Klinefelter's syndrome in the male infertility clinic

Human Reproduction vol.14 no.4 pp.946–952, 1999

Klinefelter’s syndrome in the male infertility clinic

Hiroshi Okada1, Hitoshi Fujioka, Noboru Tatsumi,Masanori Kanzaki, Yoshihiro Okuda,Masato Fujisawa, Minoru Hazama,Osamu Matsumoto, Kazuo Gohji, Soichi Arakawaand Sadao Kamidono

Department of Urology, Kobe University School of Medicine,Kobe, Japan1To whom correspondence should be addressed at: Department ofUrology, Kobe University School of Medicine, 7-5-1,Kusunoki-cho, Chuo-ku, Kobe, Japan 650-0017

The clinical features of patients with Klinefelter’s syndromeattending a male infertility clinic have been investigated inorder to consider their assisted reproduction treatmentoptions. Over 12 years, a total of 148 patients with sterilitydue to azoospermia had Klinefelter’s syndrome. Eightpatients were shown by fluorescence in-situ hybridization(FISH) on metaphase spreads to be mosaic (46,XY/47,XXY), and 140 patients showed only 47,XXY. Smalltestes were observed in 95% of patients and gynaecomastiawas seen in 12.4%. Half of the patients showed hypergona-dotrophic hypogonadism, while others showed normogon-adism (usually hypergonadotrophic). Spermatozoa wereobserved in semen from one patient with mosaicism andone without. Three-colour FISH revealed hyperploidy in2.7% and 2.3% of these spermatozoa respectively. Multiple-site testicular biopsies in five recent patients were per-formed and yielded a specimen with round and elongatedspermatids in one patient with 47,XXY karyotype. Thissample was cryopreserved for future intracytoplasmicsperm injection. At follow-up, 46% of couples had chosenartificial insemination with donor sperm, and none hadchosen adoption. Two patients developed testiculartumours, one a mature teratoma and the other a Leydigcell tumour. Two patients required androgen replacementtherapy.Key words:FISH/ICSI/infertility/Klinefelter’s syndrome/spermatozoa

Introduction

In its classic form, Klinefelter’s syndrome is characterizedby gynaecomastia, small, firm testes with hyalinization ofseminiferous tubules, hypergonadotrophic hypogonadism andazoospermia. The syndrome is not rare; its prevalence is 0.1%in the general population (Nielsen and Wohlert, 1991), andamong infertile patients up to 11% of azoospermic and 0.7%of oligozoospermic men reportedly have the 47,XXY karyotype

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(De Braekeleer and Dao, 1991; Yoshidaet al., 1996). A recentcase report indicates that a patient with Klinefelter’s syndromesometimes can father a child with the aid of assisted reproduc-tion technology (Bourneet al., 1997; Palermoet al., 1998;Reubinoffet al., 1998).

We studied the clinical features of this syndrome in menattending our clinic, and explored the feasibility of assistedreproductive technology in these patients.

Materials and methods

Patients

Between January 1985 and December 1996, a total of 1987 patientswith sterility due to azoospermia were referred by gynaecologists orurologists at other institutions to the male infertility clinic at KobeUniversity Hospital. Chromosomal analyses were performed in allpatients, and those found to have a 47,XXY karyotype or mosaicismwere enrolled into the study.

Evaluation of patients

All patients were required to complete a questionnaire concerningduration of sterility, past medical history, sexual function and resultsof gynaecological evaluation of their spouse. Measurements obtainedat physical examination included height, weight, volume of eachtestis and prostate size, as described previously (Okadaet al., 1996).The epididymis and vas deferens were examined by careful palpation.In some patients, ultrasonographic examination of the prostate wasalso performed. Hormonal analysis included serum luteinizing hor-mone (LH), follicle stimulating hormone (FSH) and testosteroneconcentrations, as described elsewhere (Okadaet al., 1996). LH andFSH concentrations were measured by immunoradiometric assay(IRMA). The World Health Organization (WHO) standards, firstinternational reference preparation (IRP) luteinizing hormone (LH)and second IRP human pituitary gonadotrophin (HPG) were used asthe reference standards of LH and FSH respectively. Values measuredby different standards were adjusted to the values measured by IRMA.Testosterone was measured by radioimmunoassay. Normal ranges atour institution for LH, FSH and testosterone were 1.1 to9.8 mIU/ml, 1.6 to 14.9 mIU/ml and 2.7 to 10.7 ng/ml respectively.

