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- Original Article -
Y chromosome microdeletions in azoospermic patients with Klinefelter's syndrome
Anurag Mitra1, Rima Dada2, Rajeev Kumar3, Narmada Prasad Gupta3, Kiran Kucheria2, Satish Kumar Gupta1
1Gamete Antigen Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India
2Department of Anatomy, 3Department of Urology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi
110029, India
Abstract
Aim: To study the occurrence of Y chromosome microdeletions in azoospermic patients with Klinefelter's syndrome
(KFS). Methods: Blood and semen samples were collected from azoospermic patients with KFS
(n = 14) and a control group of men of proven fertility
(n = 13). Semen analysis was done according to World Health Organization
(WHO) guidelines. Blood samples were processed for karyotyping, fluorescent
in situ hybridization (FISH) and measurement of plasma follicle stimulating hormone (FSH) by radioimmunoassay. To determine Y chromosome
microdeletions, polymerase chain reaction (PCR) of 16 sequence tagged sites (STS) and three genes
(DFFRY, XKRY and RBM1Y) was performed on isolated genomic DNA. Testicular fine needle aspiration cytology (FNAC) was done
in selected cases. Results: Y chromosome microdeletions spanning the azoospermia factor
(AZF)a and AZFb loci were found in four of the 14 azoospermic patients with KFS. Karyotype and FISH analysis revealed that, of the four
cases showing Y chromosome microdeletion, three cases had a 47,XXY/ 46,XY chromosomal pattern and one case
had a 46,XY/ 47,XXY/ 48,XXXY/ 48,XXYY chromosomal pattern. The testicular FNAC of one sample with Y
chromosome microdeletion revealed Sertoli cell-only type of morphology. However, no Y chromosome microdeletions
were observed in any of the 13 fertile men. All patients with KFS had elevated plasma FSH levels.
Conclusion: Patients with KFS may harbor Y chromosome microdeletions and screening for these should be a part of
their diagnostic work-up, particularly in those considering assisted reproductive techniques.
(Asian J Androl 2006 Jan; 8: 81-88)
Keywords: azoospermia; azoospermia factor; follicle stimulating hormone; Klinefelter's syndrome; Y chromosome; microdeletion
Correspondence to: Dr Satish Kumar Gupta, Gamete Antigen Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New
Delhi 110 067, India.
Tel: +91-11-2670-3651, Fax: +91-11-2616-2125
E-mail: skgupta@nii.res.in
Received 2005-01-19 Accepted 2005-06-22
DOI: 10.1111/j.1745-7262.2006.00083.x
1 Introduction
In the last few years considerable progress has been
made in understanding the pathogenesis of
spermatogenic arrest and infertility. Infertility is often due
to hypogon-adism; Klinefelter¡¯s syndrome (KFS) is the commonest
cause of hypogonadism and infertility [1]. KFS is the
most common sex chromosomal abnormality in men, with an incidence of approximately 1 in 500 newborn
phenotypic males [1]. Men with KFS have a 47,XXY chromosome complement and, of these, 15 % are
classified as mosaic, with a 46, XY/ 47, XXY chromosomal
complement. KFS is characterized by seminiferous
tubular dysgenesis, azoospermia and elevated levels of
serum gonadotropins.
Studies have shown that the deletion of azoospermia
factor (AZF) loci on the long arm of Y chromosome
results in spermatogenic failure and these loci are
recurrently deleted in infertile males [2-6]. Previously, men
with non-obstructive azoospermia had no therapeutic
fertility options except anonymous donor insemination
or adoption. The ability to extract spermatozoa from the
testes of some men with non-obstructive azoospermia
using the multi-biopsy technique of testicular sperm
extraction followed by intracytoplasmic sperm injection
(ICSI) offers an efficacious therapeutic approach [7].
The possibility of using ICSI with testicular sperm has
also been proposed in KFS patients [8]. The widespread
use of such procedures has raised some interesting and
important genetic issues about the inheritance of genetic
anomalies, such as Y chromosome microdeletions in the
offspring [9].
There are conflicting reports on the occurrence of Y
chromosome microdeletions in KFS patients [1, 10-13].
Tateno et al. [1] failed to find microdeletions of the
deleted in azoospermia (DAZ) or the Y chromosome
ribonucleic acid recognition motif (YRRM) genes in KFS
patients. On the other hand, others have reported low
incidence of Y chromosome microdeletions [11-13]. The
objective of our study was to analyze Y chromosome
microdeletions at 16 different sequence tagged sites
(STS) and three genes by polymerase chain reaction
(PCR) in Indian KFS patients attending the infertility clinic
at a tertiary care hospital.
