不良生活习惯对精子DNA碎片的影响

目的分析吸烟、喝酒、桑拿生活习惯对精子DNA碎片化的影响,寻找不育症患者的不育影响因素。方法在113例门诊患者中,77例男性不育症患者分为3组;A组为有吸烟/喝酒习惯31人,B组为有桑拿习惯21人,C组为其他不育症25人。另外D组为生育能力正常36人。应用精子染色质结构分析(SCSA)检测以上各组的精子DNA碎片化指数(DFI),分别比较各组间的差异。结果精子DNA碎片化指数(DFI):A组为(29.03±11.41)%、B组为(28.12±9.56)%、C组为(24.08±6.27)%、D组为(14.87±3.54)%。A、B组分别高于C组,A、B组间比较差异无统计意义(P0.05),A、B组分别与C组比较有差异(P0.05);A、B、C组分别与D组比较差异有显著性意义(P0.01)。结论男性不育症患者精子DNA碎片化指数(DFI)与正常生育人群有显著性差异,不良生活习惯会加重精子DNA碎片化指数(DFI),可能是引起男性不育的因素。     

精浆piRNA对精子DNA完整性及辅助生殖技术结局的影响

目的通过检测男性不育症患者精浆中存在的睾丸特异piRNA与精子DNA损伤程度的关系,探讨其对精子DNA完整性及辅助生殖技术(ART)结局的影响。方法分析149例男性不育症患者精液质量及精子DNA完整性;按精子DNA碎片指数(DFI)将不育症患者分为:A组(DFI≤15%)、B组(15%DFI30%)、C组(DFI≥30%),采用实时荧光定量聚合酶链式反应(Q-PCR)检测各组患者精浆中piRNA-013423和piRNA-023386的表达量,比较各组患者受精率、卵裂率、优胚率、生化妊娠率及临床妊娠率的差异。结果与A、B组比较,C组的精子密度、前向活动精子、精子活动率、正常形态率均明显下降(P0.05),A、B两组比较,B组精子活动率下降(P0.05)。A、B、C三组中,C组患者piRNA-013423、piRNA-023386表达均低于A、B组(P0.01);C组患者受精率、卵裂率、优胚率、生化妊娠率及临床妊娠率均低于A、B组(P0.01)。结论男性不育症患者存在精子DNA不同程度的损伤,精浆中piRNA的水平与精子DNA完整性有关,DNA完整性差的患者精浆piRNA表达降低,且影响ART临床结局。提示piRNA可能通过某些信号通路调控精子DNA损伤,但具体机制需要下一步深入研究探讨。     

精浆弹性蛋白酶与精液常规参数及精子DNA完整性的相关性研究

目的:观察男性不育患者精浆弹性蛋白酶水平与精液质量及精子DNA完整性之间的关系,探讨生殖道炎症隐性感染对男性不育的影响。方法:选择202例男性不育患者依照精浆弹性蛋白酶实验结果并结合其他生殖道感染炎症标志物将男性不育分为确认感染组和隐性感染组,选择同期因女方因素不孕前来检查的生育力评估正常,排除生殖道炎症及其他影响生育的致病因素的健康男性32例作为对照组。统计分析不育组与正常对照组、不育组中确认感染组与隐性感染组、不育组中弹性蛋白酶水平正常者与升高者之间精浆弹性蛋白酶水平与精液质量及精子DNA完整性之间的差异。精子浓度及活力用计算机辅助精子分析系统检测,精子形态检查采用Diff-Quik染色法,精子DNA完整性分析用精子染色质扩散法(SCD),精浆弹性蛋白酶用酶联免疫吸附法。结果:正常对照组和不育组的正常形态精子百分率分别为(11.9±9.2)%和(4.1±3.3)%、精子DNA碎片指数(DFI)分别为(10.9±8.7)%和(21.3±12.7)%,精浆弹性蛋白酶分别为(220.9±73.1)ng/ml和(1687.3±1621.2)ng/ml,前向运动精子百分率(PR)分别为(49.7±10.8)%和(19.1±10.3)%,两组相互比较各指标均具有统计学差异(P均0.05)。不育组中弹性蛋白酶升高者较弹性蛋白酶正常者,PR显著降低,DFI显著升高,差异均具有统计学意义(P0.05)。隐性感染和确证感染患者间精液量、精子浓度、PR、正常形态精子百分率和DFI的差异均无统计学意义(P均0.05)。结论:生殖道炎症感染无论是显性或者是隐性有可能导致DNA损伤程度增加,从而影响男性生育。     

