Fertility with testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermic men

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1 Human Reproduction vol. no.4 pp.75-70, 199 Fertility with testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermic men Semra Kahraman 1-4, Suat Ozgur 2, Cengiz Alatas. 1, Senai Aksoy 1, Murat Tasdemir 1, Alp Nuhoglu 1, I lk Ta^demir 1, Basak Balaban 1, Kutay Biberoglu 1, Robert Schoysman 3, Martine Nys 3 and Pierre Vanderzwalmen 3 'Sevgi Hospital, Assisted Reproductive Techniques and Reproductive Endocrinology Unit, Tunus Caddesi No. 2, 00 Ankara, 2 Department of Urology, Sevgi Hospital, Ankara, Turkey and 3 SIF, Von Helmont Hospital, Fertility Unit, VUvoorde, Belgium ^o whom correspondence should be addressed In non-obstructive azoospermia spermatozoa can usually only be isolated from the testicles, and thus the most promising treatment model is testicular sperm extraction (TESE). Hormone concentrations, testicular volume determinations and testicular biopsy results are not uniform enough to select potential candidates for successful TESE and intracytoplasmic sperm injection (ICSI) approaches in advance. The aim of this study was to assess the efficacy of using ICSI with testicular spermatozoa in cases of nonobstructive azoospermia and to compare the inclusion criteria and sperm existence in the testicles in sperm obtainable and non-obtainable groups. All men showed either complete or incomplete (n = 14) maturation arrest in spermatogenesis, severe hypospermatogenesis (n = 10) or Sertoli cell-only syndrome (n = 5) in their testicular biopsies. Only 14 out of a total of 29 men provided enough spermatozoa for the ICSI procedure, while no spermatozoa were found in the testicular samples of the remaining 15 men. Out of 123 oocytes obtained from 14 females, 101 were injected with the husbands' testicular sperm cells. Total fertilization failure was observed in three cases. Of 39 oocytes fertilized, 3 cleaved. The fertilization and cleavage rates were 3. and 97.4% respectively. The pregnancy rate was 20.7% per initiated cycle. In the group from whom spermatozoa were obtainable, the pregnancy rate was 42.9% per initiated cycle and 54.5% per embryo transfer. A total of six pregnancies were achieved, of which two Were twins and four were singletons. One singleton pregnancy resulted in abortion in the first trimester. There was no statistical difference concerning the serum follicle stimnlating hormone concentration, testicular volume and biopsy results in groups in which spermatozoa were obtainable or not. In conclusion, although the association of TESE with ICSI obtained pregnancies for some patients with non-obstructive azoospermia, further studies are needed to determine the inclusion criteria for successful TESE. 75 Key words: fertility/intracytoplasmic sperm injection/nonobstructive azoospermia/testicular sperm extraction Introduction In obstructive azoospermia where microsurgical epididymal sperm aspiration (MESA) is impossible because of a totally destroyed epididymis, normal fertilization and pregnancy have been achieved using testicular spermatozoa (Silber et al, 1995a,b). In non-obstructive azoospermia, fertilization, cleavage and pregnancy have been achieved in cases of almost complete spermatogenic arrest (Devroey et al, 1995). Normal follicle stimulating hormone (FSH) concentration and testicular volume suggest the presence of spermatogenesis, thus indicating the need for testicular biopsy in azoospermic men. On the other hand, high FSH concentrations are considered to be an indication of testicular failure, implying that testicular biopsy may not be recommended. Such general approaches may not always reflect the truth. There is now evidence that a high FSH concentration in azoospermia is not always a reliable indicator of testicular failure, and that FSH concentration can be normal in the case of a severe spermatogenic defect (Devroey et al, 1995). Unfortunately, criteria for predicting the presence of spermatozoa in the testicular tissue of nonobstructive azoospermic men are lacking. The aims of our study were to assess the efficacy of intracytoplasmic sperm injection (ICSI) with testicular spermatozoa in non-obstructive azoospermia and to assess the relevance of serum FSH concentration, testicular volume and testicular biopsy results as inclusion criteria for the testicular sperm extraction (TESE) procedure, in groups of men from whom spermatozoa were either obtainable or not obtainable. Materials and methods Patients TESE was performed in the male partners of 29 infertile couples with non-obstructive azoospermia