Complex cytogenetic and molecular-genetic analysis of males with spermatogenesis failure
The chromosomal anomalies, microdeletions of AZF region of Y-chromosome and CFTR gene mutations have been studied among 80 infertile men with idiopathic spermatogenetic failure: 36 (45 %) patients with aspermia, 19 (24 %) patients with azoospermia and 25 (31 %) patients with severe oligoasthenoterat...
Збережено в:
| Опубліковано в: : | Цитология и генетика |
|---|---|
| Дата: | 2010 |
| Автори: | , , , , , |
| Формат: | Стаття |
| Мова: | English |
| Опубліковано: |
Інститут клітинної біології та генетичної інженерії НАН України
2010
|
| Теми: | |
| Онлайн доступ: | https://nasplib.isofts.kiev.ua/handle/123456789/66807 |
| Теги: |
Додати тег
Немає тегів, Будьте першим, хто поставить тег для цього запису!
|
| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Цитувати: | Complex cytogenetic and molecular-genetic analysis of males with spermatogenesis failure / N. Huleyuk, D. Zastavna, M. Tyrkus , H. Makukh, S. Gavrylyshyn, M. Kurpisz // Цитология и генетика. — 2010. — Т. 44, № 5. — С. 51-56. — Бібліогр.: 31 назв. — англ. |
Репозитарії
Digital Library of Periodicals of National Academy of Sciences of Ukraine| id |
nasplib_isofts_kiev_ua-123456789-66807 |
|---|---|
| record_format |
dspace |
| spelling |
Huleyuk, N. Zastavna, D. Tyrkus, M. Makukh, H. Gavrylyshyn, S. Kurpisz, M. 2014-07-22T15:58:46Z 2014-07-22T15:58:46Z 2010 Complex cytogenetic and molecular-genetic analysis of males with spermatogenesis failure / N. Huleyuk, D. Zastavna, M. Tyrkus , H. Makukh, S. Gavrylyshyn, M. Kurpisz // Цитология и генетика. — 2010. — Т. 44, № 5. — С. 51-56. — Бібліогр.: 31 назв. — англ. 0564-3783 https://nasplib.isofts.kiev.ua/handle/123456789/66807 576.312.332:616.37–008.6–056.7:616.697–076.5 The chromosomal anomalies, microdeletions of AZF region of Y-chromosome and CFTR gene mutations have been studied among 80 infertile men with idiopathic spermatogenetic failure: 36 (45 %) patients with aspermia, 19 (24 %) patients with azoospermia and 25 (31 %) patients with severe oligoasthenoteratozoospermia. In total 30 % males with spermatogenetic failure genetic factor of infertility was observed. Karyotype anomalies were observed in 17.5 % of infertile men, within 16.2 % numerical and structural gonosomal anomalies and in 1.3 % – Robertsonian translocation were revealed. In 11 % males with spermatogenetic failure, Y-chromosome AZF region microdeletions were detected. The frequency of CFTR major mutation F508del among infertile men was 6.25 %. 5T allele of polymorphic locus IVS8polyT was detected in 7.5 % of examined men. The results obtained indicate the high complexity of cytogenetic and moleculargenetic studies of male infertility. Изучали аномалии хромосом, микроделеции AZF региона Y-хромосомы и мутации гена ТРБМ у 80 мужчин с идиопатическими нарушениями сперматогенеза, а именно: у 36 (45 %) пациентов с аспермией, 19 (24 %) пациентов с азооспермией и 25 (31 %) пациентов с олигоастенотератозооспермией IV степени. В общем у 30 % мужчин с нарушениями сперматогенеза установлены генетические факторы бесплодия. Нарушения кариотипа наблюдали у 17.5 % бесплодных мужчин, среди них у 16.2 % – количественные и структурные аномалии хромосом и у 1.3 % – робертсоновскую транслокацию. У 11 % мужчин с нарушениями сперматогенеза выявили микроделеции AZF региона Y хромосомы. Частота мажорной мутации F508del гена ТРБМ среди бесплодных мужчин составила 6.25 %. 5T аллель полиморфного локуса IVS8polyT выявили у 7.5 % обследованных мужчин. Полученные результаты свидетельствуют о высокой информативности комплексного цитогенетического и молекулярно-генетического исследования при мужском бесплодии. This work was supported of Science and Higher Education of Poland (grant no. N407 034 32/1371 [2007–2009]). en Інститут клітинної біології та генетичної інженерії НАН України Цитология и генетика Оригинальные работы Complex cytogenetic and molecular-genetic analysis of males with spermatogenesis failure Комплекс цитогенетических и молекулярно-генетических исследований мужчин с нарушеничми сперматогенеза Article published earlier |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| title |
