Human Reproduction vol.15 no.2 pp.440–444, 2000

Fluorescence in-situ hybridization of sex chromosomesin spermatozoa and spare preimplantation embryos of aKlinefelter 46,XY/47,XXY male

Magdalena Bielanska1, Seang Lin Tan1 andAsangla Ao1,2,3

Departments of 1Obstetrics and Gynecology and 2Human Genetics,Royal Victoria Hospital, Women’s Pavilion, McGill University,Montreal, QC H3A 1A1, Canada

3To whom correspondence should be addressed

It has been suggested recently that 47,XXY germ cells areable to progress through meiosis to produce hyperhaploidspermatozoa. We report on a 46,XY/47,XXY Klinefelterpatient whose spermatozoa were recovered from the ejacu-late and used for intracytoplasmic sperm injection (ICSI).Fluorescence in-situ hybridization (FISH) analysis of thepatient’s spermatozoa and of spare preimplantationembryos with DNA probes specific for chromosomes X, Yand 18 revealed sex chromosome hyperploidy in 3.9% ofthe sperm nuclei analysed (2.23% XY18, 1.12% XX18,0.56% YY18), while only three out of 10 spare embryosanalysed were normal for chromosomes tested. The abnor-malities included two diploid mosaic embryos with themajority of the blastomeres normal for the chromosomestested, and five embryos with mostly abnormal blastomeresand chaotic chromosome X, Y and 18 patterns. None ofthe embryos analysed showed a XXY1818 or XXX1818chromosome complement. The frequency of sex chromo-some hyperploidy in the spermatozoa of the mosaic Kline-felter patient was higher than the mean reported forkaryotypically normal males, supporting the hypothesisthat 47,XXY germ cells are able to complete meiosis andproduce aneuploid spermatozoa. However, most of thespermatozoa analysed were normal for sex chromosomes,and ICSI of the patient’s spermatozoa did not result in aspare embryo with a uniform 47,XXY or 47,XXX chromo-some complement. Instead, fertilization produced a highpercentage of mosaic embryos with chaotic chromosomearrangements.Key words: fluorescence in-situ hybridization/intracytoplasmicsperm injection/Klinefelter syndrome/preimplantation embryos/sex chromosomes

Introduction

With an incidence of approximately 1 in 600 in live births,and 1 in 300 in spontaneous abortions, Klinefelter syndromeis the most common sex chromosome abnormality in humans(Hassold and Jacobs, 1984; Nielsen and Wolhert, 1991). It isalso the most frequent chromosomal abnormality among infer-

440 © European Society of Human Reproduction and Embryology

tile males, accounting for 4.6% of oligozoospermic or azoo-spermic men (De Braekeleer and Dao, 1991).

Klinefelter patients present with a somatic 47,XXY, or lesscommonly with a mosaic 46,XY/47,XXY karyotype. Althoughthe clinical features are variable, the majority of patients areinfertile due to primary testicular failure. Very few cases ofnaturally conceived offspring of proven paternity have beenreported (Laron et al., 1982; Terzoli et al., 1992). The retrievalof spermatozoa from severely oligozoospermic ejaculates orfrom testicular biopsies in Klinefelter patients is possible.With recent developments in reproductive technologies, therecovered spermatozoa have been used for intracytoplasmicsperm 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-Elet al., 1999).

Recent chromosome studies have reported that Klinefeltermales have an increased incidence of 24,XY sperm cellscompared with that found in karyotypically normal men,demonstrating that 47,XXY spermatogonia may undergomeiosis to produce hyperhaploid spermatozoa (Cozzi et al.,1994; Chevret et al., 1996; Martini et al., 1996; Guttenbachet al., 1997; Hinney et al., 1997; Estop et al., 1998; Kruseet al., 1998). An increase in the frequency of sex chromosomehyperhaploidy in this population of patients may present a riskof retrieving sperm cells with an abnormal sex chromosomecomplement for ICSI protocols. This, in turn, may increasethe risk of chromosomally imbalanced embryos.

