Molecular Human Reproduction, Vol. 6, No. 2, 107-112,
February 2000
© 2000 European Society of Human Reproduction and Embryology
Genetic diagnosis |
Assessment of sex chromosome aneuploidy in sperm nuclei from 47,XXY and 46,XY/47,XXY males: comparison with fertile and infertile males with normal karyotype
Reproductive Biology Laboratory, Rouen University Hospital, Rouen, France
Abstract
Sex chromosome aneuploidy was assessed in spermatozoa from a 47,XXY male and a 46,XY/47,XXY male using three colour fluorescence in-situ hybridization (FISH) and compared with two control groups. The first group included subjects of proven fertility and the second infertile males with normal constitutional karyotype. The frequencies of XX and YY disomic, XY hyperhaploid and diploid spermatozoa were significantly increased in the 47,XXY male compared to subjects from the two control groups (P < 0.0001). For the 46,XY/47,XXY sample, the same results were observed, except that the incidence of YY disomic spermatozoa did not differ significantly from the rate obtained in infertile patients. The frequency of sex chromosome aneuploidy did not differ significantly between the 47,XXY and the 46,XY/47,XXY males, except for XX disomic sperm nuclei which was higher in the 47,XXY patient. The frequency of chromosome 12 disomy was also increased in the two XXY individuals (0.42 and 0.49% respectively; P < 0.0001). The meiotic abnormalities observed in the two XXY patients arose through segregation errors in XY germ cells. The increased number of meiotic non-disjunctions observed in the germ cells of infertile males may be a common feature of the deficient oligo- or azoospermic testis. Patients with Klinefelter's syndrome with oligozoospermia have an increased risk of both sex chromosome and autosome aneuploidy in their progeny.
infertility/in-situ hybridization/sex chromosomes/spermatozoa/XXY males
Introduction
The presence of a chromosome abnormality in the constitutional karyotype could interfere with normal spermatogenesis. Chromosome abnormalities are more frequently observed in the population of azoo- or oligozoospermic males than in the general population (Chandley, 1984
; Rivas et al., 1987). Sex chromosome aneuploidies are the most common abnormalities found, predominantly 47,XXY and 47,XYY karyotypes.
Males with Klinefelter's syndrome may have a mosaic 47,XXY or a 46,XY/47,XXY constitutional karyotype and varying degrees of spermatogenic failure. Testicular histological studies may reveal areas of atrophy and hyalinization of the seminiferous tubules as well as some areas with tubules of normal appearance with a reduced number of mature spermatozoa (Fergusson-Smith et al., 1957; Futterweit, 1967; Arce et al., 1980; Rajendra, 1981). Spermatozoa have occasionally been identified in the seminal ejaculate in some patients with Klinefelter's syndrome (Futterweit, 1967). The severity of the alterations of sperm parameters seems to be related to the gonadal chromosome constitution, and more specifically to the presence or absence of a 46,XY germ cell population (Arce et al., 1980). Rare observations of spontaneous fertility (Kaplan et al., 1963; Warburg, 1963) and proven paternity (Terzoli et al., 1992) have been reported in patients with Klinefelter's syndrome but the development of recent assisted reproductive procedures offers new hopes of paternity to these patients. Pregnancies (Bourne et al., 1995; Harari et al., 1995; Tournaye et al., 1996; Hinney et al., 1997) and births (Palermo et al., 1998; Reubinoff et al., 1998; Ron-El, 1999) have now been reported after intracytoplasmic sperm injection (ICSI) using ejaculated or testicular spermatozoa in mosaic and non-mosaic Klinefelter's patients.
