prelimi- The association of Yq11 microdeletions nary experiments). Our results provide in TMD has only recently started to be eval- strong evidence against a direct causative uated. Up to now the results are inconclu- role of Yq11 microdeletions in the develop- sive. Yq11 microdeletions have been spo- ment of TMD. radically reported in patients with a history of UT (Dada et al., 2002), while two studies B. TESTICULAR CANCER IN have failed to detect such defects in a total PATIENTS WITH A HISTORY OF of 48 patients (either infertile or not) TESTICULAR MALDESCENT (Cortes et al., 1998; Fagerli et al., 1999). Only in one study has been reported a high 1. Epidemiology of testicular tumor incidence (11/40;27.5%) of Yq11 microdele- formation tions in patients with a history of unilateral Although many risk factors have been orchidopexy presenting with clinical, hor- proposed for the etiology of testicular can- monal, seminal and testicular cytological cer in human, only a history of UT is a well features of severe bilateral testicular dam- established one (UK Testicular Cancer age (Foresta et al., 1999). Based on these Study Group, 1994a; UK Testicular Cancer findings it has been suggested that Yq11 Study Group, 1994b). UT is associated with microdeletions are responsible for the testicular neoplasia not only in humans but severe bilateral testicular damage that can also in other mammalian species such as be phenotypically expressed by unilateral horses and dogs (Boothe, 1993; Johnston UT and infertility probably because of et al., 2001; Wintzer, 1986). The magnitute altered testicular responses to mechanisms of the relative risk is a point of considerable regulating TD. In order to evaluate the prob- disagreement varying among several ability that specific genetic loci of the Y reports between 5-50 times greater and chromosome associated with the regulation 9.2-13.6 times greater in humans and dogs, of spermatogenesis (AZFα, AZFb, AZFc) respectively, compared to the general popu-

Male infertility today #4 103 lation (Cortes et al., 2001; Farrer, 1985; abnormality responsible also for the initial Johnston et al., 2001; UK Testicular Cancer TMD, rather than due to secondary dyspla- Study Group, 1994b; Whitaker, 1988; sia caused by abnormal temperature Woodhouse, 1991). No increase in risk (Giwercman et al., 1988). Although a sec- seems to exist in cases of men with a histo- ondary effect cannot be ruled out, the theory of spontaneous TD (Moller et al., 1996). ry of an intrinsic pathologic process affect- The relative frequency of a history of UT in ing both testes seems more plausible. It is men with testicular tumors (TT) is 15-fold supported by the evidence of a ~3.5-fold for unilateral and 33-fold for bilateral UT increased risk of the CDT in cases of unilat- (Stone et al., 1991), respectively. Location eral UT (~15-20% of all TT occur in the of the UT also affects the relative risk of CDT) (Batata et al., 1980; Hutson & developing a TT; the higher the position the Beasley, 1992; Johnson et al., 1968; Martin, testis, the greater the risk of developing a 1982; Prener et al., 1996). Furthermore, malignancy (Martin, 1982; Martin & Menck, among men with bilateral UT and unilateral 1975). Inta-abdominal testes have a greater TT, 15% of the contralateral to the primary risk of malignancy, compared with testes tumor testes develop malignancy (Schneck palpable in the groin (Stone et al., 1991) & Bellinger, 2002). (Campbell, 1942). The most common TT In adults, carcinoma in-situ (CIS) germ that develops from an UT is seminoma in cells are currently considered the precur- human (Abratt et al., 1992; Batata et al., sors for all germ cell tumors, except sper- 1980; Martin, 1979; Martin, 1982; Stone et matocytic seminoma (Skakkebaek et al., al., 1991), while the incidence of seminoma 1987). These cells are presumed to be and Sertoli cell tumors is almost equal in malignant gonocytes derived from primor- dogs with TMD (Johnston et al., 2001). dial germ cells that escaped normal differ- 501 Other rarer histological types encountered entiation in utero. The incidence of CIS in men with a history of TMD include is estimated ~1.7% in adults with a history embryonal carcinoma, teratocarcinoma, of TMD (Giwercman et al., 1989). CIS is and choriocarcinoma (Batata et al., 1980). more commonly detected in intra-abdomi- TT develop usually during or after puberty nal testes (Ford et al., 1985). Germ cell although there are sporadic case reports of tumors have been noted to develop in 40- development before 10 years of age (for 50% of adults during a 5-year period from review see Doi et al., 2002). In childhood, the diagnosis of CIS (von der Maase et al., 1986). The interval between the diagnosis seminoma is not as common compared to of CIS and the development of seminoma adulthood (Miller et al., 1999). Although in an UT may last more than 10 years, rare in the general population, the most while spontaneous regression of CIS has common TT associated usually with intra- not been recorded (Sigg & Hedinger, abdominal UT in childhood is mature ter- 1983). On the other hand, CIS seems to atoma (Doi et al., 2002). be an extremely rare finding before adult- 2. Pathogenesis of testicular tumor hood (Cortes et al., 2001). In a large formation series of testicular biopsies taken at sur- The cause of the increased risk for gery (before adulthood) from 1335 patients malignant testicular degeneration of the UT with UT, CIS was detected only in 6 is unclear. It has been speculated that TT patients (2 with intra-abdominal testes, 3 may arise due to an intrinsic testicular with abnormal external genitalia besides

