Human Reproduction, Vol. 15, No. 12, 2559-2562,
December 2000
© 2000 European Society of Human Reproduction and Embryology
Sex chromosome mosaicism in males carrying Y chromosome long arm deletions
1 Service d'Histologie, Biologie de la Reproduction et Cytogénétique et CECOS, Hôpital Tenon, 4 Rue de la Chine, 75020 Paris, 2 Laboratoire d'Immunogénétique Humaine, Institut Pasteur, Paris, France, 3 Andrology Unit, University of Florence, Firenze, Italy, 4 Département de Génétique, Institut Pasteur, Casablanca, Maroc, 5 CECOS Midi-Pyrénées, Centre de Stérilité Masculine and Research Group on Human Reproduction, CHU La Grave, Toulouse, 6 Service de Génétique Médicale, CHU Purpan, Toulouse and 7 Service de Biologie de la Reproduction et du Développement, Centre Hospitalier Universitaire, Clermont Ferrand, France
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Abstract |
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Microdeletions of the long arm of the Y chromosome (Yq) are a common cause of male infertility. Since large structural rearrangements of the Y chromosome are commonly associated with a 45,XO/46,XY chromosomal mosaicism, we studied whether submicroscopic Yq deletions could also be associated with the development of 45,XO cell lines. We studied blood samples from 14 infertile men carrying a Yq microdeletion as revealed by polymerase chain reaction (PCR). Patients were divided into two groups: group 1 (n = 6), in which karyotype analysis demonstrated a 45,X/46,XY mosaicism, and group 2 (n = 8) with apparently a normal 46,XY karyotype. 45,XO cells were identified by fluorescence in-situ hybridization (FISH) using X and Y centromeric probes. Lymphocytes from 11 fertile men were studied as controls. In addition, sperm cells were studied in three oligozoospermic patients in group 2. Our results showed that large and submicroscopic Yq deletions were associated with significantly increased percentages of 45,XO cells in lymphocytes and of sperm cells nullisomic for gonosomes, especially for the Y chromosome. Moreover, two isodicentric Y chromosomes, classified as normal by cytogenetic methods, were detected. Therefore, Yq microdeletions may be associated with Y chromosomal instability leading to the formation of 45,XO cell lines.
Key words: ICSI/male infertility/mosaicism/Turner syndrome/Y chromosome deletions
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Introduction |
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The Y chromosome is necessary for male germ cell development and the loss of Y chromosome sequences in the euchromatic region of the long arm (Yq) is a major cause of male infertility (Tiepolo and Zuffardi, 1976
; Vogt et al., 1992). Between 10 and 20% of phenotypically normal men with idiopathic infertility and an apparently intact Y chromosome, carry microdeletions in the euchromatic region of the long arm resulting in the loss of genes necessary for fertility (azoospermia factor, AZF) (Reijo et al., 1995, 1996; Kremer et al., 1997). These cytogenetically undetectable microdeletions define three regions of Yq11 (AZFa, AZFb, and AZFc, in proximal, middle and distal Yq respectively) (Vogt, 1998). Deletions of AZFc are the most common and are associated either with oligozoospermia or complete absence of germ cells (Reijo et al., 1995, 1996).
Structural aberrations of the Y chromosome, such as ring Y, iso or isodicentric for the short arm Yp or large cytogenetically visible deletions of the long arm Yq, are commonly associated with the occurrence of 45,XO cell lines due to a probable mitotic instability of the abnormal chromosome (Hsu, 1994). The relationship between Y microdeletions and the formation of 45,XO cell lines has not been investigated. In this study we show that some individuals with a Y chromosome microdeletion harbour a significant population of 45,XO cells in both peripheral blood lymphocytes and in germ cells. These results not only indicate a novel mechanism for the formation of 45,XO Turner's syndrome but highlight an important potential risk for offspring born to fathers carrying Y microdeletions and treated by assisted reproductive techniques such as intracytoplasmic sperm injection (ICSI). This risk includes the phenotypic anomalies frequently observed in association with sex chromosome mosacism, including ambiguous genitalia.
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Materials and methods |
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We analysed 14 infertile men (mean age 33.7 ± 4.9 years) in whom Yq microdeletions were defined in the course of genetic investigations made for cases of non-obstructive severe oligozoospermia (n = 4) or azoospermia (n = 10). Deletions were diagnosed by polymerase chain reaction (PCR) using Y-specific sequence tagged sites (STS) spreading the three different AZF loci (a,b,c). Deletions were ascertained only after repetitive negative PCR, and confirmed by Southern blotting.