Meiotic studies in these patients usually revealed meiotic arrest at the first spermatocyte level with exceptional metaphase I figures (Luciani et al., 1970; Rajendra, 1981). Other authors have suggested that only the 46,XY germ cells in 46,XY/47,XXY patients are able to go through meiosis and produce mature spermatozoa in the ejaculate (Luciani et al., 1978; Vidal et al., 1984). Only a few reports have evaluated the meiotic products of mosaic and non-mosaic Klinefelter's syndrome patients by direct analysis of ejaculated spermatozoa. The different studies performed by sperm karyotyping (Cozzi et al., 1994) or by fluorescence in-situ hybridization (FISH) analysis (Chevret et al., 1996; Martini et al., 1996; Guttenbach et al., 1997; Estop et al., 1998; Foresta et al., 1998; Kruse et al., 1998) have shown an increased frequency of sex chromosome disomy. These authors concluded that 47,XXY germ cells are able to achieve meiosis and to produce an increased number of aneuploid spermatozoa. However, these studies remain limited and further investigations are required to confirm this hypothesis.
In the present study, we evaluated the potential risk of sex chromosome aneuploidy for the progeny of a 46,XY/47,XXY male and a non-mosaic 47,XXY male by three colour FISH analysis of their spermatozoa. We attempted to determine: (i) whether this potential risk was higher than in fertile and infertile males with a normal constitutional karyotype; (ii) whether this risk was conditioned by the presence or absence of constitutional mosaicism in the sex chromosomes; and (iii) whether the meiotic abnormalities observed in spermatozoa are the consequences of normal meiosis of XXY survival germ cells or of segregation errors in XY germ cells.
Materials and methods
Subjects
Patient 1 was a 26 year old man, who presented himself at our assisted reproduction centre for ICSI due to severe oligoasthenozoospermia. Analysis of 100 metaphases from blood lymphocytes revealed a 47,XXY constitution in all cells. FISH analysis using 
-satellite centromeric probes specific for chromosomes X and Y (DXZ1 and DXZ3, Oncor®, Illkrich, France) was performed on peripheral blood cells and fibroblasts (100 nuclei analysed) and confirmed the homogenous 47,XXY constitution.
Patient 2 was a 41 year old man who was referred to our assisted reproduction centre 5 years ago for sperm donation because of a 10 year period of infertility related to asthenoteratozoospermia. A previous diagnosis of 46,XY/47,XXY mosaicism was carried out by lymphocyte karyotype. Analysis of 100 metaphases revealed five cells with a 47,XXY constitution.
Five healthy probands of proven fertility, aged 28–41 years, were included in the study as control group A. Their sperm parameters were normal and they had no history of chronic illness and professional or environmental exposures. Their constitutional karyotypes were normal 46,XY.
In addition, five males, aged 31–39 years, who were referred to our centre for exploration and treatment of a male factor infertility were included as control group B. Semen analysis spaced over 3 months revealed severe alterations of sperm parameters. Their constitutional karyotypes were also normal 46,XY.
Semen analysis was performed according to the World Health Organization (WHO, 1993) guidelines. The study was carried out on a single ejaculate. All participants gave their informed consent to participating in the study.
Spermatozoa nuclei preparation
Semen samples were collected after 3 or 5 days of sexual abstinence and were liquefied for 20 min at 37°C. A complete semen analysis was performed for each sample using WHO guidelines (1993) (Table I). The spermatozoa were washed three times by centrifugation for 10 min at 2000 rpm in phosphate-buffered saline (PBS). The pellets were resuspended in fresh fixative (3:1 methanol:acetic acid precooled to –20°C) and fixed 60 min at 4°C. The sperm suspensions were spread onto clean glass slides (SuperFrost/Plus, Menzel-Gläser®, Germany). The slides were air-dried and stored at –20°C.
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DNA probes
The probes used in this study were
-satellite centromeric probes purchased from Vysis® (Voisins Le Bretonneux, France) and directly labelled with Spectrum OrangeTM (chromosome X, CEP X; chromosome Y, CEP Y) or Spectrum GreenTM (chromosome X, CEP X; chromosome 12, CEP 12). The specificity of the different probes was verified by hybridization on lymphocyte metaphase chromosome preparations. Three colour FISH was performed, the third colour was obtained by mixing an equal volume of CEP X labelled with Spectrum OrangeTM and CEP X labelled with Spectrum GreenTM.