Semen analysis

Semen analysis was performed according to the procedures recom-mended by WHO (1992), with slight modification as describedelsewhere (Okadaet al., 1997). Semen was collected in a sterilecontainer and allowed to liquefy completely at room temperature.The whole semen was divided into several 1-ml aliquots which wereplaced into conical tubes (Falcon, Becton Dickinson, Lincoln Park,NJ, USA) and mixed thoroughly with 10 ml of phosphate-bufferedsaline (PBS). The mixture was centrifuged at 600g for 10 min atroom temperature. 10µl of the suspensions were spread onto glassslides and air-dried. After Papanicolaou staining, slides were examined

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at 3400 and 31000 magnifications with a microscope (BHS-2;Olympus, Tokyo, Japan) by one of the authors (H.O.).

Chromosomal analyses

Chromosomal analyses of peripheral blood lymphocytes (PBL) wereperformed in all patients with azoospermia by G-banding accordingto the procedures of Yuniset al. (1978). Normally, 30 metaphasespreads were evaluated, but when a marker chromosome or mosaicismwas evident, an additional repeat chromosomal analysis was per-formed. Fluorescence in-situ hybridization (FISH) was then performedon metaphase chromosomes to confirm the origin of the markerchromosome and excess chromosomes. FISH was carried out accord-ing to manufacturer’s recommendations using commercially availablekits. Briefly, PBL were incubated in RPMI 1640 medium supplementedwith 2% phytohaemaglutinin solution (Gibco-BRL, NY, USA) and15% fetal calf serum at 37°C for 72 h. The cell cycle was synchronizedby incubating with 0.04µg/ml colcemid (KARYOMAX colcemidsolution; Gibco-BRL) for 3 h. Cells then were incubated in hypotonicKCl solution and fixed in Carnoy’s solution (acetic acid:methanol,1:3). The cell suspension was spread and air-dried on precleanedglass slides, which then were immersed in denaturant solution (70%formalin/23 SSC) for 2 min. After drying the slides, a mixture offluorescence-labelled X or Y chromosome-specific probes was applied(CEP X SG/CEP Y SO, SpectrumCEP Chromosome EnumerationSystem; Vysis, Downers Grove, IL, USA). Slides were hybridized inan incubator at 42°C for 16 h. After washing, a counterstain (DAPIII; Vysis) was applied and slides were examined under a fluorescencemicroscope equipped with a double-bandpass filter (Vysis). At least100 cells were examined to evaluate the number of sex chromosomes.X and Y chromosomes were identified by green and orange fluores-cence respectively.

Meiotic segregation of sex chromosomes in sperm nuclei by FISHanalysis

Semen samples from the two patients who produced spermatozoa wereused for FISH analyses of meiotic segregation of sex chromosomes.Spermatozoa obtained from three men who fathered children withinone year were also analysed by FISH. Specimens were washed withPBS and fixed in Carnoy’s solution. The specimen was then spreadand air-dried on precleaned glass slides, which then were treated with0.005% trypsin in PBS for 5 min at room temperature, followed bywashing in PBS. The slides were dehydrated through increasingethanol concentrations and air-dried again. After denaturation, slideswere hybridized with a mixture of X-, Y- and 18-chromosome specificprobes (CEP 18 SA/X SG/Y SO; Vysis). Fluorescence was observedunder a fluorescence microscope (BX50; Olympus) with an appro-priate filter (triple-bandpass filter; Vysis). Chromosomes 18, X andY gave blue, green or orange signals respectively. If two signals ofthe same colour, size and intensity were separated by at least onedomain, disomy was diagnosed. To minimize inter-observer variability,one of us (H.O.) performed FISH analysis of sperm cells.

