Outcome of first and repeated testicular sperm extraction and ICSI in patients with non-obstructive azoospermia

Human Reproduction Vol.17, No.9 pp. 2356 2361, 2002 Outcome of first and repeated testicular sperm extraction and ICSI in patients with non-obstructive azoospermia S.Friedler 1, A.Raziel, M.Schachter, D.Strassburger, O.Bern and R.Ron-El IVF and Infertility Unit, Department of Obstetrics and Gynecology, Assaf Harofeh Medical Center, Zerifin 70300, Israel 1 To whom correspondence should be addressed. E-mail: rronel@assaf.health.gov.il BACKGROUND: It is unclear whether or not testicular sperm extraction (TESE) should be repeated for patients in whom no sperm were found during their first TESE attempt. METHODS AND RESULTS: The outcome of repeated TESE was evaluated in patients with non-obstructive azoospermia (NOA) after failing to obtain sperm in their first extraction attempt, or having used all available cryopreserved testicular tissue. Out of 83 patients with NOA, patients repeated TESE two (n 22), three (n 8), four (n 6) and five (n 3) times. Distribution of main testicular histology included germ cell aplasia (55%), maturation arrest (29%) and germ cell hypoplasia (16%). The first TESE yielded mature sperm for ICSI in 39% of patients (sp ), and failed in the remaining 61% (sp ). A second TESE yielded mature sperm in 1/4 from the sp group and in 16/18 from the sp group. At the third, fourth and fifth trials, 8/8, 5/6 and 3/3 of the original sp patients were sp again respectively. Compared with the outcome of the first trial, all further trials did not differ statistically in the rate of fertilization (54 versus 49%), implantation (9.5 versus 5.4%), or clinical pregnancy/cycle (19 versus 15%). No pregnancies were achieved among the three patients after their fifth TESE. Pregnancies occurred in all histological groups, except maturation arrest. CONCLUSIONS: The outcome of repeated TESE cycles, up to the fourth trial, justifies the procedure. Key words: ICSI/non-obstructive azoospermia/tese/testicular sperm Introduction ICSI of surgically retrieved testicular sperm has revolutionized the potential of azoospermic males to reproduce despite apparent testicular failure. Currently, there are no clinical or laboratory methods that can reliably and accurately predict the presence of sperm on testicular sperm extraction (TESE). In up to 60% of the patients suffering from non-obstructive azoospermia (NOA), no mature sperm are found that would enable ICSI to be attempted, despite several biopsies taken (Devroey et al., 1995; Kahraman et al., 1996; Friedler et al., 1997; Schlegel et al., 1997; Silber et al., 1997; Tournaye et al., 1997; Rosenlund et al., 1998; Westlander et al., 1999). Whether a repetitive TESE should be recommended in patients that had no mature sperm following their first TESE remains an unanswered question. If mature testicular sperm are found, ICSI is performed and whenever possible the residual tissue is cryopreserved to allow further ICSI cycles. Consequently, those who fail to achieve pregnancy following embryo transfer, or desire another pregnancy, usually opt for another ICSI cycle using cryopreserved thawed testicular tissue, if available after the first procedure. If all extracted testicular sperm have been injected and no more testicular tissue is available, a repetitive TESE is the only option for these patients to pursue their goal. Because testicular tissue quantity is limited and one publication (Schlegel and Su, 1997) has cautioned against possible testicular damage after TESE, the true prognosis of repetitive TESE has a paramount importance to enable responsible consultation to patients. As information in the literature regarding the outcome of repetitive TESE is scarce, we aimed to evaluate the outcome of two to five repetitive TESE procedures as opposed to the first trial in a group of patients with NOA, and the relation to their initial testicular histology. Materials and methods Study population Eighty-three patients suffering from NOA who underwent open testicular biopsy for