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。     

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。     

男性不育与Y染色体

目的:观察精子数目异常与小Y染色体及内分泌性腺激素水平。方法:对262名少精及无精症患者检测染色体,并对其中11例小Y染色体及随机抽取的15例Y染色体正常的患者运用磁性分离酶免疫测定法分别检测性腺激素。结果:小Y染色体检出率为4.19%(11/262),其内分泌性腺激素均呈高卵泡刺激素、高黄体生成素和低睾酮水平,与Y染色体正常的无精及少精症患者相比较,差异有显著性(P<0.05)。而小Y染色体不同精子数组各内分泌性腺激素比较,差异无显著性(P>0.05)。结论:精子数目异常可能与小Y染色体有关,小Y染色体基因改变可能是导致其内分泌性腺激素的变化因素。     

Y染色体单倍群与西南地区男性生精障碍的相关性

男性不育中, 原发无精、少精是最为重要的因素之一, 核型异常和无精子症因子(Azoospermia factor, AZF)微缺失能解释部分原发无精、少精的原因, 然而还有许多致病因素尚不清楚。Y染色体作为男性特有的染色体, 与男性生殖系统的正常功能密切相关。文章主要对Y染色体单倍群这一分子遗传背景与男性原发无精、严重少精症之间是否存在相关性进行探讨, 为进一步探索原发无精、严重少精症的遗传学致病原因提供依据和可行的方向。采集265名生精障碍患者(原发无精症患者193名, 原发严重少精症患者72名)以及193名正常男性样本的外周血, 进行核型分析和AZF缺失分析, 以排除有此两类异常的样本。将经过筛选的样本进行Y染色体单倍群分析, 并对其单倍群分布情况进行统计分析。分析显示, 生精障碍组和对照组分别在D1*、F*、K*、N1*和O3* 上有显著性差异(P=0.032, 0.022, 0.009, 0.009, 0.017, <0.05)。Y染色体单倍群, 这一Y染色体遗传背景与男性原发生精障碍的发生有相关性。     

＂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.     

用双色荧光愿位杂交和PCR技术鉴定无精症患者标记染色体的来源

目的：为了研究无精症患标记染色体的来源,对无精症患进行明确的遗传学诊断。方法：应用双色FISH技术和PCR技术对2例无精症患进行分子细胞遗传学和分子遗传学检测。结果：确定了两例特发性无精症患的标记染色体均来源于Y染色体,其核型为46,X,ishdel(Y)(q11)(DYZ3 ,DXZ1-)。结论：FISH技术结合PCR技术,是鉴定标记染色体来源的又一非常重要方法,对于无精症患在进行胞质内单精子注射（ICSI）或其它治疗之前,完成遗传咨询和明确的遗传学诊断也是特别必须的。     

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。     

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。     

白消安致大鼠无精子症模型的建立

目的:利用白消安建立SD(Sprague-Dawley)大鼠少精子症动物模型,为治疗人类男性不育症提供实验依据。方法:本研究取6周龄左右SD大鼠随机分为对照组和实验组,实验组通过腹腔注射不同浓度白消安(15、20、30、40、60 mg/kg体重),每个浓度分单次注射和连续10次累计注射两种方式,注射后每隔10d随机取样检测精子浓度、活力、畸形率以及曲细精管内部结构各项指标,30d出现弱精少精等不育症状后,第40d后停止取样,观察白消安对大鼠精子的作用效果。结果:单次注射15-60mg/kg剂量白消安都会使实验大鼠不同程度死亡,连续10 d累计注射剂量15 mg/kg白消安可破坏大鼠睾丸组织的部分精原干细胞,连续10 d累计注射总剂量20 mg/kg是较为理想的实验浓度,可消除全部精原干细胞和大部分支持细胞,30、40、60 mg/kg可致大鼠死亡或睾丸病变充血。结论:白消安对大鼠生殖细胞有毒害作用,通过腹腔注射一定浓度白消安可建立大鼠少精子症模型,可为外源干细胞体内转分化为生殖细胞提供准确的移植时间和实验依据。     

Screening for microdeletions in human Y chromosome--AZF candidate genes and male infertility

