Molecular Human Reproduction, Vol. 6, No. 8, 688-693,
August 2000
© 2000 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Y chromosome microdeletions and germinal mosaicism in infertile males
1 Service d'Histologie, Biologie de la Reproduction et Cytogénétique et CECOS, Hôpital Tenon, 4 Rue de la Chine, 75020 Paris, and 2 Laboratoire d'Immunogénétique Humaine, Institut Pasteur, Paris, France.
Abstract
Molecular deletions of the Y chromosome long arm are a frequent cause of male infertility. Because these deletions are thought to be inherited from fathers without Y chromosome deletions, the question arises as to whether their relatively high incidence in the male population could be due to the existence of a mosaicism in somatic and/or germinal paternal cells. This study included a total of 181 infertile men, among whom 18 were found to have an abnormal karyotype. In the other 163, polymerase chain reaction (PCR) analysis detected nine (5.5%) Y chromosome microdeletions. Blood, spermatozoa or testicular cells from 47 men (27 oligozoospermia, 20 azoospermia), including six Y-deleted patients, were screened for mosaicism using double target fluorescence in-situ hybridization (FISH) with Y centromeric and deleted in azoospermia (DAZ) gene-specific probes. Results indicated that: (i) percentages of double (intact Y chromosome) or single (deleted Y chromosome) fluorescent signals by FISH were in agreement with PCR data, thus demonstrating the reliability of the method; and (ii) a weak germ cell mosaicism was found in only two oligozoospermic patients, carrying 1.97 and 4.13% respectively of spermatozoa with a deleted Y chromosome. Further studies on larger populations are needed to evaluate precisely the incidence of Y deletion mosaicisms in infertile men.
FISH/germ cells/mosaicism/male infertility/Y deletions
Introduction
Current estimations indicate that 15–20% of couples are infertile and that a male factor is identified in 40% of these cases (Bhasin et al., 1994
; DeKretzer, 1997). Although male infertility is commonly associated with genital tract obstructions, endocrinal impairments, previous history of cryptorchidism or vascular defects such as varicocele, a genetic cause can be suspected in many cases, especially when infertility appears idiopathic. In the latter cases, microdeletions of the Y chromosome long arm (Yq) are found in >10% of men with non-obstructive azoospermia or severe oligozoospermia (for review, see McElreavey and Krausz, 1999). These microdeletions can now be diagnosed easily by polymerase chain reaction (PCR), using specific sequence-tagged sites (STS) (Vergnaud et al., 1986). They lead to the loss of genes which have been implicated in spermatogenesis and which constitute the azoospermia factor (AZF) (Tiepolo and Zuffardi, 1976). AZF is subdivided into three different loci along Yq11, i.e. AZFa, b and c (Vogt et al., 1996).
Since these deletions are thought to arise de novo from fertile fathers with an intact Y chromosome, they represent one of the most frequent structural chromosomal accidents, affecting ~ one in every 5000 men. Therefore, the question arises as to whether predisposing paternal factors, e.g. particular Y chromosome haplotypes or a germinal mosaicism for deleted and non-deleted Y chromosomes, could play a role in the occurrence of homogenous Y microdeletions in the offspring. Indeed, previous studies have shown that boys born from oligozoospermic men treated using intracytoplasmic sperm injection (ICSI) have an increased risk of carrying Y chromosome microdeletions (Kent-First et al., 1996a). These results suggest that Y chromosome deletions can exist in a mosaic state in gonads and/or somatic tissues of some infertile men, thus affecting their spermatogenesis but still permitting transmission of the deleted chromosome to their male offspring.
The use of PCR on DNA extracts from whole blood cells or germ cells fails to detect such mosaicisms. On the contrary, fluorescence in-situ hybridization (FISH) appears as a valuable tool for discriminating cells carrying an intact Y chromosome from cells with a deleted Y chromosome. Therefore, the aim of this work was: (i) to establish the frequency of Yq microdeletions in a population of infertile men using PCR; and (ii) to screen for somatic and/or a germinal mosaicism in patients considered as non-deleted by PCR techniques, using the method of double target FISH with a Y centromeric probe and a probe coding for the deleted in azoospermia (DAZ) gene located in the AZFc region.
