辅助生殖技术中锌抑制精子氧化损伤作用

目的探讨辅助生殖技术中锌对精子损伤的保护作用。方法以辅助生殖技术中常用的密度梯度离心法优选后的精子为研究对象,以过氧化氢为损伤刺激物,应用国联精子检测系统、光学显微镜以及精子DNA碎片分析试剂盒的方法比较精子的活力与存活率,精子质膜完整性以及精子DNA碎片形成,从而分析锌抑制过氧化氢对精子的损伤作用。结果过氧化氢刺激后精子的活动力与存活率差,精子质膜完整性破坏,大量精子DNA损伤,而锌能明显保护精子的损伤作用。结论精子损伤与氧化刺激密切相关,在辅助生殖技术中锌可抑制精子的损伤作用。     

硝普钠对人精子获能及顶体反应的影响

目的:探讨一氧化氮(NO)对人精子获能及顶体反应(AR)的影响。方法:在48例健康生育男性的精子悬液中加入不同浓度的NO供体硝普钠(SNP),于37℃孵育1h获能,用孕酮诱导AR15、30、45、60min,采用磷酸苯二钠法分别检测精子获能前后及AR各时间点的精子悬液上清中酸性磷酸酶(ACP),同时用计算机辅助精液分析系统(CASA)分析精子运动参数,观察NO对精子获能及AR的作用。结果:NO浓度在50、100nmol/L时精子悬液中ACP活性增高,精子运动参数明显增强,150、200nmol/L时ACP活性及精子运动参数变化不大,250、300nmol/L时精子悬液中ACP活性和精子运动参数明显降低。结论:NO对精子获能和AR具有双重性,在低浓度下有促进精子获能、AR和精子运动参数增强的作用,而在高浓度下对精子获能、AR和精子运动参数有抑制作用。精子ACP检测对精子群体获能和AR状态的评价是种较为客观、可靠的方法。     

镉离子对公牛精子功能损伤的机制

目的:重金属比如镉(Cd)作为工业污染物广泛分布在环境中,研究人员已经鉴定了这些污染物能影响男性生殖系统。本文研究的目的是测试Cd在10~1000μmol/L的浓度范围在体外对荷斯坦(Holstein)公牛精子膜和DNA完整性、活动率和精子顶体胞吐能力的影响。方法:用PBS处理公牛精液样本后进行精液分析。脂质过氧化试验评估精子膜完整性。明胶消化试验测定公牛精子顶体胞吐作用的能力。单细胞凝胶电泳(SCGE)检测单个细胞内DNA断裂和不耐碱的破坏。结果:脂质过氧化(LPO)显著增加,表明了Cd对精子膜完整性的破坏作用。这种影响在Cd浓度为1000μmol/L时特别明显。LPO与活动精子百分率之间呈负相关(r=-0.94,P<0.001)。明胶消化试验表明Cd引起公牛精子顶体胞吐作用的百分率下降。发现在LPO率和消化环百分率之间呈负相关(r=-0.97,P<0.001)。彗星试验获得的数据表明Cd能诱导精子核中的DNA断裂。接近93%的DNA损伤为双股断裂。LPO氧化率与DNA断裂百分率之间的相关性为0.95(P<0.001)。结论:总体上,Cd诱导公牛精子膜损伤、活动率降低、DNA断裂、以及顶体反应率降低而导致精子功能损伤。进入雄性性腺和精浆的Cd可能对动物精子产生了破坏作用。     