Complex cytogenetic and molecular-genetic analysis of males with spermatogenesis failure |
| spellingShingle |
Complex cytogenetic and molecular-genetic analysis of males with spermatogenesis failure Huleyuk, N. Zastavna, D. Tyrkus, M. Makukh, H. Gavrylyshyn, S. Kurpisz, M. Оригинальные работы |
| title_short |
Complex cytogenetic and molecular-genetic analysis of males with spermatogenesis failure |
| title_full |
Complex cytogenetic and molecular-genetic analysis of males with spermatogenesis failure |
| title_fullStr |
Complex cytogenetic and molecular-genetic analysis of males with spermatogenesis failure |
| title_full_unstemmed |
Complex cytogenetic and molecular-genetic analysis of males with spermatogenesis failure |
| title_sort |
complex cytogenetic and molecular-genetic analysis of males with spermatogenesis failure |
| author |
Huleyuk, N. Zastavna, D. Tyrkus, M. Makukh, H. Gavrylyshyn, S. Kurpisz, M. |
| author_facet |
Huleyuk, N. Zastavna, D. Tyrkus, M. Makukh, H. Gavrylyshyn, S. Kurpisz, M. |
| topic |
Оригинальные работы |
| topic_facet |
Оригинальные работы |
| publishDate |
2010 |
| language |
English |
| container_title |
Цитология и генетика |
| publisher |
Інститут клітинної біології та генетичної інженерії НАН України |
| format |
Article |
| title_alt |
Комплекс цитогенетических и молекулярно-генетических исследований мужчин с нарушеничми сперматогенеза |
| description |
The chromosomal anomalies, microdeletions of AZF region of Y-chromosome and CFTR gene mutations have been studied among 80 infertile men with idiopathic spermatogenetic failure: 36 (45 %) patients with aspermia, 19 (24 %) patients with azoospermia and 25 (31 %) patients with severe oligoasthenoteratozoospermia. In total 30 % males with spermatogenetic failure genetic factor of infertility was observed. Karyotype anomalies were observed in 17.5 % of infertile men, within 16.2 % numerical and structural gonosomal anomalies and in 1.3 % – Robertsonian translocation were revealed. In 11 % males with spermatogenetic failure, Y-chromosome AZF region microdeletions were detected. The frequency of CFTR major mutation F508del among infertile men was 6.25 %. 5T allele of polymorphic locus IVS8polyT was detected in 7.5 % of examined men. The results obtained indicate the high complexity of cytogenetic and moleculargenetic studies of male infertility.
Изучали аномалии хромосом, микроделеции AZF региона Y-хромосомы и мутации гена ТРБМ у 80 мужчин с идиопатическими нарушениями сперматогенеза, а именно: у 36 (45 %) пациентов с аспермией, 19 (24 %) пациентов с азооспермией и 25 (31 %) пациентов с олигоастенотератозооспермией IV степени. В общем у 30 % мужчин с нарушениями сперматогенеза установлены генетические факторы бесплодия. Нарушения кариотипа наблюдали у 17.5 % бесплодных мужчин, среди них у 16.2 % – количественные и структурные аномалии хромосом и у 1.3 % – робертсоновскую транслокацию. У 11 % мужчин с нарушениями сперматогенеза выявили микроделеции AZF региона Y хромосомы. Частота мажорной мутации F508del гена ТРБМ среди бесплодных мужчин составила 6.25 %. 5T аллель полиморфного локуса IVS8polyT выявили у 7.5 % обследованных мужчин. Полученные результаты свидетельствуют о высокой информативности комплексного цитогенетического и молекулярно-генетического исследования при мужском бесплодии.
