无精子症和严重少精子症患者Y染色体微缺失研究

目的：研究Y染色体微缺失与男性不育的关系。方法：采用多重PCR技术,研究正常男性、无精子症和严重少精子症男性不育患者Y染色体无精子因子（AZF）区域3个序列标志位点（STS）的缺失情况。结果：在93例无精子症或严重少精子症患者中,15例有Y染色体微缺失,缺失率为16%。其中,42例无精子症患者中,6例为AZFc区SY255位点缺失,2例为AZFb区SY134位点缺失;51例严重少精子症患者中,7例为AZFc区SY255位点缺失。40例正常男性无Y染色体微缺失。结论：多重PCR技术是简便而有效的对男性不育患者进行Y染色体微缺失筛查的方法;Y染色体微缺失是造成男性不育的一个重要原因,对男性不育患者进行辅助生育技术治疗前应常规进行Y染色体微缺失的检测。     

精子发生障碍的遗传学研究进展

育龄人群中约15％的夫妻被不孕不育困扰,其中男方因素导致的不孕不育约占50％.男性不育通常由精子发生障碍导致,呈现为少、弱、畸形精子症,最严重的是无精子症.本文以精子发生障碍为主线,重点综述了非梗阻性无精子症和畸形精子症的遗传学病因研究.近年来,随着高通量芯片和测序技术的快速发展,无精子症和畸形精子症的遗传学因素得以深...     

不孕症患者病因调查分析

为探讨及分析不孕不育病因情况,收集3777对门诊初诊患者的年龄、月经史、孕产史及与不孕不育相关的检查结果等资料进行统计分析,结果表明,导致女性不孕的主要病因为输卵管及盆腔疾病,其次为排卵异常,包括多囊卵巢综合征,卵巢早衰等．导致男性不育的主要因素是精液的数量及质量异常,无精子症患者在男性不育患者中占主要比重,其次为少弱畸精子症及严重少弱畸精子症．在无精子症患者中,生精功能障碍为其主要致病因素．先天性输精管缺如也是导致无精子症的重要影响因素．精液异常患者中遗传缺陷是男性不育重要的致病因素之一．     

KATNAL1 基因突变筛查及其与无精症的相关性分析

目的:探讨男性不育症患者Katnal15基因的一个突变位点与男性不育症的关系及意义。方法:运用聚合酶链反应(PCR)结合琼脂糖凝胶电泳和基因序列分析等方法,对77例原发性男性不育患者以及84名已生育的正常男性进行Katnal1基因筛查。结果:与精子形成的关键基因KATNAL1中1个致病突变位点A236G为的男性精子无力症Katnal1基因筛查的主要候选基因。结论:Katnal1基因蛋白质编码序列区A236G可能是特发性少精无精症的诱发因素之一。临床上对原发性不育患者进行A236G基因突变筛查是十分必要的。     

特发性少精子症和无精子症与Pygo2基因蛋白编码区SNPs的相关性

男性不育常伴随精子数量减少。Pygo2基因在染色质重塑的伸长精细胞中表达, 其功能受损会导致精子形成阻滞和精子生成减少而引发不育。文章旨在检测引起人特发性少精子症和无精子症的Pygo2基因突变。从77例正常生育力男性和195例特发性少精子症和无精子症患者静脉血提取DNA, 采用聚合酶链式反应-测序方法对Pygo2基因3个蛋白质编码区进行测序对比, 非同义单核苷酸多态性(Single nucleotide polymorphisms, SNPs)位点分别用SIFT、Polyphen-2和 Mutation Taster软件进行诱发蛋白质结构和表型改变的检测和分析。结果表明, 195例患者中, 178例(30例轻度或中度少精子症, 57例重度少精子症和91例无精子症)基因序列分析报告完好, 无精子症中3例患者分别在2个位点(rs61758740, rs141722381)发生了非同义突变SNPs, 重度少精子症中1例患者在位点rs61758741发生了非同义突变, 3个突变位点在SNPs基因数据库都已有报道, 轻度或中度少精子症患者以及正常生育力男性中不存在SNPs。rs61758740可使PYGO2蛋白第141位蛋氨酸(M)变为异亮氨酸(I), rs61758741使PYGO2蛋白第261位碱性赖氨酸(K)变为酸性谷氨酸(E), rs141722381使PYGO2蛋白第240位亲水侧链天冬酰胺(N)变为疏水侧链异亮氨酸(I)。软件分析表明, 在所发现的3个SNP非同义突变位点中, rs141722381引起的单个氨基酸改变会导致PYGO2蛋白空间结构破坏和诱发相关疾病。因此, Pygo2基因蛋白质编码序列区SNPs可能是特发性少精子症和无精子症的诱发因素之一, 导致男性不育。     