between January and May The mean ± SD female and male ages were 2.0 ± 3.1 and 34.0 ± 4.2 years; the mean infertility duration was.0 ± 2.0 years. Azoospermic males underwent a careful physical examination, a hormonal evaluation and a testicular volume determination by ultrasound (mean value of two testes). During the testicular biopsy, a scrotal exploration was performed to rule out the presence of any epididymal obstruction. The serum FSH concentration was determined by a radioimmunoassay. Histological examinations of testicular biopsy specimens from all patients were performed during the diagnostic work-up. Each specimen was fixed in Bouin's solution and stained with haematoxylin and eosin for the evaluation of histological details. European Society for Human Reproduction and Embryology

2 Fertility in non-obstructive azoospermic men The classification of histological findings was made according to Levin's criteria (Levin, 1979). In five patients diagnosed as having Sertoli cell-only syndrome, either an occasional single seminiferous tubule with spermatogenic activity (n = 2) was observed or no spermatogenic activity (n = 3) was seen. In all, 14 patients had complete or incomplete maturation arrest, and 10 had severe hypospermatogenesis, defined as a non-focal generalized reduction in spermatogenic activity in all seminiferous tubules. Sperm sampling and preparation Testicular tissue samples were obtained by open biopsy on the same day that the oocytes were retrieved. During the testicular biopsy, all patients were evaluated for obstructive azoospermia by a scrotal exploration. Testicular tissue was placed in Falcon tubes containing 1 ml HEPES-buffered Earle's medium. Testicular tissue samples were then progressively divided into small segments, and gently crushed between micro-needles in a Petri dish containing HEPES-buffered medium to obtain a sperm suspension. The sperm suspension was then transferred into a Falcon tube and centrifuged for 5 min at 300 g (Schoysman et al, 1993; Silber, 1995a). Another suspension containing 5 ml HEPES-buffered Earle's medium was then examined in a Petri dish at X200 magnification. If the sperm cells were identified, the suspension was centrifuged on a two-layer Percoll gradient (70 and 90%) and freed from all debris and red blood cells. The testicular tissue solution was kept in the incubator (5% CO2 and air) at 37 C until the ICSI procedure could be performed. Ovarian stimulation Ovarian stimulation was carried out with the combination of gonadotrophin-releasing hormone (GnRH) agonist (Suprefact; Hoechst, Frankfurt, Germany) and the injection of FSH (Metrodin; Serono, Rome, Italy) and human menopausal gonadotrophin (HMG; Humegon; Organon, Oss, The Netherlands). Ovulation was induced by the injection of IU human cborionic gonadotrophin (HCG; Pregnyl; Organon, Istanbul, Turkey). Oocytes were retrieved 3 h after HCG administration by vaginal ultrasound-guided puncture of the ovarian follicles. Oocyte preparation The cumulus-corona cell complexes were removed by incubating in a solution of HEPES-buffered Earle's medium containing 0 IU/ml hyaluronidase (type VHI, specific activity 320 IU/mg; Sigma Chemical Co., St Louis, MO, USA) for 30 s and by aspiration of the cell complex into and out of a hand-drawn glass pipette. The oocytes were rinsed several times in HEPES-buffered Earle's medium. They were examined under an inverted microscope at X200 magnification for nuclear maturity and cytoplasmic evaluation. Oocytes were incubated in Earle's balanced salt solution medium at 37 C in an atmosphere of 5% CO2 with air, covered by paraffin oil. Only metaphase II oocytes were used for microinjection. ICSI procedure Holding and injection pipettes were made from 30 \il borosilicate glass capillary tubes (Drummond Scientific Company, Broomall, PA, USA). After washing several times, the pipettes were pulled on a horizontal microelectrode puller (PB-7; Narishige Co. Ltd, Tokyo, Japan). The pulled capillary was opened on a microgrinder (EG-4; Narishige Co. Ltd). The inner diameter of the microinjecting pipette was 7 pm and the bevel angle was 50, which was bent by a microforge (MF-9; Narishige Co. Ltd). The holding pipette was also cut and fire-polished on a microfotge (20 im inner and 0 im outer diameter). The oocytes were placed one at a time in droplets of HEPESbuffered Earle's medium. Single sluggishly motile or immotile spermatozoa were selected and aspirated one by one with a 15 (im (tip diameter) pipette and transferred into the injection dish containing a droplet of 5 u,l HEPES-buffered Earle's medium, before being transferred into a