How successful is TESE-ICSI in couples with non-obstructive azoospermia?

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1 Human Reproduction, Vol.30, No.8 pp , 2015 Advanced Access publication on June 16, 2015 doi: /humrep/dev139 ORIGINAL ARTICLE Andrology How successful is TESE-ICSI in couples with non-obstructive azoospermia? V. Vloeberghs 1, *, G. Verheyen 1, P. Haentjens 2, A. Goossens 3, N.P. Polyzos 1, and H. Tournaye 1 1 Centre for Reproductive Medicine, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels, Belgium 2 Laboratory of Experimental Surgery and Centre for Outcomes Research, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels, Belgium 3 Department of Pathology, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels, Belgium *Correspondence address. Centre for Reproductive Medicine, Universitair Ziekenhuis Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium. Tel: ; Fax: ; veerle.vloeberghs@uzbrussel.be Submitted on February 26, 2015; resubmitted on May 11, 2015; accepted on May 21, 2015 studyquestion: What are the chances of a couple with infertility due to non-obstructive azoospermia (NOA) having their genetically own child by testicular sperm extraction combined with ICSI (TESE-ICSI)? summaryanswer: Candidate TESE-ICSI patients with NOA should be counselled that, when followed-up longitudinally, only a minority (13.4%) of men embarking for TESE eventually become a biological father. what is known already: Data available in the literature are only fragmentary because they report either on sperm retrieval rates after TESE or on the outcome of ICSI once testicular spermatozoa has been obtained, mostly in a selected subpopulation. Unfortunately, reliable data to counsel men with NOA on their chance to become a biological father are still lacking. study design, size, duration: This is a retrospective cohort study performed in the Centre for Reproductive Medicine, University Hospital of Brussel, approved by the institutional review board of the hospital. participants/materials, setting and methods: We identified all patients with NOA, based on histology, who had their first testicular biopsy between 1994 and Patients were followed longitudinally during consecutive ICSI cycles with testicular sperm. The primary outcome measure was live birth delivery. The cumulative live birth delivery rate was calculated, based only on ICSI cycles with testicular sperm (fresh and/or frozen) available for injection. When patients delivered after transfer of supernumerary frozen embryos, this delivery was tallied up to the (unsuccessful) original fresh ICSI cycle. The sperm retrieval rate and pregnancy rate were secondary outcome measures. main results and the role of chance: Among the 714 men with NOA, 40.5% had successful sperm retrieval at their first TESE. In total, 261 couples had 444 ICSI cycles and 48 frozen embryo transfer cycles, leading to 129 pregnancies and 96 live birth deliveries. Crude and expected cumulative delivery rates after six ICSI cycles were 37 and 78%. limitations and reason for caution: A retrospective cohort study design wasthe only way to study the cumulative delivery rate after TESE-ICSI in couples with NOA. Intrinsic limitations are related to the observational study design. wider implication of the finding: TESE-ICSI is a breakthrough in the treatment of infertility due to NOA, with almost 4 out of 10 (37%) couples having ICSI obtaining a delivery. However, unselected candidate NOA patients should be counselled, before undergoing TESE, that only one out of seven men (13.4%) eventually father their genetically own child. study funding and competing interests: None declared. Key words: non-obstructive azoospermia / TESE / ICSI / pregnancy / cumulative delivery rate Introduction Prior to 1995, couples in which the male partner was diagnosed with non-obstructive azoospermia (NOA) had to opt for donor sperm or adoption in order to have children, however since the introduction of testicular sperm extraction (TESE) (Devroey et al., 1995), these couples also have a chance to father their genetically own child with ICSI. TESE-ICSI has now become a routine procedure to treat male infertility due to NOA. While the initially reported retrieval rate following a first TESE attempt in a well-defined NOA population was around 50% (Tournaye et al., 1997a), retrieval rates reported later in the literature were inconsistent & The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oup.com

