Genotyping of Israeli infertile men with idiopathic oligozoospermia

Microdeletions of the long arm of the Y chromosome involving the azoospermia factor (AZF) region are associated with severe oligo- or azoospermia. Abnormal androgen receptor (AR) structure or function has also been implicated in male infertility. To assess the contribution of these genetic defects to male infertility, 61 Israeli men with severe oligo- (n = 15) or azoospermia (n = 46), were screened for Y chromosome microdeletions, and the AR-(CAG)n repeat length. Fifty fertile Israeli men were similarly analyzed. PCR amplification of 20-54 simple tag sequences (STSs) located at Yq was used to determine the rate and extent of Y chromosome microdeletions. PCR with primers flanking the AR-(CAG)n region and subsequent size fractionation on gradient acrylamide gels were used to determine AR-(CAG)n length. Five azoospermic individuals (5/61-8.2% and 5/46-10.8% of azoospermic patients) displayed Y chromosome microdeletions. The mean CAG repeat number in infertile men was 18.6 +/- 3.0 compared with 16.6 + 2.7 in fertile men (n = 50), a statistically significant difference (p = 0.003). Y chromosome microdeletions contribute to male infertility in our azoospermic population, and the mean length of the AR-CAG is significantly longer in our infertile population than in fertile men.     

Microdeletions in the Y chromosome in cases of male infertility in a population of West Bengal

IntroductionInfertility is a burning problem in gynecological, andrological, endocrine and genetic practice. Of the myriad factors responsible for male infertility, which may be manifested as oligozoospermia or azoospermia, the exact causes of the latter are still unknown or debatable. Among the known parameters, the occurrence of microdeletions in the long arm of the Y chromosome are of great importance, as they have been consistently associated with defects in spermatogenesis. The microdeletions of the Y chromosome have been mapped to three regions in interval 6 named azoospermia factor regions (AZF), AZFa, AZFb and AZFc.MethodsIn the present study 80 males suffering from oligozoospermia or azoospermia were taken from both rural and urban infertility clinics and subjected to Polymerase Chain Reaction (PCR) of DNA from blood samples using a total of 11 STS primers. These primers correspond to different segments of the AZF regions (AZFa, AZFb and AZFc) and are known as Sequence Tagged Sites (STS). This was followed by agar gel electrophoresis to look for deletions in the AZF regions corresponding to the STF primers.ResultThese tests were able to detect microdeletions in the long arm of the Y chromosome in 4 patients.DiscussionIn majority of patients PCR detects no abnormality but in cases having microdeletions, appropriate advice could be given to the patients. These patients were told to avoid the use of their sperm in assisted reproduction procedures and accept the use of donor sperm or adoption procedures as a solution to their problems of infertility.     

No association found between gr/gr deletions and infertility in Brazilian males

The Y chromosome carries several spermatogenesis genes distributed in three regions: AZFa, AZFb and AZFc. Microdeletions in these regions have been seen in 10% of sterile males with azoospermia or oligozoospermia, the most frequent of them being characterized by a complete deletion of AZFc region. A partial AZFc deletion named gr/gr has been singled out as a risk factor for spermatogenic failure. However, other authors have diagnosed it as a polymorphic deletion with no clinical relevance. We decided to investigate the association of gr/gr deletion and infertility in Brazilian males. We analysed 350 individuals (110 azoospermic, 122 fertile and 118 presumably fertile) and observed 12 g/gr deletions: five in infertile men (4.5%), three among fertile males (2.5%) and four in probably fertile individuals (3.4%). These differences were not statistically significant. Then, we decided to ascertain whether the clinical impact of the gr/gr deletion was associated with the type of Y chromosome. We have identified Y-chromosome haplogroups using 22 unique event polymorphisms (UEPs). Among the individuals with the gr/gr deletion, we found haplogroups R, K*, F*, E1, E3b2 and E3b*, all of which are common in white Brazilian males, and none revealed a particular association with infertility. Taken together, these results show no evidence of association between the occurrence of gr/gr deletion and male infertility.     

