Human Reproduction vol.11 no 10 pp 2180-2185, 1996 Fertilizing ability of immotile spermatozoa after intracytoplasmic sperm injection

1 M.Nijs , P.Vanderzwalmen, B.Vandamme, indications are also evolving with time. Initial indications were G.Segal-Bertin, B.Lejeune, L.Segal, low sperm count, poor morphology and low motility. Other E.van Roosendaal and IL Schoysman types of patient have been identified as benefiting from this technique: failure of fertilization with classic in-vitro Downloaded from https://academic.oup.com/humrep/article/11/10/2180/569871 by guest on 28 September 2021 Schoysman Infertility Management Foundation, Van Helmont Hospital, Vaartstraat 42, 1800 Vilvoorde, Belgium fertilization (TVF) or subzonal insemination (SUZI) (Van Steirteghem et al, 1993; Fishel et al, 1994), globozoospermia 'To whom correspondence should be addressed (Trokoudes et al, 1995) and azoospermia with spermatozoa Sometimes spermatozoa from ejaculate, epididymis or testis extracted from the epididymis or testis (Schoysman et al, show a total absence of motility. For some patients, however, 1993a,b). very few spermatozoa with very poor motility can be In our laboratory, we have been confronted with samples found after several hours of incubation (initially immotile from ejaculate, epididymis or testis showing no motility at all spermatozoa). Other samples show no motility at all even at the time of collection, and even after preparation and after extended culture (totally immotile spermatozoa). purification on a Percoll gradient. For some patients a few Intracytoplasmic sperm injection (ICSI) is the only method spermatozoa with very poor motility were recovered after available to select and retrieve a single immotile or initially several hours of incubation. The motility of these spermatozoa immotile spermatozoon and inject it into the oocyte. A may be impaired by a number of factors, including disorders of total of 103 patients with asthenozoospermia underwent the sperm energy metabolism, a wide variety of ultrastructural ICSI in this study. It was shown that initially immotile and abnormalities in the sperm tail (Ryder et al, 1990) or defects totally immotile spermatozoa, whatever their origin, have in the pathways of cellular signalling. The occurrence of the capacity to fertilize an oocyte after ICSI. No signi- antisperm antibodies or genital infection can also be a cause ficant difference could be observed between the fertilizing of sperm immotility (Ansbacher et al, 1971; Del Porto et al, capacity of testicular or epididymal spermatozoa. Totally 1975). Because these patients have no chance of obtaining immotile ejaculated spermatozoa, however, fertilized fertilization with classic IVF methods, it was decided to submit significantly fewer oocytes after ICSI when compared their samples for ICSI. The aim of our study was to analyse with initially immotile ejaculated spermatozoa. Embryos the possible fertilizing capacity of totally immotile spermatozoa of lower quality tended to be produced when totally and initially immotile spermatozoa from various origins, i.e. immotile spermatozoa of any origin were used, compared ejaculate, epididymis or testis, after injection into the cytoplasm with embryos resulting from initially immotile sperm- of human oocytes. atozoa. Ongoing pregnancies were conceived after ICSI One of our patients was identified as suffering from with initially immotile spermatozoa from any origin and Kartagener syndrome. The ultrastructurally defective immotile totally immotile spermatozoa retrieved from testis only. spermatozoa were used for both ICSI and SUZI because One biochemical pregnancy was the result of embryo fertilization has been obtained by the latter technique (Wolf transfer after ICSI with totally immotile ejaculated sperm- et al, 1993). atozoa. No supernumerary embryos could be cryo- Fertilization rates, embryo quality, pregnancy rates and preserved for patients with totally immotile spermatozoa ongoing pregnancy rates were analysed for all types of from ejaculate or epididymis. For a Kartagener patient, sperm origin. subzonal insemination (SUZI) seemed to be a better approach for obtaining fertilization and pregnancy than Materials and methods ICSI because no fertilization occurred after ICSI on sibling oocytes. Hence a healthy pregnancy was obtained after In our FVF laboratory, patients are treated with ICSI when the total SUZI. motile sperm count is <600 000, the number of abnormal forms in Key words: ICSI/immotile spermatozoa/Kartagener syndrome the ejaculate is >85%, when total motility is <20% or when patients have a history of fertilization failure with classic IVF. In this study, 103 pauents with severe sperm motility disorders underwent ICSI (asthenozoospermia). Introduction Ovarian stimulation After Palermo performed the first intracytoplasmic sperm As described previously by Lejeune et al (1990), ovanan stimulation injection (ICSI) m 1991 (Palermo et al, 1992), the ICSI was carried out by administering gonadotrophin-releasing hormone procedure was applied routinely all over the world. ICSI (GnRH; buserelin; Suprefact, SP; Hoechst, Brussels, Belgium) in

