1 Department of Biomedical Sciences, University of Bradford, Bradford BD7 1DP, UK, 2 Institute of Reproductive Medicine of the University, Münster and 3 Clinic of Veterinary Medicine of the Ludwig-Maximilian-University, Munich, 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: Comet assay/FISH/infertility/iRSM assay/SCSA
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Introduction |
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A recent review (Shi and Martin, 2001) cited a number of studies reporting an increase in aneuploidy in the sperm of such patients, but in most of the studies the number of individuals was low or their seminal parameters heterogeneous. In a study using the sperm chromatin structure assay (SCSA) and Comet assay (Larson et al., 2001
) it has also been suggested that infertility patients have genetically damaged sperm, but only a few patients were analysed and the study focused on teratozoospermia. Furthermore, these findings are not necessarily equivalent as different assays represent damage induced during different phases of spermatogenesis. The aim of our study was to determine whether there are differences between sperm from normozoospermic volunteers and infertile, oligozoospermic patients by fluorescence in-situ hybridization (FISH), inverse restriction site mutation assay (iRSM), Comet assay and SCSA. This may indicate whether the infertility results solely from low sperm quantity or whether poor sperm genetic quality also plays a role. Furthermore, it may also reveal whether sperm from such patients present a risk of transmitting genetic defects to the offspring.
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Materials and methods |
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Semen analysis
Semen analysis was performed according to WHO guidelines (WHO, 1999). For morphology Papanicolaou staining was used. Vitality of sperm was analysed with the eosin test in which stained cells are considered to be dead. After removal of aliquots for examination the ejaculates were frozen at 20°C.
Sperm FISH assay
In preparation for in-situ hybridization, decondensation of sperm was performed using a method previously described in detail (Baumgartner et al., 2001). Briefly, air-dried smears on microscope slides were incubated in Coplin jars containing 10 mmol/l dithiothreitol (Sigma, Poole, Dorset, UK) on ice for 30 min. They were then incubated in 4 mmol/l lithium diiodosalicylate (Sigma) for 60 min after they were allowed to air dry.
Hybridizations were performed according to a modified technique of Schmid et al. (1999). Three directly labelled probes were used simultaneously for the hybridization: D18Z1 (chromosome 18, alphasatellite, red; QBiogene-Alexis Ltd., Bingham, Notts., UK), DYZ3 (chromosome Y, alphasatellite, green; QBiogene-Alexis Ltd.) and DXZ1 (chromosome X, alphasatellite, red and green; QBiogene-Alexis Ltd.). Probes were mixed with Master Mix 2.1 (55% v/v formamide, 10% w/v dextran in 1 x SSC) and denatured at 78°C for 5 min. The sperm were denatured in 70% v/v formamide in 2 x SSC, pH 7.0, at 78°C for 5 min, dehydrated through an alcohol series (70% v/v aqueous, 90 and 100%, 2 min each) and dried at 37°C for 3 min before application of the denatured hybridization mix.
Slides were hybridized for 48 h at 37°C, then washed for 20 min in 50% formamide in 2x SSC, pH 7.0, at 45°C and 30 min in PN buffer (NaH2PO4, 0.1 mol/l; Na2HPO4, 0.125 mol/l; 1 gepal CA360, 0.1% v/v; all chemicals from Sigma) at 37°C. The nuclei were counter-stained with 4,6-diamidino-2-phenylindole (DAPI) 0.1 µg/ml in phosphate-buffered saline (PBS), for 10 min at room temperature and mounted in Vectashield (Vector Labs, Peterborough, UK). Slides were stored at 4°C in the dark.
Scoring was performed using a Leica DM Photofluorescence microscope with a triple-band filter, set for simultaneous visualization of DAPI, FITC and Texas Red. Strict scoring criteria were followed for the analysis of all slides (Robbins et al., 1995). Only single and intact sperm nuclei with homogeneous DAPI intensity were analysed. The following criteria for abnormal sperm phenotypes were used: (a) similar intensity and outline of all the signal domains, localization within the nucleus; and (b) comparability of the domains to those in the surrounding cells. Cells were scored as having two domains of the same colour if both signals were of similar size and intensity, and were separated by at least 0.5 signal diameters. Frequencies of disomies and diploidies were scored, as was the frequency of sperm with nullisomic phenotypes; however, the latter was not included in the statistical analysis. This procedure is justified because loss of a chromosome domain could be due to technical artefact.
iRSM analysis
This assay identifies the presence of mutations that convert one restriction site (HaeIII) into another (AvaII) (Jenkins et al., 1999).