2 Cumulative delivery rates of TESE-ICSI in NOA 1791 (Donoso et al., 2007; Ishikawa, 2012; Esteves et al., 2013). The selection of patients may explain these inconsistencies, e.g. inclusion of patients with hypospermatogenesis or patients without a proper histopathologic diagnosis could lead to inclusion of patients with misdiagnosed obstructive azoospermia (OA) resulting in overestimated retrieval rates. Although the first report on children born after ICSI with testicular sperm from men with NOA goes back to 1995 (Devroey et al., 1995), today data in the literature are still fragmentary; the available studies report either on sperm retrieval rates after TESE in a population of NOA men (Donoso et al., 2007; Dabaja and Schlegel, 2013) or on the outcome of ICSI in different patient populations where eventually testicular spermatozoa have been obtained (Nicopoullos et al., 2004). Few studies provide data on cumulative delivery or pregnancy rates after ICSI (Osmanagaoglu et al., 2003; Dafopoulos et al., 2005; Giorgetti et al., 2005) and these studies are prone to a strong selection bias, given that they include only patients with successful sperm retrieval and thus may overestimate pregnancy and live birth rates. In order to obtain reliable data for counselling NOA patients, this longitudinal study aimed to analyse the live birth rate in a consecutive series of patients with a histological diagnosis of NOA and having their first TESE procedure followed by ICSI with fresh or frozen testicular sperm, irrespective of whether these patients reached the sperm retrieval stage. Materials and Methods Patients In this retrospective cohort study, the outcome of 714 NOA patients having their first testicular biopsy between January 1994 and December 2009 was analysed. Patients who had a history of TESE in the past were excluded. All patients were diagnosed with azoospermia, based on the complete absence of spermatozoa in the ejaculate in at least two semen samples including high-speed centrifugation with examination of the entire pellet (World Health Organisation, 2010). All patients had a clinical work-up with physical examination, endocrine profile (follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone), genetic analysis and assessment of biochemical markers in the seminal plasma. Scrotal and transrectal ultrasounds were performed on indication. Only patients with a normal karyotype and absence of Yq deletions were included. As the World Health Organisation advises that the diagnosis of NOA should be based on the histopathological findings (Tournaye, 2002), only NOA patients with impaired or incomplete spermatogenesis as shown by histology were included. The classification as proposed by Levin et al. was used (Levin, 1979) where normal spermatogenesis refers to spermatogenic activity of the normal postpubertal male. Hypospermatogenesis refers to a generalized reduction in the number of spermatogenic cells within the seminiferous tubules. Maturation arrest is diagnosed when maturation of all germinal cells fails to progress beyond a given level (mainly the spermatocyte level). Incomplete maturation arrest is a similar condition except maturation of spermatids and eventually spermatozoa is seen focally. Germ-cell aplasia is a condition in which seminiferous tubules are completely devoid of germinal cells and lined exclusively by Sertoli cells, i.e. Sertoli-cell only syndrome (SCO). Incomplete germ-cell aplasia refers to the presence of rare foci of complete spermatogenesis adjacent to Sertoli-cell only tubuli. In tubular sclerosis, seminiferous tubuli show reduced to absent spermatogenesis with thickening of the tunica propria progressing to tubular sclerosis. The testicular histology of the patients included showed either maturation arrest with or without focal spermatogenesis, germ-cell aplasia (SCO) with or without focal spermatogenesis or tubular sclerosis and atrophy with or without focal spermatogenesis. Patients with hypospermatogenesis were excluded because these patients have complete spermatogenesis in at least some seminiferous tubules (Matsumiya et al., 1994). Testicular sperm recovery Patients had an open excisional testicular biopsy carried out under general anaesthesia or occasionally under local-regional anaesthesia. The TESE procedure was performed either before any intervention in the female partner (diagnostic), and combined with sperm cryopreservation whenever sperm retrieval was achieved (Verheyen et al., 2004), or on the day of ovum retrieval in the female partner (therapeutic). Two to five samples were taken from each testis depending on the testicular volume. In some patients, a micro- TESE procedure was performed according to Schlegel (Schlegel, 1999). During surgery, a randomly taken biopsy of each testis was sent for histological examination. The testicular cell suspension obtained after wet preparation in the laboratory was frozen for later use if at least one sperm was observed or, if after injection of the mature oocytes on the day of oocyte retrieval, sufficient supernumerary spermatozoa were available for subsequent ICSI cycle(s) (Verheyen et al., 2004). Ovarian stimulation In