1 Institute of Reproductive Medicine and 2 Institute of Human Genetics, Westphalian Wilhelms-University, D-48149 Münster, Germany
3 To whom correspondence should be addressed at: Institute of Reproductive Medicine of the University, Domagkstr. 11, D-48149 Münster, Germany. Email: eberhard.nieschl{at}ukmuenster.de
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Abstract |
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Key words: aneuploidy/FISH/hormone levels/lymphocytes/male infertility
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Introduction |
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In andrology, FISH of interphase sperm nuclei has been used successfully to determine aneuploidy rates (Luetjens et al., 2002) and has demonstrated that the aneuploidy number in lymphocytes was positively correlated with the number of aneuploid sperm (Rubes et al., 2002
). The detection of a possible low grade mosaicism in peripheral lymphocytes in Klinefelter patients by FISH implies that Klinefelter patients may have germ cells with normal 46,XY content in their testis (Westlander et al., 2001
).
This study was designed to analyse low grade mosaicism of gonosomes in Klinefelter (47,XXY), Klinefelter-like patients (46,XY) and infertile men with otherwise normal phenotype and karyotype (46,XY) by FISH on interphase and metaphase lymphocytes otherwise possibly overlooked by conventional karyotyping. Patients diagnosed were included after conventional cytogenetics was performed. In addition, the study also correlated hormone and clinical parameters with results of the FISH chromosomal status.
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Subjects and methods |
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Three-colour FISH procedure
The FISH procedure was performed according to the manufacturer's instructions and the optimization techniques published by Yan et al. (2000). We used three different DNA probes (Vysis Inc., Downers Grove, IL), tagging chromosomes X, Y and 9, each labelled with different fluorochromes (CEP-X, SpectrumAqua; CEP-Y, SpectrumOrange; and CEP-9, SpectrumGreen). Chromosome 9 was used as an internal control to evaluate binding efficiency and successful hybridization. For each slide, a hybridization reaction was prepared by mixing 7 µl of CEP hybridization buffer and 1 µl of each probe in an Eppendorf tube for a total 10 µl of hybridization solution. The slides, only two per experiment, were put in a pre-warmed (7578°C) 70% formamide/2x SSC (pH 7.0) bath for 5 min. Afterwards they were dehydrated immediately with 70, 85 (1 min) and 100% ethanol (23 min, until the hybridization solution was ready) and then dried on a warm surface (45°C). In the meantime the probes were denatured at 7578°C for 5 min. Then 10 µl of the probe mix were applied to the hybridization area (marked with a diamond pen) and covered by a 22 mm2 coverslip. Hybridization was performed at 42 °C for 4 h. After hybridization, the slides were washed for 10 min in 50% formamide/2x SSC (pH 7.0) at 45°C, for 10 min in 2x SSC at 37°C and for 10 min in 2x SSC at room temperature. Preparations were counterstained by use of 30 µl of 4',6-diamidino-2-phenylindole (0.015 µg/ml) (DAPI, Sigma) for 5 min in a dark, moist chamber and mounted in Vectashield antifade medium (Vector Laboratories Inc., Burlingame, CA) under an 18 mm2 coverslip sealed with nail polish.
Scoring
After coding the slides, FISH analysis was performed by a single observer who was blinded to the experimental status of patients and controls. For each patient, 400 interphase and 40 metaphase nuclei were scored. Except for the nuclei in clusters or overlapping, all nuclei with or without signals were counted to evaluate the hybridization specificity and sensitivity. The scoring of FISH signals in lymphocytes was performed as described in Rubes et al. (2002). Briefly, two signals of the same colour were considered to represent two individual chromosomes only when the same-coloured signals were a minimum of one diameter apart. Chromosome 9 was used as an internal control to evaluate binding efficiency and successful hybridization. Interphase and metaphase cells not showing two signals for chromosome 9 were not evaluated for FISH analyses of chromosomes X and Y as the frequency of chromosome 9 aneuploidies was considered as background noise of the method. The following FISH results were obtained using a fluorescence microscope (Axiovert, Zeiss, Jena, Germany) equipped with appropriate filters (rhodamine/FITC/Aqua/DAPI).
Chromosome analysis
Conventional karyotype analyses were performed after Giemsa banding, i.e. staining on GTP-banded metaphase peripheral blood lymphocytes according to standard methods as reported elsewhere (Therman et al., 1980).
Semen analysis and serum hormones
Semen samples were analysed according to the current World Health Organization Laboratory Manual (World Health Organization, 1999) and subjected to rigid internal (Cooper et al., 1992
) and external quality control (Cooper et al., 1999
). Only the first semen sample was evaluated for this study. In cases of extremely low sperm counts or azoospermia, the ejaculates were centrifuged and analysis was performed on the sediment. Azoospermia was defined as no sperm found after centrifugation and analysis of the pellet. The patients were requested to abstain from sexual activity for 48 h to 7 days before investigation.
