常染色体DAZL基因单核苷酸多态性与男性不育相关性研究

目的:探讨常染色体DAZL基因单核苷酸多态性(SNP)与男性不育的关系。方法:收集东北地区男性不育症患者(不育组,n=144)和健康并已育有一子女的男性(生育组,n=53)精液标本,不育组按照WHO(1999)少、弱和畸形精子分类标准分成少弱畸形精子症组(n=17)、少弱精子症组(n=33)、弱畸形精子症组(n=13)、弱精子症组(n=54)和非少弱畸形精子不育症组(n=27),应用聚合酶链反应-限制性片段长度多态性分析(PCR-RFLP)筛选SNP260多态性,应用聚合酶链反应-单链构象多态性分析(PCR-SSCP)筛选SNP386多态性,并测序验证和进行统计学分析。结果:不育组及生育组中均检测到SNP260A→G多态性,统计学分析差异无显著性(P>0.05),但在少弱畸形精子症组SNP260AG基因型比例大于其他各组;各研究组均未发现SNP386多态性。结论:SNP260和SNP386多态性与中国东北地区男性不育无相关性。前者AG基因型与少弱畸形精子的关系尚需进一步证实;后者多态性可能仅分布在台湾的一些狭小地区,两者均不能作为东北地区男性不育基因诊断的分子标志。     

浙江地区无精子症和少弱精子症患者DAZL基因A260G和A386G单核苷酸多态性分析

目的:探讨浙江地区人群中DAZL基因A260G和A386G单核苷酸多态性(SNP)与无精子症和少弱精子症的关系。方法:收集浙江地区无精子症和少弱精子症患者317例和正常生育男性246例外周血标本,利用SNa Pshot SNP分型技术对样本DAZL基因Q260G和A386G多态性位点进行分型。结果:DAZL基因A260G在浙江地区汉族人群中具有多态性,其基因频率与基因型频率分布在病例组和对照组中均符合Hardy-Weinberg平衡。AA、AG和GG的基因型频率在对照组中分别为92.3%、7.3%和0.4%,而病例组中分别为94.3%、5.7%和0%,统计学分析无显著性差异(P=0.430,OR=0.780,95%CI=0.413~1.46)。A386G突变只在正常对照组中出现1例杂合子AG,两组中都没有发现纯合子GG突变,两组之间亦无显著性差异(P=0.259,OR=0.698,59%CI:0.374~1.306)。结论:DAZL基因Q260G和A386G多态性与中国浙江地区无精子症和少弱精子症无相关性,两者都不能作为无精子症和少弱精子症遗传诊断的分子标志。     

DAZL基因多态性位点(A260G、A386G)与无精子症和少精子症的关联性Meta分析

目的:探讨DAZL基因多态性位点(A260G,A386G)与无精子症和少精子症导致男性不育的关联性。方法:计算机检索Pub Med、Science direct、Wiley online library、中国知网、维普中文科技期刊数据库、万方数据库,查找并筛选出研究DAZL基因多态性位点A260G和A386G与男性不育关系的病例对照研究,同时查阅检索结果中所附相似文献及参考文献,检索时限均为各数据库建库至2013年11月30日。由2名评价员单独进行文献筛选及资料提取,采用Stata SE12.0软件进行Meta分析以及其他相关统计学分析。结果:共纳入文献13篇(A260G位点多态性10篇,A386G位点多态性11篇);男性不育患者共2 715例,其中少精子症或无精子症患者共2 500例,健康对照1 835例。Meta分析结果显示,DAZL A260G位点多态性在等位基因模型、显性基因模型、隐性基因模型、共显性基因模型、超显性基因模型差异无统计学意义(P0.05)。而DAZL A386G位点多态性的分析表明,亚洲人中DAZL A386G位点多态性与少精子症或无精子症在等位基因模型、显性基因模型、共显性基因模型(AA/AG)和超显性基因模型下整体效应差异有统计学意义:等位基因模型优势比(OR)=0.15,95%可信区间(CI)0.07~0.34,P0.05;显性基因模型OR=0.16,95%CI 0.07~0.35,P0.05;共显性基因模型(AA/AG)OR=0.15,95%CI0.06~0.33,P0.05;超显性基因模型OR=0.15,95%CI 0.06~0.33,P0.05。地区亚组分析结果显示,中国A386G位点多态性与少精子症或无精子症患者在等位基因模型、显性基因模型和超显性基因模型下总效应差异有统计学意义:等位基因模型OR=0.11,95%CI 0.04~0.28,P0.05;显性基因模型OR=0.11,95%CI 0.04~0.28,P0.05;共显性基因模型(AA/AG)OR=0.09,95%CI 0.03~0.26,P0.05;超显性基因模型OR=0.09,95%CI 0.03~0.26,P0.05。结论:DAZL A260G位点多态性与精子生成或精子数量下降无相关性,而DAZL A386G位点多态性与精子生成或精子数量下降有相关性,而在地区亚种分析和人种亚组分析中这种相关性只在中国存在,因此探求DAZL A260G和A386G基因多态性与无精子症或少精子症导致的男性不育之间的相关性需要更多高质量的研究。     