Testicular biopsy

Conventional testicular biopsy was performed on both testes by astandard procedure under local anaesthesia. Specimens were fixed inBouin fixative and embedded in paraffin. Sections (4µm) werestained with haematoxylin and eosin and examined microscopically.Spermatogenesis was scored according to Johnsen’s scoring system(Johnsen, 1970).

Multiple site testicular biopsies have been performed since 1996in our institution. The patients’ consent was obtained before under-going this procedure. Wet preparations of the multiple-site biopsyspecimens were made according to Tournayeet al. (1996a). In brief,

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under spinal or general anaesthesia, hemiscrotomy was performedand the scrotal contents were inspected; multiple small testicularbiopsies were then performed at three or four sites. Testicular tissueobtained from each biopsy site weighed 50–100 mg. The tissue wasrinsed in PBS to remove blood as thoroughly as possible, and thenplaced in a drop of Ham’s F-10 medium in a Petri dish. The testiculartissue was minced using two no.11 blades and examined under aninverted microscope (IMT-2; Olympus) at3400 magnification todetect spermatozoa and round, elongating or elongated spermatids inthe cell suspension. One-third of the cell suspension was used toprepare a slide for cytological examination by Papanicolaou staining.Two-thirds of the cell suspension was temporarily cryopreserved. Thecriteria for identifying round spermatids were that the size of the cellwas 6.5–8.0µm in diameter, and that the developing acrosomalstructures were visible as a bright spot adjacent to the nucleus. Theelongating and elongated spermatids could easily be identified by theircharacteristic morphological features as described by Vanderzwalmenet al. (1998). When round spermatids or elongating or elongatedspermatids were found, the specimen was cryopreserved for futureintracytoplasmic sperm injection (ICSI). When no such cells werefound, cryopreserved materials were discarded after obtaining permis-sion from the couples.

Counselling

Patients with karyotypes of 47,XXY or mosaicism for 47,XXYand their spouses underwent counselling on several occasions withandrologists, gynaecologists and geneticists with respect to the aeti-ology of their infertility. Couples then were presented with thefollowing options: (i) childlessness or adoption; (ii) artificial insemina-tion with donor semen (AID); and (iii) where applicable, ICSI withspermatozoa preserved from the testicular or semen specimens.

Outcome of treatment

Patients still being seen at our hospital in 1997 were asked abouttheir plans for families. Those who did not return for appointmentswere interviewed by telephone.

Results

Among 1987 patients referred to our institution with ofazoospermia, 148 were diagnosed as Klinefelter’s syndromefrom G-banding studies of PBL showing a 47,XXY karyotypeor mosaicism and underwent further study.

The median patient age was 32 (range 24 to 44) years, andtheir median duration of sterility was 3.0 (range 0.4 to 11)years. Fifteen patients had a history of inguinal herniorrhaphy inchildhood, five had a history of gonococcal or non-gonococcalurethritis, and six had a history of prostatitis. No patientshad diabetes, hypertension or tuberculosis. Two patients hadundergone bilateral orchiopexy during infancy and one hada history of mumps orchitis. All spouses had undergonegynaecological evaluation, including monitoring of ovulation.Most had undergone tubal patency tests before referral.

Median patient height was 174 (range 158 to 188) cm andmedian body weight 68 (range 48 to 99) kg. Median testicularvolume was 4 (range 1 to 16) ml on each side. Three patientswith 46,XY/47,XXY mosaicism and two with a 47,XXYkaryotype had a testicular volume.15 ml. No spermatozoawere detected in the semen of these patients and none under-went testicular biopsy. Pubic hair distribution was classifiedas female-type in 31.5% of patients and male-type in 68.5%.

