Department of Obstetrics and Gynecology, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan
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Abstract |
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Key words: FISH/ICSI/severe oligozoospermia/sex chromosome/spermatozoa
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Introduction |
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Fluorescence in-situ hybridization (FISH) has permitted the study of aneuploid frequencies in larger numbers of human spermatozoa. This technique has been used in several studies on aneuploidy in spermatozoa from infertile men (Miharu et al., 1994; Moosani et al., 1995
; Guttenbach et al., 1997
; Pang et al., 1999
; Rives et al., 1999
; Vegetti et al., 2000
). It remains a subject of controversy, however, whether the spermatozoa of these men show significantly higher frequencies of aneuploidy. The disparate results of these studies might be related to their investigation of different types of male infertility, i.e. oligo-, astheno-, oligoastheno-, oligoterato-, oligoasthenoterato-, and asthenoteratozoospermia, unexplained infertility and antisperm antibodies. Another factor may be that several of the studies analysed a relatively small number of spermatozoa per individual. Moreover, in several of the studies, the karyotype of infertile men was not analysed; some of the subjects might thus have had an abnormal karyotype, e.g. 46,XY/47,XXY mosaicism, that might have led to a high frequency of XY disomy in spermatozoa. Therefore, in the present study, the relationship between sperm concentration and sex chromosome aneuploidy was the focus. Using three-colour FISH, the sex chromosomal aberrations were compared in a minimum of 10 000 spermatozoa per individual among severe oligozoospermic men (sperm concentration <5x106/ml) who were candidates for ICSI and had a normal karyotype, oligozoospermic men, and normospermic men. The findings of this study should be a useful step in establishing the risk of transmission of chromosomal abnormalities in ICSI.
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Materials and methods |
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Semen collection and analysis
Semen samples were produced by masturbation after 3 days of abstinence. After the ejaculates were allowed to liquefy completely, routine semen parameters (concentration, percentage total motility and volume) were analysed according to standard procedures (World Health Organization, 1992). The samples were provided by 10 severe oligozoospermic men (group S; sperm concentration <5 x106/ml), 10 oligozoospermic men (group O; sperm concentration 520 x106/ml) and seven normospermic men (group N) whose spermatozoa were used in our previous study (Samura et al., 1997
). All of the severe oligozoospermic men and oligozoospermic men were husbands of women undergoing IVF or ICSI. None of them had any history of childhood disease, environmental exposure, radiation exposure or prescription drug usage that could account for their infertility. All seven normospermic men had normal semen parameters and were confirmed to be fertile. All donors gave their informed consent prior to this study.
Slide preparation
The semen samples were washed twice with Hank's balanced salt solution and then fixed with methanol:acetic acid (3:1) for 20 min. After two rinses with fresh fixative, the sperm suspension was dropped onto clean slides and stored at 80°C until used. The slides were thawed at room temperature, and decondensation of the sperm nuclei was performed using the method of Miharu et al. (1994) with minor modifications. Slides were immersed in 30 mmol/l dithiothreitol (DTT; Sigma, St Louis, MO, USA) in a 0.1 mol/l Tris-HCl solution (pH 8.0) for 1 h and subsequently in 25 mmol/l lithium diiodosalicylate (Sigma) with 10 mmol/l DTT in a 0.1 mol/l Tris-HCl solution (pH 8.0) for 3 h. The slides were air-dried followed by ethanol dehydration series.
Chromosome-specific DNA probes
Probes labelled with either biotin or digoxigenin were purchased from Oncor, Inc. These included chromosome 18-satellite (D18Z1), X chromosome
-satellite (DXZ1) and Y chromosome classical satellite (DYZ1). For three-colour study of chromosomes X, Y and 18, a cocktail containing DXZ1 (digoxigenin-labelled), DYZ1 (biotin-labelled) and D18Z1 (biotin plus digoxigenin-labelled) was used.