104 Male infertility today #4 TMD and 1 with abnormal karyotype that may be manifested before the first year [45,X/46,XY]). Based on these results, the of life. authors recommended examination for tes- CONSERVATIVE TREATMENT ticular neoplasia by testicular biopsy at orchidopexy only in cases accompanied by There are two types of conservative these characteristics. However, the biolog- treatment for the UT, administration of hCG ic behaviour of CIS in the prepubertal and administration of GnRH-a or LHRH. testis has not yet been clarified. The pre- The administration of androgen was aban- dictive value of the identification of placen- doned because of the side effects of preco- tal-like alkaline phosphatase positive germ cious puberty. The mechanism of action of cells (a reliable immunohistochemical hCG and GnRH, is an increase in testos- marker for confirmation of the CIS histo- terone production by stimulation at different logical diagnosis) (Giwercman et al., 1991) levels of the hypothalamic-pituitary axis. in prepubertal testicular biopsy speciments This treatment is based on experimental for future malignancy has been questioned observations that the inguinoscrotal phase (Engeler et al., 2000). Therefore, the value of testicular descent is androgen mediated, of routine testicular biopsy at orchidopexy involving testicular synthesis of the active during childhood as a possible aid in the metabolite in high local concentrations identification of patients at risk for future (Rajfer & Walsh, 1977). hCC stimulates testicular neoplasia is questionable. It is Leydig cells directly to produce testos- controversial whether orchiopexy affects terone. GnRH stimulates the pituitary to the natural history of development of TT, release LH promoting the testicular produc- although there is emerging evidence to tion of testosterone. Serum testosterone support that prepubertal orchiopexy may levels during therapy for UT in prepubertal lessen the risk (Moller et al., 1996; Prener boys are much higher with hCG treatment et al., 1996; Raina et al., 1995; UK than with GnRH treatment (Rajfer et al., Testicular Cancer Study Group, 1994b).It 1986). Re- examination of boys treated with should be emphasized that orchiopexy will hormonal therapy must be performed in a allow a more thorough examination of the 6-month base, because reascent has been testis for earlier detection of futural malig- reported in up to 25% of patients. Hormonal nant degeneration. treatment is not indicated in patients with previously operated testes. 6. MANAGEMENT OF TESTICULAR Success rates of hCG treatment have MALDESCENT: HORMONAL varied considerably, from 14% to 59% TREATMENT & SURGERY (Adamsen et al., 1989; Ehrlich et al., 1969; Early management of boys referred for Job et al., 1982;). Treatment regimens have an empty scrotum is mandatory in order to also varied, and the most effective treat- maintain testicular function. Objective of ment was demonstrated to be a total dose any therapeutic intervention is the position- of at least 10,000 IU to achieve maximal ing of the gonad in the scrotum before 1 stimulation of the Leydig cells and avoid the year of age. Spontaneous descent occurs complications of doses exceeding 15000 IU in the majority of cryptorchid children by 3 (Job et al., 1982). A typical treatment months of age and it is very rare thereafter. schedule is 1500 lU injected intramuscular- Consideration for earlier intervention should ly twice per week for 4 weeks. FSH appears be made to prevent complications of UT to affect the spontaneous descent of the

Male infertility today #4 105 testis and to induce the production of LH performed a metaanalysis of 33 studies receptors; however, studies evaluating the from 1958 to 1990 and assessed the efficacy of a combined treatment with hCG results of LHRH and hCG in the treatment and FSH have shown inconsistent results of 3282 boys with UT. (Giannopoulos et al., 2001; Hoorweg SURGICAL TREATMENT Nijman et al., 1994; Saggese et al., 1989). Side effects of hormonal treatment The overall efficacy of medical treatment include increased rugation and pigmenta- is less than 20% for cryptorchid testes and tion of the scrotum. Rarely, there is an is dependent on testicular pretreatment increase in the size of the penis and devel- location. Therefore, surgery remains opment of pubic hair. These side effects are unequivocally the treatment of choice in the regressing after cessation of therapy. management of the UT. Significant increase in weight increment The analysis of surgical therapy for UT velocity was seen in 7- to 9-year-olds boys by Docimo (1995) revealed an orchiopexy treated with a dose of 10,000 IU (Adamsen success rate equal to 92% for testes below et al., 1989). Furthermore, the administra- the external ring, 89% for inguinal testes, tion of hCG in immunosuppressed patients 84% for microvascular orchiopexies, 81% should also be avoided because of a tran- for standard abdominal orchiopexies, 77% sient decrease in the absolute number of for staged Fowler-Stephens orchiopexies, total peripheral blood lymphocytes, total T and 67% for standard Fowler-Stephens cells, T helper- inducer cells, and of CD8+ orchiopexies (Docimo et al., 1995). subsets during therapy. In addition, the per- The age at which orchiopexy is best percentage of CD8+ cells and lymphocyte formed is considered to be about 12 response to the mitogen concanavalin A months, and certainly before the third year decreased significantly with hCG treatment of life, prior to morphological damage to the and returned to normal after hCG withdraw- UT. There is some preliminary evidence to al (Maghnie et al., 1991). suggest that orchopexy should be per- Results of GnRH administration trials formed earlier than 12 months (Yamanaka may have been overestimated because of et al., 1991). the inclusion of patients with retractile If a clinically evident hernia appears in testes; the reported success rates ranged an infant who has an ipsilateral UT, an from 32 to 65% (Hadziselimovic, 1982; orchiopexy should be performed at the Witjes et al., 1990). Rajfer et al. (1986) same time as the herniotomy, irrespective found that TD into the scrotum occurred in of the age of the infant. Failure to do so 19% of patients treated with GnRH and in would make subsequent orchiopexy 6% of those treated with hCG. One multi- extremely difficult, because of the produc- center study found a therapeutic gain in tion of fibrous adhesions in the region of 8.1% of children with bilateral UT and no the internal ring. effect in children with unilateral UT (Olsen et al., 1992). A success rate of 9% for A. Standard orchiopexy LHRH nasal spray and 8% for placebo was The key steps of standard orchiopexy reported in 252 prepubertal boys with 301 are: a) complete mobilization of the testis UT (De Muinck Keizer-Schrama et al., and spermatic cord, b) repair of the patent 1987). These results confirmed similar find- processus vaginalis by high ligation of the ings reported by Pyolala et al. (1995), who hernia sac, c) skeletonization of the sper-

