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 Arakawa
and Sadao Kamidono
Department of Urology, Kobe University School of Medicine,
Kobe, Japan
1To
whom correspondence should be addressed at: Department of
Urology, Kobe University School of Medicine, 7-5-1,
Kusunoki-cho, Chuo-ku, Kobe, Japan 650-0017
The clinical features of patients with Klinefelter’s syndrome
attending a male infertility clinic have been investigated in
order to consider their assisted reproduction treatment
options. Over 12 years, a total of 148 patients with sterility
due to azoospermia had Klinefelter’s syndrome. Eight
patients 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. Small
testes were observed in 95% of patients and gynaecomastia
was seen in 12.4%. Half of the patients showed hypergonadotrophic hypogonadism, while others showed normogonadism (usually hypergonadotrophic). Spermatozoa were
observed in semen from one patient with mosaicism and
one without. Three-colour FISH revealed hyperploidy in
2.7% and 2.3% of these spermatozoa respectively. Multiplesite testicular biopsies in five recent patients were performed and yielded a specimen with round and elongated
spermatids in one patient with 47,XXY karyotype. This
sample was cryopreserved for future intracytoplasmic
sperm injection. At follow-up, 46% of couples had chosen
artificial insemination with donor sperm, and none had
chosen adoption. Two patients developed testicular
tumours, one a mature teratoma and the other a Leydig
cell tumour. Two patients required androgen replacement
therapy.
Key words: FISH/ICSI/infertility/Klinefelter’s syndrome/
spermatozoa
Introduction
In its classic form, Klinefelter’s syndrome is characterized
by gynaecomastia, small, firm testes with hyalinization of
seminiferous tubules, hypergonadotrophic hypogonadism and
azoospermia. The syndrome is not rare; its prevalence is 0.1%
in the general population (Nielsen and Wohlert, 1991), and
among infertile patients up to 11% of azoospermic and 0.7%
of oligozoospermic men reportedly have the 47,XXY karyotype
946
(De Braekeleer and Dao, 1991; Yoshida et al., 1996). A recent
case report indicates that a patient with Klinefelter’s syndrome
sometimes can father a child with the aid of assisted reproduction technology (Bourne et al., 1997; Palermo et al., 1998;
Reubinoff et al., 1998).
We studied the clinical features of this syndrome in men
attending our clinic, and explored the feasibility of assisted
reproductive technology in these patients.
Materials and methods
Patients
Between January 1985 and December 1996, a total of 1987 patients
with sterility due to azoospermia were referred by gynaecologists or
urologists at other institutions to the male infertility clinic at Kobe
University Hospital. Chromosomal analyses were performed in all
patients, and those found to have a 47,XXY karyotype or mosaicism
were enrolled into the study.
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
© European Society of Human Reproduction and Embryology
Klinefelter’s syndrome in the male infertility clinic
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) 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/23 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 50–100 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 3400 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 6.5–8.0 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.
Results
Among 1987 patients referred to our institution with of
azoospermia, 148 were diagnosed as Klinefelter’s syndrome
from G-banding studies of PBL showing a 47,XXY karyotype
or mosaicism and underwent further study.
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%.
947
H.Okada et al.
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
(3106/ml)
Sperm motility
(%)
Abnormally formed
spermatozoa (%)
Eosin Y-stained
spermatozoa (%)
Semen volume
(ml)
47,XXY
46,XY/47,XXY
1
0.2
0
0
80
90
95
ND
3
2.5
ND 5 not determined.
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).
Semen analyses revealed azoospermia in 146 cases on three
consecutive occasions. However, spermatozoa were observed
in semen from one patient with a 47,XXY karyotype and from
one patient with a 46,XY/47,XXY karyotype. In the former
patient, chromosomal analyses were repeated twice to confirm
47,XXY karyotype. In the latter patient, FISH analysis confirmed the mosaicism. Spermatozoa were detected on three
out of five occasions, and on one out of four occasions
respectively. Semen analyses revealed severe oligoasthenoteratozoospermia in both patients (Table I). Eosin Y staining
revealed that only 5% of the spermatozoa in the non-mosaicism
patient were alive. One-tenth of these samples were used for
meiotic studies, and remainders were cryopreserved for future
ICSI procedures.
Conventional testicular biopsy was performed in 30 patients
between 1985 and 1988; subsequently this procedure was not
performed routinely. Instead, biopsy has been performed at
multiple sites since 1996 to facilitate ICSI. Conventional singlesite testicular biopsy showed various degrees of seminiferous
tubule deterioration. Most specimens showed germ cell aplasia
with only Sertoli cells in tubules or hyalinized tubules, with
948
Figure 2. Fluorescent in-situ hybridization (FISH) analysis of the X
chromosomes in metaphase cell. By G-banding, this patient was
diagnosed with 46,XY/47,XXY mosaicism (ratio, 2:28). An X
chromosome-specific probe was hybridized. Two green signals are
indicated by arrowheads. This patient was reclassified as having
non-mosaic Klinefelter’s syndrome.
or without mild hyperplasia of Leydig cells (Johnsen score 1
to 2). Multiple-site biopsies were performed in the five most
recent cases. Papanicolaou-stained specimens showed round
and elongated spermatids in one patient; this cell suspension
was kept frozen for possible future ICSI.