Fluorescence in-situ hybridization
The slides were thawed at room temperature for 5 min and washed in 2x sodium chloride/sodium citrate (SSC) for 5 min. Sperm nuclear partial decondensation and simultaneous denaturation was carried out by incubation for 10 min in 3 mol/l NaOH. The decondensation was controlled under a phase contrast microscope and stopped when the fixed spermatozoa were decondensed to approximately twice their original size. The slides were dehydrated by incubation in an ethanol series (70, 95 and 100%) and dried in an oven at 37°C for 5 min.
The directly labelled probes were incubated 5 min at 42°C and diluted in a hybmix buffer (Hybrisol VI, Oncor®, Illkrich, France). The probes were denatured for 5 min at 72°C and plunged into ice water before the hybridization procedure.
The hybridization mixture (15 µl) was applied to each slide under a 24x60 mm coverslip and the slides were then sealed with glass coverslip sealant (Oncor®, Illkrich, France). Hybridization was carried out for 4 h at 42°C in a moist chamber.
After hybridization, the slides were washed once in a solution of SSC 0.04x/0.3% NP 40 for 2 min at 72°C and once in a solution of SSC 2x/0.1% NP 40 at room temperature for 1 min. After the final wash, slides were air-dried in the dark.
The slides were counterstained with a solution of 4', 6-diamidino-2-phenylindole (DAPI; Cambio Biosys®,Compiègne, France) diluted in antifade mounting medium (Vectashield®, Vector, Biosys, Compiègne, France) at a final concentration of 0.5 µg/ml.
Scoring criteria, data collection and statistical analysis
The slides were examined with an epifluorescence microscope (DMRD®, Leica, Germany) at a magnification of x1000. An fluorescent isothiocyanate (FITC)/rhodamine/DAPI triple band-pass filter set was used for the count and a single band-pass filter set (DAPI or FITC or rhodamine) to differentiate a spermatozoon from a somatic cell. Images were captured at x1000 magnification with a digital imaging system (Mac probe® version 3.3; Perceptive Scientific International Ltd, Chester, England). At least 10 000 spermatozoa were scored per subject. Only slides with >95% hybridization efficiency were included in the count. Red, green and yellow spots respectively detected chromosome Y, chromosome 12 and chromosome X. A spermatozoon was considered to be haploid, if two similar fluorescent spots with comparable size and intensity, but with two different colours (red and green or yellow and green) were separated by at least one spot diameter. A sperm nucleus was considered to be disomic if two similar fluorescent spots of the same colour, with the above mentioned criteria, were observed. A spermatozoon was diploid if four fluorescent spots of different colours (one red, one yellow and two green) were present. A sperm nucleus was considered to be hyperhaploid XY when three fluorescent signals of different colours were observed (red, green and yellow). Diffuse fluorescent signals and overlapping nuclei were excluded from the count because the fluorescent signals could not be clearly distinguished. Nullisomic cells were not included in the count because they could be the result of hybridization failure.
Statistical analysis
The 
2 test was used to compare the frequency of each hybridization pattern obtained for the 47,XXY and the 46,XY/47,XXY male with those observed in the control groups A and B. P < 0.05 was considered to be significant. The statistical analysis was carried out with the logicial StatView® for Windows 95 (Abacus Concepts Inc, Berkeley, CA, USA). In the control groups, the values are noted as mean ± SE.
Results
A total of 131 481 sperm nuclei was scored, 10 123 for the 47,XXY male, 20 814 for the 46,XY/47,XXY male, 50 565 for the control group A and 49 979 for the control group B (Table II).
