Molecular analysis of Y chromosome microdeletions in idiopathic cases of male infertility in Serbia

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.     

Molecular analysis of Y chromosome microdeletions in idiopathic cases of male infertility in Serbia

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 the Y chromosome were detected in 14 of 90 cases (15.6%), nine with azoospermia and five with severe oligozoospermia. Of total number of 17 deletions, 11 (64.7%) were detected in AZFc region, three (17.6%) in AZFa region and three (17.6%) in AZFb region. Microdeletions in AZF region of the 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. The text was submitted by the authors in English.     

对法医学相关的DYS549、DYS527和DYS459基因座在男性不育症人群中的缺失、重复调查

位于Y染色体无精症因子区域(Azoospermia factor, AZF)的基因座位点DYS549、DYS527和DYS459在法医学鉴定和家系分析中被广泛应用。但是,在男性不育患者中,DYS549、DYS527和DYS459位点很可能会表现出特殊的基因型,对应用Y染色体短串联重复序列(Y chromosome short tandem repeat, Y-STR)进行个体识别的结果产生干扰。因此,文章应用14个Y-STR基因座复合扩增体系和Y染色体AZFc区DAZ、CDY1基因的拷贝数检测等方法,探讨男性不育症中法医学相关的3个Y-STR基因座的异常分型,对个体识别和家系分析中的DNA检验异常结果提供合理的解释。在240例男性非梗阻性无精、严重少精、先天性双侧输精管缺如(CBVAD)患者中,采用改良的多重PCR体系进行AZF区域微缺失的序列标签位点(Sequence tagged sites, STSs)检测,发现AZF微缺失40例(AZFa：2例;AZFb：2例;AZFc：30例;AZFb+c：6例),AZF的总缺失率为16.67%。应用14 Y-STR复合扩增体系对上述AZF微缺失的阳性患者样本进行检测,发现所有AZFb缺失患者存在DYS549等位基因缺失,AZFc缺失患者存在DYS527、DYS459等位基因缺失,AZFb+c缺失患者存在DYS549、DYS527和DYS459等位基因缺失。在AZF微缺失阴性的不育症患者中,通过检测DAZ、CDY1基因拷贝数发现10例AZFc部分复制的患者(1例为先天性输精管缺如,2例非梗阻性无精症,7例严重少精子症),占所调查不育人群的4.17%。男性不育人群AZF区域3个Y-STR基因座多态性会造成等位基因缺失或者重复,这些异常分型是由于临床遗传缺陷造成的而不是实验偏差。阐明Y-STR在男性不育人群中的异质性可以更好地完善Y-STR数据库和解释STR实验结果。     

Male infertility: Y-chromosome deletion and testicular aetiology in cases of azoo-/oligospermia

The spermatogenesis locus azoospermia factor (AZF) in Yq11 has been delineated into three microdeletion intervals designated as AZFa, AZFb andAZFc. AZFc is the most frequently deleted region. We have studied 270 male infertile patients for various genetic disorders associated with infertile phenotype. In this study, we have presented results of our studies on Y-chromosome deletions, chromosomal abnormalities (Klinefelter syndrome) and histology of testis with the objective of seeing whether there were cases of gonosomic mosaicism and a causal correlation between the genetic disorder; and testicular aetiology could be drawn. In all the 13 cases of Y-chromosome microdeletion, AZFc region and DAZ gene were deleted, while no case of AZFa deletion was detected. This result was at variance with other reports from India, where a considerable fraction of cases showed deletion in AZFa region of the Y-chromosome. Both Y-deleted and non-Y-deleted cases revealed heterogeneous testicular phenotype with comparable severity. This disparity among testicular phenotype in cases with known genetic aetiology and even in cases of unknown aetiology can be attributed to different genetic backgrounds and effect of modifiers. Since male infertility is a multifactorial disorder, the contributions of environmental and occupational insults may not be underestimated.     

Yq microdeletions--azoospermia factor candidate genes and spermatogenic arrest.