couples for whom testicular sperm were retrieved and frozen, or in couples undergoing a combined TESE-ICSI procedure, female partners underwent ovarian stimulation using urinary or recombinant FSH in combination with GnRH (Gonadotrophin Releasing Hormone) agonist or antagonist. Fertilization and embryo transfer procedure Fertilization rates were expressed as the percentage of oocytes with two distinct pronuclei per injected metaphase II oocytes. Embryos were classified according to their morphological appearance. Normally cleaving embryos with,50% fragmentation were considered eligible for transfer. Up to three embryos (exceptionally four) were transferred into the uterine cavity on Day 3 or 5 after injection. Since July 2003 onwards, the number of embryos transferred was restricted according to the Belgian law on assisted reproductive technologies (ART) (Van Landuyt et al., 2006). Supernumerary embryos of sufficient quality were cryopreserved for later use. Pregnancy follow-up Pregnancy was diagnosed by elevated serum hcg levels (.15 IU/l) on at least two consecutive occasions. A clinical pregnancy was defined by the presence of a gestational sac at transvaginal ultrasound 5 weeks after embryo transfer. Patients with an unknown outcome were considered not pregnant. The implantation rate was calculated as the number of gestational sacs with fetal heartbeat divided by the number of embryos transferred. A live birth was defined as a deliveryof a live infant of at least 20 completed weeks of gestational age or with a birthweight of 500 g (Bonduelle et al., 2002). Outcome measures The primary outcome measurewas delivery resulting in 1 live birth. The delivery of a singleton, twin or higher-order multiple pregnancies was registered as one delivery. Cumulative delivery rates have been calculated, as this is the preferred parameter in order to evaluate the effectiveness of an ART treatment (Hull et al., 1992). We calculated both crude and expected cumulative delivery rates. The real (or observed) cumulative delivery rate is defined as the observed number of live birth deliveries during a pre-determined number of cycles, divided by the total number of couples who had ICSI treatment with testicular sperm. This outcome parameter is also called the crude cumulative delivery rate. The expected cumulative delivery rate calculated according to the Kaplan Meier method assumes that patients who did not return for treatment (censored patients) had the same chance of a live

3 1792 Vloeberghs et al. birth delivery over a specific number of cycles as those who continued their treatment (Hull et al., 1992). Only ICSI cycles reaching the stage of oocyte retrieval and with testicular sperm available for injection (fresh and/or frozen) were included in this analysis. A maximum of six treatment cycles per patient were considered. Patients with an absence of testicular sperm at the time of injection were not further analysed. Patients were not re-enrolled after having reached the live birth delivery. When patients delivered following transfer of supernumerary frozen embryos, this delivery was tallied up to the (unsuccessful) fresh ICSI cycle. Statistical analysis Cumulative delivery rates were calculated by life-table analysis using the Kaplan Meier procedure. Cumulative delivery rates are expressed as cumulative probabilities with their 95% confidence intervals (95% CI). Cumulative delivery rates were compared between different subgroups based on the histological result of testis biopsy. Cox s proportional hazard model was used to perform multivariate analysis. A P-value of 0.05 or less is considered as statistically significant. Computational procedures were performed using IBM w SPSS w Statistics, version 20 (IBM Corporation, 2012). Institutional review board approval This retrospective study received approval by the local Institute s Ethics Committee. Results Testicular sperm recovery Out of the 714 consecutive NOA patients having their first testicular biopsy during the study period, 40.5% had sperm retrieved from the biopsies (Fig. 1). Demographics, histopathological findings and planning of TESE procedures are presented in Table I. Outcome of ICSI with testicular sperm There were 276 couples who started an ICSI trial either after a positive diagnostic testicular biopsy with freezing, or in combination with a fresh therapeutic testicular biopsy on the day of oocyte retrieval (Fig. 1). ICSI was performed in 261 couples with at least one mature oocyte injected with testicular sperm (Fig. 1). These 261 couples underwent 444 ICSI cycles and 48 related frozen embryo transfer (FrET) cycles leading to 129 pregnancies (positive hcg) and 96 live birth deliveries (Fig. 1). The mean number of ICSI cycles performed per couple was 1.7. For those couples who delivered, the mean number of