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。     

白藜芦醇对人精子冷冻损伤的保护作用

目的:通过在人精子冷冻保护液中添加白藜芦醇,研究其对冻融后精子质量和功能的影响。方法:选择正常精液与少弱精子症样本各50例,液化后的精液样本分别与甘油-卵黄-柠檬酸盐(GEYC)冷冻保护液或含有30μmol/L白藜芦醇的GEYC冷冻保护液混匀。冷冻复苏前后,进行精子活力、存活率及顶体反应分析。采用丙二醛(MDA)及活性氧(ROS)检测试剂盒评估精子脂质过氧化程度及ROS水平。通过罗丹明123(Rh123)染色法及TUNEL试验检测精子线粒体膜电位及DNA损伤。结果:在正常精液和少弱精子症样本组中,与各组冷冻前新鲜精液相比,冻融后前向运动精子百分率、总活力、存活率、线粒体膜电位及顶体反应率均显著下降(P0.05),而精子ROS、MDA水平和DNA碎片指数(DFI)均显著升高(P0.05)。冷冻保护液中添加30μmol/L白藜芦醇后,正常精液组前向运动精子百分率[(43.1±6.3)%]、总活力[(56.9±7.4)%]、存活率[(67.5±5.6)%]、线粒体膜电位[(63.4±7.5)%]及顶体反应百分率[(26.3±4.7)%]较冷冻对照(未加白藜芦醇)的前向运动精子百分率[(32.7±4.8)%]、总活力[(44.8±6.9)%]、存活率[(52.3±6.1)%]、线粒体膜电位[(56.5±7.0)%]及顶体反应百分率[(16.6±3.8)%]均显著提高(P0.05),而精子ROS、MDA水平和DFI较冷冻对照均显著降低(P0.05)。在少弱精子症组中,添加白藜芦醇也均显著地提高了冷冻后精子前向运动百分率、总活力百分率、存活率、线粒体膜电位及顶体反应百分率,尤其是DFI[28.5±4.8)%]较冷冻对照[(36.3±5.7)%]显著降低(P0.01)。结论:在精液冷冻保护液中添加白藜芦醇可以通过降低精子内ROS水平减少精子冷冻损伤,从而改善解冻后精子质量和功能。     

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。     

激光辅助制动对精子核DNA的影响

目的:研究激光辅助制动精子是否引起精子核DNA损伤。方法:23例行体外受精(IVF)的男性精液标本,上游法处理后用0.45ms脉冲激光照射精子尾部制动精子。采用末端转移酶介导的dUTP末端标记法(TUNEL)和单细胞凝胶电泳法(SCGE)两种方法分别检测激光辅助制动处理前后阳性精子百分率。结果:TUNEL法检测激光辅助制动处理前后阳性精子的百分率分别为(1.32±0.61)%、(1.41±0.51)%,差异无显著性(P>0.05);SCGE法检测处理前后阳性精子百分率分别为(1.59±0.70)%、(1.83±0.68)%,差异亦无显著性(P>0.05)。结论:激光照射精子尾部辅助制动精子对精子核DNA无明显损伤。     

鱼鳔补肾丸改善男性精子DNA碎片指数的120例临床观察

目的探讨临床应用鱼鳔补肾丸改善男性精子DNA完整性的效果。方法 120例男性精子DNA碎片率(DFI值)高于25%的男性患者连续给予口服鱼鳔补肾丸,于口服30d、60d、90d时复查精子DNA完整性及精液常规参数,比较DFI及精子活力的改善。结果连续口服30d、60d、90d鱼鳔补肾丸后,检测患者精子各种参数指标分析,可见随着服用时间增加DFI持续性下降,DNA完整性提高比较差异均有统计学意义;a级精子百分率、(a+b)级精子百分率、精子活动率也有持续提高,对a级精子百分率、(a+b)级精子百分率、精子活动率提升的有效率与治疗前比较差异亦有统计学意义;服用不同天数后DFI值治疗有效率呈持续性提高。结论鱼鳔补肾丸可以明显降低不育患者精子DFI值,通过修复损伤精子DNA提高精子质量,从而改善男性患者的生育能力。     

