1 Department of Biomedical Sciences, University of Bradford, UK, 2 Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, USA and 3 Institute of Reproductive Medicine of the University, Münster, Germany
4 To whom correspondence should be addressed. e-mail: m.h.brinkworth{at}bradford.ac.uk
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Abstract |
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Key words: ACM/FISH/ infertility/oligozoospermic/sperm
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Introduction |
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Aneuploidy and chromosomal aberrations can cause major morphological and biochemical defects in offspring. Most aneuploidy is attributed to errors in meiotic chromosome segregation during meiosis resulting in aneuploid gametes (Robbins et al., 1995). In the last years, several papers reported significantly elevated levels of aneuploidy in oligozoospermic patients (Bernadini et al., 1997
; Aran et al., 1999
; Pang et al., 1999
; Vegetti et al., 2000
; Schmid et al., 2003
), but relatively little is known about chromosome structural abnormalities in the sperm of these patients. Chromosome structural abnormalities are less common than aneuploidy at birth (0.25 versus 0.33%; Hassold, 1998
), but it is estimated that
80% are paternally derived (Chandley, 1991
). Hitherto, most data on chromosome structural abnormalities in sperm have been derived from studies of human spermhamster oocyte hybrids that allow analysis of the paternal chromosome complement (Rudak et al., 1978
). This is a time-consuming, though thorough, technique by which it has been estimated that such aberrations occur at a rate of 57% in normozoospermic, fertile males. Since only about a third of human conceptions are thought to result in a live birth, it has been assumed that chromosome aberrations may account for much of this failure.
In this study, a multicolour fluorescence in situ hybridization (FISH) assay (ACM) for the simultaneous detection of sperm carrying numerical chromosomal abnormalities (disomy and diploidy) as well as structural abnormalities (partial chromosomal duplications and deletions and chromosomal breaks) (Sloter et al., 2000) was used. This methodology utilizes multiple fluorescent colours to locate chromosomal domains directly in human sperm, and thus provides a direct approach to quantify abnormality levels at the loci studied. DNA probes specific for three regions of chromosome 1 are used to detect human sperm that carry numerical abnormalities and structural aberrations (duplications, deletions and breaks). The chromosome 1 region was selected because it has the longest distance between the centromere and telomere in the male (
4% of the genome). Sloter et al. (2000
) found that the incidence of breakage of chromosome 1 is representative of the frequency of sperm showing chromosomal breakage at any site across the whole genome, as measured by the hamster oocyte system.
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Materials and methods |
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Semen analysis
All patients and volunteers underwent the full diagnostic work-up routinely performed at the IRM as described (Behre et al., 2000) Semen analysis was performed according to the WHO guidelines (World Health Organization, 1999
). After removal of aliquots for examination, the ejaculates were frozen at 20°C.
ACM assay
Semen specimens were aliquoted and shipped without preservative to Livermore on dry ice. The aliquots were thawed at room temperature and a volume of 10 µl was smeared onto an ethanol-cleaned microscope slide and air-dried for 1 day. Decondensation of sperm nuclei was performed using dithiothreitol (DTT) and lithium diiodosalicylate (LIS) according to the method of Sloter et al. (2000).
The sperm ACM assay utilized DNA probes for three repetitive sequence regions on chromosome 1 [D1Z5 ( satellite or A), pUC1.77 (classical satellite or C) and D1Z2 (midisatellite or M)]. In situ hybridization was performed according to the method of Sloter et al. (2000
). Briefly, the probe mix for each slide contained 20 ng each of D1Z2 and pUC1.77 probe, 30 ng of D1Z5 probe and 10 ng of herring sperm DNA (carrier molecule) in a final concentration of 55% formamide/2x SSC and 10% dextran sulphate. Hybridization was carried out over two nights. Prior to washing, the coverslips were removed in 2x SSC at room temperature. Slides were washed in 60% formamide/2x SSC at 45°C for 5 min, followed by two 10 min washes in 2x SSC (pH 7.0) at room temperature. The biotinylated and digoxigenin-labelled probes were detected using a 1:100 dilution (in PNM buffer) of streptavidin Pacific blue (stock concentration 2.5 mg/ml; Molecular Probes) and sheep anti-digoxigeninfluorescein isothiocyanate (FITC; stock concentration 0.2 mg/ml; Boehringer Mannheim). Immunofluorescence was carried out for 30 min at room temperature in a humidity chamber, followed by two washes in 2x SSC for 3 min each. 4',6-Diamidino-2-phenylindole (DAPI), diluted to 10 ng/ml in Vectashield antifade medium (Vector), was used as counterstain. All hybridizations were performed at LLNL.