ICSI cycles needed to obtain a delivery was 4.6. Table II shows the treatment characteristics and ICSI outcome of these 444 ICSI cycles. As shown in Fig. 2 and Table III, the crude cumulative delivery rate with a maximum of six ICSI cycles with testicular sperm was 37%. However the expected cumulative delivery over six cycles was 78%. Although expected cumulative delivery rates are promising once testicular sperm is available for ICSI, the live birth delivery rate per NOA couple undergoing their first TESE is limited to 13.4% (96/714). Multivariate analysis did not reveal any significant effect of the histological diagnosis of the testicular biopsy (P ¼ 0.371), calendar year of first oocyte retrieval (P ¼ 0.182), or male age (P ¼ 0.272) on the outcome, but there was, as expected, a significant effect of female age (P ¼ 0.018). Discussion Appropriate counselling of couples undergoing infertility treatment is an important issue both from a psychological and a financial viewpoint. Although TESE-ICSI is generally considered as a successful alternative to donor insemination or adoption for patients with NOA, data allowing proper counselling are still lacking, even 20 years after introducing the use of testicular sperm for ICSI. The fertility treatment involves two major steps: first step, surgical retrieval of sperm, and second step, the use of retrieved sperm for ICSI. In the literature, there are several reports regarding the chance of retrieving testicular sperm in NOA men (Donoso et al., 2007; Ishikawa, 2012; Esteves et al., 2013). Regarding the outcome of ICSI in these NOA couples, generally lower success rates are reported when compared with couples with men showing normal spermatogenesis (OA) or hypospermatogenesis (Vernaeve et al., 2003; Nicopoullos et al., 2004; Colpi et al., 2005; Esteves and Agarwal, 2013). While these scarce studies on populations of only NOA patients report success rates per cycle, couples should also be informed about their cumulative chance of delivering a baby after a number of treatment cycles. Only a few studies present cumulative delivery rates using testicular sperm in NOA, with crude cumulative delivery rates after three ICSI cycles of 17% (Osmanagaoglu et al., 2003) and 53% (Giorgetti et al., 2005). These studies include only patients who had successful sperm retrieval, and ICSI cycles with either only fresh testicular sperm (Osmanagaoglu et al., 2003) or only frozen testicular sperm (Giorgetti et al., 2005). Despite the increased risk of not finding sperm suitable for injection, the outcome of ICSI cycles using frozen-thawed testicular sperm is not inferior to cycles with fresh testicular sperm (Verheyen et al., 2004). Also in the present study, no sperm was available for injection after freezing-thawing in 27.5% of NOA ICSI cycles scheduled with frozen-thawed testicular sperm, and a back-up fresh TESE procedure was performed on the day of oocyte retrieval (Tables I and II). Our study aimed to analyse the live birth rate in a consecutive series only including a strictly defined NOA population. According to the literature reports, sperm retrieval rates after TESE range from 16.5 to 80% (Donoso et al., 2007; Ishikawa, 2012; Esteves et al., 2013) and, unfortunately, the prediction power for a positive or negative retrieval is quite limited (Tournaye et al., 1997b; Vernaeve et al., 2004; Adamopoulos and Koukkou, 2010; Boitrelle et al., 2011; Tournaye, 2011). Only preliminary testicular histology was shown to have a limited value in predicting sperm recovery (Tournaye et al., 1997b; Sousa et al., 2002; Adamopoulos and Koukkou, 2010; Boitrelle et al., 2011; Tournaye, 2011). Our data show a sperm retrieval rate of 40.5% if only the first TESEs is considered. However, the overall retrieval rate reached 49% if we included repetitive TESE procedures in the same man (Table I). This illustrates a potential bias and explains the wide range in retrieval rates as mentioned above. A recent paper reviewing microtese in NOA patients with SCO reports a 44.5% retrieval rate (Berookhim et al., 2014), illustrating that probably, as in men with OA, the surgical technique may have a limited impact on retrieval rates (Van Peperstraten et al., 2008). For step 2 of the TESE-ICSI approach, i.e. the ICSI procedure itself, a clinical pregnancy rate per cycle of 21.7% and a live birth delivery rate per

4 Cumulative delivery rates of TESE-ICSI in NOA 1793 Figure 1 Schematic overview of the patients with non-obstructive azoospermia followed from the first testicular sperm extraction until a live birth delivery. NOA, non-obstructive azoospermia; TESE, testicular sperm extraction; neg, negative; pos, positive; COC, cumulus oocyte complex; OR, oocyte retrieval; FrET, frozen embryo transfer; IUGR, intrauterine growth restriction; TOP, termination of pregnancy.