2 Materials and methods
Male subjects with primary infertility attending the
Infertility Clinic in the Urology Department of the All
India Institute of Medical Sciences (New Delhi, India) were
enrolled in the present study. All experiments using
human samples were carried out with informed consent
following clearance from the Institutional Bio-safety and
Ethical Committee. Based on cytogenetic analysis, 14
azoospermic KFS patients and 13 fertile males (with
a child of less than 2 years; sperm count > 20
million/mL) having a normal karyotype were included in this study.
The KFS patients were enrolled over a period of two
years. Each patient was carefully examined to rule out
other causes of infertility and a detailed family,
occupational and reproductive history was collected using a
predesigned proforma. The semen analysis was done
according to World Health Organization (WHO) guidelines [14]. The plasma values of follicle stimulating
hormone (FSH) were determined by radioimmunoassay.
Whenever possible, testicular fine needle aspiration
cytology (FNAC) was performed, as testicular biopsy was
ethically not possible in these cases.
2.1 Karyotyping and fluorescent in situ
hybridization(FISH)
Peripheral blood cultures were set up for
chromosomal analysis in all the cases and five well-spread
G-banded metaphases were karyotyped using automated
karyotyping software (Cytovision 2.81, Applied Imaging
Corp., San Jose, CA, USA) [15]. In certain Klinefelter
mosaic (KFM) cases the percentage of mosaicism was
re-analyzed using FISH. This analysis was performed
on metaphase spreads and interphase nuclei using
commercially available fluorescent-labeled chromosome
enumeration X and Y probes (CEP X/CEP Y; Vysis, Downers
Grove, IL, USA) as described previously [16]. The slides
prepared from fixed cell suspension obtained during
cytogenetic analysis were denatured in 70 % formamide/
2 × SSC at 73 ºC for 2-5 min. Images were captured
using a Charged Coupled Devise (CCD) camera (Cohu Inc, Poway, CA, USA) attached to a Zeiss Axiophot
microscope (Carl Zeiss AG, Konigsallee, Gottingen, Germany) using a triple band pass filter (rhodamine,
fluorescein-isothiocyanate, and 4¡¯,6¡¯-diamidino-2-phenylindole
dihydrochloride) and analyzed using Quips Smart
Capture FISH imaging software 2.81 (Vysis, Illinois, USA).
A minimum of 200 interphase cells and 20 metaphase
spreads were scored in each case. Only those signals
well embedded in the nucleus were included for scoring.
Clumped and overlapping nuclei and those with low fluo
rescence intensity or high background intensity were
excluded. Patchy and diffused signals were included in
the evaluation only if they were well separated. The
criteria for defining a sample as "mosaic" were those in
which 20 % of the nuclei/metaphase spreads had a
variation in signal number from the majority or showed a
signal pattern other than the normal sex chromosomal XY
and XX signals. The cut-off value was calculated from
the mean ± SD of results obtained from a group of
normal control samples.
2.2 Analysis of Y chromosome microdeletions by PCR
Genomic DNA was isolated by a Genomic DNA Isolation Kit (Promega, Madison, WI, USA) using the
manufacturer¡¯s protocol. The concentration of the
isolated genomic DNA was determined by
spectrophotometric analysis at 260 nm.
All the DNA samples were processed for Yq
microde-letions analysis using PCR. Each of these samples was
analyzed using 19 sets of primers. DNA from fertile
male subjects was taken as the positive control and the
DNA from a female subject as the negative control in
each reaction.
The STS and genes used were as follows:
AZFa: sY746, sY84, sY86, DFFRY
AZFb: XKRY, sY118, sY113, sY127, sY134, sY143,
RBM1Y
AZFc: sY153, sY148, sY157, sY158, sY254, sY255,
sY160 (heterochromatin region)
The sex-determining region of Y (SRY, i.e. STS sY14)
was included as the internal control. Detailed sequences
of the primers were given in Table 1.
The samples were subjected to PCR amplification
using initial denaturation for 5 min at 94 ºC and 35 cycles
of 94 ºC for 1 min, with the annealing temperature
varying from 50 ºC to 64 ºC for 1 min and extension at 72 ºC
for 1 min. The final extension time was 7 min at 72 ºC.
The PCR products were analyzed on a 2 % agarose gel
containing ethidium bromide (0.5 µg/mL). An STS or
gene was considered absent only after at least three
amplification failures in the presence of both the internal
control (SRY) and the positive control. In addition,
deletions with respect to various STS markers were
reconfirmed by performing temperature gradient PCR in the
absence or presence of 5 % dimethyl sulfoxide.
3 Results
The karyotype analysis revealed seven cases with a
47,XXY chromosomal pattern (KFS) and seven KFM cases. Of the latter, five had a 47,XXY/ 46,XY
chromosomal pattern and two had a 46,XY/ 47,XXY/ 48,XXXY/
48,XXYY chromosomal pattern (Table 2). The fertile
male subjects had a karyotype of 46,XY chromosomes
(data not shown).