Scoring
The first author (T.E.S.) was trained at LLNL by E.S. according to the method described in Sloter et al. (2000). Scoring was performed in Bradford using a Leica DM photofluorescence microscope with a triple-band filter set for simultaneous visualization of Pacific blue, FITC and Texas red. The following criteria for abnormal sperm phenotypes were used: sperm carrying an abnormal number of same-colour domains were scored as abnormal only if the domains were of similar size and intensity (except for breaks within 1q12) and clearly separated. Each fluorescence domain had to be located within the boundary of an intact sperm nucleus. Overlapping sperm nuclei were not scored. Only cells with a flagellum (or tail attachment site) under bright-field microscopy were scored. Cells outside the normal size limits for decondensed sperm, as assessed by a microscope eyepiece graticule, were not scored.
The following describes the nomenclature and scoring criteria developed for the ACM assay (Sloter et al., 2000). One-letter abbreviations were used to denote the presence of each fluorescence domain: A (
satellite, 1cen); C (classical satellite, 1q12); and M (midisatellite, 1p36.3). The A and C regions are contiguous on chromosome 1, and the fluorescent domains are adjoined in normal sperm. A normal sperm was scored as ACM; addition or absence of letters denoted duplications or deletions. An O was used to indicate the absence of an expected domain. Thus, for example, sperm containing a duplication or deletion of M were represented by ACMM and ACO, respectively. The two M domains had to be separated by the distance of at least one normal M domain. ACACM and OOM represented centromeric duplications and deletions, respectively, of only the AC region.
Sperm carrying breaks within the 1cen1q12 (AC) region were divided into two groups based on their fluorescence phenotype. The first group included sperm containing a separation directly between the A and C regions and were denoted by A-CM. The A and C domains had to be separated by at least half the diameter of the C domain. Sperm classified into the second group carried two C domains and were represented by ACCM.
Sperm containing two copies of each domain represented chromosome 1 disomy or sperm diploidy. Each same-colour domain had to be separated by the distance of at least one full domain width. The C domain, which is larger than the others, required a half-domain separation. The absence of all three fluorescent domains was denoted by OOO (i.e. nullisomy 1). This phenotype could also represent lack of hybridization for technical reasons. In this study, no sperm with OOO were found.
Coding and statistical analysis
All slides were hybridized and encoded by a person at LLNL (F.H.) not involved in the scoring, and scoring was performed in Bradford by T.E.S. At least 5000 sperm were scored from the left half of each slide. The slides were then recoded, and a second set of 5000 sperm analysed from the right half of each slide, for a total of 10 000 sperm per slide. The CytoScore© software program developed at LLNL was used for the scoring.
The slides were decoded by the original encoder, and the first and second scoring analyses of each slide were compared using 2 analysis. Inter- and intra-donor variation in the frequencies of abnormal sperm was evaluated using contingency table analysis.
Comparisons between the oligozoospermic and normozoospermic groups were carried out with the MannWhitney U-test.
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Results |
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ACM assay
A total of 201 137 sperm were evaluated by the ACM-FISH assay. A significant increase in the (mean ± SD) frequency of sperm carrying either partial chromosomal duplications or deletions of chromosome 1 was found in the infertility patients compared with the normozoospermic controls (14.7 ± 3.6 versus 8.7 ± 2.9 per 104 sperm, P < 0.01) (Table II). Sperm carrying duplications or deletions of 1p occurred more frequently in the infertility patients than in the control group, (8.2 ± 2.2 versus 5.7 ± 1.9 per 104 sperm, P < 0.01). There was a higher frequency of sperm with duplications and deletions of 1pter in the infertile group (8.2 ± 2.2 versus 5.7 ± 1.9 per 104, P <0.01). In both groups, the frequencies of sperm carrying duplications versus deletions did not differ significantly from a 1:1 ratio, suggesting symmetrical mechanisms of formation. Sperm with duplications and deletions of 1cen were detected at average frequencies of 3.0 ± 1.0 in the control group and 6.5 ± 1.6 per 104 sperm in the infertility group (P < 0.01). There was no significant inter-donor variation for these types of sperm defects. Two types of breaks were detected by ACM sperm FISH. The average frequency of sperm carrying a break between the 1cen and 1q12 region was significantly higher in the infertility group compared with the control group (4.0 ± 1.6 versus 3.0 ± 1.3 per 104 sperm, P < 0.05). Breaks within 1q12 occurred in 12.4 ± 3.8 per 104 sperm in the oligozoospermic patients, which was significantly higher than in the control group (10.4 ± 1.2, P < 0.05). There was also no significant inter-donor variation in the total frequency of breaks among the individuals in each group studied.
