ГЕНЕТИКА, 2007, том 43, № 6, с. 850-854
ГЕНЕТИКА ЧЕЛОВЕКА
УДК 575:599.9
MOLECULAR ANALYSIS OF Y CHROMOSOME MICRODELETIONS IN IDIOPATHIC CASES OF MALE INFERTILITY IN SERBIA
© 2007 r. M. Ristanovic1, V. Bunjevacki1, C. Tulic2, I. Novakovic1, A. Nikolic3
1 Institute of Human Genetics, Medical Faculty, University of Belgrade, 11000 Belgrade, Serbia and Montenegro;
e-mail: ristanmomo@beotel.yu 2 Institute of Urology, Clinical Center of Serbia, Medical Faculty, University of Belgrade, 11000 Belgrade, Serbia and Montenegro 3 Institute of Molecular Genetics and Genetic Engineering, Belgrade, Serbia and Montenegro
Received August 24, 2006
The aim of this study was to detect frequency of microdeletions of Y chromosome in idiopathic cases of male infertility in Serbian population. Patients were subjected to detailed clinical, endocrinological and cytogenetic examinations. Ninety patients with normal cytogenetic findings with azoospermia and severe oligozoospermia were included in the study. In these patients microdeletion analysis was performed by multiplex polymerase chain reaction (PCR) method on DNA extracted from peripheral blood. In each case 6 markers in azoospermia factor (AZF) regions were tested: sY84, sY86 (AZFa); sY127, sY134 (AZFb); sY254, sY255 (AZFc). Deletions on Y chromosome were detected in 14 of 90 cases (15.6%), 9 with azoospermia and 5 with severe oligozoospermia. Of total number of 17 deletions, 11 (64.7%) were detected in AZFc region, 3 (17.6%) in AZFa region and 3 (17.6%) in AZFb region. Microdeletions in AZF region of Y chromosome, especially AZFc microdeletions, represent common genetic cause of idiopathic azoospermia and severe oligozoospremia in Serbian infertile men. Therefore, testing for Y chromosome microdeletions should be considered as an important element in diagnosis and genetic counseling of infertile men in Serbia and decisions regarding the assisted reproduction should be made based on the presence and type of AZF microdeletions.
A significant proportion of infertile men with azoospermia and severe oligospermia have a genetic etiology of reproductive failure [1]. Approximately 15% of the couples worldwide cannot conceive after one year of regular sexual intercourse, with a male factor accounting for 40% of all infertility cases. However, in up to 30% of cases the etiology is unexplained [2]. Genetic etiology of male infertility has been suspected for a long time as a cause of infertility in these idiopathic cases.
Advancements in molecular methodology have permitted precise mapping of Y chromosome microdeletions in men with azoospermia and oligospermia. Three Y chromosome regions, referred to as "azoospermia factors" (AZFa, AZFb and AZFc, from proximal to distal Yq), have been defined as spermatogenesis loci [3] (Figure). The frequency of AZF microdeletions in infertile men varies between 1% and 55%, depending on inclusion criteria, number of markers tested, chromosomal location of chosen markers and ethnic variations, but for most populations frequency of AZF microdele-tions is between 5% and 14% [4-14].
Each type of the deletion is associated with specific spermatogenic alteration, including Sertoli cell-only syndrome (SCOS), sperm maturation arrest and hy-pospermatogenesis. In general, deletions of AZFa region are mainly associated with SCOS, deletions of AZFb region with spermatogenic arrest, while absence of AZFc region is associated with a wide range of phe-
notypes, from azoospermia to oligozoospermia [15]. Segment AZFc was shown to contain the most frequently deleted gene cluster, known as DAZ (deleted in azoospermia). Several studies found AZFc deletions to
pseudoautosomal
Yp
Q
AZFc region
heterochromatin
centromere
AZFa region AZFb region
pseudoautosomal
Yq
Schematic diagram of the Y chromosome, showing the AZF regions.
Table 1. Cytogenetic findings and Y chromosome microdeletions in infertile men
Total Severe oligospermia Azoospermia Total
46, XY 32 58 90
Y chromosome microdeletions detected 5 9 14
not detected 27 49 76
be associated with successful retrieval of sperm during testicular sperm extraction (TESE), whereas deletion in AZFa was not [3, 5, 16].
The objective of this study was to detect frequency of microdeletions of Y chromosome in idiopathic cases of male infertility in Serbian population.
MATERIALS AND METHODS
Patients. The study was carried out on infertile males referred to the Department of Urology and Nephrology and Department of Assisted Reproduction -Clinical Centre of Serbia. Ninety infertile unrelated males presenting with infertility with azoospermia and severe oligozoospermia were included in this study in period June 2002 - February 2006. Written informed consent was obtained from all study patients and the investigation was approved by the hospital's ethical committee. All patients were subjected to detailed clinical, endocrinological and cytogenetic examinations. Sper-mogram has been performed in order to determine the sperm density. Diagnosis of azoospermia and severe oligozoospermia (<1 x 106 spermatozooa/mL) was made according to WHO guidelines [17]. All patients diagnosed with severe oligozoospermia had one unsuccessful attempt of intracytoplasmatic sperm injection (ICSI).