Venous blood was sampled between 08:0012:00 h at every visit. Blood samples for endocrine determinations were separated at 800 g and stored at 20°C until evaluation. Serum levels of LH, FSH, testosterone, estradiol, sex hormone-binding globulin (SHBG) and PSA were determined by highly specific routine commercial immunoassays. The normal range in our laboratory for LH is 210 IU/l, for FSH 17 U/l, for testosterone >12 nmol/l, for estradiol <250 pmol/l and for PSA <4 µg/l.
Statistical analysis
All variables were checked for normal distribution in the KolmogorovSmirnov one-sample test for goodness of fit. Variations between study groups were evaluated by one-way ANOVA followed by Tukey post hoc test. Proportions were analysed using the 2 test. Two-sided P-values of <0.05 were considered significant. All analyses were performed using the statistical software Sigma Stat for Windows version 2.03 (SPSS Inc., Chicago, IL). In general, results are given as mean ± SD.
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Results |
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Normozoospermia was diagnosed in all normal men. In all other groups, severe impairments of sperm concentration, motility and/or morphology were evident (Table I). Two Klinefelter patients had sperm in their semen. The proportion of azoospermic patients, sperm concentration, sperm motility and sperm morphology were not different between group I and group II which had a significantly higher proportion of azoospermic patients and lower sperm concentration, sperm motility and sperm morphology compared with both other groups. In addition, sperm concentration, sperm motility and sperm morphology were significantly lower in group III compared with group IV.
Karyotype
In all patients, a mean of 12 metaphases (minimum five, maximum 30) were evaluated. All patients in group II (n=11) and III (n=6) and in the normal control group (n=11) showed a normal 46,XY karyotype. Of the 18 patients with Klinefelter syndrome, 16 showed a non-mosaic 47,XXY karyotype while two patients of the Klinefelter group showed a mosaic status. In 15 metaphases of one patient, 12 were normal and three showed aneuploidy, 47,XXY[3]/46,XY[12]. In the other patient of whom 30 cells were analysed, 26 with 47,XXY, two with 46,XY and two with 46,XX were detected, 47,XY[26]/46,XY[2]/46,XX[2].
FISH analysis
For each subject, 400 interphase and 40 metaphase nuclei were scored. A total of 7920 lymphocytes were analysed in the Klinefelter group, 4840 in the Klinefelter-like group, 2640 in group III and 4400 in the normal group. The count rate of the internal control, chromosome 9, was analysed by one-way ANOVA for possibly significantly different frequencies among the four groups, but no correlation was found (Table II). The counts for chromosomes X and Y tested are presented in Table III. Fourteen of 18 Klinefelter patients showed a deviation of the expected counts above the average deviation for 46,XY patients (Figure 1). In contrast, only two out of 27 karyotyped 46,XY patients showed a deviation of the expected counts above the average deviation of counts in the Klinefelter patients. This average deviation (absolute interphase counts: 27.1 ± 5.4) of the expected counts in Klinefelter patients was significantly higher compared with the control group IV (1.0 ± 1.3%; absolute interphase counts,5.0 ± 6.9) and group III (2.8 ± 3.0%; absolute interphase counts, 9.7 ± 10.8), while group II (7.1 ± 4.0%; absolute interphase counts, 17.2 ± 18.9) showed a non-significant difference in their counts (Figure 2). Compared with all other groups, patients with Klinefelter syndrome showed mainly mosaicism with lymphocytes containing 46,XY or 46,XX (Figure 3). However, no prevalence of lymphocytes for another distinct gonosomal status could be observed in subjects with a 46,XY karyotype and, among groups IIIV, no differences in the proportion of 47,XXY cells (Figure 3) were found. The entire group of subfertile patients (IIII) shows a higher rate of gonosomal deviation from the expected counts compared with the group of normal men (group IV) (Figure 2). The infertile men with normal karyotype 46,XY (group II and III) also have a significantly higher rate of mosaicism compared with control group IV (3.9 ± 3.3 versus 1.3 ± 1.3%)
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Discussion |
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Even since the first clinical description of Klinefelter syndrome (Klinefelter et al., 1942), its prominent symptoms (small firm testes, gynaecomastia, hypogonadism, hypergonadotrophic azoospermia and tall eunochoid stature; reviewed by Lanfranco et al., 2004
) have become well known. In our study, total testicular volume was the most discriminating parameter allowing patients with Klinefelter syndrome (5.5 ± 3.4 ml) to be distinguished from all other groups (group II, 21.4 ± 11.9 ml, group III, 46.4 ± 23.9 ml; group IV, 57.9 ± 14.2 ml). In contrast to significant testicular volume differences, gonadotrophins were typically elevated in groups I and II (group I: LH, 17.3 ± 5.5 U/l; FSH, 33.0 ± 11.2 U/l; group II: LH, 12.8 ± 8.7 U/l; FSH, 28.0 ± 13.5 U/l) and did not allow discrimination of the Klinefelter syndrome from the second group. However, many of the classical symptoms are not observed exclusively in patients with Klinefelter syndrome and also occur frequently in other cases of male hypogonadism (Nieschlag et al., 2000
; Kamischke et al., 2003
).
