1 Departments of Obstetrics and Gynecology and 2 Human Genetics, Royal Victoria Hospital, Women's Pavilion, McGill University, Montreal, QC H3A 1A1, Canada
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Abstract |
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Key words: fluorescence in-situ hybridization/intracytoplasmic sperm injection/Klinefelter syndrome/preimplantation embryos/sex chromosomes
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Introduction |
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Klinefelter patients present with a somatic 47,XXY, or less commonly with a mosaic 46,XY/47,XXY karyotype. Although the clinical features are variable, the majority of patients are infertile due to primary testicular failure. Very few cases of naturally conceived offspring of proven paternity have been reported (Laron et al., 1982; Terzoli et al., 1992
). The retrieval of spermatozoa from severely oligozoospermic ejaculates or from testicular biopsies in Klinefelter patients is possible. With recent developments in reproductive technologies, the recovered spermatozoa have been used for intracytoplasmic sperm injection (ICSI), with successful fertilization of oocytes, and biochemical pregnancies and births have been achieved (Harari et al., 1995
; Staessen et al., 1996
; Bourne et al., 1997
; Hinney et al., 1997
; Tournaye et al., 1997
; Nodar et al., 1998
; Palermo et al., 1998
; Reubinoff et al., 1998
; Ron-El et al., 1999
).
Recent chromosome studies have reported that Klinefelter males have an increased incidence of 24,XY sperm cells compared with that found in karyotypically normal men, demonstrating that 47,XXY spermatogonia may undergo meiosis to produce hyperhaploid spermatozoa (Cozzi et al., 1994; Chevret et al., 1996
; Martini et al., 1996
; Guttenbach et al., 1997
; Hinney et al., 1997
; Estop et al., 1998
; Kruse et al., 1998
). An increase in the frequency of sex chromosome hyperhaploidy in this population of patients may present a risk of retrieving sperm cells with an abnormal sex chromosome complement for ICSI protocols. This, in turn, may increase the risk of chromosomally imbalanced embryos.
In the present study, to determine whether a 46,XY/47,XXY Klinefelter patient had an increased risk of a chromosomally abnormal conceptus, we used three-colour fluorescence in-situ hybridization (FISH) with probes specific to chromosomes X, Y and 18 to examine the sex chromosomes in the patient's spermatozoa and spare preimplantation embryos produced by ICSI. To our knowledge, this is the first study which reports on the sex chromosomes constitution of the sperm cells and embryos from the same Klinefelter patient.
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Materials and methods |
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Preparation of sperm nuclei
Supernumerary spermatozoa suspended in sperm washing medium (Medicult, Hopkington, USA) were obtained after ICSI had been performed according to standard protocols (Palermo et al., 1998). Spermatozoa were prepared for FISH as described previously (Martin and Ko, 1995
). Spermatozoa were washed three times with 10 mmol/l Tris0.9% NaCl. An aliquot (25 µl) of sperm suspension was smeared on glass slides and air-dried. Slides were aged at room temperature for 2 days. Sperm heads were decondensed by a 30 min immersion in dithiothreitol (DTT)/0.1 mol/l Tris, followed by a 3 h immersion in a 1 mmol/l DTT/10 mmol/l lithium diiosalicyalte/0.1 mol/l Tris solution at room temperature. Slides were rinsed in 2x SSC solution for 5 min, and air-dried.
Preparation of blastomeres
Fifteen out of 19 normally fertilized embryos were cultured under oil in Gardner's sequential media (Scandinavian IVF Science, Gothenburg, Sweden) to day 4. Ten spare, monospermic embryos, ranging from the 6- to 11-cell stages of development, were washed in phosphate-buffered saline (PBS). Each embryo was transferred in a microdroplet to a poly-L-lysine-treated slide. Blastomeres were desegregated and nuclei spread with 0.01 NHCl, 0.1% Tween 20 (Coonen et al., 1994). The slides were air-dried, washed in PBS for 5 min, and dehydrated through an ethanol series. The location of the nuclei was marked using a diamond pen.
FISH
Immediately before FISH analysis, slides with embryonic or sperm nuclei were pretreated with pepsin (100 µg/ml) in 0.01 mol/l HCl for 20 min at 37°C, rinsed in PBS, and dehydrated in an ethanol series. Three-colour FISH was performed using directly labelled alpha satellite DNA probes, specific for chromosomes X (spectrum green), Y (spectrum orange) and 18 (spectrum aqua) (Vysis, Downers Grove, IL, USA).
