Human Reproduction, Vol. 15, No. 5, 1121-1124,
May 2000
© 2000 European Society of Human Reproduction and Embryology
Chromosome analysis of spermatozoa extracted from testes of men with non-obstructive azoospermia
1 Department of Medical Genetics, Faculty of Medicine, University of Calgary, 2 Department of Genetics, Alberta Children's Hospital, 3 Department of Obstetrics and Gynecology, Faculty of Medicine, University of Calgary, Alberta, Canada and 4 Cancer Center Biometry section, Northwestern University, Chicago, Illinois, USA
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Abstract |
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Infertile men with azoospermia now have the possibility of fathering children by testicular sperm extraction combined with intracytoplasmic sperm injection. However, there are concerns about the risk of chromosomal abnormalities in their spermatozoa. We have studied aneuploidy frequencies for chromosomes 13, 21, X and Y by multicolour fluorescence in-situ hybridization (FISH) in testicular spermatozoa extracted from three men with non-obstructive azoospermia. The men were 34–37 years of age and had normal follicle-stimulating hormone (FSH) concentrations and normal 46,XY somatic karyotypes. A total of 3324 spermatozoa was analysed. The infertile patients had an elevated frequency of disomy for chromosomes 13, 21, XY disomy compared to controls but none of these reached statistical significance. Also there was no significant difference in the sex ratio or the frequency of diploidy in azoospermic patients compared to normal control donors. This first report on chromosomal aneuploidy in spermatozoa extracted from testes of patients with non-obstructive azoospermia suggests that some azoospermic men do not have a substantially increased risk of chromosomally abnormal spermatozoa.
Key words: aneuploidy/azoospermia/chromosome analysis/fluorescence in-situ hybridization/male infertility
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Introduction |
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The technique of intracytoplasmic sperm injection (ICSI) has proven to be a breakthrough in the treatment of male infertility. Even semen samples with extremely small numbers of spermatozoa can be used to produce a pregnancy with this technique. In recent years, ICSI has been extended to azoospermic men by the use of testicular biopsies to extract spermatozoa from the testes (Reubinoff et al., 1998
). However, male infertility is closely linked to chromosome abnormalities and there is concern that ICSI might lead to an increased frequency of chromosomally abnormal offspring (Martin, 1996; Meschede et al., 1998). Recent reports based on pre- and post-natal diagnosis in ICSI pregnancies have indicated an increased risk of chromosomal abnormalities of approximately 2% (Tarlatzis and Bili, 1998). Others (Van Opstal et al., 1997) have determined that the majority of these abnormalities are of paternal origin. We and others have demonstrated that men with various forms of infertility have an increased risk of chromosomal abnormalities in their spermatozoa (Moosani et al., 1995; Martin, 1996; Lähdeti et al., 1997; Bernadini et al., 1997; McInnes et al., 1998a; Pang et al., 1999) but these studies have not been extended to spermatozoa extracted from the testes. In this study, we report on sperm chromosomal analysis by multicolour fluorescence in-situ hybridization (FISH) analysis in testicular spermatozoa extracted from three men with non-obstructive azoospermia. |
Materials and methods |
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Normal control donors
Normal healthy men, as determined from information regarding childhood diseases, chronic disorders, environmental exposure, substance abuse, radiation exposure and prescription drug usage, were chosen. This group consisted of 18 men between the ages of 23–58 years with a mean of 35.6 years. All of the donors had normal semen profiles and 11 were of proven fertility and seven of unproven fertility (Kinakin et al., 1997
). Sperm samples were provided by masturbation. Sperm samples were cryopreserved until FISH analysis. We have previously demonstrated that cryopreservation has no effect on the frequency or type of chromosomal abnormalities in human spermatozoa (Martin et al., 1991).
Infertile patients
The three infertile patients all had azoospermia, normal 46,XY somatic karyotypes and normal FSH concentrations. The ages of the men were 34, 35 and 37 years, with a mean of 35.3 years. Sperm samples were retrieved by testicular biopsy and frozen in the same cryoprotective medium as control donors (Martin et al., 1991). Patients A and B had successful pregnancies after sperm retrieval and ICSI with the birth of a normal girl and normal female twins respectively. The patients were recruited from the University of Calgary Infertility Clinic. The study was approved by the institutional ethics committee and all donors gave informed consent.