Materials and methods
Patients
Our study was carried out on 181 patients referred to the laboratory for a biological investigation of their infertility and having either non-obstructive azoospermia (n = 95) or severe oligozoospermia (sperm count <3x106 spermatozoa/ml; n = 86). After genetic counselling, their karyotype was analysed using standard cytogenetic methods and genomic DNA was extracted from whole blood cells using a QIAamp Blood Midi kit (Qiagen, Germany).
Screening for Yq microdeletions was carried out in patients with normal karyotype by amplifying 12 different STS corresponding to the three AZF loci (AZFa, sY85, sY95; AZFb, sY114, sY116, sY125, sY127; AZFc, sY135, sY149, sY152, sY254; DAZ), to SRY (sY14) or to the heterochromatic distal Yq region (sY160). Briefly, 200–300 ng of genomic DNA were used as template in 50 µl of a solution containing 5µl of 10x amplification buffer (Pharmacia, Denmark), 1 mmol/l dNTPs, 25 pmol of each primer and 2.5 IU of Taq DNA polymerase (Pharmacia). After an initial denaturation step of 5 min at 95°C, 38 amplification cycles were performed on a GeneAmp 2400 thermocycler (Perkin Elmer, USA). Controls for PCR included female DNA or water as the template. PCR products were verified by electrophoresis on 1% agarose gels containing ethydium bromide. Negative PCR reactions were repeated three times to avoid false negative results. Deletions were checked by Southern blotting of the patient's DNA and hybridization of the deleted STS, labelled with [32P] as the probe. The limits of the deletions were estimated by detecting positive bordering STS.
Mosaicisms involving AZFc were detected by double FISH using both a Y centromeric probe (Oncor, USA), labelled with biotin, and a cosmid (kind gift of Professor C.Bishop, Bayor College, USA) containing a 40 kb insert coding for the DAZ gene family and labelled with digoxygenin by nick translation. This study was carried out in 47 patients among 181 (27 oligozoospermic patients with sperm counts of <3x106 spermatozoa/ml and 20 azoospermic patients) for whom germ cells or testicular cells were available. Spermatozoa from 10 normospermic men were studied as controls under the same conditions.
Somatic blood cells were analysed in each case and prepared as follows: uncultured cells were incubated in 0.56% KCl for 20 min at 37°C, recovered by centrifugation at 200 g for 5 min, fixed twice in methanol/acetic acid (3:1 v/v) for 10 min at room temperature and dropped onto glass microscope slides. The slides were then air-dried and treated directly for FISH or stored at –20°C. Prior to FISH, slides were washed in 2x sodium chloride/sodium citrate (SSC) for 1 h at 37°C, dehydrated in ethanol gradients and air-dried.
Ejaculated sperm cells were studied only for oligozoospermic patients and for controls. After sperm liquefaction for 30 min at 37°C, samples were treated differently according to the numeration. When sperm counts were >1x106/ml, the spermatozoa were washed three times in phosphate-buffered saline (PBS) and then fixed in methanol/acetic acid (3:1 v/v) for 1 h at 4°C before spreading on glass microscope slides. Sperm head decondensation was obtained by treating slides with 0.1 mol/l dithiothreitol (DTT; Sigma, St Louis, MO, USA) for 30 min at room temperature. Slides were then rinsed in 20 mmol/l diiodosalycilic acid lithium (LIS; Sigma) for 1–2 h at room temperature, washed in 2x SSC for 1 h at 37°C, dehydrated in increasing gradients of ethanol and air-dried. For sperm counts of <1x106 spermatozoa/ml, efficient recovery of sperm cells was achieved by filtering 1 ml of spermatozoa, using a syringe, onto a FHLP 02500 filter (Millipore, USA) mounted in a swinnex system (Millipore) and previously rinsed with 1 ml ethanol (Le Bourhis et al., 1999). Filters were then washed three times with 3 ml PBS, removed from the swinnex system, air-dried and fixed in methanol/acetic acid (3:1 v/v) for 1 h at 4°C. They were then treated for sperm head decondensation as described above. Prior to FISH, slides or filters were washed in 2x SSC for 1 h at 37°C, dehydrated in ethanol gradients and air-dried.