2180 © European Society for Human Reproduction and Embryology ICSI with Immotile spermatozoa

Table I. Moolity and concentrauon of sperm samples of pauents included in the study

On gin Ranges of sperm Iruual mouhty Mouhty after preparauon No of pauents included concentrauon (noJml) (%) and incubauon (%) (total = 103)

Ejaculate 10-40 X 106 0 00 12 100 000-60 X 106 0 0 1 20 Epididymis 1000-100 000 0 00 3 50 000-10 x 10* 0 01 15 Tesus 100-10000 0 00 26 1000-1 x 10* 0 0 1 27

association with human menopausal gonadotrophin (HMG; Pergonal; previously by Vanderzwalmen etal (1996). Two types of micropipette Downloaded from https://academic.oup.com/humrep/article/11/10/2180/569871 by guest on 28 September 2021 Serono Laboratories Inc., Brussels, Belgium) Human chorioruc were used. For the iniDally immotile spermatozoa a 10-13 pm pipette gonadotrophin (HCG; Pregnyl; Organon, Oss, The Netherlands; was used to retrieve those spermatozoa that showed some motility Profasi, Serono Laboratories Inc.) was given when the cohort of out of the swim-out droplet. These spermatozoa were released into follicles reached a diameter of -20 mm. Luteal phase support consisted the 3 jxl droplet in the micromanipulation dish. For some patients of administering 5000 IU HCG on days 4 and 8 after transfer with initially immotile spermatozoa, the final concentration in the swim-out droplet was sufficient to aspirate some of the sluggishly Sperm preparation motile spermatozoa on the edge of the droplet with a mouth-controlled Ejaculates were received as split samples Micro-epididymal sperm glass pipette of 30 |im diameter. These sperm sampling steps aspirations (MESA) and testicular sperm extraction (TESE) biopsies were all performed using an inverted microscope (Olympus IMT2; were prepared as described previously (Schoysman et al, 1993a, Omnilabo, Brussels, Belgium). Narashige manipulators and injectors Vanderzwalmen et al, 1994). were used to perform the ICSI procedure (Narashige; Omnilabo) For 103 patients, the absence of motihty and sperm concentra- tion were assessed on samples from ejaculate, epididymis or testis Intracytoplasmic sperm injection biopsy (Table I). Samples were centnfuged at a higher speed than No alterations were made to the ICSI procedure itself or to the normal (500 g) and/or placed on a discontinuous Percoll gradient media conditions when compared with our routine ICSI with motile (Vanderzwalmen et al, 1991) with two or three layers for 15-40 mm spermatozoa For the ICSI procedure itself, a 7 5 um (outer diameter) (depending on the concentration) After a washing step in Earle's micropipette was used to aspirate individual spermatozoa from the medium (Sigma, Bomem, Belgium) supplemented with 0.5% human sperm collection droplet and to place them into the PVP. Although serum albumin (Irvine Scientific, Zelhk, Belgium), a drop of the most of the spermatozoa were immotile, all tails were broken by sample was placed under oil (Sigma) in the cover of a Petn gentle rubbing with the micropipette. The spermatozoon was aspirated dish (Falcon, Vel, Leuven, Belgium) and incubated at 37°C in an (tail first) with some PVP into the micropipette and injected mto the atmosphere of 5% CO2 for 2-3 h. When motile spermatozoa were equatorial region of the oocyte after aspirating some ooplasm. present after preparation and incubation, they showed a poor head or The injected oocytes were washed four times and incubated in tail displacement and sometimes moved out from the central part of culture medium at 37°C in an atmosphere of 5% CO2. the drop which contained dead spermatozoa, immotile spermatozoa and blood cells ('swim-out' technique). Few moved to the edge of Subzonal sperm injection procedure the droplet. These sperm samples were defined as containing initially In all, 10-15 spermatozoa were aspirated with a 10 |im (outer immotile spermatozoa. Other samples showed no motihty at all, even diameter) pipette from the sperm collection droplet and released after extended incubation, and were classified as containing totally underneath the zona pellucida of the oocytes immotile spermatozoa. After an electron microscopy investigation, one patient was identified as carrying Kartagener syndrome. Some Assessment of fertilization and embryo cleavage of his partner's oocytes were submitted for ICSI and others for SUZI. At 18-20 h after the ICSI procedure the oocytes were checked for the presence of one, two or more pronuclei. The number of degenerated Oocyte preparation oocytes was recorded. Pnor to transfer (i.e 42 h after ICSI), the At 2-3 h after oocyte retrieval, the cumulus cells were removed by embryos were scored for their number of cells and overall quality a very brief incubation in 80 IU hyaluronidase (Type VHI; Sigma) aspect: the number and shape of the blastomeres, the percentage of in FVF 50 culture medium (Scandinavian IVF Science, GOteborg, fragments (Nijs et aL, 1993a) In general, three grade A embryos Sweden). The corona radiata was removed by aspirating the oocytes were selected for transfer. If available, grade A supernumerary in and out of a glass pipette with a diameter ranging from 180 to embryos were frozen using the slow propanediol-sucrose protocol 220 |im in IVF 50. The oocytes were washed four times and incubated (Nijs et al, 1993b) in culture medium under oil until further processing. ICSI was The transfers were performed using an Edwards-Wallace embryo performed on oocytes that had extruded their first polar body transfer catheter (International Medical, Brussels, Belgium) -42- (metaphase U oocytes), 3—4 h after their retrieval. 45 h after the ICSI procedure