All restriction enzymes were purchased from New England Biolabs (Hitchin, Herts, UK). Initial digestions were carried out in TAQ polymerase buffer (New England Biolabs) with 1 µg DNA and 20 U HaeIII overnight at 37°C. PCR amplifications were performed in a Progene cycler (Techne, Cambridge, UK) using primers obtained from Sigma-Genosys (Pampisford, Cambs, UK). The forward primer for the exon 7 was 5'-atgtgtaacagttcctgcatg-3', the reverse was 5'-ctgacctggagtcttccagtg-3'. Following digestion, the undigested (mutated) sequences were amplified by PCR using primers flanking the restriction site. Amplification was performed in TAQ polymerase buffer with 100 µmol/l deoxynucleotide triphosphates (Roche Diagnostics, Lewes, E.Sussex, UK), 10 pmol of each primer, 20 µl digested DNA (1 µg DNA) and 2.5 U Taq polymerase in a final volume of 50 µl. The thermal cycle consisted of 35 cycles at 94°C for 60 s, 60°C for 20 s and 72°C for 30 s. After amplification, the PCR product was further digested in a final volume of 20 µl overnight with 10 U of AvaII (New England Biolabs). The creation of a unique AvaII site was assessed by PAGE analysis with a 15% (w/v) gel stained with silver. The presence of AvaII restriction fragments (39 and 40 bp) and undigested sequences (79 bp) was determined by comparison with a molecular weight marker (10 bp ladder; New England Biolabs).
SCSA
The SCSA was carried out following the procedure described by Evenson and Jost (1994). Briefly, frozen semen samples were thawed in a 37°C water bath, and immediately diluted with TNE buffer (0.15 mol/l NaCl, 0.01 mol/l Tris and 0.001 mol/l EDTA, pH 7.4) to 12 x 106 sperm/ml. Four hundred microlitres of acid-detergent solution [0.08 mol/l HCl, 0.15 mol/l NaCl, 0.1% (v/v) Triton X-100, pH 1.2] were added to 200 µl of the diluted sample. After 30 s, sperm were stained by adding 1.2 ml of acridine orange (AO) staining solution containing 6 µg AO (chromatographically purified; Polysciences Inc., Warrington, PA, USA) per ml of buffer [0.037 mol/l citric acid, 0.126 mol/l Na2HPO4, 0.0011 mol/l EDTA (disodium), 0.15 mol/l NaCl, pH 6.0]. After 3 min of staining 5000 cells per sample were analysed by a FACStar Plus flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with an argon ion laser (Iona 300; Coherent, Santa Clara, CA, USA), tuned to 488 nm and operated at a power output of 200 mW in light mode. When excited with a blue light source, AO intercalated to double-stranded DNA fluoresces green (530 ± 30 nm), and AO associated with single-stranded (denatured) DNA emits red fluorescence (>630 nm).
Scattergram analysis of raw data, with each point representing the co-ordinate of red and green fluorescence intensity values for every individual spermatozoon, was carried out using standard Becton Dickinson software. The data from the eight-parameter list-mode files were transferred to a MS-DOS IBM compatible computer system by Becton Dickinson Fastfile software, and subsequent data handling was performed with Data Analysis Software DAS (Beisker, 1994). The bivariate data can be conveniently expressed by the function alpha T (
T), which is the ratio of red to total (red plus green) fluorescence intensity, thus representing the amount of denatured, single-stranded DNA over the total cellular DNA (Darzynkiewicz et al., 1975
).
T was calculated for each sperm in a sample and the results were expressed as the percentage of cells with high
T values: cells outside the main population (COMP
T).
Comet assay
DNA strand breaks were measured by the Comet assay using the method described by Anderson et al. (1997). In brief, fully frosted microscope slides were each covered with 110 µl of 0.5% normal melting-point agarose at 50°C in PBS. The slides were dried at room temperature for 3 days. Approximately 10 000 sperm were mixed with 0.5% low melting agarose to form a cell suspension and 90 µl pipetted onto the first agarose layer, spread and solidified lying flat on ice. After removal of the coverslip, a third layer of 0.5% low melting-point agarose was added, spread using a coverslip and again allowed to solidify on ice for 5 min. The slides were immersed in lysing solution [2.5 mol/l sodium chloride, 100 mmol/l EDTA, 10 mmol/l Tris, 1% Triton X-100, 10% DMSO and 0.05 mg/ml proteinase K (Roche Diagnostics)] at 37°C overnight. The slides were subjected to electrophoresis through 1 mmol/l EDTA, 300 mmol/l NaOH buffer. After 20 min incubation for unwinding of the DNA and expression of alkali-labile damage, electrophoresis was performed at 4°C for 20 min at 24 V. Tris, pH 7.5 (0.4 mol/l), was used to neutralize the alkaline buffer for 5 min, 50 µl ethidium bromide (20 µg/ml) was added and the slides coverslipped and analysed within 3 h. Slides were examined at x400 magnification on a fluorescent microscope (Leica Microsystems UK, Ltd., Milton Keynes, UK) equipped with an excitation filter of BP546/10 and a barrier filter of 590 nm. Twenty-five cells were scored from each replicate slide (50 cells total). A computerized image analysis system (Comet 3.0; Kinetic Imaging, Liverpool, UK) was used to measure the tail moment. The tail moment is the integrated value of density multiplied by migration distance; it is considered to be the most sensitive measurement (Anderson et al., 1997
) and was automatically generated by the computer.
