精子DNA碎片率与男性年龄、精子活动力和体外受精结局关系的研究

目的 探讨精子DNA碎片率(DNA fragmentation index,DFI)与男性年龄、精液检查指标、体外受精(in vitro fertilization,IVF)的受精率、优质胚胎率、周期妊娠率和胚胎着床率等关系.方法 随机选取本院生殖中心111例IVF患者,使用流式细胞仪行精子DFI测定,DFI值按不同界...     

男性不育患者年龄对精子形态及精子DNA完整性的影响分析

目的探讨男性不育患者年龄对精子形态及精子DNA完整性的影响。方法将231例男性不育患者按年龄分为A组（25—40岁）及B组（≥40岁）,对其进行精子形态学分析及精子DNA完整性检测。结果A组精子畸形率及精子DNA碎片指数（DFI）明显低于B组（P〈0．05）,两组差别有显著性意义。结论男性年龄对精子形态及精子DNA完整性有一定的影响,应引起临床关注。     

厦门地区不同职业对男性不育患者精子DNA完整性和精子参数的影响

目的探讨厦门地区不同职业对精子DNA完整性和精子参数的影响。方法将就诊的1509例男性不育患者按照职业分为司机组、计算机工作者组、工人组。将600例正常生育组做为健康对照组。采用SQA—V全自动精子分析仪进行精液常规分析,精子DNA完整性检测采用精子染色质扩散试验（SCD）,以DNA断裂指数（DFI）表示。结果司机组、计算机工作者,工人组的精子密度、活动率、前向运动率和DFI与对照组比较差异有统计学意义（P〈0．05）;司机组和计算机工作者组的精子活动率、前向运动率、精子密度及和DFI与工人组比较差异有统计学意义（P〈0．05）;4组的精液量、精液pH值差异无统计学意义（P〉0．05）。结论司机、计算机工作者和工人可以影响精液质量,从而导致男性不育。     

男性不育患者精子染色体畸变及精子DNA完整性分析

目的 探讨男性不育患者精子染色体和精子DNA完整性的改变.方法 精子染色质扩散(sperm chromatin dispersion,SCD)实验分析精子DNA碎片,正常生育男性(对照组)32名,特发性严重少弱精子症患者(idiopathic severe oligoasthenozoospermia,ISOA)19例,妻子不明原因反复自然流产(unexpbined recurrent miscarriage,URM)38例;多色荧光原位杂交(fluorescent in situ hybridization,FISH)技术检测URM妇女丈夫(n=12)、ISOA不育者(n=10)及对照组(n=5)精子13、21、18、X和Y染色体畸变.结果 对照组、ISOA不育者及URM妇女丈夫精子13、18和21染色体数目总体异常的比率分别为1.29%、4.02%和3.91%,而X和Y染色体数目总体异常的比率分别为0.61%、2.03%和1.98%,与对照组比较差异均有统计学意义(P＜0.01).SCD实验分析精子DNA碎片,ISOA不育患者(n=19)及URM妇女丈夫(n=38)精子DNA碎片比例平均为40.7%±17.8%和22.1%±10.3%,均显著性高于对照组(12.1%±5.2%,P＜0.01).FISH精子染色体(13、21、18、X和Y探针)数目畸变率与精子DNA碎片比率呈正相关(r=0.874,P＜0.01,n=27),精子DNA碎片比率与精子密度及前向运动精子率呈负相关(r=-0.571,P＜0.01,和r=-0.616,P＜0.01,n=89),与畸形精子比率呈正相关(r=0.637,P＜0.01,n=89).结论 精子染色体畸变率和精子DNA碎片比率增高,可能是ISOA和妻子URM不育男性的原因之一,精子DNA损伤筛查町能为特发性男性不育患者提供有用的信息.     