PNAS:科学家发现新的精子运动调节器

正南京医科大学生殖医学国家重点实验室、基础医学院组织胚胎学系刘明兮课题组与贝勒医学院Martin M.Matzuk课题组、大阪大学Masahito Ikawa课题组等共同解析了一个精子运动调节基因TCTE1,结果发表于《美国科学院院刊》(PNAS)。不孕不育困扰着15%的育龄夫妇,成为人们日益关注的健康问题。在不孕不育的发病中,约有一半来自男性,其中18%的出现了精子运动障碍,被称为弱精子症(asthenozoospermia)。因此了解精子的运动调节过程也成为了解决男性不育的一个关键。     

SPANX蛋白的研究进展及其与男性生育力的相关性

男性不育症病因十分复杂,遗传、环境、内分泌等许多因素都会导致男性不育。而现今临床上多依据精液常规分析对男性不育做出诊断和治疗,但仅依赖精液常规参数存在一定局限性。探寻男性生育力的潜在生物标志分子是当前男性不育的迫切需求。精子X染色体核结合精子蛋白(The sperm protein associated with the nucleus on the X chromosome,SPANX)是在精子中表达的一类小分子蛋白,SPANX蛋白家族基因定位在X染色体上,它随精子的成熟而迁徙,参与精子结构的形成,在精子成熟的不同时期,蛋白定位和蛋白表达均存在差异。在精液参数正常的不育男性和自发弱精症的男性中,SPANX表达下调;同时在活性氧自由基(reactive oxygen species,ROS)阴性的精子中,SPANXC表达降低,在DNA碎片率低的精子中,SPANX表达增高;这些表明SPANX与男性生育力存在一定的相关性,但其与生育力的影响极其相关机制还需要进一步的研究。     

基于高通量转录组测序技术的无精子症患者睾丸组织比较转录组分析

本研究探讨不同程度无精子症患者睾丸组织的转录组差异,了解差异表达基因(DEG)在功能、分类和代谢通路的不同,揭示无精子症患者精子发生分子机制,为促进男性不育研究的发展提供理论基础.选取1份非梗阻性无精子症和4份梗阻性无精子症患者睾丸组织样品(从无精子到有精子),进行RNA提取和文库构建,利用Illumina HiSeqTM2500高通量测序,构建无精子症患者睾丸组织转录组文库,并用生物信息学方法进行分析.结果发现,样品比对基因组数据库的平均比对率为94.38%,共检测2 242个属于预测新的蛋白质编码基因的转录本.得到差异表达基因统计结果为:NOA vs. OA1基因上调8 045,下调1 150;OA1 vs. OA2基因上调1 538,下调420;OA2 vs. OA3基因上调1 275,下调1 690;OA3 vs. OA4基因上调1 834,下调1 853.比较5例无精子症睾丸组织的差异基因KEGG,主要富集在RNA降解通路、基底细胞瘤通路、癌通路、黑色素生成通路和调节干细胞多能性等信号通路. PRM1、PRM2、TNP1、UBXN6、CXCL16、NUPR2、CCDC136和CRISP2等基因的表达呈递增趋势,并具有时序特异性.此外,5例无精子症睾丸组织的表达基因有不同程度的基因融合.综上,基因融合可能和无精子症相关,并且不同程度无精子症患者睾丸组织的差异表达基因数量、功能、分类和代谢通路不同.本研究筛选出精子发生、精子运动等差异表达基因,丰富了无精子症患者睾丸组织转录组信息,为开展无精子症患者睾丸组织相关基因及分子调控机制的研究奠定基础.     

严重寡精症ICSI精子供体的DAZ基因拷贝缺失研究

DAZ基因拷贝缺失与人类的生精障碍有关。为了解中国正常生精男性和ICSI中严重寡精症精子供体DAZ基因拷贝缺失的分布, 探讨DAZ基因拷贝数检测在严重生精障碍精子供体遗传缺陷筛查中的意义, 本研究运用多重PCR和PCR-RFLP技术, 对128例严重寡精症ICSI精子供体和287个正常生精男性的DAZ基因缺失进行了研究。发现DAZ1/DAZ2、DAZ3/DAZ4和全部4个拷贝缺失等3种拷贝缺失类型, 其中全部4个拷贝缺失仅见于严重寡精症患者, 频率为11.7%; DAZ1/DAZ2缺失的频率在严重寡精症患者中显著高于正常男性(9.4% vs 2.8%, P = 0.004); 在严重寡精症患者中DAZ基因拷贝完全缺失与DAZ1/DAZ2缺失的总发生率为21.1%。DAZ3/DAZ4缺失的频率在两组人群中无显著差异(7.0% vs 3.8%, P > 0.05)。这些结果提示, DAZ基因全部拷贝缺失是严重寡精症患者生精障碍的常见遗传病因, 而DAZ1/DAZ2缺失则可能是一种高风险因素。鉴于上述DAZ基因缺失在严重生精障碍精子供体中较高的发生率, 在应用ICSI进行辅助生育前, 建议对严重寡精症的精子供体进行DAZ基因全缺失与DAZ1/DAZ2共缺失筛查, 以评估其男性后代患病的风险。     