Statistical analysis
In the sperm FISH assay the 2-test with Yates correction was used for a statistical overall comparison of the frequencies of disomic or diploid sperm, and the comparisons between the oligozoospermic and normozoospermic groups were carried out using the MannWhitney U-test. Pair-wise comparisons of the percentage denatured DNA measured by SCSA of sperm from oligozoospermic patients and normozoospermic volunteers were analysed with the MannWhitney U-test. Tail moment values in the Comet assay are not normally distributed and violate the requirements for analysis by parametric statistics. To resolve this problem, we used the slide as the unit of measure rather than the cell. The mean tail moment of each slide was determined and the averaged mean tail moments obtained from each group were compared by a one-way analysis of variance test (ANOVA) as described in Anderson and Plewa (1998)
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Results |
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Figure 2 shows typical SCSA flow cytometry data from an oligozoospermic patient. The COMP T indicates the percentage of abnormal cells (cells outside the main population). A significant increase (P < 0.01) in COMP
T (representing the amount of denatured, single-stranded DNA over the total cellular DNA, mean ± SD) was found in the infertility patients (61.2 ± 16.6%), compared with 42.6 ± 12.8% in the control group, using the MannWhitney U-test.
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Discussion |
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To understand the mechanisms affecting the production of chromosome abnormalities, it is necessary to study human sperm directly.
The iRSM assay detects the creation of new restriction sites from pre-existing ones (Jenkins et al., 1999). This method differs from restriction fragment length polymorphism analysis in that rare mutant alleles can be detected, due to selective removal of the wild-type sequences by restriction digestion of the pre-existing site and specific amplification of mutant sequences by PCR. The iRSM technique was used to analyse mutations in the p53 gene because of its central role in mediating responses to genetic damage. Furthermore, the p53 gene contains a number of mutation hotspots, for example codon 249 in an HaeIII site (GGCC) in exon 7, which has been used previously for the detection of mutation (Jenkins et al., 2001
).
The iRSM data demonstrate that the successful detection of point mutations in a mutation hotspot within the p53 gene is possible in sperm DNA. Nudell et al. (2000), using sequence analysis of a polymorphic marker, found an increased level of mutations in testicular DNA from azoospermic infertile men. However, the increase in mutations in the sperm of oligozoospermic patients was not significant in our study. Since the mutations found by Nudell et al. (2000)
were from the testes of men with meiotic arrest, it may be that they arose as a result of the length of time that the pre-meiotic cells had been arrested in the testis. Given that the other assays in the present study detect genetic damage induced in meiotic or post-meiotic cells and were all positive, it may be that the damage that correlates with oligozoospermia is induced during and after meiosis, but not in spermatogonia.
The SCSA measures the susceptibility of sperm chromatin to acid denaturation. During sperm maturation, somatic-type, nuclear histones are replaced by arginine- and cysteine-rich protamines, followed by chromatin stabilization through interprotamine crosslinking via disulphide bridges (Balhorn, 1982). The result is a highly condensed sperm nucleus that is more resistant to DNA cleavage and denaturation than that of a somatic cell. The SCSA measures the ratio of the amounts of single-stranded DNA (red fluorescence) to double-stranded DNA (green fluorescence) after acid treatment, and is ideally suited to assess sperm DNA integrity in terms of fertility potential (Evenson and Jost, 2000
). The Comet assay is also a useful and rapid method for examining DNA damage in sperm (as well as in somatic cells) (Anderson et al., 1997
). In this assay, broken DNA strands migrate towards the anode and form a comet tail under electrophoresisthe larger the tail, the greater the extent of damage. This is a sensitive technique, which can be used to detect both single- and double-stranded DNA breaks. Although the origin and biological significance of these events are not clear, they may be related to the levels of damaging, oxidative agents in the ejaculate or in the male reproductive tract (Hughes et al., 1996
; Haines et al., 2001
).