Serum follicle-stimulating hormone (FSH) level was determined using radio-immune assay (RIA). Peripheral blood cultures were set up for chromosomal analysis and 11 well spread G banded metaphases were karyotyped using standard protocols. Histology of a testis biopsy obtained by testicular sperm extraction and Y chromosome microdeletion analysis were performed in patients with normal karyotype findings. Control group of 100 age-matched men who had fathered at least two children was also analyzed for the presence of Y chromosome microdeletions.
Y chromosome analysis. Peripheral blood samples were collected and DNA was isolated using standard protocols. Six markers in AZF region were amplified by PCR to determine the presence of 3 types of deletions (AZFa, AZFb and AZFc). Primers and PCR conditions used for amplification were recommended by Simoni et al. [7]: sY84 and sY86 for AZFa; sY127 and sY134 for AZFb; sY254 and sY255 for AZFc. Amplifications were carried out in two multiplex PCR reactions. Multiplex A consisted of sY86, sY127 and
sY254, with SRY and ZFX/ZFY as internal controls. Multiplex B consisted of sY84, sY134 and sY255, also with SRY and ZFX/ZFY as internal controls. Samples were subjected to 30 cycles at 94°C for 1 minute, 55°C for 45 seconds and 72°C for 60 seconds. Initial denatur-ation was 5 minutes at 94°C and a final extension 7 minutes at 72°C. All products of amplification were analyzed by electrophoresis on 10% (29 : 1) polyacry-lamide (PAA) gel, followed by silver staining. Deletion of specific region was detected by absence of PCR product. Multiplex amplifications found to be negative for one or more markers were repeated twice to confirm the obtained results. Markers found to be deleted in multiplex PCR were also amplified in single reactions to confirm the presence of deletion.
RESULTS
A total of 90 infertile men with normal kariotype (58 azoospermic and 32 oligozoospermic) and 100 age-matched fertile controls were analyzed for Y microde-letions. Microdeletions were not found in any of controls. Deletions on Y chromosome were detected in 14 cases, in 9 of 58 men with azoospermia and in 5 of 32 men with severe oligozoospermia. The total frequency of microdeletions was 15.6%, and it was the same in both subgroups (Table 1). Types of microdele-tions, as well as clinical findings for each patient are shown in Table 2.
Complete AZFa and partial AZFb microdeletions were detected in two patients, while complete AZFa microdeletion was detected in one patient. The presence of AZFc microdeletion with partial AZFb microdeletion was detected in 1 patient, while AZFc microdeletion was detected in 10 patients. Of total number of 17 deletions, 11 (64.8%) were detected in AZFc region, 3 (17.6%) in AZFa region and 3 (17.6%) in AZFb region.
Testis biopsy analysis has shown the presence of SCOS in both patients with complete AZFa and partial AZFb microdeletion. Hypospermatogenesis was detected in 3 patients, two with AZFc microdeletion and one with partial AZFb and AZFc microdeletions.
Among patients with microdeletions, two had elevated FSH levels (>11 mlU/ml), while in others the FSH level was normal (0.7-11 mlU/ml). The mean FSH level among patients with microdeletions (4 mIU/ml) did not
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Table 2. Clinical and laboratory findings in infertile men with Y chromosome microdeletions
Patient Semen analysis Testis biopsy result FSH (mIU/ml) AZF microdeletions
1 Azoospermia SCOS 30 AZFa + Partial AZFb
2 Azoospermia SCOS 26 AZFa + Partial AZFb
3 Azoospermia Normal result 9 AZFa
4 Azoospermia Hypospermatogenesis 4 Partial AZFb + AZFc
5 Azoospermia Normal result 2 AZFc
6 Azoospermia Normal result 4 AZFc
7 Azoospermia Hypospermatogenesis 1 AZFc
8 Azoospermia Normal result 10 AZFc
9 Azoospermia Normal result 5 AZFc
10 Oligozoospermia Normal result 2 AZFc
11 Oligozoospermia Hypospermatogenesis 4 AZFc
12 Oligozoospermia Normal result 3 AZFc
13 Oligozoospermia Normal result 3 AZFc
14 Oligozoospermia Normal result 2 AZFc
differ significantly from the mean FSH level in patients without microdeletions (4.6 mlU/ml).
DISCUSSION
Many studies have supported the concept of genetic basis of male infertility and it has been shown that there is association between Y chromosome microdeletions and idiopathic infertility [2, 3, 8, 9, 15, 18]. This is the first study to investigate the pattern and prevalence of Y chromosome microdeletions in AZF subregions in Serbian men with idiopathic male infertility. We detected Y chromosome microdeletions in 14 out of 90 infertile patients (15.6%), which is within the range of frequencies obtained for many populations [11, 12, 14, 19, 20]. The obta
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