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Figure 1. Profiles of luteinizing hormone (LH), follicle stimulating hormone (FSH) and testosterone in patients with Klinefelter’s syndrome.Shading represents the normal range at our institution.

Table I. Spermatozoa in the semen from patients with non-mosaic and mosaic Klinefelter’s syndrome

Karyotype Sperm concentration Sperm motility Abnormally formed Eosin Y-stained Semen volume(3106/ml) (%) spermatozoa (%) spermatozoa (%) (ml)

47,XXY 1 0 80 95 346,XY/47,XXY 0.2 0 90 ND 2.5

ND 5 not determined.

Prostate size by palpation was atrophic in 36.7% of patientsand normal in 63.3%; gynaecomastia was observed in 12.4%.

Median serum concentrations of LH, FSH and testosteronewere 14.8 (range 1.8 to 33.5) mIU/ml, 32.0 (range 3.1 to 110)mIU/ml and 2.4 (range 0.2 to 7.1) ng/ml respectively. Thepercentage of patients with elevated serum concentrationsof LH and/or FSH with decreased concentrations of serumtestosterone (hypergonadotrophic hypogonadism), the percent-age with elevated serum concentrations of LH and/or FSHwith normal concentrations of serum testosterone (hyper-gonadotrophic normogonadism), and the percentage with nor-mal concentrations of serum LH, FSH and testosterone (normo-gonadotrophic normogonadism), were 52.8, 44.3 and 2.9%respectively (Figure 1).

Semen analyses revealed azoospermia in 146 cases on threeconsecutive occasions. However, spermatozoa were observedin semen from one patient with a 47,XXY karyotype and fromone patient with a 46,XY/47,XXY karyotype. In the formerpatient, chromosomal analyses were repeated twice to confirm47,XXY karyotype. In the latter patient, FISH analysis con-firmed the mosaicism. Spermatozoa were detected on threeout of five occasions, and on one out of four occasionsrespectively. Semen analyses revealed severe oligoasthenotera-tozoospermia in both patients (Table I). Eosin Y stainingrevealed that only 5% of the spermatozoa in the non-mosaicismpatient were alive. One-tenth of these samples were used formeiotic studies, and remainders were cryopreserved for futureICSI procedures.

Conventional testicular biopsy was performed in 30 patientsbetween 1985 and 1988; subsequently this procedure was notperformed routinely. Instead, biopsy has been performed atmultiple sites since 1996 to facilitate ICSI. Conventional single-site testicular biopsy showed various degrees of seminiferoustubule deterioration. Most specimens showed germ cell aplasiawith only Sertoli cells in tubules or hyalinized tubules, with

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Figure 2. Fluorescent in-situ hybridization (FISH) analysis of the Xchromosomes in metaphase cell. By G-banding, this patient wasdiagnosed with 46,XY/47,XXY mosaicism (ratio, 2:28). An Xchromosome-specific probe was hybridized. Two green signals areindicated by arrowheads. This patient was reclassified as havingnon-mosaic Klinefelter’s syndrome.

or without mild hyperplasia of Leydig cells (Johnsen score 1to 2). Multiple-site biopsies were performed in the five mostrecent cases. Papanicolaou-stained specimens showed roundand elongated spermatids in one patient; this cell suspensionwas kept frozen for possible future ICSI.