Sperm collection Follow-up Prior to micromampulation, a micromanipulation dish was prepared Luteal phase support consisted of 5000 IU HCG given on days 4 and containing one central droplet of \0°h polyvinylpyrrohdone (PVP, 8 after transfer Pregnancy was diagnosed on the basis of an HCG Sigma), five droplets of 10 joJ culture medium-1% HEPES and one concentration >30 mlU/ml in serum on day 15, with confirmation droplet of 3 ul culture medium-1% HEPES under oil on days 20 and 25 after transfer Vaginal ultrasound sonography was Details of the preparation of the micropipettes have been described earned out in week 6 after transfer The presence of a yolk sac, the 2181 M.Nijs et aL

Table II. Subzonal insemination (SUZI) and uitracytoplasmic sperm Table IV. The use of initially rmmotile spennatozoa from ejaculate, epididymis or injection (ICSI) with totally immotile spennatozoa from a patient suffering testis in an lntracytoplasmic sperm injection programme 1:erulizing capacity, pregnancy from Kartagener syndrome rate, ongoing pregnancy rates, patients entering the freezing programme

SUZI ICSI Ejaculate Epididymis Testis

No of oocytes manipulated 12 4 No of patients 20 15 27 No of two-pronuclear/marupulated oocytes 3/12 0/4 No of two-pronuclear/injected oocytes 105/162 100/160 143/238 No of embryos transferred 3 - Fertilization rate (%) 65 63 60 Pregnancy 1 No. of transfers 20 15 26 Patients with transfers (%) 100 100 100 Mean no of embryos/transfer 26 3 3.3 No. of pregnancies/transfer 6720" 9/15** mi* Pregnancies (%) 30 60 30 Miscarriages/biochemical pregnancy 3(50) 1(11) 1(13) Table HL The use of totally immotile spermatozoa from ejaculate, epididymis Downloaded from https://academic.oup.com/humrep/article/11/10/2180/569871 by guest on 28 September 2021 or testis in an lntracytoplasmic sperm injection programme: fertilizing No of ongoing pregnancies 3(15) 8(53) 7(26) capacity, pregnancy rate, ongoing pregnancy rales, patients entering the Patients with freezing programme 3/20(15) 3/15 (20) 4/26 (15) freezing programme Values in parentheses are percentages Ejaculate Epididymis Testis •^Values with the same superscripts were significantly different (P < 0 05)