精子DNA完整性检测在男性不育诊断中的应用价值

目的分析精子DNA完整性与精液主要参数的相关性,探讨其在男性不育诊断中的临床应用价值.方法收集2010年6月至2012年2月在门诊就诊的465例男性患者的精液标本,参照第5版《世界卫生组织人类精液检查与处理实验室手册》严格标准进行精液常规及精子形态学分析,然后采用精子染色质扩散法检测精子DNA完整性,计算DNA碎片指数(DNA fragmentation index,DFI),按DFI值分为3组,分别为A组DFI≤15%(n=183)、B组15%0.05).结论精子DNA完整性检测结合精液常规分析有助于评估男性精液质量及生育力.     

334例男性不育症患者精子DNA完整性与精液常规参数间的相关性分析

目的探讨男性不育症患者精子DNA完整性与精液常规参数的相关性,研究精子DNA完整性在评估男性生育力方面的应用价值。方法回顾性分析2015年1月至2016年12月于西北妇女儿童医院生殖中心男性不育门诊就诊的334例患者的临床资料,按照精子DNA碎片指数(DFI)不同进行分组,Ⅰ组(192例):DFI≤15%;Ⅱ组(98例):15%DFI30%;Ⅲ组(44例):DFI≥30%。比较各组患者的精液常规参数(精子浓度、前向运动精子率、正常形态率、年龄和禁欲时间),分析DFI与精液常规参数的相关性。结果不同DFI组间比较,年龄、精子浓度及正常形态率无统计学差异(P0.05),但禁欲时间和前向运动精子率组间差异有统计学意义(P0.05)。DFI与前向运动精子率和正常形态率呈显著负相关(r分别为-0.457、-0.147,P0.05),与禁欲时间呈显著正相关(r=0.222,P0.05),与年龄及精子浓度无明显相关性(P0.05)。结论精子DNA完整性与精液常规参数(前向运动精子率、正常精子形态率、禁欲时间)具有一定的相关性,精子DNA完整性检测可作为精液常规分析的补充。     

不育患者精子DNA链损伤类型的研究及临床意义

目的:观察精子DNA单、双链损伤(SSB、DSB)在男性不育中的特征,探讨DSB与男性不育的关系,为男性不育的诊疗提供新的观察指标与思路。方法:选择男性不育患者60例以及同期因女方因素不孕前来检查的生育力评估正常的健康男性30例作为对照组,分析两组精子DNA损伤及精液主要参数的差异。精子浓度及活力采用计算机辅助精子分析系统,精子存活率分析采用低渗膨胀试验,精子形态采用Diff-Quik染色法,精子DNA损伤采用双尾彗星实验。结果:双尾彗星实验检测精子DNA完整性共有9种彗星模型。不育患者精子DNA损伤指数(DFI)为(33.8±13.1)%,单链损伤指数(SSB-DFI)为(19.2±11.4)%、SSB占所有损伤精子的比率(SSB-DFI/DFI)为(56.8±32.4)%,双链损伤指数(DSB-DFI)为(23.9±13.4)%、DSB占所有损伤精子的比率(DSB-DFI/DFI)为(70.8±19.5)%;与对照组比较[分别为(16.3±7.9)%、(14.9±7.6)%、(91.4±27.8)%、(6.1±2.7)%、(37.4±11.3)%)],SSB-DFI无显著性差异(P0.05),DSB-DFI、DFI和DSB-DFI/DFI显著高于对照组(P均0.01),SSB-DFI/DFI显著降低于对照组(P0.01)。绘制ROC曲线,DSB-DFI/DFI、DSB-DFI及DFI诊断男性不育最佳截断值分别为39.5%、15.85%和18.65%,ROCAUC、敏感度和特异性分别为(0.969,98.3%,90.0%)、(0.912,86.7%,80.0%)、(0.861,90.0%,70.0%)。不育组精子SSB-DFI及SSB-DFI/DFI与精液常规参数均无相关关系(P均0.05),DFI与前向运动精子百分率、精子存活率及正常形态精子百分率呈负相关(P0.05或P0.01),与精子浓度无相关关系(P0.05),精子DSB-DFI及DSB-DFI/DFI与精子浓度、精子存活率、前向运动精子百分率及正常形态精子百分率均呈负相关(P0.05或P0.01)。结论:影响男性不育的DAN损伤因素可能是DSB,而与SSB相关性不大,精子DNA链的损伤类型对男性生殖能力的评估有较大的参考价值。     

