Department of Urology, Kobe University School of Medicine, Kobe, Japan
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Abstract |
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Key words: FISH/ICSI/infertility/Klinefelter's syndrome/spermatozoa
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Introduction |
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We studied the clinical features of this syndrome in men attending our clinic, and explored the feasibility of assisted reproductive technology in these patients.
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Materials and methods |
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Evaluation of patients
All patients were required to complete a questionnaire concerning duration of sterility, past medical history, sexual function and results of gynaecological evaluation of their spouse. Measurements obtained at physical examination included height, weight, volume of each testis and prostate size, as described previously (Okada et al., 1996). The epididymis and vas deferens were examined by careful palpation. In some patients, ultrasonographic examination of the prostate was also performed. Hormonal analysis included serum luteinizing hormone (LH), follicle stimulating hormone (FSH) and testosterone concentrations, as described elsewhere (Okada et al., 1996
). LH and FSH concentrations were measured by immunoradiometric assay (IRMA). The World Health Organization (WHO) standards, first international reference preparation (IRP) luteinizing hormone (LH) and second IRP human pituitary gonadotrophin (HPG) were used as the reference standards of LH and FSH respectively. Values measured by different standards were adjusted to the values measured by IRMA. Testosterone was measured by radioimmunoassay. Normal ranges at our institution for LH, FSH and testosterone were 1.1 to 9.8 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 recommended by WHO (1992), with slight modification as described elsewhere (Okada et al., 1997). Semen was collected in a sterile container and allowed to liquefy completely at room temperature. The whole semen was divided into several 1-ml aliquots which were placed into conical tubes (Falcon, Becton Dickinson, Lincoln Park, NJ, USA) and mixed thoroughly with 10 ml of phosphate-buffered saline (PBS). The mixture was centrifuged at 600 g for 10 min at room temperature. 10 µl of the suspensions were spread onto glass slides and air-dried. After Papanicolaou staining, slides were examined at x400 and x1000 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) were performed in all patients with azoospermia by G-banding according to the procedures of Yunis et al. (1978). Normally, 30 metaphase spreads were evaluated, but when a marker chromosome or mosaicism was evident, an additional repeat chromosomal analysis was performed. Fluorescence in-situ hybridization (FISH) was then performed on metaphase chromosomes to confirm the origin of the marker chromosome and excess chromosomes. FISH was carried out according to manufacturer's recommendations using commercially available kits. Briefly, PBL were incubated in RPMI 1640 medium supplemented with 2% phytohaemaglutinin solution (Gibco-BRL, NY, USA) and 15% fetal calf serum at 37°C for 72 h. The cell cycle was synchronized by incubating with 0.04 µg/ml colcemid (KARYOMAX colcemid solution; Gibco-BRL) for 3 h. Cells then were incubated in hypotonic KCl solution and fixed in Carnoy's solution (acetic acid:methanol, 1:3). The cell suspension was spread and air-dried on precleaned glass slides, which then were immersed in denaturant solution (70% formalin/2x SSC) for 2 min. After drying the slides, a mixture of fluorescence-labelled X or Y chromosome-specific probes was applied (CEP X SG/CEP Y SO, SpectrumCEP Chromosome Enumeration System; Vysis, Downers Grove, IL, USA). Slides were hybridized in an incubator at 42°C for 16 h. After washing, a counterstain (DAPI II; Vysis) was applied and slides were examined under a fluorescence microscope equipped with a double-bandpass filter (Vysis). At least 100 cells were examined to evaluate the number of sex chromosomes. X and Y chromosomes were identified by green and orange fluorescence respectively.