No of patients 12 3 26 Fertilization No of two-pronuclear/injected oocytes 75/141 18/30 142/220 Fertilization rate (%) 53' 60 65' Whatever the origin, it is clear that totally immotile and No of transfers 12 3 26 initially immotile spermatozoa have the capacity to fertilize Patients with transfers (%) 100 100 100 an oocyte by ICSI (Table HI and J7V): fertilization ranged from Mean no of embryos/transfer 3 0 4 3 3 0 No of pregnancies/transfer l/12b 0/3 8/26b 53 to 65%. Pregnancies (%) 8-31 When initially immotile spermatozoa were used for ICSI, c Miscarriages/biochemical pregnancy l (100) - 3 (38) the origin of the spermatozoa did not seem to play an important No of ongoing pregnancies 0(0) - 5(19) Patients with freezing programme - - 4/26 (15) role: 65% of the oocytes injected with ejaculated spermatozoa showed two pronuclei after 18 h (105/162). Sluggishly motile Values in parentheses are percentages spermatozoa from epididymis or testis showed a 63 and •"•"Values with the same superscripts were significantly different (P < 0 05) cBiochemical pregnancy 60% fertilizing capacity respectively (100/160 and 143/238). Comparable fertilization rates were obtained when totally immotile spermatozoa from epididymis or testis were injected fetus and heart activity were evaluated and registered as a clinical into metaphase II oocytes: 18 of the 30 oocytes (60%) and pregnancy. 142 out of 220 oocytes (65%) respectively were fertilized after injection. Results When totally immotile spermatozoa from ejaculates were used, fertilization rates dropped to 53%, compared with totally Kartagener syndrome immotile spermatozoa from epididymis or testis (the latter For the patient suffering from Kartagener syndrome, three out difference was significant; P < 0.05) and with the fertilization of 12 oocytes fertilized after SUZI. None of the oocytes rate of initially immotile ejaculated spermatozoa (65%; 105/ fertilized after ICSI. Three good quality embryos were replaced 162 versus 75/141;/> < 0.05) into the partner, who gave birth to one healthy baby boy (Table II). Embryo quality and freezing programme Figure 1 represents the quality scoring of the embryos 42 h Immotile spermatozoa after ICSI. When totally immotile spermatozoa were injected A total of 103 patients with immotile spermatozoa in their into oocytes, the overall embryo quality of the embryos was initial sperm samples were included in this study (Table I): 41 lower than when initially immotile spermatozoa were used of them had sperm samples showing no motility at all, even Fewer grade A embryos resulting from totally immotile after prolonged culture, details of which are shown in Table spermatozoa (and subsequently more grade B embryos) were HI. For 62 patients, initially immotile spermatozoa were available for transfer. Hence, fewer embryos were available obtained in the fresh samples of ejaculate, epididymis or testis. for freezing when totally immotile spermatozoa were used However, after preparation and incubation for several hours, (Tables ffl-IV). Of the patients with 'initially immotile' some of their sperm cells showed a very poor sluggish motility. spermatozoa, 15-20% had embryos that could enter the freezing Details of these samples are given in Table TV. programme. When totally immotile testicular spermatozoa Of the retrieved oocytes, 84% were at the metaphase II were used, freezing was possible for the embryos from 15% stage (951/1132). An average of 9.2 oocytes per patient were of the patients. injected with spermatozoa. The outcome of the study using totally immotile spermatozoa Transfer and pregnancy rates and initially immotile spennatozoa from different origins for All 103 patients in this study had an embryo transfer (Tables ICSI is presented in Tables HI and IV. in and IV). A mean number of 3.2 embryos was transferred 2182 ICSI with immotile spermatozoa