PRM1基因SNP位点-190C-＞A多态性对精子畸形率的影响

目的:探讨鱼精蛋白基因1(protamine 1,PRM1)单核苷酸多态性(SNP)与畸形精子症的关系。方法:收集畸形精子症不育患者(病例组,n=157)和精子形态正常男性(对照组,n=37)精液样本,进行形态学分析并提取基因组DNA,应用Sequenom MassARRAY SNP分型技术对PRM1基因-190C->A SNP位点(rs2301365)进行基因分型,比较病例组与对照组基因型的分布差异及病例组不同基因型间精子形态参数的差异。结果:病例组中,基因型CC、CA、AA的分布频率及个体数分别为38.9%(61)、44.6%(70)、16.6%(26),对照组为45.9%(17)、51.4%(19)、2.7%(1),病例组AA基因型分布频率显著高于对照组(P<0.05)。病例组等位基因C、A的分布频率分别为57.6%、42.4%,对照组为71.6%、28.4%,病例组等位基因A的频率与对照组差异无显著性(P>0.05)。病例组基因型CC与CA、AA、CA+AA在精子形态学参数的比较,差异无统计学意义(P>0.05)。结论:PRM1基因SNP位点-190C->A可能与中国汉族畸形精子症男性不育存在相关,该位点可能导致精子形态异常,但所致形态异常并无部位上的特异性。     

精子Tektin-2基因单核苷酸多态性与特发性弱精子症的相关性研究

目的探讨Tektin-2基因的单核苷酸多态性(SNP)位点rs12043423与特发性弱精子症的相关性。方法采用病例对照法,随机选取特发性弱精子症患者192例作为弱精子症组,另募集同期208例精子活力正常的不育男性作为不育症组和213例精液正常的已生育男性作为正常对照组,所有研究对象均进行精液分析,对三组患者Tektin-2基因的SNP位点rs12043423进行基因分型,比较三组间的基因型和等位基因频率,并且进行与特发性弱精子症的关联分析。结果(1)弱精子症组Tektin-2基因的SNP位点rs12043423的CC基因型频率显著低于正常对照组及不育症组,TT基因型频率则显著增加(P<0.05),而CT基因型频率在三组间的分布频率无显著性差异(P>0.05)。弱精子症组C等位基因的分布频率显著低于正常对照组和不育症组,而T等位基因的频率显著高于正常对照组和不育症组(P<0.05)。不育症组和正常对照组比较,不同基因型的分布频率及等位基因的频率在两组间均无显著性差异(P>0.05)。(2)弱精子症组与正常对照组比较,Tektin-2基因突变(杂合子[CT]和纯合子[TT])的发生率为61.5%vs.50.2%,TT基因型与弱精子症的风险因素分析结果为[OR=1.968,95%CI(1.041,3.723),P=0.035];弱精子症与不育症组比较,Tektin-2基因突变(CT+TT)的发生率为61.5%vs.51.5%,TT基因型与弱精子症的风险因素分析结果为[OR=1.918,95%CI(1.014,3.630),P=0.043]。结论Tektin-2基因rs12043423的多态性位点TT基因型和T等位基因增加特发性弱精子症的易感性,在特发性弱精子症的发展中可能是危险因素。     

