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Analysis of Meiosis in Intratesticular Germ Cells from Subjects Affected by Classic Klinefelter’s Syndrome

University of Padua, Department of Medical and Surgical Sciences, Clinica Medica 3, 35128 Padua, Italy

Address correspondence and requests for reprints to: Dr. Carlo Foresta, University of Padua, Department of Medical and Surgical Sciences, Clinica Medica 3, 35128 Padua, Italy. E-mail: forestac{at}protec.it

	Abstract

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Azoospermic subjects affected by Klinefelter’s syndrome may occasionally show the presence of intratesticular residual foci of spermatogenesis, and the retrieval of mature spermatozoa from the testis may permit fertility and paternity by means of intracytoplasmic sperm injection.

Previous studies have demonstrated that these subjects show the presence of an increased incidence of hyperaploid spermatozoa. Here we analyzed, by fluorescence in situ hybridization using specific probes for chromosomes 8, X, and Y, the spermatogenic process and the meiotic progression of 47,XXY germ cells retrieved by fine needle aspiration of the testis in ten azoospermic patients affected by classic Klinefelter’s syndrome. All patients had lower testicular volume, higher gonadotropins, and lower testosterone plasma levels compared with control subjects. Cytological analysis of the testicular cells retrieved by fine needle aspiration showed the presence of Sertoli cells only in eight subjects, while germ cells were observed in two patients. In each patient Sertoli cells showed a 47,XXY karyotype, and the same chromosome pattern was observed in spermatogonia and primary spermatocytes of patients presenting a residual spermatogenesis. Secondary spermatocytes, spermatids, and mature spermatozoa showed different sex chromosome patterns, reflecting their origin from 47,XXY spermatogonia.

In conclusion, this study demonstrated that, in subjects affected by Klinefelter’s syndrome, residual germ cells may be present in the testis and that 47,XXY spermatogonia are able to undergo and complete the spermatogenic process leading to mature spermatozoa. These data further suggest the need to evaluate the sex chromosome status of sperm from patients affected by Klinefelter’s syndrome undergoing assisted reproductive techniques.

	Introduction

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Klinefelter’s syndrome (KS) is the most frequent sex-chromosome abnormality found in human males, with an incidence of approximately 1:500 newborn infants, and it is frequently associated with azoospermia (1). Men with 46,XY/47,XXY mosaicism may occasionally present with severe oligozoospermia, and there have been reports of fertility (2, 3) and proven paternity (4, 5, 6) in such patients. On the contrary, only few subjects with the classic KS possess spermatogenic cells (7).

Previous studies, using either direct sperm chromosome analysis or fluorescence in situ hybridization (FISH) on sperm nuclei, have shown an increased incidence of hyperaploid 24,XY spermatozoa both in men with 46,XY/47,XXY mosaicism and in complete KS (8, 9, 10, 11). In patients with mosaicism it has been assumed that only 46,XY germ cells can complete meiosis (12) and also in men with the classic KS a testicular mosaicism with some 46,XY germ lines has not been excluded. However, in 1969, Skakkebaek et al. (13) suggested that 47,XXY germ cells may achieve meiosis and may produce mature spermatozoa, and more recent reports confirmed this observation (9). From these studies, obtained by sperm karyotyping or DNA in situ hybridization, it appears that in 46,XY/47,XXY mosaicism there is a significant increase in hyperaploid, 24,XY-bearing sperm, while the corresponding 22,0 hypoaploid cells expected from meiosis I nondisjunction in 46,XY cells are not increased. These findings suggest that 47,XXY spermatogonia are able to undergo and complete spermatogenesis and produce hyperaploid spermatozoa (9, 14). Recently, we confirmed this hypothesis, demonstrating that the distribution of sex chromosomes in ejaculated sperm from two patients with KS agrees with the possibility that 47,XXY germ cells can complete the meiotic process (15).

In the present study, we strengthen this hypothesis by analyzing, with a multicolor FISH approach, the spermatogenic process and the meiotic phases of 47,XXY germ cells retrieved by fine needle aspiration (FNA) of the testis in ten azoospermic subjects affected by classic KS.

	Subjects and Methods

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Patients

We studied ten subjects, in an age range of 28–37 yr, who consulted our clinic because of infertility and were found to have nonmosaic KS. This pathology was demonstrated by peripheral lymphocyte karyotyping on 200 metaphases (performed by GTG and QFQ banding) and by FISH (using X- and Y-specific probes), which revealed a 47,XXY constitution in all examined cells. Ten normal fertile men with normal 46,XY karyotype represented the control group.

Physical, hormonal, and seminal analyses were performed in both patients and control subjects; FSH, LH, and testosterone plasma levels were measured by RIA using a double antibody (Ares-Serono, Milan, Italy). Semen samples were collected on two different occasions, separated by a 3-week interval, after 3 days of sexual abstinence, and were analyzed as recommended by the World Health Organization.