In the present study, to determine whether a 46,XY/47,XXYKlinefelter patient had an increased risk of a chromosomallyabnormal conceptus, we used three-colour fluorescence in-situhybridization (FISH) with probes specific to chromosomes X,Y and 18 to examine the sex chromosomes in the patient’sspermatozoa and spare preimplantation embryos produced byICSI. To our knowledge, this is the first study which reportson the sex chromosomes constitution of the sperm cells andembryos from the same Klinefelter patient.

Materials and methods

Patients

The couple, a 36-year-old female and her 35-year-old husband, werereferred to the McGill Reproductive Centre for infertility arising fromprimary testicular failure associated with a karyotype consistent withKlinefelter syndrome. Two peripheral blood chromosome analyseshad been performed. One revealed a 47,XXY karyotype in all 50cells analysed. The second investigation, at a different medical

Hyperploidy in spermatozoa and embryos in a Klinefelter male

laboratory, disclosed the probable presence of low-level mosaicism,with one cell in 60 showing a 46,XY karyotype. The patient had anelevated concentration of follicle stimulating hormone (FSH). Pastseminal analysis demonstrated severe oligozoospermia or azoosper-mia. Analysis of semen used for ICSI showed severe oligozoospermia,with a sperm count of 0.1�106 per ml of ejaculate and motility of60%. The wife had no evidence of fertility problems. The coupleopted for preimplantation genetic diagnosis (PGD). However, due totechnical difficulties, FISH results could not be obtained on thebiopsied blastomeres. After consultation with the patients, threeembryos of unknown sex chromosome constitution were transferred,and resulted in an ongoing twin pregnancy.

Preparation of sperm nuclei

Supernumerary spermatozoa suspended in sperm washing medium(Medicult, Hopkington, USA) were obtained after ICSI had beenperformed according to standard protocols (Palermo et al., 1998).Spermatozoa were prepared for FISH as described previously (Martinand Ko, 1995). Spermatozoa were washed three times with 10 mmol/l Tris–0.9% NaCl. An aliquot (2–5 µl) of sperm suspension wassmeared on glass slides and air-dried. Slides were aged at roomtemperature for 2 days. Sperm heads were decondensed by a 30 minimmersion in dithiothreitol (DTT)/0.1 mol/l Tris, followed by a 3 himmersion in a 1 mmol/l DTT/10 mmol/l lithium diiosalicyalte/0.1mol/l Tris solution at room temperature. Slides were rinsed in 2�SSC solution for 5 min, and air-dried.

Preparation of blastomeres

Fifteen out of 19 normally fertilized embryos were cultured underoil 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 washedin phosphate-buffered saline (PBS). Each embryo was transferred ina microdroplet to a poly-L-lysine-treated slide. Blastomeres weredesegregated and nuclei spread with 0.01 NHCl, 0.1% Tween 20(Coonen et al., 1994). The slides were air-dried, washed in PBS for5 min, and dehydrated through an ethanol series. The location of thenuclei was marked using a diamond pen.

FISH

Immediately before FISH analysis, slides with embryonic or spermnuclei were pretreated with pepsin (100 µg/ml) in 0.01 mol/l HCl for20 min at 37°C, rinsed in PBS, and dehydrated in an ethanol series.Three-colour FISH was performed using directly labelled alphasatellite 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 containing70% formamide/2� 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, andimmediately applied to the specimen. The probe was hybridized tosperm DNA by incubation in a moist chamber at 37°C for 4 h. Slideswere washed in 0.04� SSC/0.3% Tween 20 solution for 3 min,followed by 2� SSC, at room temperature, for 1 min. Slides wererinsed in PBS and counterstained with 2 ng/ml of 4�,6-diamidino-2-phenylindole (DAPI) for 20 s, rinsed and mounted in anti-fademedium (Vector, Burlingame, CA, USA).