TESE during the period of October 1995 to December 1999, at the Assaf Harofeh Medical Center s IVF Unit, were included in the study. When viable sperm were found, ICSI was performed. All male partners in the group underwent andrological investigation suitable for azoospermic patients. This included physical examination and patients history-taking along with evaluation of their hormonal profile, peripheral blood karyotype and transrectal ultrasound. Extensive search by the Extended Sperm Preparation (ESP) method (Ron-El et al., 1997) of several ejaculates (at least three) prior to surgical sperm retrieval revealed no sperm in any ejaculate provided. Diagnosis of NOA was based upon a histological report, taken during the current procedure. Nineteen patients had a diagnostic testicular biopsy prior to their first TESE. Outcome was analysed according to the main histological diagnosis on the testicular biopsies, including severe hypospermatogenesis, complete and incomplete maturation arrest (MA) and germ cell aplasia (GCA). As 2356 European Society of Human Reproduction and Embryology

Repetitive TESE ICSI in non-obstructive azoospermia Table I. Patients characteristics Female age Male age FSH Testosterone a (years) (years) (miu/ml) (nmol/l) sp (32 patients) 30.3 5.6 b 33.8 5.9 b 23.3 13.4 b 11.6 6.1 b sp (51 patients) 29.9 5.4 b 33.7 5.6 b 23.6 11.6 b 11.8 6.1 b Values are mean SD. a Normal range: 9.4 37 nmol/l. b sp versus sp : not significant. sp sperm positive; sp sperm negative. none of the patients diagnosed as Klinefelter s syndrome underwent repetitive TESE, this group was not included in our study. Patients in whom mature testicular sperm were found after the first TESE were grouped as sperm positive (sp ). Those in whom TESE yielded no sperm for ICSI were grouped as sperm negative (sp ). When no cryopreserved testicular tissue was available to allow further ICSI cycles, repetition of the TESE procedure was offered to the patients. A minimum interval of 6 months between the surgical sperm retrievals was recommended, a policy that was later verified (Schlegel and Su, 1997). Twenty-two patients chose to undergo at least one repetitive TESE cycle, and eight, six and three patients underwent three, four and five surgical retrievals respectively. Patients characteristics, including female and male partners age, male patients serum FSH and testosterone levels are presented in Table I. Sperm retrieval and preparation On the day of the oocyte retrieval, the male partners produced fresh ejaculates and ESP confirmed that no sperm were found in the specimens. Methodology of the TESE procedure The technique of surgical testicular sperm retrieval in patients with NOA, sperm preparation and ICSI have been described in detail elsewhere (Friedler et al., 1997). Once sperm were found, the surgical procedure was terminated. If sperm were not observed, up to three biopsies were taken, in different areas in the same testicle and also from the contralateral one. During repetitive TESE, the scrotum was opened and adhesions, if present, were dissected. No specific technical problems were encountered in the repetitive TESE cases that could disturb the exposure of testicular tissue. Ovarian stimulation was performed using the routine long protocol of pituitary suppression followed by ovarian stimulation. Oocytes were retrieved by vaginal ultrasound-guided follicular puncture. Embryo transfer was performed on day 2 or 3 following oocyte retrieval. Recently, the policy of transferring fewer embryos has been emphasized at our Unit. However, in our population, when a couple go through a complete IVF cycle including surgical sperm aspiration, they frequently ask to have more embryos transferred and prefer to take the risk of multiple pregnancy, despite our explanations concerning the risks involved. However, no more than three embryos were transferred, except in women 38 years old or in cases with recurrent failures of implantation, where up to five embryos were transferred if available. Following embryo transfer, all patients received