Molecular Human Reproduction, Vol. 6, No. 7, 665-668,
July 2000
© 2000 European Society of Human Reproduction and Embryology
Genetic diagnosis |
Fluorescence in-situ hybridization analysis of chromosomal constitution in spermatozoa from a mosaic 47,XYY/46,XY male
1 Division of Genetics, New England Medical Center, Boston and 2 Fertility Center of New England, Reading, MA, USA
Abstract
Sex-chromosome mosaicism in spermatozoa from a mosaic 47,XYY[20%]/46,XY[80%] male with fertility problems was assessed using triple-probe fluorescence in-situ hybridization (FISH) studies. Chromosome-specific probes for X, Y and 18 were used, and the possible outcomes were deduced. In normal haploid spermatozoa of the patient and a normal 46,XY male control, the X:Y ratio was close to 1:1. There was a significant difference in the total incidence of karyotypically abnormal spermatozoa between the patient and the 46,XY male control (2.31% versus 1.46%, P < 0.0001). The incidence of some types of disomic spermatozoa X+Y+18 (24,XY) and X+18+18 (24,X, +18), or diploid X+Y+18+18 (46,XY) spermatozoa was significantly increased in the patient's semen sample. There was, however, no significant difference in the incidence of disomic Y+Y+18 (24,YY) spermatozoa. Because the majority of the patient's spermatozoa was karyotypically normal, the aetiology of his fertility problems was unclear. These results add to the growing body of information regarding chromosome abnormalities in spermatozoa from men who are mosaic for sex chromosome abnormalities. In these men, FISH analysis of spermatozoa may be warranted to determine the relative percentages of abnormal cells, and to determine if in-vitro fertilization with preimplantation genetic diagnosis may increase the likelihood of a successful pregnancy.
47,XYY/46,XY male/chromosome analysis/FISH/mosaicism
Introduction
47,XYY is a common chromosomal disorder, affecting ~1:1000 live-born males (Autio-Harmainen et al., 1980
). The majority of 47,XYY males are phenotypically normal, but a pattern of variable clinical features has been appreciated, including tall stature, large teeth, and occasionally other physical findings. The majority of 47,XYY males are fertile and have chromosomally normal offspring. However, an increased risk for offspring with chromosomal abnormalities as well as miscarriage and perinatal death has been suggested for these men (Jones, 1997). Although an early report did not reveal a significant difference in human sperm chromosomal anomalies between non-mosaic 47,XYY male and normal controls using IVF of hamster oocytes with human spermatozoa (Benet and Martin, 1988), more recent studies using the fluorescence in-situ hybridization (FISH) technique have suggested that non-mosaic XYY males have an increased frequency of sex chromosome disomic spermatozoa (Mercier et al., 1996; Blanco et al., 1997; Chevret et al., 1997; Martin et al., 1999; Morel et al., 1999).
To date, only two cases of FISH analysis in spermatozoa from males with 47,XYY/46,XY mosaicism have been reported in the literature (Lim et al., 1999). These authors demonstrated an increased incidence of sperm disomy XY in a patient with 90% mosaicism in the blood, and in a patient with 19% mosaicism in the blood. Disomy YY was shown only in the spermatozoa from the patient with 90% mosaicism. The present study was designed to analyse the sex chromosome constitution of spermatozoa in our patient, a mosaic 47,XYY/46,XY male with infertility, and to see if his fertility problems were related to an increase in the number of chromosomally abnormal spermatozoa. In doing so, we have added data to the growing body of information regarding sex chromosome abnormalities in spermatozoa from men who are mosaic for sex chromosome aneuploidy (Bielanska et al., 2000; Rives et al., 2000).
Materials and methods
Case report
The study subject was a 31 year old healthy male with infertility. The sperm parameters of the subject were normal (motility 60%; sperm concentration 70x106/ml; morphology 17% normal by strict criteria). He had been married for 2 years. The couple had twice had intrauterine insemination and both pregnancies were spontaneously aborted at very early gestational ages. Due to their fertility problems, the couple had chromosome analyses performed on their peripheral blood. The study subject had a mosaic karyotype of 47,XYY[20%]/46,XY[80%] and his wife had a normal karyotype (46,XX). He was 6 feet tall, non-dysmorphic, and had normal-sized teeth. The control subject used in this study was a 30 year old healthy male with a normal peripheral blood chromosome karyotype of 46,XY.
Spermatozoa preparation
The semen samples were collected into plastic tubes from both the patient and normal control, and were processed immediately. The semen was diluted 1:2 with Ca2+–Mg2+-free Dulbecco's phosphate-buffered saline (PBS) (Sigma Chemical Co., St Louis, MO, USA). Diluted semen was washed twice at 400 g for 10 min at room temperature. The sperm suspension was thin-smeared on the slides. The slides were air-dried overnight and fixed with 3:1 methanol–acetic acid at room temperature for 20 min.