男性不育患者精子DNA完整性与精浆氧化应激水平的关系及其对体外受精的影响

目的:探讨男性不育患者精子DNA完整性与精液常规参数、精浆氧化应激水平的关系,以及对体外受精(IVF)结局的影响。方法:采用精子染色质扩散法(SCD)检测433个IVF周期中精子DNA损伤性,根据精子DNA碎片指数(DFI)的不同,分为低DFI组(DFI30%)、高DFI组(DFI≥30%),比较2组精子浓度、前向运动精子百分率、快速前向运动精子百分率、精浆丙二醛(MDA)、总抗氧化能力(TAC)水平及受精率、卵裂率、优胚率的差异。结果:高DFI组与低DFI组比较,前向运动精子百分率及快速前向运动精子百分率显著降低[(29.2±16.8)%vs(48.6±16.7)%、(9.4±6.6)%vs(19.0±9.1)%,P0.01],高DFI组精子浓度与低DFI组相比差异无显著性[(52.3±32.4)×106/ml vs(51.4±30.9)×106/ml,P0.05];高DFI组精浆中的MDA含量明显高于低DFI组[(2.28±0.26)nmol/L vs(0.95±0.18)nmol/L,P0.01],高DFI组精浆中的TAC含量明显低于低DFI组[(10.2±3.5)U/L vs(33.2±7.9)U/L,P0.01];高DFI组IVF受精率明显低于低DFI组[(58.9±30.0)%vs(77.2±25.0)%,P0.01],两组间卵裂率[(70.7±35.6)%vs(80.4±15.6]、优胚率[(40.4±31.3)%vs(41.7±29.4)]比较差异均无显著性(P均0.05)。结论:精子DNA损伤与氧化应激有关,同时精子DNA损伤可降低IVF受精率,影响IVF结局。     

乙肝病毒对人精液参数和精子DNA完整性的影响

目的:探讨精液中乙肝病毒存在对精液参数和精子DNA完整性的影响。方法:采用实时荧光定量PCR技术对153例血清乙肝表面抗原阳性患者精液进行乙肝DNA定量检测,采用精子染色质扩散法检测精子DNA损伤指数(DFI),对比分析精液HBV DNA阳性组(A组,n=43)与阴性组(B组,n=110)精液参数包括精液体积、精子浓度、存活率、前向运动精子百分率、平均直线速率(VSL)、平均路径速率(WAP)结果,以及HBV DNA拷贝数与精子DNA损伤指数的关系。结果:乙肝感染者精液HBV DNA检出率为28.1%(43/153);两组年龄、精液体积、精子浓度无统计学差异(P>0.05),A组精子存活率显著低于B组[(51.4±17.1)%vs(58.0±18.8)%,P>0.05],前向运动精子率显著低于B组[(24.5±10.1)%vs(29.6±13.3)%,P>0.05],VSL显著低于B组[(19.9±4.5)μm/s vs(23.7±4.0)μm/s,P<0.01],WAP显著低于B组[(23.4±5.3)μm/s vs(26.5±7.0)μm/s,P<0.01];A组精子DNA碎片指数显著高于B组[(24.2±9.4)%vs(19.3±8.0)%,P<0.01];精液中HBV DNA拷贝数与精子DNA碎片指数呈正相关(r=0.819,P<0.01)。结论:精液中HBV DNA存在对精子数量尚未构成显著影响,但可能改变一些精液参数,包括精子活力和运动速度以及精子DNA完整性,精液中乙肝病毒载量和精子DNA损伤之间呈现病毒载量-效应关系。     