In the last few years considerable progress has been made in the study of sperm physiology and the biology of gamete interaction, furthering our understanding of the pathophysiology of male infertility. With the advent of assisted reproductive technology and intracytoplasmic sperm injection, study of the various factors leading to spermatogenic impairment has become a major focus of scientific research. Understanding the genetic factors that lead to infertility has taken on a certain urgency, as we have learned not only of the transmission to male offspring of spermatogenic impairment, but that these offspring may also be born with a secondary, larger deletion with worsening of phenotype and genital ambiguity.Ten to twenty-five percent of couples encounter difficulty procreating. Microdeletions of the long arm of the Y chromosome are associated with spermatogenic failure and have been used to define three regions on Yq (AZFa, AZFb, and AZFc) that are critical for spermatogenesis. This study was conceived in order to identify the frequency of submicroscopic interstitial deletions in azoospermia factor loci in infertile Indian males. One hundred and seventy five males with nonobstructive oligozoospermia and azoospermia were included in this study. Semen analysis was done in each case to determine the spermatogenic status-normospermic, oligozoospermic (< 20 million sperm/mL), or azoospermic (no sperm in the semen). Detailed medical, clinical, reproductive, and family histories were taken of each patient. Thirty G-banded metaphases were analyzed in each case and polymerase chain reaction microdeletion analysis was done in 133 cytogenetically normal cases. For this genomic, DNA was extracted using peripheral blood. The sequence tagged site primers tested in each case were sY84, sY86 (AZFa); sY113, sY116, sY127, sY134 (AZFb); sY254, sY255 (AZFc). Polymerase chain reaction amplifications found to be negative were repeated at least three times to confirm the deletion of a given marker. The polymerase chain reaction products were analyzed on a 1.8% agarose gel. Eight of the 133 cases showed deletion of at least one of the sequence tagged site markers. Review of the literature has shown that the overall frequency of microdeletions varies from 1% to 55%. In the present study the frequency of microdeletion was 6.01%. Deletions were detected in cases with known and unknown etiology with bilateral severe testiculopathy.     

Y chromosome microdeletions in Brazilian fertility clinic patients

Microdeletions in Yq are associated with defects in spermatogenesis, while those in the AZF region are considered critical for germ cell development. We examined microdeletions in the Y chromosomes of patients attended at the Laboratory of Human Reproduction of the Clinical Hospital of the Federal University of Goiás as part of a screening of patients who plan to undergo assisted reproduction. Analysis was made of the AZF region of the Y chromosome in men who had altered spermograms to detect possible microdeletions in Yq. Twenty-three patients with azoospermia and 40 with severe oligozoospermia were analyzed by PCR for the detection of six sequence-tagged sites: sY84 and sY86 for AZFa, sY127 and sY134 for AZFb, and sY254 and sY255 for AZFc. Microdeletions were detected in 28 patients, including 10 azoospermics and 18 severe oligozoospermics. The patients with azoospermia had 43.4% of their microdeletions in the AZFa region, 8.6% in the AZFb region and 17.4% in the AZFc region. In the severe oligozoospermics, 40% were in the AZFa region, 5% in the AZFb region and 5% in the AZFc region. We conclude that microdeletions can be the cause of idiopathic male infertility, supporting conclusions from previous studies.     

Y chromosome micro-deletions in idiopathic infertility from Northern India

Azoospermia factor locus (AZF) is assumed to contain the genes responsible for spermatogenesis. Deletions in these genes are thought to be pathologically involved in some cases of male infertility associated with azoospermia or oligozoospermia. An attempt was made to establish the prevalence of micro-deletions on the Y chromosome in 79 infertile North Indians with azoospermia and oligozoospermia. Detail clinical examinations as well as endocrinological parameters were also done. Polymerase chain reaction (PCR) micro-deletion analysis was done in 79 infertile men. For this, genomic DNA was extracted from the peripheral blood. Seven sets of primers were used encompassing AZFa, AZFb and AZFc regions. Micro-deletions in five of the 79 cases (6.3%) showed deletions of at least one of the STS markers. Deletions were detected with known and unknown aetiology and at least in one of the infertile male with varicocele. AZF micro-deletions seen in idiopathic infertile males suggest the need for molecular screening in non-idiopathic cases.     

AZF microdeletions on the Y chromosome of infertile men from Turkey

Intervals V and VI of Yq11.23 regions contain responsible genes for spermatogenesis, and are named as "azoospermia factor locus" (AZF). Deletions in these genes are thought to be pathogenetically involved in some cases of male infertility associated with azoospermia or oligozoospermia. The aim of this study was to establish the prevalence of microdeletions on the Y chromosome in infertile Turkish males with azoospermia or oligozoospermia. We applied multiplex polymerase chain reaction (PCR) using several sequence-tagged site (STS) primer sets, in order to determine Y chromosome microdeletions. In this study, 61 infertile males were enrolled for the molecular AZF screening program. In this cohort, one infertile male had 46,XX karyotype and the remaining had 46,XY karyotypes. Forty-eight patients had a diagnosis of azoospermia and 13 had oligozoospermia. Microdeletions in AZFa, AZFb and AZFc (DAZ gene) regions were detected in two of the 60 (3.3%) idiopathic infertile males with normal karyotypes and a SRY translocation was determined on 46,XX male. Our findings suggest that genetic screening should be advised to infertile men before starting assisted reproductive treatments.     