Sperm nuclei FISH
Slides with sperm nuclei were denatured in a Coplin jar containing 70% formamide/2x saline sodium citrate (SSC) at 72°C, for 2 min. Slides were immediately dehydrated in a cold ethanol series, and air-dried. The probe mixture was denatured at 75°C for 5 min, and immediately applied to the specimen. The probe was hybridized to sperm DNA by incubation in a moist chamber at 37°C for 4 h. Slides were washed in 0.04x SSC/0.3% Tween 20 solution for 3 min, followed by 2x SSC, at room temperature, for 1 min. Slides were rinsed in PBS and counterstained with 2 ng/ml of 4',6-diamidino-2-phenylindole (DAPI) for 20 s, rinsed and mounted in anti-fade medium (Vector, Burlingame, CA, USA).
Embryonic nuclei FISH
The probe mixture was applied to the slide with embryonic nuclei and covered with a plastic coverslip. Probe and target nuclear DNA were co-denatured by heating the slide to 78°C for 6 min. Hybridization was carried out in a moist chamber at 37°C for 2 h. Following hybridization, the slides were washed using 0.04x SSC/0.3% Tween 20 solution, at 73°C, for 3 min. The slides were air-dried, and mounted in DAPI (II) counterstain (Vysis).
Analysis
Following FISH, the nuclei and fluorescence signals were viewed using a fluorescence Olympus DX 60 microscope equipped with appropriate filters: FITC/Texas red/DAPI (Applied Imaging, Santa Clara, CA, USA) and Aqua (Vysis). Only intact, non-overlapping embryonic nuclei and sperm heads, with clear fluorescence signals were scored. 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. Images were captured using a CCD camera and Cytovision software (Applied Imaging).
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Results |
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Discussion |
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Multi-colour FISH using chromosome-specific DNA probes has been successfully used to determine the frequencies of numerical chromosome abnormalities in the spermatozoa of both normal and infertile men (Moosani et al., 1995; Martin et al., 1996
; Rademaker et al., 1997
; Finkelstein et al., 1998
; Storeng et al., 1998
; Pang et al., 1999
). Despite the scarcity of spermatozoa in many Klinefelter syndrome males, a number of studies were able to analyse from 24 to 27 097 spermatozoa per patient (Martini et al., 1996
; Hinney et al., 1997
; Estop et al., 1998
; Kruse et al., 1998
). In the present study, we were able to screen chromosomes X, Y and 18 in a total of 358 available spermatozoa.
The total incidence of sex chromosome disomy in our patient's spermatozoa was 3.9%. This value is higher than that determined by FISH (0.14% to 0.43%) for karyotypically normal fertile men (Williams et al., 1993; Wyrobek et al., 1994
; Martin et al., 1996
; Spriggs et al., 1996
; Rademaker et al., 1997
; Storeng et al., 1998
). It is also higher than the reported mean values of sex chromosome disomy for males with abnormal seminal parameters (0.33% to 2.95%) (Moosani et al., 1995
; Storeng et al., 1998
; Pang et al., 1999
). The total frequency of sex chromosome hyperploidy for our patient falls in the range ascertained in other mosaic (0.92% to 5.0%) and non-mosaic (1.36% to 25%) Klinefelter syndrome individuals (Cozzi et al., 1994
; Chevret et al., 1996
; Martini et al., 1996
; Estop et al., 1998
; Kruse et al., 1998
).
Non-disjunction in normal 46,XY spermatogonia in meiosis I should produce the same number of 24,XY and 22,-X,-Y sperm cells. Non-disjunction in meiosis II should produce equal frequencies of spermatozoa with 24,XX, 24,YY and sex chromosome hypohaploidy. In contrast to the increased frequency of spermatozoa with sex chromosome disomy (3.9%), only 0.56% of nuclei from our patient's spermatozoa did not contain gonosomes. The lack of corresponding sperm cells nullsomic for sex chromosomes in our patient suggests that the 24,XY and 24,XX spermatozoa probably arose from the abnormal 47,XXY spermatogonia, thus supporting the evidence from previous studies that these germ cells can complete meiosis.