Fluorescence in-situ hybridization (FISH) and analysis
The spermatozoa were decondensed according to previously reported methods with minor modifications (Samura et al., 1997). The slides containing spermatozoa were immersed in 25 mmol/l dithiothreitol (DTT; Sigma) for 30 min. Slides containing spermatozoa were then rinsed in 2xsaline sodium citrate (SSC; Gibco BRL, Grand Island, NY, USA), dehydrated in an ethanol series (70%, 80%, 97%), and air-dried. The slides were then hybridized using directly labelled probes for chromosome 18 (D18Z1) with fluorescein-dUTP (green colour), chromosome Y (PHY10) with Cy3-dCTP (red colour), and chromosome X (DXZ1) with both fluorescein-dUTP and Cy3-dCTP (yellow colour) according to previously reported methods (Zhen et al., 1998). Only spermatozoa with an attached tail were scored to avoid the inadvertent inclusion of other cell types. Overlapping nuclei were not included. Sperm nuclei were scored as having two fluorescent signals if they were of equal size and intensity and were separated by at least the diameter of the domain of one signal. The frequencies of the mosaic haploid, diploid, hyperploid, disomic and monosomic karyotypes in the semen samples were counted. The potential karyotypes of offspring, assuming fertilization of a normal haploid oocyte, were deduced. The hybridization efficiencies of the probes in both subject and control were >99% in our experiments. A total of 10 000 spermatozoa was scored to assess the percentage mosaicism in both the subject and control semen samples. Data were analysed using the 
2-square and Fisher exact tests where appropriate. P < 0.05 was considered to be statistically significant.
Results
Figure 1 shows sperm nuclei of the subject processed with three-colour FISH. The frequencies of different chromosome combinations in spermatozoa were counted, their karyotypes were deduced, and a statistical analysis was performed (Table I). In normal haploid spermatozoa of the subject and control, the X:Y ratio was observed to be close to 1:1. There was a significant difference in the incidence of total karyotypically abnormal spermatozoa between the subject and control (2.31% versus 1.46%, P < 0.0001). The incidence of some disomic X+Y+18 (24,XY) and X+18+18 (24,X +18) spermatozoa, or diploid X+Y+18+18 (46,XY) spermatozoa was significantly increased in the subject. There were no significant differences in the incidence of disomic Y+Y+18 (24,YY) spermatozoa or in the incidence of monosomic spermatozoa between the subject and control. The incidence of diploid Y+Y+18+18 (46,YY) spermatozoa was significantly lower in the subject than in the control.
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Discussion
The main advantage of three-probe FISH (X, Y and autosome) over two-probe FISH (X and Y) is that it is possible to distinguish sex chromosome disomy from diploidy and thus to calculate the frequencies of the different populations of sperm cells observed (Williams et al., 1993). Two previous studies used only a two-colour FISH technique to analyse the frequency of sex chromosome abnormalities in spermatozoa from a 47,XYY male (Han et al., 1994; Mercier et al., 1996). In the present study the spermatozoa samples from both the patient and the normal control were analysed using three-colour FISH with probes for chromosomes X, Y and 18.
Our results demonstrated that there was an increased incidence of disomic X+Y+18 (24,XY) spermatozoa in a mosaic 47,XYY/46,XY male. However, there was no significant difference in the incidence of disomic Y+Y+18 (24,YY) and X+X+18 (24,XX) spermatozoa. These findings are consistent with a previous report of semen analysis in a low level (19%) mosaic 47,XYY/46,XY male (Lim et al., 1999). These authors reported that the incidence of disomy XY in spermatozoa was significantly elevated in both low level and high level (90%) mosaic 47,XYY/46, XY males compared with the controls (0.23 and 1.02%, respectively, versus 0.10%). Disomy YY was increased only in the high level mosaic male. In a non-mosaic 47,XYY case analysed by three-colour FISH, two studies demonstrated that the number of spermatozoa with XY disomy was increased in semen samples (Blanco et al., 1997; Martin et al., 1999). The 47,XYY/46,XY mosaicism might also result in karyotypically abnormal spermatozoa, especially with regard to XY disomy.
The percentage of diploid X+Y+18+18 (46, XY) cells in our patient was also increased. This finding is similar to that of the previous case with a high percentage of mosaicism, but not in the case with a low percentage of mosaicism (Lim et al., 1999). One three-colour FISH study of a non-mosaic 47,XYY male reported that the percentage of diploid XY spermatozoa was increased in 47,XYY men, as compared with controls (Chevret et al., 1997); other studies did not note an increase (Blanco et al., 1997; Martin et al., 1999). Thus, it appears that there is clinical variation in the percentage of diploid XY spermatozoa in 47,XYY males. Regarding the rate of monosomic spermatozoa, there was no significant difference between the control and the subject. This suggests that mosaic 47,XYY/46,XY males do not have a higher rate of monosomic spermatozoa.
In the present case, we noted a difference in the percentage of cells with an abnormal karyotype between peripheral blood (20%) and spermatozoa (2.31%). This difference might result from a relative disadvantage of spermatozoa with an abnormal karyotype during spermatogenesis.
In conclusion, this report adds to the growing literature on analysis of sex chromosome mosaicism in spermatozoa in mosaic 47,XYY/46,XY males with fertility problems. An increased percentage of cells with an abnormal karyotype was observed as compared with a normal control. The clinical significance of the increased frequency of abnormal mosaic spermatozoa, and its relationship to the fertility problems of the patient and his wife, are unclear, because the majority of his spermatozoa were normal and haploid. However, an assessment of the frequencies of abnormal spermatozoa in men with sex chromosome aneuploidy may be warranted, particularly if the couple will go on to IVF, in which preimplantation genetic analysis may increase the likelihood of a successful outcome of pregnancy.
Acknowledgments
This study was supported by a grant to the Molecular Cytogenetics Core Facility from New England Medical Center.
Notes
3 To whom correspondence should be addressed at: Division of Genetics, Department of Pediatrics, New England Medical Center, Tufts University School of Medicine, NEMC #394, 750 Washington Street, Boston, MA 02111, USA. E-mail: DBianchi{at}Lifespan.org 
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Submitted on January 31, 2000; accepted on May 4, 2000.
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