Idiopathic cases of male infertility from a region in India show low incidence of Y-chromosome microdeletion

Chromosomal and Y-chromosomal microdeletion analysis has been done in cases of idiopathic infertility with the objective of evaluating the frequency of chromosomal and molecular anomaly as the causal factor of infertility. Barring a few cases of Klinefelter syndrome (XXY or XY/XXY mosaics), no chromosomal anomaly was encountered. Y-microdeletion was analysed by PCR-screening of STSs from different regions of the AZF (AZFa, AZFb, AZFc) on the long arm of the Y, as well as by using DNA probes of the genes RBM, DAZ (Yq), DAZLA (an autosomal homologue of DAZ) and SRY (Yp; sex determining gene). Out of 177 cases examined, 9 (azoospermia -8 and oligoasthenospermia -1) showed partial deletion of AZF. The size of deletion varied among patients but AZFc was either totally or partially removed in all of them. In contrast, no deletion was detected in AZFa. Testis biopsy done on a limited number of cases (50) showed diverse stages of spermatogenic arrest with no specific correlation with the genotype. The frequency of Y-chromosome microdeletion in our samples (∼ 5%) is much lower than the frequency (∼ 10%) reported globally and the two previous reports from India. We contend that the frequency may be affected by population structures in different geographical regions.     

Screening of Y chromosome microdeletion which contains AZF regions in 71 Turkish azoospermic men

Screening of Y chromosome microdeletion which contains AZF regions in 71 turkish azoospermic men: In 71 Turkish men Y chromosome microdeletions have been studied before intracytoplasmic sperm injection (ICSI). DNA samples were amplified with 18 STS primers of the azoospermia factor (AZF) region on the Y chromosome by using multiplex polymerase chain reaction (PCR). Microdeletions were detected in 4 azoospermic men (5.6 %); one with a deletion in the AZFb region, while the 3 others had a large deletion extending over multiple chromosomal regions (AZFb+c+d and AZFa+b+c+d). In the patients with microdeletion, no spermatogenetic activity could be detected in testis biopsies. This result confirms the idea that Y chromosome microdeletion analysis is important in investigating the possibility of finding sperm in testicular sperm extraction (TESE). Therefore, we point out the importance of genetic testing and counselling regarding Y chromosome microdeletion for couples requesting ICSI.     

Y chromosome microdeletions in infertile males from Andhra Pradesh, South India

Studies on the frequency of Y chromosome microdeletions were carried out in 70 idiopathic infertile males with normal karyotypes. Genomic DNA was isolated from blood and PCR analysis was carried out with AZFa, AZFb, and AZFc STS markers SY 84, SY 87, SY 127, SY 254, and SY 158 to detect the deletions. In 9/70 (12.8%) subjects AZF deletions were observed. In 4/9 (44.4%) subjects were azoospermic, 4/9 (44.4%) of cases were severe oligozoospermic, and 1/9 (11.1%) cases was oligozoospermic.     

Y-chromosome microdeletions as a prognostic marker of male infertility

The results of molecular-genetic study of Y-chromosome microdeletions in men with spermatogenesis failure and in patients with cryptorchism are presented. The molecular-genetic studies of regions AZFa, AZFb, AZFc in STS loci - sY84, sY86, sY127, sY134, sY254, sY255 and SRY gene have been performed. Y-chromosome microdeletions were detected in 13,3% infertile men with spermogram failure. The frequency of genetic (cyto- and molecular) abnormalities among boys with isolated cryptorchism was 4%. The results show the necessity of additional study ofgenetic factors ofcryptorchism development.     

Microdeletions in the Y chromosome as a predictive marker of infertility in males

Results of a molecular-genetics study of microdeletions in the Y chromosome among males with disturbances in spermatogenesis and among patients with cryptorchism are presented. A study of subregions AZFa, AZFb and AZFc with the use of DNA analysis in the STS loci sY84, sY86, sY127, sY134, sY254, sY255, and the gene SRY is performed. Microdeletions in the Y chromosome were found in 13.3% of infertile males studied who exhibited failed spermogram indicators, attesting to the significant information value of the study. The frequency of genetic (cyto- and molecular-genetic) damage among young boys with isolated cryptorchism amounted to 4%, which points to a need for further study of the genetic basis of cryptorchism. Management and optimization of the molecular-genetics study of microdeletions in the Y chromosome are of great importance for medical practice.     