葡萄糖调节蛋白78基因3'UTR多态性与男性弱精子症的相关性研究

目的:探究葡萄糖调节蛋白78(GRP78)基因3'UTR单核苷酸多态性(rs12009、rs1140763和rs16927997位点)与弱精子症的关系。方法:选取400例弱精子症不育男性为弱精子症组,400例有生育史的健康男性为对照组,运用多重单碱基延伸PCR(SNa Pshot)对所有研究对象GRP78基因的rs12009、rs1140763和rs16927997位点进行基因分型,再分析此3个位点多态性与男性弱精子症的相关性。结果:弱精子症组和对照组的前向运动精子百分率分别为(20.09±8.18)%、(57.16±13.45)%,两者差异具有统计学意义(P0.01)。GRP78基因rs12009和rs1140763存在CC、CT、TT 3种基因型和C、T 2种等位基因;rs16927997存在GG、GA、AA 3种基因型和G、A 2种等位基因。在弱精子症组中,rs12009位点C、T等位基因频率分别为47.3%、52.7%;rs1140763位点C、T等位基因频率分别为52.0%、48.0%;rs16927997位点G、A等位基因频率分别为4.4%、95.6%。而在对照组中,rs12009位点C、T等位基因频率分别为44.3%、55.7%;rs1140763位点C、T等位基因频率均为50.0%;rs16927997位点G、A等位基因频率分别为6.0%、94.0%。3个位点基因型及等位基因频率在弱精子症组和对照组中的分布差异均无统计学意义(P0.05);进一步单倍型分析,发现在弱精子症组和对照组中主要为C-C-A、T-C-G、T-T-A 3种单倍型,但也未发现单倍型与男性弱精子症存在相关性。结论:GRP78基因3'UTR多态性与男性弱精子症的发病风险不存在相关性。     

谷胱甘肽S-转移酶基因遗传多态性与特发性男性不育症相关性研究

目的该研究旨在探讨谷胱甘肽S-转移酶基因(GST)M1、T1及P1基因多态性与特发性男性不育症的相关性。方法该病例-对照研究包括246例特发性少弱精子症男性不育患者及117例正常健康有生育史男性对照。采用聚合酶链反应(PCR)、聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法分别对GSTM1、GSTT1和GSTP1基因进行分型。结果 GSTM1基因缺失型在正常对照组和特发性少弱精子症不育患者组基因型频率分别为41.88%和60.57%,具有显著差异(P=0.001);GST T1基因缺失型在正常对照组和特发性少弱精子症不育患者组基因型频率分别为47.86%和62.60%,具有显著差异(P=0.008);GSTM1/T1基因缺失型在正常对照组和特发性少弱精子症不育患者组基因型频率分别为14.53%和38.62%,具有显著差异(P0.001);GSTP1基因突变型在正常对照组和特发性少弱精子症不育患者组基因型频率分别为27.35%和32.11%,无明显差异(P=0.847)。结论GSTM1、GSTT1基因缺失型分别是特发性男性不育症的危险因素。GSTP1基因突变型与特发性男性不育症患者无明显相关性。     

186例少弱精子症患者MTHFR C677T基因多态性观察

目的观察186例少弱精子症男性不育患者的亚甲基四氢叶酸还原酶(MTHFR)C677T基因多态性的分布及频率。方法以186例少弱精子症的男性不育患者为不育组,同期就诊的有正常生育史和精液参数正常的男性131例为对照组,比较两组男性MTHFR C677T基因多态性的分布及频率差异。结果不育组中MTHFR基因CC基因型分布频率显著低于对照组(36.6%vs.55.0%,P<0.05),TT型分布频率则显著高于对照组(20.4%vs.13.7%,P<0.05),T等位基因的分布频率亦显著高于对照组(41.9%vs.29.4%,P<0.05)。结论 MTHFR基因C677T多态性可能与男性少弱精子症的发生相关,可能是造成男性不育的一个遗传学因素。但因本研究纳入的观察对象数量有限,结论存在一定的局限性,后续尚需更深入的研究探讨。     