广西地区男性不育患者Y染色体微缺失结果分析及遗传效应

目的探讨广西地区男性不育患者Y染色体微缺失结果情况和临床遗传效应。方法采用染色体技术、PCR技术等对2180例男性不育患者进行外周血染色体分析及Y染色体微缺失6个系列标签位点检测。结果 2180例样本Y染色体AZF基因检测和Y染色体异常分别为总缺失率为6%（130/2180）和3.3%（71/2180）。同时发现Y染色体AZF基因缺失在生精障碍组与正常组、不孕产史组具有统计学意义;Y染色体核型以小Y和染色体多态性常见。结论男性不育患者与Y染色体微缺失及细胞核型所表现临床遗传效应密切相关,对于生殖异常的男性行外周血染色体检查和AZF微缺失检测有助于明确其遗传学病因,更好的为患者提供病因诊断、遗传咨询和治疗方案。     

广州地区不育男性Y染色体无精子因子微缺失的筛查

目的探讨Y染色体无精子因子(azoospermia factor,AZF)区域微缺失与原发无精、严重少精症之间的关系。方法采用多重聚合酶链反应技术对广州地区103例原发无精子症、72例原发严重少精症患者及60名正常生育男性进行AZFa、AZFb、AZFc3个区域微缺失分析。结果60名正常生育男性未发现Y染色体AZF区域微缺失,175例生精障碍患者中发现AZF微缺失19例,总缺失率为10.9%。其中11例无精症患者和4例少精症患者的缺失发生在AZFc区域,缺失率为8.6%;1例无精症患者和2例少精症患者发生AZFb、AZFc双重缺失,缺失率为1.7%;1例无精症患者发生AZFa、b、c3个区域同时微缺失,缺失率0.6%。生精障碍组与正常生育男性组比较Y染色体AZF区域微缺失率差异具有统计学意义(P<0.01)。结论Y染色体AZF区域微缺失是引起男性无精、少精子症的重要原因之一,对原发无精、少精子症患者在单精子注射之前进行微缺失筛查是必要的。     

男性不育患者Y染色体AZF基因微缺失检测

目的探讨原发性无精子症、严重少精子症及少精子症患者与Y染色体无精子因子（azoospermia factor,AZF）区微缺失的关系。方法采用多重PCR方法对对照组192例已正常生育男性和实验组448例男性不育患者进行AZF区域内的15个序列标签位点（STS）的检测。结果对照组未发现AZF基因微缺失,实验组448例患者检测出五种AZF微缺失类型共41例,总缺失率为9.2%（41/448）,其中无精子症、严重少精子症和少精子症患者的缺失率分别为12.0%（19/158）、10.8%（17/157）、3.8%（5/133）,无精子症和严重少精子症患者Y染色体AZF微缺失率明显高于少精子症组,差别有统计学意义（P〈0.05）。使用15个STS位点进行检测其检出率较利用欧洲男科学会（European Academy of Andrology,EAA）推荐的6个STS位点提高约14%（5/36）。结论AZF微缺失是引起原发性无精子症、严重少精子症和少精子症的重要原因之一;增加STS位点检测数有利于提高AZF微缺失的检出率。     

Y chromosome microdeletions, in azoospermic or near-azoospermic subjects, are located in the AZFc (DAZ) subregion

