D e n iz S e v in ç , M .S .* / A h m e t Z e h ir , P h .D .* *
A n ıl B ir ic ik , M .S .* * * / S e m ra S e r t y e l, M .S .* * *
S e m ra K a h r a m a n , M . D . * * * / İl t e r G ü n e y , M . D . , P h .D .* * * *
* D e p a r t m e n t o f M e d ic a l B io lo g y a n d G e n e tic s , S c h o o l o f M e d ic in e , M a lte p e U n iv e rs ity , Is ta n b u l, T u rk e y . * * D e p a r t m e n t o f B io lo g y , S c h o o l o f A rt a n d S c ie n c e , M a r m a r a U n iv e rs ity , Is ta n b u l, T u rk e y . * * * A R T a n d G e n e tic s C e n te r, Is t a n b u l M e m o r ia l H o s p ita l, Is ta n b u l, T u rk e y . * * * * D e p a r t m e n t o f M e d ic a l B io lo g y , S c h o o l o f M e d ic in e , M a r m a r a U n iv e rs ity , Is ta n b u l, T u rk e y . A B S T R A C T
O b je c t iv e :
Recent
investigations
have
supported the importance of Y chromosome
microdeletions in
male infertility.
Besides
different factors, sperm morphology is also a very
important component of the clinical evaluation of
male fertility potential. The aim of our study was
to
understand
the
relation
between
Y
chromosome
microdeletions
and
sperm
morphology in different risk groups.
M aterial and M e th o d s:
In this study, 34 infertile
men were selected and examined in terms of
sperm
morphology
and
Y
chromosome
microdeletions. Semen analyses of the patients
were performed by using Strict criteria and the
samples were grouped according to the sperm
abnormalities. Peripheral blood samples were
examined for Y chromosome microdeletions by a
multiplex polymerase chain reaction amplification
of sequence-tagged sites (STS) of the Y
chromosome.
R e s u lt s :
The incidence of deletions in the group
was 14.7 % (5/34). The rates of Y chromosome
microdeletion were 33.3% for megalohead,
33.3% for elongehead, 0% for roundhead, 100%
for pinhead, 14.3% for severe teratozoospermia,
33.3% for severe neck abnormalities and 0% for
tail-stump Pregnancy could not be obtained from
Y chromosome deleted cases.
C o n c lu s io n :
It seems that especially patients
with sperm head and neck
abnormality have
increased microdeletion risk. It is suggested that
a detailed sperm morphology examination and
classification is needed to clarify the Y
chromosome deletion-sperm morphology relation.
K e y W o rd s : Y chromosome microdeletions,
Sperm morphology, Male infertility
IN T R O D U C T IO N
Y chromosome is one of the sex chromosomes
in males. It is known that this chromosome has
an important role in spermatogenesis. A defect in
any step of spermatogenesis can cause infertility
( 1 - 6 ).( A c c e p t e d 1 7 J u n e . 2 0 0 2 ) M a r m a r a M e d ic a l J o u r n a l 2 0 0 2 ; 1 5 ( 4 ) : 2 2 7 - 2 3 2 Correspondence to: Deniz Sevinç, M.S. - Department of Medical Biology and Genetics,
One of the important factors in spermatogenic
failure is Y chromosome microdeletions (1,2,7-
14).
Although
the
relation
between
microdeletions and spermatogenic failure has
been known for a long time, the defective regions
were identified at the molecular level only in
recent years. There are a total
of four
AZoospermic Factor (AZF) regions on the Y
chromosome AZFa, AZFb, AZFc and AZFd (1-
12). These deletions in the Yq11 region are
mostly undetectable by cytogenetic analysis.
Molecular
analysis
can
detect
the
presence/absence
of
these
deletions
by
polymerase chain reaction (PCR). In spite of this,
there is no agreement about which factors play a
role in male infertility due to unknown causes.
Different studies on male infertility cases showed
a wide range
(1%-55%) of Y chromosome
microdeletion rates (3,15-32). It is suggested that
different factors related to the usage of different
techniques can affect these rates.