100

80

60 D grade A B grade B 40 a grade C

20

0 ejac epld testis Downloaded from https://academic.oup.com/humrep/article/11/10/2180/569871 by guest on 28 September 2021 Initially immotile spermatozoa Totally immotile spermatozoa

Figure 1. Embryo quality at the moment of transfer (day 2) after intracytoplasmic sperm mjection with initially and totally immoule spermatozoa from ejaculate (ejac), epididymis (epid) or testis. Y axis = percentage of embryos X axis = quality/grade of embryo, grade A, B.C.

per patient. Encouraging pregnancy rates were obtained after formation has been described after the injection of sperm ICSI with initially immotile spermatozoa from any origin and nuclei or intact dead spermatozoa from several species (human, totally immotile spermatozoa retrieved from the testis (from hamster, mouse, rabbit and bull) into hamster oocytes (Uehara 30 to 60%). For the initially immotile sperm group, six out of and Yanigamachi, 1976, 1977; Martin et al, 1988; Perreault 20 patients became pregnant with ejaculated spermatozoa et al, 1988). Gearon et al. (1995) injected non-viable human (30%). Of the 'epididymaT patients, 60% obtained a pregnancy spermatozoa into aged human oocytes and observed fertiliza- (9/15) and 30% of the 'testicular' ones became pregnant (8/27) tion and early cleavage. Ghunaim et al. (1995) injected totally As mentioned, 31% of the patients who had totally immotile immotile human spermatozoa stored for 2 days at 4°C and testicular spermatozoa used for ICSI (8/26) had positive HCG obtained fertilization and development. Other authors did values 15 days after transfer. One patient became pregnant not find any fertilization after ICSI with totally immotile when using totally immotile spermatozoa from the ejaculate spermatozoa (Liu et al, 1995). The reason is probably because (8%), but unfortunately this pregnancy turned out to be a of a technical problem rather than the fertilizing potential of biochemical one. No pregnancy was obtained for the 3 patients the spermatozoon. Because these spermatozoa are immotile, in the totally immotile epididymal sperm group. one might think that it is not necessary to immobilize them In all sperm groups miscarriages occurred, ranging from 11 before injection. However, their tail must still be touched (initially immotile epididymal spermatozoa) to 50% (initially before injection (Vanderzwalmen et al, 1996) to induce a immotile ejaculated spermatozoa). destabilization in the plasma membrane with the release of an Ongoing pregnancies were obtained in 15, 53 and 26% of activating factor 'oscillin' into the ooplasm (Dozortsev et al, the cycles using ejaculate, epididymal and testicular initially 1995; Parrington et al, 1996). Another possibility is that depolymerization of the sperm membrane is necessary for immotile spermatozoa respectively When totally immotile ooplasmic enzymes to reach the sperm nucleus in order to spermatozoa were used, ongoing pregnancies were only start chromatin decondensation (Tesarik et al, 1994). Totally obtained with testicular spermatozoa. No ongoing pregnancies immotile spermatozoa from ejaculates fertilized significantly followed ICSI with totally immotile spermatozoa from ejaculate fewer oocytes than all the other sperm types. Perhaps the or epididymis. patient had a long period of abstinence and hence spermatozoa had a long transit time through the epididymis. This increases Discussion the presence of degenerating or dead spermatozoa in the sperm ICSI has been introduced for patients suffering from severe sample (Bedford, 1994). These aged spermatozoa contain ohgoasthenoteratozoospermia, previous fertilization failure or fragile DNA, the protamine packaging has become unstable and pronucleus formation is subsequently hampered. azoospermia (Catt et al, 1995). Our study has identified another important group of patients who could clearly benefit Overall, 95% of the two-pronuclei oocytes developed into from microinjection of their (initially) immotile spermatozoa 4- or 8-cell