吸烟对精子染色质结构完整性影响的探讨

目的:探讨吸烟对精子染色质结构完整性的影响。方法:784例男性不育患者从病例库中选取,根据吸烟与否及每日吸烟支数(≤10、11~19、≥20)和吸烟年限(≤5、6~9、≥10)进行分组,比较各组患者精液常规检测参数及精子染色质结构受损和精子核成熟情况。精子染色质结构完整性采用流式细胞仪检测,DNA损伤程度和不成熟精子指数分别以DNA损伤指数(DFI)和高DNA着色性(HDS)来表示。结果:吸烟≥20支/日和吸烟年限≥10年的患者组精液量、前向运动精子比例低于其他组而精子畸形率高于其他组(P<0.05);吸烟组男性的DFI和HDS值均升高(P<0.05);HDS与前向运动精子比例呈负相关(r=-0.18,P<0.05),DFI与HDS均与精子畸形率呈正相关(r=0.31与r=0.39,P均<0.05)。结论:日吸烟量≥20支或吸烟年限≥10年对男性的精液量、前向运动精子比例和精子畸形率都有影响,吸烟影响男性精子DNA完整性和精子核成熟。     

精子DNA完整性对体外授精-胚胎移植妊娠结局的影响

目的 探讨精子DNA完整性对体外受精-胚胎移植(IVF-ET)临床结局的影响。方法 采用染色质扩散实验(SCD)对接受203个周期IVF-ET治疗的男方精液进行精子DNA完整性检测。因女方卵巢反应差取消10个周期,剩余193个周期。根据精子DNA碎片指数(DFI)是否超过30%和是否药物治疗分为3组:DFI异常组55例(DFI30%),DFI正常组(DFI30%)78例和DFI治疗组60例,其中DFI治疗组指患者DFI30%,经药物治疗3个月,降至30%以下。比较DFI与受精率、2PN受精率、卵裂率及优胚率的相关性;比较3组间的种植率、妊娠率和流产率。结果 精子DFI与受精率、2PN受精率及卵裂率均无显著相关性(P0.05),与优胚率具有显著负相关(P0.05)。DFI正常组和DFI治疗组(治疗后DFI30%)的种植率及妊娠率均高于DFI异常组,差异有统计学意义(P0.05)。DFI正常组与DFI治疗组的种植率及妊娠率差异均无统计学统计(P0.05)。3组流产率表现相当,差异均无统计学意义(P0.05)。结论 精子DNA完整性对受精率、2PN受精率及卵裂率无影响,对优胚率、种植率及妊娠率有负面影响,药物改善精子DNA完整性后可提高优胚率、种植率及妊娠率。     

乙肝病毒携带者不育男性精液质量分析

目的:分析携带乙肝病毒(HBV)的不育男性的精液质量,探讨HBV感染对男性精液质量的影响。方法:选择2018年门诊初诊的782例不育男性,年龄25~35岁,根据HBV感染情况分为小三阳组(血清学检查乙肝表面抗原、e抗体、核心抗体阳性,n=286)和大三阳组(血清学检查乙肝表面抗原、e抗原、核心抗体阳性,n=230),以未感染者作为对照组(n=266),对上述3组进行精液常规、精子顶体酶活性及精子染色质结构分析,比较3组结果是否有差异。结果:①小三阳组精子浓度[(71.49±60.03)×10^6/ml]、前向运动精子百分率[(30.70±14.79)%]、精子活率[(42.67±17.23)%]、精子存活率[(81.07±10.19)%]、正常形态精子百分率[(5.72±3.47)%]均低于大三阳组[(88.20±82.62)×10^6/ml、(34.88±15.60)%、(45.77±16.58)%、(82.55±7.55)%、(6.93±4.45)%]和对照组[(89.29±53.80)×10^6/ml、(37.82±13.63)%、(48.16±14.03)%、(85.26±6.39)%、(7.27±4.43)%],除精子存活率以外差异均有统计学意义(P<0.05);大三阳组精子浓度、前向运动精子百分率、精子活率、精子存活率、正常形态精子百分率均低于对照组,其中前向运动精子百分率、精子存活率的差异有统计学意义(P<0.05);②小三阳组的精子顶体酶活性[(57.07±26.38)μIU/10^6精子]显著低于大三阳组[(63.03±28.75)μIU/10^6精子,P<0.05]和对照组[(78.00±33.49)μIU/10^6精子,P<0.01];大三阳组的精子顶体酶活性显著低于对照组(P<0.01);③小三阳组精子DNA碎片指数[DFI,(14.79±9.46)%]和高可染性[HDS,(9.62±6.20)%]均高于大三阳组[(12.95±7.29)%、(8.43±4.72)%]和对照组[(11.60±5.98)%、(8.41±4.59)%],差异有统计学意义(P<0.05);大三阳组的DFI和HDS均高于对照组,仅DFI的差异有统计学意义(P<0.05)。结论:HBV携带者的男性精液质量显著低于未感染者,HBV感染可能是引起男性生育力降低的原因之一。     