不明原因男性不育人群中睾丸特异性乳酸脱氢酶基因突变的发现及其意义

为了研究睾丸特异性乳酸脱氢酶,即乳酸脱氢酶C4(LDH-C4)基因突变在男性不育发病中的作用,利用LDH-C4特异性底物对100名不明原因男性不育症患者的精子LDH-C4进行活性显色,用变性高效液相色谱(DHPLC)技术对LDH-C4活性低下的患者进行LDHC基因PCR产物的突变筛查,对DHPLC峰形异常的PCR产物进行序列测定.筛选到一组精子LDH-C4活性明显下降的患者,其中1名患者的LDHC基因PCR产物在DHPLC中呈异常洗脱峰.对这一PCR产物进行序列测定,发现患者LDHC基因第5外显子的115位碱基发生了T→A的杂合改变(GenBank登录号GU479375),该突变使LDHC基因的178位密码子由原来的TTG(编码亮氨酸)变为TAG(终止密码子),形成截短的C亚基.T克隆-测序进一步证实了该无义突变的杂合状态.这是在人类LDHC基因上发现的第一个突变,提示LDHC基因突变可能是男性不育发病的原因之一.     

Towards an understanding of the genetics of human male infertility: lessons from flies

It has been argued that about 4–5% of male adults suffer from infertility due to a genetic causation. From studies in the fruitfly Drosophila, there is evidence that up to 1500 recessive genes contribute to male fertility in that species. Here we suggest that the control of human male fertility is of at least comparable genetic complexity. However, because of small family size, conventional positional cloning methods for identifying human genes will have little impact on the dissection of male infertility. A critical selection of well-defined infertility phenotypes in model organisms, combined with identification of the genes involved and their orthologues in man, might reveal the genes that contribute to human male infertility.     

Génétique de l’infertilité chez l’homme, nouvelles approches

15% of couples worldwide present with reproduction difficulties related to infertility. To date, very few genetic causes have been associated with male or female infertility. The identification of single-gene mutations causing male infertility is not a field of intense research at the present time, although they are probably responsible for a large number of so-called idiopathic cases of infertility. Murine models were created several years ago by gene knock-out by genetic recombination: more than 200 genes have been shown to be responsible for isolated syndromic infertility. This is the case for genes controlling meiosis. The course of meiosis and the genes associated with this process have been largely characterized in yeasts. Mammalian homologues were recently cloned and knocked out in mice, demonstrating their essential roles during meiosis and gametogenesis. The gonadal phenotype of these mutant animals is similar to that of certain patients with unexplained infertility. The search for possible mutations in meiosis genes, genes that have been highly preserved during evolution, is currently underway. These murine models are very useful to study and dissect the various steps of normal and pathological gametogenesis in mammals. This progress should lead, in the near future, to more precise diagnosis and therefore informed genetic counselling in these infertile couples.     

A single nucleotide polymorphism within the novel sex-linked testis-specific retrotransposed PGAM4 gene influences human male fertility

BACKGROUND: The development of novel fertilization treatments, including in vitro fertilization and intracytoplasmic injection, has made pregnancy possible regardless of the level of activity of the spermatozoa; however, the etiology of male-factor infertility is poorly understood. Multiple studies, primarily through the use of transgenic animals, have contributed to a list of candidate genes that may affect male infertility in humans. We examined single nucleotide polymorphisms (SNPs) as a cause of male infertility in an analysis of spermatogenesis-specific genes. METHODS AND FINDING: We carried out the prevalence of SNPs in the coding region of phosphoglycerate mutase 4 (PGAM4) on the X chromosome by the direct sequencing of PCR-amplified DNA from male patients. Using RT-PCR and western blot analyses, we identified that PGAM4 is a functional retrogene that is expressed predominantly in the testes and is associated with male infertility. PGAM4 is expressed in post-meiotic stages, including spermatids and spermatozoa in the testes, and the principal piece of the flagellum and acrosome in ejaculated spermatozoa. A case-control study revealed that 4.5% of infertile patients carry the G75C polymorphism, which causes an amino acid substitution in the encoded protein. Furthermore, an assay for enzymatic activity demonstrated that this polymorphism decreases the enzyme's activity both in vitro and in vivo. CONCLUSION: These results suggest that PGAM4, an X-linked retrogene, is a fundamental gene in human male reproduction and may escape meiotic sex chromosome inactivation. These findings provide fresh insight into elucidating the mechanisms of male infertility.     