droplet of 10% polyvinylpyrrolidonc (PVP; P52; Sigma Chemical Co.). The ICSI procedure (Van Steirteghem et al, 1993) was performed on the heated stage of an inverted microscope (IMT-2; Olympus Corporation, Tokyo, Japan). The microscope was equipped with two coarse positioning manipulators (MM-1; Narishige Co. Ltd). A selected spermatozoon was immobilized by touching the tail before it was aspirated, tail first, into the injection pipette. Touching of the tail was performed even in the presence of a totally immotile sperm sample. The oocyte was held with a holding pipette, with the polar body at the 12 or o'clock position. The injecting pipette containing the spermatozoon was introduced across the zona pellucida and into the oocyte's cytoplasm. A small part of the cytoplasm was aspirated as cytoplasmic breakage was observed. The spermatozoon and the cytoplasm were ejected, and the injecting pipette was withdrawn gently. The injected oocytes were placed into a four-well Nunc dish for incubation. Assessment of fertilization, embryo cleavage and establishment of pregnancy The oocytes were observed for the presence of pronuclei 1-1 h after ICSI. Fertilization was assessed as normal when two clearly distinct pronuclei containing nucleoli were visible. The state of embryo cleavage and quality were assessed after a further 24 h of in-vitro culture. Embryos were evaluated according to the blastomere size equality and the relative proportion of anucleate fragments. A maximum of four embryos were transferred in exceptional cases when all the available embryos had >50% fragmentation. Pregnancy was confirmed by increased serum HCG concentrations 12 and 1 days after embryo transfer. Clinical pregnancy was diagnosed by ultrasonography at 7 weeks of pregnancy. All couples were counselled and agreed to have prenatal diagnosis. Prenatal diagnosis was performed by amniocentesis at 1 weeks of pregnancy. Couples were also informed about a prospective follow-up study of the children bom after ICSI. Statistical analysis Statistical tests were performed using the StatView 4.0 package (BrainPower Inc., Calabasas, CA, USA) on a Macintosh PowerBook 10 personal computer. A statistical analysis between the different groups was performed using the Mann-Whitney U and % 2 tests, as needed. The correlation between FSH concentration, testicular volume, testicular tissue biopsy result and the success of TESE was analysed by multiple and logistic regressions. Results No spermatozoa could be retrieved in 15 patients. In 14 of the 29 patients, spermatozoa were retrieved by testicular biopsy. Comparisons of the groups with or without spermatozoa concerning serum FSH concentrations, testicular volume and histological findings in the biopsy specimens are demonstrated in Table L The mean ± SD values for serum FSH concentration and testicular volume respectively in the two groups were 15.4 ± 7.4 IU/1 and 10.1 ± 3.4 ml, and 13.2 ± 9. IU/1 and 9. ± 3.2 ml. Five patients diagnosed as having Sertoli cells only had either an occasional single seminiferous tubule with spermatogenic activity {n = 2) or there was no spermatogenic 757

3 S.Kahraman et al Table L Follicle stimulating hormone (FSH) concentrations, tesiicular volume and histological analysis of testicular tissue in non-obstructive azoospermic patients Sperm obtainable (i = 14) Sperm non-obtainable (n = 15) Patient no. FSH (IU/1) Testicular volume (ml)* Histological analysis Patient no. FSH (IU/1) Testicular volume (ml) Histological analysis l b 2" C C C 14 C Mean Range (7.4) (3.4) Mean Range (9.) (3.2) There was no significant difference in FSH concentration or tcsticular volume between the two groups. = hypospermatogenesis; = maturation arrest; = Sertoli cell-only syndrome. Mean value of two testes. b-c Twin and singleton pregnancies achieved respectively. Table H. Outcome of intracytoplasmic sperm injection and embryo transfer Total no. of patients No. of patients from whom spermatozoa were obtainable No. of oocytes retrieved No. of oocytes injected No. of oocytex degenerated No. of intact oocytes (unfertilized) No. of fertilized oocytes (with two pronuclei) No. of cleaved embryos No. of embryos transferred No. of embryos implanted No. of-embryo transfers No. of pregnancies for sperm-providing patients Pregnancy rate per embryo transfer No Proportion _ 14/29-101/123 /101 51/101 39/101 3/39 3/3 /3 /14 /14 / Percentage _ activity (n = 3). Complete and incomplete maturation arrest was apparent in 14 patients, and 10 patients had severe hypospermatogenesis, denned as a non-focal generalized reduction in spennatogenic activity in all seminiferous tubules. The FSH concentration, testicular volume and biopsy findings were not significantly different between the two groups. Moreover, no association was found between the criteria mentioned above and the success of TESE. As is shown in Table n, in 14 cycles with successful TESE, 123 oocytes were retrieved, i.e.. oocytes/cycle. A total of 101 metaphase II oocytes (2.1%) were injected The numbers of intact and degenerated oocytes were 51 and respectively. Of the 39 oocytes fertilized (fertilization rate 3.