Denaturation and FlSH
In-situ hybridization and detection were performed according to the manufacturer's instructions. For detection, incubations at 37°C were performed for 60min with fluorescein-labelled avidin (avidin-FITC; Vector Laboratories, Burlingame, CA, USA) and anti- digoxigenin-rhodamine (Boehringer-Mannheim Biochemica, Mannheim, Germany). The signal was amplified with additional rounds of anti-avidin antibody (Vector Laboratories) and an avidin-FITC. Counterstaining was performed with DAPI (Sigma).
Fluorescence microscopy and analysis
Hybridization signals in sperm nuclei were examined using a Nikon Axiophot microscope fitted with a triple-band pass filter (Chroma Technology, Bratteboro, VT, USA). Only slides that had >98% hybridization efficiency were used for this study. Slides were coded and a minimum of 10 000 sperm nuclei per individual was scored in cases of severe oligozoospermia and oligozoospermia, and a minimum of 6000 sperm nuclei per individual in cases of normospermia. Sperm nuclei were scored as having two signals when there were two discrete signals of equal size and intensity separated by at least one diameter of the domain of one signal. Because of the large domain diameter for the Y chromosome probe, cells were scored as having two signals when the signals were separated by at least one-half of a domain diameter. Sperm nuclei with two distinct signals for X and/or Y chromosomes and one yellow signal were scored as sex chromosome disomy. Sperm nuclei with two distinct yellow signals and one signal for an X or Y chromosome were scored as chromosome 18 disomy. Sperm nuclei with two distinct yellow signals and also two signals for X and/or Y chromosomes were scored as diploidy. Certain populations of sperm nuclei were eliminated from scoring: overlapping nuclei, very large nuclei possibly due to over- decondensation or large nuclei that were 1.5 times larger than decondensed normal sperm nuclei and were round in shape (possibly somatic cells). Nuclei without a signal, either nullisomic nuclei for the chromosomes or non-hybridized nuclei resulting from technical failure, have not been included in this study.
Statistical analysis
Data for aneuploid frequencies of each group were compared statistically using the Mann-Whitney U-test. P 0.05 was considered statistically significant.
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Results |
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The mean frequency of 18 disomy was 0.20% (range 0.090.52%) in group S, 0.13% (range 0.080.20%) in group O and 0.17% (range 0.110.23%) in group N. The mean frequency of XX disomy was 0.07% (range 0.04-0.11%) in group S, 0.07% (range 0.030.13%) in group O and 0.11% (range 0.060.19%) in group N. The mean frequency of YY disomy was 0.07% (range 0.040.12%) in group S, 0.08% (range 0.020.15%) in group O and 0.11% (range 0.050.18%) in group N. There were no statistically significant differences among the three groups in the frequencies of either 18, XX or YY disomy.
The mean frequency of total diploidy was 0.49% (range 0.151.43%) in group S, 0.22% (range 0.070.37%) in group O and 0.21% (range 0.120.30%) in group N. There was a significantly higher frequency of total diploidy in group S than in group O (P = 0.026) or group N (P = 0.017). There was no significant difference in the frequency of total diploidy between group O and group N.
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Discussion |
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In assessing the association between semen parameters and aneuploidy rate, Rives et al. (1999) reported that a relationship was observed between aneuploidy rate and sperm concentration, although no relationship was observed between aneuploidy rate and sperm motility or morphology. Vegetti et al. (2000) reported an inverse correlation between aneuploidy rate and total progressive motility and no correlation between aneuploidy rate and abnormal morphology. Taking into account data from the present study and from the two studies mentioned here, sperm concentration seems to be the most important factor affecting aneuploidy rate.