Embryonic nuclei FISH

The probe mixture was applied to the slide with embryonic nucleiand covered with a plastic coverslip. Probe and target nuclear

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Table I. Results from fluorescence in-situ hybridization of patient’sspermatozoa with probes specific for chromosomes X, Y and 18

FISH Presumed No. of % of spermatozoaresults karyotype spermatozoa

X18 23,X 180 50.28Y18 23,Y 158 44.13XX18 24,XX 4 1.12YY18 24,YY 2 0.56XY18 24,XY 8 2.23XY1818 46,XY 3 0.84XXY18 24,XXY,-18 1 0.2818 22, -X or -Y 2 0.56Total 358 100

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 0.04� 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 viewedusing a fluorescence Olympus DX 60 microscope equipped withappropriate filters: FITC/Texas red/DAPI (Applied Imaging, SantaClara, CA, USA) and Aqua (Vysis). Only intact, non-overlappingembryonic nuclei and sperm heads, with clear fluorescence signalswere scored. Two signals of the same colour were considered torepresent two individual chromosomes only when the same-colouredsignals were a minimum of one diameter apart. Images were capturedusing a CCD camera and Cytovision software (Applied Imaging).

Results

Spermatozoa

FISH results from a total of 358 spermatozoa available foranalysis are presented in Table I. In total, 338 (94.4%) spermcells were normal with an X18 (50.28%) or a Y18 (44.13%)chromosome pattern. The difference in 23Y- and 23X-bearingspermatozoa was not statistically significant. An abnormalchromosome complement was detected in 5.6% of spermato-zoa. Sex chromosome disomy was detected in 3.9% of nucleiand included cells with XY18 (2.23%), XX18 (1.12%) andYY18 (0.56%) signals (Figure 1a). Other abnormal chromo-some patterns detected included XY1818 (0.84%), XXY18(0.28%) and 018 (0.56%).

Embryos

A total of 80 nuclei, obtained from 75 blastomeres, werespread from ten day 4 embryos. Upon analysis, 67 (84%)yielded intact nuclear material with large, bright fluorescencesignals. The results of FISH using probes specific for chromo-somes X, Y and 18 are shown in Table II. Of ten normallyfertilized spare embryos analysed, three were normal forchromosomes tested, one with an XX1818, and two with anXY1818 pattern. Two embryos were diploid mosaic, withthe majority of blastomeres uniformly normal for the threechromosomes tested (Figure 1b). Five embryos consisted ofmostly abnormal blastomeres, with a variable number of XY18

M.Bielanska, S.L.Tan and A.Ao

Figure 1. Fluorescence in-situ hybridization of Klinefelter syndrome patient’s spermatozoa and spare preimplantation embryo with CEP®

alpha satellite DNA probes specific for chromosomes X (green), Y (red) and 18 (aqua). (a) An abnormal sperm nucleus showing an XY18chromosome complement. (b) Normal nucleus of a blastomere from a mosaic embryo showing XY1818 chromosome signals. (c–f)Abnormal nuclei from four blastomeres of a chaotic embryo showing varied number of XY18 signals, (c) Y181818, (d) XXYY1818, (e)X1818, (f) Y18. (Original magnification,�1000.)

signals in the nuclei, consistent with chaotic chromosomesegregation (Figure 1c–f). None of the seven spare embryoswith chromosomally abnormal blastomeres showed a uniformXXY1818 or XXX1818 complement. Three embryos ofunknown sex chromosome constitution which had been trans-ferred resulted in an ongoing twin pregnancy. Amniocentesisshowed both fetal karyotypes to be 46,XX.

Discussion

Most Klinefelter patients with a pure 47,XXY somatic karyo-type are azoospermic, and spermatozoa for ICSI are retrievedvia testicular biopsy (Staessen et al., 1996; Tournaye et al.,1996; Nodar et al., 1998; Palermo et al., 1998; Reubinoffet al., 1998; Ron-El et al., 1999). In contrast, in cases with a46,XY/47,XXY mosaic karyotype, spermatozoa can usuallybe recovered from the ejaculate (Harari et al., 1995; Martiniet al., 1996; Guttenbach et al., 1997; Hinney et al., 1997;Kruse et al., 1998; Okada et al., 1999). Our patient’s cyto-genetic analysis had shown a dominantly 47,XXY karyotype,with only one 46,XY cell in 60. The proband was diagnosedwith primary testicular failure; however, his ejaculate containedenough spermatozoa for ICSI and for subsequent FISH analysis.This finding demonstrates the presence of limited spermato-genesis in this patient. Furthermore, it indicates testicularmosaicism, and suggests that the one 46,XY cell observed wasnot an artefact. Perhaps the mosaicism also extended to, and

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was present in various percentages in, other non-investigatedtissues.