Sperm preparation for FISH analysis
For each patient, one straw of frozen material from the testicular biopsy was thawed, washed with 5 ml of Biggers–Whitten–Whittingham (BWW) medium (Martin et al., 1982) centrifuged at 1200 g for 15 min, and the supernatant was discarded. The pellet was resuspended in 1 ml of pentoxifylline (Sigma P1784, 4 mg/ml; Sigma-Aldrich Canada Ltd., Oakville, ON, Canada), and the suspension was incubated at 37°C for 15 min. At the end of the incubation time, 3 ml BWW medium was added to the suspension, the sample was centrifuged at 600 g for 10 min, and the supernatant was discarded. A final wash with 3 ml BWW medium and centrifugation at 600 g for 10 min was carried out, the supernatant was discarded, and 250 µl of BWW was used to resuspend the pellet. Approximately 100 µl of this suspension was pipetted, a little off-centre, into the lid of a 60x15 mm Petri dish, spread to a circle approximately 3 cm in diameter, an approximately equal volume of fetal bovine serum (ICN Flow no. 29–161–49; ICN Biomedicals Inc., Costa Mesa, CA, USA) was pipetted and mixed into the spread drop, and paraffin oil was added to cover all. Viewing the drip under an interference-contrast microscope, a micromanipulator arm (fitted with a needle fashioned from 0.5 mm glass tubing pulled on a pipette puller, broken to an appropriate diameter by brushing the tip against the frosted end of a microscope slide, and filled with fetal bovine serum to prevent sticking of spermatozoa to the needle) was used to extract spermatozoa from the debris and expel them onto a microscope slide. The sperm collecting process described above was repeated until sufficient spermatozoa were deposited into the slide, the sperm-covered area was etched from below, and the slide was dried at room temperature for 1 day. In an effort to conserve as many spermatozoa as possible during the decondensation process, slides were positioned flat in a humidified box at room temperature. A drop of 10 mmol/l dithiothreitol (DTT) in Tris (5 µl 1 mol/l DTT in 0.5 ml 0.1 mol/l Tris) was centred over the sperm area and left for 25 min. The edge of a tissue was touched to the edge of the drop to blot the liquid, then a drop of 10 mmol/l LIS (3,5-diioiosalicylic acid, lithium salt, Sigma D-3635)/1 mmol/l DTT (dithiothreitol, Sigma D-9779) in Tris (tris-hydroxy-aminomethane, Sigma T-1503) (0.25 ml 20 mmol/l LIS, 0.5 µl 1 mol/l DDT, 0.25 ml 0.1 mol/l Tris, pH = 80) was used to cover the sperm area, and left for 2.5 h, checking from time to time and adding LIS/DTT solution as necessary to ensure that the drip did not evaporate. The LIS/DTT was blotted as described above, a few drops of 2xSSC were gently rinsed over the area, and the slide was air dried. Slides with decondensed spermatozoa were hybridized with probes specific to chromosome 13 and 21 [Vysis Inc., LSI 13 (RB-1) SpectrumGreen and LSI 21 SpectrumOrange] or to probes specific to chromosome X, Y and 1 (X specific 
-satellite probe generously provided by E. Jabs, Johns Hopkins University, Baltimore, MD, USA (Jabs et al., 1989) and chromosome 1 specific satellite II sequence, pUC1.77 kindly provided by H.J. Cooke, Edinburgh, UK (Cooke and Hindley, 1979) labelled directly with Fluorogreen3TM and Fluoroblue3TM (Amersham Pharmacia Biotech Inc., Baie d'Urfe, QC, Canada) respectively, and SpectrumOrange Yq (Vysis Inc.). Spermatozoa were scored using a Zeiss Axioplan epifluorescence microscope fitted with fluorescein isothiocyanate (FITC) and AMCA single bandpass filter sets, a rhodamine/FITC dual bandpass filter set and rhodamine/FITC/DAPI (4',6-diamidino-2-phenylindole) triple bandpass filter set, using scoring criteria described previously (Martin et al., 1995).
Statistical analysis
Statistical analysis was performed by a two-tailed Z statistic (Rosner, 1995).
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Results |
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A total of 3324 spermatozoa was analysed from the three infertile men: 1535 spermatozoa were analysed for the chromosome 13/21 hybridizations with 1035 from patient A, 400 from patient B and 100 from patient C; 1779 spermatozoa were analysed for the sex chromosome hybridization with 214 from patient A, 1236 from patient B, and 329 from patient C. Data from these three men were compared to results from 363 157 spermatozoa from 18 normal control donors (Kinakin et al., 1997
; McInnes et al., 1998a).
