Molecular and cytogenetic evaluation of Y chromosome in

Journal of Cell and Molecular Biology 7(2) & 8(1): 45-52, 2010
Haliç University, Printed in Turkey.
http://jcmb.halic.edu.tr
Research Article
Molecular and cytogenetic evaluation of Y chromosome in
spontaneous abortion cases
Gülşah KOÇ1, Korkut ULUCAN*,1, Deniz KIRAÇ2, Deniz ERGEÇ1, Tufan TARCAN3, A.
İlter GÜNEY1.
1
Marmara University, Faculty of Medicine, Department of Medical Genetics, Istanbul, Turkey.
Yeditepe University, Faculty of Medicine, Department of Biochemistry, Istanbul Turkey.
3
Marmara University, Faculty of Medicine, Department of Urology, Istanbul, Turkey.
(* author for correspondence; [email protected])
2
Received: 21 April 2010; Accepted: 05 May 2010
Abstract
Infertility is defined as not being able to get pregnant despite having frequent, unprotected sex for at least a year.
Several conditions contribute to infertility and 50% is considered to be caused by a male-related factor.
Spontaneous abortion (SAB) is noninduced embryonic or fetal death or passage of products of conception before
the 20th week of pregnancy and is the most common complication of early pregnancy. SAB can occur by
teratogenic factors, advanced maternal age, genetic factors such as Y chromosome microdeletions and
chromosomal anomalies. In order to investigate the etiology of recurrent pregnancy loss (RPL) and to develop an
appropriate therapeutic strategy, it is necessary to ascertain the molecular and cytogenetic basis of these defects. In
this study, we aimed to reveal the relations between male infertility, Y chromosome microdeletions and SAB.
Thirty couples with a spontaneous abortion history and thirty fertile men were recruited to the study. All the
women were 46, XX and men were 46, XY. We couldn’t detect any Y chromosome microdeletion that could be the
reason for SAB. In order to evaluate effect of chromosome anomalies and Y chromosome microdeletions on SAB,
further studies with increased number of cases and controls need to be carried on.
Keywords: Infertility, spontaneous abortion, Y chromosome microdeletions.
Spontan düşük vakalarında Y kromozomunun moleküler ve sitogenetik incelemesi
Özet
Çiftlerin çocuk sahibi olma isteklerine ve düzenli cinsel ilişkiye rağmen, bir yıl içerisinde gebelik elde
edilmemesine infertilite (kısırlık) adı verilmektedir. İnfertiliteye etki eden birçok faktör bulunmaktadır ve bunların
%50’sinde etken erkek infertilitesidir. Gebeliğin ilk 20 haftası içinde, dışarıdan herhangi bir müdahale olmadan,
doğal nedenlerle, embriyo veya fetus ve eklerinin tamamının veya bir kısmının uterus kavitesi dışına atılması
olayına spontan düşük (abortus) denilmektedir ve gebeliğin erken döneminde en çok gözlenen komplikasyondur.
Spontan düşükler, teratojenik faktörler, ileri anne yaşı gibi nedenlerin yanında, Y kromozomu mikrodelesyonları ve
kromozomal anomaliler gibi genetik faktörlere bağlı olarak da oluşabilmektedir. Tekrarlayan gebelik kayıplarının
etyolojisini belirlemek ve uygun bir tedavi yöntemi geliştirmek için bu defektlerin moleküler ve sitogenetik
temellerinin incelenmesi gerekmektedir. Bu çalışmada, erkek infertilitesi, Y kromozom mikrodelesyonları ve
spontan düşükler arasındaki ilişkinin ortaya çıkarılması amaçlanmıştır. Spontan düşük hikayesi bulunan 30 çift ve
fertil 30 erkek çalışmaya dahil edilmiştir. Çalışmaya dahil olan bireylerin kromozom analizi sonuçlarına göre, tüm
kadınlar 46,XX ve erkekler ise 46,XY‘dir. Çalışmamızda spontan düşüklere neden olabilecek herhangi bir Y
kromozom mikrodelesyonu belirlenememiştir. Kromozom anomalilerinin ve Y kromozomu mikrodelesyonlarının
spontan düşükler üzerindeki etkisinin değerlendirilebilmesi için vaka ve kontrol sayılarının arttırılarak başka
çalışmalar yapılması gerekmektedir.
Anahtar Sözcükler: İnfertilite, spontan düşük, Y kromozom mikrodelesyonu.
46
Gülşah Koç et. al.
Introduction
Infertility is the inability of being pregnant after
one year of unprotected sexual intercourse.
Infertility comprises up to 15% of couples of
reproductive age in which 50% is caused by a male
factor (Noordam and Repping, 2006). Several
factors contribute to male infertility, such as gene
defects, hormonal milieu, chromosomal aberrations
and genital infections (Stipoljev et al., 2006).