G-banding studies in PBL indicated mosaicism (46,XY/47,XXY) in 10 patients. FISH analysis confirmed mosaicismin six; however, in PBL from the remaining four patients, twoX chromosomes and one Y chromosome were demonstrated(Figure 2). These patients were karyotyped as 47,XXY. Thepercentage of mosaicism by G-banding among PBL from the

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Table II. Mosaicism in Klinefelter’s syndrome by G-banding and FISH

Karyotype (G-banding) Ratio of mosaicism

LH FSH Testosterone Right testis Left testisG-banding FISH (mIU/ml) (mIU/ml) (ng/ml) volume (ml) volume (ml)

46,XY/47,XXY 10:20 25:75 11 33.8 6.9 8 846,XY/47,XXY 10:20 20:80 14 26.4 2.9 8 846,XY/47,XXY 5:25 25:75 16.9 24.4 4.1 10 1046,XY/47,XXY 5:25 25:75 15 18.3 3.4 15 1546,XY/47,XXY 10:20 30:70 10.5 9.6 3.2 16 1646,XY/47,XXY 10:20 25:75 12.4 16.5 3.5 16 1647,XXY/47,XYY/48,XXYY 3:20:7 5:80:15 11 32.1 1.0 3 346,XY/47,XXY/48,XXYY 5:20:5 25:50:25 10.5 15.9 1.9 3 3

G-banding was performed on 30 metaphase cells as described in Materials and methods. Fluorescence in-situ hybridization (FISH) was performed on 100blood lymphocyte metaphase chromosomes using an X chromosome-specific probe, and a Y chromosome-specific probe.FSH 5 follicle stimulating hormone; LH5 luteinizing hormone.

four re-designated patients was,10%; in the other six patientsin whom mosaicism was present in.10% of PBL byG-banding, 46,XY/47,XXY mosaicism was confirmed by FISHanalysis. Three of these patients showed normal testicularsize (ù15 ml). Hormonal profiles of six patients indicatedhypergonadotrophic normogonadism (Table II). When PBLfrom patients with 46,XY/47,XXY mosaicism were assessedby G-banding and FISH, the percentage of PBL with a normal46,XY karyotype varied from 17% to 33% by G-banding, andfrom 20% to 30% by FISH (Table II).

Mosaicism for 47,XXY/47,XYY/48,XXYY and 46,XY/47,XXY/48,XXXY were observed in one patient each. Thesepatients had small testes (,4 ml) and hormonal profilesshowing hypergonadotrophic hypogonadism.

Meiotic segregation of the sex chromosomes was analysedin 623 spermatozoa obtained from a patient with 46,XY/47,XXY mosaicism and 597 spermatozoa from a patient withnon-mosaic 47,XXY karyotype using three-colour FISH. Inboth patients, karyotyping was done on two distinct occasionsin a total of 60 metaphase spreads by two different technicians.Most spermatozoa from the former (90.6%) and the latter(88.5%) patient were proven to have a normal haploid karyo-type (23,X or 23,Y). X-bearing sperm cells represented 43.1%and Y-bearing sperm cells represented 47.5% of the total inthe 46,XY/47,XXY patient; in the 47,XXY patient these valueswere 42.2% and 46.3%. Sex-chromosomal hyperploidy wasrespectively observed in 2.5% and in 2.7% of spermatozoa inthese two patients. Prevalences of 24,XX, 24,XY, and 24,YYwere 1.1, 1.0 and 0.2%, and 1.0, 1.3 and 0.2% in the twopatients. These rates of sex chromosome hyperploidy with24,XX and 24,XY were slightly higher than the rates of 0.11–0.24% and 0.06–0.42% respectively observed in the presentseries in normal fertile men. The occurrence rates of diploidcells were 0.8% and 0.34% respectively—almost the same rateas in fertile men (0.17–0.41%). Disomy 18 was not detectedin patients. No signals were detected in 5.3% and 7.7% ofsperm nuclei in the respective patients (Table III).

In our patient with non-mosaic Klinefelter’s syndrome, thecouple declined the option of ICSI, which was planned in thecase of mosaic Klinefelter’s syndrome using cryopreservedspermatozoa, but cancelled due to severe ovarian hyperstimula-tion syndrome (OHSS). Round and elongated spermatids

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extracted from testicular tissue in one case have been frozenfor possible future ICSI, pending approval of the local ethicscommittee.