Chromosomal Abnormality and Y Chromosome Microdeletion in Chinese Patients with Azoospermia or Severe Oligozoo-spermia

Chromosomal abnormality and Y chromosome microdeletion are regarded as two frequent genetic causes associated with spermatogenic failure in Caucasian population. To investigate the distribution of the two genetic defects in Chinese patients with azoospermia or severe oligozoospermia, karyotype analysis by G-banding was carried out in 358 idiopathic infertile men, including 256 patients with azoospermia and 102 patients with severe oligozoospermia, and screening of AZF region microdeletion of Y chromosome by multiplex PCR was performed in those patients without detectable chromosomal abnormality and 100 fertile controls. Of 358 patients, 39(10.9%) were found to have chromosomal abnormalities in which Klinefelters syndrome (47, XXY) was the most common chromosomal aberration. The incidence of sex chromosomal abnormality in patients with azoospermia was significantly higher than that in patients with severe oligozoospermia (12.1% vs 1%). Among the rest of the 319 patients with normal karyotype, 46 (14.4%) were found to have microdeletions in AZF region. The prevalence rates of AZF microdeletion was 15% and 13.1% in patients with azoospermia and severe oligozoospermia respectively. The microdeletion in AZFc was the most frequent deletion and all the microdeletions in AZFa were found in azoospermic patients. No microdeletion in AZF region was detected in fertile controls. In conclusion, chromosomal abnormality and AZF region microdeletion of Y chromosome might account for about 25% of Chinese infertile patients with azoospermia or severe oligozoospermia, suggesting the two abnormalities are important genetic etiology of spematogenic failure in Chinese population and it is essential to screen them during diagnosis of male infertility before in vitro assisted fertilization by introcytoplasmic sperm injection.     

A cost-effective screening test for detecting AZF microdeletions on the human Y chromosome

PCR-based screening of microdeletions in the azoospermic factor (AZF) on the Yq chromosome is an accepted means of identifying a common genetic cause of male infertility, responsible for 5-15% of cases associated with a low sperm count (相似文献   

中国无精症、严重寡精症患者的染色体异常和Y染色体微缺失

染色体异常和Y染色体微缺失被认为是两个白种人群中常见的生精障碍相关遗传因素。为了解中国无精症、严重寡精症患者中的染色体异常和Y染色体微缺失,运用染色体G显带技术,在358个原发无精症（256人）和严重寡精症（102人）不育患者中进行染色体核型分析;同时运用多重PCR技术,在核型正常的患者和100个正常生育男性中,对Y染色体AZF区微缺失进行筛查。在358个患者中,39人（10．9％）发现有染色体异常,Klinefelter（47,XYY）最为常见。无精症患者性染色体异常频率明显高于严重寡精症患者（12．1％VS1％）。在319个核型正常的患者中,46（14．4％）发现有AZF区微缺失,无精症和寡精症患者中Y染色体微缺失频率分别为15％和13．1％,AZFc区的微缺失最为常见,AZFa区的微缺失只见于无精症患者,正常生育男性中未发现AZF区的微缺失。结果显示,在中国无精症、严重寡精症患者中,大约25％的患者有染色体异常或Y染色体AZF区微缺失,提示这两种遗传异常是中国人群生精障碍的重要相关遗传病因,有必要在男性不育的诊断以及利用细胞浆内精子注射技术进行辅助生育时,对患者的这些遗传异常进行筛查。     

Molecular Genetic Analysis of Y-Chromosome Microdeletions in Men with Severe Spermatogenetic Defects

Microdeletions of the Y-chromosomal AZF loci were revealed in 10 (12%) of 82 patients with severe idiopathic spermatogenetic defects. Deletions involved AZFc in six patients, AZFa in one patient, AZFb+c in two patients, and AZFa+b+c in one patient. Microdeletion analysis employed multiplex PCR with 22 pairs of primers directed to Y-specific STS of deletion intervals 5, 6, and 7 (Yq11). Spermatogenesis in men with AZF microdeletions was assessed with semen analysis, microscopic examination of testicular aspirate, and quantitative karyotypic analysis of immature germline cells in ejaculate or aspirate. The character of spermatogenetic defects was correlated with the size and location of microdeletions in order to study the genotype–phenotype relationship.     