The clinical parameters of the patients with KFS were
summarized in Table 2. The age of the KFS patients
ranged from 18 years to 32 years (Table 2), whereas the
mean age of the normal samples was 31 years. All cases
presented with primary infertility. Except for
one patient having aspermia, all other cases showed the absence of
sperm in the semen. The FSH levels in the KFS cases
were found to be very high ([39.56 ± 20.73] mIU/mL).
The samples taken from fertile men had mean 5 mIU/mL
of FSH level (range [2-7] mIU/mL).
Four of the 14 (28.6 %) KFS patients showed deletions
in both the AZFa and AZFb regions (Table 2, Figures 1 and
2). Deletions in STS represented by sY86, sY746 and sY127
were present in all four patients (Table 2, Figure 1). Two
of these four patients also had a deletion in STS sY84 of
the AZFa region (Figure 1). In addition, deletions in the
AZFb regions were also found in STS sY134 in one
subject (Table 2), and STS sY113 (Figure 2B) and sY118
(Table 2) in another. In comparison, using similar
conditions of PCR, none of the fertile men showed any Y
chromosome microdeletions. FNAC was carried out in two
patients with KFS. Case 240/03 with multiple cell lines
(47,XXY [87 %]/46,XY [7 %]/ 48,XXYY [3 %]/48,XXXY [3 %]) and
AZFa and AZFb microdeletions had Sertoli cell only type II syndrome (SCO II, isolated foci
of spermatogenesis along with Sertoli cells) and case
288/03 with a 47,XXY chromosomal complement had Sertoli
cell-only type I syndrome (SCO I, complete absence of
germ cells in the seminiferous tubules and presence of
only Sertoli cells, see Table 2). The latter case had no
AZF microdeletions.
4 Discussion
The role of Y chromosome microdeletions in male
infertility has been well established. Microdeletions on the
long arm of human Y chromosome, postulated as the
AZF factor, are associated with spermatogenic
failure [2-6]. The reported frequency of Y chromosome microdeletions
varies from 1 % [3] to 55 % [2], which is largely related
to different inclusion criteria [13].
Cytogenetic abnormalities are known factors for
spermatogenic failure, most of which are numerical
or structural. KFS is a numerical chromosomal aberration,
in which most patients have a 47,XXY chromosomal component or are mosaic (46,XY and 47,XXY), or are
mosaic variant cases with additional cell lines (48,XXYY
and 48,XXXY). Surprisingly, in the present study we
observed a very high incidence of KFM (seven out
of 14), of which two were variants. An incidence of KFM or
mosaic variant of approximately 15 % in KFS patients
has been reported previously [17]. Conventional
karyotyping of metaphase spreads of peripheral
blood lymphocytes, when up to 20 cells are counted, may miss low-grade
mosaicism. In a recent study, 18 KFS patients with a
47,XXY chromosomal complement, as per conventional
karyotyping, were further analyzed by FISH [18]. Scoring
of 400 interphase and 40 metaphase lymphocyte nuclei per
patient revealed low-grade mosiacism in this group [18].
No significant differences were observed between
interphase nuclei and metaphase spreads, suggesting that FISH
on interphase nuclei can be routinely used to screen
aneuploidy status. Interestingly, two KFS patients with a
high prevalence of normal 46,XY lymphocytes had sperm
in their ejaculate [18]. However, in the present study,
FISH and cytogenetic analysis did not reveal any
differences in the percentage of mosaic cell lines. Increased
gonosomal aneuploidy frequencies in spermatozoa of KFS
patients has also been reported [19]. This knowledge
may help in the genetic counseling of KFS patients prior
to ICSI treatment, because a low level of mosaicism in
their lymphocytes may also be reflected in the germ cells.
It may just be a chance phenomenon that we observed
an incidence of 50 % of KFM or its variants in our study
of KFS patients. A larger series might determine the true
incidence of such cases. It might be stated that, being a
tertiary care referral hospital with full diagnostic facilities,
we are able to identify KFS cases with the slightest
phenotypic abnormality.
Though the studies on microdeletions in Y
chromosomes have been very common in cases of idiopathic
infertility, there have been very few studies on Y
chromosome microdeletions in KFS patients. Tateno
et al. [1] investigated deletions of Yq interval 6,
DAZ and YRRM genes in 21 KFS patients with
(n = 1) and without (n = 20) spermatogenesis. Their findings suggested no deletions
of Yq interval 6 in these patients. Similarly, in another
study of combined cytogenetic and Y chromosome microdeletion screening, no deletions in the Y chromo
some were found in KFS patients [10]. In a clinical
study of 42 infertile men positive for AZFc deletion, it was
observed that one case could be categorized as KFM [11].