Submicroscopic deletions of the Y chromosome and polymorphisms of the androgen receptor (AR) gene in the X chromosome have been observed in men with defective spermatogenesis. To further define the subregions/genes in the Y chromosome causing male infertility and its relationship to polymorphisms of the AR polyglutamine tract, we screened the genomic DNA of 202 subfertile males and 101 healthy fertile controls of predominantly Chinese ethnic origin. Y microdeletions were examined with 16 sequence-tagged site (STS) probes, including the RBM and DAZ genes, spanning the AZFb and AZFc subregions of Yq11, and related to the size of trinucleotide repeat encoding the AR polyglutamine tract. Y microdeletions were detected and confirmed in three out of 44 (6.8%) of azoospermic and three out of 86 (3.5%) severely oligozoospermic patients. No deletions were detected in any of the patients with sperm counts of >0.5 x 10(6)/ml, nor in any of the 101 fertile controls. All six affected patients had almost contiguous Y microdeletions spanning the entire AZFc region including the DAZ gene. The AZFb region, containing the RBM1 gene, was intact in five of the six subjects. Y deletions were not found in those with long AR polyglutamine tracts. Our study, the first in a Chinese population, suggest a cause and effect relationship between Y microdeletions in the AZFc region (possibly DAZ), and azoospermia or near-azoospermia. Y microdeletions and long AR polyglutamine tracts appear to be independent contributors to male infertility.      

男性不育患者Y染色体微缺失的研究

目的研究无精子症和少精子症患者与Y染色体位点缺失的相关性，建立Y染色体微缺失的分子诊断方法。方法采用多重PCR技术对53例染色体核型正常的无精子症和少精子症患者以及5例正常男性的无精子因子（azoospermia factor，AZF）区域的6个STS位点进行检测。结果5例精液正常男性未检出Y染色体微缺失；53例患者中6例有AZF区域的微缺失，总缺失率为11．3％。结论Y染色体微缺失是严重生精障碍的重要原因之一，无精子因子（AZF）候选基因在精子发生过程中可能起重要作用。     

桂西地区壮族不育患者Y染色体无精子因子筛查

目的探讨桂西地区壮族不育患者Y染色体无精子因子(azoospermia factor,AZF)微缺失与原发无精子、严重少精子症之间的关系。方法采用多重聚合酶链反应技术对桂西地区52例原发无精子症、76例原发严重少精子症患者及40名正常生育男性进行4个区域15个位点微缺失分析。结果 40名正常生育男性未发现Y染色体AZF微缺失,128例生精障碍患者中发现AZF微缺失13例,总缺失率为10.2%。生精障碍组与正常生育男性组比较Y染色体AZF微缺失率差异具有统计学意义(P0.01)。结论 Y染色体AZF微缺失是男性无精子、少精子症的要重原因之一。     

Identification of a Y chromosome haplogroup associated with reduced sperm counts

In man, infertility is associated with microdeletions of specific regions of the long arm of the Y chromosome. This indicates that factors encoded by the Y chromosome are necessary for spermatogenesis. However, the majority of men with either idiopathic azoospermia or oligozoospermia have grossly intact Y chromosomes and the underlying causes of their infertility are unknown. We hypothesized that some of these individuals may carry other rearrangements or sequence variants on the non-recombining region of the Y chromosome that may be associated with reduced spermatogenesis. To test this hypothesis, we typed the Y chromosome in a group of Danish men with known sperm counts and compared the haplotype distribution with that of a group of unselected Danish males. We found that one class of Y chromosome, referred to as haplogroup 26+, was significantly overrepresented (27.9%; P < 0.001) in the group of men with either idiopathic oligozoospermia (defined as <20 x 10(6 )sperm/ml) or azoospermia compared to the control Danish male population (4.6%). This study defines, for the first time, a class of Y chromosome that is at risk for infertility in a European population. This observation suggests that selection may be indeed active on the Y chromosome, at least in the Danish population, raising the possibility that it could alter the pattern of Y chromosome haplotype distribution in the general population.     