Male age is more critical to sperm DNA integrity than routine semen parameters in Chinese infertile males

This retrospective study evaluated the correlation between the sperm DNA integrity results and infertile male age or sperm motility in 654 infertile men undergoing infertility evaluations from 2013 to 2016. The correlation between the results of sperm DNA integrity and male age was positive, while a negative correlation was detected between sperm DNA integrity and sperm motility in all subjects. According to age (≤30, 30–35 and ≥35), men with normozoospermia or abnormal semen parameters were, respectively, divided into groups 1, 2 and 3, or groups A, B and C. The sperm DNA fragmentation index (DFI) and DFI abnormality rates in groups 3 and C were highest among their respective cohorts. But they were not significantly different between groups within the same age range. Statistically significant differences were found in male age, progressive motility, as well as total motility between patients with normal DFIs and those with abnormal DFIs in group C, but not in group 3. Older (≥35 years) infertile men have increased sperm DNA fragmentation, independent of conventional semen parameters. Male age is more critical to sperm DNA integrity than routine semen parameters.     

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。     

Flow cytometric measurement of sperm nuclear DNA fragmentation in infertile men with normal standard sperm parameters

BackgroundMale-factor infertility plays a role in approximately 50% of infertile couples. In at least 30% of cases, repeated standard semen analyses of the male partner of an infertile couple reveal normal results. When diagnostic work-up of the female partner is also normal, they are classified as idiopathic. The objective of this study was to evaluate the levels of sperm nuclear DNA fragmentation in a population of infertile men with normal standard semen parameters and to compare their results with those from men who had abnormal semen parameters, as well as with a control group of fertile men.MethodsSemen samples were obtained from 202 infertile men and 30 fertile donors. Standard semen analysis was performed according to the World Health Organization guidelines. Flow cytometry has been extensively used to study sperm DNA fragmentation and the results are expressed as the percentage of sperm DNA fragmentation index (DFI).ResultsOf the 202 patients, 48 (23.8%) had normal standard sperm parameters, while 154 (76.2%) had an abnormality in one or more of these parameters. DFI in infertile men with normal sperm parameters was significantly higher than in fertile donors (p = 0.03), but not significantly different from infertile men with abnormal sperm parameters (p = 0.10). There were statistically significant negative correlations between DFI and the percentage of motile sperm from infertile men with abnormal and normal semen parameters, but not in fertile donors (r = ?0.26, p = 0.001 and r = ?0.48, p = 0.0001, respectively).ConclusionSperm from infertile men with normal standard sperm parameters may have significant levels of DNA fragmentation that are comparable to levels in infertile men with abnormal sperm parameters. Sperm DNA fragmentation analysis is an independent test of sperm quality and has an important diagnostic value in the evaluation of male infertility.     

益肾活血法对精子DNA损伤及自然妊娠结局的影响

目的:探讨益肾活血法治疗男性不育症的疗效,观察其对精子DNA损伤、自然妊娠结局的影响。方法:伴有精子DNA碎片指数(DFI)异常的50例男性不育症患者为观察对象,随机分为2组,最后42例纳入统计分析,其中益肾活血法组(试验组)22例,对照组20例。试验组予口服中药治疗,对照组予口服他莫昔芬片和维生素E胶丸,3个月为1个疗程。治疗前后检测精液常规参数、精子形态、DFI变化情况,并对自然妊娠情况随访。结果:与治疗前比较,治疗后试验组精子浓度[(36.82±29.16)×10^6/ml vs(50.00±39.16)×10^6/ml,P<0.05]、前向运动精子百分率[(20.62±9.10)%vs(36.82±13.45)%,P<0.05]、正常形态精子百分率[(1.28±1.00)%vs(3.44±1.33)%,P<0.05]显著升高,精子DFI显著降低[(29.07±11.52)%vs(15.51±11.31)%,P<0.05];对照组精子浓度[(34.56±37.03)×10^6/ml vs(40.72±47.37)×10^6/ml,P<0.05]、前向运动精子百分率[(21.25±9.11)%vs(26.18±10.60)%,P<0.05]、正常形态精子百分率[(1.48±0.91)%vs(2.57±1.32)%,P<0.05]也显著升高,精子DFI也显著降低[(24.43±8.46)%vs(18.53±10.44)%,P<0.05];两组比较,试验组在提高精子总活力、前向运动精子百分率、正常形态精子百分率方面显著优于对照组(P<0.05),而在增加精子浓度和降低DFI差异无统计学意义(P>0.05)。试验组妊娠率(18.2%vs 15.0%)和活产率(18.2%vs 10.0%)均高于对照组,但差异无统计学意义(P>0.05)。结论:益肾活血法类中药有助于降低精子DNA损伤,改善自然妊娠结局。     

