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- Original Article -
Routine screening for classical azoospermia factor deletions
of the Y chromosome in azoospermic patients with
Klinefelter syndrome
Jin Ho Choe, Jong Woo Kim, Joong Shik Lee, Ju Tae Seo
Department of Urology, Cheil General Hospital, Kwandong University College of Medicine, Seoul 100-380, Korea
Abstract
Aim: To evaluate the occurrence of classical azoospermia
factor (AZF) deletions of the Y chromosome as a routine
examination in azoospermic subjects with Klinefelter syndrome (KS).
Methods: Blood samples were collected from
95 azoospermic subjects with KS (91 subjects had a 47,XXY karyotype and four subjects had a mosaic
47,XXY/46,XY karyotype) and a control group of 93 fertile men. The values of testosterone, follicle stimulating hormone (FSH)
and luteinizing hormone (LH) were measured. To determine the presence of Y chromosome microdeletions,
polymerase chain reaction (PCR) of five sequence-tagged site primers (sY84, sY129, sY134, sY254, sY255) spanning the
AZF region, was performed on isolated genomic DNA.
Results: Y chromosome microdeletions were not found in
any of the 95 azoospermic subjects with KS. In addition, using similar conditions of PCR, no microdeletions
were observed in the 93 fertile men evaluated. The level of FSH in KS subjects was higher than that in fertile
men (38.2 ± 10.3 mIU/mL
vs. 5.4 ± 2.9 mIU/mL,
P < 0.001) and the testosterone level was lower than that in the
control group (1.7 ± 0.3 ng/mL
vs. 4.3 ± 1.3 ng/mL,
P < 0.001).
Conclusion: Our data and review of the published
literature suggest that classical AZF deletions might not play a role in predisposing genetic background for the
phenotype of azoospermic KS subjects with a 47,XXY karyotype. In addition, routine screening for the classical
AZF deletions might not be required for these subjects. Further studies including partial
AZFc deletions (e.g. gr/gr or
b2/b3) are necessary to establish other mechanism underlying severe spermatogenesis impairment in KS.
(Asian J Androl 2007 Nov; 9: 815_820)
Keywords: Y chromosome; chromosome deletion; Klinefelter syndrome; azoospermia
Correspondence to: Dr Ju Tae Seo, Department of Urology, Cheil
General Hospital, Kwandong University College of Medicine,
1-19 Mukjeong-dong, Jung-gu, Seoul 100-380, Korea.
Tel: + 82-2-2000-7585 Fax: +82-2-2000-7787
E-mail: jtandro@cgh.co.kr
Received 2007-04-09 Accepted 2007-05-18
DOI: 10.1111/j.1745-7262.2007.00315.x
1 Introduction
Infertility is a major health problem that affects
approximately 15% of the population of reproductive age;
a male factor can be identified in approximately half of
these cases [1]. Moreover, a significant proportion of
infertile males are affected by either oligospermia or
azoospermia. These conditions have many causes, such
as varicocele, infection, cryptorchidism,
endocrinological disorders or obstruction/absence of a seminal
pathway [2]. However, up to 66% of all infertile men have
idiopathic azoospermia or severe oligospermia [3, 4].
Because infertility is largely a result of impaired
gametogenesis, in which a number of genes participate,
it is reasonable to predict that deletions in spermatogenic
genes result in impaired spermatogenesis, leading to
infertility. Therefore, evaluation of microdeletions might
be useful for identifying molecular defects of the Y
chromosome.
Microdeletions of the Y chromosome represent the
most frequent cause of male infertility, and are
responsible for 10%_15% of cases of azoospermia or severe
oligozoospermia. Such deletions have been localized to
one or more loci referred to as azoospermia factors
(AZF) a, b and c. These three non-overlapping regions are
mapped within intervals five and six of the Y
chromosome and lie within Yq11.21. to Yq11.23 [5].
