Analysis of chromosomal abnormalities in testicular and epididymal spermatozoa from azoospermic ICSI patients by uorescence in-situ hybridization

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1 Human Reproduction Vol.19, No.1 pp. 118±123, 2004 DOI: /humrep/deh012 Analysis of chromosomal abnormalities in testicular and epididymal spermatozoa from azoospermic ICSI patients by uorescence in-situ hybridization L.Rodrigo 1, C.Rubio 1, E.Mateu 1, C.SimoÂn 1,2, J.RemohõÂ 1,2, A.Pellicer 1,2 and M.Gil-Salom 1,3,4 1 Instituto Valenciano de Infertilidad, Valencia and Departments of 2 Pediatrics, Obstetrics and Gynecology and 3 Surgery, University of Valencia, Valencia, Spain 4 To whom correspondence should be addressed at: Instituto Valenciano de Infertilidad, Plaza PolicõÂa Local 3, Valencia, Spain. Manuel.Gil-salom@uv.es BACKGROUND: An increased incidence of numerical chromosomal abnormalities has been reported in the ejaculated spermatozoa of infertile patients. However, there are few cytogenetic studies of testicular and epididymal spermatozoa, and their results are still controversial. METHODS: Fluorescence in-situ hybridization (FISH) analysis of chromosomes 13, 18, 21, X and Y was performed on seven testicular samples and two epididymal samples from patients with obstructive azoospermia (OA), and on 13 testicular samples from patients with non-obstructive azoospermia (NOA). Five ejaculated sperm samples from normozoospermic fertile donors were evaluated as a control group. RESULTS: Both epididymal sperm samples showed normal FISH results for the parameters analysed when compared with those of the control group. FISH results were abnormal in 29% (two of seven) of testicular samples from OA patients and in 54% (seven of 13) of those from NOA patients, although this difference was not statistically signi cant. Testicular samples from OA patients showed a signi cant increase of disomy for sex chromosomes (P < 0.01), whereas NOA patients displayed signi cantly higher rates of diploidy (P < ) and disomy for chromosomes 13 (P < ), 21 (P < 0.001) and sex chromosomes (P < ) than the control group. CONCLUSIONS: Testicular spermatozoa from azoospermic patients present increased rates of chromosomal abnormalities, mainly of the sex chromosomes, which are particularly high in NOA patients. Key words: azoospermia/chromosomal abnormality/epididymal spermatozoa/ uorescence in-situ hybridization/testicular spermatozoa Introduction In recent years, the use of ICSI has signi cantly improved the fertility prognosis of infertile couples affected by severe oligozoospermia (Palermo et al., 1992; Van Steirteghem et al., 1993) or azoospermia, in the latter case using spermatozoa retrieved from the epididymis (Tournaye et al., 1994) or testicle (Schoysman et al., 1993; Devroey et al., 1995). However, prenatal diagnosis following ICSI has shown statistically signi cant increases in de-novo sex chromosomal abnormalities and structural autosomal aberrations (Bonduelle et al., 2002; Van Steirteghem et al., 2002), most of which seem to be of paternal origin (Van Opstal et al., 1997; Meschede et al., 1998), thus highlighting the need for a strict genetic evaluation of ICSI candidates. Analyses of ejaculates and testicular biopsies from infertile patients with normal blood karyotypes have shown a variable incidence of meiotic anomalies in 6±37.9% of patients, with a particularly high incidence among patients with < sperm/ml and high serum FSH values (Egozcue et al., 1983; Vendrell et al., 1999; Egozcue et al., 2000). Desynapsis is considered the most common anomaly restricted to the germ cell line, producing partial or complete meiotic arrest, which results in oligozoospermia or azoospermia, respectively (Vendrell et al., 1999). The development of uorescence insitu hybridization (FISH) techniques has made it possible to analyse the cytogenetics of large numbers of spermatozoa. FISH studies reveal a signi cantly increased incidence of numerical chromosomal abnormalities, mainly of the sex chromosomes, in oligoasthenoteratozoospermic patients (Moosani et al., 1995; Bernardini et al., 1998, 2000; AraÂn et al., 1999; Colombero et al., 1999; Pang et al., 1999; Pfeffer et al., 1999; Nishikawa et al., 2000; Ushijima et al., 2000; Vegetti et al., 2000; Calogero et al., 2001a,b; Rubio et al., 2001; Martin et al., 2003). However, the results of cytogenetic studies carried out on testicular spermatozoa are controversial. Most FISH studies report a higher incidence of chromosomal abnormalities in testicular spermatozoa, particularly in that of non-obstructive azoospermic patients (Bernardini et al., 2000; Levron et al., 2001; Burrello et al., 2002; Mateizel et al., 2002; Palermo et al., 2002), than in ejaculated spermatozoa from normozoospermic donors. These results, however, have not been con rmed by 118 Human Reproduction vol. 19 no. 1 ã European Society of Human Reproduction and Embryology 2004; all rights reserved