胰岛素样生长因子-1对氧化损伤精子的保护作用

目的探讨胰岛素样生长因子-1(Insulin-like growth factor-1,IGF-1)对过氧化氢(H2O2)造成的人精子氧化损伤的保护作用。方法用H2O2处理人精子悬液建立氧化损伤模型,28例精子悬液平均分成4份,实验分4组:对照组(仅精子悬液)、H2O2组、25ng/mL IGF-1组、50ng/mL IGF-1组。分别于培养后12h及24h,检测各组精子活力及前向运动率(PR)、正常形态精子比率、DNA碎片率及丙二醛(MDA)含量。结果培养后12 h及24 h,50ng/mL IGF-1组精子活力、PR高于H2O2组,精子DNA碎片率、MDA水平低于H2O2组,差异均有统计学意义(P0.05);培养后24 h,25 ng/mL IGF-1组精子PR高于H2O2组、DNA碎片率、MDA水平低于H2O2组,差异均有统计学意义(P0.05);培养后12 h及24 h,各组间正常形态精子比率差异无统计学意义(P0.05)。结论适量浓度的IGF-1对活性氧导致的精子运动功能下降及DNA损伤具有一定的保护作用。     

生精片对男性少弱精子症患者精液参数的影响及作用机制研究

目的:观察生精片治疗男性少弱精子症患者的临床有效性,并对其作用机制进行探讨。方法:选取少弱精子症患者120例,采取随机对照研究的方法,治疗组口服生精片,对照组服用维生素E,服用12周后,观察治疗前后两组精子浓度、精子活力及正常形态精子百分率,血清FSH、T、LH水平,DNA碎片指数,低渗肿胀精子百分率,精浆弹性蛋白酶、α-葡糖苷酶、精子顶体酶、果糖及精浆锌等指标变化。结果:生精片能显著改善精子浓度、活力和形态(P<0.01),血清FSH水平明显降低,T明显升高(P<0.01),DNA碎片指数明显降低,低渗肿胀精子百分率显著升高,弹性蛋白酶明显降低,α-葡糖苷酶、精子顶体酶、果糖、精浆锌等水平明显升高。结论:生精片通过多层次、多环节、多途径改善少弱精子症患者的精液参数,具有良好的临床疗效。     

Ⅲ型前列腺炎患者精液参数、锌浓度及抗菌活性的变化

目的:探讨Ⅲ型前列腺炎(CP/CPPS)患者精液常规参数、锌浓度及抗菌活性的关系。方法:对60例CP/CPPS患者和20例健康男性进行精液常规参数、锌浓度及抗菌活性的检测。结果:CP/CPPS患者精液液化时间、精子活力、精浆抗菌活性及精浆锌离子浓度与对照组相比,差异有显著性(P均<0.01)。CP/CPPS患者精子活力与精浆锌离子浓度间有显著相关性(r=0.272,P=0.015)。精浆抗菌活性与精浆锌离子浓度间有显著相关性(r=0.449,P<0.01)。结论:CP/CPPS患者精液液化时间延长,精子活力下降,精浆锌浓度和抗菌活性降低。精浆抗菌活性与精浆锌浓度、精子活力间有显著相关性。     

Effects of nitric oxide on human spermatozoa activity, fertilization and mouse embryonic development

This study was conducted to investigate the effects of nitric oxide (NO) on human sperm activity, human sperm-oocyte fusion and mouse embryonic development. Results showed that various concentrations of NO synthase blocker, N(omega)-nitro-L-arginine methyl ester, did not affect sperm cell motility at 0, 1, 2 or 4 hr, respectively. In contrast, sodium nitroprusside (SNP) significantly inhibited sperm cell motility and caused apoptosis. The adversely dose-dependent effect was only observed if SNP was freshly prepared. Adenosine triphosphate reversed the hazardous effect of SNP on sperm activity/viability. Hemoglobin neutralized the adverse effect of SNP. In hemi-zona sperm fusion test, the number of sperm bound to the zona in the presence of 10(-4) M SNP was significantly less than the control group. SNP at 10(-4) M caused all mouse embryonic development arrest. 46% and 56% of zygote reached the blastocyst stage with the treatment of 10(-6) M and 10(-8) M SNP, respectively, while the control reached 70%. NO adversely affected human sperm activity, human sperm-zona binding and embryonic development. It would appear that high concentration of NO may potentially decrease fertility.     