Effect of varicocele on chromatin condensation and DNA integrity of ejaculated spermatozoa using cytochemical tests

Varicocele occurs in approximately 15% to 20% of the general male population and it is the most common cause of poor semen production and decreased semen quality. It has been demonstrated that patients with varicocele have a significantly higher DNA fragmentation index (DFI) and spermatozoa with nuclear anomalies than healthy fertile men. Therefore, the aim of this study was to evaluate sperm chromatin integrity in these patients. Sixty men referring to the andrology laboratory were categorised into three different groups: 20 infertile men with varicocele, 20 infertile men with abnormal semen parameters and 20 fertile men who had normal spermatogram were considered as control group. Semen analysis was performed according to WHO criteria. To evaluate sperm chromatin quality and DNA integrity, after fixation of sperm smears, aniline blue, toluidine blue, chromomycin A₃ and acridine orange staining were applied in three groups. The slides were analysed by light and fluorescent microscopy and to determine the percentage of mature or immature spermatozoa, 200 spermatozoa were counted in each slide. The results showed that the rates of aniline blue-reacted spermatozoa were significantly higher in infertile and varicocele patients than in the normal group ( P  < 0.001). In addition, with regard to chromomycin A₃, acridine orange and toluidine blue staining, there was a significant difference between the three groups ( P  < 0.001). The results showed that the varicocele samples contain a higher proportion of spermatozoa with abnormal DNA and immature chromatin than those from fertile men as well as infertile men without varicocele. Therefore, varicocele results in the production of spermatozoa with less condensed chromatin and this is one of the possible causes of infertility due to varicocele.     

肿瘤坏死因子α-308多态性与弱精子症关系的研究

目的:探讨肿瘤坏死因子α(TNFα)基因启动子区-308位点基因型多态性与弱精子症的关系。方法:采用等位基因特异多聚酶链式反应(ASPCR)和聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)法分别对187例男性不育患者(分3组:A组,60例,为弱精子症组;B组,65例,为少弱精子症组;C组,62例,为正常精液不育组)的TNFα基因-308位点基因型进行分析。采用放射免疫分析法测定患者精浆中的TNF-α水平,并对各组数据进行统计学分析。结果:观察各组TNFα-308位点基因型GA/AA频率,与C组(8.06%)相比,A组(21.67%)和B组(26.15%)显著增高,差异均有统计学意义(P<0.05)。采用Spearman方法分析,各组间TNFα-308位点等位基因GA+AA类型与a+b级精子百分率呈显著负相关关系(r=-0.690,P<0.05)。观察各组精浆TNF-α水平,与C组[(4.03±0.66)ng/ml]相比,A组[(4.23±0.45)ng/ml]和B组[(4.29±0.47)ng/ml]显著升高,差异均有统计学意义(P<0.05)。而A组与B组相比,差异无统计学意义(P>0.05)。观察TNFα-308位点不同等位基因(GA+AA、GG)间的精浆TNFα水平,与GG类型组[(4.06±0.45)ng/ml]相比,GA+AA类型组[(4.61±0.29)ng/ml]显著升高,差异有统计学意义(P<0.05)。结论:不论精子密度如何,TNFα-308GA/AA的频率与a+b级精子百分率呈显著负相关关系,其机制之一可能与精浆TNF-α水平有关。据此推测,治疗弱精子症,抗TNFα方案可能有效;另外,精浆TNFα水平的检测可作为男性不育症的辅助诊断指标。     