5 1794 Vloeberghs et al. Table I Demographics, histopathological findings and planning of the testicular biopsies in the 714 patients. All patients Positive sperm Negative sperm (n 5 714) retrieval retrieval... Demographics Age, years 35.0 (+7.6) 34.8 (+6.1) (mean + SD) TESE First TESE 289 (40.5%) 425 (59.5%) Planning Diagnostic 103 (34.3%) 197 (65.7%) Therapeutic 186 (44.9%) 228 (55.1%) Histopathology MA 80 (45.7%) 25 (54.3%) SCO 178 (38.4%) 286 (61.6%) Sclerosis and/or 31 (41.3%) 44 (58.7%) atrophy All TESE 432 (49%) 450 (51%) (1st + consecutive) Time interval (6.6) 12.2 (5.0) Back-up TESE 2 25 (50%) 25 (50%) TESE, testicular sperm extraction; MA, maturation arrest; SCO, Sertoli cell-only. 1 Time interval means the interval between two TESE procedures; data are presented as mean (median) months. 2 New rescue TESE that was performed if frozen-thawed suspensions could not be used on the day of oocyte retrieval. cycle of 20.6% were observed (Table II). These results are acceptable given that they are partially based on cycles performed in the mid-1990s and ever since there has been a substantial evolution in both IVF laboratory techniques and controlled ovarian stimulation protocols. Taking into account the low numbers of frozen embryos in our series, it is questionable whether embryo vitrification would increase the success rates in this patient population. Furthermore, from 2003 the Belgian law enforces a single embryo transfer (SET) policy. For couples with NOA, who are known to have lower implantation rates, this policy may limit success rates in the fresh cycle. The crude cumulative delivery rate per couple having ICSI was 34% after three cycles and further increased to only 37% after six cycles (Fig. 2). In the present study, better results were obtained than in a former study from our IVF unit reporting a crude cumulative delivery rate of 17% after three treatments cycles (Osmanagaoglu et al., 2003); the difference may be explained by improvements in the IVF laboratory procedures and because the present series also includes pregnancies from subsequent FrET cycles. The expected cumulative delivery rate per couple having ICSI was 52% after three cycles and 78% after six cycles. However, expected cumulative delivery rates using life table analysis may overestimate the chances of success, as very few patients continue their treatment after four cycles (Table II). To the best of our knowledge, this is the first study reporting cumulative delivery rates in a well-defined cohort of patients with NOA (men with a diagnosis based on histological findings, with normal karyotype, Table II Treatment characteristics and ICSI outcome of patients with 1 oocyte injected with testicular sperm. 7 (1.6%) All couples (n ¼ 261) Demographics Age female, years (+5.6) 444 ICSI cycles Injection with fresh testicular sperm 293 (66%) Injection with frozen testicular sperm 142 (32%) Injection with fresh and frozen testicular sperm 2 (0.4%) Injection with testicular sperm (fresh/frozen) and donor sperm ICSI cycles (with only injection of testicular sperm) Embryologic characteristics Number of COCs (+7.4) Number of MII oocytes (+6.1) % 2PN (+26.5) Cycles with fertilization failure (n) 35 Cycles with ET (n) 356 Embryos (n)/et (+1.3) Reproductive outcome Positive hcg/cycle (%) 121/437 (27.7) Positive hcg/et (%) 121/356 (34.0) Clinical PR/cycle (%) 95/437 (21.7) Clinical PR/ET (%) 95/356 (26.7) Implantation rate (%) 123/814 (15.1) Live birth delivery rate/cycle (%) 90/437 (20.6) Live birth delivery rate/et (%) 90/356 (25.3) COC, cumulus oocyte complex; ET, embryo transfer; PR, pregnancy rate. 1 As recorded on the day of the oocyte retrieval at the first ICSI cycle, mean years (+SD). 2 Injection was done partially with testicular sperm and partially with donor sperm; these cycles were included since there was injection of oocytes with testicular sperm, but in the calculation of the cumulative delivery rate only live birth delivery resulting from injection with testicular sperm was included. 3 Data are presented as mean (+SD). 4 Presented value is the average of fertilization calculated per cycle (+SD). Figure 2 Crude and expected cumulative delivery rates in nonobstructive azoospermia patients.