luteal support, including i.m injections of hcg, or of progesterone in oil. Only clinical pregnancies including ultrasonographic demonstration of a gestational sac were counted. Statistical analysis Statistical evaluation was performed using Student s t-test, χ 2 -test and Fisher s exact test, where appropriate. Differences were considered significant at P 0.05. Table II. Successful sperm retrieval rate, according to the testicular sperm extraction (TESE) trial (a) Repetitive TESE ICSI cycles, by the number of trial TESE trial 2nd TESE 3rd TESE 4th TESE 5th TESE No. of patients 22 8 6 3 Sperm found (%) 17 (77) 8 (100) 5 (83) 3 (100) (b) Repetitive TESE ICSI cycles, by the number of trial. Correlation with the outcome in the previous TESE Sperm retrieval on TESE Previous TESE Repetitive TESE 2nd TESE sp : 4 sp : 3;sp : 1 sp : 18 sp : 2;sp : 16 3rd TESE sp : 8 sp : 8 4th TESE sp : 6 sp : 1;sp : 5 5th TESE sp : 3 sp : 3 sp sperm positive; sp sperm negative. a Only sp patients underwent 3rd, 4th and 5th TESE attempts. (c) First and repetitive TESE ICSI cycles TESE trial 1st TESE Repetitive TESE No. of patients 83 39 Sperm found (%) 32 (39) 33 (85) Results Among the 83 patients with NOA included in the study, female and male partners age was 29.8 5.9 and 33.5 6.3 years respectively (mean SD). Male serum levels of FSH and testosterone were 23.4 12.1 miu/ml and 11.7 7.6 nmol/l (mean SD) (normal range for males: 9.4 37 nmol/l). Following their first TESE, no mature sperm were found in 51 (61%) patients and ICSI was not performed (sp group). In 32 (39%) patients TESE enabled performance of ICSI using testicular sperm (sp group). Comparison between the groups, regarding female or male average age, male serum levels of FSH and testosterone, rendered no statistically significant difference (see Table I). Successful testicular sperm retrieval rate, enabling performance of ICSI, according to the TESE trial is presented in Table IIa c. Twenty-two patients underwent a second TESE (Table 2357

S.Friedler et al. Table III. (a) Outcome of repetitive testicular sperm extraction (TESE) ICSI cycles, by the number of trial TESE trial 2nd TESE 3rd TESE 4th TESE 5th TESE No. of ICSI cycles 17 8 5 3 No. of ova injected/cycle 7.7 5 8.7 5 11 7 11.3 2.5 No. of ova fertilized/cycle 5.3 3.5 5 4 6.3 6.3 5.7 1.5 Fertilization rate (%) 48 50 44 54 No. of embryos cleaved/cycle 4.6 2.9 5 4.8 5 6.4 5 1.0 No. of embryos replaced/ 3.4 1.8 5 4.8 4.5 1.4 4.7 0.6 embryo transfer Implantation rate (%) 3/56 (5.3) 2/32 (6.3) 1/10 (10) 0/14 (0) Clinical pregnancy rate/ 3/17 (17.6) 1/7 (14.3) 1/4 (25) 0/3 (0) embryo transfer (%) Clinical pregnancy rate/ 3/17 (17.6) 1/8 (12.5) 1/5 (20) 0/3 (0) ICSI cycle (%) Pregnancy outcome 3 (delivered) 1 (missed twin, 1 (IUFD at 34th singleton week of delivered) gestation) Table III. (b) Outcome of first and repetitive testicular sperm extraction (TESE) ICSI cycles TESE trial 1st TESE Repetitive TESE No. of ICSI cycles 32 33 No. of ova injected/cycle 9.2 4.6 9.0 4.9 No. of ova fertilized/cycle 5.0 3.4 5.4 2.8 Fertilization rate (%) 54 49 No. of embryos cleaved/cycle 4.7 3.4 4.8 3.0 No. of embryos replaced/embryo transfer 3.0 1.5 4.0 1.8 Implantation rate (%) 10/105 (9.5) 6/112 (5.4) Clinical pregnancy rate/embryo transfer (%) 6/31 (19) 5/31 (16) Clinical pregnancy rate/icsi cycle (%) 6/32 (19) 5/33 (15) Pregnancy outcome 4 (delivered) 1 (missed twin, 2 (early miscarriages) singleton delivered) 3 (delivered) 1 (IUFD at 34th week of gestation) IUFD intrauterine fetal death. IIa). Altogether, during the second TESE procedure, in 1/4 patients from the sp group and in 16/18 from the sp group, sperm was found to enable ICSI (Table IIb). Further trials were performed on patients from the sp group only. At the third trial, in all eight patients, at the fourth trial in 5/6 and at the fifth trial in all three patients, testicular sperm was obtained enabling ICSI, as presented in Table IIa,b. Overall, in repetitive TESE, 33/39 procedures were sp (85%) (Table IIc). The outcome of the ICSI cycles according to the TESE order is presented