Although the number of Klinefelter syndrome cases with a known chromosomal constitution of the zygote following ICSI is limited, no affected progeny have been reported to date. A normal karyotype, following fertilization with a Klinefelter patient's spermatozoon, was detected as a 9-week fetus (Hinney et al., 1997). To date, the 11 liveborn in-vitro-conceived Klinefelter patients' offspring have also been found to be chromosomally normal (Bourne et al., 1997
; Tournaye et al., 1997
; Nodar et al., 1998
; Palermo et al., 1998
; Reubinoff et al., 1998
; Ron-El et al., 1999
).
Two recent studies used FISH to analyse preimplantation embryos following ICSI with a Klinefelter patient's spermatozoa. In the first (Staessen et al., 1996), PGD was performed with probes for chromosomes X,Y on five embryos, from a total of three patients. All five embryos were diagnosed with a normal chromosome content. The second study (Reubinoff et al., 1998
) reported on PGD with probes for chromosomes X, Y and 18 in three embryos from two patients. One embryo was diagnosed as normal, was transferred, and resulted in the birth of a karyotypically normal neonate. The biopsied blastomere from the second embryo had an abnormal XX18 constitution. The blastomere from the third embryo was found also to contain an abnormal pattern, XXY1818. Spreading of the remainder of the cells from the above two embryos revealed nuclei with various X and Y patterns, consistent with chaotic cell division. Therefore, among all the abnormal embryos combined, no uniform XXY or XXX chromosome complement had been detected. Our observations in 10 spare preimplantation embryos are very similar. Although only three (33%) of the spare embryos produced by ICSI using our patient's spermatozoa were uniformly normal, none of the abnormal embryos analysed was uniformly aneuploid for the sex chromosomes. Instead, as in the above reports, the abnormal embryos were mosaics with chaotic chromosome segregation patterns.
Intracytoplasmic fertilization of oocytes with a Klinefelter male's 24,XY- or 24,XX-bearing spermatozoa, could theoretically produce a 47,XXY or a 47,XXX embryo. This chromosomal status of ICSI-induced conceptions cannot be excluded from the three transferred embryos among the six reported (Reubinoff et al., 1998), though chromosome analysis was not performed. Similarly, the gonosome constitution of one of the three embryos that had been transferred to our patient remains unknown. Furthermore, diagnosis of six embryos reported earlier by one group (Reubinoff et al., 1998
) and four embryos reported by another group (Staessen et al., 1996
) was based on only single blastomeres. The incidence of chromosomal mosaicism in in-vitro-fertilized human preimplantation embryos is substantial (Coonen et al., 1994
; Munné et al., 1994
; Harper et al., 1995
; Handyside, 1996
; Delhanty et al., 1997
). Among the seven abnormal embryos produced from our patient's spermatozoa, all contained at least one normal XX1818 or XY1818 blastomere in addition to those which were chromosomally abnormal. Therefore, mitotic non-disjunction and/or sex chromosome loss in one blastomere of a 47,XXY or 47,XXX embryo, followed by a biopsy of this now `rescued' blastomere in the embryos diagnosed by PGD is possible. Such an event would lead to a misdiagnosis. It is therefore important to advise PGD patients to undergo prenatal diagnosis to ensure the normality of the fetus.
In conclusion, despite an increased frequency of sperm hyperhaploid for sex chromosomes, FISH analysis of our patient's spermatozoa indicated that the majority of spermatozoa (94.4%) were normal for the chromosomes tested. This agrees with findings in other Klinefelter cases, and confirms that the karyotype in peripheral blood in this population of patients is not indicative of the chromosome constitution of other tissues such as the testis. Furthermore, ICSI of the patient's spermatozoa induced fertilization, produced a high percentage of cleavage-stage embryos, and led to implantation and ongoing pregnancy. None of the spare embryos analysed had a Klinefelter syndrome karyotype. In order to have a clear understanding of the risk of affected progeny in Klinefelter males procreating via ICSI, further screening for gonosomes in spermatozoa, preimplantation spare embryos, concepti and neonates of this population of patients is necessary.