Multi-colour FISH using chromosome-specific DNA probeshas been successfully used to determine the frequencies ofnumerical chromosome abnormalities in the spermatozoa ofboth normal and infertile men (Moosani et al., 1995; Martinet al., 1996; Rademaker et al., 1997; Finkelstein et al., 1998;Storeng et al., 1998; Pang et al., 1999). Despite the scarcityof spermatozoa in many Klinefelter syndrome males, a numberof studies were able to analyse from 24 to 27 097 spermatozoaper patient (Martini et al., 1996; Hinney et al., 1997; Estopet al., 1998; Kruse et al., 1998). In the present study, we wereable to screen chromosomes X, Y and 18 in a total of 358available spermatozoa.

The total incidence of sex chromosome disomy in ourpatient’s spermatozoa was 3.9%. This value is higher than thatdetermined by FISH (0.14% to 0.43%) for karyotypicallynormal fertile men (Williams et al., 1993; Wyrobek et al.,1994; Martin et al., 1996; Spriggs et al., 1996; Rademakeret al., 1997; Storeng et al., 1998). It is also higher than thereported mean values of sex chromosome disomy for maleswith abnormal seminal parameters (0.33% to 2.95%) (Moosaniet al., 1995; Storeng et al., 1998; Pang et al., 1999). The totalfrequency of sex chromosome hyperploidy for our patient fallsin the range ascertained in other mosaic (0.92% to 5.0%) andnon-mosaic (1.36% to 25%) Klinefelter syndrome individuals

Hyperploidy in spermatozoa and embryos in a Klinefelter male

Table II. Results of fluorescence in-situ hybridization of patients’ spare embryos with probes specific forchromosomes X, Y and 18

Embryo No. of No. of FISH results (No. of nuclei) Embryo diagnosisnumber blastomeres nuclei

1 8 8 XX1818 (5) Diploid2 6 6 XY1818(1)/Y(1)/XXYY(1)/X(1)/ Chaotic mosaic

XY18 (1)/Y1818 (1)3 7 7 XX 1818(2) / X1818(3) / X18(2) Chaotic mosaic4 6 8 XX1818(3) / 18 (1) Diploid mosaic5 11 11 XY1818(8) Diploid6 7 7 XX1818(2)/XXX18181818(3)/ Chaotic mosaic

XXXX18181818 (1)/XX18(1)7 7 7 XY1818(2)/XXY181818 (2)/ Chaotic mosaic

XXXY18181818(2)/18(1)8 8 10 XY1818(6)/XXYY181818(1)/ Diploid mosaic

XX1818(1)/X(1)9 10 10 XY1818(1)/ X1818(3)/Y18(1) Y(1)/ Chaotic mosaic

Y181818(1)/XXYY18181818(1)/XXYY1818(1)

10 5 6 XY1818(5) Diploid

Total 75 80 (67)

(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 Ishould produce the same number of 24,XY and 22,-X,-Ysperm cells. Non-disjunction in meiosis II should produceequal frequencies of spermatozoa with 24,XX, 24,YY andsex chromosome hypohaploidy. In contrast to the increasedfrequency of spermatozoa with sex chromosome disomy(3.9%), only 0.56% of nuclei from our patient’s spermatozoadid not contain gonosomes. The lack of corresponding spermcells nullsomic for sex chromosomes in our patient suggeststhat the 24,XY and 24,XX spermatozoa probably arose fromthe abnormal 47,XXY spermatogonia, thus supporting theevidence from previous studies that these germ cells cancomplete meiosis.