Deletions have been shown to more frequently involve the deleted
in azoospermia (DAZ) gene in
AZFc, and are associated with both azoospermia and severe oligospermia; however,
men with deletions involving the proximal regions
(AZFa and AZFb) have also been shown to present with
azoospermia or severe oligospermia [2, 6, 7]. Since 1995,
based on the results of a large number of studies, Y
chromosome microdeletion screening has become part of the
routine diagnostic work-up for severe male factor
infertility [8, 9]. The screening for Yq deletions has
provided an etiology for spermatogenic disturbances, and
has also provided a prognosis for testicular sperm
retrieval based on the type of deletion. Assisted
reproductive techniques have offered an efficient therapy for
men bearing Y microdeletions; however, this genetic
defect is then transmitted to male offspring in cases of
successful reproduction.
Klinefelter syndrome (KS) is the most common
sex-chromosome abnormality in men, and results in
testicular failure, variable degrees of androgen deficiency and
infertility. It affects approximately one in 500 newborn
boys and accounts for up to 11% of azoospermic men
[10]. KS results from an extra X chromosome in male
karyotypes (47,XXY) or a combination of normal and
extra X karyotypes (mosaic pattern, 47,XXY/46,XY) in
somatic and germ cells. Most men with KS have a nonmosaic karyotype; only about 10% of men are mosaic.
KS is the most common chromosomal abnormality associated with male infertility and azoospermia. The
mechanism by which the chromosome abnormality leads
to the spermatogenic defect remains unknown. Studies
aiming to define the predisposing genetic background for
the KS phenotype have not been successful. Several
investigators have hypothesized that Y chromosome
deletions might affect the phenotypic expression of KS in
regard to spermatogenesis. However, there have been
very few studies on Y chromosome microdeletions in
subjects with KS and the reports have been conflicting
regarding the occurrence of microdeletions [11_15]. The
aim of the present study was to evaluate the occurrence
of classical AZF deletions of Y chromosome in
azoospermic subjects with KS, and to determine whether
routine screening for this examination would be useful in
the clinical setting.
2 Materials and methods
2.1 Study population
From September 2002 to December 2005, male subjects with primary infertility attending the fertility clinic
were enrolled in the present study. The study protocol
was approved by the Institutional Review Board and
informed consent was obtained from all subjects. Each
subject provided a detailed family, occupational and
reproductive history. A general physical
examination with particular attention to the scrotal contents, including
measurement of the testicular volume, using an orchidometer,
was performed. Semen analysis was performed twice
according to the World Health Organization Guidelines
[16]. Hormonal assays that reflected activity of the
spermatogenic axis (follicle-stimulating hormone [FSH])
and the androgenic axis (testosterone and luteinizing hormone
[LH]) were drawn.
Cytogenetic analysis was performed on peripheral
lymphocytes that were cultured for 72 h. Karyotypes
were analyzed by GTG banding (G-bands by trypsin using
Giemsa). For each case, at least 30 metaphase spreads
were examined.
Finally, based on cytogenetic analysis, 95
azoosper-mic subjects with KS and a control group of 93 fertile
men with a normal 46,XY karyotype were evaluated. Of
all the subjects with KS, 91 subjects had a 47,XXY
(pure or nonmosaic) chromosomal pattern and 4 had a
47,XXY/46,XY (mosaic) chromosomal pattern.
2.2 Genomic DNA preparation
Genomic DNA was obtained from peripheral lymphocytes using the QIAamp Blood Kit (QIAZEN,
Chatsworth, TN, USA) or the Aquapure Genomic DNA Isolation Kit (Bio-Rad, Hercules, CA, USA). After 10 mL
of peripheral blood was collected in an EDTA vacuum
tube, procedures were performed as recommended by the manufacturer.
2.3 Polymerase chain reaction
A set of five Y specific sequence-tagged sites (STS,
accessed from Bioneer, Seoul, Korea) were amplified
using genomic DNA isolated from the subjects by
polymerase chain reaction (PCR). According to the
recommendations of the European Academy of Andrology
guidelines [17], the representative STS spanning the
AZF region were selected for use; a total of five loci, sY84
(AZFa region), sY129, sY134 (AZFb region), and sY254,
sY255 (AZFc region) covering the euchromatic region
between Yp 11.31 and distal Yq11.23 were examined. The
sequences of one set of primers for each gene are shown
in Table 1. Of these, sY254 and sY255 are encoded in
the region of the DAZ gene in subinterval 6 of Yq11.