embryos, and all asthenozoospermic patients had into oocytes. It was shown that initially immotile and totally embryos transferred. immotile spermatozoa, whatever their origin (ejaculate, epidi- The observation that totally immotile human spermatozoa dymis or testis) have the capacity to fertilize an oocyte can support the early embryo and fetal development confirms after ICSI. However, totally immotile ejaculated spermatozoa studies m the cow by Goto et al. (1990). They described the fertilized significantly fewer oocytes than the other sperm birth of a calf after ICSI with immobilized killed spermatozoa groups. In our study, a difference in embryo quality according to the The potential of an immotile human spermatozoon to activate type of immotihty, however, was observed: fewer good quality a mammalian oocyte is not a novel observation: pronucleus embryos were obtained when totally immotile spermatozoa (of 2183 any origin) were used compared with embryos derived from either degenerative (dead) or mature but structurally affected. initially immotile spermatozoa. Consequently, no embryos Testicular totally immotile spermatozoa may have delayed could be cryopreserved for patients with totally immotile maturation and have not yet switched on their energetic spermatozoa from ejaculate or epididymis. As already pathways for motility. This latter maturation process is perhaps explained, agemg of spermatozoa can result in DNA strand time and/or culture dependent. We are currently evaluating the breaks and, although fertilization and first cleavage stages can influence of a long-term culture (up to 48 h) and different occur, the genome of the spermatozoon is not capable of culture conditions on totally immotile spermatozoa of any completing embryogenesis. Defective protamine packaging in origin. The use of the swelling test (Van der Ven et al., 1986) sperm DNA and their incorrect replacement by histones was applied on some samples but did not prove to be efficient during fertilization and cleavage could create problems like because most of the samples contained few spermatozoa after asynchrony and delays during cell cycles just after fertilization. preparation. We are currently investigating in more detail, Some of the anomalies arising in embryos after the use of however, if this test (in combination with ICSI) could be used immotile spermatozoa could be due to abnormal spindle to identify those spermatozoa that are viable but have not yet Downloaded from https://academic.oup.com/humrep/article/11/10/2180/569871 by guest on 28 September 2021 formation because the centrioles were abnormal (Asch et al, acquired any form of motility. These spermatozoa could then 1995). Because for these groups not enough supernumerary be injected into oocytes and could prove to be better candidates embryos of good quality were available for further culture for ICSI: in this way, better fertilization rates, better embryo in vitro up to the blastocyst stage, we could not confirm this quality and more ongoing pregnancies could be obtained. If hypothesis of complete embryo blockage as yet. It confirms, longer incubation times of totally immotile ejaculated or however, the studies of Janny and Me'ndzo (1994), who epididymal spermatozoa still do not result in an improvement, identified a strong paternal effect on human embryo develop- one could try to extract sperm cells from the testis of these ment and blastocyst formation. The presence of only one patients and determine whether these spermatozoa show the biochemical pregnancy (for the totally immotile ejaculated same pathology. sperm group), the absence of any pregnancy for totally immotile epididymal spermatozoa and the relatively high miscarriage rate (50%) in the initially immotile ejaculated Acknowledgements sperm group can again be explained by the low potential of We thank Mr N Junker from Olympus-Optical co-GnBH, Germany and Mr TGielens from Omnilabo, Belgium for the use of their the embryonal genome. Olympus EX 70 inverted microscope with