%), 3 were cleaved (cleavage rate 97.4%). In all, 3 embryos were eligible for replacement In three couples from whom spermatozoa were obtainable, no fertilization occurred. Thus, of the 14 couples with spermatozoa, had transferable embryos. Six 75 pregnancies were achieved, including two twins and four singletons. One singleton pregnancy resulted in a clinical abortion in the first trimester. In all, 3 embryos were transferred and on eight occasions positive fetal heart activity was confirmed by ultrasound, giving a 22.2% implantation rate. The pregnancy rates per initiated cycle and per sperm-obtainable cycle were 20.7 and 42.9% respectively. Discussion With the reporting of fertilization and pregnancy using testicular spermatozoa, a new era of treatment for the azoospermic man commenced (Schoysman et al, 1993). Using testicular tissue as the source of sperm cells for ICSI is a new approach. At first, the TESE procedure was applied to men with obstructive azoospennia in whom the epididymis was entirely destroyed or in the case of bilateral absence of the vas deferens (Devroey et al, 1994; Silber, 1995a). TESE was described by Silber etal (1994) and Devroey et al (1995) after they first performed this procedure in More recently testicular sperm aspiration and direct sperm aspiration have been described as alternative sperm recovery methods which may be more likely to yield spermatozoa in patients with focal spennatogenesis. If multiple sites are to be sampled, an open biopsy might not include an active focal area. However, if a gauge butterfly needle is passed directly into the testis through the scrotal skin under local sedation or general anaesthesia, using a 10 ml attached syringe to create a strong negative pressure, multiple biopsies can be carried out in this way, making an open biopsy unnecessary (Craft and Tsirigotis, 1995; Craft et al., 1995a,b; Tsirigotis et al., 1995). In these cases the spennatogenesis was normal and it was shown that motile spermatozoa were present in the testicular tissue.

4 Fertility in non-obstructive azoospermic men In non-obstructive azoospermic or extreme oligoasthenoteratozoospermic men who have severe hypospermatogenesis, bilateral maturation arrest in spermatogenesis, Sertoli cell-only syndrome or tubular fibrosis, the testicles are the only source of sperm cells. Therefore, the most promising treatment model is the combination of TESE and ICSI (Devroey et al, 1995). In non-obstructive azoospermia, normal fertilization and pregnancy can be achieved by using testicular spermatozoa (Zukerman et al, 197; Silber, 191, 1995b; Silber et al, 1995a,c). We wished to study whether the serum FSH concentration, the testicular volume and the testicular biopsy result could be used to predict which patients in this group should be selected for a successful outcome from the TESE and ICSI procedures. Classically, normal testicular volume and FSH concentration have indicated that spermatozoa will be present in the testicular biopsy specimen. Conversely, a high FSH concentration and a decreased testicular volume have indicated that spermatozoa will not be found in testicular tissue after biopsy. This classical approach should be questioned. It has been shown that 4% of azoospermic men undergoing a testicular biopsy and having FSH concentrations >30 mtu/ml had mature spermatozoa in the biopsy (Gilbaugh et al, 1994). Thus, an elevated FSH concentration does not always indicate a damaged germinal epithelium but may also reflect a compensatory adaptation to partial destruction resulting in (sub)normal sperm production (Martin-du-Pan and Bischof, 1995). Of the 29 non-obstructive azoospermic males confirmed as non-obstructive by an exploration of the scrotum during testicular tissue biopsy, and who were accepted for the TESE procedure, spermatozoa were obtainable in only 14. It appeared that in the other 15 patients, no sperm cells were seen even on wet preparations, except in three patients who had been diagnosed as having severe hypospermatogenesis following the histological examination. These false-negative results may be explained by the focal