|
| issn |
0564-3783 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/66807 |
| citation_txt |
Complex cytogenetic and molecular-genetic analysis of males with spermatogenesis failure / N. Huleyuk, D. Zastavna, M. Tyrkus , H. Makukh, S. Gavrylyshyn, M. Kurpisz // Цитология и генетика. — 2010. — Т. 44, № 5. — С. 51-56. — Бібліогр.: 31 назв. — англ. |
| work_keys_str_mv |
AT huleyukn complexcytogeneticandmoleculargeneticanalysisofmaleswithspermatogenesisfailure AT zastavnad complexcytogeneticandmoleculargeneticanalysisofmaleswithspermatogenesisfailure AT tyrkusm complexcytogeneticandmoleculargeneticanalysisofmaleswithspermatogenesisfailure AT makukhh complexcytogeneticandmoleculargeneticanalysisofmaleswithspermatogenesisfailure AT gavrylyshyns complexcytogeneticandmoleculargeneticanalysisofmaleswithspermatogenesisfailure AT kurpiszm complexcytogeneticandmoleculargeneticanalysisofmaleswithspermatogenesisfailure AT huleyukn komplekscitogenetičeskihimolekulârnogenetičeskihissledovaniimužčinsnarušeničmispermatogeneza AT zastavnad komplekscitogenetičeskihimolekulârnogenetičeskihissledovaniimužčinsnarušeničmispermatogeneza AT tyrkusm komplekscitogenetičeskihimolekulârnogenetičeskihissledovaniimužčinsnarušeničmispermatogeneza AT makukhh komplekscitogenetičeskihimolekulârnogenetičeskihissledovaniimužčinsnarušeničmispermatogeneza AT gavrylyshyns komplekscitogenetičeskihimolekulârnogenetičeskihissledovaniimužčinsnarušeničmispermatogeneza AT kurpiszm komplekscitogenetičeskihimolekulârnogenetičeskihissledovaniimužčinsnarušeničmispermatogeneza |
| first_indexed |
2025-11-24T05:45:15Z |
| last_indexed |
2025-11-24T05:45:15Z |
| _version_ |
1850841111292018688 |
| fulltext |
УДК 576.312.332:616.37–008.6–056.7:616.697–076.5
N. HULEYUK 1, D. ZASTAVNA 1, M. TYRKUS 1,
H. MAKUKH 1, S. GAVRYLYSHYN 2, M. KURPISZ 3
1 Institute of Hereditary Pathology of Academy of Medical Sciences
of Ukraine, Lviv
2 Prycarpatian Center of Human Reproductions, Ivano�Frankivsk
3 Institute of Human Genetics, Polish Academy of Sciences, Poznan
E�mail: huleyuk@yahoo.com
COMPLEX CYTOGENETIC AND
MOLECULAR�GENETIC ANALYSIS
OF MALES WITH SPERMATOGENESIS
FAILURE
The chromosomal anomalies, microdeletions of AZF
region of Y�chromosome and CFTR gene mutations have been
studied among 80 infertile men with idiopathic spermatoge�
netic failure: 36 (45 %) patients with aspermia, 19 (24 %)
patients with azoospermia and 25 (31 %) patients with severe
oligoasthenoteratozoospermia. In total 30 % males with sper�
matogenetic failure genetic factor of infertility was observed.
Karyotype anomalies were observed in 17.5 % of infertile
men, within 16.2 % numerical and structural gonosomal
anomalies and in 1.3 % – Robertsonian translocation were
revealed. In 11 % males with spermatogenetic failure, Y�
chromosome AZF region microdeletions were detected. The
frequency of CFTR major mutation F508del among infertile
men was 6.25 %. 5T allele of polymorphic locus IVS8polyT
was detected in 7.5 % of examined men. The results obtained
indicate the high complexity of cytogenetic and molecular�
genetic studies of male infertility.
Introduction. Infertility is the problem of almost
15 % of married couples. Half of the cases is
caused by a «male factor». Commonly, idiopathic
oligo� and azoospermia are diagnosed in these
cases. Genetic reasons of spermatogenesis failure,
such as numerical or structural chromosome
anomalies and gene mutations that are responsible
for fertility often depend on ethnic background of
patients [1–10].
Karyotype abnormalities are observed in 4.6 %
of men with oligospermia and in 13.7 % patients
with azoospermia. Structural chromosomal anom�
alies are detected in 5.1 % of infertile men, besides,
autosomal translocations are the most common in
men with oligospermia and changes in gonosomes
(sex chromosomes) are more characteristic for
persons with azoospermia [10].
The genes located on the Y chromosome play
an essential role in the control and regulation of
spermatogenesis. Microdeletions of AZF locus are
one of the most widespread genetic causes of infer�
tility in men with severe spermatogenetic failure:
microdeletions in AZF are diagnosed in 5–11 %
individuals with azoospermia, while in oligosper�
mia – in 2–8 % of cases [4, 7, 9]. In 12 % of infer�
tile men mutations of CFTR (Cystic Fibrosis
Transmembrane Conductance Regulator) gene are
detected. Furthermore, in these cases uni� or bilat�
eral congenital absence of the vas deferens
(CAVD) can be revealed [11].
Consequently, previously�described genetic
factors have the leading role in etiology dysfunc�
tions of male reproductive system. That is why the
purpose of the study was to set the frequency and
the spectrum of the chromosomal anomalies,
microdeletions of AZF region of Y chromosome
and CFTR gene mutations among infertile men
from Ukraine.