KITLG基因rs995030和rs4474514位点单核苷酸多态性与男性不育相关性研究

目的:探讨酪氨酸激酶受体的特异性配体(KITLG)基因rs995030和rs4474514位点单核苷酸多态性与男性不育之间的相关性。方法:收集南京周边地区特发性男性不育患者360例(病例组),已生育的健康男性338例(对照组);按照WHO《人类精液检查与处理实验室手册》第5版,将病例组分为无精子症组(n=143)、严重少精子症组(n=159)和少精子症组(n=58)。收集所有研究对象的临床基本资料,并采集病人外周血提取基因组DNA,用Sequence Mass-Array技术检测KITLG基因rs995030和rs4474514位点的基因型,通过Logistic回归分析两位点基因多态性与男性不育的相关性。结果:病例组与对照组比较,精子浓度[(13.23±24.52)×10~6/ml vs(78.74±61.25)×10~6/ml]、前向运动精子百分率[(18.71±15.19)%vs(39.36±9.75)%]、FSH[(16.09±17.31) IU/L vs(4.56±2.41) IU/L]差异显著(P0.01)。经Logistic回归分析,未发现各基因型与男性不育有统计学意义的相关性,亚组分析也未发现相关性。结论:KITLG基因rs995030和rs4474514位点的单核苷酸多态性与男性不育无显著相关性,后续可以通过扩大样本量以及样本选取范围来进一步研究验证。     

TP53基因rs1042522单核苷酸多态性与男性不育相关性研究

目的:探讨肿瘤蛋白p53(TP53)基因rs1042522位点单核苷酸多态性与男性不育的关系。方法:采用病例-对照研究,收集南京地区特发性男性不育患者380例(病例组),有生育史的健康男性398例(对照组);并将病例组分为无精子症组(n=140)和少精子症组(n=240)。用Sequence Mass Array技术检测TP53基因rs1042522位点的基因型,通过Logistic回归分析该位点多态性与男性不育的相关性。结果:病例组与对照组前向运动精子百分率[(10.38±5.57)%vs(42.55±9.57)%]、精子浓度[(13.13±24.96)×106/ml vs(77.34±49.24)×106/ml]、T[(14.07±5.36)nmol/L vs(11.89±4.50)nmol/L]、FSH[(16.80±18.20)U/L vs(4.55±7.17)U/L]存在显著性差异(P0.05)。经Logistic回归分析,未发现各基因型与男性不育有统计学意义的相关性,亚组分析也未发现相关性。结论:TP53基因rs1042522位点的单核苷酸多态性与男性不育无显著相关性。     

A to G transitions at 260, 386 and 437 in DAZL gene are not associated with spermatogenic failure in Indian population

The autosomal DAZL (Deleted-in-Azoospermic-Like) gene, mapped to the short arm of the human chromosome 3, is the precursor for the Y-chromosomal DAZ cluster, which encodes for putative RNA-binding proteins. Mutations in the DAZL have been reported to be associated with spermatogenic failure in Taiwanese population but not in Caucasians. As there was no study on Indian populations, we have analysed the entire coding sequences of exons 2 and 3 of DAZL in a total of 1010 men from Indian subcontinent, including 660 infertile men with 598 non-obstructive azoospermia, 62 severe oligozoospermia and 350 normozoospermic fertile control men, to investigate whether mutation(s) in the DAZL is associated with male infertility. Interestingly, none of our samples (1010) showed A386G (T54A) mutation, which was found to be associated with spermatogenic failure in Taiwanese population. In contrast, A260G (T12A) mutation was observed in both infertile and normozoospermic fertile control men, without any significant association with infertile groups (chi2= 0.342; p = 0.556). Similarly, we have found a novel A437G (I71V) mutation, which is also present in both infertile and normozoospermic fertile control men without any significant difference (chi2 = 0.476; p = 0.490). Our study clearly demonstrates the complete absence of the A386G (T54A) mutation in Indian subcontinent and the other two mutations --A260G (T12A) and A437G (I71V)--observed are polymorpic. Therefore, we conclude that these mutations in the DAZL gene are not associated with male infertility in Indian subcontinent.     

