磷酸化组蛋白H2AX介导的DNA损伤精子受精卵内修复

成熟精子易受外界影响产生DNA损伤,却缺乏DNA损伤修复系统。由于DNA损伤精子仍具有受精能力和发育潜力,其损伤修复可能发生在受精后。遗憾的是,DNA损伤精子受精后的修复机制尚不清楚。研究发现磷酸化组蛋白H2AX(histone H2AX phosphorylation,γH2AX)参与了DNA损伤精子受精后的修复。本综述主要探讨DNA损伤精子受精后受精卵内经由γH2AX介导修复的可能机制。     

反复自然流产与精子DNA完整性的相关性研究

目的 探讨反复自然流产与精子DNA完整性的相关性。方法 按纳入标准将研究对象分成反复自然流产患者丈夫组（n=58）和正常生育男性对照组（n=50），采用吖啶橙实验（AOT）对研究对象精子DNA完整性进行检测，并对研究对象精液进行常规分析。砖果两组研究对象年龄、吸烟史、饮酒史无统计学差异（P〉0．05）；反复自然流产患者丈夫组精液的精子密度、精子活力显著低于正常生育对照组（P〈O．05）；反复自然流产患者丈夫组精子DNA的完整性低于正常生育对照组，差异有统计学意义（P〈O．05）。砖论精子DNA完整性损伤是反复自然流产的可能原因，精子DNA完整性损伤导致反复自然流产的致病机理尚需进一步研究。     

Etiologies of sperm DNA damage and its impact on male infertility

Male factor is responsible for up to 50% of infertility cases in the world. Semen analysis is considered the cornerstone of laboratory evaluation of male infertility, but it has its own drawbacks and fails to predict the male fertility potential with high sensitivity and specificity. Different etiologies have been linked with male infertility, of which sperm DNA damage has gained significant attention with extensive research on sperm function tests. The associations between sperm DNA damage and a variety of disorders such as varicocele, obesity, cancer, radiation and lifestyle factors are explored in this review. Furthermore, we discuss the mechanisms of DNA damage as well as its impact in different scenarios of male infertility, associated with spontaneous and assisted reproduction. Finally, we review the clinical applicability of sperm DNA fragmentation testing in the management of male infertility.     

Sperm chromatin structure and male fertility： biological and clinical aspects

Aim:Sperm chromatin/DNA integrity is essential for the accurate transmission of paternal genetic information,andnormal sperm chromatin structure is important for sperm fertilizing ability.The routine examination of semen,whichincludes sperm concentration,motility and morphology,does not identify defects in sperm chromatin structure.Theorigin of sperm DNA damage and a variety of methods for its assessment are described.Evaluation of sperm DNAdamage appears to be a useful tool for assessing male fertility potential both in vivo and in vitro.The possible impactof sperm DNA defects on the offspring is also discussed.(Asian J Androl 2006 Jan;8:11-29)     

精子DNA损伤与辅助生殖技术

随着辅助生殖技术的广泛开展,精子评估已由传统的精液常规分析向细胞、分子水平深入发展。精子DNA损伤是反映男性生育力的一个新指标。精子DNA损伤的发生机制包括精子染色质包装与分离异常、氧化应激、细胞凋亡异常等。精子染色质结构分析是目前检测精子DNA损伤最常用的方法之一。精子DNA损伤可能与辅助生殖技术治疗结局、复发性自然流产、增加ICSI后代遗传风险相关。采取口服抗氧化药物、取睾丸精子行ICSI、预冻存精子、去除病因以及中医中药等治疗对策可能会降低精子DNA损伤程度,进而提高辅助生殖技术成功率。本文主要就精子DNA损伤的机制与检测方法、DNA损伤与生殖结局以及辅助生殖技术中与DNA损伤相关的治疗对策作一综述。     

高原低氧环境对男性精子浓度影响研究进展

近年来研究表明长期暴露在高原低氧环境下严重影响成年男性生殖健康,导致精子浓度的降低,而且这种影响随着海拔高度的增加而加重。导致男性精子浓度降低这一结果的产生与高原低氧环境下精子发生减少、精子DNA的损伤、精子凋亡增加及生殖激素水平的变化等密切相关。本文就国内外关于高原低氧环境影响男性精子浓度的机制研究进展做一综述。     

精子DNA完整性检测技术研究进展

氧化胁迫、精子染色质包装或分离异常、凋亡异常发生等因素可引起精子DNA损伤。精子DNA完整性异常,会影响受精和胚胎发育,最终导致不孕不育、流产、死产、子代智力低下和染色体疾病。传统的精液检查往往不能找到精子DNA完整性异常患者的不育原因,因此对这部分患者进行精子DNA完整性检查具有更重要的意义。目前已经建立了很多方法来检测精子DNA的完整性,现就常用的精子DNA完整性检测技术的原理、方法、步骤及其临床应用作一综述。     