不育厨师精液质量参数及精子DNA完整性分析

目的探讨不育厨师精液常规质量参数和精子畸形率、精子DNA碎片率等指标。方法对生殖门诊52名不育厨师（观察组）和49名育前体检者（对照）行精液常规和DNA碎片率等检测。结果观察组精液液化异常率、精子畸形率、DNA碎片率都显著高于对照组;精子浓度、前向运动精子比率显著低于对照组。结论厨师职业暴露会影响精液质量,导致男性不育,应加强保护和防范。     

吸烟对精子DNA碎片的影响

目的探讨男性不育症患者吸烟对精子DNA碎片的影响。方法采用精子染色质扩散（SCD）实验检测383例男性不育患者精子DNA碎片的比率,根据吸烟多少分组并进行比较研究。结果吸烟〉20支/天组患者与吸烟≤20支/天组患者及不吸烟组患者相比DNA碎片率显著升高（P〈0.05）。吸烟≤20支/天组患者与不吸烟组患者相比DNA碎片率没有明显差别。结论吸烟对DNA碎片率有影响,且吸烟量越大、时间越长、精子DNA碎片率越高。     

不明原因复发性流产与精子DNA完整性的关系

目的探讨不明原因复发性流产与男方精子DNA完整性的关系。方法精子DNA完整性检测采用精子染色质扩散试验（sperm chromatin dispersion,SCD）,以DNA断裂指数（DNA fragmentation index,DFI）表示。分别对不明原因复发性流产男方与无流产史男方精子进行精液常规分析和DFI分析。结果流产组精子密度、活动率、前向运动率和DFI与生育组比较差异有统计学意义（P〈0.05）。结论不明原因复发性流产可能与精子DNA损伤存在一定关系。     

过氧化氢对生育男性精子凋亡及核DNA完整性的影响

目的研究体外过氧化氢对生育男性精子凋亡及精子DNA完整性的影响。方法58例正常生育男性均来自吉林大学第二临床医院泌尿男科,精子Percoll优选后制作精子悬液,用伊红Y染色分析精子活率,用瑞-姬染色分析精子凋亡率,用精子核吖啶橙荧光染色检测精子DNA完整性。结果外加过氧化氢浓度为0.02mmol/l时,精子活率显著低于对照组（P〈0.05）。当浓度为0.20mmol/l时,精子活率显著低于对照组（P〈0.05）,精子凋亡率显著高于对照组（P〈0.05）。外加过氧化氢浓度为0.02mmol/l、0.2mmol/l时,精子DNA完整性均显著低于对照组（P〈0.05）,且精子DNA完整性随实验浓度增加而下降。结论体外过氧化氢对精子活率、凋亡率与DNA完整性有影响,高浓度的过氧化氢可促使精子凋亡,损伤精子DNA完整性,导致男性不育。     

司机职业对男性精子DNA完整性的影响分析

目的探讨司机职业对男性精子DNA完整性的影响。方法选择91例司机职业的男性不育患者为司机组,根据驾龄分为2组：A组驾龄1～10年,B组驾龄大于10年。对照组为117例非司机职业的健康正常男性,对其进行精子DNA完整性检测。结果司机组内A组精子DNA碎片指数（DFI）与正常对照组比较差异有显著性（P〈0．05）,而司机组内B组精子DNA碎片指数（DFI）与正常对照组比较差异有非常显著性（P〈0．01）。结论司机职业会对男性精子DNA完整性产生一定的影响,长期开车可能是引起男性不育的原因之一。     