106 Male infertility today #4 matic cord without sacrificing vascular ment of a traction transparenchymal suture integrity in order to achieve tension-free (Jarow, 1991). placement of the testis within the depend- B. Techniques for the highly ent position of the scrotum, and d) creation undescended testis of a superficial pouch within the hemiscro- Occasionally, greater mobilization of the tum to receive the testis. proximal spermatic cord structures does Experimental evidence has demonstrat- not provide adequate length to allow ten- ed that transparenchymal suture fixation sion-free placement of the testis within the causes testicular damage by inducing an scrotum. Greater cord length can be inflammatory reaction regardless of suture obtained by mobilizing the spermatic ves- size and material. In comparison, dartos- sels medially. The spermatic vessels are fixed testes demonstrated complete cir- usual the limiting factor in these circum- cumferential adherence with only 5% or the stances. The Prentiss manoeuvre was animals in the dartos pouch group demon- described in 1960 and occasionally is help- strating an inflammatory response; normal ful in adding length to the spermatic ves- spermatogenesis was found in 94% of the sels by positioning the spermatic vessels animals (Bellinger et al., 1989; Dixon et al., medially and thereby choosing the 1993). Based on these studies, trans- hypotenuse of the triangle, or most direct parenchymal suture should be avoided. course to the scrotum, created by the natu- There are only two exceptions: for tethered ral course of the vessels laterally through testis when its position is tenuous after the internal ring. complete mobilization of the spermatic cord and short-term external fixation to a C. Management of intra-abdominal nylon button is required, and for testicular testis fixation of the ipsilateral and contralateral I. Laparoscopy testes due to clinical torsion of the sper- Laparoscopy is a safe and effective matic cord. A significant variation of the method for localization a nonpalpable technique, however, is to completely avoid testis. The accuracy of transperitoneal deep transparenchymal placement of the laparoscopy to locate a nonpalpable testis sutures by superficial placement of the approaches 100% and subsequently sutures just under the tunica albuginea. defines the management options. Once the The sutures used in these cases are fine, presence or absence of the testis is verified permanent monofilarnent suture, such as by diagnostic laparoscopy, an orchiopexy or 5-0 or 6-0 Prolene, on a noncutting needle. orchiectomy may be performed with laparo- It is important that the surgeon has to be scopic assistance. The initial purpose of familiar with the intragonadal vascular diagnostic laparoscopy is to plan the subse- anatomy in such cases, especially when quent surgical approach or to avoid an ligation of the spermatic vessels is contem- open exploration. The three most frequent plated. The collateral arterial circulation findings at diagnostic laparoscopy for a through the deferential artery, which com- nonpalpable testis are: a) blind-ending ves- municates with the internal spermatic arte- sels above the internal ring, b) cord struc- rial system both in the spermatic cord and tures entering the internal ring, and c) an at the lower pole of the testis, was found to intra-abdominal testis. If blind-ending sper- be compromised in all testes with place- macic vessels are found, no further surgical

Male infertility today #4 107 investigation is necessary. The testis is con- stages. If an one-stage repair is decided to sidered “vanished” as the result of a prena- be performed, it is critical early in the distal vascular accident. When spermatic ves- section that a wide pedicle of peritoneum sels, even hypoplastic ones, are visualized be preserved with the vas deferens in order entering the internal ring, inguinal explo- to maintain collateral blood flow. Careful ration is mandatory. The intra-abdominal inspection is required to identify the primary testis can be managed by either an “open” and collateral vessels to the testis, as well surgical or laparoscopic orchiopexy proce- as the course of the vas deferens. It is very dure or by orchiectomy. The orchiopexy can common than anatomic variations of the either be staged or performed with com- vascular supply to the testis are present in plete mobilization of the testis into the scro- associated cases of a long-looping vas. tum in one procedure. The choice of repair This (a long-looping vas) most important technique depends on the viability of the requirement for successful Fowler- testis, the anatomy of the paratesticular Stephens orchiopexy is present in less than structures, the distance of the testis from one third of boys with true intra-abdominal the scrotum, the status of the contralateral testes. Children with a long-looping vas that testis, and, most important the surgeon’s extends down to the inguinal canal are the experience and ability. A micro-surgical ideal candidates for an one-stage repair. autotransplantation of the intra-abdominal True intra-abdorninal testes lie at least 1 cm testis is an extreme measure with few indi- above the internal ring and have shortened cations. spermatic vessels. If a long-looping vas is present, it is carefully mobilized. Once it II. Fowler-Stephens orchiopexy has been determined that the testis cannot The management of the high inguinal or be placed in its scrotal position, the sper- intra-abdominal testis occasionally matic vessels are divided. The Fowler- demands ligation of the testicular vessels. Stephens test is a method to evaluate the The testicular artery and veins often limit collateral blood supply to the testis. It is per- the distal mobility of these testes. It was not formed by temporarily occlusion of the tes- until 1959 that Fowler and Stephens stud- ticular artery with a vascular clamp for 5 ied the vascular anatomy of the testis and minutes and inspection of the testis for devised a means to repair the highly unde- color as well as after a small incision in the scended testis preserving its blood supply tunica albuginea of the testis to document via collateral circulation. The testis has arterial bleeding. A carefully inspection is three arteries for blood supply: the testicu- needed to distinguish the tunical blood sup- lar artery (primary), the deferential artery of ply which may be preserved despite testicu- the vas deferens, and the cremasteric arter- lar arterial occlusion from the blood supply ies. When the spermatic vessels are divid- to the testicular parenchyma. If the blood ed, blood supply to the testis is dependent supply is at this point considered tenuous on collateral circulation from the deferential the performance of a two-stage repair allow artery, a branch of the inferior vesical the development of the collateral circula- artery, and the cremasteric system, a tion. branch of the inferior epigastric artery. The anticipated advantages of a two- The technique described by Fowler and stage orchiopexy with spermatic vessel lig- Stephens was originally a one-stage proce- ation are: a) the development of the collat- dure, but it may also be performed in two eral blood supply to compensate for divi-