in Table IIIa,b. The average number of oocytes injected, fertilized and cleaved did not differ significantly among the trials (Table IIIa). Whereas in the first trial of all 32 patients treated by ICSI three cases had no fertilizations, in the repetitive TESE ICSI cycles only one case had fertilization failure (on the third trial). The average number of embryos replaced increased from 3.0 1.5 in the first trial to 4.0 1.8 in the repetitive TESE cycle (Table IIIb). Compared with the outcome of the first trial, implantation rates per embryos transferred and clinical pregnancy rates per cycle or embryo transfer did not differ significantly, although the number of cycles in each group was rather small (Table IIIa). As seen in Table IIIa, pregnancies occurred in each trial up to the fourth; however, only three patients underwent a fifth TESE, resulting in no pregnancies. Overall, comparing first 2358 TESE with repetitive TESE, the difference in fertilization rate (54 versus 49%), embryo implantation rate (9.5 versus 5.4%), and pregnancy rate per ICSI cycle (19 versus 15%) or per embryo transfer (19 versus 16%) was not statistically significant (Table IIIb). Delivery of healthy infants occurred in 4/6 and 4/5 clinical pregnancies after the first and repetitive TESE procedures respectively (Table IIIb). Distribution of testicular histology (main finding) among all 83 patients included GCA (55%), MA (29%) and germ cell hypoplasia (HS) (16%). The frequency of sp according to the initial main testicular diagnosis and the occurrence of pregnancies in each group is presented in Table IVa. Testicular sperm could be identified and ICSI could be performed in all histological groups. The frequency of sp according to histology group at the first trial was 33, 29 and 77% for GCA, MA and HS respectively. Distribution of testicular histology (main finding) among the 22 patients who underwent repetitive TESE included GCA (59%, 13 patients), MA (23%, five patients) and HS (18%, four patients) (Table IVb). At the second TESE the frequency of sp was 77, 60 and 100% for GCA, MA and HS respectively. More specifically, in one patient with HS and one patient with MA, who were sp in the first TESE, became sp in the second TESE. In one patient with MA, who was

Repetitive TESE ICSI in non-obstructive azoospermia Table IV. Sperm availability after testicular sperm extraction (TESE) and occurrence of pregnancies according to testicular histology among the group of patients Histology GCA MA SH All cycles sp sp sp sp sp sp sp sp (a) At their first TESE 1st TESE trial in all 83 patients 15 31 7 17 10 3 32 51 No. of clinical pregnancies 5 0 1 6 Repetitive TESE trial 22 3 6 2 5 1 33 6 No. of clinical pregnancies 4 0 1 5 (b) Among repetitive TESE 1st TESE trial in (22 patients) 11 a 2 3 a 2 b 4 0 No. of clinical pregnancies 2 2nd TESE trial (22 patients) 10 3 3 2 4 0 No. of clinical pregnancies 2 1 3rd TESE trial (8 patients) 6 0 1 0 1 a 0 No. of clinical pregnancies 1 4th TESE trial (6 patients) 4 0 0 0 0 1 No. of clinical pregnancies 1 5th TESE trial (3 patients) 15 0 1 0 No. of clinical pregnancies a One sp patient became sp. b One sp patient became sp. GCA germ cell aplasia; MA maturation arrest; SH severe hypospermatogenesis. sp in the first TESE, sperm was found (sp ) in the second TESE (Table IVb). Including all repetitive TESE cycles, the frequency of sp was 88, 75 and 83% for GCA, MA and HS respectively (Table IVa). Among patients with GCA, 5/15 achieved a pregnancy (33%) at the first TESE and 4/22 (18%) after repetitive TESE (Table IVa). Among patients with HS, the corresponding rates were 1/10 (10%) and 1/5 (20%). No additional pregnancies were achieved after repetitive TESE in patients with main testicular histological diagnosis of MA (Table IV). Complications included 4/83 (4.8%) cases with self-resolved extratunical haematomata, all occurring at the first trial. During the performance of repetitive TESE, adhesions were rarely found, in contrast with the findings of another study (Amer et al., 1999), and no specific technical problems or