Removal of zinc from subcellular regions of human spermatozoa by EDTA treatment studied by X-ray microanalysis

The distribution of zinc in the head, neck and mid-piece of human ejaculated spermatozoa was investigated with energy-dispersive X-ray microanalysis. EDTA treatment within 1 h after ejaculation caused removal of 80–90% of the zinc in these regions. Storage for 24 h in own seminal plasma did not alter sperm zinc content. However, these aged spermatozoa only lost 35% of their zinc content on EDTA treatment. The stronger binding of zinc, occurring upon storage in seminal plasma, developed in all 3 parts of the cell investigated. These results are discussed in the light of the role of zinc in nuclear chvomatin decondensation ability and sperm head-tail connection.     

Insulin and leptin enhance human sperm motility, acrosome reaction and nitric oxide production

Aim： To investigate the in vitro effects of insulin and leptin on human sperm motility, viability, acrosome reaction and nitric oxide （NO） production. Methods： Washed human spermatozoa from normozoospermic donors were treated with insulin （10 μIU） and leptin （10 nmol）. Insulin and leptin effects were blocked by inhibition of their intracellular effector, phosphotidylinositol 3-kinase （PI3K）, by wortmannin （10 μmol） 30 min prior to insulin and leptin being given. Computer-assisted semen analysis was used to assess motility after 1, 2 and 3 h of incubation. Viability was assessed by fluorescence-activated cell sorting using propidium iodide as a fluorescent probe. Acrosome-reacted cells were observed under a fluorescent microscope using fluorescein-isothiocyanate-Pisum sativum agglutinin as a probe. NO was measured after treating the sperm with 4,5-diaminofluorescein-2/diacetate （DAF-2/DA） and analyzed by fluorescence-activated cell sorting. Results： Insulin and leptin significantly increased total motility, progressive motility and acrosome reaction, as well as NO production. Conclusion： This study showed the in vitro beneficial effects of insulin and leptin on human sperm function. These hormones could play a role in enhancing the fertilization capacity of human spermatozoa.     

解脲支原体引起男性不育的机理研究——Ⅰ.精子形态学观察

本文对精液解脲支原体(Ureaplasma urealyticum)培养阳性的不明原因不育男性及正常生育男性的精子分别进行扫描电镜及免疫金电镜观察。结果发现在不育男性组;部分精子上有较多解脲支原体吸附;精子畸形率明显增加,主要为严重的卷尾畸形和头尾-成角畸形,此外尚有较多尖头畸形及多精子凝集现象;在解脲支原体附着的部位,精子膜缺损乃至严重破坏;精子的活力也较正常生育组明显下降;部分脱落的生精细胞胞浆内发现解脲支原体样结构,生精细胞胞质崩解。提示解脲支原体感染是男性不育的因素之一,而其对精子形态学的影响是造成男性不育的重要机制。     

Zinc levels in seminal plasma of fertile and infertile men

Zinc levels were measured in seminal plasma from 78 men classified on the basis of spermogram analyses into five groups: normo-, oligo-, astheno-, oligoastheno- and azoospermia. Higher zinc levels were found in seminal plasma from the group of asthenozoospermia men in comparison to normo-, oligoastheno- (p less than 0.001), oligo- and azoospermia men (p less than 0.01), while no significant differences appeared when other group pairs were compared. Seminal plasma zinc levels were positively correlated with sperm density (r = 0.6358, p less than 0.01) in asthenozoospermia men, whereas a significant negative correlation was seen in all groups between percentage forms showing normal progressive motility and zinc concentration in seminal plasma. Although zinc is required in seminal plasma for normal spermatozoon functionality, excessively high levels of this ion may be related with defective motility in asthenozoospermia samples.     