不育夫妇中男性精子DNA碎片指数变异的研究

目的 探讨不育夫妇中男性精子DNA碎片指数(DNA fragmentation index,DFI)的变异程度.方法 2009年4月至2012年4月,将539对不育夫妇中的539例男性纳入本研究.根据世界卫生组织标准(1999年),应用精子染色质扩散(sperm chromatin dispersion,SCD)试验对每名男性进行至少1次的重复精液常规分析和DFI检测,计算DFI的变异系数(coefficient of variation,CV),对DFI的变异程度及其影响因素进行统计分析.结果 539例男性中,有473例、59例、6例和1例分别行1次、2次、3次和4次重复精液常规分析和DFI检测,共计613次重复检测,重复检测和首次检测的间隔时间为0.5～34个月,中位数3.0(1.0～11.0)个月.539份首次检测样本间的DFI变异系数(between-sample coefficient of variation,CVB)为71.2％.与首次检测间隔0.5～3个月、3～12个月和12～34个月的DFI与首次检测的DFI比较,差异均有统计学意义(P＜0.01).以18％作为男性生育力的DFI阈值,539例男性中有142例(26.3％,95％CI:22.6％～30.0％)的首次和重复检测DFI值分别位于阈值两侧.539例男性自身DFI的变异系数(within-subject coefficient of variation,CVw)为0.0～110.5％,中位数为26.0％(12.6％～42.8％),精子总数、精子浓度、精子活动力和精子形态的CVw的中位数分别为41.9％(21.8％～72.6％)、31.9％(15.1％～62.5％)、29.4％(13.2％～55.4％)和46.0％(22.4％～87.1％),各精液常规参数的CVw分别与DFI的CVw比较,差异均有统计学意义(P＜0.01).DFI的CVw与精子总数、精子浓度和DFI重复检测的间隔时间密切相关(P＜0.05).结论 不育夫妇中的男性精子DFI是一个变异较大的参数,应对每例男性的DFI进行重复检测才能反映其精子DNA损伤的水平.     

The effects of male age on sperm DNA damage in healthy non-smokers

BACKGROUND: The trend for men to have children at older ageraises concerns that advancing age may increase the productionof genetically defective sperm, increasing the risks of transmittinggerm-line mutations. METHODS: We investigated the associationsbetween male age and sperm DNA damage and the influence of severallifestyle factors in a healthy non-clinical group of 80 non-smokers(mean age: 46.4 years, range: 22–80 years) with no knownfertility problems using the sperm Comet analyses. RESULTS:The average percentage of DNA that migrated out of the spermnucleus under alkaline electrophoresis increased with age (0.18%per year, P = 0.006), but there was no age association for damagemeasured under neutral conditions (P = 0.7). Men who consumed>3 cups coffee per day had 20% higher percentage tail DNAunder neutral but not alkaline conditions compared with menwho consumed no caffeine (P = 0.005). CONCLUSIONS: Our findingsindicate that (i) older men have increased sperm DNA damageassociated with alkali-labile sites or single-strand DNA breaksand (ii) independent of age, men with substantial daily caffeineconsumption have increased sperm DNA damage associated withdouble-strand DNA breaks. DNA damage in sperm can be convertedto chromosomal aberrations and gene mutations after fertilization,increasing the risks of developmental defects and genetic diseasesamong offspring.     

Influence of global sperm DNA methylation on IVF results

BACKGROUND: In cases of male infertility, routine analysis for sperm characteristics is a poor predictive factor for the segmentation rate and embryo development in assisted reproductive technologies. It is assumed that epigenetic factors could have an influence on the embryo's quality. The aim of this work was to determine the relationship between sperm DNA methylation level and fertilization and pregnancy rates according to the assisted reproduction technique performed. METHODS: A prospective study was undertaken. Ejaculates were obtained from men (n = 63) undergoing an assisted reproduction procedure. 5-Methylcytosine was immunostained with a polyclonal antibody and revealed by fluorescein isothiocyanate. The DNA methylation level was then quantified by flow cytometry. RESULTS: Sixty-three conventional IVF cycles were performed, 760 oocytes were retrieved, an average of 8.1 +/- 4.8 embryos was obtained, and 2.4 embryos were transferred. Neither the fertilization rate nor the rate of good quality embryos was correlated with the DNA methylation level (r = -0.1 and r = -0.08 respectively; not significant). When sperm DNA methylation was >555 arbitrary units, the pregnancy rate was 33.3% compared with 8.3% in the lower (<555) group (P<0.05). CONCLUSION: DNA methylation level in human sperm could represent a new approach to study the ability of sperm to lead to pregnancy in an assisted reproduction procedure, especially when sperm samples with normal characteristics are used.     