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Control populations A and B
The rate of YY- and XY-bearing spermatozoa were higher in the group of infertile males than the rate obtained in the group of fertile males (P < 0.0001 and P = 0.001 respectively). The frequency of XX disomic spermatozoa did not significantly vary between the two control groups (P = 0.37). The mean frequency of diploidy was moderately increased in infertile group but with no significant statistical difference (P = 0.14).
The mean frequency of chromosome 12 disomy was significantly increased in infertile males compared to the control group A (P < 0.0001).
47,XXY sample
The proportion of X- and Y-bearing spermatozoa was 49.60 and 48.33% respectively and did not significantly differ from the 1:1 ratio expected. The frequency of disomic sperm was 0.45% for the chromosome X, 0.37% for the chromosome Y and 0.42% for the chromosome 12. The frequency of XY hyperhaploid sperm nuclei was 0.54% (Figure 1). The frequency of diploid spermatozoa was 0.23%. The frequency of XX, YY and 1212 disomic, XY hyperhaploid and diploid spermatozoa was significantly increased in the 47,XXY male as compared to control group A (fertile males) (P < 0.0001).
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The same data were observed when the frequencies if sex chromosome and chromosome 12 disomy were compared with those obtained in the control group B (infertile males) with P < 0.0001 for XX, YY and 1212 disomic and XY hyperhaploid spermatozoa and P < 0.0046 for the diploidy frequency.
46,XY/47,XXY sample
The rate of X- and Y-bearing spermatozoa (1.01) did not significantly differ from the 1:1 expected ratio (49.68 and 48.90% respectively). Whatever control population was tested, the frequency of XX disomic, XY hyperhaploid and diploid spermatozoa in the tested sample was also increased (XX disomy: P = 0.001 and P = 0.016 for control groups A and B respectively; XY disomy and diploidy: P < 0.0001 for control groups A and B). The same data was observed for the frequency of chromosome 12 disomy (P < 0.0001 for control groups A and B). However, the difference in the percentage of YY disomic spermatozoa between the tested sample and the control group B was not significant (P = 0.18).
The frequency of XY hyperhaploid and diploid sperm cells were close to those observed in the non-mosaic 47,XXY sample (P = 0.47 and P = 0.07 respectively), whereas the frequency of XX and YY disomic spermatozoa was significantly increased in the 47,XXY sample (P = 0.002 and P = 0.011 respectively).
The frequency of chromosome 12 disomy did not significantly vary between the two individuals with Klinefelter's syndrome (P = 0.41).
Discussion
The incidence of sex chromosome aneuploidy was estimated, in our study, in sperm nuclei of two patients with Klinefelter's syndrome: the first one had an homogenous 47,XXY constitutional karyotype and the second one a mosaic 46,XY/47,XXY constitution. To our knowledge, only a limited number of reports have analysed sex chromosome non-disjunction in sperm nuclei from patients with Klinefelter's syndrome. These studies were generally performed in mosaic 46,XY/47,XXY patients (Cozzi et al. 1994; Chevret et al., 1996; Martini et al., 1996; Araki et al., 1997; Lim et al., 1999). Only three studies have previously investigated spermatozoa from non-mosaic 47,XXY patients (Guttenbach et al., 1997; Estop et al., 1998; Foresta et al., 1998).
The clinical phenotype and the altered sperm parameters could be directly related to the presence of normal 46,XY cells. Cozzi et al. (1994) investigated a patient with a mosaicism of 60% 46,XY and 40% 47,XXY in lymphocytes who had normal semen parameters. The patient explored by Chevret et al. (1996) had 90% 46,XY cells and 10% 47,XXY cells in lymphocyte karyotype but the sperm parameters indicated oligoasthenoteratozoospermia with a sperm count of 9.6x106/ml. In a 45 year old infertile man, Martini et al. (1996) detected five cells with an XXY constitution after FISH analysis in peripheral blood cells and the semen analysis revealed oligoasthenozoospermia with a sperm count of 11.8x106/ml and a motility of 1%. The two non-mosaic patients respectively investigated by Guttenbach et al. (1997) and Estop et al. (1998) presented with severe oligoasthenozoospermia and a sperm count of <1x106/ml. In comparison, our mosaic patient, with 5% of 47,XXY cells, had a normal sperm count and asthenoteratozoospermia. Our homogenous patient appeared to be more affected as he presented with severe oligoasthenoteratozoospermia. The degree of the somatic mosaicism has a variable effect on altered sperm parameters and on the incidence of aneuploid spermatozoa, including diploid spermatozoa (Lim et al., 1999). In our study, this incidence did not vary significantly between the mosaic and non-mosaic Klinefelter males (1.42 and 1.6% respectively; P = 0.32). However, the degree of gonadal mosaicism could be an interesting factor that may directly interfere with the normal process of meiosis.