Estimate of oxygen consumption and intracellular zinc concentration of human spermatozoa in relation to motility

Aim: To investigate the human sperm oxygen/energy consumption and zinc content in relation to motility. Methods: In washed spermatozoa from 67 ejaculates, the oxygen consumption was determined. Following calculation of the total oxygen consumed by the Ideal Gas Law, the energy consumption of spermatozoa was calculated. In addition, the zinc content of the sperm was determined using an atomic absorption spectrometer. The resulting data were correlated to the vitality and motility. Results: The oxygen consumption averaged 0.24μmol/106 sperm×24 h, 0.28μmol/106 live sperm×24 h and 0.85μmol/106 live & motile sperm×24 h. Further calculations revealed that sperm motility was the most energy consuming process (164.31 mJ/106 motile spermatozoa×24 h), while the oxygen consumption of the total spermatozoa was 46.06 mJ/106 spermatozoa×24 h. The correlation of the oxygen/ energy consumption and zinc content with motility showed significant negative correlations (r= -0.759; P<0.0001 and r=-0.441; P<0.0001, res     

The biological significance of zinc (author's transl)]

Zinc takes part in the catalytic function of many metalloenzymes. In others it plays a role in conformational stability. In zinc deficient animals protein synthesis is disturbed. Conversely zinc metabolism is influenced by protein deficiency. Zinc takes part in drug metabolism, in mobilizing vitamin A from the liver, and in a system defending the organism against free radical damage. Zinc distribution in the organism is influenced by steroid hormones and leucocytic endogenous mediators. Of the intracellular zinc only a small part is bound to metalloenzymes, most being coordinated to binding sites of nonspecific proteins. Thus the organism defends itself against conformational changes of irritable enzymes which may bind excess zinc to side chains. Zinc can protect the organism against cadmium toxicity. In the serum the smaller part of zinc is firmly bound to several specific proteins, the majority being loosely bound to albumin. Some aspects of human zinc metabolism in health and disease are reviewed. Zinc deficiency in man is rare. In Iran and Egypt a syndrome of iron and zinc deficiency associated with anaemia, hepatosplenomegaly, dwarfism, and hypogonadism is known. In wound healing and tissue repair substitution of zinc is beneficial only if a zinc deficiency exists. For purposes of long term parenteral nutrition zinc should be added to the different infusion solutions.     

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。     

Zinc concentrations and total amount of zinc in seminal plasma of infertile men with special reference to prostatic secretory function

The exact relationship between seminal plasma zinc and fertility is not known. Zinc is secreted mainly by the prostate, and zinc concentration in seminal plasma is regarded as an excellent indicator of prostatic secretory function. However, low zinc concentration may result not only from poor secretory function of the prostate but also from dilution due to excessive secretion of seminal vesicular fluid. This assumption is supported by the present result that zinc concentration was inversely correlated with fructose concentration. Therefore zinc concentration is thought to reflect prostatic function in proportion to seminal vesicular function. Total amount of zinc in seminal plasma seems to be an appropriate indicator for prostatic secretory function. In the present study, concentrations and total amount of zinc were examined in seminal plasma of men with various fertility problems. There were no significant differences between men with normal spermodiagram and those with abnormal spermodiagram, seminal inflammation or varicocele in concentration or total amount of zinc. No changes were observed in any of them after various therapies including oral zinc sulfate. However, the percentage of men with normal spermodiagram was low in the group with extremely low or high zinc concentration and total sperm count tended to increase with increase in total amount of zinc. Furthermore, the spermatozoal motility was better in the prostatic fraction than in the vesicular fraction of split ejaculates, and the percentage of men with decreased motility and normal sperm concentration was significantly high in the group with lower zinc concentration or decreased total amount of zinc. These observations indicate that prostatic secretion has a stimulatory effect on spermatozoal motility. The secretory activities of the prostate and the seminal vesicle are generally known to be closely controlled by androgens, but our findings indicate that the secretory functions of these accessory organs are independent because there was no correlation between total amount of zinc and fructose. Analysis of the relative concentrations in prostatic secretion, split ejaculates, and seminal plasma confirmed an almost exclusively prostatic origin of zinc. As part of the routine andrologic examination, measurement of concentration and total amount of zinc in seminal plasma is useful for evaluating prostatic function, but measurement of acid phosphatase, magnesium, calcium or potassium will provide almost as much information, since they also seem to be secreted primarily by the prostate.