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
Klinefelter’s syndrome in the male infertility clinic
Table II. Mosaicism in Klinefelter’s syndrome by G-banding and FISH
Karyotype (G-banding)
46,XY/47,XXY
46,XY/47,XXY
46,XY/47,XXY
46,XY/47,XXY
46,XY/47,XXY
46,XY/47,XXY
47,XXY/47,XYY/48,XXYY
46,XY/47,XXY/48,XXYY
Ratio of mosaicism
G-banding
FISH
LH
(mIU/ml)
FSH
(mIU/ml)
Testosterone
(ng/ml)
Right testis
volume (ml)
Left testis
volume (ml)
10:20
10:20
5:25
5:25
10:20
10:20
3:20:7
5:20:5
25:75
20:80
25:75
25:75
30:70
25:75
5:80:15
25:50:25
11
14
16.9
15
10.5
12.4
11
10.5
33.8
26.4
24.4
18.3
9.6
16.5
32.1
15.9
6.9
2.9
4.1
3.4
3.2
3.5
1.0
1.9
8
8
10
15
16
16
3
3
8
8
10
15
16
16
3
3
G-banding was performed on 30 metaphase cells as described in Materials and methods. Fluorescence in-situ hybridization (FISH) was performed on 100
blood lymphocyte metaphase chromosomes using an X chromosome-specific probe, and a Y chromosome-specific probe.
FSH 5 follicle stimulating hormone; LH 5 luteinizing hormone.
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 (ù15 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).
Mosaicism for 47,XXY/47,XYY/48,XXYY and 46,XY/
47,XXY/48,XXXY were observed in one patient each. These
patients had small testes (,4 ml) and hormonal profiles
showing hypergonadotrophic hypogonadism.
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 0.11–
0.24% and 0.06–0.42% respectively observed in the present
series in normal fertile men. The occurrence rates of diploid
cells were 0.8% and 0.34% respectively—almost the same rate
as in fertile men (0.17–0.41%). 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).
In our patient with non-mosaic Klinefelter’s syndrome, the
couple declined the option of ICSI, which was planned in the
case of mosaic Klinefelter’s syndrome using cryopreserved
spermatozoa, but cancelled due to severe ovarian hyperstimulation syndrome (OHSS). Round and elongated spermatids
extracted from testicular tissue in one case have been frozen
for possible future ICSI, pending approval of the local ethics
committee.
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.
Discussion
Classic Klinefelter’s syndrome is characterized by gynaecomastia, small, firm testes with hyalinization of seminiferous
tubules, hypergonadotrophic hypogonadism and azoospermia,
though these features are reported to be variable (Paulsen
et al., 1968). In the present series, only azoospermic patients
were karyotyped, and the prevalence of this syndrome among
them was 7.4%. This was slightly lower than the value of
10% reported in Western countries (De Braekeleer and Dao,
1991), but was similar to that reported previously in Japanese
azoospermic patients (7.8%) (Matsuda et al., 1992).
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
949
H.Okada et al.
Table III. Chromosome analysis of spermatozoa by three-colour FISH
No. of spermatozoa
Hybridization
signals
Presumed
karyotype
18/X
18/Y
18/X/X
18/X/Y
18/Y/Y
18/18/X
18/18/Y
18/18/X/X
18/18/X/Y
18/X/X/Y
18/18
23,X
23,Y
24,XX
24,XY
24,YY
24,X118
24,Y118
46,XX
46,XY
25,XXY
44,-XX, -XY
OR -YY
22, -X OR -Y
18
Null
Total
46,XY/47,XXY
47,XXY
Fertile man 1
Fertile man 2
Fertile man 3
269
296
7
6
1
4
1
2
3
1
0
252
277
6
8
1
3
1
1
1
1
0
2665
2438
12
10
0
7
10
0
23
0
0
3560 (44.42)
3689 (46.03)
9 (0.11)
5 (0.06)
2 (0.02)
11 (0.14)
12 (0.15)
1 (0.01)
13 (0.16)
15 (0.19)
0 (0)
2369
2590
13
23
0
23
30
0
15
9
2
0 (0)
698 (8.71)
8015 (100)
1 (0.02)
357 (6.57)
5432 (100)
(43.18)
(47.51)
(1.12)
(0.96)
(0.16)
(0.64)
(0.16)
(0.32)
(0.48)
(0.16)
(0)
0 (0)
33 (5.30)
623 (100)
(42.21)
(46.40)
(1.01)
(1.34)
(0.17)
(0.50)
(0.17)
(0.17)
(0.17)
(0.17)
(0)
0 (0)
46 (7.71)
597 (100)
(46.94)
(42.94)
(0.21)
(0.18)
(0)
(0.12)
(0.18)
(0)
(0.41)
(0)
(0)
0 (0)
513 (9.03)
5678 (100)
(43.61)
(47.68)
(0.24)
(0.42)
(0)
(0.42)
(0.55)
(0)
(0.28)
(0.17)
(0.04)
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 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 Gbanding is likely to be an artefact, requiring re-evaluation
by FISH.
In general, the percentage of mosaicism detected by Gbanding 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.
950
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-
Klinefelter’s syndrome in the male infertility clinic
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 threecolour 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 nonmosaicism 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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Received on July 17, 1998; accepted on December 16, 1998
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