The effects of diabetes on male fertility and epigenetic regulation during spermatogenesis

The effects of diabetes mellitus include long-term damages, dysfunctions, and failures of various organs. An important complication of diabetes is the disturbance in the male reproductive system. Glucose metabolism is an important event in spermatogenesis. Moreover, glucose metabolism is also important for maintaining basic cell activity, as well as specific functions, such as motility and fertilization ability in mature sperm. Diabetic disease and experimentally induced diabetes both demonstrated that either type 1 diabetes or type 2 diabetes could have detrimental effects on male fertility, especially on sperm quality, such as sperm motility, sperm DNA integrity, and ingredients of seminal plasma. Epigenetic modifications are essential during spermatogenesis. The epigenetic regulation represents chromatin modifications including DNA methylation, histone modifications, remodeling of nucleosomes and the higher-order chromatin reorganization and noncoding RNAs. If spermatogenesis is affected during the critical developmental window, embryonic gonadal development, and germline differentiation, environmentally-induced epigenetic modifications may become permanent in the germ line epigenome and have a potential impact on subsequent generations through epigenetic transgenerational inheritance. Diabetes may influence the epigenetic modification during sperm spermatogenesis and that these epigenetic dysregulation may be inherited through the male germ line and passed onto more than one generation, which in turn may increase the risk of diabetes in offspring.     

精子DNA损伤与保护

精子DNA损伤是引起不育的重要原因,携有DNA损伤的幸存精子可能逃避体内精子选择机制而成熟并将遗传缺陷传递于后代。因此对精子DNA损伤的研究已成为生殖医学的热点之一。损伤精子DNA的因素主要包括氧化应激、微量元素、精子毒性物质、放射线等,而机体则凭借高度压缩精子DNA、抗氧化系统等机制保护精子DNA的完整性。此外,一些药物如抗氧化剂、黑茶提取物等也可以促使这种保护机制的健全与重建。     

Poor sperm quality and advancing age are associated with increased sperm DNA damage in infertile men

With increasing evidence for faulty paternal contribution to reproduction, there has been a steady increase in studies highlighting an association between sperm DNA damage, failed/delayed fertilisation and aberrant embryo development. Owing to prevailing ambiguity, the aims of the study were to analyse the genetic integrity of the male gamete and then to understand its association with age, standard semen parameters, lifestyle and occupational factors. The study included 504 subjects, attending university infertility clinic for fertility evaluation and treatment. Semen characteristics were analysed by standard criteria; terminal deoxynucelotidyl transferase-mediated nick end-labelling assay was employed for DNA damage assessment. The average incidence of sperm DNA damage in patients with normozoospermic semen parameters was <10%. Patients with oligozoospermia, severe oligozoospermia, oligoasthenoteratospermia, asthenoteratozoospermia and necrozoospermia had significantly higher level of sperm DNA damage (P < 0.001). Patients above 40 years of age had significantly high levels of DNA damage (P < 0.001) compared with their counterparts. Patients with varicocele and a history of alcohol consumption had higher incidence of spermatozoa with DNA damage (P < 0.01). Poor sperm characteristics in the ejaculate are associated with increased sperm DNA damage. Age-related increase in sperm DNA damage and association of the same with varicocele and alcohol consumption are also demonstrated.     

人精子染色质损伤及其检测的研究进展

人精子染色质损伤是常见的影响男性生殖能力的重要原因之一,受到遗传、环境和生活习惯等多因素的影响,且与男性不育、复发性流产等有着密切的联系。随着人们对精子染色质结构和功能认识的加深,对精子染色质结构分析技术的不断改进和推广,以及辅助生殖技术越来越广泛的应用,精子DNA损伤逐渐被认识到是一个新的评价精子质量的重要检测指标,对男性生殖力的评估和辅助生殖技术的选择具有重要的临床意义。     

Obesity leads to higher risk of sperm DNA damage in infertile patients

There has been a growing interest over the past few years in the impact of male nutrition on fertility. Infertility has been linked to male overweight or obesity, and conventional semen parameter values seem to be altered in case of high body mass index （BMI）. A few studies assessing the impact of BMI on sperm DNA integrity have been published, but they did not lead to a strong consensus. Our objective was to explore further the relationship between sperm DNA integrity and BMI, through a 3-year multicentre study. Three hundred and thirty male partners in subfertile couples were included. Using the terminal uridine nick-end labelling （TUNEL） assay, we observed an increased rate of sDerm DNA damage in obese men （odds ratio （95% confidence interval）： 2.5 （1.2-5.1））.     