Materials and methods. Eighty idiopathically
infertile males, selected out of 260 infertile men,
attending the Prycarpatian Center of Human
Reproductions, were included in the study. The
diagnosed cases of anatomic defects, infectious dis�
eases, endocrine, immunological infertility were
excluded from the studied group. The age of these
individuals ranged from 25 to 43 years. An experi�
enced urologist carried out a detailed anamnesis and
clinical examination of every patient. Sperm analysis
was performed at least twice at appropriate interval.
Based on spermatological analysis results, infertile
were subdivided into three groups: 36 (45 %)
patients with aspermia (AS), 19 (24 %) patients
ІSSN 0564–3783. Цитология и генетика. 2010. № 6 51
© N. HULEYUK, D. ZASTAVNA, M. TYRKUS, H. MAKUKH,
S. GAVRYLYSHYN, M. KURPISZ, 2010
with azoospermia (AZ) and 25 (31 %) patients
with severe oligoasthenoteratozoospermia (OAT).
Cytogenetic analyses of the chromosomes from
PHA�stimulated peripheral blood leukocyte in
vitro culture were performed according to standard
protocols [12]. Ethidium bromide (10 μg/ml) was
added simultaneously with colchicine in order to
obtain high�quality chromosomes of early and
middle mitotic stages. GTG and CBG [12, 13]
banded chromosomes were analyzed at the
550 bands resolution level [13, 14].
DNA of these samples was isolated using the
salting out method in own modification [15].
Extracted DNA was amplified by PCR [16]. Pre�
sence and specificity of the PCR reaction products
was verified by means of electrophoresis in 2.5 %
agarose gel. Microdeletions of Y chromosome AZF
region were analyzed using two multiplex PCR: in
each reaction fragments of three AZF regions
(AZFa, AZFb, AZFc) were amplified [17]. Multi�
plex reaction A (Fig. 1, a) allows to analyze follow�
ing loci: SRY (472 bp), sY254 (400 bp), sY86
(320 bp), sY127 (274 bp). Multiplex reaction B
(Fig. 1, b) allows to analyze following loci SRY
(472 bp), sY84 (326 bp), sY134 (301 bp), sY255
(126 bp). The absence of specific fragments indi�
cated the presence of microdeletions in respective
loci. Whenever failure of amplification in any sam�
ple was detected, 2 additional PCRs were performed
to confirm the absence of the unamplified STSs.
To detect alleles in tri�allelic polymorphic sequence
IVSpolyT�5T, 7T or 9T of CFTR gene, allele�spe�
cific PCR was used (Fig. 2) [18]. For detection
CFTR mutation �I507, F508C, F508del, 2184insA,
2143delT heteroduplex and for CFTRdele21kb
deletion analyses have been used. Restriction frag�
ment length polymorphism analysis was used to
detect CFTR gene mutations: G542X, N1303K,
W1282X, G551D, R553X, 1717–1G>A, R117H,
R347P, R347H, R347L, R347C, I336K, R334W,
R560T, G551S, Q552X, Y122X, D1270N, 621+
1G�T, S549I, S549N, 1898–1G>A, 3849+10kbC/T.
PCR products were separated on 2–3 % agarose or
10 % polyacrylamide gels stained with ethidium
bromide on the basis of the size of the product
obtained.
Results and discussion. Cytogenetic analysis
allowed to determining karyotype anomalies in
14 carriers (17.5 %). In general, numerical (13.7 %)
and structural (2.5 %) gonosomal anomalies (Table)
were observed. In particular, regular disomy of X
chromosome (karyotype 47,XXY, Klinefelter’s
syndrome) was detected in 11 cases, deletion of
long arm of Y chromosome – in 2 cases: karyotype
46,Xdel(Y)(q12) in one case and 46,Xdel(Y)
(q11)[23]/45,X[10] – in the second one.
Autosomal changes were observed in one carri�
er (1.3 %) in the form of a Robertsonian transloca�
tion (RT) – karyotype 45,XY,t(13;14)(p11;q11).