2 Chromosomal abnormalities in testicular and epididymal spermatozoa Table I. Age, aetiology, serum FSH values, and aneuploidy and diploidy rates in fresh (NOAF) and frozen-thawed (NOAf) testicular spermatozoa from NOA patients Patient Age (years) Aetiology Pathology FSH Scored Sex chromosome disomies 18 Scored Total NOAF1 26 orchitis sclerosis a NOAF2 30 cryptorchidism SCO a NOAF3 32 cryptorchidism Hypo a 8.33 a a a NOAF4 40 idiopathic Hypo a NOAF5 34 varicocele MA a NOAF6 36 varicocele MA NOAF7 29 chemotherapy Hypo ± a NOAF8 41 chemotherapy MA ± NOAf9 34 idiopathic SCO NOAf10 36 idiopathic Hypo a a 0.87 a 0.89 a 0.88 a NOAf11 40 idiopathic Hypo ± NOAf12 40 idiopathic MA NOAf13 32 idiopathic SCO Mean SD Median Range 26±41 2.6±38 8±477 0±50 0±8.3 5±544 0±0.74 0±0.94 0±25 0±0.89 0±9.68 a P < 0.05 versus control group. SCO = Sertoli-cell only syndrome; Hypo = severe hypospermatogenesis; MA = maturation arrest. other authors (Martin et al., 2000a; Viville et al., 2000). Likewise, a higher incidence of chromosomal abnormalities in epididymal sperm than in ejaculated spermatozoa has been discovered in some studies (Bernardini et al., 2000; Burrello et al., 2002), but not in others (Palermo et al., 2002). The objective of the present study was to analyse the rates of numerical chromosomal abnormalities in testicular and epididymal spermatozoa obtained from azoospermic ICSI candidates, and to compare FISH results in epididymal and testicular spermatozoa with those of ejaculated spermatozoa from a control group of fertile donors. Materials and methods Patients A total of 22 sperm samples taken from azoospermic ICSI patients at our institution were evaluated. All showed a normal, 46,XY, blood karyotype. The origin of the spermatozoa was as follows. (a) Testicular sperm samples were obtained from thirteen patients with non-obstructive azoospermia (NOA) whose mean age (6 SD) was (range: 26±41). Testicular histopathological evaluation of these patients revealed severe hypospermatogenesis in ve, maturation arrest in four, Sertoli cell-only syndrome in three and tubular sclerosis in one. Serum FSH levels are shown in Table I. (b) Testicular sperm samples were acquired from seven obstructive azoospermic (OA) patients with a mean age (6 SD) of (range: 32±39). All patients showed normal serum FSH values and normal spermatogenesis at histopathology. The aetiology of obstructions is shown in Table II. (c) Epididymal sperm samples were obtained from two patients with OA caused by congenital bilateral absence of the vas deferens (Table II). Mean age (6 SD) of this group was (range: 29± 30). All of the testicular and epididymal spermatozoa evaluated were fresh samples, except for ve samples from NOA patients, which were frozen-thawed. Ejaculated spermatozoa from ve normozoospermic fertile donors, classi ed according to World Health Organization (1999) criteria, were evaluated as control group. The mean age of this group was (range: 22±46, Table III). Age of controls, OA and NOA patients were not signi cantly different (unpaired t-test). The study was approved by the local ethical committee and all patients signed a consent form prior to being included in the study. Samples extraction and preparation Testicular sperm were retrieved by testicular sperm extraction using open testicular biopsies. The tissue was placed in a Petri dish with 1 ml of Sperm Medium (Medicult, Copenhagen, Denmark) and was dissected using two sterile surgical blades. Epididymal sperm samples were obtained employing a modi ed macroscopic epididymal aspiration technique (Cha et al., 1997) and were placed in a conic tube containing 1 ml of Sperm Medium. Both testicular and epididymal sperm suspensions were frozen by adding 1 ml of Sperm Freezing Medium (Medicult, Jyllinge, Denmark) containing glycerol and human serum albumin. The mixture was homogenized and placed in a 4 C bath for 45 min. It was then homogenized once again and distributed with an automatic pipette in 100 ml droplets onto a dry ice surface for 1 min. Frozen droplets (`pills') were transferred to labelled cryotubes, which were subsequently submerged in liquid nitrogen (±196 C) and stored for future ICSI attempts (Romero et al., 1996). An aliquot was collected from 17 samples for FISH analysis prior to the freezing process. In the remaining ve testicular samples, FISH analysis was performed after thawing a variable number of frozen sperm pills depending on the sample quality. Pills were left to thaw in a conic tube for 10 min at room temperature and were then washed in 5 ml of Sperm Medium. Fresh and frozen-thawed samples were centrifuged for 5 min at 600 g and suspended for 20 min at 37 C in 10 ml of KCl M. Following hypotonic treatment, samples were centrifuged for 10 min at 600 g and pellets were xed in methanol:acetic acid (3:1). After two consecutive xations, sperm dilutions were spread on several slides and air-dried. Slides were stored at ±20 C until FISH analysis was performed. Ejaculated samples were prepared for FISH analysis as previously described (Rubio et al., 2001). FISH protocol For FISH analysis of testicular, epididymal and ejaculated samples, sperm nuclei were decondensed by slide incubation for 5±7 min at 119