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Acknowledgments |
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Notes |
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References |
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Chevret, E., Rousseaux, S., Monteil, M. et al. (1996) Increased incidence of hyperhaploid 24,XY spermatozoa detected by three-colour FISH in a 46,XY/47,XXY male. Hum. Genet., 97, 171175.[ISI][Medline]
Coonen, E., Harper, J.C., Ramaekers, F.C.S. et al. (1994) Presence of chromosomal mosaicism in abnormal human preimplantation embryos detected by fluorescent in situ hybridization (FISH). Hum. Genet., 54, 609615.
Cozzi, J., Chevret, E., Rousseaux, S. et al. (1994) Achievement of meiosis in XXY germ cells: study of 543 sperm karyotypes from an XY/XXY mosaic patient. Hum. Genet., 93, 3234.[ISI][Medline]
De Braekeleer, M. and Dao, T.N. (1991) Cytogenetic studies in male infertility: a review. Hum. Reprod., 6, 245250.[ISI][Medline]
Delhanty, J.D., Harper, J.C., Ao, A. et al. (1997) Multicolour FISH detects frequent chromosomal mosaicism and chaotic division in normal preimplantation embryos from fertile patients. Hum. Genet., 99, 755760.[ISI][Medline]
Estop, A.M., Munné, S., Cieply, K.M. et al. (1998) Meiotic products of a Klinefelter 47,XXY male as determined by sperm fluorescence in-situ hybridization analysis. Hum. Reprod., 13, 124127.[Abstract]
Finkelstein, S., Mukamel, E., Yavetz, H. et al. (1998) Increased rate of nondisjunction in sex cells derived from low-quality semen. Hum. Genet., 102, 129137.[ISI][Medline]
Guttenbach, M., Michelmann, H.W., Hinney, B. et al. (1997) Segregation of sex chromosomes into sperm nuclei in a man with 47,XXY Klinefelter's karyotype: a FISH analysis. Hum. Genet., 99, 474477.[ISI][Medline]
Handyside, A.H. (1996) Mosaicism in the human preimplantation embryo. Reprod. Nutr. Dev., 36, 643649.[ISI][Medline]
Harari, O., Bourne, H., Baker, G. et al. (1995) High fertilization rate with intracytoplasmic sperm injection in mosaic Klinefelter's syndrome. Fertil. Steril., 63, 182184.[ISI][Medline]
Harper, J.C., Coonen, E., Handyside, A.H. et al. (1995) Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic preimplantation human embryos. Prenat. Diagn., 15, 4149.[ISI][Medline]
Hassold, T. and Jacobs, P.A. (1984) Trisomy in man. Annu. Rev. Genet., 6, 555565.
Hinney, B., Guttenbach, M., Schmid, M. et al. (1997) Pregnancy after intracytoplasmic sperm injection with sperm from a man with a 47,XXY Klinefelter's karyotype. Fertil. Steril., 68, 718720.[ISI][Medline]
Kruse, R., Guttenbach, M., Schartmann, B. et al. (1998) Genetic counseling in a patient with XXY/XXXY/XY mosaic Klinefelter's syndrome: estimate of sex chromosome aberrations in sperm before intracytoplasmic sperm injection. Fertil. Steril., 69, 482485.[ISI][Medline]
Laron, Z., Dickerman, Z., Zamir, R. et al. (1982) Paternity in Klinefelter's syndrome a case report. Arch. Androl., 8, 149151.[ISI][Medline]
Martin, R.H. and Ko, E. (1995) Fluorescence in situ hybridization in human sperm. In: Verma, R.S. and Babu, A. (eds), Human Chromosomes: Principles and techniques. 2nd edn. McGraw-Hill, New York, pp. 223231.
Martin, R.H., Spriggs, E. and Rademaker, A.W. (1996) Multicolor fluorescence in situ hybridization analysis of aneuploidy and diploidy frequencies in 225,846 sperm from 10 normal men. Biol. Reprod., 54, 394398.[Abstract]
Martini, E., Geraedts, J.P., Liebaers, I. et al. (1996) Constitution of semen samples from XYY and XXY males as analysed by in-situ hybridization. Hum. Reprod., 11, 16381643.[Abstract]
Moosani, N., Pattinson, H.A., Carter, M.D. et al. (1995) Chromosomal analysis of sperm from men with idiopathic infertility using sperm karyotyping and fluorescence in situ hybridization. Fertil. Steril., 64, 811817.[ISI][Medline]
Munné, S., Weier, H.U., Grifo, J. et al. (1994) Chromosome mosaicism in human embryos. Biol. Reprod., 51, 373379.[Abstract]
Nielsen, J. and Wohlert, M. (1991) Chromosome abnormalities found among 34,910 newborn children: results from a 13-year incidence study in Arhus, Denmark. Hum. Genet., 87, 8183.[ISI][Medline]
Nodar, F., De Vincentiis, S., Olmedo, S.B. et al. (1998) Birth of twin males with normal karyotype after intracytoplasmic sperm injection with use of testicular spermatozoa from a nonmosaic patient with Klinefelter's syndrome. Fertil. Steril., 71, 11491152.[ISI]
Okada, H., Fujioka, H., Tatsumi, N. et al. (1999) Klinefelter's syndrome in the male infertility clinic. Hum. Reprod., 14, 946952.