One case with 46,XX karyotype in male was
identified. It is noteworthy that karyotype 47,XXY
is, after chromosome 21 trisomy, the second most
frequent numerical anomaly and is observed in
ISSN 0564–3783. Цитология и генетика. 2010. № 652
N. Huleyuk, D. Zastavna, M. Tyrkus et al.
Fig. 1. Examples of both Multiplex PCR: a – lane 1, marker
of MW 50 bp ladder; lane 2, DNA of AZFa�deleted
patient; lane 3, DNA of AZFb�deleted patient; lane 4,
DNA of AZFc�deleted patient; lane 5, DNA of AZFb+c�
deleted patient; lane 6, DNA of AZFa+b+c�deleted patient;
lane 4, DNA of normal male; b – lane 1, marker of MW
50 bp ladder; lane 2, DNA of AZFa�deleted patient; lane
3, DNA of AZFb�deleted patient; lane 4, DNA of AZFc�
deleted patient; lane 5, DNA of AZFb+c�deleted patient;
lane 6, DNA of AZFa+b+c�deleted patient; lane 4, DNA of
normal male
1 out of 1000 newborn boys [19, 20]. In the study,
this gonosomal anomaly was detected in every
sixth patient with aspermia and azoospermia but
only in every twelfth patient with OAT. This
matches the data from other studies [1, 2, 21] that
deal severity of spermatogenesis failure with given
karyotype anomaly. Two cases of Y chromosome
deletion were observed among patients suffering
from AZ and OAT.
A single case of autosomal rearrangement in
the form of Robertsonian translocation was
detected in a patient with OAT. The frequency of
RT is 1/1000 and is, consequently, is the most
often occurring balanced rearrangement [22]. It
shall be emphasized that in male RT carriers the
failure of spermatogenesis and infertility are
observed more often that in female carriers [1, 23,
24]. Cytogenetic analysis of spermatozoa using
FISH with different chromosome�specific probes
indicates the presence of segregation disturbance
during meiosis in RT carriers [25–27].
Genes of AZF regions located on the long arm
of the Y chromosome and SRY (the sex determin�
ing gene) located on the short arm of the Y chro�
mosome play an essential role in spermatogenesis.
In this study, the detection of AZFa�, AZFb�,
AZFc�regions and the SRY gene deletions was
performed. Two multiplex reactions for three
above mentioned regions and for SRY control
fragment were carried out. Among infertile men
with karyotype 46,XY (64 individuals), Y chromo�
some microdeletions were detected in 10.9 %
(7/64) males: microdeletions of AZFa subregion
in 1 patient (14.2 %), AZFb subregion – 2
(28.6 %), AZF (b + c) subregions – 2 (28.6 %),
AZFc subregion – 2 (28.6 %). Total frequency of
detection of abnormalities in AZF region among
idiopathic infertile men reached 11.25 % (9/80)
(Table). Generally, AZFc subregion was most fre�
quently altered. It is important to note that only
among individuals with AS and AZ the whole
spectrum of microdeletions was observed, where�
as in OAT males microdeletions of subregion
AZFc only detected. Obtained results confirm,
firstly, that the most severe clinical presentations
are observed in patients with AZFa and AZFb
subregion microdeletions and secondly, that
AZFc subregion microdeletions are more frequent
in idiopathically infertile men, however this muta�
tion is observed in patients with different degree of
severity of spermatogenetic failure. The results of
our study coincide with data obtained by other
researchers [28, 29].
In the male with 46,XX karyotype the presence
of SRY gene and the deletion of AZFa, AZFb, and
AZFc regions were detected that allowed its classi�
fication as a de la Chapelle syndrome. Most fre�
quently males with de la Chapelle syndrome have
SRY gene located on the X chromosome – this is
the result of abnormal recombination between loci
Xp22.3 and Yp11.3 during spermatogenesis in
father [30]. Such rearrangements are detected by
FISH with probes that are specific for short arm of
Y chromosome where SRY gene is located.
ІSSN 0564–3783. Цитология и генетика. 2010. № 6 53
Complex cytogenetic and molecular�genetic analysis of males with spermatogenesis failure
Fig. 2. CFTR intron 8 alleles characterized by allele�specific PCR. Outermost lanes: marker of MW 100 bp ladder. Each patient
is characterized with three reactions, labelled 5, 7, or 9, representing allele�specific PCRs for the 5T, 7T, or 9T alleles. The
amplification products are 136, 138, and 140 bp in size, respectively. A band of 800 bp in each lane represents amplification
of the control fragment. Genotypes are shown below each triad
Notably, in all fathers with 46, XX sex inversion a
paracentric Yp inversion is detected. Similar inver�
sion polymorphism is found in approximately one
third of European males and, probably, leading to
susceptibility for ectopic Xp-Yp recombination [31].
Mutations in CFTR gene are the most frequent
causes of male sterility associated with unior bilat�
eral CAVD. The first stage of CFTR gene muta�
tions screening in infertile men was the detection
of major F508del mutation (Fig. 2). The frequen�
cy of the major F508del mutation in the studied
group of infertile men was 6.25 % (Table), but
rather higher than frequency of F508del mutation
in the group of women – donors of oocytes (1 %).