Genetic variants in antioxidant genes are associated with sperm DNA damage and risk of male infertility in a Chinese population

To test the hypothesis that polymorphisms in antioxidant genes are more susceptible to sperm DNA damage and male infertility, we examined 11 single-nucleotide polymorphisms from six antioxidant genes (GPX1, CAT, PON1, NQO1, SOD2/MnSOD, and SOD3) in 580 infertility cases and 580 controls from a Chinese population-based case-control study (NJMU Infertility Study). Genotypes were determined using the OpenArray platform. Sperm DNA fragmentation was detected using the Tdt-mediated dUTP nick-end labeling assay, and the level of 8-hydroxydeoxyguanosine (8-OHdG) in sperm DNA was measured using immunofluorescence. The adjusted odds ratio and 95% confidence interval (CI) were estimated using unconditional logistic regression. The results indicated that the PON1 Arg192Glu (rs662) and SOD2 Val16Ala (rs4880) variant genotypes were associated with a significantly higher risk of male infertility. In addition, subjects carrying variant genotypes of both loci had a twofold (95% CI, 1.42-2.90) increase in the risk of male infertility, indicating a significant gene-gene interaction between these two loci (P for multiplicative interaction=0.045). Moreover, linear regression analysis showed that individuals carrying the PON1 Arg192Glu (rs662) or SOD2 Val16Ala (rs4880) variants have significantly higher levels of sperm DNA fragmentation and 8-OHdG. These data suggest that genetic variations in antioxidant genes may contribute to oxidative sperm DNA damage and male infertility.     

Anomalies génétiques et infertilité masculine

Fifteen percent of couples are infertile and in about 50% of cases the cause is of male origin. The aetiology is still unknown in more than 90% of cases and there may be genetic or environmental causes. Three approaches are used to detect genetic causes for male infertility: 1) cytogenetics, resulting in particular from progress made in molecular cytogenetics and the direct analysis of gametes by in situ molecular hybridation techniques. When a chromosome anomaly, the most common cause of infertility, including deletion of the Y chromosome, is discovered, it is not easy to distinguish between gene anomalies resulting from change and mechanical anomalies that are an integral part of meiosis; 2) the analysis of candidate genes, which often uses data obtained from animal, usually murine, models. This approach, frequently described in the literature, tends to be lengthy, expensive and rarely results in the discovery of an abnormal gene, as is the case, for example, with meiotic genes; 3) Mendel’s approach is clearly the preferred choice, studying as it does cases of inherited infertility, which is much more widespread than we might think.     

Anomalies génétiques de la spermatogenèse

The important role of genetic abnormalities in the causation of human male infertility is increasingly recognized. Considerable progress has been achieved over the past years both in the clinical delineation of genetic forms of male infertility and in the characterization of the responsible genes and their mutations. We review the current state of knowledge on genetic disorders where male infertility is a major and regular feature.     

＂Micro-deletions＂ of the human Y chromosome and their relationship with male infertility

