ICSI Cycles. Sperm FISH Studies. Statistical Analysis RESULTS. MATERIALS AND METHODS Patients

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1 Testicular sperm from patients with obstructive and nonobstructive azoospermia: aneuploidy risk and reproductive prognosis using testicular sperm from fertile donors as control samples Lorena Rodrigo, B.Sc., a Carmen Rubio, Ph.D., a Vanessa Peinado, B.Sc., a Rafael Villamon, M.D., d Nasser Al-Asmar, B.Sc., a Jose Remohı, Ph.D., b Antonio Pellicer, Ph.D., b Carlos Simon, Ph.D., b and Manuel Gil-Salom, Ph.D. c,e a Preimplantation Genetic Diagnosis Unit, b Medical Reproduction Unit, and c Andrology Unit, Instituto Valenciano de Infertilidad; d Andrology Unit, Hospital Malvarrosa; and e Universidad de Valencia, Valencia, Spain Objective: To establish a baseline incidence of chromosomal abnormalities in testicular sperm of fertile men and to determine the best control sample for comparisons with azoospermic males to estimate their reproductive prognosis. Design: Prospective study. Setting: Infertility clinic. Patient(s): Sixteen obstructive azoospermic (OA) and 19 nonobstructive azoospermic patients (NOA). Control samples were ejaculated sperm from ten fertile donors and testicular sperm from ten other fertile donors. Intervention(s): Fluorescence in situ hybridization (FISH) in sperm. Main Outcome Measure(s): Sperm numerical abnormalities for chromosomes 13, 18, 21, X, and Y; ongoing implantation and pregnancy rates in intracytoplasmic sperm injection (ICSI) cycles. Result(s): In control samples, testicular sperm showed higher incidences of diploidy (0.27% vs. 0.10%) and disomy for chromosomes 13 (0.16% vs. 0.07%), 21 (0.25% vs. 0.12%), and sex chromosomes (0.34% vs. 0.21%) than ejaculated sperm. Comparisons with ejaculated control samples showed 12.5% OA and 68.4% NOA patients having significantly higher incidence of sperm chromosomal abnormalities. Compared with testicular control subjects, fewer OA (6.3%) and NOA (42.1%) patients had chromosomally abnormal sperm. NOA patients had lower ongoing implantation and pregnancy rates than OA patients, particularly those with abnormal FISH compared with testicular control samples. Conclusion(s): Sperm FISH analysis using testicular sperm control samples better identifies NOA patients with a lower likelihood of reproductive success. (Fertil Steril Ò 2011;95: Ó2011 by American Society for Reproductive Medicine.) Key Words: FISH, chromosomal abnormalities, testicular sperm Intracytoplasmic sperm injection (ICSI) has been successfully used in couples with severe male factor infertility, and it currently assists azoospermic men in contributing to pregnancy by using spermatozoa retrieved from the epididymis or testis (1, 2). However, questions surround the success rates of the ICSI technique in obstructive (OA) and nonobstructive (NOA) azoospermic males. Some authors describe similar and high fertilization and pregnancy rates (3 5), whereas others report lower ICSI outcomes in NOA than in OA patients (6, 7). In prenatal testing after ICSI, the percentage of de novo abnormal karyotypes has been reported to range from 1% to 2.4% for pregnancies with testicular sperm in OA patients, and from 7% to 11.1% in Received April 21, 2010; revised September 15, 2010; accepted October 13, 2010; published online November 11, L.R. has nothing to disclose. C.R. has nothing to disclose. V.P. has nothing to disclose. R.V. has nothing to disclose. N.A-A. has nothing to disclose. J.R. has nothing to disclose. A.P. has nothing to disclose. C.S. has nothing to disclose. M.G-S. has nothing to disclose. Supported in part by IMPIVA (project nos. IMIDTZ/2008/106, IMIDTZ/ 2008/25, IMIDTF/2009/135) and IZASA, CH-Werfen Company, Spain. Reprint requests: Lorena Rodrigo, B.Sc., Preimplantation Genetic Diagnosis Unit, Instituto Valenciano de Infertilidad, Plaza de la Policıa Local, 3, 46015, Valencia, Spain ( lrodrigo@ivi.es). NOA