108 Male infertility today #4 sion of the main blood supply to the testis, sion (either iatrogenic or spontaneous), and b) a greater mobilization of the testis in damage to the vas deferens, and testicular order to place it within the scrotum (Elder, atrophy. Atrophy of the testis is the most 1989; Elder, 1992). After 6 months or longer devastating complication, but it is a rare re-operation must be performed to com- finding after the performance of standard plete the vasal peritoneal pedicle mobiliza- orchiopexies. Devascularization with atro- tion using the standard Fowler Stephens phy of the testis can result from skele- technique. tonization of the cord, from overzealous III. Microvascular autotransplantation electrocautery, from inadvertent torsion of Silber & Kelly (1976) first reported the the spermatic vessels during passage of performance of a microvascular anastomo- the testis into the scrotum or as a result of sis to bring extra blood supply to the testicle ligation and division of the spermatic ves- after mobilization of a high inta-abdominal sels during a Fowler-Stephens orchiopexy. testicle in a child with prune-belly syn- Devascularization may also be caused by drome. Initial series were limited to older excessive axial tension on the spermatic boys whose internal spermatic artery was vessels, particularly if the collaterals are large enough to be anastomosed to the poor. Some degree of post-haematoma is inferior epigastric artery, but the technique common. In contrast, the development of a was demonstrated to be successful in boys large haematoma is an uncommon finding younger than 2 years of age (Harrison et after the performance of standard al., 1990). Overall success rates are in orchiopexies. Large haematoma increases excess of 80% (Bianchi, 1984; Bukowski et the risk of infection and abscess formation. al., 1995a; Bukowski et al., 1995b; Harrison Although retraction is usually a result of et al., 1990; Wacksman et al., 1982). inadequate retroperitoneal dissection, Bukowski and associates reported a 96% insufficient cord length precludes success- success rate for testicular autotransplanta- ful orchiopexy no matter how much dissection over a 17-year period (Bukowski et al., tion is performed. If the patent processus 1995a). vaginalis is ligated within the canal and not D. Complications of orchiopexy at the level of the internal ring, the peri- Complications of orchiopexy include tes- toneum remains adherent to the spermatic ticular retraction, hematoma formation, vessels, and complete retroperitoneal mobi- ilioinguinal nerve injury, postoperative tor- lization of vessels is not feasible.

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Male infertility today #4 135 Wensing CJ, Colenbrander B, van Straat- is there still an indication? A clinical study in 78 en HW. boys with 103 undescended testicles. Normal and abnormal testicular descent in Eur Urol 17: 226-228, 1990. some mammals. Wolfson A. In: Clinics in Andrology: Descended and Sperm storage at lower-than-body temperature Cryptorchid Testis. ESE Hafez (ed), outside the body cavity of some passerine The Hague, Martinus Nijhoff, pp. 125-137 (vol birds. 3), 1980. Science 120: 68-71, 1954. Werdelin L & Nilsonne A. Woodal PF. The evolution of the scrotum and testicular The male reproductive system and the phyloge- descent in mammals: a phylogenetic view. ny of elephant-shrews. J Theor Biol 196: 61-72, 1999. Mammal Rev 25: 87-93, 1995. Whitaker RH. Neoplasia in cryptorchid Woodhouse CJ. men. Undescended testes. Semin Urol 6: 107-109, 1988. In: Long-Term Paediatric Utology. Woodhouse Whitfield LS, Lovell Badge R, Goodfellow CJR (ed), PN. Oxford, Blackwell Scientific, pp. 167-175, 1991. Rapid sequence evolution of the mammalian Woolveridge I, de Boer-Brower M, Taylor sex-determining gene SRY. MF, Teerds KJ, Wu FCW, Morris ID. Nature 364: 713-715, 1993. Apoptosis in the rat spermatogenic epithelium Wiener JS, Marcelli M, Gonzales ET, Roth following androgen withdrawal: changes in DR, Lamb DJ. apoptosis-related genes. Androgen receptor gene alterations are not Biol Reprod 60: 461-470, 1999. associated with isolated cryptorchidism. Wyllie A, Kerr J, Curie A. J Urol 160: 863-865, 1998. Cell death the significance of apoptosis. Wilkerson ML, Bartone FF, Fox L, Int Rev Cytol 68: 251-306, 1980. Hadziselimovic F. Xu J, Xu Z, Jiang Y, Qian X, Huang Y. Fertility potential: a comparison of intra-abdomi- Cryptorchidism induces mouse testicular germ nal and intracanalicular testes by age groups in cell apoptosis and changes in bcl-2 and bax children. protein expression. Horm Res 55: 18-20, 2001. J Environ Pathol Toxicol Oncol 19: 25-33, 2000. Williams MP & Hutson JM. Yamamoto CM, Sinha Hikim AP, Huynh The phylogeny of testicular descent. PN, Shapiro B, Lue Y, Salameh WA, Wang C, Pediatr Surg Int 6: 162-166, 1991a. Swerdloff RS. Williams MP & Hutson JM. Redistribution of Bax is an early step in an The history of ideas about testicular descent. apoptotic pathway leading to germ cell death in Pediatr Surg Int 6: 180-184, 1991b. rats, triggered by mild testicular hyperthermia. Biol Reprod 63: 1683-1690, 2000. Wintzer HJ. Equine diseases. Berlin, Verlag Paul Parey, 1986 Yamanaka J, Baker M, Metcalfe S, Hutson JM. Wislocki GB. Serum levels of Mullerian inhibiting substance Observations on the gross and microscopic in boys with cryptorchidism. anatomy of the sloths (Bradypus griseus Gray J Pediatr Surg 26 :621-623, 1991. and Choloepus hoffmanni Peters. Yamanaka J, Metcalfe SA, Hutson JM, J Morph 46: 317-328 1928. Mendelsohn FA. Witjes JA, de Vries JD, Lock MT, Testicular descent. II. Ontogeny and response Debruyne FM. to denervation of calcitonin gene-related pep- Use of luteinizing-hormone-releasing hormone tide receptors in neonatal rat gubernaculum. nasal spray in the treatment of cryptorchidism: Endocrinology 132: 280-284, 1993.