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Discussion |
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Our data represent the first demonstration with sperm FISH that oligozoospermic infertility patients show higher frequencies of chromosomal structural aberrations of chromosome 1 in their sperm than normozoospermic men. The ACM assay detects damage in specific regions of just one chromosome and it is possible that similar aberrations or rates of damage will not be seen on all chromosomes. However, extrapolation of the ACM data for the control group (244 sperm with chromosomal structural aberrations/110 619 sperm analysed) to the haploid genome yields an estimate of 4.46.1% sperm carrying structural aberrations. This is in line with what has been reported using the hamster oocyte assay for healthy donors (Sloter et al., 2000). These results suggest that structural aberrations may be a widespread phenomenon in the sperm of infertile, oligozoospermic men and may provide an explanation for some of the failures of assisted conception cycles in infertile patients and highlight the need for investigations of heritable chromosome damage in the offspring that are produced by assisted reproductive techniques.
An average karyotype abnormality rate of 6% of azoospermic and oligozoospermic men has been reported in a review covering almost 10 000 men (Johnson, 1998
). Among 432 infertile men requiring treatment for infertility by ICSI in Münster (Meschede et al., 1998
), the rate was 2.1%. However, only two of the patients investigated here showed reductions in sperm parameters requiring ICSI treatment, and neither of them showed karyotype abnormalities. It is therefore highly unlikely that such abnormalities could explain the increase in chromosome 1 structural aberrations reported here.
The frequencies of sperm with duplications, deletions and breaks in our normal reference were similar to those reported by Sloter et al. (2000), demonstrating that the assay is robust for inter-laboratory comparisons. Our results also confirm the findings of Sloter et al. (2000
) that the spontaneous frequencies of sperm with structural chromosomal abnormalities are higher than those of numerical abnormalities and that chromosome breaks are more prevalent than partial duplications and deletions.
The formation of chromosome aberrations requires a DNA double strand break, which may be followed by rearrangement onto another chromosome. When DNA strand breakage occurs before or during male meiosis, it can lead to sperm carrying partial duplications and deletions of chromosomal regions (Van Hummelen et al., 1996). Our study showed significantly higher frequencies of sperm with duplications, deletions and breaks in the infertility patients compared with the controls. This can be associated with the recent finding that oligozoospermic patients also have significantly higher levels of chromatin disturbances and DNA strand breaks using SCSA and the Comet assay, respectively, than normozoospermic controls (Schmid et al., 2003
). It was assumed previously that Comet and SCSA damage in sperm arise during spermiogenesis. However, it was shown recently that X-irradiation of spermatogenic stem cells, proliferating spermatogonia and spermatocytes can induce Comet assay damage in resulting sperm (Haines et al., 2002
). This suggests that the Comet sperm assay could also be an indicator of pre-chromosomal lesions in pre-meiotic and meiotic male germ cells. Thus, it is possible that our group of infertile men had DNA damage in their meiotic or pre-meiotic germ cells that led to the induction of chromosome aberrations. In support of this suggestion, the Comet assay data (Schmid et al., 2003
) showed the highest levels of damage in the sperm of the same patients in which the highest rates of structural aberrations were found in the present study. There was a slight negative correlation between the sperm concentration of the oligozoospermic patients and the number of breaks in the 1cen1q12 region of chromosome 1, suggesting that the mechanism that leads to a low sperm count could also lead to a higher rate of breaks (Figure 1). Interestingly, there was no correlation of aberrations with motility or morphology.
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Finally, our study indicates that oligozoospermia is associated with chromosomal structural abnormalities and that the ACM assay is a robust approach for assessing the genetic integrity of the male germline
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Acknowledgements |
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Submitted on May 15, 2003; accepted on March 29, 2004.