主要精液参数及精子DNA完整率与不明原因习惯性流产的相关性

目的 研究主要精液参数和精子DNA完整率与习惯性流产的相关性.方法 收集配偶具有习惯性流产史的患者85例,其精液标本与正常生育的男性(20名)进行对比研究.精子DNA检测采用精子染色质扩散试验法.结果 在精液常规方面,精子活力与习惯性流产存在相关性(P＜0.05),习惯性流产组精子DNA断裂指数为(34.99±14.62)％,对照组为(10.82±4.80)％,两者比较差异具有统计学意义(P＜0.01).结论 精子活力与习惯性流产存在相关性,习惯性流产组患者与正常生育组比较,具有较高精子DNA损伤.     

Nuclear staining identifies two populations of human sperm with different DNA fragmentation extent and relationship with semen parameters

BACKGROUND: Sperm DNA fragmentation is a possible predictive parameter formale fertility status. The occurrence of M540 bodies in semenof subfertile subjects affects flow cytometric investigationsin sperm. We set up a new method to evaluate DNA fragmentationexcluding M540 bodies. METHODS: DNA fragmentation was evaluated by flow cytometry in semen of75 subjects both by terminal deoxynucleotidyl transferase-mediatedfluorescein-dUTP nick end labeling (TUNEL, traditional method)and by double staining with TUNEL and propidium iodide (PI,new method). RESULTS: The use of the new method revealed that TUNEL underestimatessperm DNA fragmentation in flow cytometry and showed two spermpopulations stained with low (PI^dim) and high (PI^br) avidityfor PI. The PI^dim population is entirely composed of DNA fragmentedsperm and its incidence shows highly significant negative correlationswith morphology, motility, sperm count and concentration (respectively,r = –0.51, –0.52, –0.46 and –0.32, n= 75). DNA fragmentation in the PI^br sperm population is independentfrom semen quality. CONCLUSIONS: The correlations between sperm DNA breakage and semen qualitypreviously reported are mainly driven by the occurrence of thePI^dim population. DNA fragmented sperm in this population aremore likely to have poorer morphology, reduced motility andthus a reduced chance to fertilize an oocyte than DNA damagedsperm in PI^br population. Distinguishing between the two typesof sperm DNA fragmentation appears to be important in clinicalinvestigations.     

Study of mitochondrial membrane potential, reactive oxygen species, DNA fragmentation and cell viability by flow cytometry in human sperm

BACKGROUND: Sperm cell death appears to be a cause of male infertility. The objective of this study was to determine the most reliable method for the evaluation of sperm quality in semen samples during sperm preparation for IVF. METHODS: Conventional analysis of semen samples was compared with several cytofluorometric methods detecting death-associated changes. Neat semen from infertile patients and sperm prepared by PureSperm gradient were studied by conventional microscopy and analysed for mitochondrial membrane potential (Delta Psi(m)), generation of reactive oxygen species, DNA fragmentation and cell viability. RESULTS: In neat semen, a positive correlation was found between the percentage of Delta Psi(m)(high) sperm cells and standard semen parameters (concentration/motility). Sperm cells depicting Delta Psi(m)(high) and cells with low DNA fragmentation displayed high fertilization rate after IVF. The only changes that could be detected in prepared sperm were changes in Delta Psi(m), with Delta Psi(m)(high) sperm positively correlated with forward motility and also with high fertilization rates after IVF. CONCLUSION: Analysis of mitochondrial membrane potential is the most sensitive test by which to determine sperm quality. These findings promise development of a test that may help to predict successful IVF.     