Fine needle aspiration cytology (FNAC) of the testis

In Klinefelter subjects the analysis of testicular structure was performed with conventional FNAC of the testis by a puncture in the middle portion of the testis, in the side opposite to the epidydimis, as previously described (16). Briefly, bilateral FNAC was performed using 23 gauge (0.6 mm) butterfly needles and aspirating with a 20 mL syringe. The cellular material was placed on two or more microscope slides for each testis and air-dried for 24 h. One slide was stained with May Grünwald-Giemsa and examined under a light microscope at X 25, X400, and X1250 magnifications. The following cell types were identified using criteria previously described (16): spermatogonia, primary spermatocytes, secondary spermatocytes, early and late spermatids, corresponding to Sa-Sb and Sc-Sd steps of spermatogenesis, respectively, spermatozoa, and Sertoli cells.

Fluorescence in situ hybridization (FISH)

Numerical chromosome alterations of testicular cells were evaluated by multicolor FISH, as previously reported (15). Cellular nuclei were decondensed according to Martin et al. (17), and May Grünwald-Giemsa staining was performed successively to recognize each specific cell type. After decondensation, slides were immediately used for the successive steps or were stored in a refrigerator (2–4 days, 4 C).

DNA hybridization was performed using human -satellite probes specific for chromosomes X, Y, and 8 (Amersham Pharmacia Biotech) directly labeled using fluorochromes FluorX (chromosome X, green) and Cy3 (chromosome Y, orange). To detect chromosome 8, a mixture (1:1) of FluorX and Cy3 directly labeled specific probes was used, resulting in a yellow signal.

DNA denaturation of testicular cells and probes, incubation, and post-hybridization washing were performed according to the Amersham Pharmacia Biotech protocol. Cellular nuclei were successively counterstained (1 min at room temperature) in a Coplin jar containing a phosphate-buffered saline (pH 7.4)-4',6-diamidine-2'-phenylindole dihydrochloride solution (20 ng/mL). Slides were then rinsed in distilled water, air dried in the dark, mounted using an antifade solution (glycerol-distilled water; 9.1–1.4 diazabicycle-[2.2.2] octane; 2% wt/vol), and stored (1–4 days, 4 C) or immediately observed using a Leica Diaplan epifluorescence microscope (Leica Corp., Wetzlar, Germany) fitted with a 100-watt mercury lamp and a triple bandpass filter suitable for the fluorochromes in use. This procedure allowed the detection of all probes as bright, compact, and uniformly sized spots.

Each spot was evaluated and scored as specific for the chromosome corresponding to its color only when the intensity and size were similar to those of spots of the same color in the surrounding cells. Furthermore, if two spots of the same color were located in the same cell, the distance had to be more than their diameter for them to be considered distinct chromosomes.

DNA probes were provided by Amersham Pharmacia Biotech (Milan, Italy). 4',6-diamidine-2'-phenylindole dihydrochloride was purchased from Roche Molecular Biochemicals (Milan, Italy). All other chemicals were purchased from Sigma (Milan, Italy).

Statistical analysis

Student’s t test was performed to compare results from 47,XXY males and controls, and a difference was considered significant at P < 0.05.

	Results

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Table 1 reports clinical and hormonal parameters in our patients affected by KS compared with those in the controls. All patients showed lower testicular volume, higher FSH and LH plasma levels, with reduced testosterone plasma levels. Seminal analysis repeatedly revealed azoospermia.

Table 2 shows the results of chromosomal arrangement analysis relative to chromosomes X, Y, and 8 observed in testicular cells. In each subject Sertoli cells showed a 47,XXY karyotype. The same chromosome pattern was observed in all spermatogonia and primary spermatocytes of the two subjects presenting intratesticular residual spermatogenesis. Secondary spermatocytes, spermatids, and spermatozoa showed a sex chromosome distribution of hyperaploides reflecting their origin from 47,XXY spermatogonia. Figs. 1 and 2 show, as examples, a 47,XXY spermatogonium and a 24,XY spermatozoon. The identification of each cell type was confirmed by staining with May Grünwald-Giemsa after the FISH analysis, using morphological criteria such as the cellular and nuclear diameter and the chromatin pattern, as previously described (16).

View larger version (111K): [in this window] [in a new window]   Figure 1. A, Cytologic appearance of a spermatogonium retrieved by fine needle aspiration of the testis from a nonmosaic 47,XXY Klinefelter’s subject (May Grünwald-Giemsa staining). B, Three-color FISH of a spermatogonium exhibiting a 47,XXY chromosome pattern.

View larger version (83K): [in this window] [in a new window]   Figure 2. Three-color FISH of intratesticular mature spermatozoa from an oligozoospermic subject affected by nonmosaic Klinefelter’s syndrome. It is possible to observe one sperm with a normal sex chromosome set (23,X) and an aneuploid sperm with 24,XY chromosome pattern.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

KS is a form of primary testicular failure with testicular hypotrophy and elevated gonadotropin plasma levels (1). Subjects affected by KS are usually azoospermic, and in the classic form, spermatozoa are only rarely demonstrated in the ejaculate (7, 10).

