弱精子症病人精子线粒体DNA4977bp缺失研究

应用聚合酶链反应（ＰＣＲ）技术检测线粒体ＤＮＡ４９７７ｂｐ缺失在弱精子症病人和精子活力正常人中的发生情况,以探讨其与精子活力的关系。材料与方法４１例病人来自本院男性门诊,年龄２２～４５岁。按ＷＨＯ推荐的实验方法及标准将４１例病人分为两组：弱精子症组２...     

精子线粒体功能障碍在弱精子症中的研究进展

世界范围内大约有15%的夫妇受不育症困扰,其中男性不育者的精液异常多表现为精子活力低下。研究表明,精子中线粒体膜电位、活性氧、线粒体内Ca2+含量、线粒体酶以及蛋白组活性、线粒体超微结构、mtDNA等的异常可引起精子活力下降。本文通过回顾线粒体与弱精子症的相关文献,阐述精子线粒体功能障碍在弱精子症中可能的作用,为弱精子症发生发展机制的研究提供新的思路。     

离子通道与弱精子症

弱精子症是引起男性不育的常见病因之一。随着以精子自身为对象的研究深入,发现离子跨膜转运对精子的生理活动起着重要的作用。离子通道病于20世纪90年代提出,现已逐渐发现临床上许多先天性和/或后天获得性疾病与异常的离子通道有关。因此,关于精子的生理和疾病与离子通道的关系研究逐渐成为当前研究的热点之一。现就近年来与精子运动相关的离子通道,包括阳离子通道和阴离子通道的研究进展作一综述。     

弱精子症相关基因与蛋白研究进展

男性不育病因复杂,其中弱精子症是导致男性不育的常见原因之一。然而,弱精子症的发病机制仍然不明确。近年来对弱精子症的研究取得了一定进展,一些基因(如tejin-2、DNAI1、DNAH5、DNAH11、AKAP4、SEPT4和Smcp)和蛋白(如精子蛋白ACTB、ANXA5、PRM1、PRM2、SABP等和精原蛋白Tf、PSA、PAP、Fractalkine等)被证实与弱精子症的发生有关,这些分子标记物的发现将为阐述弱精子症的分子机制提供基础,同时可能成为弱精子症诊断和治疗的靶标。     

弱精子症、少弱精子症患者血清、精浆和精子锌含量分析

目的:检测弱精子症和少弱精子症患者血清、精浆和精子锌的含量,分析锌含量的变化与精子密度和精子运动之间的关系。方法:按照WHO《人类精液及精子-宫颈粘液相互作用实验室检验手册》第四版的标准进行精液质量分析,随机筛选出90例弱精子症、60例少弱精子症患者以及20例精液质量正常的生育者作为研究对象,利用原子吸收光谱法检测其血清、精浆、精子的锌含量并进行统计学分析。结果:3组间血清锌含量没有显著差异;弱精子症、少弱精子症患者精浆锌含量均显著低于正常生育者(P<0.05);少弱精子症患者精子锌含量显著高于弱精子症患者和正常生育者(P<0.01)。结论:弱精子症、少弱精子症患者精子的发生及运动功能下降可能与精浆锌含量的低下呈正相关;但过高的精子锌含量与精子的发生和运动功能的关系尚不十分明了,有待进一步研究。     

黄精赞育胶囊对弱精子症患者精子线粒体膜电位的影响

目的:观察黄精赞育胶囊对弱精子症患者精子线粒体膜电位的影响及临床疗效。方法:弱精子症患者70例分药物治疗组(n=39)和空白对照组(n=31)做队列观察研究。药物治疗组予黄精赞育胶囊口服3个月,1次4粒,每日3次。服用时可备孕,期间若配偶怀孕则停止用药。空白对照组不予药物干预。观察初次就诊和3个月后精液参数变化,及JC-1染色后流式细胞仪检测精子线粒体膜电位(MMP)值的变化。结果:两组各失随访1例。药物治疗组总有效率为71.05%,服药过程中3例患者女方自然妊娠。药物治疗组35例患者完成观察期结束后检查,MMP%、精子总活力、前向运动精子百分率(PR%)、精子形态均有改善,其变化值较空白对照组30例患者的测定值有显著差异(P0.01),分别为△MMP%:(20.28±14.21)%vs(1.19±10.36)%;△PR%:(17.58±12.73)%vs(2.26±8.29)%;△总活力:(20.68±14.12)%vs(3.46±8.67)%;△正常形态精子百分率:(3.37±3.99)%vs(0.23±3.48)%。MMP%与精子总活力(r=0.69)、PR%(r=0.75)、正常形态精子百分率(r=0.26)具有显著相关性(P0.01)。结论:黄精赞育胶囊能提高精子MMP值,改善线粒体功能,从而有效增加精子活力及PR%并改善精子形态,对治疗弱精子症安全有效。     

蛋白质组学在弱精子症中的研究进展

男性不育是不孕不育的重要原因之一,这一疾病也严重困扰着患者和医生.据统计,不育症夫妇中50%是男性不育所致,其中,弱精子症占有相当大的比例.研究发现,精子活动能力下降与一氧化氮和超氧化物岐化酶、钙离子通道、微量元素异常以及蛋白质络氨酸磷酸化和膜流动性等很多因素相关.近来,蛋白质组学在弱精子症中的研究日趋兴起.     