精子染色质扩散实验检测精索静脉曲张及不明原因不育患者精子DNA碎片

目的了解精索静脉曲张(VC)及不明原因不育患者精子DNA碎片的发生比例。方法改进的精子染色质扩散(SCD)实验分析精子DNA碎片。检测VC不育患者39例,不明原因不育患者57例。以生育健康成年男性32例为对照组。结果VC不育患者SCD小光晕和无光晕精子(精子DNA碎片)比值平均为(36.6±18.9)%,VC不育组明显高于对照组(12.1±5.2)%(P<0.001),而大光晕和中光晕精子比值VC不育组明显低于对照组(P<0.01);不明原因不育患者精子DNA碎片比值平均为(26.8±10.2)%,与对照组[(12.1±5.2)%]比较有显著性差异(P<0.001)。结论SCD实验表明,VC及不明原因不育患者精子DNA碎片比值增高。     

不同年龄对精子凋亡率及DNA完整性影响的研究

目的:探讨不同年龄阶段精子凋亡率、精子DNA完整性及精液基本参数与年龄是否存在相关性。方法:随机选取在我科因女方因素行体外受精-胚胎移植男性精液标本104例,按男方年龄分为3组,年龄<35岁组43例、35～39岁组31例、≥40岁组30例,按WHO手册第4版手工分析精液常规参数,流式细胞术(FCM)结合FITC-Annexin V/PI荧光染色检测精子凋亡率,吖啶橙荧光染色检测精子DNA完整性。结果:①3组精液量[(2.87±0.89)ml vs(2.98±1.09)ml vs(2.65±0.95)ml]、精子浓度[(60.40±25.43)×106/ml vs(69.74±28.33)×106/ml vs(55.97±27.22)×106/ml]无显著性差异(P>0.05),≥40岁组前向运动精子百分率[(39.00±8.35)%]减少,与<35岁组[(48.72±9.89)%]和35～39岁组[(45.65±10.55)%]相比,差异有显著性(P<0.01);≥40岁组正常形态精子百分率[(11.11±8.26)%]与<35岁组[(16.43±8.75)%]相比,差异有显著性(P<0.01)。②精子凋亡在不同年龄组人群均有一定发生率,≥40岁组精子凋亡率为[(11.82±5.77)%],高于<35岁组[(7.04±3.50)%]和35～39岁组[(9.42±4.73)%],与<35岁组相比结果有统计学意义(P<0.01);≥40岁组DNA完整精子百分率降低[(75.52±10.60)%],与<35岁组[(86.55±5.60)%]和35～39岁组[(81.39±8.94)%]相比,差异有显著性(P<0.01);③男性年龄与精子凋亡百分率呈正相关(P<0.01),与前向运动精子、DNA完整精子百分率呈负相关(P<0.01)。结论:男性年龄增加可能导致前向运动精子百分率减少,正常形态精子百分率降低,精子凋亡百分率增加,DNA完整精子百分率性降低,在生育过程中,男性年龄应引起关注。     

Development of the NBT assay as a marker of sperm oxidative stress

Oxidative stress is a well-established cause of male infertility, with reactive oxygen species (ROS) causing infertility principally by impairing sperm motility and DNA integrity. Currently, most clinics do not test their infertile patients for the presence of oxidative stress because the available tests are expensive or difficult to perform. As antioxidant therapy may improve sperm DNA integrity and pregnancy outcomes, it has become apparent that there is an unmet clinical need for an inexpensive and easy-to-perform assay to identify sperm oxidative stress. The aim of this study was to develop a standardized protocol for performing a photometric nitro blue tetrazolium (NBT) assay for the measurement of seminal ROS production via production of coloured formazan, whilst correlating these results with impaired sperm function (motility and DNA integrity). Semen samples from 21 fertile and 36 male aetiology infertile men were assessed for ROS production (NBT assay), sperm DNA integrity (TUNEL), apoptosis (Annexin V) and sperm motility. Infertile men's semen contained on average fourfold higher levels of ROS than fertile men. The production of ROS by sperm was positively correlated with sperm DNA fragmentation and apoptosis, whilst being negatively correlated with sperm motility. Receiver-operating characteristic plot analysis established a cut-off point of 24 μg formazan/10⁷ sperm having a sensitivity of 91.7% and a specificity of 81% for determining the fertility status of an individual. This study has been successful in establishing a standardized protocol for performing a photometric seminal NBT assay that has significant clinical utility in identifying men with impaired fertility because of oxidative stress.