Since the deleted regions involve the genes
which are responsible for sperm production,
sperm number, structure and function can be
affected (17-22,33,34). The association between
the phenotypic characteristics of sperm and the
genetic structure of Y chromosome is being
examined by ongoing studies. Especially, after
understanding
that
Y
chromosome
microdeletions can be a factor in infertile men
and they can be transmitted by ICSI from father
to son, more interest has been focused on
deletion and sperm parameters including
morphology, number and function (35-39).
Flowever, the possible relation between Y
chromosome
microdeletions
and
sperm
morphology is not yet clear.
The goal of the study was to determine the sperm
morphology and Y chromosome microdeletion
correlation in order to understand its influence on
male infertility.
M A T E R IA L S A N D M E T H O D S
Thirty-four infertile men with sperm morphology
defects who referred to Istanbul Memorial
Hospital IVF Center were selected for this study.
Semen analysis was done by using WFIO and
Kruger Strict Morphology Criteria and samples
were grouped according to sperm abnormalities.
Semen Analysis
Sperm
concentration
and
movement
characteristics were detected by Makler counting
chamber. The evaluation of motility and
progression was performed by counting at least
100 sperm under the light microscope. Kruger
Strict Morphology Criteria were used to evaluate
sperm morphology by light microscopy.
DNA
Isolation
and
Polymerase
Chain
Reaction
After the isolation of DNA from peripheral blood
by using Promega Wizard Genomic DNA
Purification Kit, Promega Deletion Detection Kit
Version 1.1 was used to detect 18 loci on Y
chromosome by multiplex polymerase chain
reaction.
Analyses of PCR products were performed by
agarose gel electrophoresis.
RESULTS
The results of the sperm analysis of 34 patients
are shown in Table I. As can be seen from the
table, apart from two cases, all the cases were
found to have low sperm number. In addition, it
was found that the sperm volume and the sperm
numbers did not correlate. Although all samples
had different structural sperm defects, Y
chromosome microdeletion was found in only 5
patients. Twelve patients gave healthy birth by
the help of assisted reproductive techniques.
Y chromosome microdeletion rates in pinhead,
(megalohead-elonge head and severe neck
abnormality) and severe teratozoospermia were
100%, 33% and 14% respectively (Table II). No
microdeletion was detected in tail-stump and
round head abnormalities.
The products of multiplex PCR were evaluated
by using positive and negative controls on
agarose gel electrophoresis (Fig 1)
D IS C U S S IO N
Recent studies have confirmed the importance of
Y chromosome microdeletions in male infertility
(1,2,7-14,31,40). Flowever, genotype-fenotype
correlation was not clearly explained in these
Table I: The clinical and molecular findings of 34 infertile men
SAMPLE No
AGE SEMEN VOLUME (ml)
SPERM NUMBER (/ml) MOTILITY (%)
SPERM MORPHOLOGY YCHR. MICDEL NORMAL BIRTH 1 34 6 6 million (Oligozoospermie) 50 Roundhead, Severe neck abnormality
2 36 2,5 22 million (Normozoospermie) 10 Round head
-3 28 4,5 30.000 (Oligozoospermie) 20 Severe teratozoospermia +
4 35 3 8 million (Oligozoospermie) 52 Round head - +
5 39 2 26.000 (Oligozoospermie) 12 Severe teratozoospermia +
6 43 3 9 million (Oligozoospermie) 13 Round head - +
7 37 3,5 49 million (Normozoospermie) 34 Elongehead, Severe neck abnormality - +
8 21 1,5 200.000 (Oligozoospermie) 32 Megalohead, Pinhead +
-9 30 1 3000 (Oligozoospermie) 11 Severe teratozoospermia
10 36 3 17 million (Oligozoospermie) 11 Megalohead, Elongehead - +
11 31 1 1000 (Oligozoospermie) 0 Megalohead, Elongehead, Pinhead +
-12 34 1,5 700.000 (Oligozoospermie) 42 Megalohead, Elongehead -
-13 37 3 30.000 (Oligozoospermie) 63 Megalohead, Roundhead +
14 23 2 10.000 (Oligozoospermie) 30 Megalohead
15 28 1 Azoospermie - Severe teratozoospermia - +