不同程度弱精子症的精子蛋白质差异表达分析

目的研究不同程度弱精子症蛋白质表达差异,以期揭示蛋白质改变在弱精子症发生、进展中的作用.方法用iTRAQ标记以及二维高效液相色谱/串联质谱联用的方法研究人类正常精子以及轻、中、重度弱精子症患者的精子蛋白表达,通过相关软件分析数据,比较正常精子与不同程度弱精子症精子蛋白间的变化情况.结果本研究发现1073个蛋白质改变与弱精子症相关,而这些蛋白中,蛋白表达是以下调表达占主要形式的.通过Panther进行弱精子症中蛋白质组的蛋白质进行分类,结果发现这些升高蛋白主要涉及15个生物过程;降低的蛋白质中除了涉及上升表达的15个生物过程外,还涉及内环境稳态生物过程蛋白.这些生物过程中,与生殖过程密切相关的蛋白有26个,其中上调有10个,降低的蛋白为16个.结论精子蛋白表达是以下调表达占主要形式的,而这种情况可能是弱精子症发生的一个重要原因.在弱精子症的发生、发展中,多蛋白通过不同的生物学作用和途径交互影响,可能是最终影响精子的发生和活动力的重要原因.     

生育男性和白细胞精子症患者精子线粒体基因缺失和含量的研究

目的:测定生育男性与白细胞精子症患者精子线粒体DNA(mtDNA)含量、mtDNA4977bp缺失及精浆活性氧(ROS)水平,探讨精液中白细胞增加、ROS水平升高与精子mtDNA变化的相关性。方法:选取门诊白细胞精子症患者78例(病例组)、正常生育男性31例(对照组),采用Percoll梯度离心法将精液样本分为上清层、30%层和80%精子层,分别应用荧光实时定量PCR技术对每个精子mtDNA含量及mtDNA4977bp缺失进行定量分析,同时应用流式细胞术测定精浆ROS水平。结果:病例组精浆中ROS含量显著高于对照组(P<0.01)。与对照组比较,病例组各层精子mtDNA含量均显著升高,其P值均<0.01。在上清层和80%层,病例组mtDNA4977bp缺失与对照组差异不显著;而在30%层,病例组mtDNA4977bp缺失明显高于对照组(P<0.01)。对照组与病例组各样本中ROS含量分别与30%层精子和80%层精子的mtDNA含量间呈显著正相关(r=0.347,P<0.01;r=0.456,P<0.01),与上清层精子mtDNA含量无相关性。结论:白细胞增多和ROS升高可能是导致精子mtDNA含量显著增高的原因之一,但是对于mtDNA4977bp缺失没有显著影响。     

4977‐bp mitochondrial DNA deletion in infertile patients with varicocele

Varicocele is the abnormal inflexion and distension of veins of the pampiniform plexus within spermatic cord and is one of the amendable causes of male infertility. It can increase reactive oxygen species (ROS) production in semen and cause oxidative stress. The purpose of this study was to analyse spermatozoa mtDNA 4977‐bp deletion in infertile men with varicocele. To detect 4977‐bp deletion in spermatozoa mtDNA, semen samples of 60 infertile patients with clinical varicocele and 90 normal men from northern Iran were prepared. After extraction of spermatozoa total DNA, Gap polymerase chain reaction (Gap PCR) was performed. 4977‐bp deletion was observed in 81.66% of patients with varicocele, while approximately 15.55% of controls had this deletion. As spermatozoa from patients with varicocele had a high frequency of occurrence of 4977‐bp deletion in mtDNA [OR = 24.18, 95% confidence interval (CI) = 10.15–57.57, P < 0.0001], varicocele may induce mtDNA deletion in spermatozoa and cause infertility in north Iranian men. However, to determine the relation between sperm mtDNA 4977‐bp deletion and varicocele‐induced infertility, larger population‐based studies are needed. It is concluded that there is an association between sperm mtDNA 4977‐bp deletion and varicocele‐induced infertility in the population studied.     

MicroRNA profiling in spermatozoa of men with unexplained asthenozoospermia

Asthenozoospermia (AZS) which is characterised by decreased sperm motility is one of the main causes of male infertility. Recent studies demonstrated altered microRNAs (miRNAs) in total semen, seminal plasma and spermatozoa of asthenozoospermic men. In line with these studies, it was aimed to evaluate the miRNA expression profile in spermatozoa of unexplained asthenozoospermic men. Thirty‐nine cases with idiopathic AZS and 35 fertile and healthy men as control were included. After total RNA extraction from spermatozoa, high‐throughput sequencing technology was employed to display miRNA profiles in spermatozoa samples pooled from AZS cases and healthy controls. Relative quantification by real‐time PCR was performed to validate RNA‐seq results. SNORD48 was used as normaliser gene, and fold change was calculated by 2^?ΔΔCt method. Profiling results showed that 18 altered miRNAs in AZS men in comparison to controls. Subsequently, seven miRNAs were selected to validate by RT‐PCR that showed MiR‐888‐3p significantly overexpressed in AZS cases (p = 0.014) in comparison with controls. It seems upregulation of miR‐888‐3p was associated with idiopathic AZS. This finding paves the way to the future investigation on the actual molecular role of miR‐888‐3p in aetiology of AZS.     