patients (8, 9), with 1.5% of pregnancies having sex chromosome abnormalities (10). These data suggest that NOA patients could pass to their offspring a higher percentage of aneuploidies in their sperm than OA patients. Several studies have proposed that a compromised testicular endocrine environment could increase the incidence of meiotic errors (11, 12 15). Furthermore, immunocytogenetic studies of human spermatogenesis have revealed lower frequencies of meiotic recombination in NOA than in OA males, with mechanisms linked to aneuploid sperm production (16 20). Indeed, most of the cytogenetic fluorescence in situ hybridization (FISH) studies on testicular sperm from normal karyotype azoospermic patients confirm an increase of numeric abnormalities mainly for sex chromosomes compared with control subjects (21 26), more remarkable in NOA than in OA men (22, 24, 26). Recently, our group observed that increases in aneuploid and diploid sperm lead not only to elevated rates of abnormal and mosaic embryos that could be responsible for implantation failures and abortions (27 29), but also to potentially viable abnormal embryos mainly with sex chromosome aneuploidies (30). Additionally, elevated rates of aneuploid and mosaic embryos derived from testicular sperm more remarkably in NOA men have been /$36.00 Fertility and Sterility â Vol. 95, No. 3, March 1, doi: /j.fertnstert Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 reported, suggesting that preimplantation genetic diagnosis for aneuploidy screening (PGS) to select euploid embryos could offer higher reproductive success to these couples (31 35). Several reports in ejaculated sperm have confirmed the potential role for aneuploidy testing in the work-up of infertile patients for genetic reproductive advice (28, 36 41) and as a predictor of ICSI success (42, 43). For a correct diagnosis, FISH analysis should use appropriate control samples. Currently, most of the groups use ejaculated sperm from normozoospermic donors, considering the rates of sperm chromosomal abnormalities as the baselines for fertile men, and they are compared with those of infertile men regardless of the origin of sperm being analyzed (ejaculate, epididymis, or testis) (21 24, 26, 27, 44, 45). Apoptosis is a hormone- and gene-regulated mechanism (46 50) used by the testis to remove redundant germ cells (48, 51, 52) and to eliminate chromosomally aberrant germ cells (53) during the spermatogenesis. The presence of apoptotic germ cells seems to be low in the ejaculate of fertile men with normal sperm parameters (54). However, infertile men with poor sperm parameters oligozoospermia or azoospermia show a wide variable incidence of apoptotic germ cells (51, 52, 55). Furthermore, recent studies have described a relationship between the frequencies of numeric chromosome abnormalities in sperm and immaturity (56 59) or DNA fragmentation (60 62), features that are positively correlated with apoptotic sperm markers (63). Apoptotic sperm in the ejaculate are suggested as abortive apoptotic sperm coming from apoptotic spermatocytes or spermatids that escaped the removal mechanisms in the testis and completed the meiosis and spermiogenesis (64, 65). If this occurs, we hypothesize that in fertile men with normal sperm parameters, just a low number of apoptotic sperm some of them carrying chromosomal alterations should escape the removal process, and therefore a higher frequency of aneuploid sperm should be in the testis than in the ejaculate. The lack of information on the baseline incidence of testicular sperm aneuploidies in fertile men presents a problem when studying azoospermic males by FISH, from which only testicular sperm are available. To confirm our hypothesis, and to evaluate which type of spermatozoa (ejaculated or testicular) from fertile men should be used as controls for FISH analysis in azoospermic men, the objectives of the current study were: 1) to