Karyotypes were previously performed by analysing 20 cells using classical cytogenetic methods and allowed the patients to be divided into two groups: group 1, six azoospermic males (cases A, B, C, D, E, F) each with a 45,XO cell line determined by standard karyotype analysis; group 2, four oligozoospermic (cases 1–4) and four azoospermic (cases 5–8) men whose karyotypes were defined as 46,XY by routine karyotype analysis. In the latter group, sex chromosome mosaicism was determined by a re-analysis of at least 300 somatic cells by fluorescence in-situ hybridization (FISH) using the centromeric probes DYZ3 (Y chromosome) and DXZ1 (X chromosome; ONCOR, Gaithersburg, MD, USA). Briefly, lymphocytes from fresh or frozen buffy coat samples were prepared for hybridization by incubating in hypotonic KCl (0.56%) for 15 min at 37°C and then fixed three times in methanol/acetic acid 3:1 (vol/vol) at room temperature. Cells were spread on glass slides, air dried and hybridized according to manufacturer's recommendations.
In three oligozoospermic patients (cases 1, 2, 3), sperm samples were available for a screening by FISH of a gonosome mosaicism in germinal cells. After sperm liquefaction for 30 min at 37°C, spermatozoa were washed three times in phosphate-buffered saline (PBS) and then fixed in methanol/acetic acid 3:1 (vol/vol) 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 LIS (di-iodosalicylic acid lithium) (Sigma) for 1–2 h at room temperature, washed in 2xsaline sodium citrate/standard saline citrate (SSC) for 1 h at 37°C, dehydrated in increasing gradients of ethanol and air dried. FISH was performed by incubating slides in 50 µl of hybridization buffer (Hybrizol buffer VII; Oncor) containing 1.5 µl of each X, Y and 18 centromeric probes (Oncor) labelled with rhodamin (Y), fluorescein isothiocyanate (FITC) (18) or a mixture of both dyes (X). After sealing of cover glasses with rubber cement, slides were placed in a thermocycler (Omnislide, Hybaid, Ashford, UK) and allowed to denature for 10 min at 73°C. The hybridization was performed at 37°C overnight. Slides were then washed in 0.4xSSC/0.1% Tween 20 for 4 min at 73°C and then in 0.1xSSC/0.1% Tween 20 for 2 min at room temperature. Staining was performed using 30 µl of a mixture containing an antibody to digoxigenin coupled to rhodamine (Oncor) and avidin/FITC (Oncor). Slides were incubated at 37°C for 10 min and then rinsed three times in 1xSSC/0.1% Tween 20 at room temperature.
After mounting and counterstaining in Vectashield/4,6-diamidino-2-phenylindole (DAPI) (Vector, Burlingame, CA, USA), a double-blind analysis of FISH signals was performed under UV light on a photomicroscope (Zeiss Axiophot, Oberkochen, Germany) equipped with epifluorescence optics and an appropriate filter set. At least 200 somatic cells were counted for each case. Respectively 500 and 700 spermatozoa were counted for patients 1 and 3. In patient 2, 150 spermatozoa and 100 round sperm cells were analysed. As controls, 1759 somatic cells from 11 fertile men (mean age 33.7 ± 6.6 years) and 104 spermatozoa from 11 other men with normal sperm counts (mean age 35.4 ± 4.7 years) were prepared and analysed in the same conditions. Statistical analysis of differences between patients of group 2 and controls was made using the non-parametric Wilcoxon test (PC StatView 5.0 Program).
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Results |
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Details of the bioclinical characteristics of patients and percentages of abnormal somatic cells are shown in Table I
. In group 1, all patients showed an apparently normal Y chromosome in their 46,XY cell line when analysed by standard cytogenetic methods. All patients had a very high percentage of 45,XO cells, ranging from 24 to 82%. Other chromosomes were normal except for patient C, who carried a pericentric inversion of chromosome 10. However, in two men (cases A and F), these Y chromosomes exhibited two fluorescent signals after FISH when hybridized with the DYZ3 centromeric probe. Therefore, these karyotypes were reclassified as isodicentric, 45,XO/46,Xidic(Y) (q11.2). In case B, FISH revealed that the Y chromosome was monocentric. Unfortunately, for patients C, D and E, lymphocytes were no longer available to perform FISH. Two patients (C, D) carried large terminal deletions encompassing loci AZFa, b and c while the four others had terminal deletions restricted to AZFb and c (A, B, F) or AZFc (E).
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In group 2, karyotype revealed a single case (no. 2) with a detectable deletion and a complex mosaicism by FISH [45,XO (8%) and 47,XXY (4%)]. In other patients, Y chromosomes were normal by cytogenetic methods; however, molecular deletions were interstitial and limited to AZFb (cases 3, 8) or AZFc (cases 1, 4, 5, 6, 7). In this group as a whole, percentages of 45,XO cells in lymphocytes were lower than in group 1 but statistically different from controls (P < 0.05).
Screening by FISH for mosaicism in germ cells of the three oligozoospermic patients (cases 1, 2, 3) revealed statistically significant percentages (respectively 18.7, 15.1 and 17.7; P < 0.001) of cells with Y chromosome loss (Table II). Interestingly, in these three patients, sex ratio between normal Y/18 and X/18 spermatozoa was abnormal with a low percentage of Y/18 cells in comparison with the observed 
50% rate of X bearing spermatozoa. In case no. 2, the existence of a 45,XO germ cell lineage was confirmed by the observation of the numerous round germ cells present in the ejaculate which revealed a percentage of 36.3% of cells lacking the Y chromosome.