Meiotic segregation of sex chromosomes in sperm nuclei by FISH analysis
Semen samples from the two patients who produced spermatozoa were used for FISH analyses of meiotic segregation of sex chromosomes. Spermatozoa obtained from three men who fathered children within one year were also analysed by FISH. Specimens were washed with PBS and fixed in Carnoy's solution. The specimen was then spread and air-dried on precleaned glass slides, which then were treated with 0.005% trypsin in PBS for 5 min at room temperature, followed by washing in PBS. The slides were dehydrated through increasing ethanol concentrations and air-dried again. After denaturation, slides were hybridized with a mixture of X-, Y- and 18-chromosome specific probes (CEP 18 SA/X SG/Y SO; Vysis). Fluorescence was observed under a fluorescence microscope (BX50; Olympus) with an appropriate filter (triple-bandpass filter; Vysis). Chromosomes 18, X and Y gave blue, green or orange signals respectively. If two signals of the same colour, size and intensity were separated by at least one domain, 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 a standard procedure under local anaesthesia. Specimens were fixed in Bouin fixative and embedded in paraffin. Sections (4 µm) were stained 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 1996 in our institution. The patients' consent was obtained before undergoing this procedure. Wet preparations of the multiple-site biopsy specimens were made according to Tournaye et al. (1996a). In brief, under spinal or general anaesthesia, hemiscrotomy was performed and the scrotal contents were inspected; multiple small testicular biopsies were then performed at three or four sites. Testicular tissue obtained from each biopsy site weighed 50100 mg. The tissue was rinsed in PBS to remove blood as thoroughly as possible, and then placed in a drop of Ham's F-10 medium in a Petri dish. The testicular tissue was minced using two no.11 blades and examined under an inverted microscope (IMT-2; Olympus) at x400 magnification to detect spermatozoa and round, elongating or elongated spermatids in the cell suspension. One-third of the cell suspension was used to prepare a slide for cytological examination by Papanicolaou staining. Two-thirds of the cell suspension was temporarily cryopreserved. The criteria for identifying round spermatids were that the size of the cell was 6.58.0 µm in diameter, and that the developing acrosomal structures were visible as a bright spot adjacent to the nucleus. The elongating and elongated spermatids could easily be identified by their characteristic morphological features as described by Vanderzwalmen et al. (1998). When round spermatids or elongating or elongated spermatids were found, the specimen was cryopreserved for future intracytoplasmic sperm injection (ICSI). When no such cells were found, cryopreserved materials were discarded after obtaining permission from the couples.
Counselling
Patients with karyotypes of 47,XXY or mosaicism for 47,XXY and their spouses underwent counselling on several occasions with andrologists, gynaecologists and geneticists with respect to the aetiology of their infertility. Couples then were presented with the following options: (i) childlessness or adoption; (ii) artificial insemination with donor semen (AID); and (iii) where applicable, ICSI with spermatozoa preserved from the testicular or semen specimens.
Outcome of treatment
Patients still being seen at our hospital in 1997 were asked about their plans for families. Those who did not return for appointments were interviewed by telephone.
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Results |
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The median patient age was 32 (range 24 to 44) years, and their median duration of sterility was 3.0 (range 0.4 to 11) years. Fifteen patients had a history of inguinal herniorrhaphy in childhood, five had a history of gonococcal or non-gonococcal urethritis, and six had a history of prostatitis. No patients had diabetes, hypertension or tuberculosis. Two patients had undergone bilateral orchiopexy during infancy and one had a history of mumps orchitis. All spouses had undergone gynaecological evaluation, including monitoring of ovulation. Most had undergone tubal patency tests before referral.
Median patient height was 174 (range 158 to 188) cm and median body weight 68 (range 48 to 99) kg. Median testicular volume was 4 (range 1 to 16) ml on each side. Three patients with 46,XY/47,XXY mosaicism and two with a 47,XXY karyotype had a testicular volume >15 ml. No spermatozoa were detected in the semen of these patients and none underwent testicular biopsy. Pubic hair distribution was classified as female-type in 31.5% of patients and male-type in 68.5%. Prostate size by palpation was atrophic in 36.7% of patients and normal in 63.3%; gynaecomastia was observed in 12.4%.
Median serum concentrations of LH, FSH and testosterone were 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. The percentage of patients with elevated serum concentrations of LH and/or FSH with decreased concentrations of serum testosterone (hypergonadotrophic hypogonadism), the percentage with elevated serum concentrations of LH and/or FSH with normal concentrations of serum testosterone (hypergonadotrophic normogonadism), and the percentage with normal concentrations of serum LH, FSH and testosterone (normogonadotrophic normogonadism), were 52.8, 44.3 and 2.9% respectively (Figure 1).
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G-banding studies in PBL indicated mosaicism (46,XY/47,XXY) in 10 patients. FISH analysis confirmed mosaicism in six; however, in PBL from the remaining four patients, two X chromosomes and one Y chromosome were demonstrated (Figure 2). These patients were karyotyped as 47,XXY. The percentage of mosaicism by G-banding among PBL from the four re-designated patients was <10%; in the other six patients in whom mosaicism was present in >10% of PBL by G-banding, 46,XY/47,XXY mosaicism was confirmed by FISH analysis. Three of these patients showed normal testicular size (
15 ml). Hormonal profiles of six patients indicated hypergonadotrophic normogonadism (Table II
). When PBL from patients with 46,XY/47,XXY mosaicism were assessed by G-banding and FISH, the percentage of PBL with a normal 46,XY karyotype varied from 17% to 33% by G-banding, and from 20% to 30% by FISH (Table II
).