establish the baseline incidence of ejaculated and testicular sperm chromosomal abnormalities in fertile men; 2) to classify testicular sperm FISH results from NOA and OA patients as normal or abnormal when using both ejaculated and testicular sperm from fertile males as controls; 3) to evaluate the predictive potential of sperm FISH analysis on ICSI outcome in NOA and OA patients, when using ejaculated or testicular sperm as controls. MATERIALS AND METHODS Patients The present prospective study was carried out from January 2001 to October 2009 and approved by the Institutional Review Board of the Instituto Valenciano de Infertilidad. Informed consent was obtained from each of the patients and control subjects. Chromosomal abnormalities in spermatozoa were analyzed by FISH in 55 normal-karyotype men distributed in four groups: Ejaculated control group (EC): ejaculated sperm samples from ten normozoospermic donors with proven fertility. Mean (SD) number of previous children from spontaneous pregnancies was Testicular control group (TC): testicular sperm samples from ten men of proven fertility, for whom a testicular sperm aspiration was performed at the time of vasectomy. Mean number of previous children from spontaneous pregnancies with ejaculated sperm was Obstructive azoospermia group (OA): testicular sperm samples from 16 OA infertile patients. Clinical diagnoses of the obstruction were epididymal obstruction (n ¼ 2), congenital bilateral absence of the vas deferens (n ¼ 2), bilateral obstruction of the vas deferens (n ¼ 1), and vasectomy (n ¼ 11), with FISH analysis performed a mean years after vasectomy. Nonobstructive azoospermia group (NOA): testicular sperm samples from 19 nonobstructive azoospermic infertile patients. Histologic diagnoses are summarized in Table 1. ICSI Cycles Twenty-one ICSI cycles in 16 patients with OA and 42 ICSI cycles in 16 patients with NOAwere performed using spermatozoa retrieved from the testis. After ovarian stimulation, oocyte retrieval was carried out via transvaginal aspiration of follicles under ultrasound guidance. ICSI was performed according to the protocol of our clinic, fertilization was assessed hours after ICSI (day 1) and then embryo cleavage hours later (days 2 3). Embryos were transfered into the uterus hours after ICSI. Female age in both ICSI groups was %37 years. Sperm FISH Studies FISH analysis on ejaculated and testicular spermatozoa from fertile men was exclusively performed to determine basal levels of chromosomal abnormalities in control groups. Testicular sperm from the NOA and OA patients were retrieved to be used in future ICSI attempts (66), and before the freezing process, an aliquot was collected for FISH analysis. Spermatozoa were obtained by macroscopic testicular aspiration technique in the TC group and by testicular sperm extraction using open testicular biopsies in the NOA and OA groups (67). Sperm samples were prepared for FISH to analyze chromosomes 13, 18, 21, X, and Y (Vysis, Downers Grove, IL), as previously described (26, 27), and spermatozoa with disomy and diploidy for these chromosomes were scored as abnormal. Hybridization efficiency was R93% in testicular samples and R98% in ejaculated samples. Statistical Analysis Chi-square test with Yate correction and Fisher exact test were used to compare sperm FISH results between the study groups. Fisher exact test, Student t test, and Welch t test were used to compare embryo quality and ICSI outcomes between NOA and OA groups. The pregnancy outcome of ICSI in relation to number of cycles were compared between NOA and OA by Kaplan-Meier survival analysis, followed by log rank, Breslow, and Tarone-Ware tests to evaluate the equality of the survival distributions. The level of significance was selected as P<.05. RESULTS Similar percentages of X-bearing and Y-bearing sperm were found in EC and TC (50.8% and 49.2% vs. 49.0% and 51.0% respectively). TC showed significantly