ISSN 0564–3783. Цитология и генетика. 2010. № 654
N. Huleyuk, D. Zastavna, M. Tyrkus et al.
Frequency and spectrum of chromosomal abnormalities, Y chromosome microdeletions and CFTR gene
mutations among Ukrainian males with spermatogenetic failure
*Identification of CFTR mutations or 5T IVS8 polyT allele only does not allow doing certain conclusions about CFTR�
related disease in these patients. These cases require additional rare CFTR gene mutation testing.
Number
of samples
Karyotype Y chromosome AZF locus microdeletions CFTR mutations
Aspermia (n = 36)
Azoospermia (n = 19)
Y�69
Y�71
Y�171
Y�225
Y�252
Y�318
Y�342
Y�364
Y�371
Y�386
Y�429
Y�444
Y�452
Y�471
Y�474
Y�537
Y�565
Y�39
Y�40
Y�65
Y�67
Y�162
Y�402
47,ХХY
47,ХХY
47,ХХY
47,ХХY
47,ХХY
46,XX
47,ХХY
46,ХYqh�[23] /45,Х[10]
47,ХХY
47,ХХY
47,ХХY
AZFb:sY127,sY134; AZFc: sY254,sY255
AZFb:sY127,sY134; AZFc: sY254,sY255
AZFb:sY127,sY134
AZFa: sY84, sY86 AZFb:sY127,sY134
AZFc: sY254,sY255
AZFb:sY127,sY134; AZFc: sY254,sY255
AZFa: sY84, sY86
AZFb: sY134
5T/7T IVS8 polyT*
F508del/N*
5T/7T IVS8 polyT*
F508del/5T/7TIVS8polyT
F508del/N*
F508del/N*
G542X/5T/7T IVS8polyT
Severe oligoasthenoteratozoospermia (n = 25)
Y�41
Y�43
Y�54
Y�64
Y�72
Y�80
Y�90
Y�189
Y�500
Total
47,ХХY
46,Х,delYq12
47,ХХY
45,ХY,t(13,14) (p11;p11)
14/80 (17,5 %)
AZFc:sY254,sY255
AZFc: sY254,sY255
9/80 (11,25 %)
F508del/N*
5T/7T IVS8 polyT*
5T/7T IVS8 polyT*
10/80 (12,5 %)
Obtained results testify the presence of G542X
mutation in one patient with aspermia. The CFTR
mutations �I507, F508C, CFTRdele21kb,
2184insA, 2143delT, N1303K, G551D, W1282X,
R553X, 1717–1G>A, 621+1G�T, 1898–1G>A,
R117H, R347P, R347H, R347L, R347C, I336K,
R334W, R560T, G551S, Q552X, Y122X, D1270N,
S549I, S549N, 3849+10kbC/T were not detected
in studied group. 5T allele of IVS8polyT polymor�
phic locus was detected in 7.5 % of males. 5T allele
acts as a mild mutation. In one case 5T allele was
combined with F508del mutation, in the other
case – with G542X mutation. These two cases
were diagnosed as CFTR related disease. In
remaining patients second CFTR gene mutation
was not identified and these cases require addi�
tional rare CFTR gene mutation testing. The distri�
bution of CFTR IVS8polyT genotypes in the stud�
ied group of infertile men was the following:
7T/9T alleles were observed in 62.5 %, 7T/7T –
28.75 %, 5T/7T – 7.5 %, 9T/9T – 1.25 %. It
should be to mentioned that mutations of CFTR
gene were not detected in patients with AZ, and
the highest frequency of F508del mutation was in
the group of individuals with aspermia. Results
show the value of information for CFTR gene
mutations and IVS8polyT polymorphic locus
analysis in infertile men.
Conclusions. Totally, in 28,75 % (23/80) of
males with spermatogenetic failure the genetic fac�
tor of infertility was detected. Karyotype anomalies
were observed in 17.5 % of infertile men, within
16.2 % of cases the numerical and structural gono�
somal anomalies were detected. In 11,25 % males
with spermatogenetic failure Y chromosome AZF
region microdeletions were found. The frequency
of CFTR major mutation F508del among infertile
men was 6.25 %. 5T allele of polymorphic locus
IVS8polyT was observed in 7.5 % of examined
men. Identification of CFTR mutations or 5T
IVS8 polyT allele only does not allow to do certain
conclusions about CFTR�related disease in such
patients. These cases require additional rare CFTR
gene mutation testing. The results obtained indi�
cate the high complexity of cytogenetic and molec�
ular�genetic studies underlying male infertility.
This work was supported of Science and Higher
Education of Poland (grant no. N407 034 32/1371
[2007–2009]).