Testicular biopsy specimens of infants affected by this disorder reveal a reduced number of germ cells (1). After puberty the testicular structure shows the pathologic alterations characteristic of KS, such as hyalinization and fibrosis of the seminiferous tubules (1). However, the presence of tubules with residual foci of spermatogenesis have also been reported, with meiotic arrest at primary spermatocytic or spermatidic stages and foci of normal spermatogenesis (2, 3, 4, 5).

In oligozoospermic and azoospermic men affected by KS, the fertility prognosis was absolutely hopeless before the introduction of the intracytoplasmic sperm injection (ICSI) technique, which offers to such patients the opportunity for procreation, even when spermatozoa in the ejaculate are lacking (20). In these cases ICSI may allow fertilization and pregnancy by extracting mature spermatozoa from the testicular tissue. In fact, ICSI has already been performed successfully in patients with KS using both ejaculated and testicular sperm, and pregnancies and live births have been reported (21, 22, 23).

Recently, we have had the opportunity to analyze the testicular cells obtained by FNAC in ten azoospermic subjects affected by KS, who consulted our clinic for an ICSI program. The testicular cytologic analysis showed in all cases the presence of Sertoli cells and, in two of the ten, the presence of residual spermatogenic cells. These results suggest that, more frequently than we have known to date, azoospermic subjects affected by classic KS may have seminiferous tubules containing both Sertoli cells and residual spermatogenesis.

The aim of this study was to investigate if spermatogenesis in patients affected by classic KS may origin from 47,XXY spermatogonia or, alternatively, if a testicular mosaicism exists. To clarify this aspect, a 3-color FISH was performed on testicular cells retrieved by FNA in these ten patients. Three-color FISH was performed using X and Y DNA probes to study the percentages of sex chromosome aneuploides and a probe for chromosome 8 as an internal control of efficient hybridization and to distinguish between diploidy and hyperaploidy. This analysis showed that in each subject all Sertoli cells had Klinefelter 47,XXY constitution as well as all spermatogonia and primary spermatocytes, when present. These findings demonstrated that all subjects were affected by classic KS, allowing us to exclude a mosaicism confined to the testicular tissue. Furthermore, the presence of 47,XXY spermatogonia and primary spermatocytes associated with the presence of spermatids and spermatozoa demonstrate, for the first time, that in nonmosaic Klinefelter’s subjects germ cells may undergo and complete the mitotic and meiotic processes.

The distribution of hyperaploides in spermatids and spermatozoa seemed to reflect their origin from 47,XXY spermatogonia, and agreed with the frequency and distribution of sperm sex aneuploides recently observed in ejaculated spermatozoa of Klinefelter’s subjects (15). The low number of retrieved spermatogenic cells in our Klinefelter’s subjects did not allowed us to investigate the maturation process of XXY-bearing germ cells, even if the higher percentage of X-bearing with respect to Y-bearing secondary spermatocytes, spermatids, and mature spermatozoa suggested a preferential pairing during the meiotic process of homologous sex chromosomes, when three gonosomes are present. In fact, regular meiosis in a 47,XXY spermatogonium should lead to the same proportion of 23,X- and 24,XY-bearing sperm cells. However, the frequency of 23,X sperm observed in this study was twice that of 23,Y and 24,XY. This observation suggests the preferential pairing of homologous sex chromosomes, as previously reported (13, 15). Furthermore, the high percentages of spermatids and mature spermatozoa carrying a normal sex chromosome pattern suggest that 47,XXY germ cells show a difficult progression through the meiotic process, such as in 47,XYY males. In fact, it has been demonstrated that XY pairing, associated with a univalent Y, resulted in a high level of spermatocytic death in these patients (12).

The normal karyotype of infants born after ICSI using sperm obtained by Klinefelter’s subjects (22, 23) agrees with the presence of high percentages of chromosomically normal spermatozoa in these subjects (15, 21). However, in patients affected by KS the percentage of spermatozoa bearing sex chromosome aneuploidy is remarkable, and therefore the evaluation of the sex chromosome status of intratesticular or ejaculated sperm should be performed before using these cells in assisted reproductive techniques. Furthermore, each patient affected by KS undergoing in vitro fertilization techniques should be informed about the possibility of transmitting sex chromosome abnormalities. Genetic counseling and informed consent should be obtained, and important ethical considerations should be borne in mind as to the possiblities of interruption of pregnancy on the basis of prenatal tests.

In conclusion, the results of this study demonstrated for the first time that in subjects affected by classic KS a residual spermatogenesis may exist and that 47,XXY spermatogonia are able to complete the spermatogenic process leading to the formation of mature spermatozoa.

Received February 16, 1999.

Revised April 21, 1999.

Accepted June 10, 1999.

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