Narashige manipulators Why do some samples show totally immotile spermatozoa We thank Dr W.Ombelet and the Organizing Committee from the and others obtain some form of motility after a short period7 International Symposium on Human Reproduction and Male Sub- One of the causes of immotility is the presence of antisperm fertihty 'Andrology in The Nineties, 1995, Genk, Belgium' for the Award of the Best Free Communication antibodies. Their presence interferes considerably with preg- nancy outcome because they cause a developmental block just before genomic activation (Naz, 1992). Another type of defect References is structural abnormality. Kartagener syndrome patients suffer Ansbacher, R., Manarang-Pangan, S and Snvannaboon, S. (1971) Sperm from the absence of dynein arms. However, in the ejaculate antibodies in infertile couples. Feral Stenl, 22, 298-302. Asch, R , Simerly, C, Ord, V and G. Schatten, G (1995) The stages at which of our patient, dead spermatozoa could also be present. When human fertilization arrests microtubule and chromosome configurations in performing the micromanipulation, no distinction can be made inseminated oocytes which failed to complete fertiuzanon and development between these types of sperm cell. When SUZI was applied, in humans. Mol Hum. Reprod, 1, 1897-1906. the theoretical probability that one mature spermatozoon was Bedford, J (1994) Epididymal physiology its implications for epididymal microsurgery. In Schoysman, R. (ed.)t Microsurgery of Male Infertility present in the >10 spermatozoa injected was obviously higher Fondazione per gli studi sulla riproduzione umana, Palermo, Italy, pp than when only one spermatozoon is retrieved for ICSI. This 71-100. could explain the difference in fertilization rates. Aldiough Catt, J , Ryan, J , Pike, L et al (1995) Fertilization rates using mtracytoplasmic sperm injection are greater than subzonal insemination but are dependent only one patient was included in this study, SUZI seems to on prior treatment of sperm FemL Stenl, 64, 764—769. be the method of choice for this specific condition (Wolf Del Porto, G , Derrick, F and Bannister, E. (1975) Bacterial effects on sperm et al., 1993). motility. Urology, 5, 638-639 Some initially immotile spermatozoa obtained some form Dozortsev, D, Rybouchkw, A., De Sutter, P et al (1995) Human oocyte activauon following intracytoplasmic injection the role of the sperm cell of motility after culture. Bedford (1994) described that during Hum. Reprod., 10, 403-^07. transit through the testis and epididymis, spermatozoa undergo Fishel, S , Tunson, J , Lisi, F. et al (1994) Microassisted fertilization who several biophysical alterations: S-S bonds are stabilized in the have failed subzonal insemination Hum. Reprod, 9, 501-505 Gearon, C , Taylor, A and Fonnan, R. (1995) Factors affecting activation and sperm nucleus, in the perinuclear matrix and in various tail fertilization of human oocytes following mtracytoplasmic injection. Hum. structures. Perhaps preparation, purification and incubation of Reprod, 10, 896-902. these immotile spermatozoa can circumvent these in-vivo Ghunaim, S., Nijs, M., Keilani, S et al (1995) Fertilization with two-day old maturation processes (Schoysman et al., 1995). The initially lmmonle sperm after mtracytoplasmic sperm injection in a human IVF program. J Assist. Reprod., 12, Abstr OC-232. immotile spermatozoa (from ejaculate, epididymis or testis) Goto, K., Kinosmta, A., Takuma, Y. and Ogawa, K. (1990) Fertilisation of have the capacity to recover sluggish motility because most bovine oocytes by the injection of immobilised, killed spermatozoa. Vet. of this sperm population is probably immature. For the totally Rec, 517-519 immotile sperm cells, however, it is thought that for epididymal Janny, L. and Menizo, Y (1994) Evidence for a strong paternal effect on human pTeimplantation embryo development and blastocyst formation Mol and ejaculated cells the majority of the spermatozoa are Reprod. Dev, 38, 36-42. 2184 ICSI with immotile spermatozoa