Karyotyping of metaphase spreads of peripheral blood lymphocytes is still the gold standard for the diagnosis of Klinefelter syndrome and differential diagnosis of other numerical and structural gonosomal aberrations, although several studies have verified deficits of the technique (Okada et al., 1999; Kurková et al., 1999
; Pettenati et al., 1999
; Blanco et al., 2001
; Westlander et al., 2001
; Kamischke et al., 2003
). The main problem is that conventional karyotyping, when up to 20 cells are counted, may miss low grade mosaicsism. FISH of interphase cells allows the direct visualization of the sex chromosomes and hence the detection of low grade mosaicsism. In patients clinically suspected of having Klinefelter syndrome but normal karyotype, we found the highest rate of gonosomal mosaicism when performing FISH. In this group, one might speculate that a higher incidence of mosaic sex chromosomal aneuploidies is causally related to infertility and other clinical symptoms (i.e. elevated gonadotrophins). Meschede et al. (1998)
have revealed by conventional karyotype that the gonosomal mosaicism in infertile patients was 7.6% per couple or 3.8% per individual studied and is believed to be a major contributor to the genetic risks of infertility treatment by ICSI. To compare our results with data published so far, we have compared results of six other studies (see Table IV). Although the aims of these studies differ from those of our study, and therefore are mainly incomparable, the data are inconsistent. In all lymphocytes of Klinefelter patients, a low grade mosaicism was diagnosed but, among the very few studies with subjects karyotyped as 46,XY, three reveal a low grade mosaicism (Kurková et al., 1999
; Gazvani et al. 2000
; Rubes et al., 2002
).
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Our data support the idea that gonosomal mosaicism in lymphocytes is associated with increased sperm aneuploidy in men with idiopathic infertility (Rubes et al., 2002). It is shown that proper control of the cell cycle in mitosis and meiosis is a crucial component of the spermatogonia and the somatic cells (reviewed by Wolgemuth, 2003
). The direct correlation of the rate of gonosomal mosaicism in somatic cells and spermatozoa in all men, fertile or infertile, suggests that a common mechanism controls the genesis of both cell types (Wolgemuth, 2003
; Critchlow et al., 2004
). Overall, the entire group of subfertile patients (IIII) show hints of suboptimal mitotic precision in the cell cycle of peripheral blood lymphocytes (Table III and Figure 2). However, a correlation of aneuploidy rates in lymphocytes and spermatozoa cannot be shown in the present study because two groups (group I and II) were mainly azoospermic. Nor did we find a definite correlation between sperm parameters, hormone parameters and aneuploidy frequency in any group.
As is true for conventional karyotyping, tissue-specific mosaicism can also be overlooked by FISH. However, the two Klinefelter patients with sperm in their ejaculate also had an increased number of cells with a normal XY content (6.0 and 7.0%). This is in agreement with a study by Westlander et al. (2001) with karyotyped non-mosaic Klinefelter patients, of which two patients turned out to have aneuploidy rates >10% after FISH analyses of peripheral lymphocytes. FISH of peripheral blood lymphocytes offers additional information to estimate the sex chromosome aneuploidy rate in germ cell nuclei of mosaic and non-mosaic Klinefelter patients, but only FISH in biopsies of testis will answer the question of mosaicism in germ cells.
This may enhance genetic counselling of the patients prior to entering an ICSI programme because Klinefelter patients with low level mosaicism in their lymphocytes may be likely to have such mosaicism in their germ cells as well. Present day advanced reproductive technology stimulates the hope of infertile men for paternity, as several successful pregnancies of Klinefelter patients have been reported (reviewed by Lanfranco et al., 2004; Vernaeve et al., 2004
). In both groups, Klinefelter and Klinefelter-like patients, we found increased gonosomal aneuploidy frequencies (Figure 2). Interestingly, several groups also found increased gonosomal aneuploidy frequencies in spermatozoa of Klinefelter patients (Eskenazi et al., 2002
) as well as in OAT patients (Rives et al., 1998
; Pang et al., 1999
; Rubes et al., 2002
; Schmid et al., 2003
). Gazvani et al. (2000)
found increased aneuploidy rates among infertile oligozoospermic men in peripheral lymphocytes and spermatozoa which were significantly correlated.
In conclusion, infertile men are likely to have low grade mosaicism. Klinefelter patients with sperm and infertile 46,XY patients with a similar phenotype demonstrate increased aneuploidy frequencies in peripheral blood lymphocytes; genetic counselling should be offered to these couples. The correlation between the chromosomal status of the testicular cells and other easily accessible tissues (buccal mucosa, peripheral lymphocytes) should be the subject of future investigations. In particular, FISH might help to identify further factors such as mitotic instability concerning the origin of the disease and the probability of successful sperm retrieval in patients with azoospermia.
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Acknowledgements |
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References |
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Submitted on August 19, 2004; resubmitted on November 22, 2004; accepted on December 13, 2004.