Prevalence of 4977bp Deletion in Mitochondrial DNA from Patients with Chronic Kidney Disease Receiving Conservative Treatment or Hemodialysis in Southern Brazil

Background. Damage to mitochondrial DNA (mtDNA) has been described in patients with chronic kidney disease (CKD). The presence of mtDNA 4977bp deletion in many different tissues can serve as a marker of this damage. However, no attempt has been made to detect the presence of mtDNA 4977bp in blood cells of patients with CKD. Methods. Polymerase chain reaction techniques (PCR) were used to detect mtDNA 4977bp deletion in blood samples of 94 CKD patients. Results. The prevalence of 4977bp deletion in mtDNA was 73.1% (38/52) in patients with CKD undergoing hemodialysis, 57.1% (27/42) in patients with CKD receiving conservative treatment, and 27.8% (15/54) in control samples (p < 0.001). Higher prevalence of this mutation was not associated with patient age (p?=?0.54) or time on hemodialysis (p?=?0.70). Conclusion. The higher prevalence of mtDNA 4977bp deletion in patients in this study indicates that the CKD can induce damage to mtDNA in blood cells and could be exacerbated by hemodialysis.     

Low copy number and high 4977 deletion of mitochondrial DNA in uterosacral ligaments are associated with pelvic organ prolapse progression

Introduction and hypothesis  The pathophysiology of pelvic organ prolapse (POP) is related to aging in the pelvic organ support, and mitochondrial dysfunction is one of the major contributors to aging. Therefore, the objective of this study was to investigate the correlation between alternations of mitochondrial DNA and progression of POP. Methods  Polymerase chain reaction (PCR) was applied in the present study. Uterosacral ligaments (UL) were obtained from 45 POP patients and 38 myoma patients without POP. Chi-square test, Student’s t-test, Mann–Whitney U test, and Spearman correlation analysis were applied in the comparison between POP and non-POP patients. Results  The results revealed that significant depletion of mitochondrial DNA (mtDNA) and an increase in the incidence of 4977 deletion of mtDNA (mtDNA⁴⁹⁷⁷) in the UL tissue of POP patients. Conclusions  The alternations of mtDNA may play an important role in the molecular pathogenesis and process of POP formation.     

父源印记基因H19印记控制区域DNA甲基化与少、弱精子症相关性分析

目的:研究印记基因H19印记控制区域的DNA甲基化程度与男性少、弱精子症的相关性。方法:通过染色体核型分析和Y染色体微缺失检测排除染色体因素干扰,进一步借助精液常规参数、伊红染色以及精子形态等指标,筛选出18例单一因素少精子症患者(浓度<15×106/ml,其余指标均正常)和20例单一因素弱精子症患者(前向运动精子百分率<32%,其余指标均正常)用于DNA甲基化检测;提取精子样本全基因组DNA,进行亚硫酸氢盐处理、PCR扩增目的基因片段并测序;通过BIQ Analyzer软件对测序结果进行质控和DNA甲基化程度分析。20例正常生育男性精液标本作为对照组。结果:与对照组甲基化丢失率[(0.30±0.06)%]相比,少精子症患者的H19印记控制区域的DNA甲基化丢失程度显著增高[(9.19±2.45)%],尤其当精子浓度<3×106/ml时,差异达到极显著水平(P<0.01)。在弱精子症患者中H19印记控制区域的DNA甲基化丢失程度[(0.30±0.07)%]和模式均与对照组无显著差异(P=0.62)。进一步重点分析了CTCF6位点的DNA甲基化状态,少精子症患者的DNA甲基化丢失程度[(2.67±0.75)%]显著高于对照组[(0.05±0.03)%]和弱精子症组[(0.03±0.02)%],而后两者之间无显著差异(P=0.35)。结论:H19印记控制区域的DNA甲基化程度的降低与少精子症密切相关,且降低程度与精子浓度呈显著负相关,而与精子活力无关。     

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...     

Effect of repeated sequential ejaculation on sperm DNA integrity in subfertile males with asthenozoospermia

The aim of this work was to study the possible beneficial effect of repeated sequential ejaculation on sperm DNA integrity in subfertile males and its possible implementation in assisted reproduction. The study included 20 infertile males with idiopathic asthenozoospermia or oligoasthenozoospermia. They underwent detailed history taking, complete clinical assessment and hormonal assessment. Patients were asked to bring two semen samples (taken within 1-3 h). Two consecutive samples were assessed with regard to semen volume, sperm count, motility grading, and morphology and sperm DNA integrity using the comet assay. There was a significant improvement in the sperm motility pattern and DNA integrity in the second sample in comparison with the first sample. Therefore, it is concluded that due to its positive impact on sperm motility and DNA integrity, repeated sequential ejaculation is recommended in subfertile males with idiopathic asthenozoospermia who pursue assisted reproduction.