3 L.Rodrigo et al. Table II. Age, aetiology, and aneuploidy and diploidy rates in testicular (toa) and epididymal (eoa) spermatozoa from OA patients Patient Age Aetiology Scored Sex chromosome disomy 18 Scored Total diploidy toa1 37 EO a a toa2 37 EO toa3 32 CBAVD a toa4 35 vasectomy toa5 39 vasectomy toa6 39 vasectomy toa7 34 vasectomy eoa8 29 CBAVD eoa9 30 CBAVD Mean SD Median Range 29±39 35±2232 0±1.49 0± ±2098 0±0.34 0±1.15 0±0.59 0±0.50 0±0.54 a P < 0.05 versus control group. EO = epididymal obstruction; CBAVD = congenital bilateral absence of the vas deferens. Table III. Age, seminal parameters, and aneuploidy and diploidy rates in ejaculated spermatozoa from control group Age Volume (ml) Count Total Normal Scored (310 6 /ml) motility forms (%) (%) Sex chromosome 18 disomies Scored Total diploidy C C C C C5 46 2, Mean SD Median Range 22±46 2±5 49±120 50±75 16± ± ± ± ± ± ± ± ± ± C in 5 mmol/l dithiothreitol and 1% Triton X-100. DNA was denatured for 5 min at C in a water bath in 70% formamide. Numerical abnormalities for chromosomes 13, 18, 21, X and Y were evaluated in different slides from the same sample, using triple-colour FISH for chromosomes 18, X and Y, and dual-colour FISH for chromosomes 13 and 21. Centromeric DNA probes for chromosome 18 (locus D18Z1, CEP 18 Spectrum Aqua; Vysis, Downers Grove, IL), chromosome X (locus DXZ1, CEP X Spectrum Green; Vysis) and chromosome Y (locus DYZ1, CEP Y Spectrum Orange; Vysis) were used for the triple-colour FISH analysis. Locus-speci c DNA probes for chromosome 13 (locus RB, LSI 13 Spectrum Green; Vysis) and chromosome 21 (loci D21S259, D21S341, D21S342, LSI 21 Spectrum Orange; Vysis) were used for dual-colour FISH analysis. FISH incubation and detection were performed according to the manufacturer's instructions. Analysis was carried out using an Olympus AX70 epi uorescence microscope equipped with a triple-band pass lter for 4 6-diamidino- 2-phenylindole/Texas Red/ uorescein isothiocyanate (FITC), and single-band pass lters for FITC, Texas Red and Aqua Blue. Due to the dif culty of differentiating the spermatozoa by its shape, only tailed spermatozoa in testicular and epididymal samples were analysed, and sperm nuclei scoring was performed according to established strict criteria (Blanco et al., 1996). Spermatozoa with disomy and diploidy for the analysed chromosomes were identi ed and scored. Nullisomic spermatozoa were not directly assessed because of the dif culty of differentiating them from a hybridization failure (for details, see Egozcue et al., 1997). About cells per control patient and 2000 sperm cells per epididymal sample were scored at each hybridization. In testicular samples, only a small 120 number of tailed spermatozoa could be detected and evaluated, the number varying depending on the quality of the sample. Hybridization ef ciency was >93% in testicular samples and >98% in epididymal and ejaculated samples. Statistical analysis FISH results were compared among the OA and NOA groups and with the control group. Individual FISH results were also compared with those of the control group and were considered abnormal when statistically signi cant increases in any of the analysed parameters were observed. Statistical analysis was performed using the two-tailed c 2 test (with Yates' correction when necessary) and Fisher's exact test. The unpaired t-test with Welch's correction was used for comparing age of controls and OA and NOA patients. Spearmen test was used for correlation analysis. A P value of <0.05 was considered to be statistically signi cant. Analysis was carried out using Graphpad Instat version 2.05a (Graphpad Software, San Diego, CA). Results Tables I and II show FISH results in each individual patient compared with those of the control group (Table III). Individual FISH results were normal for all the analysed parameters in both epididymal sperm samples. Individual FISH results were abnormal in 29% (two of seven) of testicular samples from OA patients and in 54% (seven of 13) of testicular samples from NOA patients. These differences were not statistically signi cant.