higher incidences of diploidy and disomy for chromosomes 13, 21, X, and Y than EC (Table 2). The incidence of aneuploid and diploid sperm from OA and NOA was compared with both EC and TC (Table 1). OA compared as a total with EC showed a significantly higher incidence of spermatozoa with disomy for sex chromosomes. However, no differences in the percentages of abnormal sperm were observed when compared with TC. The individual analysis showed an abnormal FISH diagnosis in two out of 16 OA patients (12.5%) when using EC, but only one abnormal OA patient (6.3%) when compared with TC. The total group of NOA patients when compared with EC showed a significantly higher percentage of spermatozoa with diploidy and disomy for chromosomes 13, 18, 21, X, and Y. Significant differences were also observed when compared with TC, with an increase of diploid sperm and disomies for chromosomes 13, 21, X, and Y. Individual comparisons with EC resulted in 13 out of 19 NOA patients 1006 Rodrigo et al. Sperm aneuploidy and ICSI outcome Vol. 95, No. 3, March 1, 2011

3 Fertility and Sterility â 1007 TABLE 1 Age, etiology, pathology, and sperm fluorescence in situ hybridization (FISH) results in patients with obstructive azoospermia (OA) and nonobstructive azoospermia (NOA). Patient Age (y) Etiology Pathology Scored XY18 Haploid sperm Chr. 18 Disomic sperm Sex chr. Scored 13/21 Haploid sperm Chr. 13 Disomic sperm Chr. 21 Diploid sperm FISH result OA-1 37 EO g A N OA-2 37 EO N N OA-3 32 CBAVD a,h A A OA-4 35 VAS N N OA-5 39 VAS N N OA-6 39 VAS 2, , N N OA-7 34 VAS N N OA-8 38 VAS 1, N N OA-9 47 VAS N N OA VAS N N OA VAS N N OA CBAVD N N OA VAS N N OA VAS N N OA VAS N N OA BOVD N N Total OA (mean SD) , (n ¼ 6,752) 0.00 (n ¼ 0) 0.35 e (n ¼ 24) 3, (n ¼ 3,418) 0.15 (n ¼ 5) 0.23 (n ¼ 8) 0.13 (n ¼ 13) NOA-1 26 ORCH Sclerosis b,h A A NOA-2 30 CRYPT SCO g 0.18 A N NOA-3 32 CRYPT Hypo d,h d,h d,h A A NOA-4 40 ID Hypo f A N NOA-5 34 VAR MA f A N NOA-6 36 VAR MA d,h 6.25 e f A A NOA-7 34 ID SCO a,g 1.24 b,h A A NOA-8 36 ID Hypo c,h f 0.88 a,h A A NOA-9 40 ID Hypo N N NOA ID SCO N N NOA ID MA N N NOA CHEM Hypo N N NOA CHEM MA e A N NOA CRYPT SCO d,h d,h A A NOA ID MA e A N NOA ID SCO b,h 0.85 g 0.57 g A A NOA ID Hypo N N NOA VAR MA N N NOA ID Hypo d,h c,h d,h A A Total NOA (mean SD) , (n ¼ 4,799) 0.14 f,i (n ¼ 7) 1.13 d,h,j (n ¼ 55) 4, (n ¼ 4,181) 0.54 c,h,i (n ¼ 23) 0.52 a,h (n ¼ 22) 0.56 c,h,j (n ¼ 51) Note: A ¼ abnormal; BOVD ¼ bilateral obstruction of the vas deferens; CBAVD ¼ congenital bilateral absence of the vas deferens; CHEM ¼ chemotherapy; CRYPT ¼ cryptorchidism; EO ¼ epididymal obstruction; VAR ¼ varicocele; VAS ¼ vasectomy; ORCH ¼ orchitis; ID ¼ idiopathic; N ¼ normal. a P<.05; b P<.01; c P<.001; d P<.0001 vs. total TC (Table 2); e P<.05; f P<.01; g P<.001; h P<.0001 vs. total EC (Table 2); i P<.01; j P<.0001 vs. OA group (chi-square test and Fisher exact test). Rodrigo. Sperm aneuploidy and ICSI outcome. Fertil Steril vs. EC A A vs. TC N A

4 1008 Rodrigo et al. Sperm aneuploidy and ICSI outcome Vol. 95, No. 3, March 1, 2011 TABLE 2 Sperm fluorescence in situ hybridization results from ejaculated (EC) and testicular (TC) control samples. Sample Age (y) Scored XY18 Haploid sperm Disomic sperm Disomic sperm Chr. 18 Sex chr. Scored 13/21 Haploid sperm Chr. 13 Chr. 21 Diploid sperm EC , , EC , , EC , , EC , , EC , , EC , , EC , , EC , , EC , , EC , , Total EC (mean SD) , (n ¼ 102,052) 0.03 (n ¼ 30) 0.21 (n ¼ 210) 102, (n ¼ 101,895) 0.07 (n ¼ 73) 0.12 (n ¼ 126) 0.10 (n ¼ 203) TC TC , TC TC , TC TC TC TC , , TC TC , Total TC (mean SD) , (n ¼ 6,782) a P<.05. b P<.01. c P<.0001 vs. total EC (chi-square test). Rodrigo. Sperm aneuploidy and ICSI outcome. Fertil Steril (n ¼ 3) 0.34 a (n ¼ 23) 7, (n ¼ 7,412) 0.16 a (n ¼ 12) 0.25 b (n ¼ 19) 0.27 c (n ¼ 39)