Genetic damage detected by the SCSA and the Comet assay results from effects on elongating or maturation phase spermatids, which contrasts with the meiotic effects seen with FISH (see below). Our data represent the first demonstration using FISH, iRSM, Comet and SCSA on sperm from the same semen samples, that oligozoospermic infertility patients show genetic damage in their sperm arising in several different ways. There was an increase in denatured, single-stranded DNA and of DNA strand breaks in the sperm of these patients. It is assumed that chromatin-packaging anomalies in human sperm can arise because of defective protamination and the presence of breaks in the DNA molecule (Balhorn et al., 1988). The mechanisms responsible for producing abnormal sperm are still poorly understood. It is possible that DNA damage in the sperm is related to problems in nuclear remodelling, resulting from problems during protamine deposition in spermatogenesis (Sakkas et al., 2002
). A number of studies have shown that sperm with abnormal nuclear chromatin are more frequent in subfertile and infertile men than in fertile men (Evenson et al., 1980
; Engh et al., 1992
; Spano et al., 2000
). The increased frequencies of chromatin disturbances and DNA strand breaks found here by SCSA and Comet assay also extend the limited results of Larson et al. (2001)
in globozoospermic, infertile men. Larson et al. reported that sperm with chromatin disturbances measured by SCSA are strongly correlated with DNA fragmentation in the Comet assay. If chromatin fragmentation is related to DNA strand breaks (Balhorn, 1988
), this could explain that correlation and account for the positive results in both assays in the present study. It would also indicate that either test could be used to indicate fertility potential, but confirmation awaits similar studies in fertile men with sperm counts below 20 x 106/ml. The pregnancies (not carried to term) achieved by the partners of patients 2, 7, 9 and 10 showed no apparent trend in the Comet assay or SCSA data of those individuals.
Application of the sperm-FISH assay has opened the way for studies on numerical chromosome abnormalities in human sperm. Most aneuploidy is attributed to errors in chromosome segregation during meiosis, resulting in aneuploid gametes (Robbins et al., 1995). The relatively simple sperm-FISH approach to detecting aneuploidy in human sperm used in the present study allows rapid screening of thousands of sperm for the detection of particular types of aneuploidy (Shi and Martin, 2001
). This permits the detection of particular types of aneuploidy and could thus indicate the risk of specific defects, for example the risk of Klinefelter syndrome (47,XXY).
The oligozoospermic infertility patients were found to have an elevated level of XY aneuploidy and of XY diploidy in the germ-line. It is likely that such an event stems from non-disjunction at meiosis I, when the homologous chromosomes should separate. It is interesting that an increase in autosomal disomy 18 was not found. This may be related to the differing size of the X and Y chromosomes, which may lead to a higher probability of non-disjunction than in homologous autosomes. The reason we studied chromosome 18 is that along with the sex chromosomes, aneuploidies for chromosome 18 are clinically significant, because offspring with trisomy 18 can sometimes survive for a limited time (as can offspring with trisomy 13 or 21). However, our data do not indicate any increased risk for autosomal trisomy among the offspring of oligozoospermic men who succeed in having (or are helped to have) children.
The relative risk of sex chromosome aneuploidy is 1.5 times higher than in normozoospermic men, which may explain the frequency of 1% sex chromosomal abnormalities observed from prenatal diagnosis after ICSI (Liebers et al., 1995). Using multi-colour FISH for chromosomes 1, 12, X and Y in five men with oligo-, astheno- or teratozoospermia, Moosani et al. (1995)
found a significant increase in disomy, particularly XY disomy. Since we have found comparable results for men with only oligozoospermia, we suggest that such abnormalities may be related to the mechanism responsible for the deficient sperm production, rather than to the mechanisms by which astheno- or teratozoospermia are produced.
The only other study to date to use an X,Y,18 multi-colour FISH assay (Ohashi et al., 2001) found a high frequency of XY disomy in the sperm of severely oligozoospermic men. The patients in that study had sperm counts below 5 x 106/ml, whereas in the present study they ranged from 3.1 x 106/ml to 17.6 x 106/ml, and only three were below 5 x 106/ml. Thus, we demonstrate that the association of oligozoospermia and gonosomal aneuploidy is not confined to severely affected men and seems to correlate with the likelihood of having fertility problems with a sperm concentration below 20 x 106/ml.
One of the main conclusions of Ohashi et al. (2001) was that ICSI may be associated with an increased risk of Klinefelters syndrome as a result of the increased levels of gonosomal disomy in the sperm of patients who would be candidates for such treatment. However, the prevalence of sperm with relatively high levels of DNA strand breaks and defective chromatin packaging in infertile, oligozoospermic patients in the present study suggests that many of the sperm could be non-viable. It is therefore likely that some or all of the aneuploid sperm also have these defects, which might ensure that they could not participate in fertilization. Thus, the increase in gonosomal disomy need not indicate an increased risk of Klinefelters syndrome. We are planning further studies to determine whether a significant proportion of aneuploid sperm do indeed also have SCSA- and/or Comet-type damage. Such work is important to help clarify whether there is any genetic risk posed by the use of ICSI in cases of idiopathic oligozoospermia.
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Acknowledgements |
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Submitted on August 19, 2002; resubmitted on December 24, 2002; accepted on March 4, 2003.