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Meiotic segregation of the sex chromosomes was analysed in 623 spermatozoa obtained from a patient with 46,XY/47,XXY mosaicism and 597 spermatozoa from a patient with non-mosaic 47,XXY karyotype using three-colour FISH. In both patients, karyotyping was done on two distinct occasions in 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 karyotype (23,X or 23,Y). X-bearing sperm cells represented 43.1% and Y-bearing sperm cells represented 47.5% of the total in the 46,XY/47,XXY patient; in the 47,XXY patient these values were 42.2% and 46.3%. Sex-chromosomal hyperploidy was respectively observed in 2.5% and in 2.7% of spermatozoa in these two patients. Prevalences of 24,XX, 24,XY, and 24,YY were 1.1, 1.0 and 0.2%, and 1.0, 1.3 and 0.2% in the two patients. These rates of sex chromosome hyperploidy with 24,XX and 24,XY were slightly higher than the rates of 0.110.24% and 0.060.42% respectively observed in the present series in normal fertile men. The occurrence rates of diploid cells were 0.8% and 0.34% respectivelyalmost the same rate as in fertile men (0.170.41%). Disomy 18 was not detected in patients. No signals were detected in 5.3% and 7.7% of sperm nuclei in the respective patients (Table III).
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Follow-up telephone interviews were successful in 52 patients: 58% of couples did not pursue pregnancy, while 42% underwent AID, which was successful for 82% of cases. No couple adopted a child. Two patients requested androgen replacement therapy because of feelings of fatigue and muscular weakness, and symptoms improved with testosterone enanthate (250 mg every 3 weeks). No patient reported a diagnosis of osteoporosis or frequent fractures, despite low testosterone concentrations in many instances.
Two patients developed testicular tumours; one was a mature teratoma in the right testis and the other a Leydig cell tumour in the right testis (Okada et al., 1994). High inguinal orchiectomy was performed in both patients who are alive without evidence of recurrence 8 and 6 years after surgery.
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Discussion |
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Although gynaecomastia has been reported previously in 50% of patients (Tournaye et al., 1996b), it was observed in only 12.4% of our cases. Median height and weight indicated that patients were slightly taller and thinner than average Japanese men of the same age. Over 95% of patients had small testes; notably, three patients with 46,XY/47,XXY mosaicism had normal-sized testes. Pubic hair distribution showed a female pattern in one-third of patients, and the prostate was atrophic in one-third. While half of the patients showed low testosterone concentrations with elevated gonadotrophins, half had normal concentrations of testosterone. Compared with classic Klinefelter's syndrome, our patients often showed different clinical features. As they were sufficiently virile to become married, the differences can be explained by this selection bias. Thus, our patients may represent one end of a spectrum in Klinefelter's syndrome.
The percentage of patients on androgen replacement therapy was extremely low in the present series, possibly because their insurance did not cover androgen replacement in the earlier period of this study. In addition, this may be because most patients in this series were sufficiently virile to be married and perform sexual intercourse, despite low concentrations of serum androgen, and thus had no need for hormone replacement therapy. Moreover, since the median follow-up period was as short as 3 months and most patients were referred from remote institutions, they seldom returned to our institution after a diagnosis of absolute sterility due to male factor. The success rate of interview by telephone was less than one-third. Taken together, these factors may lead to a failure to identify other patients receiving androgen replacement therapy.
In the present series, 10 out of 148 patients had been diagnosed with mosaicism, but upon re-evaluation by FISH, four of these were proven not to have mosaicism. It is noteworthy that in each of these four cases the rate of mosaicism in G-banding studies of PBL was <10%. From these data we suspect that mosaicism in <10% of PBL by G-banding is likely to be an artefact, requiring re-evaluation by 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 of PBL showed a 47,XXY karyotype. Hormonally, they showed hypergonadotrophic normogonadism. Other types of mosaicism seen in two patients (47,XXY/47,XYY/48,XXYY; 46,XY/47,XXY/48,XXYY) were associated with both small testes and hypergonadotrophic hypogonadism.
In our series, two patients with 47,XXY or 46,XY/47,XXY karyotype had spermatozoa in the semen on several occasions. Azoospermia is not a consistent feature of Klinefelter's syndrome. Most 47,XXY Klinefelter patients show germinal aplasia histologically, while some 46,XY/47,XXY patients focally show spermatogenesis (Gordon et al., 1972). Only a few cases of apparently non-mosaic 47,XXY individuals have shown focal spermatogenesis histologically, but in one recent report four out of nine such patients were found to have spermatozoa in ejaculates or in the testes (Tournaye et al., 1996b
).