Palermo, G., Joris, H., Devroey, P. et al. (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet, 340, 1718.[ISI][Medline]
Palermo, G.D., Schlegel, P.N., Sills, E.S. et al. (1998) Births after intracytoplasmic injection of sperm obtained by testicular extraction from men with nonmosaic Klinefelter's syndrome. N. Engl. J. Med., 338, 588590.
Pang, M.G., Hoegerman, S.F., Cuticchia, A.J. et al. (1999) Detection of aneuploidy for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X and Y by fluorescence in-situ hybridization in spermatozoa from nine patients with oligoasthenoteratozoospermia undergoing intracytoplasmic sperm injection. Hum. Reprod., 14, 12661273.
Rademaker, A., Spriggs, E., Ko, E. et al. (1997) Reliability of estimates of diploid human spermatozoa using multicolour fluorescence in-situ hybridization. Hum. Reprod., 12, 7779.[ISI][Medline]
Reubinoff, B.E., Abeliovich, D., Werner, M. et al. (1998) A birth in non-mosaic Klinefelter's syndrome after testicular fine needle aspiration, intracytoplasmic sperm injection and preimplantation genetic diagnosis. Hum. Reprod., 13, 18871892.[Abstract]
Ron-El, R., Friedler, S., Strassburger, D. et al. (1999) Birth of a healthy neonate following the intracytoplasmic injection of testicular spermatozoa from a patient with Klinefelter's syndrome. Hum. Reprod., 14, 368370.
Spriggs, E.L., Rademaker, A.W. and Martin, R.H. (1996) Aneuploidy in human sperm: the use of multicolor FISH to test various theories of nondisjunction. Am. J. Hum. Genet., 58, 356362.[ISI][Medline]
Staessen, C., Coonen, E., Van Assche, E. et al. (1996) Preimplantation diagnosis for X and Y normality in embryos from three Klinefelter patients. Hum. Reprod., 11, 16501653.[Abstract]
Storeng, R.T., Plachot, M., Theophile, D. et al. (1998) Incidence of sex chromosome abnormalities in spermatozoa from patients entering an IVF or ICSI protocol. Acta Obstet. Gynecol. Scand., 77, 191197.[ISI][Medline]
Terzoli, G., Lalatta, F., Lobbiani, A. et al. (1992) Fertility in a 47,XXY patient: assessment of biological paternity by deoxyribonucleic acid fingerprinting. Fertil. Steril., 58, 821822.[ISI][Medline]
Tournaye, H., Staessen, C., Liebaers, I. et al. (1996) Testicular sperm recovery in nine 47,XXY Klinefelter patients. Hum. Reprod., 11, 16441649.[Abstract]
Tournaye, H., Camus, M., Vandervorst, M. et al. (1997) Surgical sperm retrieval for intracytoplasmic sperm injection. Int. J. Androl., 20 (suppl. 3), 6973.[ISI][Medline]
Williams, B.J., Ballenger, C.A., Malter, H.E. et al. (1993) Non-disjunction in human sperm: results of fluorescence in situ hybridization studies using two and three probes. Hum. Mol. Genet., 2, 19291936.[Abstract]
Wyrobek, A.J., Robbins, W.A., Mehraein, Y. et al. (1994) Detection of sex chromosomal aneuploidies X-X, Y-Y, and X-Y in human sperm using two-chromosome fluorescence in situ hybridization. Am. J. Med. Genet., 53, 17.[ISI][Medline]
Submitted on July 2, 1999; accepted on November 4, 1999.