There are two possible explanations for the increase of sex chromosome aneuploidy in spermatozoa of Klinefelter individuals: (i) XXY germ cells are able to go through meiosis and produce both normal and aneuploid spermatozoa; or (ii) XY germ cells are present but deficiencies in the XXY testis are responsible for meiotic non-disjunction. The meiotic segregation of the sex chromosomes in our 47,XXY and 46,XY/47,XXY patients produced an increased number of 24,XY spermatozoa compared with the rate observed in the control group A (fertile males). As regards to previous pachytene stage studies performed in mosaic 46,XY/47,XXY males, only normal 46,XY germ cells were able to initiate meiosis and complete spermatogenesis (Kjessler et al., 1966; Luciani et al., 1970; Vidal et al., 1984). The 24,XY sperm cells may also result from first meiotic sex chromosome non-disjunction in the normal 46,XY germ cell line. If this hypothesis is correct, the number of hypohaploid 22,–X,–Y complements should have been present in the same proportion. These complements were not detected in our two patients because in our scoring criteria, the absence of one hybridization signal could be due to the failure of the hybridization procedure. In fact, the rate of 24,XY spermatozoa could not only have been the consequence of non-disjunction of 46,XY cells at the first meiotic division but also from the regular meiosis of 47,XXY germ cells after a preferential pairing of the sex chromosomes (XX bivalent and Y univalent). Chevret et al. (1996) suggested that the other possibility of pairing between sex chromosomes, i.e. XY bivalent and X univalent, could lead to primary spermatocyte death and consequently to a premature arrest of the spermatogenesis. The hypothesis of a preferential pairing between homologous sex chromosomes has previously been suggested in other reported studies using sperm karyotypes (Cozzi et al., 1994) or FISH (Chevret et al., 1996; Guttenbach et al., 1997). Foresta et al. (1998) reported major increases in both XY and XX disomy and a 2:1 ratio of X- to Y-bearing spermatozoa in two Klinefelter individuals and these data are consistent with the normal meiosis of XXY germ cells. In our study, we did not observe any difference in the percentage of X- and Y-bearing spermatozoa. The capability of the 47,XXY spermatocytes to achieve meiosis appears also to be related to the number of 46,XY spermatocytes present in the germ cell line population (Sarkar and Marimuthu, 1983). We suggest that our homogenous 47,XXY male may have a mosaicism confined to testicular tissue. The 46,XY germ cell line is probably the most numerous because the population of haploid spermatozoa (98.4%) was the most frequent.
The increased frequency of YY and XX disomic sperm nuclei observed in our two patients, compared with the group of fertile males, could be the consequence of 46,XY spermatocyte non-disjunction occurring at the second meiotic division because similar rates were obtained regardless of whether the patients with Klinefelter's syndrome were mosaic or non-mosaic (P = 0.348 and P = 0.976 respectively). Guttenbach et al. (1997) did not observe the same findings in an homogenous 47,XXY patient and they estimated the incidence of XX disomic spermatozoa to be 1.36% which was significantly higher than the rate of YY disomic sperm nuclei (0.09%). Mroz et al. (1999) observed a significant increase in sex chromosome disomy and other numerical abnormalities in spermatozoa from XXY mice. They concluded that meiotic abnormalities observed in spermatozoa from XXY males are attributable to segregation errors in XY germ cells, rather than to the survival of XXY germ cells in the testis. Our two patients with Klinefelter's syndrome also had an increased incidence of disomy 12 and diploidy frequency but no alteration in the proportion of X/Y ratio among euploid spermatozoa. We postulate that numerical chromosome abnormalities observed in the two individuals with Klinefelter's syndrome are compatible with the `testicular environment' model of Mroz et al. (Mroz et al., 1999).