The Y chromosome evolves from an autochromosome and accumulates male-related genes including sex-determining region of Y-chromosome (SRY) and several spermatogenesis-related genes.The human Y chromosome (60 Mb long) is largely composed of repeti-tive sequences that give it a heterochromatic appearance,and it consists of pseudoautosomal,euchromatic,and heterochromatic regions.Located on the two extremities of the Y chromosome,pseudoautosomal regions 1 and 2 (PAR1 and PAR2,2.6 Mb and 320 bp long,re-spectively) are homologs with the termini of the X chromosome.The euchromatic region and some of the repeat-rich heterochromatic parts of the Y chromosome are called "male-specific Y" (MSY),which occupy more than 95% of the whole Y chromosome.After evolu-tion,the Y chromosome becomes the smallest in size with the least number of genes but with the most number of copies of genes that are mostly spermatogenesis-related.The Y chromosome is characterized by highly repetitive sequences (including direct repeats,inverted repeats,and palindromes) and high polymorphism.Several gene rearrangements on the Y chromosome occur during evolution owing to its specific gene structure.The consequences of such rearrangements are not only loss but also gain of specific genes.One hundred and fifty three haplotypes have been discovered in the human Y chromosome.The structure of the Y chromosome in the GenBank belongs to haplotype R1.There are 220 genes (104 coding genes,111 pseudogenes,and 5 other uncategorized genes) according to the most recent count.The 104 coding genes encode a total of about 48 proteins/protein families (including putative proteins/protein families).Among them,16 gene products have been discovered in the azoospermia factor region (AZF) and are related to spermatogenesis.It has been dis-covered that one subset of gene rearrangements on the Y chromosome,"micro-deletions",is a major cause of male infertility in some populations.However,controversies exist about different Y chromosome haplotypes.Six AZFs of the Y chromosome have been discov-ered including AZFa,AZFb,AZFc,and their combinations AZFbc,AZFabc,and partial AZFc called AZFc/gr/gr.Different deletions in AZF lead to different content spermatogenesis loss from teratozoospermia to infertility in different populations depending on their Y hap-lotypes.This article describes the structure of the human Y chromosome and investigates the causes of micro-deletions and their relation-ship with male infertility from the view of chromosome evolution.After analysis of the relationship between AZFc and male infertility,we concluded that spermatogenesis is controlled by a network of genes,which may locate on the Y chromosome,the autochromosomes,or even on the X chromosome.Further investigation of the molecular mechanisms underlying male fertility/infertifity will facilitate our knowledge of functional genomics.     

Detection of Y chromosome microdeletions and mitochondrial DNA mutations in male infertility patients

Infertility affects about 10-15% of all couples attempting pregnancy with infertility attributed to the male partner in approximately half of the cases. Proposed causes of male infertility include sperm motility disturbances, Y chromosome microdeletions, chromosomal abnormalities, single gene mutations, and sperm mitochondrial DNA (mtDNA) rearrangements. To investigate the etiology of decreased sperm fertility and motility of sperm and to develop an appropriate therapeutic strategy, the molecular basis of these defects must be elucidated. In this study, we aimed to reveal the relationships between the genetic factors including sperm mtDNA mutations, Y chromosome microdeletions, and sperm parameters that can be regarded as candidate factors for male infertility. Thirty men with a history of infertility and 30 fertile men were recruited to the study. Y chromosome microdeletions were analyzed by multiplex PCR. Mitochondrial genes ATPase6, Cytb, and ND1, were amplified by PCR and then analyzed by direct sequencing. No Y chromosome microdeletions were detected in either group. However, a total of 38 different nucleotide substitutions were identified in the examined mitochondrial genes in both groups, all of which are statistically non-significant. Fifteen substitutions caused an amino acid change and 12 were considered novel mutations. As a conclusion, mtDNA mutations and Y chromosome microdeletions in male infertility should be examined in larger numbers in order to clarify the effect of genetic factors.     

Association of T/A polymorphism in miR-1302 binding site in CGA gene with male infertility in Isfahan population

Infertility occurs in 10–15% of couples worldwide and close to half of it is caused by male factors. One of the genes that can affect male infertility is CGA. Polymorphisms in CGA gene may affect gene expression, therefore affecting male infertility by disrupting the regulation of this gene. One of the polymorphisms is the substitution of T with A in the miR-1302 binding site in the 3′ untranslated region of the CGA gene. In this study, we explored this polymorphism in Isfahan population. In this case-control study, by the use of Tetra primer-ARMS–PCR technique, rs6631 has been investigated in 224 infertile men and 196 controls. Infertile men were recruited from Isfahan Fertility and Infertility Center. Analysis of genotype and allele frequencies indicated that the differences between case and control populations were significant for rs6631 because P?=?0.00 which is above the threshold. We found a significant relationship between this polymorphism and male infertility. This study which performed for the first time in Iran suggests that polymorphism in CGA gene can affect male infertility. Also, this polymorphism has high heterozygosity, so it can be used for further studies in different populations.     

Genetics of male infertility: the new players

Approximately 10-15% of couples experience infertility and male factors contribute to half of these cases. It was usually thought that infertility cannot be transmitted, but accumulating evidence indicates that many cases are indeed caused by genetic defects, some inherited. The use of single nucleotide polymorphisms (SNP) arrays allowing to genotype the totality of the genome recently led to identify several genes which, when mutated, generate specific infertility phenotypes. With the tremendous progresses in high throughput sequencing techniques, we can expect many more new genes involved in fertility to be identified in the next years. For the patients concerned, these findings mean the possibility of an accurate diagnosis and improved prognosis. Furthermore, these data will lead to a better understanding of the molecular mechanisms underlying spermatogenesis and thus should contribute to identify and offer new therapeutic strategies for the treatment of infertility.