Prevalence of Y chromosome microdeletions in infertile men with severe oligozoospermia in Serbia

AIM: The aim of this study was to determine the prevalence and type of microdeletions of the Y chromosome of men with severe oligozoospermia-ICSI candidates in the Serbian population and to compare our findings with those from other parts of the world. METHODS: In all patients spermiogram has been performed in order to determine the sperm concentration. Patients were subjected to detailed clinical, endocrinological and cytogenetic examinations. Microdeletion analysis was performed by polymerase chain reaction (PCR) on 203 patients with normal cytogenetic findings. The STS markers tested in each case were sY84, sY86 (AZFa); sY127, sY134 (AZFb); sY254, sY255 (AZFc). RESULTS: at least one of the STS markers was deleted in 11 of the 203 cases (5.4%). CONCLUSION: AZFc microdeletions were identified with a rather high prevalence in men with severe oligozoospermia ICSI candidates in Serbian population.     

Defining regions of the Y-chromosome responsible for male infertility and identification of a fourth AZF region (AZFd) by Y-chromosome microdeletion detection

Cytogenetic and molecular deletion analyses of azoospermic and oligozoospermic males have suggested the existence of AZoospermia Factor(s) (AZF) residing in deletion intervals 5 and 6 of the human Y-chromosome and coinciding with three functional regions associated with spermatogenic failure. Nonpolymorphic microdeletions in AZF are associated with a broad spectrum of testicular phenotypes. Unfortunately, Sequence Tagged Sites (STSs) employed in screening protocols range broadly in number and mapsite and may be polymorphic. To thoroughly analyze the AZF region(s) and any correlations that may be drawn between genotype and phenotype, we describe the design of nine multiplex PCR reactions derived from analysis of 136 loci. Each multiplex contains 4-8 STS primer pairs, amplifying a total of 48 Y-linked STSs. Each multiplex consists of one positive control: either SMCX or MIC2. We screened four populations of males with these STSs. Population I consisted of 278 patients diagnosed as having significant male factor infertility: either azoospermia, severe oligozoospermia associated with hypogonadism and spermatogenic arrest or normal sperm counts associated with abnormal sperm morphology. Population II consisted of 200 unselected infertile patients. Population III consisted of 36 patients who had previously been shown to have aneuploidy, cytological deletions or translocations involving the Y-chromosome or normal karyotypes associated with severe phenotype abnormalities. Population IV consisted of 920 fertile (control) males. The deletion rates in populations I, II and III were 20.5%, 7% and 58.3%, respectively. A total of 92 patients with deletions were detected. The deletion rate in population IV was 0.87% involving 8 fertile individuals and 4 STSs which were avoided in multiplex panel construction. The ability of the nine multiplexes to detect pathology associated microdeletions is equal to or greater than screening protocols used in other studies. Furthermore, the data suggest a fourth AZF region between AZFb and AZFc, which we have termed AZFd. Patients with microdeletions restricted to AZFd may present with mild oligozoospermia or even normal sperm counts associated with abnormal sperm morphology. Though a definitive genotype/phenotype correlation does not exist, large deletions spanning multiple AZF regions or microdeletions restricted to AZFa usually result in patients with Sertoli Cell Only (SCO) or severe oligozoospermia, whereas microdeletions restricted to AZFb or AZFc can result in patients with phenotypes which range from SCO to moderate oligozoospermia. The panel of nine multiplexed reactions, the Y-deletion Detection System (YDDS), provides a fast, efficient and accurate method of assessing the integrity of the Y-chromosome. To date, this study provides the most extensive screening of a proven fertile male population in tandem with 514 infertile males, derived from three different patient selection protocols.     

Molecular genetic analysis of Y-chromosome micro deletions in men with severe spermatogenic defects

Microdeletions of the Y-chromosomal AZF loci were revealed in 10 (12%) of 82 patients with severe idiopathic spermatogenetic defects. Deletions involved AZFc in six patients, AZFa in one patient, AZFb + c in two patients, and AZFa + b + c in one patient. Microdeletion analysis employed multiplex PCR with 22 pairs of primers directed to Y-specific STS of deletion intervals 5, 6, and 7 (Yq11). Spermatogenesis in men with AZF microdeletions was assessed with semen analysis, microscopic examination of testicular aspirate, and quantitative karyotypic analysis of immature germline cells in ejaculate or aspirate. The character of spermatogenetic defects was correlated with the size and location of microdeletions in order to study the genotype-phenotype relationship.