Mechanisms and clinical correlates of sperm DNA damage

Among the different DNA anomalies that can be present in the male gamete, DNA fragmentation is the most frequent, particularly in infertile subjects. There is now consistent evidence that a sperm containing fragmented DNA can be alive, motile, morphologically normal and able to fertilize an oocyte. There is also evidence that the oocyte is able to repair DNA damage; however, the extent of this repair depends on the type of DNA damage present in the sperm, as well as on the quality of the oocyte. Thus, it is important to understand the possible consequences of sperm DNA fragmentation (SDF) for embryo development, implantation, pregnancy outcome and the health of progeny conceived, both naturally and by assisted reproductive technology (ART). At present, data on the consequences of SDF for reproduction are scarce and, in many ways, inconsistent. The differences in study conclusions might result from the different methods used to detect SDF, the study design and the inclusion criteria. Consequently, it is difficult to decide whether SDF testing should be carried out in fertility assessment and ART. It is clear that there is an urgent need for the standardisation of the methods and for additional clinical studies on the impact of SDF on ART outcomes.     

高原环境对青年男性精子DNA损伤的影响

目的:探讨长期高原作业是否能造成精子DNA的损伤。方法:以53例驻守低海拔地区的部队官兵为对照组,51例高海拔地区的部队官兵为观察组进行调查和对照研究,通过单细胞凝胶电泳技术和染色质扩散实验,检测精子DNA的损伤情况。结果:观察组在进入高原前的各项指标与对照组之间均无明显差异(P>0.05)。观察组的总彗星细胞发生率、各级彗星细胞发生率(G1、G2、G3)和DNA异常精子百分率在进入高原地区前分别为:(5.56±3.98)%、(3.72±1.85)%、(1.57±1.07)%、(0.27±0.34)%和(16.59±12.07)%,进入高原地区半年后均有升高,分别为:(11.15±8.59)%、(5.97±3.26)%、(3.83±2.13)%、(1.35±1.53)%和(22.03±15.33)%,与半年前相比差异显著(P<0.05);二级彗星细胞发生率(G2)和DNA异常精子百分率在1年后有所下降,分别为(3.32±1.83)%和(20.54±15.52)%,但仍比进入高原地区前有明显升高(P<0.05)。结论:长期在高原地区作业可引起精子DNA损伤,但对生育潜能的影响有待进一步研究。加强高原地区作业人员的男性生殖健康预防保健工作仍然十分重要。     

Sperm chromatin structure assay （SCSA）： a tool in diagnosis and treatment of infertility

Diagnosis of male infertility has mainly been based on the World Health Organization (WHO) manual-based semen parameter's concentration, motility and morphology. It has, however, become apparent that none of these parameters are reliable markers for evaluation of the fertility potential of a couple. A search for better markers has led to an increased focus on sperm chromatin integrity testing in fertility work-up and assisted reproductive techniques. During the last couple of decades, numerous sperm DNA integrity tests have been developed. These are claimed to be characterized by a lower intraindividual variation, less intralaboratory and interlaboratory variation and thus less subjective than the conventional sperm analysis. However, not all the sperm chromatin integrity tests have yet been shown to be of clinical value. So far, the test that has been found to have the most stable clinical threshold values in relation to fertility is the sperm chromatin structure assay (SCSA), a flow cytometric test that measures the susceptibility of sperm DNA to acid-induced DNA denaturation in situ. Sperm DNA fragmentation as measured by SCSA has shown to be an independent predictor of successful pregnancy in first pregnancy planners as well as in couples undergoing intrauterine insemination, and can be used as a tool in investigation, counseling and treatment of involuntary childlessness. More conflicting data exist regarding the role of sperm DNA fragmentation in relation to fertilization, pre-embryo development and pregnancy outcome in in vitro fertilization and intracytoplasmic sperm injection (ICSI).     

Effect of varicocele repair on sperm DNA fragmentation: a review

Varicocele, the leading cause of male infertility, can impair sperm quality and fertility via various oxidative stress mechanisms. An imbalance between excessive reactive oxygen species production and antioxidant protection causes alterations in nuclear and mitochondrial sperm DNA, thus rendering a subset of varicocele men less fertile. In particular, sperm DNA fragmentation is usually elevated in men with clinical varicocele in both abnormal and normal semen parameters by the current World Health Organization criteria. In this review, we discuss the evidence concerning the association between varicocele, oxidative stress, and SDF, and the possible mechanisms involved in infertility. Furthermore, we summarize the role of varicocele repair as a means of alleviating SDF and improving fertility. Lastly, we critically appraise the evidence-based algorithm recently issued by the Society for Translational Medicine aimed at guiding urologists on the use of SDF testing in men with varicocele seeking fertility. Current evidence based on careful review of published studies confirms the effectiveness of varicocelectomy as a means of both reducing oxidatively induced sperm DNA damage and potentially improving fertility. Varicocele repair should be offered as part of treatment option for male partners of infertile couples presenting with palpable varicoceles.     