Н.Л. Гулеюк, Д.В. Заставна, М.Я. Тыркус,
Г.В. Макух, С.В. Гаврилишин, М. Курпиш
КОМПЛЕКС ЦИТОГЕНЕТИЧЕСКИХ
И МОЛЕКУЛЯРНО�ГЕНЕТИЧЕСКИХ
ИССЛЕДОВАНИЙ МУЖЧИН
С НАРУШЕНИЯМИ СПЕРМАТОГЕНЕЗА
Изучали аномалии хромосом, микроделеции AZF
региона Y�хромосомы и мутации гена ТРБМ у 80 муж�
чин с идиопатическими нарушениями сперматогенеза,
а именно: у 36 (45 %) пациентов с аспермией, 19 (24 %)
пациентов с азооспермией и 25 (31 %) пациентов с оли�
гоастенотератозооспермией IV степени. В общем у 30 %
мужчин с нарушениями сперматогенеза установлены
генетические факторы бесплодия. Нарушения кариоти�
па наблюдали у 17.5 % бесплодных мужчин, среди них
у 16.2 % – количественные и структурные аномалии
хромосом и у 1.3 % – робертсоновскую транслокацию.
У 11 % мужчин с нарушениями сперматогенеза выяви�
ли микроделеции AZF региона Y�хромосомы. Частота
мажорной мутации F508del гена ТРБМ среди бесплод�
ных мужчин составила 6.25 %. 5T аллель полиморфного
локуса IVS8polyT выявили у 7.5 % обследованных муж�
чин. Полученные результаты свидетельствуют о высо�
кой информативности комплексного цитогенетического
и молекулярно�генетического исследования при муж�
ском бесплодии.
REFERENCES
1. Bорсанова С.Г., Юров В.Б., Чернышов В.Н. Меди�
цинская цитогенетика. –М.: Медпрактика, 2006. –
299 с.
2. Курило Л.Ф., Гришина Е.М. Роль структурных хро�
мосомных аномалий в развитии патозооспермии
у мужчин с бесплодием // Андрология и генит. хи�
рургия. – 2006. – № 4. – С. 36–40.
3. Созанский О.А., Гулеюк Н.Л., Акопян Г.Р. и др. Не�
которые аномалии хромосом при азооспермии у
мужчин // Генетика. – 1988. – 24, № 7. – С. 1209–
1303.
4. Черных В.Б., Курило Л.Ф., Штлейко Л.В. и др. Анализ
микроделеций в локусе AZF у мужчин с бесплоди�
ем : совместный опыт исследований // Мед. гене�
тика. – 2003. – 2, № 8. – С. 367–379.
5. Howards S.S. Treatment of male infertility // N. Engl.
J. Med. – 1995. – 332. – P. 312–317.
6. Bache I., Assche E.V., Cingoz S. et al. An excess of chro�
mosome 1 breakpoints in male infertility // Eur. J.
Hum. Genet. – 2004. – 12. – P. 993–1000.
7. De Kretser D.M. Male infertility // Lancet. – 1997. –
349. – P. 787–790.
8. Etem E.Ö., Yüce H., Erol D. et al. Cytogenetic analysis
in infertile males with sperm anomalies // Marmara
Med. J. – 2009. – 22, № 3. – P. 217–224.
9. Tiepolo L., Zuffardi O. Localization of factors control�
ІSSN 0564–3783. Цитология и генетика. 2010. № 6 55
Complex cytogenetic and molecular�genetic analysis of males with spermatogenesis failure
ling spermatogenesis in the nonfluorescent portion of
the human Y chromosome long arm // Hum. Genet. –
1976. – № 34. – P. 119–124.
10. Van Assche E., Bonduelle M., Tournaye H. et al. Cyto�
genetics of infertile men // Hum. Reprod. – 1996. – 11
(Suppl 4). – P. 1–24.
11. Bernardino A., Lima C., Zatz M. Analysis of mutations
in the cystic fibrosis transmembrane regulator (CFTR)
gene in patients with obstructive azoospermia // Genet.
Mol. Biol. – 2003. – 26. – Р. 1–3.
12. Hungerford D.A. Leucocytes culture from small inocula
of whole blood and the preparation of metaphase chro�
mosomes by treatment with hypotonic KCl // Stain.
Technol. – 1965. – 40, № 6. – P. 333–338.
13. Зерова�Любимова Т.Е., Горовенко Н.Г. Цитогене�
тичні методи дослідження хромосом людини : Ме�
тод. рекомендації. – Київ, 2003. – 23 с.
14. ISCN (2005): An International System for Human
Cytogenetic Nomenclature / Еds L.G. Shaffer, N.
Tommerup. – Basel : Karger, 2005. – 131 p.