As an internal control, coamplification of the
sex-determining gene SRY and the autosomal gene GAPDH
were routinely performed on all genomic DNA samples.
PCR reactions were performed in 10 mmol/L Tris-HCl,
pH 8.3, 50 mmol/L KCl, 1.5 µmol/L
MgCl2, and 200 µmol/L dNTPs containing 100 ng of genomic
DNA, 4_20 µmol/L of each primer, and 0.3 units of
Taq polymerase in a final volume of 20 µL. After an
initial denaturation step at 94ºC for 2 min, the cycle
parameters were: 35 cycles at 94ºC for 40 s, 58ºC for
80 s and 72ºC for 60 s. This protocol was followed by
the final extension step at 72ºC for 10 min. The reaction
products were then analyzed by electrophoresis at 76 V
on 2%_4% agarose gels (Sigma Chemical, St. Louis, MO,
USA) containing ethidium bromide (0.1 mg/mL), and
visualized under ultraviolet light. A positive control (sample
from a normal fertile male) and two negative controls
(normal female sample and every constituent except
DNA), were included in every PCR assay. We confirmed
a deletion at the loci studied when the product of the
expected size was not obtained after at least three PCR
experiments with a single primer pair. P value < 0.05
was used to define statistical significance. Results are
presented as the mean ± SD unless otherwise indicated.
3 Results
The clinical and seminal parameters of the subjects
are summarized in Table 2. The mean age of the KS
subjects and normal fertile men were 32.9 ± 3.9 years
and 33.5 ± 4.9 years, respectively. The level of FSH in
KS subjects was higher than in fertile men
(38.2 ± 10.3 mIU/mL vs. 5.4 ± 2.9 mIU/mL,
P < 0.001) and the testosterone level was lower than in the control
group (1.7 ± 0.3 ng/mL
vs. 4.3 ± 1.3ng/mL,
P < 0.001).
Y chromosome microdeletions using a total of five
representative STS spanning the AZFa,
AZFb and AZFc loci were not found in any of the 95 azoospermic
subjects with KS. In addition, using similar conditions of
PCR, none of the 93 fertile men showed any Y
chromosome microdeletions.
4 Discussion
The etiology of the defects of spermatogenesis in
KS might involve many factors that remain to be defined:
the underlying mechanisms of testicular degeneration are
poorly understood. Many hypotheses regarding the
underlying mechanism of depletion of germ cells in Klinefelter
men have been reported and include insufficient
supernumerary X-chromosome inactivation, Leydig cell
insufficiency and disturbed regulation of apoptosis of Sertoli
and Leydig cells. Lee et al. [11] suggest that X
chromosome over-dose might interfere with the function of the
Y chromosome in non-mosaic type. In contrast, altered
dosage of some genes on the X chromosome might affect the development and/or degeneration of germ cells
in men with 47,XXY. However, at present, the exact
mechanism remains unclear. To date, little data is
available on the frequency of Y chromosome microdeletions
in subjects with KS. There are conflicting reports on the
occurrence of Y chromosome microdeletions in subjects
with KS (Table 3). Tateno et al. [12] failed to find
microdeletions of the DAZ or YRRM genes in 21
nonmosaic KS subjects with (n = 1) and without
(n = 20) spermatogenesis. In another study, Y chromosome
microdeletions were observed in one of nine (11.1%)
subjects with idiopathic azoospermia using 60 STS,
whereas no deletions were found in subjects with the
non-mosaic type of KS. However, others have reported
a low incidence of Y chromosome microdeletions. In an
independent study of the prevalence of Y chromosome
microdeletions in 186 oligospermic and azoospermic men
opting for intracytoplasmic sperm injection, only one man
belonged to the Klinefelter mosaic category and also had
AZFc microdeletions [14]. In another screening study
for Y chromosome microdeletions in 226 Slovenian subfertile men, it was observed that five subjects had
low-level mosaicism 46,XY/47,XXY (abnormal karyotype
< 2.5%) and of these, only one subject had an
AZFc microdeletion [15]. However, because of the small
number of subjects analyzed in the three studies, no firm
conclusion could be drawn regarding this issue. Recently,
Y chromosome microdeletions using 19 sets of primers