postoperative complications were encountered. Follow-up of the patients was performed on a clinical basis. Testicular sonography was not performed on a routine basis. Discussion Information regarding the outcome of repetitive TESE procedures is scarce in the literature. To enable valuable counselling, data regarding the actual prognosis in repetitive TESE ICSI cycles is of utmost importance, and of value even if the groups involved are small. We found only two papers reporting information regarding outcome of repetitive open TESE. Schlegel and Su recommended that TESE should be repeated at an interval of 6 months, because the chances of retrieving sperm went up to 80% compared with 25% when TESE was repeated after a shorter interval (Schlegel and Su, 1997). Amer et al. reported their experience in repeated TESE for 27 patients with NOA (Amer et al., 1999). Performing multiple biopsies per testicle, they were able to find sperm in 24/27 (88.9%) of their patients who had sperm at their first trial. Of 19 patients who underwent the procedure after an interval of 3 months, 18 (94.7%) were positive at the repeated trial, whereas when the repeated TESE was performed after an interval of 3 months, only 6/8 (75%) were positive. Obviously the amount of testicular tissue in each patient is limited and is not regenerating after biopsies are taken and repetitive trauma to the testicles may inflict irreparable damage upon them. Ultrasonographic changes after open testicular biopsies do occur (Harrington et al., 1996; Schlegel and Su, 1997; Ron El et al., 1998; Amer et al., 2000); however, the clinical meaning of these findings remains to be elucidated. We found no data in the literature regarding testicular sonography after repetitive TESE. It should be recognized that absence of apparent clinical complication does not exclude possible intratesticular damage that may be demonstrated by ultrasound or that will appear at a longer interval of follow-up. Therefore, caution should be taken when counselling patients regarding repetitive TESE. Our policy of repetition of TESE at intervals of 6 months seems in concordance with the reported experience in the literature. The result of 85% success of testicular sperm retrieval is in the range appearing in these earlier reports. Furthermore, performance of three biopsies per testicle will screen the majority of the positive cases for sperm presence in testicles of men with NOA (Hauser et al., 1998; Amer et al., 1999). It should be mentioned that in NOA, testicular sperm may be retrieved also by performing testicular sperm aspiration (TESA); however, as sperm is produced focally, the chance of finding sperm is related to the amount of testicular tissue extracted. Therefore, in several comparative studies TESE was found to be more efficient compared with TESA in successfully of extracting sperm in these cases (Friedler et al., 1997; Ezeh et al., 1998; Mercan et al., 2000). Alternative 2359

S.Friedler et al. methods of testicular sperm retrieval that avoid open biopsy were also proposed, including testicular biopty gun needle (Tuuri et al., 1999) or Trucut needle and milking of seminiferous tubules (Steele et al., 2000). Presently there is no consensus over the ultimate method of testicular sperm retrieval in NOA and each centre tends to specialize in its method of preference. Regarding repeated surgical sperm retrievals, experience with repeated TESA has been reported (Westlander et al., 2001). Out of 34 patients with NOA, 34, 14, five, three and one patients underwent a second, third, fourth, fifth and sixth TESA procedure respectively. The fertilization rate remained similar, and pregnancies were achieved up to the fifth attempt, but only five men underwent more than three retrievals. There were no sperm recovery failures in any of the repeated TESA procedures and the time interval did not seem to affect the outcome. No post-operative complications were reported. Whether these presumably less aggressive sperm retrieval methods should be proposed in repeated cases is to be evaluated by a