136 Male infertility today #4 Yin Y, DeWolf W, Morgentaler A. Zimmermann S, Schottler P, Engel W, Experimental cryptorchidism induces testicular Adham IM. germ cell apoptosis by p53-dependent and Mouse Leydig insulin-like (Ley I-L) gene: struc- –idependent pathways in mice. ture and expression during testis and ovary Biol Reprod 58: 492-496, 1998. development. Yin Y, Hawkins KL, Dewolf WC, Morgen- Mol Reprod Dev 47: 30-38, 1997. taler A. Heat stress cause testicular germ cell apopto- Zimmermann S, Schwarzler A, Buth S, sis in adult mice. Engel W, Adham IM. J Androl 18: 159-165, 1997. Transcription of the Leydig insulin-like gene is Yin Y, Stahl BC, Dewolf WC, Morgentaler A. mediated by steroidogenic factor-1. p53 and Fas are sequential mechanisms of tes- Mol Endocrinol 12: 706-713, 1998. ticular germ cell apoptosis. J Androl 23: 64-70, 2002. Zimmermann S, Steding G, Emmen JM, Brinkmann AO, Nayernia K, Holstein AF, Young GP, Goldstein M, Phillips DM, Sun- Engel W, Adham IM. daram K, Gunsalus GL, Bardin CW. Sertoli cell-only syndrome produced by cold Targeted disruption of the Insl3 gene causes testicular ischemia. bilateral cryptorchidism. Endocrinology 122: 1074-1082, 1988. Mol Endocrinol 13: 681-691, 1999.k

Male infertility today #4 137 138 Male infertility today #4 MALE ACCESSORY GLAND INFECTIONS AND INFERTILITY

Enzo Vicari, Sandro La Vignera, Alessandro Arancio, Aldo Eugenio Calogero

Section of Endocrinology, Andrology and Internal Medicine, Department of Biomedical Sciences, University of Catania, Catania

Corrispondenza: Prof. Enzo Vicari, Catt. Endocrinologia, Sez. Endocrinologia, Andrologia e Medicina Interna, Dip. Scienze Biomediche, Università di Catania, Osp. Garibaldi, P.zza S.M. Gesù, 95123 Catania

MALE ACCESSORY GLAND (Mehik et al., 2000) than that reported in INFECTIONS : BACKGROUND other parts of Europe (5-9%) (Weidner et al., 1999) or in the world. Male accessory gland infections (MAGI) Physicians, urologists and pathologists are of worldwide distribution in both indus- have reported a broad range of epidemio- trialized and developing countries. Except logical prevalence of prostatitis. Particularly, the uncommonly acute, symptomatic condi- a physician’s diagnosis of prostatitis ranges tions, MAGI have an underhand beginning, from 1% to 11%, using data from the and a chronic, symptomless or rather pau- Olmsted County Study of Urinary cisymptomatic course. Clinically, MAGI Symptoms and Health Status among men (Roberts et al., 1998). In this community- include: a) the “uncomplicated MAGI”, based study, only 4% of the nearly 2 million known as prostatitis or prostatitis syn- visits for prostatitis per year were recorded dromes, when the inflammation is limited to as “acute prostatitis”, suggesting that the urethro-prostatic regions; b) the “compli- chronic prostatitis is quite common. On the cated MAGI”, when the inflammatory other hand, 8% (Collins et al., 1998), 11.5% process involves more accessory glands (Nickel et al., 2001) or 16% (Collins et al., and, thus, it is better defined as prostato- 2002) of men have symptoms that urolo- vesiculitis (PV), prostato-vesiculo-epi- gists have diagnosed as prostatitis over a didymitis (PVE) or epididymo-orchitis (EO). year. Furthermore, the histopathologic Prostatitis prevalence of prostatitis ranged from 6% to Prostatitis is the most common prostate 44% in patients undergoing prostate biopsy, disease, resulting in more physician visits according to Roberts et al. (1998). than either benign prostatic hyperplasia or Prostatitis is today more frequently prostate cancer (NIH, 1990). Prostatitis is referred to as “prostatitis syndrome”. also the most common urologic diagnosis According to the duration of the symptoms, in men under 50 years of age, and the third this condition is described as either acute most common in older men (Collins et al., or chronic, when they are present for at 1999). least 3 months. The predominant symptoms In men living in northern Finland, the include pain at various localizations occurence of prostatitis is higher (14.2%) (prostate/perineum 46%; scrotum and/or