Although the number of Klinefelter syndrome cases with aknown chromosomal constitution of the zygote following ICSIis limited, no affected progeny have been reported to date. Anormal karyotype, following fertilization with a Klinefelterpatient’s spermatozoon, was detected as a 9-week fetus (Hinneyet al., 1997). To date, the 11 liveborn in-vitro-conceivedKlinefelter patients’ offspring have also been found to bechromosomally normal (Bourne et al., 1997; Tournaye et al.,1997; Nodar et al., 1998; Palermo et al., 1998; Reubinoffet al., 1998; Ron-El et al., 1999).

Two recent studies used FISH to analyse preimplantationembryos following ICSI with a Klinefelter patient’s spermato-zoa. In the first (Staessen et al., 1996), PGD was performedwith probes for chromosomes X,Y on five embryos, from atotal of three patients. All five embryos were diagnosed witha normal chromosome content. The second study (Reubinoffet al., 1998) reported on PGD with probes for chromosomesX, Y and 18 in three embryos from two patients. One embryowas diagnosed as normal, was transferred, and resulted inthe birth of a karyotypically normal neonate. The biopsiedblastomere from the second embryo had an abnormal XX18constitution. The blastomere from the third embryo was found

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also to contain an abnormal pattern, XXY1818. Spreading ofthe remainder of the cells from the above two embryos revealednuclei with various X and Y patterns, consistent with chaoticcell division. Therefore, among all the abnormal embryoscombined, no uniform XXY or XXX chromosome complementhad been detected. Our observations in 10 spare preimplantationembryos are very similar. Although only three (33%) ofthe spare embryos produced by ICSI using our patient’sspermatozoa were uniformly normal, none of the abnormalembryos analysed was uniformly aneuploid for the sex chromo-somes. Instead, as in the above reports, the abnormal embryoswere mosaics with chaotic chromosome segregation patterns.

Intracytoplasmic fertilization of oocytes with a Klinefeltermale’s 24,XY- or 24,XX-bearing spermatozoa, could theoretic-ally produce a 47,XXY or a 47,XXX embryo. This chromo-somal status of ICSI-induced conceptions cannot be excludedfrom the three transferred embryos among the six reported(Reubinoff et al., 1998), though chromosome analysis was notperformed. Similarly, the gonosome constitution of one of thethree embryos that had been transferred to our patient remainsunknown. Furthermore, diagnosis of six embryos reportedearlier by one group (Reubinoff et al., 1998) and four embryosreported by another group (Staessen et al., 1996) was basedon only single blastomeres. The incidence of chromosomalmosaicism in in-vitro-fertilized human preimplantationembryos is substantial (Coonen et al., 1994; Munne et al.,1994; Harper et al., 1995; Handyside, 1996; Delhanty et al.,1997). Among the seven abnormal embryos produced fromour patient’s spermatozoa, all contained at least one normalXX1818 or XY1818 blastomere in addition to those which werechromosomally abnormal. Therefore, mitotic non-disjunctionand/or sex chromosome loss in one blastomere of a 47,XXYor 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 thereforeimportant to advise PGD patients to undergo prenatal diagnosisto ensure the normality of the fetus.

M.Bielanska, S.L.Tan and A.Ao

In conclusion, despite an increased frequency of spermhyperhaploid for sex chromosomes, FISH analysis of ourpatient’s spermatozoa indicated that the majority of spermato-zoa (94.4%) were normal for the chromosomes tested. Thisagrees with findings in other Klinefelter cases, and confirmsthat the karyotype in peripheral blood in this population ofpatients is not indicative of the chromosome constitution ofother tissues such as the testis. Furthermore, ICSI of thepatient’s spermatozoa induced fertilization, produced a highpercentage of cleavage-stage embryos, and led to implantationand ongoing pregnancy. None of the spare embryos analysedhad a Klinefelter syndrome karyotype. In order to have a clearunderstanding of the risk of affected progeny in Klinefeltermales procreating via ICSI, further screening for gonosomesin spermatozoa, preimplantation spare embryos, concepti andneonates of this population of patients is necessary.

AcknowledgementsThe authors wish to thank the couple for the donation of thespermatozoa and spare preimplantation embryos for the study, andthe embryologists and clinicians at the McGill Reproductive Centrefor their assistance and cooperation.

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Received on July 2, 1999; accepted on November 4, 1999