精子DNA完整性在男性不育中的作用

精子DNA完整性对自然受精或人工授精 ,胚胎、胎儿和婴儿的发育至关重要。研究表明 ,夫妇中丈夫精子DNA受损率超过 30 %时 ,自然受精率很低。精子DNA完整性被认为是优于传统精液参数的男性不育指标 ,已经有多种方法可用于检测 ,而且监测精子DNA完整性可用于评估欲行辅助生殖技术 ,如卵细胞胞质内单精子注射 (ICSI)治疗的夫妇。最后对精液冷冻和制备技术对精子DNA完整性的影响进行了总结。     

Sperm DNA fragmentation affects epigenetic feature in human male pronucleus

To evaluate whether the sperm DNA fragmentation affects male pronucleus epigenetic factors, semen analysis was performed and DNA fragmentation was assessed by the method of sperm chromatin structure assay (SCSA). Human‐mouse interspecies fertilisation was used to create human male pronucleus. Male pronucleus DNA methylation and H4K12 acetylation were evaluated by immunostaining. Results showed a significant positive correlation between the level of sperm DNA fragmentation and DNA methylation in male pronuclei. In other words, an increase in DNA damage caused an upsurge in DNA methylation. In the case of H4K12 acetylation, no correlation was detected between DNA damage and the level of histone acetylation in the normal group, but results for the group in which male pronuclei were derived from sperm cells with DNA fragmentation, increased DNA damage led to a decreased acetylation level. Sperm DNA fragmentation interferes with the active demethylation process and disrupts the insertion of histones into the male chromatin in the male pronucleus, following fertilisation.     

Sperm DNA damage in men from infertile couples

Aim： To investigate the prevalence of high levels of sperm DNA damage among men from infertile couples with both normal and abnormal standard semen parameters. Methods： A total of 350 men from infertile couples were assessed. Standard semen analysis and sperm chromatin structure assay （SCSA） were carried out. Results： Ninety-seven men （28% of the whole study group） had a DNA fragmentation index （DFI） 〉 20%, and 43 men （12%） had a DFI 〉 30%. In the group of men with abnormal semen parameters （n = 224）, 35% had a DFI 〉 20%, and 16% had a DFI 〉 30%, whereas these numbers were 15% and 5%, respectively, in the group of men with normal semen parameters （n = 126）. Men with low sperm motility and abnormal morphology had significantly higher odds ratios （ORs） for having a DFI 〉 20% （4.0 for motility and 1.9 for morphology） and DFI 〉 30% （6.2 for motility and 2.8 for morphology） compared with men with normal sperm motility and morphology. Conclusion： In almost one-third of unselected men from infertile couples, the DFI exceeded the level of 20% above which, according to previous studies, the in vivo fertility is reduced. A significant proportion of men with otherwise normal semen parameters also had high sperm DNA damage levels. Thus, the SCSA test could add to explaining causes of infertility in cases where semen analysis has not shown any deviation from the norm. We also recommend running the SCSA test to choose the appropriate assisted reproductive technique （ART）.     

DNA fragmentation assessment by flow cytometry and Sperm-Bos-Halomax (bright-field microscopy and fluorescence microscopy) in bull sperm

The aim of this study was to find the relationship between fertility (as 90-day non-return rates) and DNA fragmentation assessed by two techniques [sperm chromatin structure assay (SCSA) and Sperm-Bos-Halomax (SBH)]. Furthermore, other quality parameters were achieved (motility, morphological abnormalities, cytoplasmic droplets, viability, capacitation and acrosomal and mitochondrial status) and their correlations with fertility were analysed. Bulls were divided into three fertility groups: high [non-return rate (NRR) >or= 80], medium (80 < NRR >or= 70) and low (70 < NRR > 40). The results of this study indicate that there is a good correlation between fertility and different parameters of sperm quality (SBH and SCSA parameters, % of spermatozoa with head, neck and total abnormalities, and % of spermatozoa with proximal cytoplasmic droplets) and differences between fertility groups were observed in some of them (SBH and SCSA parameters and % of spermatozoa with head, neck and total abnormalities). In this sense, SBH parameters rendered good correlations with fertility (r = -0.42 using bright light microscope and r = -0.47 with fluorescence). Also, standard deviation of DNA fragmentation index (SD-DFI) and DFIh (cells with High DNA fragmentation index) showed good correlations with fertility (r = -0.41 and r = -0.29). No correlations were observed between SCSA and SBH parameters. A multiple regression shows that four parameters (% of proximal cytoplasmic droplets, % of intact acrosomes in total population, SD-DFI and percentage of fragmented DNA detected by bright light microscope) present a good predictive value of the fertility of sperm samples (r(2) = 0.34, p < 0.001).