Genetic factors are considered to affect almost 30%
of severe male infertility cases (Noordam and
Repping, 2006). The diagnosis of male infertility
include anamnesis, physical examination, semen
analysis, hormonal screening and genetic factors of
somatic cells (Stipoljev et al., 2006).
Spontaneous abortion (SAB) is the expulsion of
an embryo or fetus due to accidental trauma or
natural causes before approximately 22nd week of
gestation. It effects up to 15% clinically recognized
pregnancies and considered to be the most common
adverse outcome of pregnancy. Although several
studies tried to explain the etiology of SAB, the
results are still controversial. Beside the teratogenic
factors and advanced maternal age, genetic factors
such as Y chromosome microdeletions and
chromosomal anomalies are considered to be the
main reason of SAB (Dewan et al., 2006; Pryor et
al., 1997).
Y chromosome is essential not only for human
sex determination but also for maintenance of sex
cells and sex cell development. Y chromosome
(Yq) microdeletions represent the most frequent
molecular genetic cause of severe infertility,
observed with a prevalence of 10-15% in nonobstructive azoospermia and severe oligozoospermia (Sinclair et al.,1990). The regions
responsible for male infertility of Y chromosome
are located on the long arm of chromosome and are
termed as AZFa, AZFb, AZFc (AZF: Azoospermia
Factor) ( Burgoyne, 1998) (Stouffs et al., 2009).
The AZFa locus is located on proximal Yq11
(Yq11.21), while AZFb and AZFc are located on
distal Yq11 (Yq11.23). These AZF genes code
RNA binding proteins and may be involved in the
regulation of gene expression, RNA metabolism,
RNA packaging and RNA transportation from
nucleus to cytoplasm (Li et al., 2008). Deletions of
these regions result in spermatogenic arrest and are
associated with oligozoospermia, azoospermia and
also with a extended testis histological profile range
from Sertoli cell only (SCO), maturation arrest and
hypospermatogenesis (Vollrath, 1992) (Vogt et al.,
1996) (Briton-Jones and Haines, 2000).
The prevalence of the Y chromosome
microdeletions in the proximal AZFc region was
found higher in men from recurrent pregnancy loss
(RPL) couples than from fertile or infertile couples.
Although these patients are from a tertiary referral
center that may not reflect the population
informations, one may consider proximal AZFc
region detecting in the evaluation of RPL couples
when all other tests fail to reveal the etiology
(Dewan et al., 2006).
Before performing a molecular test, cytogenetic
analysis is necessary for an accurate approach to
elucidate the causes of spontaneous abortion.
Chromosomal anomalies which may cause male
infertility can be determined by cytogenetic
techniques. It is also known that approximately
50% of recurrent spontaneous abortions in the first
trimester is caused by chromosomal anomalies.
Besides these, recent data show that Y chromosome
microdeletions can also be a major factor in these
cases. These findings suggest a potential relation
between RPL and microdeletions in AZF regions.
In order to investigate the etiology of RPL and
to develop an appropriate therapeutic strategy, it is
necessary to ascertain the molecular and
cytogenetic basis of these defects. So in this study,
we aimed to reveal the relations between male
infertility, Y chromosome microdeletions and
recurrent spontaneous abortions.
Material and methods
Patient and Control Groups
Thirty couples that applied to Marmara University,
Department of Urology and Kartal Education and
Research Hospital with a spontaneous abortion
history were recruited to the study. Thirty fertile
men, at least having one child, were examined as
the control group. Written informed consent was
taken from all cases.
Chromosome Analyses from Peripheral Blood
Cell Culture
Lymphocytes from 400 µl peripheral blood were
cultured for 72 hours at 37ºC culture medium
containing 8.5 ml RPMI, 1.5 ml fetal bovine serum,
Y chromosome microdeletions in spontaneous abortions 47
200 µl L-Glutamin, 20 µl penicillin- streptomycin
and 200 µl phytohaemagglutinin. After incubation
at 37ºC for 72 hours, 200 µl Colchicine was added
to arrest the cells at metaphase. Following an
additional incubation at 37ºC for 30 minutes and
centrifugation at 20ºC for 8 min. at 1500 rpm the
supernatant was removed. The pellet was resuspended with up to 10 ml hypotonic solution
(0.4% KCl solution) vortexed immediately. All the
samples were kept at 37ºC for 20 minutes and
again centrifuged at the same condition. After
removing supernatant from the samples, the pellet
which contains cells at metaphase, was
homogenised. Fixative solution (methanol and
acetic acid mixed with 3:1 ratio) was added and the
tubes were vortexed for the fixation of
chromosomes. Then samples were centrifuged after
adding up to 5 ml of fixative solution. Supernatant
was discarded from the samples and fresh fixative
solution was added to the tubes. This procedure
was repeated until the samples were clarified.