Male infertility today #4 139 testes 39%; penis 6%; urinary bladder 6%; complicated by the presence of inhibitory lower back (2%) (Zermann et al., 1999) and substances present in the prostatic secre- lower urinary tract syndromes (LUTS) (pol- tion, and of previous courses of antibiotics. lakiuria, difficulty urinating, pain/increased In patients complaining of prostatitis, the pain on urination). quality of health is similar to that of patients Prostatitis is diagnosed and classified by with unstable angina, recent myocardial symptoms, microbiological and cytological infarction, or active Crohn’s disease testing of expressed prostatic secret and/or (Schaeffer et al., 2002). Therefore, the segmented urine samples (Meares & National Institutes of Health Chronic Stamey, 1968). Prostatitis Symptom Index (NIH-CPSI), avail- Prostatitis actually may be classified able online at http://www.QLMed.org/nih-cpsi, according to the NIDDK Workshop, US accurately measures three major domains of National Institutes of Health (Krieger et al., the chronic prostatitis syndrome: pain, void- 1999) in four categories: ing dysfunction, and impact/quality of life. The Category I Acute bacterial prostatitis utility of this symptom index has been con- Category II Chronic bacterial prostatitis firmed by three years of clinical and research Category III Chronic abacterial prostatitis use (Nickel et al., 2001). /chronic pelvic pain syndrome: IIIA. Inflammatory chronic PROSTATO-VESICULITIS AND pelvic pain syndrome PROSTATO-VESICULO-EPIDIDYMITIS (significant leukocytes in Chronic prostato-vesiculitis (PV) and semen; expressed pro- prostato-vesiculo-epididymitis (PVE) may static secretion; third be regarded as complicated MAGI, due to voided urine specimen); an ascending spread of urethral pathogens IIIB. Non-Inflammatory chronic via the ejaculatory duct into the vas defer- pelvic pain syndrome ens up to the epididymes. PV or PVE epi- (non-significant leuko- demiological data are scanty. These condi- cytes in semen; tions have been defined as “occult patholo- expressed prostatic gy” (Doble & Carter, 1989) o “enigmatic secretion; third voided syndromes” (Nickel et al., 2002) because of urine specimen); the multiple clinical challenges. Category IV Asyntomatic inflammation of In patients with PV, there is no clear prostate (histological prostatitis) relationship between the extention of the inflammation changes visualised by ultra- Since bacterial prostatitis (acute or sound and the severity of the symptoms or chronic) accounts for a low percentage (5- the presence of leukocytospermia. 10%), the most significant proportion of the However, patients with PV represent an urologic population studied is affected by important clinical MAGI subgroup for sever- chronic nonbacterial prostatitis (60-65%) or al reasons: prostatodynia (30%) (Weidner et al., 1991). a) they complain of symptoms even in uni- Recent data suggest that chronic nonbacte- lateral forms (Christiansen & Purvis, rial prostatitis may actually have a cryptic, 1991) with a frequency higher than that nonculturable (biofilm bacteria) microrgan- observed in patients with prostatitis; ism-based etiology: the microbiological b) some symptoms overlap with those of workup of these specimens are further patients with prostatitis (spermatorrhea,

140 Male infertility today #4 haematospermia, lower abdominal dis- enterobacteriaceae, gram positives aer- comfort or perineal/pelvic discomfort), obes, C. Trachomatis or Ureaplasma ure- others with those of patients with PVE alyticum) (Christiansen & Purvis, 1991; (funicolar pain); Krishnan & Heal, 1991). c) since PV represents mainly a complication of prostatitis, its presence is associ- PREVALENCE OF MAGI ated with post-flogistic organic and/or AND INFERTILITY functional sequelae of the lower repro- It is known that MAGI is identified by ductive genital tract, up to severe dys- WHO among diagnostic categories affecting permia in almost 40% of the patients male reproductive function and fertility affected (Purvis & Christiansen, 1993); (WHO, 1993). Chronic, mainly symptomless d) a correct diagnosis of PV prevents a MAGI may contribute to infertility to a various possible further extension of the inflam- extent, depending on the site of inflammation matory process to the epididymus (PVE) (Weidner et al., 1999; Ochsendorf, 1999; with a more severe sperm impairment. Vicari, 1999; Agarwal et al., 2003) and/or on Indeed, a careless clinical and ultra- the inflammatory response in terms of leuko- sound approach to patients with MAGI cytospermia and its products: reactive oxy- results in an understimed incidence of gen species (Vicari, 1999; Vicari, 2000; PV or PVE. In the last 20 years, the Vicari & Calogero, 2001; Vicari et al., 2002) number of items recorded in the and/or cytokines (Depuydt et al., 1996; PubMed are 3368 for prostatitis and Diemer et al., 2003). only 59 for PV and 1724 for PVE (con- The presence of MAGI is diagnosed in sidered secondary to PV). The greater patients with oligo-, astheno- or terato- number of citation for PVE compared to zoospermia who fulfil the WHO convention- the scanty citations of PV may likely al criteria (WHO,1993) (Fig. 1). reflects the onset of the most complicat- MAGI has been reported to account for ed form of MAGI due to a lack of a cor- a wide percentage (1.6 - 15%) of male rect precocious diagnosis of PV. infertility in various infertility clinic settings (Comhaire et al., 1986; Andreeßen et al., Epididymitis 1993; Vicari, 2000; Diemer et al., 2003). The incidence of epididymitis is approxi- The broad range of prevalence reported in mately 600,000 cases per year. The highest the literature reflects various factors: prevalence is in young men (19-35 years of 1. difference in the consulting physician age). The disorder is a major cause of hos- speciality. This has a different impact on pital admission among soldiers (causing the work-up of the infertile patient with approximately 20% of the admissions). The MAGI; condition, described as discomfort or pain 2. incomplete and not well defined diagno- in the scrotum, testicle or epididymis (one sis (“sperm infection” is an improperly or both sides) of at least 3 month duration, used laboratory word and it should be is usually associated with a past or concur- correctly replaced by the male diagnos- rent history of sexually transmitted urethritis tic category defined conventionally as (N. Gonorrhoeae or C. trachomatis) in MAGI); young men. In older men, it is more often 3. lack of clinical characterization in associated with bacterial prostatitis or PV patients initially fulfilling the conventional (infections with typical uropathogens: i.e. WHO criteria (combined: history and

Male infertility today #4 141 physical signs; prostatic fluid and ejacu- European Association of Urology (EAU) late signs) in diagnostic MAGI cate- working group (Weidner et al., 2002). Table gories (i.e. prostatitis; PV, PVE, EO); 1 summarizes the account (balance 4. the greatest prevalence may be found in between values vs. limits) about MAGI and studies which include patients who infertility, generated by scientific communi- require specific andrological clinical ties (WHO; NIH consensus) and by ultra- counselling for persistent infection (even sonographic-based evidence in this after antimicrobials) and/or previous IVF research area. programme failure. Whereas WHO included MAGI among An open debate with pros and cons on the the diagnostic categories of infertile role of MAGI in male inefertility is going on. patients and recommended some com- It has been recently evaluated by an bined conventional criteria for its identifica-

Figure 1. MAGI: Conventional WHO criteria (WHO, 1993)

142 Male infertility today #4 Table 1. Account (values and limits) about MAGI and male infertility

VALUES LIMITS

WHO criteria Recognize and establish a combi- • Neglet the significance to define nation of factors (clinical and the site of inflammation (of one seminological) or more sexual glands); • Seminal leukocyte assessment can be understimated by the routinary staining; • Have inspired studies based mainly on seminal data, and poorly on the clinical expression of inflammed glands.