424例非梗阻性无精子症和严重少精子症患者的细胞与分子遗传学研究

目的探讨非梗阻性无精子症和严重少精子症患者的细胞与分子遗传学特点。方法应用染色体核型分析、Y染色体微缺失检测和荧光原位杂交（FISH）、PCR等技术对非梗阻性无精子症（n=291）和严重少精子症患者（n=133）男性不育患者（共424例）进行细胞和分子遗传学检测。结果424例患者中有98例明确为遗传异常引起的,其中66例检测到染色体畸变,44例Y染色体微缺失检测见缺失,12例患者染色体核型和微缺失检测均见异常。部分AZF缺失患者精液或睾丸中有精子,但其生精功能呈进行性下降的特点。结论男性不育最常见的遗传学病因为K linefelter综合征和Y染色体AZFc缺失。Y染色体微缺失检测对Y染色体长臂异染色质区缺失是否为多态性具有明确诊断的作用。细胞与分子遗传学检测为男性不育的诊断、治疗和预后以及ICSI治疗前遗传咨询提供重要依据。     

165例男性不育染色体核型及AZF基因缺失分析

目的探讨男性不育患者染色体核型异常及无精症因子（AZF）基因缺失与男性不育的关系。方法对2012年5月-2014年5月来本院就诊的（重庆地区）原发性男性不育患者165例,进行外周血G显带核型分析并采用多重PCR对无精症因子区域的15个标签序列位点进行检测。结果165例生精障碍患者中染色体异常共检出5例,1例为男性性反转（46,XX）,1例为克氏综合征（47,XXY）,1例为47,XY,＋mar,1例为46,XY,Y≥18,1例46,XY,in（9）,其余均为正常核型,总异常率为3.03％（5／165）;AZF基因位点发生微缺失患者共检出25例,总缺失率为15.15S。结论染色体异常和AZF微缺失是男性不育的重要原因,对男性不育诊断时有必要进行检查。     

Study of AZFc partial deletion gr/gr in fertile and infertile Japanese males

A recurrent partial azoospermia factor C (AZFc) deletion, called gr/gr, has been reported to be a male infertility risk factor. A specific type of Y chromosome observed in approximately 30% of Japanese males (haplogroup D derived at YAP+) is believed to have a fixed gr/gr deletion. A recent study claimed that spermatogenic failure is more likely in males with D Y chromosomes, because of the gr/gr deletion, the presence of which is not well characterized among D haplogroup chromosomes. We therefore decided to perform a systematic study of the frequency of the gr/gr deletion in the Japanese. We studied fertile and infertile males to investigate the possibility of different gr/gr frequencies. The deletions were detected by use of single tagged-sequences (STSs) and the D haplogroup sub-lineages typing were done by use of the biallelic markers M174, M64, M116.1, 12f2.2, M15, M151, and M125. Analysis of gr/gr deleted Y chromosomes showed that all are classified as haplogroup D2, suggesting a lineage association. The subtype D2b1 was most frequent among the Japanese, in control and infertile samples. The haplogroups D2b2, D*, and D1 were not found in any population group. Remarkably, we observed no statistical difference between haplogroup D sub-lineages of the infertile and control groups, although the statistical power of this study is low. This study suggests lack of significant evidence of increased infertility risk in haplogroup D Japanese males. We were also able to establish the ancestral chromosome that suffered a gr/gr deletion, and propose a new Y chromosome phylogeny for haplogroup D and its derivatives. In summary, we were able to define the frequency of gr/gr deletion in Japanese males and show that the gr/gr deletion was probably present in the ancestral Y chromosome that entered Japan at least 12,000 years ago.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Molecular analysis of microdeletions of the Y chromosome in Venezuelan males with idiopathic infertility

Today infertility is a major health problem affecting about 10-20% of couples. A male factor is assumed to be responsible in about 50% of the infertile couples. The origin of reduced testicular sperm function is unknown in about 60-70% of cases. There are several causes of male infertility such as varicocele, spermatic duct obstruction, and endocrine disorders. Micro-deletions in the Yq are known to represent the pathogenic mechanisms for infertile males. Three different non-overlapping regions designated as AZFa, AZFb, and AZFc are located in interval 5-6 of Yq, and are associated with impaired spermatogenesis in humans. To determine the prevalence of Y chromosomal microdeletions in Venezuelan males with idiopathic infertility, chromosomal, seminal, histological and molecular analyses were carried out in 29 Venezuelan males with idiopathic azoospermia or oligoospermia. Y-microdeletions analyses were performed using a multiplex polymerase chain reaction (PCR)-based technique with 22 sequences-tagged-sites (STSs). One of 29 patients (3.4%) had Yq microdeletions on AZFc. The frequency of AZF microdeletions in Venezuelan patients was similar to other populations with different ethnical or geographical origin.     