The frequency of disomy for chromosomes 13, 21, and the sex chromosomes as well as the proportion of X- and Y-chromosome-bearing spermatozoa and diploidy are presented in Table I. The infertile patients had an elevated frequency of disomy for chromosomes 13, 21 and XY disomy. In particular, patient A had aneuploidy frequencies considerably higher than controls. However none of these reached statistical significance. The only statistically significant difference between the infertile patients and control donors was for the proportion of YY disomy in which patients had 0% compared to 0.06% in controls (P < 0.001, two-tailed Z statistic).
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Discussion |
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A number of studies have demonstrated that infertile men have a 10-fold increase in the incidence of constitutional chromosomal abnormalities and that this is particularly pronounced for azoospermic men (Egozcue et al., 1983
; Luciani et al., 1987). Even among infertile men with a normal somatic karyotype, the frequency of chromosomal abnormalities restricted to the germ line is considerable at 6–18% (De Braekeleer and Dao, 1991; Vendrell et al., 1999). Furthermore, infertile males have an increased frequency of chromosomal pairing problems during spermatogenesis (Egozcue et al., 1983) so that even men with chromosomally normal somatic and germinal tissue could be at risk for an elevated frequency of aneuploidy in spermatozoa. This has been reflected in chromosomally normal infertile men shown to have an increased incidence of aneuploid spermatozoa by FISH analysis (for example, Moosani et al., 1995; McInnes et al., 1998a,b). Thus, azoospermic men, who require testicular sperm extraction for ICSI, could be particularly at risk for chromosomally abnormal spermatozoa. This was the impetus for our study as there have been no reports on this subject. This is a research endeavour fraught with difficulties since it is necessary to get an adequate sample of spermatozoa to assess aneuploidy frequency and yet it will be impossible to get a large sample of spermatozoa from azoospermic men. Our sample of over 3000 spermatozoa in three men is inadequate to uncover a small increase in the frequency of aneuploidy compared with controls but is robust enough to detect a 2.4–5.4-fold increase (depending on the specific chromosome). Although there was an increase in autosomal and sex chromosomal disomy in azoospermic men compared to controls, this failed to reach statistical significance.
To our knowledge, there has only been one report of FISH analysis in testicular biopsies from infertile men (Huang et al., 1999). This study was quite different from ours since it was performed on archival paraffin-embedded testicular tissue samples whereas ours comprised the spermatozoa that would be used in ICSI after testicular sperm extraction. The study by Huang et al. (1999) analysed diploid and haploid germ cells but spermatozoa were excluded from examination. Thus their study was more an examination of the earlier stages of spermatogenesis whereas ours was one of the end products which would be used for ICSI. Huang et al. (1999) found an alarmingly high frequency of sex chromosomal aneuploidy ranging from 39–43.5% in men with impaired spermatogenesis compared to 29.1% in controls. The majority of the abnormalities were observed in diploid cells. In our study, there was no significant difference in the frequency of sex ratio, diploidy, or aneuploidy for autosomes or sex chromosomes in azoospermic patients compared to controls. The difference in the two studies is striking with abnormality frequencies 20–30-fold higher in the study by Huang et al. (1999). Possible explanations for this include differences in methodology: there has been a paucity of FISH studies on archival fixed tissue and this may affect detection of fluorescent signals; also tissue sectioning could disturb the integrity of cells and promote spurious FISH signals. It is also possible that there is significant selection against chromosomally abnormal spermatozoa during spermatogenesis and that the frequency of aneuploidy in the spermatozoa of the patients of Huang et al. (1999) would have been much lower if it had been studied. Our studies of more than 30 reciprocal translocation carriers contradict a process of sperm selection against chromosomally abnormal spermatozoa since we have found that 19–77% (mean of 54%) of spermatozoa are chromosomally unbalanced demonstrating that even spermatozoa with major chromosomal imbalances complete spermatogenesis and fertilization processes (Martin, 1995). It would certainly be very interesting to study spermatozoa from the patients of Huang et al. (1999) to see if there is a dramatic decrease in the frequency of aneuploidy compared to germ cells. Finally, it is certainly possible that there are individual differences in the causes of non-obstructive azoospermia in infertile men with some men having a substantial risk for chromosomal abnormalities in meiotic cells and others having minimal risk. This should be a fruitful area of future research to determine which groups of infertile men are most at risk for chromosomal abnormalities in their offspring.
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Acknowledgments |
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Sincere thanks to the patients who participated in this study, and to D. Cole who prepared the manuscript. This research was supported by the Medical Research Council of Canada and the Alberta Children's Hospital Foundation.
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Notes |
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5 To whom correspondence should be addressed at: Medical Genetics Clinic, 1820 Richmond Road SW, Calgary, Alberta, Canada T2T 5C7
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Submitted on November 4, 1999; accepted on February 1, 2000.
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