5 TABLE 3 ICSI outcomes in NOA compared to OA patients. NOA (n [ 16) OA (n [ 16) Female age (y), mean SD No. of ICSI cycles Cycles/patient, mean SD Fertilization rate, % 55.5 a 67.0 Blastomeres on day 3, mean SD b Fragmentation on day 3, mean SD c No. of transfers 36 (85.7) 18 (85.7) No. of embryos transferred, mean SD Implantation rate, % Ongoing pregnancy rate/cycle, % (n) 21.4 (9) 38.1 (8) a,b P<.01. c P<.0001 vs. OA patients (Fisher exact test, Student t test, and Welch t test). Rodrigo. Sperm aneuploidy and ICSI outcome. Fertil Steril (68.4%) having an abnormal FISH diagnosis, and comparisons with TC resulted in 8 out of 19 NOA patients (42.1%) with an abnormal FISH. Additionally, we observed significantly higher incidences of diploidy and disomy for chromosomes 13, 18, X, and Y in NOA compared with OA. ICSI outcomes in NOA compared with OA resulted in a significantly lower fertilization rate and poorer embryo quality, in terms of mean blastomere number and fragmentation degree on day 3 (Table 3). Importantly, the ongoing pregnancy rate per ICSI cycle was lower for NOA than in OA patients (21.4% vs. 38.1%), and this was demonstrated by the survival analysis, in which we observed that the numbers of cycles needed to achieve pregnancy were significantly higher in NOA than in OA patients, with medians of five versus two cycles, respectively (log rank test: P<.001). NOA patients with normal sperm FISH results showed similar ICSI success in terms of implantation and pregnancy rates regardless of the control group used for FISH comparisons (Table 4). In general, NOA patients with abnormal FISH results showed lower success rates than those with normal FISH, particularly when sperm FISH was diagnosed using TC, with the worst ICSI results (9.1% of implantation rate and 11.1% of ongoing pregnancy rate versus 18.6% and 18.5%, respectively, when using EC). In the 16 NOA patients undergoing ICSI cycles, five of them had a normal sperm FISH compared with both EC and TC, with three ongoing pregnancies (60%) achieved after a mean of 3.7 ICSI cycles. Another five NOA patients had an abnormal sperm FISH when using EC but a normal result when using TC, resulting in three ongoing pregnancies (60%) after a mean of 4.5 ICSI cycles. However, in the six NOA patients that were diagnosed as abnormal FISH only when using TC, one ongoing pregnancy was achieved (16.7%) after a mean of 9 ICSI cycles. DISCUSSION We sought to establish a baseline incidence of chromosomal abnormalities in testicular sperm of fertile men, and to determine the best control sample for comparisons with azoospermic infertile men. To our knowledge, this is the first FISH study performed on sperm retrieved from the testis of fertile men before vasectomy TABLE 4 ICSI outcomes in NOA patients according to their sperm FISH diagnosis when using the testicular (TC) or the ejaculate (EC) control samples. Using TC Using EC Abnormal FISH (n [ 6) Normal FISH (n [ 10) Abnormal FISH (n [ 11) Normal FISH (n [ 5) Female age (y), mean SD No. of ICSI cycles Cycles/patient, mean SD Fertilization rate 68.2 a 51.4 a Blastomeres on day 3, mean SD Fragmentation on day 3, mean SD b b No. of transfers 7 (77.8) 29 (87.9) 25 (92.6) 11 (73.3) No. of embryos transferred, mean SD c c Implantation rate Ongoing pregnancy rate/cycle a,b,c P<.01 compared with other values with same letter (Fisher exact test and Welch t test). Rodrigo. Sperm aneuploidy and ICSI outcome. Fertil Steril Fertility and Sterility â 1009

6 and, therefore, with a physiologically normal testicular environment. Confirming the hypothesis of our study, we found a higher incidence of numeric chromosomal abnormalities in spermatozoa retrieved from the testis of fertile men than from the ejaculate. Earlier FISH studies in testicular sperm from fertile men undergoing vasectomy reversal have shown slightly higher percentages of aneuploidies than those observed in our study (0.46% versus 0.34% disomy for sex chromosomes, 0.10% versus 0.04% disomy 18, 0.21% versus 0.16% disomy 13 and 0.40% versus 0.25% disomy 21) (18). These differences could be