Follow-up telephone interviews were successful in 52patients: 58% of couples did not pursue pregnancy, while 42%underwent AID, which was successful for 82% of cases. Nocouple adopted a child. Two patients requested androgenreplacement therapy because of feelings of fatigue and muscu-lar weakness, and symptoms improved with testosteroneenanthate (250 mg every 3 weeks). No patient reported adiagnosis of osteoporosis or frequent fractures, despite lowtestosterone concentrations in many instances.

Two patients developed testicular tumours; one was a matureteratoma in the right testis and the other a Leydig celltumour in the right testis (Okadaet al., 1994). High inguinalorchiectomy was performed in both patients who are alivewithout evidence of recurrence 8 and 6 years after surgery.

Discussion

Classic Klinefelter’s syndrome is characterized by gynaeco-mastia, small, firm testes with hyalinization of seminiferoustubules, hypergonadotrophic hypogonadism and azoospermia,though these features are reported to be variable (Paulsenet al., 1968). In the present series, only azoospermic patientswere karyotyped, and the prevalence of this syndrome amongthem was 7.4%. This was slightly lower than the value of10% reported in Western countries (De Braekeleer and Dao,1991), but was similar to that reported previously in Japaneseazoospermic patients (7.8%) (Matsudaet al., 1992).

Although gynaecomastia has been reported previously in50% of patients (Tournayeet al., 1996b), it was observed inonly 12.4% of our cases. Median height and weight indicatedthat patients were slightly taller and thinner than averageJapanese men of the same age. Over 95% of patients hadsmall testes; notably, three patients with 46,XY/47,XXYmosaicism had normal-sized testes. Pubic hair distributionshowed a female pattern in one-third of patients, and theprostate was atrophic in one-third. While half of the patientsshowed low testosterone concentrations with elevated gona-dotrophins, half had normal concentrations of testosterone.Compared with classic Klinefelter’s syndrome, our patients

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Table III. Chromosome analysis of spermatozoa by three-colour FISH

Hybridization Presumed

No. of spermatozoa

signals karyotype 46,XY/47,XXY 47,XXY Fertile man 1 Fertile man 2 Fertile man 3

18/X 23,X 269 (43.18) 252 (42.21) 2665 (46.94) 3560 (44.42) 2369 (43.61)18/Y 23,Y 296 (47.51) 277 (46.40) 2438 (42.94) 3689 (46.03) 2590 (47.68)18/X/X 24,XX 7 (1.12) 6 (1.01) 12 (0.21) 9 (0.11) 13 (0.24)18/X/Y 24,XY 6 (0.96) 8 (1.34) 10 (0.18) 5 (0.06) 23 (0.42)18/Y/Y 24,YY 1 (0.16) 1 (0.17) 0 (0) 2 (0.02) 0 (0)18/18/X 24,X118 4 (0.64) 3 (0.50) 7 (0.12) 11 (0.14) 23 (0.42)18/18/Y 24,Y118 1 (0.16) 1 (0.17) 10 (0.18) 12 (0.15) 30 (0.55)18/18/X/X 46,XX 2 (0.32) 1 (0.17) 0 (0) 1 (0.01) 0 (0)18/18/X/Y 46,XY 3 (0.48) 1 (0.17) 23 (0.41) 13 (0.16) 15 (0.28)18/X/X/Y 25,XXY 1 (0.16) 1 (0.17) 0 (0) 15 (0.19) 9 (0.17)18/18 44,-XX, -XY 0 (0) 0 (0) 0 (0) 0 (0) 2 (0.04)

OR -YY18 22, -X OR -Y 0 (0) 0 (0) 0 (0) 0 (0) 1 (0.02)Null 33 (5.30) 46 (7.71) 513 (9.03) 698 (8.71) 357 (6.57)Total 623 (100) 597 (100) 5678 (100) 8015 (100) 5432 (100)

Fertile man 1: sperm concentration, 583106/ml; sperm motility, 75%; abnormal spermatozoa, 35%; semen volume, 3.5 ml.Fertile man 2: sperm concentration, 1023106/ml; sperm motility, 63%; abnormal spermatozoa, 25%; semen volume, 3.3 ml.Fertile man 3: sperm concentration, 333106/ml; sperm motility, 82%; abnormal spermatozoa, 28%; semen volume, 4.2 ml.Null 5 no signal detected. Values in parentheses are percentages.

often showed different clinical features. As they were suffi-ciently virile to become married, the differences can beexplained by this selection bias. Thus, our patients mayrepresent one end of a spectrum in Klinefelter’s syndrome.