About 30% of couple infertilities are of male origin, some of them caused by genetic abnormalities of the Y chromosome. Deletions in AZF region can cause severe spermatogenic defects ranging from non-obstructive azoospermia to oligospermia. The intracytoplasmatic sperm injection technique (ICSI) is rapidly becoming a versatile procedure for human assisted reproduction in case of male infertility. The use of ICSI allows Y chromosome defects to be passed from father. The goal of our study is to evaluate the frequency of microdeletions in the long arm of Y chromosome, within the AZF regions, in these cases of infertilities, using molecular genetics techniques. Thirty infertile men with azoospermia or oligozoospermia, determined by spermogram, were studied after exclusion of patients with endocrine or obstructive causes of infertility. Peripheral blood DNA was extracted from each patient, then amplified by multiplex PCR with STS genomic markers from the Y chromosome AZF zones. Each case was checked by multiplex PCR through coamplification with the SRY marker. Three men with microdeletions of the long arm of the Y chromosome were diagnosed among the 30 patients, corresponding to a proportion of 10%. The relatively high proportion of microdeletions found in our population suggest the need for strict patient selection to avoid unnecessary screening for long arm Y chromosome microdeletions. The molecular diagnostics was performed according to the current European Academy of Andrology laboratory guidelines for molecular diagnosis of Y chromosomal microdeletions.     

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.     

Embryo outcome in Y-chromosome microdeleted infertile males after ICSI

A prospective study involving 118 infertile Japanese couples to assess the embryo outcomes in both azoospermic and oligoasthenoteratoazoospermic (OAT) patients with Y-chromosome microdeletion. The men were divided into two groups; azoospermia (n = 27), and OAT, sperm concentration <5 x 10(6)/ml (n = 91). They were investigated for Y-chromosome microdeletions by a polymerase chain reaction (PCR) amplification of the Y-chromosome-specific sequence tag site (STS). The embryo outcomes of patients found to have Y-microdeletion were determined. The frequency of microdeletion was 8.8% (9) and two had microdeletions distal to DAZ. The mean fertilization rate and the cleavage rate in the eight cycles of both azoospermic and oligospermic patients were 59.3 and 87.5%, respectively. The percentages of grade 1 & 2 embryos, > or =6 cells embryos, and blastocyts were 51.7, 65.6, and 45.3%, respectively. Three pregnancies resulted from the eight cycles (37.5%). CONCLUSION: in Y-chromosome microdeletion cycles in which sperm cells were available for intracytoplasmic sperm injection (ICSI), embryo outcome was comparable to conventional IVF.     

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.     

Two DNA sequences specific for the canine Y chromosome

Data are presented on the characterization of two nucleotide sequences found exclusively in the DNA of male dogs. In polymerase chain reactions (PCRs) of canine genomic DNA with a decanucleotide primer of arbitrary sequence (OP-W17), two nucleotide segments (650 and 990 bp) were amplified only from male samples, whereas a number of other fragments between 400 and 2500 bp in size were amplified from both male and female samples. The two male-specific segments were cloned and sequenced, and terminal 24mer oligonucleotide primer pairs were synthesized. PCR with these specific primer pairs resulted in amplification of the two male-specific sequences only from DNA samples of 34 male dogs; no product was amplified from 42 samples of females. A segment of the SRY gene previously localized on the Y chromosome could be amplified in DNA samples that had the two new sequences. Eco RI digested genomic male DNA when hybridized with the 650 bp or the 990 bp sequences, resulted in a single band for each on Southern analysis; DNA from females did not yield any bands. Comparisons between the two new sequences and the SRY gene segment revealed no homologies. We concluded that the two new sequences are specific for the canine Y chromosome and do not contain the short characterized segment of the SRY gene.     

Molecular screening for Yq microdeletion in men with idiopathic oligozoospermia and azoospermia

Infertility affects 15% couples attempting pregnancy and in 40–50% of these cases the male partner has qualitative or quantitative abnormalities of sperm production. Microdeletions in the azoospermia factor (AZF) region on the long arm of the Y chromosome are known to be associated with spermatogenic failure and have been used to define three regions on Yq (AZFa, AZFb and AZFc) which are critical for spermatogenesis and are recurrently deleted in infertile males. Semen analysis was carried out on one hundred and twenty five infertile males with oligozoospermia and azoospermia. Cytogenetic analysis was done for all the cases and in all cytogenetically normal cases (n = 83) microdeletion analysis was carried out on DNA extracted from peripheral blood using PCR. The sequence tagged sites (STS) primers sY84, sY86 (AZFa); sY127, sY134 (AZFb); sY254, sY255 (AZFc) were used for each case. Eight of the eighty three cases (9.63%) showed deletion of at least one of the STS markers. Correlation of phenotype with microdeletion was done in each case to determine any phenotype association with deletion of particular AZF locus. Based on the present study, the frequency of microdeletion in the Indian population is 9.63%. This study emphasizes the need for PCR analysis for determining genetic aetiology in cases with idiopathic severe testiculopathy.