nature of the pathological alterations in the testes and underline the importance of multiple sampling (Tournaye et al, 1995). Thus, a preliminary diagnostic biopsy with wet preparation and definitive histological examination may be advisable but may still not be conclusive because of the focal nature of the pathological alterations. Spermatozoa were obtainable from men who had moderately or highly elevated serum FSH concentrations and abnormal testicular biopsy findings, including some degree of hypomaturation, spermatogenic arrest or Sertoli cell-only syndrome. No statistically significant difference was found in the serum FSH concentrations, the testicular volumes or the biopsy results between the groups with or without obtainable spermatozoa. There was also no association between the criteria mentioned above and the success of TESE. Recently, Devroey et al (1995) reported that patients with severe spermatogenic defects may have normal FSH concentrations. Similarly our study demonstrated that neither the FSH concentration nor the testicular volume can be used as a predictive factor for success in patients whose testicular spermatozoa are to be used in ICSI. A singleton pregnancy was achieved in a couple where the male had a small testicular volume (4.4 ml) and a high FSH concentration (29.4 IU/1). Thus the outcome of our study contradicts the classical view in which patients with nonobstructive azoospermia, elevated FSH and small testes are considered sterile. The use of TESE with ICSI enabled us to establish that these patients are potentially fertile. A common observation for all the sperm samples obtained by TESE was that most of the spermatozoa were totally immotile or had a sluggish twitching motion. After an incubation period of h, spermatozoa showed increased motility. Nagy et al. (1995) had the same experience and suggested the existence of a testicular motility blocking factor. The maturational status of testicular spermatozoa could be confirmed by using a contrast modulator, but as yet we have not used one in routine clinical practice. In our experience, using immature testicular spermatozoa in ICSI increases the time taken for the procedure because they stick to the tip of the injecting pipette and require skilful manipulation. In three cases, fertilization was not achieved by ICSI. The number of non-fertilized intact oocytes was rather high (n = 51), i.e. the non-fertilization rate was 50.5%. The number of degenerated oocytes was acceptable (/i = ). Six pregnancies have been established. One of the singleton pregnancies aborted in the first trimester. Of the five ongoing pregnancies, two are twins and three are singletons. In our study, the combination of TESE with ICSI yielded a rather high implantation rate (22.2%) and pregnancy per embryo transfer rate (54.5%) in the sperm-obtainable group. Although the number of cases is very limited, we believe that the achievement of high implantation and pregnancy rates by using testicular spermatozoa will initiate new discussions. Furthermore, these high rates corroborate the findings of Silber et al (1994, 1995c), who pointed out that it is likely for the women in this population to be more fertile than in, say, cases of oligozoospermia or normal sperm counts. In such cases, a certain number would achieve pregnancy anyway, including couples in whom the male suffered from severe oligozoospermia because the wife was extremely fertile (Emperaire etal, 192). A possible objection to the results of our study is that the number of patients from whom the spermatozoa were not obtained is rather high compared with results published recently by Devroey et al. (1995). The Percoll sperm separation technique could be a possible reason. Alternatively, a high proportion of biopsy specimens with maturation arrest could also explain the high number of non-obtainable sperm cases. The focal nature of the pathological alteration in these cases again emphasizes the importance of multiple sampling. Because only one biopsy specimen was obtained from our patients, this could account for the high number of patients from whom spermatozoa were not obtainable. Although all biopsy material was examined meticulously for 2-3 h until enough spermatozoa were found for ICSI, failure to obtain spermatozoa in some cases might have resulted from the unavailability of a contrast modulator, which facilitates the identification of single spermatozoa hidden between layers of Sertoli cells. We have shown that the classic assumptions regarding the outcome of TESE do not necessarily apply in cases of nonobstructive azoospermia. Criteria to predict the success of TESE combined with ICSI in these cases remain to be 759