In azoospermic patients, testicular biopsies were treated as described previously (Luciani et al., 1984); samples were incubated in a solution of KCl (8.8 g/l) for 10 h at room temperature and then fixed in methanol/acetic acid (3:1 v/v) for 24–72 h at room temperature. They were then dilacerated in the same fixative using two fine needles. After centrifugation at 200 g for 10 min, pellets were dissociated in 45% acetic acid before spreading on microscope glass slides. The different cell types were then visualized and localized after Giemsa staining. After bleaching, slides were rinsed in 2x SSC for 1h at 37°C, dehydrated in gradients of ethanol and air-dried before performing FISH.
In all cases, FISH was performed by incubating slides or filters, set on a microscope glass slide, in 50 µl of hybridization buffer (Hybrizol buffer VII; Oncor) containing 1.5 µl of the Y centromeric biotin labelled probe and 15 ng/µl of the DAZ-specific digoxigenin labelled cosmid, under cover glass sealed with rubber cement. Slides were set up onto a thermocycler (Omnislide; Hybaid, UK) and allowed to denature for 4 min at 73°C for blood and testicular cells and for 10 min at 73°C for spermatozoa. The hybridization programme then consisted of an incubation at 37°C overnight. Slides were washed first in 0.4x SSC/0.1% Tween 20 for 4 min at 73°C and then in 0.1x SSC/0.1% Tween 20 for 2 min at room temperature. Probes were visualized using 30 µl of a mixture containing an antibody to digoxygenin, coupled to rhodamine (Oncor), and avidin/fluorescein isothiocyanate (FITC; Oncor). Slides or filters were incubated under cover glass for 10 min at 37°C and then rinsed three times in 1x SSC/0.1% Tween 20 at room temperature. After mounting and counterstaining in Vectashield/DAPI (Vector, USA), preparations were observed under UV light on a photomicroscope (Zeiss Axiophot, Germany) equipped with epifluorescence optics and a filter set including three single pass band filters, respectively for DAPI, FITC and rhodamine, and a triple pass band filter for the simultaneous detection of fluorescent signals.
A minimum of 300 blood cells were analysed in each case. According to the patient's sperm count, 5000–10 000 spermatozoa in oligozoospermic patients and 40–300 testicular cells in azoospermic patients were counted. Only nuclei or sperm heads showing separate fluorescent spots, with at least the Y centromeric signal, were scored. In azoospermic patients, FISH results in testicular cells were compared to those obtained in somatic cells of controls. Statistical analysis was performed using the 
2 test.
Results
Cytogenetic and molecular analysis results are summarized in Figure 1. Of the 181 patients, 18 presented with an abnormal karyotype (9.9%); four Klinefelter syndromes, three Robertsonian translocations [two t (13q;14q), one t (14q;21q)], three reciprocal translocations [t (1;3), t (Y;4), t (Y;13)], two 45,X / 46,XY mosaicisms, one supernumary marker and five structural aberrations of the Y chromosome (three pericentric inversions, one dicentric, one ring Y) were found.
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Of the 163 infertile men with a normal karyotype, 9 (5.5%) carried a microdeletion of the Y chromosome long arm (Figure 2
). Three oligozoospermic patients had an interstitial deletion of the AZFb locus and the others six exhibited different molecular deletions of the AZFc locus and were either azoospermic (n = 2) or severely oligozoospermic (n = 4). Two of these deletions were terminal and four were interstitial. The 47 patients (out of 163) screened for somatic and germinal Y deletion mosaicism, included six of the nine Y chromosome microdeletion carriers (three AZFb and three AZFc deletions including the DAZ locus). Representative FISH results are shown in Figure 3.
