Mosaic status in lymphocytes of infertile men with or without Klinefelter syndrome

P. Lenz1, C.M. Luetjens1, A. Kamischke1, B. Kühnert1, I. Kennerknecht2 and E. Nieschlag1,3

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


    Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Gonosomal aneuploidies such as Klinefelter syndrome (47,XXY) are the most frequent chromosomal aberration in infertile men. Normally the chromosomal status of patients is detected by karyotyping of up to 20 metaphase spreads of lymphocyte nuclei, whereby low grade mosaicism may be overlooked. To test whether Klinefelter patients with 47,XXY karyotype or infertile men with 46,XY karyotype represent gonosomal mosaicisms, we performed meta- and interphase fluorescence in situ hybridization (FISH) on 45 men. METHODS AND RESULTS: A total of 400 interphase and 40 metaphase lymphocyte nuclei per patient were scored after hybridization with DNA probes specific for chromosomes X and Y, and chromosome 9 as a control. On the basis of conventional karyotype, hormone levels and clinical appearance, patients were subdivided into 18 Klinefelter syndrome patients with 47,XXY (group I), 11 Klinefelter syndrome-like patients with normal karyotype, 46,XY (group II) and six non-Klinefelter-like infertile patients with normal 46,XY karyotype (group III). Ten normal men (group IV) served as controls. Testicular volume in the Klinefelter group I was smaller compared with group II (P=0.016), group III (P<0.001) and group IV (P<0.001). In addition, testicular volumes in group II were lower compared with group III and group IV (P<0.004). No significant differences between the aneuploidy rate analysed by FISH in interphase nuclei and metaphases were found in either single patients or groups. Patients with Klinefelter syndrome, 47,XXY (group I) or with symptoms similar to those in Klinefelter patients 46,XY (group II) showed a similar aneuploidy rate (group I 7.1 ± 4.0% and group II 4.6 ± 3.4%) and two 47,XXY patients with a high prevalence for normal 46,XY lymphocytes had sperm in their ejaculate. However, in general, no correlations between FISH mosaic status and serum hormone parameters, nor with ejaculate parameters were found. CONCLUSIONS: The results suggest that 47,XXY patients with an increased incidence of XY cells (average of 4.2 ± 2.3) may have a higher probability of germ cells as we found sperm only in the ejaculate of Klinefelter syndrome patients with mosaic 46,XY cells (6.0 and 7.0%). On the other hand, 46,XY patients with mosaic sex chromosome aneuploidies detected by FISH analysis more often show symptoms of hypogonadism phenotypically resembling Klinefelter syndrome.

Key words: aneuploidy/FISH/hormone levels/lymphocytes/male infertility


    Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
In its classic form, Klinefelter syndrome is characterized by gynaecomastia, small, firm testes with hyalinization of seminiferous tubules, hypergonadotrophic hypogonadism and azoospermia (Klinefelter et al., 1942Go). In the general population, the syndrome prevalence is 0.1–0.2% (Nielsen and Wohlert, 1991Go; Lanfranco et al., 2004Go), and among infertile patients up to 11% of azoospermic and 0.7% of oligozoospermic men reported have a 47,XXY karyotype (De Braekeleer and Dao, 1991Go; Yoshida et al., 1996Go). Karyotyping 15–20 metaphase spreads in human genetic departments, a procedure considered the gold standard, defines the chromosomal status of a patient (Zang, 1984Go; Kamischke et al., 2003Go). The relatively low numbers of cells investigated by karyotyping may miss such low grade aneuploidy rates (Westlander et al., 2001Go). However, for genetic screening purposes, fluorescence in situ hybridization (FISH) reveals low grade mosaicism because a greater number of cells can be studied. Using commercially available chromosome-specific probes, FISH can be applied to detect chromosome number aberrations in metaphase spreads, as well as in interphase nuclei. In the interphase nuclei, FISH is also especially useful for clinical application when rapid results are desired and cells are difficult to culture in vitro to induce metaphases. Interphase cytogenetics are now widely used to study a variety of cell types: bone marrow, buccal mucosa, sperm, amniocytes, chorionic villi and uncultured blood cells (Yan et al., 2000Go).

In andrology, FISH of interphase sperm nuclei has been used successfully to determine aneuploidy rates (Luetjens et al., 2002Go) and has demonstrated that the aneuploidy number in lymphocytes was positively correlated with the number of aneuploid sperm (Rubes et al., 2002Go). 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., 2001Go).

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.


    Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Subjects
For the study, 35 age-matched patients attending the Institute of Reproductive Medicine of the University (Münster, Germany), mainly for infertility, suspected Klinefelter syndrome or hypogonadism, and 10 age-matched normal controls were examined. Based on routine karyotype analyses and results of clinical and hormonal investigations (Kamischke et al., 2003Go), the heterogeneous group of infertile patients was subdivided into 18 Klinefelter syndrome patients (group I), eleven 46,XY Klinefelter syndrome-like patients (highly elevated gonadotrophins, azoospermia or severe oligozoospermia, low to normal serum testosterone and small testicular volumes) (group II) and six 46,XY severely infertile oligoasthenoteratozoospermia (OAT) patients (normal gonadotrophins, azoospermia or severe oligozoospermia, low to normal testosterone values and normal testicular volume) (group III). As controls, 10 normal men were recruited from male contraceptive studies prior to any treatment (group IV) (see Table I). The Klinefelter syndrome group (group I) and the Klinefelter syndrome-like group (group II) showed elevated FSH and LH values compared with both other groups which had gonadotrophins within the normal range. The testosterone values differed significantly between the Klinefelter syndrome group I and the control group IV (P<0.001). No significant difference was found in the estradiol levels between the groups. Prostate-specific antigen (PSA) levels were normal in all groups and a significant difference could only be seen between groups I and III (data not shown). Testicular volume (5.5 ± 3.3 ml) in the Klinefelter group was significantly smaller compared with group II (P=0.016), group III (P<0.001) and group IV (P<0.001). In addition, testicular volumes (20.6 ± 10.9 ml) in group II were significantly lower compared with group III and group IV (P<0.004). The age of all groups investigated did not differ significantly. Heparinized peripheral blood samples were obtained from all men to perform three-colour FISH. Further examinations (some not performed in all men) included general and genital examination, reproductive hormone and semen analyses, and clinical and sonographic evaluation of the scrotal content. Informed consent was obtained from all subjects.


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Table I. Hormone values, sperm parameters and testes size of all subjects

 
Lymphocyte preparation and fixation
Heparinized peripheral blood from all subjects was obtained and gradient separation was carried out as described by Bhat et al. (1993)Go. After centrifugation, the lymphocyte ring was collected and cultured in Dulbecco's medium (2% penicillin–streptomycin, 15% fetal calf serum) with 15 µl of phytohaemagglutinin solution (PHA-M, 20 mg/ml, Roche, Mannheim, Germany) at 37°C for up to 72 h. Prior to harvesting, the cells were treated for 4 h with colcemid (Karyomax, Gibco-BRL, Karlsruhe, Germany) to enrich metaphase cells. After a 10 min hypotonic treatment (0.075 mol/l KCl) at 37°C and three changes of fixative 3:1 of methanol:glacial acetic acid, nuclei were spread onto slides by dropping the solution onto pre-cleaned slides (polylysine coated, Langenbrück, Germany). The anonymized slides were kept in the dark prior to analysis at –20°C.

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)Go. 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 (75–78°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 (2–3 min, until the hybridization solution was ready) and then dried on a warm surface (45°C). In the meantime the probes were denatured at 75–78°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)Go. 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., 1980Go).

Semen analysis and serum hormones
Semen samples were analysed according to the current World Health Organization Laboratory Manual (World Health Organization, 1999Go) and subjected to rigid internal (Cooper et al., 1992Go) and external quality control (Cooper et al., 1999Go). 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 08:00–12:00 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 2–10 IU/l, for FSH 1–7 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 Kolmogorov–Smirnov 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 {chi}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.


    Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Semen analysis
All patients underwent sonographic evaluation of the testes; two were excluded from the comparative analyses because of previous hemicastration due to testicular cancer. A total of 41 patients provided an ejaculate (Table I).

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 II–IV, no differences in the proportion of 47,XXY cells (Figure 3) were found. The entire group of subfertile patients (I–III) 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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Table II. Chromosome 9 aneuploidy rates in the Klinefelter group (group I), the Klinefelter-like group (group II), the non-Klinefelter-group (group III) and the normal group (group IV)

 

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Table III. Lymphocyte aneuploidy rates in the Klinefelter-group (group I), the Klinefelter-like group (group II), the non-Klinefelter-group (group III) and the normal group (group IV)

 


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Figure 1. Dot plot of the deviation from the expected karyotype of each diagnosed subject. The Klinefelter patients have the highest deviation from the expected counts indicated by the dotted line at their average deviation. Only three Klinefelter-like subjects are below the average aneuploidy rate for all 46,XY subjects (lower dotted line). In only one subject was no deviation from the normal non-mosaic sex chromosomal karyotype found.