5 S.Kahraman et al established. In conclusion, this study confirms previous reports that high implantation and pregnancy rates can be achieved using testicular spermatozoa in combination with ICSI in cases of non-obstructive azoospermia. after retrieval of spermatozoa by testicular biopsy from an azoospermic male with testicular tubular atrophy. FertiL SteriL, 3, Zukerman, Z., Rodriguez-Rigau, U., Weiss, D.B. et al. (197) Quantitative analysis of the seminiferous epithelium in human testicular biopsies, and the relation of spermatogenesis to sperm density. FertiL SteriL, 30, 44. Received on September 4, 1995; accepted on February 5, 1995 References Craft, I. and Tsirigotis, M. (1995) Simplified recovery, preparation and cryopreservation of testicular spermatozoa. Hum. Reprod., 10, Craft, I., Khalifa, Y., Boulos, A. et at. (1995a) Factors influencing the outcome of in-vitro fertilization with percutaneous aspirated epididymal spermatozoa and intracytoplasmic sperm injection in azoospermic men. Hum. Reprod., 10, Craft, I., Tsirigotis, M. and Shrivastav, P. (1995b) Value of percutaneous epididyma] sperm aspiration? FertiL Steril., 3, Devroey, P., Liu, J., Nagy, Z. et at. (1994) Normal fertilisation of human oocytes after testicular sperm extraction and intracytoplasmic sperm injection. Fertil. Steril., 2, Devroey, P., Liu, J., Nagy, Z. et at. (1995) Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Hum. Reprod., 10, Emperaire, J.C., Gauzere-Sonmireu, E. and Audebert, AJ. (192) Female fertility and donor insemination. Fertil. SteriL, 37, 90. Gilbaugh, J.H., Patil, V.R., Turek, PJ. and Lipshultz, L.I. (1994) Testis biopsy findings in azoospermic patients with markedly elevated serum FSH levels. Presented at the 50th Annual Meeting of the American Fertility Society, San Antonia, 5 10 November, p. S3. Levin, H.S. (1979) Testicular biopsy in the study of male infertility. Its current usefulness, histologic techniques, and prospects for future. Hum. PathoL, 10, Martin-du-Pan, R.C. and Bischof, P. (1995) Increased follicle stimulating hormone in infertile men. Is increased plasma FSH always due to damaged germinal epithelium? Hum. Reprod., 10, Nagy, Z., Liu, J., Cecile, J. et at. (1995) Using ejaculated, fresh, and frozenthawed epididymal and testicular spermatozoa gives rise to comparable results after intracytoplasmic sperm injection. FertiL SteriL, 3, Schoysman, R., Vanderzwalmen, P., Nijs, M. et al (1993) Pregnancy after fertilisation with human testicular spermatozoa. Lancet, 342, Silber, SJ. (1995a) Sertoli cell only revisited. Hum. Reprod, 10, Silber, SJ. (1995b) What forms of male infertility are there left to cure? Hum. Reprod., 10, Silber, SJ. and Rodriguez-Rigau, I. (191) Quantitative analysis of testicle biopsy: determination of partial obstruction and prediction of sperm count after surgery for obstruction. FertiL SteriL, 3, Silber, S J., Nagy, Z.P., Liu, J. et al (1994) Conventional in-vitro fertilization versus intracytoplasmic sperm injection for patients requiring microsurgical sperm aspiration. Hum. Reprod., 9, Silber, SJ., Devroey, P., Tournaye, H. and Van Steirteghem, A.C. (1995a) Fertilizing capacity of epididymal and testicular sperm using intracytoplasmic sperm injection (ICSI). Reprod Fertil. Dev., 7, Silber, SJ., Nagy, Z., Liu, J. et al. (1995b) The use of epididymal and testicular spermatozoa for intracytoplasmic sperm injection: the genetic implications for male infertility. Hum. Reprod., 10, Silber, SJ., Van Steirteghem, A.C, Liu, J. et al (1995c) High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa obtained from testicle biopsy. Hum. Reprod., 10, Toumaye, P., Devroey, P., Liu, J. et al (1994) Microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection: a new effective approach to infertility as a result of congenital bilateral absence of the vas deferens. FertiL SteriL, 1, Toumaye, H., Camus, M., Goossens, A. et al (1995) Recent concepts in the management of infertility because of non-obstructive azoospermia. Hum. Reprod., 10 (Suppl.), 5-9. Tsirigotis, M., Pelekanos, M., Foster, C. and Craft, I. (1995) Testicular sperm aspiration (TESA). Simplified recovery and preparation of testicular spermatozoa for ICSI cycles. Contracept FertiL Sex., 23 (Suppl. 9), 3. Van Steirteghem, A.C., Nagy, Z., Joris, H. et al (1993) High fertilization and implantation rates after intracytoplasmic sperm injection. Hum. Reprod., 10, Yemini, M., Vanderzwalmen, P., Mukaida, T., Schoengold, S. and Birkenfeld, 70 A. (1995) Intracytoplasmic sperm injection, fertilization, and embryo transfer