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Discussion |
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These results demonstrate that microdeletions of the Y chromosome long arm can be associated with somatic and germinal gonosomal mosaicisms. Structural cytogenetically visible aberrations of the Y chromosome in humans are frequently associated with aneuploid karyotypes. In a review of 600 cases of Y aneuploidy, it was found (Hsu, 1994
) that 45,XO cell lines were present in more than half of the cases of postnatally diagnosed carriers of aberrant Y chromosomes. Our findings support the close association between rearranged or large Yq deletions and the occurrence of a 45,XO cell line and extend these findings to include Yq microdeletions, frequently observed in infertile men, associated with a significant gonosomal mosaicism in both somatic cells and in germ cells. Loss of gonosomes, X in females and Y in males, is a normal and common event observed in peripheral lymphocytes or bone marrow cells in elderly people (Guttenbach et al., 1995; Stone and Sandberg, 1995). The mean age of the patients in this study did not differ significantly from controls, suggesting that sex chromosome mosaicism might be related to the presence of the abnormal Y chromosome itself. The human Y chromosome is often the first chromosome lost from tumour cells (bladder cancer, renal cell carcinoma, renal oncocytoma, oesophageal carcinoma, non-small lung cell cancer) (Riske et al., 1994) and is highly susceptible to intrachromosomal deletions associated with male infertility. The majority of Y microdeletions are de novo and neither their cellular origin, nor the underlying mechanism has been determined. The relatively high frequency of Y deletions suggests that this chromosome is susceptible to the spontaneous loss of genetic material. Deletions may be caused by different mechanisms: aberrant recombination between areas of homologous or similar sequence repeats between the X and Y chromosomes, aberrant intrachromosomal recombination by unbalanced sister chromatid exchange, or by slippage during DNA replication (McElreavey et al., 1999). It is possible that Y rearrangements or deletions may reflect a more general genomic instability, leading ultimately to the loss of the entire chromosome in some cell populations. Such an instability could be more pronounced in germ cells than in somatic ones, as suggested by the higher rate of nullisomic spermatozoa for gonosomes than 45,X0 lymphocytes found in our study.
The data reported here are of clinical significance. We observed significant loss of the Y chromosome in men with AZFc deletions. This deletion, estimated to be 2x106 bp in size is the most frequent deletion associated with infertility. The presence of 45,XO cells particularly in the germ line may be a contributing factor in the phenotypic variability associated with AZFc deletions. More importantly, it is possible that microdeleted Y chromosomes may contribute to the development of 45,XO Ulrich–Turner syndrome in the offspring of deleted men treated by ICSI. Turner syndrome patients usually lack the paternal X chromosome and an increased frequency of X-Y non-disjunction in meiosis I has been shown in some fathers of affected girls (Martinez-Pasarell et al., 1999). The presence of a 45,XO/46,XY mosaicism in the father's gonads could also lead either to the formation of a monosomic X embryo or to the transmission of a potentially unstable Y chromosome to a male fetus. In the latter event, a 45,XO/46,XY mosaicism could occur in the early steps of embryo development, leading to major clinical consequences in the new-born boy, such as ambiguous external genitalia or mixed gonadal dysgenesis (Hsu, 1994; Lazebnik et al., 1996). An increased risk for Turner's syndrome and gonosomal mosaicism, 45,XO/46,XY, has already been described in babies conceived by ICSI (In't Veld et al., 1995; Van Opstal et al., 1997). Although molecular deletions of the Y chromosome have not been screened in these cases, the parental origin of the missing X chromosome was always paternal (Van Opstal et al., 1997).
This hypothesis, if confirmed by other studies, has an impact on genetic counselling given to couples seeking ICSI and in which the male partner carries a Y chromosome microdeletion. On the basis that these deletions do not trigger any risk other than a sperm production failure in male progeny, ICSI has already been successfully proposed to such couples (Mulhall et al., 1997; Rossato et al., 1998). To our knowledge, the few babies born after ICSI from Y-deleted men are phenotypically normal. However, we recommend that every patient carrying a Y chromosome microdeletion and asking for ICSI undergoes screening for the detection of a gonosomal mosaicism by multicolour FISH performed on somatic and, if available, germinal cells.
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Acknowledgments |
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The authors thank Professor Jean Marie Antoine, Drs Jerome Pfeffer, Jean Paul Taar and Simone Zerah for their contribution to the recruitment of patients. They also thank Brigitte Fontaine, Sylvie Giacuzzo and Maryline Perdereau for their technical assistance. This work has been supported by grants from AP-HP (PHRC AOM96142 and CRC 96053), the Association pour la Recherche sur la Cancer (ARC) and Telethon Italy (Grant n281/b).
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Notes |
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8 To whom correspondence should be addressed at: Hôpital Tenon, 4 Rue de la Chine, 75020 Paris France. E-mail: jean-pierre.siffroi{at}tnn.ap-hop-paris.fr
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Submitted on May 15, 2000; accepted on August 2, 2000.
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