Association of polymorphisms of A260G and A386G in DAZL gene with male infertility: a meta-analysis and systemic review

To investigate the association of single nucleotide polymorphism 260 and 386 (SNP260 and SNP386) gene with male infertility, an electronic search was performed to identify case-control studies evaluating the relationship of SNP260 or SNP386 of deleted in azoospermia-like (DAZL) and male infertility. Review Manager 5 was used to process the meta-analysis and other statistical analysis. A total of 139 records were retrieved, of which 13 case-control studies with total 2715 patients and 1835 normozoospermic men were included. SNP260 was found not to play a functional role in male oligo/azoospermia either for Caucasians or for Asians. But for SNP386, models of allele (A/G), dominant (AA/AG + GG), co-dominant (AA/AG) and super-dominant (AA + GG/AG) had a strong correlation to spermatogenic failure with related odds ratio being 0.15 (95% confidence interval [95% CI] 0.07 to 0.34, P < 0.00001), 0.16 (95% CI 0.07 to 0.35, P < 0.00001), 0.15 (95% CI 0.06 to 0.33, P < 0.00001) and 0.15 (95% CI 0.06 to 0.33, P < 0.00001), respectively. Moreover, this correlation was only found in the Chinese Han population (decreasing around 85% risk of oligo/azoospermia infertility) and not found in India, Japan, and Caucasian countries. Our analysis demonstrated that SNP260 of DAZL did not contribute to oligo/azoospermia while SNP386 was correlated to male infertility. However, this correlation was only found in China with a country-specific and ethnicity-specific manner.     

DAZL polymorphisms and susceptibility to spermatogenic failure: an example of remarkable ethnic differences

Polymorphisms in genes involved in spermatogenesis are considered potential risk factors for male infertility. Recently a polymorphism in the deleted in azoospermia-like (DAZL) gene (T54A) was reported as susceptibility factor to oligo/azoospermia in the Chinese population. DAZL is an autosomal homologue of the Y chromosomal DAZ (deleted in azoospermia) gene cluster and both are considered master regulators of spermatogenesis. The aim of the present study was to screen (i) for mutations of the entire coding sequence of the DAZL gene in patients lacking of the DAZ gene cluster, in order to evaluate if DAZL polymorphisms may influence the AZFc deletion phenotype; (ii) for the two previously described (and eventually newly identified) single nucleotide polymorphisms (SNPs) in a large group of infertile and normospermic men of Italian origin. We failed to detect new mutations. We confirmed previous results showing no evidence for a functional role of the T12A mutation. Surprisingly, the T54A polymorphism, which was present in 7.4% of the Chinese patients was absent in our Caucasian population. This remarkable difference represent an example of how ethnic background is important also for polymorphisms involved in spermatogenesis and contributes to better select clinically relevant tests, specifically based on the ethnic origin of the infertile patients.     

Mutation screening and association study of the TSSK4 Gene in Chinese infertile men with impaired spermatogenesis

The testis-specific serine/threonine kinase (TSSK) family is a specific kinase group with exclusive or dominant expression in testis and involvement in spermatogenesis and male infertility. TSSK4 is a newly identified member of the TSSK family. In order to investigate the possible relationships between variations, including mutations and polymorphisms of the TSSK4 gene and impaired spermatogenesis in humans, mutation screening of this gene in 372 patients with azoospermia or severe oligospermia and 220 controls was performed. In total, 4 novel single nucleotide changes including c.679G>A, c.987+108G>A, c.-155C>G and c.765C>A were discovered. The latter 2 variations were found only in patients, not in controls. Bioinformatics analysis suggested that allele A of c.765C>A could decrease the activity of pre-mRNA splicing of TSSK4. The frequency of allele A of c.679G>A was significantly higher in controls than in patients. On the contrary, allele A of c.987+108G>A was remarkably increased in patients compared with controls. Our investigation of TSSK4, a potentially important testicular gene, in Chinese infertile and control men identified the association of some single nucleotide polymorphisms in this gene with male infertility.     

XPC基因多态性与男性不育的相关性研究

目的:位于XPC基因外显子区域Ala499Val(C>T)和Lys939Gln(A>C)两个非同义突变的单核苷酸多态性位点在人群中研究广泛,具有潜在的功能性,其多态的变化影响到XPC基因的结构和功能,进而影响到DNA损伤修复率。本文探讨了这两个位点基因多态性在中国汉族人群中的分布及其与男性不育发病风险的关联。方法:采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法,分析318例男性不育患者和228例正常对照男性中XPC基因两个多态性位点的基因分型和等位基因频率,以及这两个位点单独和联合作用与男性不育的相关性。结果:在Ala499Val(C>T)多态性位点中,CC、CT、TT三种基因型频率在病例和对照组中的分布存在显著性差异(P=0.020)。携带TT基因型的个体罹患男性不育的风险是CC基因型个体的0.49倍(95%CI=0.23～0.88),是(CC+CT)基因型个体的0.39倍(95%CI=0.22～0.71)。Lys939Gln(A>C)多态性位点与男性不育的患病风险无显著性关联。联合两个位点分析,个体携带1～4个危险位点患男性不育的风险是携带零个的2.75倍(95%CI=1.50～5.04)。结论:XPC基因Ala499Val(C>T)基因多态性与男性不育的发病风险存在关联,可能是我国汉族人群男性不育的遗传易感因素之一。     