NIH Consensus Gives a new classification of pro- • Neglets complicated MAGI (PV; statitis, including the prostatitis PVE): this results in a lower syndromes. number of items in PubMed (59 and 1724 citations for PV and PVE, respectively, compared to 3368 for prostatitis); • The 3 major domains of symptom index (NIH-CPSI) pain, void- ing dysfunction, and impact/quality of life are not much applicable to an andrological, infertile population.

Reports about scrotal and Various ultrasound criteria have • Neglets to perform additional rectal ultrasound testings been considered indicative of seminal data about host inflam- chronic inflammation and can dif- matory response (leukocyte ferentiate prostatitis, PV, PVE. assessment through immunocy- tochemistry; ROS hyperproduction) in different infertile MAGI categories (prostatitis, PV, PVE).

tion, the lack of “site-diagnosis”, based on b) the disproportionate number of items in the clinical characterization of the adnex PubMed among putative clinical MAGI glands, has determined contrasting and categories: 3368, 59 and 1724 items uncertain conclusions, this has also origi- about prostatitis, PV and PVE, respec- nated several negative influences, such as: tively; a) the enrollment of non-homogenous c) understatement of ultrasound features in cohorts of infected patients, which in patients with chronic MAGI; turn has provoked contrasting and not d) lack of synergy and application between comparable effects on the main sperm the recent knowledge of basic research parameters (Weidner et al., 1999); of inflammatory response (leukocy-

Male infertility today #4 143 tospermia and ROS hyperproduction) The promoted “actions” for each level and a clinical ultrasound-based are based from the following evidences: approach. This is enough to lead us to • In the first level, the “actions” are identi- think that for many years the above fied by conventional WHO criteria for parameters or measurements of inflam- MAGI (Figure 1) (WHO, 1993). In partic- matory response have been preferen- ular, the presence of oligo (sperm con- tially evaluated in patients with idiopath- centration 5-19 millions/ml), astheno ic infertility rather than in patients with (forward progressive motility a+b, after 1 proven MAGI; h = <25%) and/or terato-zoospermia e) Finally, a careless diagnosis of MAGI (normal oval forms: 14-29%) in combi- has not allowed to make an early diag- nation with other factors (see Figure 1), nosis and managemnent of PV, which allows to establish a suspected (ethio- may be regarded as an intermediate logical) diagnosis of MAGI (microbial or condition of MAGI, before it becomes amicrobial); PVE. • In the second level, the localization of the inflammatory process is based on HOW TO MAKE A CORRECT the presence of a significant number of DIAGNOSIS ultrasound abnormalities for each infect- The algorithm for the assessment and ed gland (i.e. >2 in the prostatitis; >4 in treatment of MAGI is based, in our the PV; >6 in the PVE) (Table 2). These approach, into three sequential levels abnormalities have been found stronger (Figure 2). Each level includes relative associated with elevated (>105 CFU/ml) “process” broken down into specific “actions” bacteriospermia and ROS hyperproduc- and “outcomes”. tion in recent studies on selected infer- a) To suspect/identify an aspecific MAGI tile infected patients (Vicari, 1999; “first-line testing” is enough (i.e. history, Vicari, 2000); physical uro-genital examination, sperm • In the third levels, in patients with com- analyses including seminal leukocytes); b) When an initial diagnosis of MAGI is plicated MAGI, the extension of infec- suspected, “second-line testing” (i.e. tious /inflammaion process to more sex- microbiological tests and ultrasound ual glands potentially enhances the scans - didymo-epididymal and rectal host inflammtory response, in terms of prostato-vesicular ultrasonography) leukocytospermia and production of allows to confirm and assess MAGI, metabolic spermiotoxyc leukocyte-relat- both from the ethiological and localiza- ed products (ROS, cytokine) tion point of view, respectively; (Ochsendorf, 1999; Vicari, 1999; Vicari, c) In patients with complicated (microbial or 2000; Agarwal et al., 2003). Since the amicrobial) MAGI (PV or PVE), “third-line above sperm oxidative stress seems to testing”, i.e. leukocytospermia (accurately persist even following antimicrobials assessed by immunocytochemical stain- (Omu et al., 1998; Vicari, 2000), the ing), reactive oxygen species production presence of an alteration of the above (by chemioluminescence analysis) aids parameters suggests a subsequent to explore the host inflammatory rational pharmacological treatment response and to direct the therapeutical (Figure 3) (Vicari & Calogero, 2001; course in a more rational manner. Vicari et al., 2002).

144 Male infertility today #4 Figure 2. Diagnostic algorithm to make a clinical decision in the assessment of patients with MAGI.

Male infertility today #4 145 Table 2. Ultrasound (US) findings considered indicative of MAGI (from Vicari, 1999)

1. PROSTATITIS alone It was suspected in presence of 2 or more of the following US signs: a.1) glandular asymmetry; b.1) hypoechogenicity associated with oedema; c.1) hyperechogenicity associated with areas of calcification; d.1) dilation of the periprostatic venous plexus.