According to the cell density, up to 0.5 ml fixative
solution was added to the samples. Then samples
were homogenized and cells were lied onto slide
glasses, which were kept at 4ºC in distilled water
till they are used. After spreading the cells on the
slides, the samples were dried at room temperature
and kept overnight at 60ºC.
Karyotyping
GTG (Giemsa-Trypsin) banding technique was
performed. When the banding of the chromosomes
was not successful, the protocol was repeated.
After staining, at least 20 metaphase plaques were
analysed for each sample (Figure 1).
Detection of Y chromosome microdeletions
DNA isolation from blood
DNA was extracted from 200 µl peripheral blood by
using High Pure PCR Template Preparation Kit
(Roche-Germany) according to the manufacturer’s
protocol.
Multiplex
polymerase
(multiplex PCR)
chain
reaction
For detection of Y chromosome microdeletions,
isolated DNA was amplified by multiplex PCR. AB
ANALITICA–The AZF Extension Kit, which is
recommended by European Andrology Association
was used in multiplex PCR. By using this kit, 13
different regions could be investigated at the same
time by performing 3 multiplex PCRs for each
sample. Three primer sets, each containing primers
that is unique to ZFX/Y locus which also exist in X
chromosome are shown in Table 1.
Table 1. Primers used for multiplex PCR and the length of amplicons.
MIX1
Amlicon
length (bp)
MIX2
Amlicon
length (bp)
MIX3
Amlicon
length (bp)
ZFX/Y
495
ZFX/Y
495
DBY
689
SRY
472
SRY
472
ZFX/Y
495
sY 254
380
sY 95
303
SRY
472
sY 86
320
sY 117
262
sY 84
326
sY 127
274
sY 125
200
sY 134
301
sY 255
120
DFFRY
155
48
Gülşah Koç et. al.
In addition to the mixtures which are found in
the AZF Extension Kit, 0.3µl Taq DNA polymerase
and 8µl DNA sample were added to each tube
during multiplex PCR. The conditions of PCR
amplification were as follows: a denaturation step at
94˚C for 5 min followed by 35 cycles at 94˚C for 1
min, 60˚C for 1 min, 72˚C for 1 min and a final
extension at 72˚C for 7 min and stop at 4˚C. After
multiplex PCR, products were electrophoresed on
2% agarose gel.
After performing lymphocyte cell culture,
metaphase plaques were analyzed for the detection
of karyotypes of patient and control groups.
According to karyotype analyses, all the males and
females were found as 46, XY and 46, XX
respectively in the patient group, whereas all the
males were found as 46, XY in the control group
(Figure 1).
Results
After multiplex PCR, PCR products were examined
by electrophoresis on 2% agarose gel. Y
chromosome microdeletions were not found in
patient and control groups.
Karyotyping
Figure 1. Karyotype analyses of a male (46, XY) patient.
Detection of Y chromosome microdeletions
Y chromosome microdeletions in spontaneous abortions 49
Figure 2. Multiplex PCR analyses of Y chromosome microdeletions (M: 50 bp ladder (Fermentas,
Germany); Mix1a, Mix1b and Mix1c: 3 sets of PCR reactions that amplify different loci on Y chromosome
for sample a; Mix1b, Mix2b, Mix3b for sample b; boxes indicate the region and the length of the amplicons.
Discussion
Chromosomal abnormalities, including translocations and deletions, are higher in infertile men and
are recognized as one of the main causes of
spontaneous abortions with an estimated frequency
of 50–70% (Svetlana et al., 2005)
In couples experiencing RPL, the incidence of
chromosomal translocations is higher than the
incidence present in newborn series (De Braekeleer
and Dao 1991). There is also evidence which
indicates that the presence of translocations
changes the spermatogenic process. It has been
found that the incidence of reciprocal translocation
carriers is seven times more than in newborn series.
As a general rule reciprocal translocation carriers
produce more unbalanced sperm than normal or
balanced sperm. The proportion of unbalanced
forms depends on the characteristics of the
reorganization. Also deletions which remove Y
chromosomal genes required for spermatogenesis
may effect infertility and susceptibility of RPL
(Byrne and Ward, 1994) (Simpson, 1981). As the
severity of the spermatogenic defect increases, the
frequency of the microdeletions also increases.
In this study, primarily, cytogenetic evaluation
was performed from peripheral blood samples of
the couples in spontaneous abortion cases. 30
couples who had a spontaneous abortion history
were karyotyped to detect the chromosome
anomalies. According to karyotype analyses, all the
women and men were found to be 46, XX and 46,
XY, respectively. In our study we couldn’t detect
any numerical and structural chromosome
anomalies that can be detected by karyotype
analyses. Other genetic abnormalities such as Y
chromosome microdeletions may effect spermatogenesis, fertilization and post-zygotic metabolism
and may influence male infertility and RPL.