15. Makukh H., Zastavna D., Tyrkus M., Tretjak B., Chor�
na L. The manner of DNA extaction from blood leuco�
cytes. Patent of serviceable model. Ukraine, 32044,
UA. МПК (2006) G01N33/49. State institution
«Institute of Hereditary Pathology». U200801896,
14.02.2008, 25.04.2008, Bull. № 8.
16. McPherson M.J., Quirke P., Taylor G.R. PCR a
Practical Approach. – New York : Oxford Univ. Press,
1993. – 253 р.
17. Simoni M., Bakker E., Krausz C. EAA/EMQN best prac�
tice guidelines for molecular diagnosis of Y�chromoso�
mal microdeletions // Int. J. Andrology. – 2004. –
№ 27. – P. 240–249.
18. Friedman K.J., Heim R.A., Knowles M.R. et al. Rapid
сharacterization of the variable length polythymidine
tract in the cystic fibrosis (CFTR) gene: Association of
the 5T allele with selected cftr mutations and its inci�
dence in atypical sinopulmonary disease // Hum.
Mutat. – 1997. – 10. – P. 108–115.
19. De Braekeleer M., Dao T�N. Cytogenetic studies in
male infertility: a review // Hum.Reprod. – 1991. – 6. –
P. 245–250.
20. Yoshida A., Kazukiyo M., Shirai M. Chromosome
abnormalities and male infertility // Ass. Reprod. Rev. –
1995. – 6. – P. 93–99.
21. Tachdian G., Frydman N., Morichon�Delvallez N. et al.
Reproductive genetic counseling in non mosaic 47,
XXY patients: implications for preimplantation or pre�
natal diagnosis. Case report and review // Hum.
Reprod. – 2003. – 18. – P. 271–275.
22. Nielsen J., Wohlert M. Chromosome abnormalities
found among 34,910 newborn children: results from a
13�year incidence study in Aarhus, Denmark // Hum.
Genet. – 1991. – 87. – P. 81–83.
23. Гулеюк Н.Л., Гуменецький І.Є., Заставна Д.В., Гель�
нер Н.В., Біль І.А. Результати цитогенетичних до�
сліджень подружніх пар з первинним та вторин�
ним непліддям // Проблеми екологічної та медич�
ної генетики і клінічної імунології : Зб. наук. пр. –
Київ; Луганськ; Харків, 2009. – Вип. 1–2 (88–89). –
С. 43–51.
24. Kim S.R., Shaffer L.G. Robertsonian translocations:
mechanism of formation aneuploidy and uniparental
disomy and diagnostic considerations // Genet.
Testing. – 2002. – 6. – Р. 163–168.
25. Baccetti B., Collodel G., Marzella R., Moretti E.,
Piomboni P., Scapigliati G., Serafini F. Ultrastructural
studies of spermatozoa from infertile males with
Robertsonian translocations and 18, X, Y aneuploi�
dies // Hum. Reprod. – 2005. – 20, № 8. – P. 2295–
2300.
26. Biricik A., Guneu I., Berkil H. et al. A male (15;15)
Robertsonian translocation case with 11 previous con�
secutive recurrent spontaneous abortions // Marmara
Med. J. – 2004. – 17, № 1. – P. 25–38.
27. Ogur G., Van Assche E., Vegetti W. et al. Chromosomal
segregation in spermatozoa of 14 Robertsonian translo�
cation carriers // Mol. Hum. Reprod. – 2006. – 12,
№ 3. – P. 209–215.
28. Фесай О.А., Пампуха В.М., Соловйов О.О. та ін. Мо�
лекулярно�генетичний аналіз дефекта гена AZF Y�
хромосоми та гена ТРБМ при чоловічому без�
плідді // Біополімери і клітина. – 2008.– 24, № 3.–
P. 231–237.
29. Simoni M., Tüttelmann F., Gromoll J. et al. Clinical
consequences of microdeletions of the Y chromosome:
the extended Münster experience // Reprod. Bio Med.
Online. – 2008. – 16, № 2. – P. 289–303.
30. Черных В.Б. Макро� и микроструктурные пере�
стройки Y�хромосомы // Мед. генетика. – 2007. –
6, № 10. – С. 45–52.
31. Hopps C.V., Mielni A., Goldstein M. et al. Detection of
sperm in men with Y chromosome microdeletions of
the AZFa, AZFb and AZFc regions // Hum. Reprod. –
2003. – 18. – Р. 1660–1665.
Received 11.01.10
ISSN 0564–3783. Цитология и генетика. 2010. № 656
N. Huleyuk, D. Zastavna, M. Tyrkus et al.
|