Late, but not early, paternal effect on human embryo development is related to sperm DNA fragmentation

BACKGROUND: It is known that repeated failure of assisted reproduction treatment (ART) can be due to a paternal effect. This study was undertaken to analyse the possible relationship between ART failure and sperm DNA fragmentation. METHODS: Zygote morphology and the percentage of spermatozoa with fragmented DNA (assessed by TUNEL) were compared in two groups using donor oocytes for ICSI attempts. The experimental group consisted of 18 infertile couples who had each undergone three previous failed ART attempts. The control group included 18 randomly selected infertile couples undergoing their first ICSI attempt. Both groups used sibling oocytes from the same donors. RESULTS: In 10 couples of the experimental group, the adverse paternal effect was evident as early as the zygote stage. This early paternal effect was not associated with sperm DNA fragmentation. In eight couples of the experimental group, the adverse paternal effect did not produce any perceptible deterioration of zygote morphology. However, this late paternal effect was associated with an increased percentage of spermatozoa with fragmented DNA. CONCLUSIONS: Early paternal effect can compromise ART outcomes in the absence of increased sperm DNA fragmentation. Evaluation of sperm DNA integrity is useful to detect late paternal effect, which is not associated with morphological abnormalities at the zygote and early cleavage stages.     

Mitochondrial DNA deletions and nuclear DNA fragmentation in testicular and epididymal human sperm

BACKGROUND: There are still concerns about the safety of intracytoplasmic sperm injection (ICSI) due to its brief clinical record and lack of animal testing. Testicular and epididymal sperm are now used routinely for ICSI in patients with obstructive azoospermia. The use of such immature sperm compounds fears, since little is known of their mitochondrial and nuclear DNA quality. METHODS: A modified long polymerase chain reaction (LPCR) was employed to study mitochondrial DNA (mtDNA) and a modified alkaline Comet assay to determine nuclear DNA (nDNA) fragmentation in testicular and epididymal sperm from men with obstructive azoospermia (n = 25) attending the Regional Fertility Centre. RESULTS: Testicular sperm displayed significantly more wild-type mtDNA (45% of patients) than epididymal sperm (16% of patients). They also had a lower incidence of multiple deletions and smaller mtDNA fragments. Epididymal sperm harboured more large-scale deletions (P < 0.05). There was a strong correlation between nuclear DNA fragmentation, the number of mtDNA deletions (r = 0.48, r = 0.50, P < 0.001) and their size (r = 0.58, r = 0.60, P < 0.001) in both epididymal and testicular sperm. CONCLUSION: This study suggests that mtDNA and nDNA of testicular sperm have fewer mutations and fragmentation than epididymal sperm and should be used in preference for ICSI in clinical treatment.     

Increased sperm mitochondrial DNA content in male infertility

BACKGROUND: There is increasing evidence that mitochondrial DNA (mtDNA) anomalies in sperm may lead to infertility. Point mutations, deletions and the presence of a specific mtDNA haplogroup have been associated with poor sperm quality, but little attention has been paid to the role of mtDNA content. METHODS: Using density gradient separation and swim-up methods, we selected motile sperm from 32 normal and 35 abnormal sperm samples. The mtDNA/beta-globin gene ratio was determined by real-time quantitative PCR. RESULTS: The average mtDNA/beta-globin ratio of sperm collected from 100% density layers was 1.4 for normal sperm, 6.1 for sperm samples presenting at least one abnormal criterion [among the three criteria established by World Health Organization (1999), i.e. sperm count, motility and morphology], and 9.1 for sperm samples presenting two or more of these abnormal criteria. These differences are very highly significant (P < 0.0001). The mtDNA numbers were also much greater in sperm collected from the 40% density gradient layers (mean: 17.1, P < 0.001), known to contain the most abnormal sperm of the sperm samples, than in those collected from the 100% layers known to contain sperm with the best fertilizing ability. CONCLUSION: Our results showed significant mtDNA amplification in sperm collected from abnormal sperm samples.