In an independent study of the prevalence of Y
chromosome microdeletions in 186 oligospermic and azoospermic
males opting for ICSI, it was observed that only one
male belonged to the KFM category and also had
AZFc microdeletions [12]. In another screening study for Y
chromosome microdeletions in 226 Slovenian sub-fertile
men, it was observed that five patients had low-level
mosaicism (abnormal karyotype < 2.5 %) and of these
five patients only one had AZFc microdeletion [13].
These studies describe very weak mosaicism in comparison to ours, where the mosaicism found in the cells
is as high as 87 % in some cases (Table 2). Our study is
the first detailed one on Y chromosome microdeletions in
KFS patients with 19 sets of primers. This study
illustrated deletions in the AZFa and
AZFb regions, in contrast to the deletions in the
AZFc region observed in KFS patients by others [11-13]. The deletions were further
confirmed by running temperature gradient PCR. The
AZFa and AZFb combined deletions were found
in 28.6 % of the patients. The high incidence of Y chromosome
microdeletions observed by our group may be a chance
phenomenon. In parallel studies carried out by our group,
using the same sets of PCR primers, idiopathic infertile
azoospermic men (n = 67) showed an incidence of Y
chromosome microdeletions in 19.4 % (unpublished observations). Of the 13 patients who were positive for
Y chromosome microdeletions, three had deletions only
in AZFa, three only in AZFb, one in
AZFa and AZFb, one in AZFb and
AZFc, and five in AZFc alone. At this
stage, the reasons for the high incidence of Y
chromosome microdeletions observed in AZFa and
AZFb loci in the Indian population is not clear. An independent study
of Y chromosome deletions in 340 azoospermic Indian
men, which used 30 STS markers, revealed an overall
deletion of 8.5 % [4]. Those showing deletions had
AZFc deletion in 82.8 %, AZFb in 55.2 % and
AZFa in 24.1 %. However, the authors failed to see any deletions in the
sY86 and sY84 STS, commonly recommended for analysis. On the other hand, the missing STS were sY746,
sY741, sY742, sY615 and the gene DFFRY, which are inter-spread between or around sY86 and sY84. Hence,
the chances of finding a deletion is higher if more sets of
primers are used, as suggested by Thangaraj et
al. [4]. But this contrasts with the recommendations of the
European Academy of Andrology guidelines [20], which
suggest that over 90 % of microdeletions can be detected with the use of only two STS markers for each
AZF loci.
Although the nature of the histological changes at the
testicular level still needs to be defined, patients with KFS
are known to have progressive deterioration of their
testicular architecture. After the onset of puberty, their
testes usually shrink and become firm. Observed high levels
of FSH in our study was in agreement with very low
levels of inhibin-B and high levels of FSH reported in KFS
patients [21]. The KFS case finding by Oates et
al. [11] shows SCO II syndrome, which is in agreement with
the prevailing notion. Though it was not possible to take
the FNAC of all the patients in our study as it is clinically
not recommended, the FNAC report from two patients
showed an SCO II type of morphology in one case and
SCO I in another. The findings of Kamp et
al. [22] suggest a high frequency of
AZFa deletions in men with SCO syndrome. It can also be interpreted that deletions
in AZFa give rise to more severe phenotypes such as SCO
syndrome, which is in agreement with our findings. In this
study we found that case 240/03 with AZFa and
AZFb deletions had few spermatogenesis loci in the testes (SCO
type II). Determination of telomerase activity in
therapeutic testicular biopsies of KFS patients have revealed that
those with high telomerase activity (> 39.0 units/µg protein)
have a high probability of the presence of sperm [23].
In a study of 24 non-mosaic KFS patients, it was observed that
men who had spermatozoa in their testicular
tissues (50 %), were positive for both 46,XY and 47,XXY spermatogonia
in their seminiferous tubules [24]. In contrast, KFS
patients without spermatozoa in their testicular tissues were
positive only for 47,XXY spermatogonia.
It has been shown that sperm can be retrieved from
pure and mosaic KFS patients for use in ICSI, leading to
successful pregnancy [8, 25] and in such cases the
consequences of vertical transmission of deletions may be
further studied. Follow-up of 42 infertile men with
deletion in the AZFc region and 18 children conceived through
the use of ICSI revealed that though the offspring were
healthy, the sons inherited the AZFc deletion with no
increase in length [11]. In cases where sperm could be
retrieved, the presence of Y chromosome microdeletions
had no obvious impact on fertilization or pregnancy rate.
Due to observed Y chromosome microdeletions in KFS patients, the analysis may be imperative in routine
clinical follow-up of such cases, followed by genetic
counseling with respect to the risk of transmitting Y chro
mosome microdeletions to the male progeny, if the
patients opts for assisted reproductive techniques.
Acknowledgment
This study was conducted under the Task Force on
`Male Genomics¡¯ constituted and financed by the Indian
Council of Medical Research, Government of India.
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