2. PROSTATO-VESICULITIS (PV) In additing, to the above mentioned US inflammatory iuxta-prostate changes, fulfilled 2 or more of the following US signs: a.2) enlargment and asymmetry; b.2) tickening and calcification of the glandular epithelium; c.2) policiclic areas separated by hyperechogenic septs

3. PROSTATO-VESICULO-EPIDIDYMITIS (PVE) The PV-group specific US signs associated with 2 or more of the following US signs: a.3) an increased size of the head (diameter cranio-caudal >12 mm) and/or tail (diameter cranio- caudale >6 mm) of the epididymis; b.3) presence of multiple microcyst lesions confined to the epididymal head and/or tail; c.3) oedematous hyperechoic epididymis; d.3) a large hydrocele

WHEN AND HOW TO TREAT lems of couple’s fertility is summarized in INFERTILE PATIENTS WITH MAGI the Figure 3. A prolonged exposition/interaction Only patients with a clear clinical evi- between the inflammatory noxae on one dence of MAGI require a full scale treat- hand, and sperm and sexual glands on ment. Symptomatic or commonly asympto- the other hand, enhances an inflammato- matic MAGI should be treated with antibi- ry response through the release of semi- otics when a morphological and/or the func- nal reactive oxygen species (and tional sperm alteration is present, or when cytokines), which may persisted even fol- there is a sub-obstruction of the seminal lowing antibiotic treatment in patients with tract or a proven mono or multiple glandular complicated MAGI (PV and PVE) (Vicari, infection. On the other hand, the presence 2000). This justify a multi-step treatment of bacteriospermia or leukocytospermia of patients with PVE, who have a signifi- alone does not necessarily mean that there cantly higher production of ROS com- is a glandular infection, since temporary pared to patients with prostatitis or PV, inflammatory episodes are likely present in using as first-line therapy, antibiotics (fluo- the majority of sexually active men (Purvis roquinolones, macrolydes, or doxycicline & Christiansen, 1993). Furthermore, bacte- are potentially effective); as second-line riospermia may represent contamination, therapy, non-steroidal antiflammatory colonization or infection. The treatment (NSAI) compounds and finally as third-line algorithm of patient with MAGI and prob- therapy, antioxidants (combined carnitine

146 Male infertility today #4 plus acetilcarnitine) (Vicari & Calogero, and ROS production) and reproductive 2001; Vicari et al., 2002). When this (pregnancy rate) parameters has been sequential pharmacological management recently observed in patients with PVE is adopted, a significant increase of (Vicari & Calogero, 2001; Vicari et al., sperm (forward motility and viability; WBC 2002).

Figure 3. Treatement of the infetile patient with MAGI

Male infertility today #4 147 IMPACT OF INFECTED SPERM ON CONCLUSIONS ASSISTED REPRODUCTIVE TECH- Chronic symptomatic, or more common- NIQUES (ART) ly asymptomatic MAGI may lead to male The influence of seminal bacteria and/or infertility when the noxae, acting for long leukocyte on the outcome of IVF or ICSI periods of time, produce sperm and extend- (Michelmann, 1998) is influenced by three ed (to more accessory glands) glandular factors which have little in common with in- phlogosis/infection -related effects, enhanc- vivo conditions: ing a host inflammatory response, in terms 1. sperm process (swim-up, Percoll) with of semen ROS and pro-inflammatory antibiotic buffered media; cytokine (IL-1β; IL-6; IL-8; TNFα) hyperpro- 2. the small amount of inseminated sper- duction. These bioactive substances may matozoa (100,000) in IVF; persist even following antimicrobials, since 3. the short cultivation time. the initial antioxidant capacity (mainly based on epididymal biological micronutri- In contrast to viruses (i.e HIV, HBV, ents of the seminal plasma) is progressively HCV), the presence of bacteria in the ejcu- exhausted, thereby impairing sperm func- late does not influence ART program. The tion by inducing DNA damage and/or reason is that ejaculate preparation tech- apoptosis (Agarwal et al., 2003; Sanocka et niques, performed immediately after semen al., 2003). liquefaction, not only separate motile sper- The approach to the infertile patients matozoa but also wash them to get rid of with MAGI requires a comprenhensive bacterial impurities through various mecha- clinical investigation, including the charac- nisms: a) antibiotics (penicillin and strepto- terization of the different MAGI infertile mycin) as an additive to the medium; b) subgroups by ultrasound scans, microbio- washing procedure with centrifugation and logical investigation, immunocytological swim-up; c) high dilution factor. leukocyte determination, and biochemical Thus, if at the beginning of IVF the 29% measurement of ROS production (Vicari, of all ejaculates is contaminated, after 1999). Only when infertile men show a insemination medium exposure as well as clear clinical evidence of MAGI, a sequen- in the culture media no bacteria were tial step-wise pharmacological treatment detectable (Forman et al., 1987). The same (antibiotics NSAI antioxidants) may result was found by Cottell et al. (1996) who be established. did not find bacteria in the culture medium Microbiological investigations and sup- of embryos on the day of embryo transfer. pletive cytological (immunocitochemistry Furthermore, in this study, fertilization, leukocyte assessment; ROS analysis) are cleavage, and pregnancy rates were inde- not necessary if the suspected pathogens pendent of the microbial presence. (microbiotes, leukocytes) are not able to Short-term antibiotic treatment of asymp- induce severe and extented glandular infec- tomatic patients before ART should be done tions, proven through the lack of a significa- with caution, since the pregnancy rate after tive number of didymo-epididymal and in-vitro fertilization and embryo transfer does prostato-vesicular post-phlogistic abnormal- not improve (De Geyter et al., 1994), and it ities at the ultrasound scans. These condi- may also be detrimental to IVF outcome in tions may be defined as transitory phlogis- some cases (Liversedge et al., 1996). tic processes, due to noxae with low glan-

148 Male infertility today #4 dular reactive impact and have a negligible conditions. During ART, sperm processing impairment on fertility. (wash and swim-up) in an antibiotic rich cul- Taken in these terms, infertile patients ture medium effectively eliminates organ- with proven clinical MAGI represent a group isms and seems to be devoid of effects in of patients who may be treated rationally. IVF or ICSI outcome. However, the addition Furthermore, a carefull approach including of antioxidant compounds to culture media ultrasound (didymo-epididymal and rectal or during sperm preparation (Aitken et al., prostato-vesicular) investigation always 1991; Guerin et al., 2001) could be useful helps to prevent a further extension of the to reduce oxidative stress during sperm inflammatory process, which in turn may centrifugation, even in the specimens lead to a worst reproductive prognosis. obtained from patients with well-diagnosed A difference must be made when the PV or PVE, in whom ART is indicated fol- influence of bacteria is evaluated in-vitro lowing in vivo unsuccessfull treatment.

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