different study. Currently, there are no clinical or laboratory methods that can predict reliably and accurately the presence of sperm on TESE. It was shown that even the presence of sperm in a preliminary testicular biopsy may fail to predict the presence of mature testicular sperm at the actual TESE ICSI in up to 30% of cases (Vanderzwalmen et al., 1997). According to our results and those of others (Amer et al., 1999), consultation prior to repetitive TESE should consider that even performing TESE after the recommended interval may fail to produce mature sperm for ICSI in up to 10% of cases. Our results indicate that for patients with available sperm (sp ) at their first TESE, failure to obtain sperm may occur during repetitive TESE [in our experience at a rate of 11% (2/28) up to 33% (2/6) during the second and fourth TESE respectively]. Therefore, finding of mature sperm for ICSI in the first fresh TESE offers a good prognosis of an 85% chance of finding sperm in the following trial, but patients should be cautioned that it still may not completely assure success in further TESE trials. On the other hand, in our experience 1/4 sp patients at the first trial did become sp at their second trial. When testicular sperm are found, performance of ICSI after a repetitive TESE using the fresh testicular sperm results in a similar outcome with regards to fertilization, embryo cleavage and implantation rates. However, in our study, no pregnancies were achieved using sperm from patients with MA as their main testicular histological diagnosis. Others have reported normal pregnancies resulting from such cases (Silber et al., 1996), and therefore repetition of TESE in these patients may still be considered with caution, as lack of success may result from other contributing factors. Also, no pregnancies were achieved among the three patients who chose to undergo a fifth TESE in spite of transfer of 14 embryos. In conclusion, repetition of TESE has clinical value because pregnancies may be achieved in each repetitive trial. Special difficulties or complications during or after performing the procedure were not encountered at any higher rate than after the first TESE, based on clinical examination and judgement. Due to the group s size it is difficult to draw significant conclusions regarding recommendations to perform more than 2360 four repetitive TESE procedures. New clinical tools to predict testicular mature sperm availability with proven accuracy are awaited ardently. New sperm retrieval techniques, such as micro-tese (Schlegel, 1999) should also be tested in repeated TESE procedures in patients with NOA. References Amer, M., El Haggar, S., Moustafa, T., Abd El-Naser, T. and Zohdy, W. (1999) Testicular sperm extraction: impact of testicular histology on outcome, number of biopsies to be performed and optimal time for repetition. Hum. Reprod., 14, 3030 3034. Amer, M., Ateyah, A., Hany, R. and Zohdy, W. (2000) Prospective comparative study between microsurgical and conventional testicular sperm extraction in non-obstructive azoospermia: follow-up by serial ultrasound examinations. Hum. Reprod., 15, 653 656. Devroey, P., Liu, J., Nagy, Z., Goossens, A., Tournaye, H., Camus, M., Van Steirteghem, A. and Silber, S. (1995) Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Hum. Reprod., 10, 1457 1460. Ezeh, U.I.O, Moore, H.D.M. and Cooke, I.D. (1998) A prospective study of multiple needle biopsies versus open biopsy for testicular sperm extraction in men with non-obstructive azoospermia. Hum. Reprod., 13, 3075 3080 Friedler, S., Raziel, A., Strassburger, D., Soffer, Y., Komarovsky, D. and Ron-El, R. (1997) Testicular sperm retrieval by percutaneous fine needle sperm aspiration compared with testicular sperm extraction by open biopsy in men with non-obstructive azoospermia. Hum. Reprod., 12, 1488 1493. Harrington, T.G., Schauer, G. and Gilbert, B.R. (1996) Percutaneous biopsy: an alternative to open testicular biopsy in the evaluation of the subfertile man. J. Urol., 156, 1647 1651. Hauser, R., Botchan, A., Amit, A., Ben