Novel mutations in ubiquitin-specific protease 26 gene might cause spermatogenesis impairment and male infertility

Aim： To study the incidence of single nucleotide polymorphisms in ubiquitin-specific protease 26 （USP26） gene and its involvement in idiopathic male infertility in China. Methods： Routine semen analysis was performed. Infertility factors such as immunological, infectious and biochemical disorders were examined to select patients with idiopathic infertility. DNA was isolated from peripheral blood of the selected patients and control population, which were examined for mutations using polymerase chain reaction-single strand conformation polymorphism analysis. Furthermore, nucleotide sequences were sequenced in some patients and controls. Results： Of 41 infertile men, 9 （22.0%, P = 0.01） had changes in USP26 gene on the X chromosome. A compound mutation （364insACA; 460G→A） was detected in 8 patients （19.5%, P = 0.01） and a 1044T→A substitution was found in 1 patient （2.4%, P 〉 0.05）. All three variations led to changes in the coding amino acids. Two substitutions predict some changes： 460G→ A changes a valine into an isoleucine, and 1044T → A substitutes a leucine for a phenylalanine. Another insertion of three nucleotides ACA causes an insertion of threonine. No other changes were found in the remaining patients and fertile controls. Conclusion： The USP26 gene might be of importance in male reproduction. Mutations in this gene might be associated with male infertility, and might negatively affect testicular function. Further research on this issue is in progress.     

Level of neutral alpha‐1,4‐glucosidase in seminal plasma of Chinese men

Neutral alpha‐1,4‐glucosidase (NAG) is a crucial biomarker for the function of epididymis and is reported to be associated with semen quality. However, the correlation between NAG and Chinese semen quality has never been reported. This study aimed to investigate the level of NAG in the seminal plasma of Chinese men. A total of 394 cases of seminal plasma samples from normal, subfertile and infertile men were enrolled in this study. Male subfertility was caused by teratozoospermia, asthenospermia, severe oligozoospermia, asthenoteratozoospermia, oligoasthenospermia and oligoasthenoteratozoospermia. Male infertility was resulted from azoospermatism. The level of NAG was detected by spectrophotometry. Results showed that the level of NAG in normal men was significantly higher than that in subfertile and infertile men (p = .000). Meanwhile, the level of NAG in subfertile men was significantly greater than that in infertile men (p = .000). In addition, a significant difference was observed in normozoospermia, teratozoospermia, asthenospermia, severe oligozoospermia, asthenoteratozoospermia, oligoasthenospermia, oligoasthenoteratozoospermia and azoospermatism (p < .05). In conclusion, these data indicate that NAG is a crucial marker for assessing seminal plasma quality in Chinese men, which might be helpful for the assistant diagnosis of male infertility.     

Luteinising hormone β-subunit gene Gly102Ser variant and oxidative stress biomarkers in Egyptian infertile males

The aim of the present study was to evaluate the association of Glycine102Serine molecular variant of luteinising hormone (LH) β-subunit gene with infertility in Egyptian males and to assess the impact of oxidative stress in association with Glycine102Serine molecular variant on male infertility. Fifty men with idiopathic oligozoospermia were enrolled in the study. The control group consisted of 39 men with normal semen parameters and proven fertility. Variable oxidative status markers were evaluated. Glycine102Serine variant was investigated using restriction fragment length polymorphism-polymerase chain reaction. The study concluded that Glycine102Serine variant of LH β-subunit gene is relatively common among infertile Egyptian males and might be implicated in some Egyptian infertility cases. In addition, the association of this variant, in some cases, with disturbed oxidative status (decreased seminal plasma antioxidants' level and increased sperm lipid peroxidation) probably have a significant role in accentuation of infertility in those particular cases.