Lejeune, B , Bariow, P, Puissant, F and Leroy, F (1990) BusereUn acetate in an m vitro fertilisation program, a comparison with classical clomiphene citrate-human menopausal gonadotrophin treatment Fertil StenL, 54, 475-^81 Liu, J, Nagy, Z., Jons, H et al (1995) Analysis of 76 fertilization failure cycles out of 2732 lntracytoplasmic sperm injection cycles Hum. Reprod., 10, 2630-2636 Martin, R H., Ko, E and Rademaker, A (1988) Human sperm chromosome complements after rrucroinjection of hamster eggs J Reprod. Feml, 84, 179-186 Naz, R. (1992) Effects of antisperm antibodies on early cleavage of fertilized ova. Bio/. Reprod., 46, 130-139. Nijs, M , Geerts, L., van Roosendaal, E et al (1993a) Prevention of multiple pregnancies in an m vitro fertilization program Fertil Stenl, 59,1245-1250 Nijs, M , Vanderzwalmen, P, Segal-Bertin, G et at (1993b) A monozygonc twin pregnancy after application of zona rubbing on a frozen-thawed Downloaded from https://academic.oup.com/humrep/article/11/10/2180/569871 by guest on 28 September 2021 blastocyst Hum. Reprod., 8, 127-129 Palermo, G., Joris, H, Devroey, P et al (1992) Pregnancies after lntracytoplasmic injection of a single spermatozoa mto an oocyte Lancet, 340, 17-18. Pamngton, J, Swann, K., Shevchenko, V et al (1996) Calcium oscillations in mammalian eggs triggered by a soluble sperm protein. Nature, 379, 364-368 Perreault, S , Barbee, R., Elstein, K et al (1988) Interspecies differences in the stability of mammalian sperm nuclei assessed in vivo by sperm microinjection and in vitro by flow cytometry BioL Reprod., 39, 157-167. Ryder, T, Mobberley, M, Hughes, L. et al (1990) A survey of the ultrastmctural defects associated with absent or impaired human sperm moulity Feml Stenl, 53, 556-560 Schoysman, R, Vanderzwalmen, P, Nijs, M et al (1993a) Pregnancy after fertilisation with human testicular spermatozoa. Lancet, Ml, 13 Schoysman, R, Segal, L, Vanderzwalmen, P et al (1993b) Assisted fertilization with epididymal spermatozoa. Acta Eur Fen., 24, 1 Schoysman, R, Vanderzwalmen, P., Nijs, M et al (1995) Indications for use of deferential sperm in IVF programs In Proceedings of the World Congress of In Vitro Fertilization. Vienna, Austria, Abstr 1-4, p 4. Tesank, J , Sousa, M and Testart, J (1994) Human oocyte activation after lntracytoplasmic sperm injection Hum. Reprod., 9, 511-518. Trokoudes, K, Danos, N, Kalogirou, L et al (1995) Pregnancy with spermatozoa from a globozoospermic man after lntracytoplasrruc sperm injection treatment. Hum. Reprod., 10, 880-882 Uehara, T and Yarugamachi, R. (1976) Microsurgical injection of spermatozoa into hamster eggs with subsequent transformation of sperm nuclei mto male pronuclei BioL Reprod., 15, 467-470 Uehara, T and Yanigamachi, R (1977) Behavior of nuclei of testicular, caput and cauda epididymal spermatozoa injected into hamster eggs Bwl Reprod., 16, 315-321 Van der Ven, H, Jeyendran, R, Al-Hasani, S et al (1986) Correlation between human sperm swelling in hypo-osmotic medium (hypo-osmotic swelling test) and in vitro fertilization J Androl, 7, 190-1% Van Steuteghem, A., Liu, J, Joris, H et al (1993) Higher success rate by lntracytoplasmic sperm injection than by subzonal insemination Report of a second series of 300 consecutive treatment cycles. Hum. Reprod., 8, 1055-1066 Vanderzwalmen, P, Bertin, G, Geerts, I* et al (1991) Sperm morphology and IVF pregnancy rate a comparison between Percoll gradient centnfugation and swim-up procedures Hum. Reprod., 6, 581-588 Vanderzwalmen, P, Nijs, M., Segal-Bertin, G et al (1994) Epididymal sperm aspiration for in vitro fertilisation In Schoysman, R. (ed.). Microsurgery of Male InfeniUty Fondazione per gli studi suUa nproduzione umana, Palermo, Italy pp 229-248 Vanderzwalmen, P, Berlin, G, Lejeune, B et al (1996) Two essential steps for a successful lntracytoplasmic sperm injection injection of immobilized spermatozoa after rupture of the oolema. Hum. Reprod., 11, 540-547 Wolf, J., Feneux, D, Escalier, D et al (1993) Pregnancy after subzonal insemination with spermatozoa lacking dynein arms J Reprod. Feml, 97, 487-492

Received on Apnl 29, 1996, accepted on July 27, 1996

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