The percentage of patients on androgen replacement therapywas extremely low in the present series, possibly because theirinsurance did not cover androgen replacement in the earlierperiod of this study. In addition, this may be because mostpatients in this series were sufficiently virile to be married andperform sexual intercourse, despite low concentrations ofserum androgen, and thus had no need for hormone replacementtherapy. Moreover, since the median follow-up period was asshort as 3 months and most patients were referred from remoteinstitutions, they seldom returned to our institution after adiagnosis of absolute sterility due to male factor. The successrate of interview by telephone was less than one-third. Takentogether, these factors may lead to a failure to identify otherpatients receiving androgen replacement therapy.

In the present series, 10 out of 148 patients had beendiagnosed with mosaicism, but upon re-evaluation by FISH,four of these were proven not to have mosaicism. It isnoteworthy that in each of these four cases the rate ofmosaicism in G-banding studies of PBL was,10%. Fromthese data we suspect that mosaicism in,10% of PBL by G-banding is likely to be an artefact, requiring re-evaluationby FISH.

In general, the percentage of mosaicism detected by G-banding and FISH was almost identical (approximately 30%).In all patients with 46,XY/47,XXY mosaicism the majority ofPBL showed a 47,XXY karyotype. Hormonally, they showedhypergonadotrophic normogonadism. Other types of mosaicismseen in two patients (47,XXY/47,XYY/48,XXYY; 46,XY/47,XXY/48,XXYY) were associated with both small testesand hypergonadotrophic hypogonadism.

In our series, two patients with 47,XXY or 46,XY/47,XXYkaryotype had spermatozoa in the semen on several occasions.

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Azoospermia is not a consistent feature of Klinefelter’s syn-drome. Most 47,XXY Klinefelter patients show germinalaplasia histologically, while some 46,XY/47,XXY patientsfocally show spermatogenesis (Gordonet al., 1972). Only afew cases of apparently non-mosaic 47,XXY individuals haveshown focal spermatogenesis histologically, but in one recentreport four out of nine such patients were found to havespermatozoa in ejaculates or in the testes (Tournayeet al.,1996b).

A small number of patients with Klinefelter’s syndromehave been reported to succeed in fathering a child before theera of assisted reproduction technology (Kaplanet al., 1963;Laron et al., 1982; Terzoliet al., 1992). At present, assistedreproduction techniques using seminal or testicular spermato-zoa have enabled some patients with non-mosaic Klinefelter’ssyndrome to fertilize eggs (Hinneyet al., 1997) and fatherchildren (Bourneet al., 1997; Palermoet al., 1998; Reubinoffet al., 1998). Among our patients, one with 46,XY/47,XXYmosaicism and one with a 47,XXY karyotype had a fewspermatozoa in the semen, and one patient with 47,XXYkaryotype proved to have round and elongated spermatids inthe testis.

To determine whether these spermatozoa or spermatids canbe used to father a normal child requires a meiotic study. Toassess the karyotype of spermatozoa, the zona-free hamsteroocyte penetration system can be used to provide materialfor chromosomal analysis (Cozziet al., 1994), though thisprocedure requires special techniques and is very time-consum-ing. Moreover, as only fertilized oocytes can be used foranalysis, bias is introduced in selecting spermatozoa. Giventhese drawbacks and the paucity of spermatozoa, only a fewreports have shown the segregation of the sex chromosome ofspermatozoa from patients with Klinefelter’s syndrome usingsuch methods (Cozziet al., 1994).