in 4 of 14 azoospermic KS subjects studied was reported
[13]. All 4 cases with microdeletions were of mosaic
type (a 47,XXY/46,XY pattern in three subjects and a
46,XY/47,XXY/48,XXXY/48,XXYY pattern in one subject), whereas 11 subjects without microdeletions
were all nonmosaic type 47,XXY. Although the present
study showed that no microdeletions were present in four
subjects with mosaic KS, further studies are needed to
clarify the association between the mosaic pattern and
microdeletions. Based on the findings of the present study
and review of published reports it appears that classical
AZF deletions might not play a role in predisposing
genetic background for the phenotype of azoospermic KS
subjects with a 47,XXY. Partial AZFc deletion might
cause spermatogenic failure, and several types of partial
AZFc deletions have been proposed and designed as
gr/gr, g1/g2, b1/b3,
b2/b3, rg/gr, g1/g3 and
b3/b4 deletions [19, 20]. In the present study, the
AZFc subdeletions were not evaluated because they had not yet made an
issue of the subdeletions when this study was designed.
Therefore, we could not get the results of subdeletions.
Additional further studies would resolve this
issue.
In the present study, screening of Y chromosome
microdeletion assay was performed in a clinical practice.
The use of only one or two STS for each AZF region in
this study might be considered a limitation of the study
design. There is no consensus concerning which and
how many loci should be analyzed; as has been reported,
detected microdeletion frequencies are not dependent on
the number of STS analyzed [21]. Several studies that
use a variable number of STS have report that the
prevalence of deletions does not increase with more STS used.
Therefore, at present it appears that it is acceptable to
use two or three STS markers for each AZF region, as
is suggested by Simoni et al. [17]. In addition, the choice
of STS/gene markers used to identify Yq deletions is
unrestricted. In terms of the described Y-specific STS,
different panels of STS may cause different results; this
is because some show differences in their reported map
locations, others have multiple loci, and several
represent naturally occurring polymorphisms. Taking into
consideration these patterns and the extent of published
Yq deletion intervals, we selected the most widely used
five STS that would unequivocally determine the
presence or absence of Y-specific sequences across the three
known AZF regions.
Some clinicians have performed genetic screening,
including karyotyping and an assessment of Y
chromosome microdeletions simultaneously to save time for the
couples. The reasoning behind this is that KS has a
considerably different clinical presentation and in
practice is difficult to distinguish from other causes
of hypergonadotrophic hypogonadism. Although we agree
with this opinion in part, the work up for infertility should
proceed step by step. We suggest that the chromosomal
analysis should be performed first in azoospermic men
with a suspected KS. And then further genetic
screening is indicated. Moreover, clinicians need to consider
minimizing costs to reduce patient burden from
unnecessary testing in fertility clinics. In our center, the cost
is KRW116 448 (approximately $US124) for
high-resolution chromosomal analysis and KRW 64 420
(approxi-mately $US69) for microdeletions of
AZF.
In conclusion, classical AZF deletions might not play
a role in predisposing genetic background for the
phenotype of azoospermic KS subjects with a 47,XXY. In
addition, routine screening for the classical
AZF deletions might not be required for these subjects. Further
studies, including partial AZFc deletions
(e.g. gr/gr or b2/b3), might be necessary to establish other
mechanisms underlying severe spermatogenesis impairment in
KS. We expect that the recent progress in genomic
analysis of the X and Y chromosome as well as improved
understanding of the regulation of gene expression will
lead to a better understanding of the mechanisms involved
in germ-cell depletion.
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