6 Cumulative delivery rates of TESE-ICSI in NOA 1795 Table III Crude and expected cumulative delivery rates after ICSI with testicular sperm in NOA patients. Treatment cycle number Patients (n) Non-pregnant and discontinued (n) Deliveries (n) Non-pregnant (n) Dropout rate 48% 49% 30% 58% 33% 100% Crude cumulative delivery rate 22% 30% 34% 36% 36% 37% Low 95% CI 17% 25% 28% 30% 31% 31% High 95% CI 27% 36% 40% 41% 42% 43% Expected cumulative delivery rate 22% 38% 52% 61% 70% 78% Low 95% CI 18% 31% 44% 51% 58% 63% High 95% CI 27% 44% 61% 70% 83% 92% Delivery rates per cycle 22% 20% 23% 17% 25% 25% CI, confidence interval; NOA, non-obstructive azoospermia. without AZF deletions and no preselection by previous TESE, as re-tese patients were not included) undergoing the TESE-ICSI procedure. It is of utmost importance when counselling patients with NOA who decide to embark upon TESE and eventually ICSI-TESE procedure, to give a correct and realistic estimation of their actual chances of parenthood. The cohort of patients studied is as homogenous as possible and this makes our data even more valid. Our study, however, has some limitations. Data are prone to bias owing to the observational study design, which is inevitable for this type of longitudinal study aiming at including a large study population. More specifically, outcome might have been influenced by improvements in laboratory conditions or the introduction of a SET policy from July 2003 onwards. However, even if this would increase outcome events, the prognosis would still remain limited for a population with strictly defined NOA. In conclusion, although TESE-ICSI is a major breakthrough in the treatment of infertility in NOA men, with almost 4 out of 10 (37%) couples who have ICSI reaching a delivery, candidate TESE-ICSI patients with NOA should be counselled that when followed-up longitudinally, only one out of seven men embarking on TESE eventually father their genetically own child. This is important information for the fertility specialist in order to counsel NOA couples towards the TESE-ICSI treatments or its alternatives, e.g. donor insemination or adoption. Authors roles V.V. was involved in the set-up of the database, analysed the data and drafted the manuscript. H.T., G.V. and V.V. were involved in the design of the study and conception of the study. P.H. performed the statistical analysis. N.P.P., A.G., P.H., H.T and G.V. contributed in revising of the manuscript. All authors have approved the final manuscript. Funding No external funding was used for this study. Conflict of interest None declared. References Adamopoulos DA, Koukkou EG. Value of FSH and inhibin-b measurements in the diagnosis of azoospermia a clinician s overview. Int J Androl 2010; 33: Berookhim BM, Palermo GD, Zaninovic N, Rosenwaks Z, Schlegel PN. Microdissection testicular sperm extraction in men with Sertoli cell-only testicular histology. Fertil Steril 2014;102: Boitrelle F, Robin G, Marcelli F, Albert M, Leroy-Martin B, Dewailly D, Rigot JM, Mitchell V. A predictive score for testicular sperm extraction quality and surgical ICSI outcome in non-obstructive azoospermia: a retrospective study. Hum Reprod 2011;26: Bonduelle M, Liebaers I, Deketelaere V, Derde MP, Camus M, Devroey P, Van Steirteghem A. Neonatal data on a cohort of 2889 infants born after ICSI ( ) and of 2995 infants born after IVF ( ). Hum Reprod 2002;17: Colpi GM, Piediferro G, Nerva F, Giacchetta D, Colpi EM, Piatti E. Sperm retrieval for intra-cytoplasmic sperm injection in non-obstructive azoospermia. Minerva Urol Nefrol 2005;57: Dabaja AA, Schlegel PN. Microdissection testicular sperm extraction: an update. Asian J Androl 2013;15: Dafopoulos K, Griesinger G, Schultze-Mosgau A, Orief Y, Schöpper B, Nikolettos N, Diedrich K, Al-Hasani S. Cumulative pregnancy rate after ICSI with cryopreserved testicular tissue in non-obstructive azoospermia. Reprod Biomed Online 2005;10: Devroey P, Liu J, Nagy Z, Goossens A, Tournaye H, Camus M, Van Steirteghem A, Silber S. Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Hum Reprod 1995;10: Donoso P, Tournaye H, Devroey P. Which is the best sperm retrieval technique for non-obstructive azoospermia? A systematic review. Hum Reprod Update 2007;13: Esteves SC, Agarwal A. Reproductive outcomes, including neonatal data, following sperm injection in men with obstructive and nonobstructive

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