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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Key words: aneuploidy/azoospermia/chromosome analysis/fluorescence in-situ hybridization/male infertility
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Introduction |
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Materials and methods |
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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 BiggersWhittenWhittingham (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. 2916149; 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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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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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 3943.5% 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 2030-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 1977% (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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Notes |
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References |
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Submitted on November 4, 1999; accepted on February 1, 2000.