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In blood cells from the 41 men with no Y deletions and from the three patients carrying an AZFb deletion, the mean percentage of normal cells, exhibiting both Y centromeric and DAZ fluorescent signals, was 99.7% and was not significantly different from that observed in controls. On the other hand, in the three men deleted for AZFc region, as detected by PCR, constitutional deletion of this locus was affirmed by FISH, showing a double fluorescent signal in <0.33% of nuclei (Table I
).
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The 27 patients, screened for Y deletion mosaicism by FISH in spermatozoa, included 23 non-deleted men, three AZFb deletions and one AZFc (DAZ-negative) deletion. In the latter case, 99.7% of the spermatozoa exhibited no specific DAZ fluorescent signal. Other patients, who were had either no Y deletions or had AZFb deletions, had a similar rate of spermatozoa with two fluorescent spots as the controls, except two men with no Y deletions who exhibited 1.97% (P < 0.001) and 4.13% (P < 0.001) of spermatozoa lacking the DAZ signal (Table I
). These two patients presented with severe oligozoospermia with 0.2 and 0.4x106 spermatozoa/ml respectively.
Analysis by FISH of testis biopsies in the 20 azoospermic patients failed to detect any mosaicism involving the DAZ locus. All cases had apparently normal Y chromosomes with two fluorescent spots except the two patients who were deleted for DAZ by PCR and who had 100% of their testicular cells showing only the Y centromeric signal (Table I).
Discussion
Genetic investigations are now an obligatory step in the screening of male infertility aetiologies. Karyotypic abnormalities are the most common finding in this screening, affecting 5–10% of men with idiopathic severe oligozoospermia or azoospermia (Guichaoua et al., 1991; Johnson, 1998). The rate of chromosomal aberrations in our study (9.9%) is consistent with these findings.
Since the description of infertile men carrying a deletion of the Y chromosome long arm (Tiepolo and Zuffardi, 1976), the existence of genes localized in Yq11 and involved in the normal spermatogenesis process has now been well studied. In patients with non-obstructive azoospermia or severe oligozoospermia, the frequency of Yq microdeletions varies from 1% (Van der Ven et al., 1997) to 55% (Foresta et al., 1998). Such a variation is due mainly to differences in the choice of STS used for PCR analyses and to the clinical and biological criteria for patient's inclusion. In our population of 181 infertile patients, the frequency of 5.5% for deleted Y chromosomes is in agreement with most published studies (Reijo et al., 1995, 1996; Stuppia et al., 1996; Vogt et al., 1996; Kremer et al., 1997; Pryor et al., 1997; Simoni et al., 1997).
Relationships between the size of the deletions, their localization with respect to the three AZF loci and the severity of spermatogenesis impairment in carriers, have been proposed (Vogt et al., 1996): deletions of AZFa are related to the absence of germ cells in seminiferous tubules, resulting in Sertoli cell-only (SCO) syndromes, deletions of the AZFb locus are preferentially associated with a maturation arrest in meiosis, and those affecting AZFc lead to more variable phenotypes ranging from SCO to hypospermatogenesis. This explains why some AZFc deleted patients present only severe oligozoospermia rather than azoospermia and why deletions may be transmitted from subfertile fathers to their male offspring after ICSI (Kent-First et al., 1996b; Cram et al., 1999; Kamischke et al., 1999; Page et al., 1999) or, in very rare cases, from fertile fathers without any assisted reproduction technology (Chang et al., 1999). Large deletions are commonly associated with more severe spermatogenetic defects (McElreavey and Krausz, 1999). In our patients, correlations between genotypic and phenotypic data coincide with these observations: most deletions included the AZFc locus (six out of nine) and were observed either in oligozoospermic or azoospermic men while others covered the AZFb (three out of nine) locus and were found in oligozoospermic patients. These results agree with the expected frequencies of Yq deletions in infertile patients.