4 Chromosomal abnormalities in testicular and epididymal spermatozoa Table IV. Chromosomal abnormalities in fresh and frozen-thawed testicular sperm samples from NOA patients Fresh samples (n =8) Frozen-thawed samples (n =5) No. sperm scored Sex chromosome disomies (%) 20 (1.14) 12 (0.93) 18 (%) 2 (0.11) 2 (0.16) No. sperm scored (%) 8 (0.40) 3 (0.29) 21 (%) 8 (0.40) 5 (0.49) Total sperm scored (%) 12 (0.32) 9 (0.39) Table V. Chromosomal abnormalities in sperm samples from OA and NOA patients Control OA Non-obstructive Azoospermia Ejaculated sperm (n =5) Epididymal sperm (n =2) Testicular sperm (n =7) Total OA (n =9) Testicular sperm (n =10) No. sperm scored Sex chromosome disomies (%) 122 (0.24) 8 (0.19) 21 (0.46) a,b 29 (0.33) 26 (0.86) a±d 18 (%) 14 (0.03) 3 (0.07) 0 (0.00) 3 (0.03) 3 (0.10) No. sperm scored (%) 49 (0.10) 5 (0.12) 4 (0.15) 9 (0.13) 11 (0.37) a,d 21 (%) 70 (0.14) 3 (0.07) 7 (0.26) 10 (0.15) 13 (0.44) a,b,d Total sperm scored (%) 110 (0.11) 13 (0.16) 9 (0.12) 22 (0.14) 18 (0.30) a,c,d a P < 0.05 versus control. b P < 0.05 versus epididymal sperm. c P < 0.05 versus testicular sperm from OA. d P < 0.05 versus total OA. Fresh and frozen-thawed testicular sperm samples showed similar incidences of chromosomal abnormalities for chromosomes 13, 18, 21 and sex chromosomes in NOA patients. These results allowed us to form a single group including all NOA patients (Table IV). Table V shows FISH results in the different groups based on the cause of azoospermia (obstructive or non-obstructive) and on the origin of the spermatozoa. Some patients with NOA (NOAF3, NOAF6 and NOAf9) were not included in this analysis because of the low number of spermatozoa analyzed in these cases ( ve to 19 spermatozoa). Overall, sperm samples from OA patients (retrieved either from testicle or epididymis) showed similar results as those of the control group. However, testicular samples from OA patients displayed an increased incidence of sex chromosome disomies when compared to the control group (P = ). Testicular samples from NOA patients showed signi cantly higher rates of diploidy (P < ), and disomy for chromosomes 13 (P < ) and 21 (P < 0.001), and for sex chromosomes (P < ), than those of the control group. In addition, signi cant increases of diploidy (P = 0.04) and disomy for sex chromosomes (P = 0.02) were observed in testicular samples from NOA patients when compared to testicular samples from OA patients. There was no signi cant correlation between individual FISH results in NOA patients and serum FSH levels (normal 2± 10 miu/ml). Results after correlation analysis (Spearman) were as follows: sex chromosome disomies: r = , P = ; disomy 18: r = , P = ; disomy 13: r = ±0.507, P = ; disomy 21: r = ±0.3262, P = ; and total diploidy: r = , P = Discussion Our study con rms an increased incidence of numerical chromosomal abnormalities, mainly of sex chromosomes, in testicular sperm samples from OA and NOA patients when compared to ejaculated spermatozoa from normozoospermic donors. These results re ect those observed in most other related studies (Bernardini et al., 2000; Levron et al., 2001; Burrello et al., 2002; Mateizel et al., 2002; Palermo et al., 2002), although statistical differences between NOA and OA patients and controls were not found by Martin et al. (2000a) and Viville et al. (2000). We have also detected a higher incidence of chromosomal abnormalities in NOA patients than in OA patients. Similar results have previously been reported by Levron et al. (2001) and Burrello et al. (2002), but Mateizel et al. (2002) recently found similar incidences of aneuploidy and diploidy in testicular sperm samples from these two groups of patients. Several factors may explain the discrepancy in the results of these studies: the sizes of the series are small; the number of spermatozoa available for analysis in testicular samples is low; the subjects of these studies lack homogeneity, with differing histopathological patterns among NOA patients and with differing types of obstruction (congenital or acquired) among OA patients. The highest incidence of aneuploidies in testicular samples from OA and NOA patients was found for the sex chromosomes and chromosome 21. The production of aneuploid gametes, leading to abnormal embryos, could be attributed to non-disjunction during gametogenesis. During the male meiotic process, chromosome 21 and sex chromosomes are more susceptible to non-disjunction than other autosomes 121

