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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Key words: ICSI/male infertility/mosaicism/Turner syndrome/Y chromosome deletions
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Introduction |
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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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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 14) and four azoospermic (cases 58) 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 12 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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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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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 UlrichTurner 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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Notes |
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References |
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Submitted on May 15, 2000; accepted on August 2, 2000.