attributed to the endocrine environment which, modified as a consequence of the physical obstruction, could lead to meiotic defects (11) resulting in aneuploid sperm. Additionally, immunocytogenetic studies on testicular samples from OA infertile men have described a decrease in recombination frequency and a modest increase in aneuploid sperm (22, 26, 16 18). When we used EC for sperm FISH comparisons, OA patients showed a slight increase of spermatozoa with disomy for sex chromosomes. However, this increase was not observed when compared with TC. None of our 11 OA patients with vasectomy had increased sperm chromosomal abnormalities, consistent with earlier results of infertile OA patients and fertile men undergoing vasectomy reversal (18, 19). Moreover, good ICSI pregnancy and implantation rates were observed in our OA patients, similar to those described in the literature (3, 5), indicating that this level of sperm aneuploidy would not affect reproductive outcomes. Alternatively, a large proportion of the NOA patients showed increases of sperm abnormalities when using both EC and TC. These data reflect earlier reports of aneuploidy in NOA patients (21 24, 26, 45, 68). Furthermore, Shi et al. (69) observed a low frequency of recombination in 24,XY aneuploid human sperm, prompting subsequent reports relating decreased recombination in spermatocytes to an increased frequency of aneuploidy in sperm from NOA men (18, 20, 70, 71). Our NOA patients with abnormal FISH diagnosis using both EC and TC had lower pregnancy and implantation rates than NOA patients with normal FISH. More interestingly, those with abnormal FISH using TC showed the poorest ICSI outcomes. Therefore in NOA patients, sperm FISH comparisons with TC better identify patients with the poorest reproductive prognosis. Embryo quality in ICSI cycles from NOA patients was similar regardless of the sperm FISH results, suggesting that the sperm chromosomal abnormalities would contribute to their low reproductive success. Our previous studies of PGS in NOA patients showed elevated rates of aneuploid (69.7%) and mosaic (31.2%) embryos, higher than those observed in OA patients (52.6% and 18.3%, respectively) (33) and consistent with other reports (31, 32, 72). Furthermore, cytogenetic studies of preimplantation embryos from patients with abnormal sperm FISH have described not only an increased incidence of abnormal embryos, but also a strong correlation between different patterns of sperm chromosomal abnormalities and embryo aneuploidy and triploidy rates (28, 30). These findings support that sperm aneuploidies can be responsible for implantation failures and abortions in some couples (27 29), because embryo morphology does not completely select against chromosomal abnormalities (73, 74). In the present study, a lower number of testicular sperm cells per patient were examined compared with ejaculated, and this fact could bias the estimation of aneuploidy. However, it would be more representative of the whole sample than the analysis of a higher number of sperm in ejaculated samples. This would be supported by the correlation observed between the sperm FISH results in OA and NOA patients and the reproductive ICSI outcome when using a proper TC. In conclusion, the baseline incidence of sperm chromosomal abnormalities is higher in the testis than in the ejaculate of fertile men. In a clinical setting, spermatozoa from both origins could be used as control samples for FISH studies. However, TC seems to be more appropriate when performing FISH to analyze testicular sperm samples to better identify NOA patients with higher sperm aneuploidy rates and lower potential reproductive success. Acknowledgments: The authors thank the clinicians, IVF and PGS embryologists, and technicians of the Instituto Valenciano de Infertilidad clinics for their cooperation in the development of this study. 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