In the group of infertile males, the rate of XY and YY sperm nuclei was significantly higher than in fertile males (P < 0.0001 and P = 0.001 respectively). Our results suggest that non-disjunction for chromosome X and Y can occur, in patients with poor semen quality parameters, at the first meiotic division and also for chromosome Y at the second meiotic division between its sister chromatids. However, with infertile men, the possibility of sex chromosome testicular mosaicism, which is not detected by standard lymphocyte karyotyping, cannot be excluded. Although the incidence of sex chromosome aneuploidy was significantly higher in the sperm nuclei of mosaic and non-mosaic patients with Klinefelter's syndrome than in control group B (infertile males), this increase was smaller than the one observed with the fertile subjects in group A (5.6 times higher with control group A versus 2.4 times higher with control group B). The risk of sex chromosome abnormalities for the progeny of patients with Klinefelter's syndrome, when spermatozoa are isolated for ICSI, is potentially enhanced. However, this risk is close to the potential risk of infertile males with oligoasthenoteratozoospermia and normal constitutional karyotype. In a previous study, we evaluated the frequency of sperm nuclei disomy for chromosomes 1, 13, 14, 18, 21, 22, X and Y in 50 infertile males with a normal constitutional kayotype (Rives et al., 1999). We demonstrated that during spermatogenesis, males with sperm parameter alterations had a decreased frequency of meiotic pairing and crossing over that could arrest spermatogenesis at the meiotic stage and/or increase meiotic non-disjunction. Meiotic arrest may be responsible for oligozoospermia while non-disjunction may cause aneuploidy in mature male gametes. These different results suggest that the increased incidence of meiotic errors observed in germ cells of infertile males are probably a common feature of deficient oligo- or azoospermic testis.
Studies based on prenatal diagnosis in ICSI pregnancies indicated an increased risk of sex chromosome abnormalities (Bonduelle et al., 1995, 1998). The risk did not originate only from infertile males with abnormal constitutional karyotypes. (i) Moosani et al. identified an elevated frequency of sex chromosomal abnormalities in spermatozoa from a patient who fathered a pregnancy with a 47,XXY karyotype detected after prenatal diagnosis (Moosani et al., 1999). (ii) Blanco et al. observed in two patients who fathered two individuals with Down's syndrome of paternal origin a significant increase of chromosome 21, X and Y disomy (Blanco et al., 1998). Normal fertilization and embryo development, pregnancies and births have been achieved in mosaic and non-mosaic Klinefelter's syndrome patients after ICSI using testicular extracted spermatozoa (Bourne et al., 1995; Tournaye et al., 1996; Palermo et al., 1998; Ron-El, 1999) or ejaculated spermatozoa (Bourne et al., 1997; Hinney et al., 1997). An estimation of sex chromosome aneuploidy in sperm nuclei of mosaic and non-mosaic Klinefelter patients could be useful firstly to evaluate the risk of chromosome abnormality for their progeny before entering an ICSI programme, secondly to inform the patients and thirdly, depending on the potential risk, to propose preimplantation or prenatal genetic diagnosis.
Acknowledgments
The authors thank R.Medeiros for his editorial assistance. The experiments performed in this study comply with the current French legislation.
Notes
1 To whom correspondence should be addressed
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Submitted on April 12, 1999; accepted on November 5, 1999.
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