With the introduction of FISH, however, the sex chromosomecan be visualized in an ordinary laboratory setting, and three-

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colour FISH can detect simultaneously the presence of threedistinct chromosomes in a single cell (Martiniet al., 1996;Chevretet al., 1997; Estopet al., 1998). Using this method,Guttenbachet al. (1997) have reported that over 92% of spermnuclei in a man with a 47,XXY karyotype can be presumedto have a 23,X or 23,Y karyotype, but a significantly increasedrate of sex-chromosome hyperploidy such as 24,XX or 24,XYoccurs (Guttenbachet al., 1997). We used the same three-colour FISH with specific probes for chromosomes X, Y and18 instead of Guttenbach’s probes for chromosomes X, Y and1. We analysed the chromosomes of spermatozoa from onenon-mosaic and one mosaic Klinefelter’s syndrome patient,our results supporting the previous observations (Chevretet al.,1995; Guttenbachet al., 1997). We can speculate that germcells in patients with Klinefelter’s syndrome with either non-mosaicism or mosaicism can undergo meiosis, but infrequentlycan produce sex chromosome hyperploid spermatozoa. How-ever, we can also postulate that the increased incidence of sexchromosome hyperploid spermatozoa can be attributed to theincreased rate of non-disjunction of normal XY germ cells.

Theoretically, if spermatozoa from these patients are usedfor assisted reproduction techniques, including ICSI, the riskof XXY or XXX progeny is increased. However, the karyotypesof delivered offspring, an ectopic pregnancy and pre-implanta-tion embryo were all normal in previous reports (Staessenet al., 1996; Bourneet al., 1997; Palermoet al., 1998).We discussed the small but persisting possibility of sexchromosome hyperploid children with two patients and theirspouses. One patient with non-mosaic Klinefelter’s syndrome,who had spermatozoa in the ejaculated semen, could not acceptthis possibility. The wife of the other patient (with mosaicKlinefelter’s syndrome) whose semen contained spermatozoadeveloped OHSS. As ICSI with spermatids has not yet beenapproved by the ethics committee in Japan, we have not yetsucceeded in obtaining a pregnancy. Successful pregnancy anddelivery at other institutions has encouraged patients withKlinefelter’s syndrome to undergo evaluation, including mul-tiple-site testicular biopsy. Genetic counselling for thesecouples is needed in parallel with the advancement of assistedreproduction techniques. ICSI procedures performed success-fully in these couples should be followed up by prenataldiagnosis. The most practical method to obtain a normalfetus is by pre-implantation diagnosis (Straessenet al., 1996;Tournayeet al., 1997). However, the clinical application ofthis method is not permitted in Japan at present because of alack in general consensus.

Patients with Klinefelter’s syndrome are known to be atincreased risk of malignant tumours (Hasleet al., 1995). Inour interview series, two intrascrotal tumours had developedand had been excised successfully. Half of the patients withKlinefelter’s syndrome in our series had low concentrationsof serum testosterone, but did not receive androgen replacementtherapy. Their endocrine deficit may lead to osteoporosis,and patients should be given this information together withappropriate treatment options.

In conclusion, our study population of Klinefelter’s syn-drome patients drawn from a male infertility clinic includeda few patients with some spermatozoa in the semen, or

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spermatogenic cells appropriate for ICSI in multiple-site tes-ticular biopsy specimens, whether the individual showedmosaicism or not. While anecdotal reports have not shownsex chromosome hyperploidy in offspring of patients with thesyndrome, we found this chromosomal anomaly in over 2%of sperm cell nuclei studied. Therefore, genetic counsellingand prenatal diagnosis become important when assisted repro-duction techniques are applied to these patients.

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Received on July 17, 1998; accepted on December 16, 1998

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Klinefelter's syndrome in the male infertility clinic