16 35 3 6 million (Oligozoospermie) 30 Round head +
17 53 3 4 million (Oligozoospermie) 31 Severe teratozoospermia -
-18 45 3 3 million (Oligozoospermie) 13 Severe teratozoospermia +
19 29 1,5 700.000 (Oligozoospermie) <1 Severe teratozoospermia
-20 38 4 7 million (Oligozoospermie) 14 Severe teratozoospermia - +
21 31 3 400.000 (Oligozoospermie) 33 Severe teratozoospermia -
-22 39 2,5 700.000 (Oligozoospermie) 14 Severe teratozoospermia -
-23 39 2 2 million (Oligozoospermie) 10 Severe teratozoospermia
24 29 3 2 million (Oligozoospermie) 20 Severe teratozoospermia
-25 32 2 300.000 (Oligozoospermie) 5 Severe teratozoospermia - +
26 25 5 500.000 (Oligozoospermie) 2 Round head +
27 31 3 200.000 (Oligozoospermie) 40 Round head
-28 25 3 600.000 (Oligozoospermie) 5 Round head
29 25 3 156.000 (Oligozoospermie) 23 Elongehead - +
30 34 2,5 600.000 (Oligozoospermie) 16 Severe neck abnormality, Elongehead +
31 35 1 2000 (Oligozoospermie) 0 Tail-stump
32 32 3 5.5 milyon (Oligozoospermie) 1 Tail-stump
33 32 4 Azoospermie 0 Tail-stump
-34 38 3 3000 (Oligozoospermie) 3 Severe teratozoospermia +
Table II: The abnormalities of sperm mophology and Y chromosome microdeletions.
Megalohead Elongehead Roundhead Pinhead Severe teratozoospermia Severe Neck abnormalities Tail-stump
Total Number of Samples 6 6 9 2 14 3 3
Y Chromosome
Microdeletion Positive Samples 2 2 0 2 2 1 0
Y Chromosome Microdeletion Rate (%) 33,3 33,3 0 100 14,3 33,3 0
studies. Only the relation between certain
microdeletions
and
numerical
sperm
abnormalities
such
as
azoospermia
and
oligozoospermia was tried to be examined. In this
study we found Y chromosome microdeletions in
five
oligozoospermie
patients
(15%).
In
accordance with previous studies (20,23,37,41-
43), one patient out of five exhibited total deletion
of AZF region, while the remaining four patients
showed AZFc deletions.
F i g . l : Agarose gel electrophoresis of multiplex PCR (in four sets) products correspond to oligozoospermie or azoospermie patients (lanes: 2,3,4,5 and 6) and normal control (lane 1). S= size marker. Deleted regions can be seen easily in patient no:2.
In assisted reproduction units, Y chromosome
microdeletion detection is commonly applied for
azoospermia and oligozoospermia in infertility
cases. The other parameters of sperm analysis
such as functional and structural defects are not
generally considered as test criteria. Shortly,
structural sperm defects which are accepted as a
test criteria in this study are not used for routine
deletion analysis.
Because of keeping the numerical defects of the
sperm in the foreground during examination and
due to insufficient structural evaluation, the
reliability of the spermiogram results can be
affected. Morphology can be accepted as a test
criteria for deletion detection whenever the
relation between sperm structure and deletion
becomes clear.
In our study group with structural sperm
abnormality, the microdeletion rate is 15%
supporting the genotype-phenotype correlation.
This result is in the range of the previous studies
(1% -55%) which were designed generally
according to the numerical criteria in infertile men
(3,15-32).
The relation is not clear when we take three
parameters (Y chromosome microdeletions,
numerical and structural abnormalities) into
account but it is found that in this preliminary
group Y chromosome microdeletion rates are
higher in sperm head and neck abnormalities.
Especially when we take the pinhead group into
account, both samples are Y chromosome
deleted and this data seems to give an idea for
future studies as pinhead is not so frequent as
the other head abnormalities. Although these
preliminary findings suggest that there can be an
association especially between these two
morphological abnormalities and Y chromosome
microdeletions, a detailed structural sperm
examination and more samples are needed to
obtain a significant conclusion.
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