5 L.Rodrigo et al. (Downie et al., 1997). This could be related to a reduction of recombination in acrocentric chromosomes such as number 21 (Warren et al., 1987; Nicolaidis et al., 1998), and to the presence of a single and terminal chiasma between the X and Y chromosomes at meiosis I (Hassold et al., 1991). Reviewing the literature, a negative correlation has been reported between sperm aneuploidy rate and sperm concentration (Bernardini et al., 1998, 2000; Pang et al., 1999; Pfeffer et al., 1999; Nishikawa et al., 2000; Ushijima et al., 2000; Vegetti et al., 2000; Calogero et al., 2001a; Rubio et al., 2001; Martin et al., 2003), and between sperm aneuploidy rate and the percentage of spermatozoa with normal forms (In't Veld et al., 1997; Calogero et al., 2001a; Devillard et al., 2002), affecting mainly the sex chromosomes. In these patients, severe meiotic arrest due to synaptic anomalies would explain the production of spermatozoa with a high incidence of chromosomal abnormalities (Egozcue et al., 1983). In recent years, it has been suggested that there are several checkpoints that control the meiosis process (Nicklas et al., 1997; Woods et al., 1999; Roeder et al., 2000). Chaganti and German (1979) suggested that male infertility was due to mutations in genes that regulate meiotic progression, and, more recently, a relationship between mutations in mismatch repair genes and sperm aneuploidy rate has been reported (Martin et al., 2000b). Sperm immaturity has also been correlated with the presence of chromosomal abnormalities in spermatozoa. Kovanci et al. (2001) demonstrated a close relationship between the incidence of immature spermatozoa and disomies, indicating that the latter are caused primarily by the former. Considering these observations, it seems that testicular samples obtained from OA and NOA patients contain a signi cant proportion of immature spermatozoa with an increased incidence of chromosomal abnormalities. In cases of NOA patients with abnormal spermatogenesis, synaptic anomalies and meiotic errors could explain the higher incidence of chromosomal aneuploidies and diploidies than that in OA patients. The incidence of aneuploidy and diploidy in epididymal sperm obtained from patients with OA is still controversial. An increased incidence of chromosomal abnormalities has been reported by some investigators (Bernardini et al., 2000; Burrello et al., 2002), but not by others (Palermo et al., 2002). Although in our study aneuploidy and diploidy rates in epididymal sperm were comparable to those of controls, our results should be interpreted with caution, since we have only analyzed epididymal sperm in two patients. The varying incidences of abnormalities observed in different studies may be due to the low number of samples and spermatozoa analysed and/or the effect of inter-individual variation on FISH results (Burrello et al., 2002). Furthermore, the clinical consequences of using sperm samples with an abnormal FISH result in ICSI programs have been evaluated by several authors. It seems that sperm chromosomal abnormalities may adversely affect ICSI outcome in oligoasthenoteratozoospermic, microepididymal sperm aspiration and testicular sperm aspiration patients, decreasing fertilization (Pfeffer et al., 1999) and pregnancy rates (Pang et al., 1999; Pfeffer et al., 1999; Bernardini et al., ; Calogero et al., 2001b; Rubio et al., 2001) and increasing miscarriage rates (Rubio et al., 2001), at least in some cases. Moreover, Gianaroli et al. (2000) and Silber et al. (2003) analysed the incidence of chromosomal abnormalities in embryos originating from azoospermic patients participating in a preimplantation genetic diagnosis program. Embryos from these patients suffered higher rates of abnormalities than those obtained from normozoospermic or oligozoospermic patients, with high incidences of embryos with aneuploidies for sex chromosomes (Gianaroli et al., 2000) and mosaic embryos (Silber et al., 2003). In conclusion, our results show an increased incidence of chromosomal abnormalities in testicular sperm from azoospermic patients, particularly in that of NOA patients. Therefore, these patients should be informed of their genetic risks before being accepted onto an ICSI program. Several alternatives could be offered, including prenatal testing, preimplantation genetic diagnosis or sperm donation. 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