Genetic risk factors in infertile men with severe oligozoospermia and azoospermia.

BACKGROUND: Male infertility due to severe oligozoospermia and azoospermia has been associated with a number of genetic risk factors. METHODS: In this study 150 men from couples requesting ICSI were investigated for genetic abnormalities, such as constitutive chromosome abnormalities, microdeletions of the Y chromosome (AZF region) and mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. RESULTS: Genetic analysis identified 16/150 (10.6%) abnormal karyotypes, 8/150 (5.3%) AZFc deletions and 14/150 (9.3%) CFTR gene mutations. An abnormal karyotype was found both in men with oligozoospermia and azoospermia: 9 men had a sex-chromosomal aneuploidy, 6 translocations were identified and one marker chromosome was found. Y chromosomal microdeletions were mainly associated with male infertility, due to testicular insufficiency. All deletions identified comprised the AZFc region, containing the Deleted in Azoospermia (DAZ) gene. CFTR gene mutations were commonly seen in men with congenital absence of the vas deferens, but also in 16% of men with azoospermia without any apparent abnormality of the vas deferens. CONCLUSIONS: A genetic abnormality was identified in 36/150 (24%) men with extreme oligozoospermia and azoospermia. Application of ICSI in these couples can result in offspring with an enhanced risk of unbalanced chromosome complement, male infertility due to the transmission of a Y-chromosomal microdeletion, and cystic fibrosis if both partners are CFTR gene mutation carriers. Genetic testing and counselling is clearly indicated for these couples before ICSI is considered.     

Genetic markers of male infertility: Y chromosome microdeletions and cystic fibrosis transmembrane conductance gene mutations

Today, approximately 15% of couples have reduced fertility. In most cases the reason is male infertility, usually of genetic origin. Thus, in the context of research in genes involved in reproduction and sex determination, genetic defects in gametogenesis are being extensively studied. The most frequent pathogenic causes of male infertility are Y chromosomal microdeletions and obstructive azoospermia due to congenital absence of the vas deferens (CAVD) in the presence of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We have investigated the most common CFTR gene alterations in Croatian men with CAVD, using Roche research prototype assays. Results revealed that the 5T variant was present in 27% of the subjects. The F508 deletion was found in 21% of the subjects. It was the most frequent mutation, although its incidence was much lower than among patients with cystic fibrosis. The prevalence of microdeletions in the azoospermia factor region (AZF) of the Y chromosome in Croatia was 4.5%. This is the first report of Y microdeletions in the Croatian population. Genetic counseling of all couples with the diagnosis of male infertility is recommended before intrauterine insemination, in vitro fertilization, and intracytoplasmic sperm injection, and should also include AZF and CFTR genotyping. Couples requesting assisted reproductive treatment should be offered molecular analysis of the CFTR gene, if male infertility due to obstructive azoospermia is the underlying cause. Also, men with severe oligozoospermia or non-obstructive azoospermia seeking assisted reproductive treatment should be screened for deletions in the Y chromosome.     