Since abnormal Y chromosomes are inherited from fertile fathers, microdeletions can arise in offspring either from de-novo random meiotic errors in paternal germ cell lineages or from paternal constitutional mosaics as a result of mitotic accidents. These accidents are thought to occur prior to testis differentiation during embryogenesis, thus leading to various levels of infertility in adults according to the percentage of deleted cells in testis. Therefore, the question arises as to whether some infertile patients may carry a testicular and/or a somatic Y deletion mosaicism.
Such mosaicisms have already been inferred by Kent-First et al. (1996a) and then confirmed by the same author using PCR reactions in isolated blood and sperm cells from different groups of fertile and infertile men (Kent-First et al., 1999). In that study, the authors found Y-deleted cells both in blood cells and spermatozoa from two infertile ICSI-treated fathers of sons carrying homogenous Y deletions. This is not surprising, but they failed to detect such mosaicisms in three other fertile men who had affected sons. In five infertile males suspected of carrying cryptic deletions after PCR, they found some somatic and germ cells with an apparently deleted Y chromosome in tandem with other cells presenting an intact Y chromosome. However, for these latter patients, no percentages of deleted and non-deleted cells were given.
The detection of STS from a single cell is a time and labour consuming method which requires a non-specific amplification of the whole genome in order to obtain sufficient DNA material for subsequent amplifications of each STS. Although this technique offers a good screening of the different AZF loci, the number of cells which can be analysed is limited and DNA could possibly contaminate the procedure. For these reasons, we chose to investigate the existence of Y deletion mosaicisms in infertile patients using FISH with a fluorescent probe coding for one of the most frequently deleted regions on the Y chromosome, the DAZ gene family in AZFc. This method enables many hundreds or thousands of cell nuclei to be examined quickly and the percentage of mosaicism calculated.
Our results indicate that FISH is a valuable tool for detecting specific deletions of the Y chromosome long arm because it gave hybridization results which were in agreement with PCR data. The percentage of Y chromosomes with two fluorescent signals, corresponding to the centromeric and the DAZ regions, were >99% in controls and in the three patients carrying AZFb deletions. Similarly, >99% of cells from the three men with a proven AZFc (DAZ-negative) deletion exhibited only a fluorescent signal corresponding to the Y centromere. Therefore, FISH appears to be a sensitive and specific method of exploring the Y chromosome constitution of both somatic and germinal cells in fertile and infertile men.
In the group of PCR patients which did not harbour Y-deletions, FISH analysis failed to detect somatic mosaicism for the DAZ locus. Two oligozoospermic men showed a low mosaicism with 1.97 and 4.13% of spermatozoa carrying a deleted Y chromosome while, in azoospermic patients, hybridization of the DAZ probe gave results in agreement with PCR data, and showing no detectable mosaicism. These results suggest that mosaicisms involving Y deletions are not common in infertile men and, if they do occur, the percentage of Y-deleted cells is low. Discrepancy between our results and those presented by Kent-First et al. (1999) could be due to artefactual results in PCR reactions on isolated blood and/or sperm cells. On the contrary, FISH analysis permits accurate and sensitive evaluation of Y microdeletions in a large number of cells. Further studies are needed to evaluate more precisely the frequency of Y deletion mosaicism, both in fertile and infertile men. For this purpose, FISH (with probes coding respectively for the different AZF loci) appears to be the technique of choice.
Acknowledgments
The authors thank Dr Samia Kanafani, Sylvie Giacuzzo and Maryline Perdereau for their technical assistance. This work has been supported by grants from AP-HP ( PHRC AOM 96142 and CRC 96053) and from the Association pour la Recherche contre le Cancer (ARC).
Notes
3 To whom correspondence should be addressed at: Service d'Histologie, Biologie de la Reproduction et Cytogénétique et CECOS, Hôpital Tenon, 4 Rue de la Chine, 75020 Paris, France. E-mail: jean-pierre.dadoune{at}tnn.ap-hop-paris.fr 
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Submitted on January 4, 2000; accepted on May 23, 2000.
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