50
Gülşah Koç et. al.
So we used multiplex PCR for the detection of
microdeletions on the long arm of the Y
chromosome.
In this study, “AB ANALITICA–The AZF
Extension Kit” used for the analysis of
microdeletions rather than AZF-MX Extension kit.
Diagnostic sensitivity is considered to be the
capacity of the device to correctly identify the
deleted samples with reference to AZF locus under
investigation. The results obtained from an
experimental investigation show that the diagnostic
sensitivity of the system is 100%.
The kit is in premix format as all the reagents
for the amplification are pre-mixed and aliquoted in
single dose tubes in which only additional Taq
polymerase and the extracted DNA should be
added. This premix format allows the reduction of
the manipulation in preamplification steps, with
considerable time saving for the operator, the
repeated freezing/thawing of reagents (that could
alter the products’ performances) is avoided and,
above all, this form minimizes the risk of sample
contamination and the risk of false positive results.
The amplified regions of the Y chromosome are
not polymorphic and are well known to be deleted
specifically in men affected by oligo/azoospermia
according to the known, clinically relevant
microdeletion pattern (Viswambharan, 2007).
Based on the experience of many laboratories and
the results of external quality control and
considering the multiplex PCR format, the first
choice of STS primers recommended in the first
version of the guidelines remains basically valid.
These primers include the regions:
For AZFa: sY84, sY86
For AZFb: sY127, sY134
For AZFc: sY254, sY255
The usage of this primer set will enable the
detection of almost all the clinically relevant
deletions and of over 95% of the deletions reported
in the literature in the three AZF regions and is
sufficient for routine analysis (Simoni, 2004).
In this study, the set of PCR primers as best
choice for the diagnosis of microdeletion of the
AZFa, AZFb and AZFc region (sY14 (SRY),
ZFX/ZFY, sY84, sY86, sY127, sY134, sY254,
sY255) used in multiplex PCR reactions. We
couldn’t detect any Y chromosome microdeletions
in AZFa, AZFb and AZFc regions.
Genes that are located on Y chromosome and
responsible from spermatogenesis have a mosaic
structure at somatic and/or germ cells. When
leukocytes from blood were used, usually the
results can not be suitable for Y chromosome
microdeletion analysis because there may have
been deletions in germ cells (Martin, 2008).
There may be a mosaicism between
seminiferous tubules in terms of the expression of
genetic material. Some seminiferous tubules have
aplasia whereas some tubules can be normal or
mutant arrest at testes. In the identification of
deletions this situation may show different
outcomes when cells from blood or semen were
used. When fibroblasts or leukocytes are used in
genetic analysis, the proportion of a detection of Y
chromosome microdeletion is slightly low because
the deletions occuring in germ line cells have an
independent nature from other tissues.
In this study, we used peripherial blood
leukocytes for the detection of Y chromosome
microdeletions, however we couldn’t find any
deletions. But the possibility of having deletions in
germline cells shouldn’t be omitted. We are looking
forward to extend our study by adding spermial Y
chromosome microdeletion analysis from the same
individuals.
Dewan et al. (2006) reported the relation
between RPL and proximal AZFc deletions and
found a significant correlation. Although, they
detected proximal Y chromosome AZFc
microdeletions in 14 of 17 patients (82%), they
couldn’t find any deletion in control group.
Karaer et al. (2008) reported 43 infertile men
among which 7 of them have sY 220 (AZFb)
deletions (16%) of the 4 examined region, stating
the importance of AZF deletions in the aetiology of
RPL.
In the previous studies, sequenced tagged site
(STS) numbers which were selected for detection
of Y chromosome microdeletions are different from
each other. After physical mapping of Y
chromosome, more than 300 STS were produced. It
was stated that, analysing of low number of STS
can be insufficient for detection of deletion regions
also high number of STS can give false-positive
results as polymorphic regions may identified as
deletions (Simoni, 2001).
One of the most important criteria for the
detection of Y chromosome microdeletions is the
selected STS. For this reason, European Andrology
Association and European Molecular Genetics
Y chromosome microdeletions in spontaneous abortions 51
Quality Network improved a standardization to
distinguish the differences of deletion proportions
between different laboratories. So they proposed 6
STS for detecting of AZFa, AZFb ve AZFc regions.
In the present study, although 13 STS including 6
STS which were suggested by European Molecular
Genetics Quality Network were analyzed, we
couldn’t detect any microdeletions on Y
chromosome. We propose the evolution of the
results by increasing the analysed STS.
Due to limited knowledge of the metabolism
and the progress of the genes on Y chromosome,
we can not predict the answers of the questions
including Y chromosome microdeletion’s effect on
RPL. For this reason researches should be focused
on the relationship of Y chromosome microdeletions, male infertility and RPL.
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