810例严重少(无)精子症患者Y染色体微缺失筛查与分析

目的筛查原发无精子症与重度少精子症患者Y染色体微缺失情况,探讨Y染色体微缺失与男性不育的关系。方法采用改良多重PCR方法对810例男性不育患者(457例原发无精子症和353例严重少精子症)基因组DNA进行Y染色体微缺失筛查。结果810例患者中发现77例Y染色体微缺失患者,缺失率为9.5%,其中少精子症31例,均为AZFc微缺失,无精子症46例,缺失类型呈多样化。缺失类型包括AZFa微缺失3例(3.90%),AZFb微缺失2例(2.60%),AZFc微缺失63例(81.82%),AZFb+c微缺失4例(5.19%),AZFa+b+c微缺失5例(6.49%)。结论Y染色体微缺失是原发无精子症和少精子症的重要原因之一,AZFc缺失为最常见的缺失类型,对此类患者进行Y染色体微缺失的常规筛查是有必要的,尤其是拟行辅助生殖技术助孕的不育患者。     

AZF微缺失与生精障碍症的研究分析

目的探讨Y染色体AZF微缺失与生精障碍症(男性原发性无精子症和少精子症)之间的关系。方法采用多重聚合酶链反应技术对158例生精障碍症患者(包括健康男性50例)进行AZFa、AZFb、AZFc和AZFd四个区域微缺失分析。结果 158例生精障碍症患者中发现AZF微缺失21例,总缺失率为13.3%。结论 Y染色体AZF区域微缺失与男性生精障碍症有着明显的相关性,因此有必要对生殖门诊及准备行辅助生育技术的生精障碍症患者行Y染色体AZF微缺失检测,其对该症的诊断及治疗具有重要的意义。     

No AZF deletion in 160 patients with testicular germ cell neoplasia

Testicular germ cell cancer is aetiologically linked to genital malformations and male infertility and is most probably caused by a disruption of embryonic programming and gonadal development during fetal life. In some cases, germ cell neoplasia is associated with a relative reduction of Y chromosomal material (e.g. 45,X/46,XY) or other abnormalities of the Y chromosome. The euchromatic long arm of the human Y chromosome (Yq11) contains three azoospermia factors (AZFa, AZFb, AZFc) functionally important in human spermatogenesis. Microdeletions encompassing one of these three AZF loci result in the deletion of multiple genes normally expressed in testis tissue and are associated with spermatogenic failure. The aim of our study was to investigate whether AZF microdeletions, in addition to causing infertility, predispose also to germ cell neoplasia, since subjects with poor spermatogenesis have an increased risk of testicular cancer. We screened for putative deletions of AZF loci on the Y chromosome in DNA isolated from white blood cells of 160 Danish patients with testicular germ cell neoplasia. Interestingly, although AZF microdeletions are found frequently in patients with idiopathic infertility, in all cases studied of testicular germ cell cancer the Yq region was found to be intact. We conclude that the molecular aetiology of testicular germ cell neoplasia of the young adult type most likely does not involve the same pathways as male infertility caused by AZF deletions. Malignant transformation of germ cells is thus caused by the dysfunction of some other genes that still need to be identified.     

The prognostic role of the extent of Y microdeletion on spermatogenesis and maturity of Sertoli cells

Substantial involvement of the Y chromosome in sexual development and spermatogenesis has been demonstrated. Over the last decade, varying extent of Y chromosome microdeletions have been identified among infertile patients with azoospermia or oligozoospermia. These microdeletions were clustered in three main regions named AZFa, AZFb, and AZFc. Analysis of the Y chromosome microdeletion was found to be of prognostic value in cases of infertility, both in terms of clinical management as well as for understanding the aetiology of the spermatogenesis impairment. However, the accumulated data are difficult to analyse, due to the variable extent of these deletions, the different sequence-tagged sites (STS) used to detect the microdeletions, and the non-uniformity of the histological terminology used by different investigators. This debate discusses the chances of finding testicular spermatozoa in men with a varying extent of Y chromosome microdeletions. The genotype and germ cell findings in men with AZFa microdeletions as well as those that include more than a single AZF region are reviewed, as is the effect of Y chromosome AZF microdeletions on the maturity of the Sertoli cells.