A small number of patients with Klinefelter's syndrome have been reported to succeed in fathering a child before the era of assisted reproduction technology (Kaplan et al., 1963; Laron et al., 1982
; Terzoli et al., 1992
). At present, assisted reproduction techniques using seminal or testicular spermatozoa have enabled some patients with non-mosaic Klinefelter's syndrome to fertilize eggs (Hinney et al., 1997
) and father children (Bourne et al., 1997
; Palermo et al., 1998
; Reubinoff et al., 1998
). Among our patients, one with 46,XY/47,XXY mosaicism and one with a 47,XXY karyotype had a few spermatozoa in the semen, and one patient with 47,XXY karyotype proved to have round and elongated spermatids in the testis.
To determine whether these spermatozoa or spermatids can be used to father a normal child requires a meiotic study. To assess the karyotype of spermatozoa, the zona-free hamster oocyte penetration system can be used to provide material for chromosomal analysis (Cozzi et al., 1994), though this procedure requires special techniques and is very time-consuming. Moreover, as only fertilized oocytes can be used for analysis, bias is introduced in selecting spermatozoa. Given these drawbacks and the paucity of spermatozoa, only a few reports have shown the segregation of the sex chromosome of spermatozoa from patients with Klinefelter's syndrome using such methods (Cozzi et al., 1994
).
With the introduction of FISH, however, the sex chromosome can be visualized in an ordinary laboratory setting, and three-colour FISH can detect simultaneously the presence of three distinct chromosomes in a single cell (Martini et al., 1996; Chevret et al., 1997
; Estop et al., 1998
). Using this method, Guttenbach et al. (1997) have reported that over 92% of sperm nuclei in a man with a 47,XXY karyotype can be presumed to have a 23,X or 23,Y karyotype, but a significantly increased rate of sex-chromosome hyperploidy such as 24,XX or 24,XY occurs (Guttenbach et al., 1997
). We used the same three-colour FISH with specific probes for chromosomes X, Y and 18 instead of Guttenbach's probes for chromosomes X, Y and 1. We analysed the chromosomes of spermatozoa from one non-mosaic and one mosaic Klinefelter's syndrome patient, our results supporting the previous observations (Chevret et al., 1995
; Guttenbach et al., 1997
). We can speculate that germ cells in patients with Klinefelter's syndrome with either non-mosaicism or mosaicism can undergo meiosis, but infrequently can produce sex chromosome hyperploid spermatozoa. However, we can also postulate that the increased incidence of sex chromosome hyperploid spermatozoa can be attributed to the increased rate of non-disjunction of normal XY germ cells.
Theoretically, if spermatozoa from these patients are used for assisted reproduction techniques, including ICSI, the risk of XXY or XXX progeny is increased. However, the karyotypes of delivered offspring, an ectopic pregnancy and pre-implantation embryo were all normal in previous reports (Staessen et al., 1996; Bourne et al., 1997
; Palermo et al., 1998
). We discussed the small but persisting possibility of sex chromosome hyperploid children with two patients and their spouses. One patient with non-mosaic Klinefelter's syndrome, who had spermatozoa in the ejaculated semen, could not accept this possibility. The wife of the other patient (with mosaic Klinefelter's syndrome) whose semen contained spermatozoa developed OHSS. As ICSI with spermatids has not yet been approved by the ethics committee in Japan, we have not yet succeeded in obtaining a pregnancy. Successful pregnancy and delivery at other institutions has encouraged patients with Klinefelter's syndrome to undergo evaluation, including multiple-site testicular biopsy. Genetic counselling for these couples is needed in parallel with the advancement of assisted reproduction techniques. ICSI procedures performed successfully in these couples should be followed up by prenatal diagnosis. The most practical method to obtain a normal fetus is by pre-implantation diagnosis (Straessen et al., 1996; Tournaye et al., 1997
). However, the clinical application of this method is not permitted in Japan at present because of a lack in general consensus.
Patients with Klinefelter's syndrome are known to be at increased risk of malignant tumours (Hasle et al., 1995). In our interview series, two intrascrotal tumours had developed and had been excised successfully. Half of the patients with Klinefelter's syndrome in our series had low concentrations of serum testosterone, but did not receive androgen replacement therapy. Their endocrine deficit may lead to osteoporosis, and patients should be given this information together with appropriate treatment options.
In conclusion, our study population of Klinefelter's syndrome patients drawn from a male infertility clinic included a few patients with some spermatozoa in the semen, or spermatogenic cells appropriate for ICSI in multiple-site testicular biopsy specimens, whether the individual showed mosaicism or not. While anecdotal reports have not shown sex chromosome hyperploidy in offspring of patients with the syndrome, we found this chromosomal anomaly in over 2% of sperm cell nuclei studied. Therefore, genetic counselling and prenatal diagnosis become important when assisted reproduction techniques are applied to these patients.
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Notes |
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References |
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Submitted on July 17, 1998; accepted on December 16, 1998.