Yosef, D., Gamzu, R., Paz, G., Lessing, J.B., Yogev, L. and Yavetz, H. (1998) Multiple testicular sampling in non-obstructive azoospermia: is it necessary? Hum. Reprod., 13, 3081 3085. Kahraman, S., Ozgur, S., Alatas, C., Aksoy, S., Balaban, B., Evrenkaya, T., Nuhoglu, A., Tasdemir, M., Biberoglu, K., Schoysman, R. et al. (1996) High implantation and pregnancy rates with testicular sperm extraction and intracytoplasmic sperm injection in obstructive and non-obstructive azoospermia. Hum. Reprod., 11, 673 676. Mercan, R., Urman, B., Alatas, C., Aksoy, S., Nuhoglu, A., Isiklar, A. and Balaban, B. (2000) Outcome of testicular sperm retrieval procedures in non-obstructive azoospermia: percutaneous aspiration versus open biopsy. Hum. Reprod., 15, 1548 1551. Ron-El, R., Strassburger, D., Friedler, S., Komarovski, D., Bern, O., Soffer, Y. and Raziel, A. (1997) Extended sperm preparation: an alternative to testicular sperm extraction in non-obstructive azoospermia. Hum. Reprod., 12, 1222 1226. Ron-El, R., Strauss, D., Friedler, S., Strassburger, D., Komarovsky, D. and Raziel, A. (1998) Serial sonography and colour flow Doppler imaging following testicular and epididymal sperm extraction. Hum. Reprod., 13, 3390 3393. Rosenlund, B., Kvist, U., Ploen, L., Rozell, B.L., Sjoblom, P. and Hillensjo, T. (1998) A comparison between open and percutaneous needle biopsies in men with azoospermia. Hum. Reprod., 13, 1266 1271. Schlegel, P.N. ( 1999) Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision. Hum. Reprod., 14, 131 135. Schlegel, P.N. and Su, L.M. (1997) Physiological consequences of testicular sperm extraction. Hum. Reprod., 12, 1688 1692. Schlegel, P.N., Palermo, G.D., Goldstein, M., Menendez, S., Zaninovic, N., Veeck, L.L. and Rosenwaks, Z. (1997) Testicular sperm extraction with intracytoplasmic sperm injection for non-obstructive azoospermia. Urology, 49, 435 440. Silber, S.J., Van Steirteghem, A., Nagy, Z., Liu, J., Tournaye, H. and Devroey, P. (1996) Normal pregnancies resulting from testicular sperm extraction and intracytoplasmic sperm injection for azoospermia due to maturation arrest. Fertil. Steril., 66, 110 117. Silber, S.J., Nagy, Z., Devroey, P., Camus, M. and Van Steirteghem, A.C. (1997) The effect of female age and ovarian reserve on pregnancy rate in male infertility: treatment of azoospermia with sperm retrieval and intracytoplasmic sperm injection. Hum. Reprod., 12, 2693 2700. Steele, E.K., Kelly, J.D., Lewis, S.E., McNally, J.A., Sloan, J.M. and McClure, N. (2000) Testicular sperm extraction by Trucut needle and milking of seminiferous tubules: a technique with high yield and patient acceptability. Fertil. Steril., 74, 380 383.

Repetitive TESE ICSI in non-obstructive azoospermia Tournaye, H., Verheyen, G., Nagy, P., Ubaldi, F., Goossens, A., Silber, S., Van Steirteghem, A.C. and Devroey, P. (1997) Are there any predictive factors for successful testicular sperm recovery in azoospermic patients? Hum. Reprod., 12, 80 86. Tuuri, T., Moilanen, J., Kaukoranta, S., Makinen, S., Kotola, S. and Hovatta, O. (1999) Testicular biopty gun needle biopsy for collecting spermatozoa for intracytoplasmic injection, cryopreservation and histology. Hum. Reprod., 14, 1274 1278. Vanderzwalmen, P., Zech, H., Birkenfeld, A., Yemini, M., Bertin, G., Lejeune, B., Nijs, M., Segal, L., Stecher, A., Vandamme, B. et al. (1997) Intracytoplasmic injection of spermatids retrieved from testicular tissue: influence of testicular pathology, type of selected spermatids and oocyte activation. Hum. Reprod., 12, 1203 1213. Westlander, G., Hamberger, L., Hanson, C., Lundin, K., Nilsson, L., Söderlund, B., Werner, C. and Bergh, C. (1999) Diagnostic epididymal and testicular sperm recovery and genetic aspects in azoospermic men. Hum. Reprod., 14, 118 122. Westlander, G., Rosenlund, B., Soderlund, B., Wood, M. and Bergh, C. (2001) Sperm retrieval, fertilization and pregnancy outcome in repeated testicular sperm aspiration. J. Assist. Reprod. Genet., 18, 171 177. Submitted on July 7, 2000; resubmitted on April 16, 2002; accepted on May 10, 2002 2361