 


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Figure 2. Comparison of the signal counts of four different subject groups for interphase, metaphase and the total of both lymphocyte nuclei. In none of the groups does the expected signal count for both nuclei phases differ significantly, but the Klinefelter group differs significantly from the non-Klinefelter-like and the normal subject group. Data are means ± SD. ***P<0.001; **P<0.01; *P<0.05.

 


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Figure 3. Mean distribution of the various sex chromosomal aneuploidy types found among the subjects' lymphocyte nuclei. None of the three 46,XY karyotyped groups showed a tendency of >1% to accumulate two X chromosomal counts and one Y chromosomal count per lymphocyte. Data are means ± SD.

 
Aneuploidy correlated with sperm and serum parameters
No correlation between the rate of mosaicism and hormone serum parameters was found. Furthermore, no correlation between gonosomal counts and sperm parameters was found.


    Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Because Klinefelter syndrome shows a great variety of different phenotypes, diagnosis is often delayed or patients remain undiagnosed (Abramsky and Chapple, 1997Go; Smyth and Bremner, 1998Go; Amory et al., 2000Go; Wilkes, 2000Go; Bojesen et al., 2003Go; Kamischke et al., 2003Go; Lanfranco et al., 2004Go). However, Klinefelter syndrome is not rare among infertile patients suffering from azoospermia or severe oligozoospermia and presenting with signs of androgen deficiency (Nielsen and Wohlert, 1991Go). Early diagnosis of the syndrome by karyotyping allows initiation of testosterone substitution therapy to avoid symptoms and sequelae of androgen deficiency (Nielsen et al., 1988Go; Nieschlag et al., 2000Go). It also may allow future fertility to be preserved for young Klinefelter patients, because germ cell depletion can progress with age (Damani et al., 2001Go).

Even since the first clinical description of Klinefelter syndrome (Klinefelter et al., 1942Go), its prominent symptoms (small firm testes, gynaecomastia, hypogonadism, hypergonadotrophic azoospermia and tall eunochoid stature; reviewed by Lanfranco et al., 2004Go) 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., 2000Go; Kamischke et al., 2003Go).

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., 1999Go; Kurková et al., 1999Go; Pettenati et al., 1999Go; Blanco et al., 2001Go; Westlander et al., 2001Go; Kamischke et al., 2003Go). 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)Go 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., 1999Go; Gazvani et al. 2000Go; Rubes et al., 2002Go).


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Table IV. Lymphocyte gonosomal aneuploidy rates from the literature and from this study in interphase cells

 
The general question of this study is whether the detected variable chromosomal counts reflect true mosaicsism of the subjects. The FISH technique is considered to show false-negative and false-positive counts and therefore overestimates the number of aneuploid cells. We also found a mean of 4.1 ± 5.6% (median:7%) aberrant counts of chromosome 9 signals in our population, but there was no correlation with the aberrant counts of chromosome X or Y signals as seen in the different groups (Table II). There was also no correlation of the aberrant counts of the chromosome 9 signal with the four subject groups. Nor do we assume that an increase of aneuploidy rates may occur in the cultured cells due to the culture duration because such a correlation has been excluded by Bortolai and Melaragno (2001)Go. Moreover, the lymphocytes of all subjects were cultured and harvested according to the same protocol so that such a systematic artefact would be manifest equally in all groups. To avoid overestimating gonosomal aneuploidies, only cells with chromosome 9 diploidy were studied for gonosomal signals. The similar FISH signal counts found in the metaphase and interphase cells demonstrate that optical or steric hindrances do not cause false-negative results (Figure 2). None of our patients had major differences between the counts in interphase nuclei and metaphase spreads, suggesting that FISH on interphase nuclei is an appropriate technique for an andrology clinic to screen the aneuploidy status of their patients. Definite check of the chromosomal status should still be done by conventional cytogenetics. Even if we consider the gonosomal counts of the control group (IV) as the baseline frequency, the count number of the Klinefelter patients remains significantly different from that of the infertile men of group III.

Our data support the idea that gonosomal mosaicism in lymphocytes is associated with increased sperm aneuploidy in men with idiopathic infertility (Rubes et al., 2002Go). 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, 2003Go). 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, 2003Go; Critchlow et al., 2004Go). Overall, the entire group of subfertile patients (I–III) 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)Go 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., 2004Go; Vernaeve et al., 2004Go). 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., 2002Go) as well as in OAT patients (Rives et al., 1998Go; Pang et al., 1999Go; Rubes et al., 2002Go; Schmid et al., 2003Go). Gazvani et al. (2000)Go 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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
We are grateful to Dr D.Meschede for critical review of the paper and thank I.Upmann for technical assistance and Susan Nieschlag for language editing of the manuscript.


    References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on August 19, 2004; resubmitted on November 22, 2004; accepted on December 13, 2004.