The data presented here show that most severe oligozoospermic men with normal karyotype have a higher incidence for XY disomy in spermatozoa, which has significance for couples who are candidates for ICSI. In fact, Van Opstal et al. (1997) reported that prenatal cytogenetic analysis of 71 fetuses conceived by ICSI resulted in the detection of nine (12.7%) chromosome aberrations. These aberrations included two cases of 47,XXY, which were revealed to be paternally derived by molecular analysis. Devroey (1998) found eight de-novo sex chromosomal aberrations including four cases of 47,XXY (0.4%) in 977 cases of prenatal diagnosis. All of the eight de-novo sex chromosomal aberrations were conceptuses fertilized by spermatozoa from men with oligoasthenoteratozoospermia. More recently, Bonduelle et al. (1999) reported that a slight increase of sex chromosomal aberrations (0.83%), predominantly 47,XXY (0.37%), was found in 1082 cases of prenatal diagnosis after ICSI. Therefore, it should be considered that there can be an increased risk of 47,XXY conceptuses, especially when infertile men with severe oligozoospermia undergo ICSI.
Severe oligozoospermia is accompanied with increased frequency for chromosomal abnormalities, especially for XY disomy, in spermatozoa. To date, two possible causes have been posited: Klinefelter mosaicism and chromosome paring abnormality in the first meiotic chromosomes. A case of a 46,XY/47,XXY male with oligozoospermia was reported (Chevret et al., 1996). The case showed a significantly increased frequency of XY disomy (2.09%) in spermatozoa compared to controls (0.36%), yet the frequencies of XX disomy, YY disomy and disomy for chromosome 1 were close to those obtained from controls. Other investigators have also reported that spermatozoa in Klinefelter mosaicism showed an increase in the frequency of XY disomy (Cozzi et al., 1994
; Martini et al., 1996
; Bielanska et al., 2000
; Morel et al., 2000
; Rives et al., 2000
). Thus, Klinefelter mosaicism is considered to be one of the possible causes leading to both high frequency of XY disomy and oligozoospermia. However, the cytogenetic analysis performed in the current study with the FISH technique showed a normal XY constitution karyotype in 200 interphases of lymphocytes from the peripheral blood of severe oligozoospermic men, although a germinal mosaicism could not be excluded. On the other hand, a generalized pairing abnormality in the meiotic chromosomes has been thought to contribute to both non-disjunction and oligozoospermia (Martin, 1996
). In particular, the sex chromosome bivalent is susceptible to pairing abnormalities because there is generally only one cross-over in the pseudoautosomal region (Martin et al., 1996). In an electron microscopic study of spermatocytes, it was postulated that XY synapsis was lower in infertile men than controls (Speed and Chandley, 1990
). In mouse spermatocytes, the paring of X and Y-chromosomes seems to be a prerequisite for the completion of meiosis and for the formation of spermatozoa (Rapp et al., 1977
). Moreover, in a cytogenetic study of spermatozoa in the XXY mouse it was found that spermatozoa from the XXY mouse had a markedly higher incidence of XY disomy, resulting from non-disjunction of the sex chromosomes in the first meiosis (Mroz et al., 1998
). They suggested that the meiotic abnormalities observed in spermatozoa even from XXY males are attributable to segregation errors in XY germ cells, rather than to the survival of XXY germ cells in the testis, at least in the mouse. This suggestion was based on their studies that almost all spermatogonial cells observed in the testis of the XXY mouse had an XY sex chromosome complement like the testis of normal males. In the current study, severe oligozoospermic men showed a significantly higher frequency of XY disomy than either oligozoospermic men or normospermic men (P = 0.004 and P = 0.019 respectively), whereas the frequencies for XX disomy and YY disomy were almost the same between these two groups. The data indicate that non-disjunction of the sex chromosomes in the first rather than the second meiosis could cause an increase of the frequency for XY disomy in some cells capable of completing spermatogenesis and cause severe oligozoospermia in other cells incapable of completing spermatogenesis.
In summary, the results of the present study indicated that men with severe oligozoospermia tended to be at greater risk for XY disomy in their spermatozoa, due to non-disjunction of first meiosis. Such an increase in XY disomy could lead to a slight increase in 47,XXY concepti after ICSI. Therefore, when men with low sperm concentrations of 5x106/ml undergo ICSI, even if they have a normal karyotype, it is important to inform them and their partners of the possible risks of aneuploidy in their fetus.
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Acknowledgements |
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Notes |
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References |
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Submitted on September 13, 2000; accepted on January 4, 2001.