弱精症男子与正常人精子膜尿激酶型纤溶酶原激活因子含量的研究

按ＷＨＯ精液参数标准，分别选择精子活力低下的男性不育症患者和正常生育力男子精液标本各２０例，将洗涤后的精子用含１％ＴｒｉｔｏｎＸ－１００的０．１ＭＴｒｉｓ－ＨＣｌ缓冲液处理，提取精子膜结合尿激酶型纤溶酶原激活因子（ｕＰＡ），然后采用抗人尿激酶多克隆抗体，进行各样品ｕＰＡ含量的测定（夹心法ＥＬＩＳＡ）。结果显示：弱精症男子精子膜结合ｕＰＡ（２３．１０±７．３５ｍｕ／１０６ｃｅｌｓ）显著性低于正常人精子膜ｕＰＡ（２９．８９±９．４８ｍｕ／１０６ｃｅｌｓ），Ｐ＜０．０２５。且精子膜ｕＰＡ含量与精子活率呈直线正相关（ｒ＝０．６４，Ｐ＜０．０００１），与精子活力（直线前向运动精子百分率）亦呈线性相关（ｒ＝０．４８，Ｐ＜０．００５）。说明精子膜结合ｕＰＡ可能与精子活力及生育力之间有一定关联。     

腺苷脱氨酶在人精子膜上的定位及其单克隆抗体对人精子穿卵能力的影响

利用本实验室制备的抗人精子膜结合腺苷脱氨酶（ＡＤＡ）单克隆抗体（ＭｃＡｂ）和间接荧光免疫技术，分析了该酶在正常人精子膜上的位置。同时，利用该酶单抗分析了该酶活性对人精子穿透去透明带卵的影响。上述两个实验的结果显示：（１）ＡＤＡ结合于人精子中段、尾部膜的外侧；（２）利用抗ＡＤＡＭｃＡｂ抑制ＡＤＡ活性可明显抑制正常人精子穿透去透明带卵的能力。这一研究结果提示，ＡＤＡ作为精子的膜蛋白组分，可能参与对精子生理功能的调节过程。     

腺苷脱氨酶在人精子膜上的定位及单克隆抗体对人精子穿卵能力的影响

利用本实验室制备的抗人精子膜结合腺苷脱氨酶（ＡＤＡ）单克隆抗体和间接荧光免疫技术，分析了该酶在正常人精子膜上的位置。同时，利用该酶单抗分析了该酶活性对人精子穿透去透明带卵的影响。上述两个实验的结果显示：（１）ＡＤＡ结合于人精子中段，尾部膜的外侧；（２）利用抗ＡＤＡＭｃＡｂ抑制ＡＤＡ活性可明显抑制正常人精子穿透去透明带卵的能力。这一研究结果提示，ＡＤＡ作为精子的膜蛋白组分，可能参与精子生理功能的调节     

胆道梗阻后肝细胞线粒体钙含量变化和肝脂质过氧化损害的实验研究

为了解胆道梗阻对肝脏的损害机理,在复制大鼠胆道梗阻模型基础上,分离肝细胞线粒体,动态检测肝细胞线粒体钙含量,肝组织ＭＤＡ、ＳＯＤ含量,血清ＴＢｉｌ、ＡＬＴ、ＡＬＰ及ＧＧＴ含量。结果：肝细胞线粒体钙含量、肝组织ＭＤＡ含量和血清ＴＢｉｌ、ＡＬＴ、ＡＬＰ及ＧＧＴ水平均随梗阻时间延长而逐渐升高（Ｐ＜０．０５）,肝组织ＳＯＤ含量则逐渐减少（Ｐ＜０．０５）;肝细胞线粒体钙含量与肝组织ＭＤＡ含量、血清ＡＬＴ及ＡＬＰ含量变化呈明显正相关,ｒ分别为０．９６７、０．９２４和０．９１９（Ｐ＜０．０１）;肝组织ＭＤＡ含量与血清ＡＬＴ和ＡＬＰ含量变化呈明显正相关,ｒ分别为０．９４９和０．８４３（Ｐ＜０．０１）。结论：肝细胞线粒体钙超载和脂质过氧化损伤密切相关,在胆道梗阻所致肝损害过程中起重要作用。     

弱精症男子与正常人精浆和精子膜尿激酶酶活性的研究

应用琼酯糖－纤维蛋白－平板法和抗人尿激酶多克隆抗体的免疫阻断法，分别测定了２０名正常生育力男子和２２例精子活力低下（弱精症）的男性不育症患者精浆中及精子膜尿激酶型纤溶酶原激活因子（ｕＰＡ）的酶活性。结果：弱精症组精浆ｕＰＡ活性为２１３４±１５８１ＩＵ／Ｌ，正常生育组精浆ｕＰＡ活性为３３６５±１８５９．５ＩＵ／Ｌ，两组间差异有显著性意义（Ｐ＜０．０５）。弱精症组精子膜ｕＰＡ活性为５．１３±３．８２ｍＵ／１０６ｃｅｌｓ，正常生育组精子膜ｕＰＡ活性为１０．１７±６．１８ｍＵ／１０６ｃｅｌｓ，两组间差异有极显著性意义（Ｐ＜０．００５）。精子膜ｕＰＡ活性值与精子活率、活力亦呈线性相关。提示精子膜ｕＰＡ酶活性与精子活力及生育力可能有一定关系。     

慢性细菌性前列腺炎对精液质量和精子功能的影响

为了探讨慢性细菌性前列腺炎引起不育的机理，本文对１７例经细菌学检查诊断为慢性细菌性前列腺炎（ＣＢＰ）的不育患者和７例正常生育力者进行了研究。包括精液常规检查、ＳＶＴ和精子低渗肿胀试验，精液丙二醛（ＭＤＡ）和精浆ＳＯＤ测定，抗精子抗体与精浆Ｔ细胞ＣＤ４／ＣＤ８比例测定等。结果提示：１．ＣＢＰ可通过多方面机制影响精液质量和男性生育力；２．ＣＢＰ患者精液中白细胞及其活性产物在精子膜过氧化机制中发挥重要作用，并影响精子功能。     

慢性细菌性前列腺炎对精液质量和精子功能的影响

为了探讨慢性细菌性前列腺炎引起不育的机理，本文对１７例经细菌学检查诊断为慢性细菌性前列腺炎（ＣＢＰ）的不育患者和７例正常生育力者进行了研究，包括精液常规检查、ＳＶＴ和精子低涌肿胀试验，精液丙二醛（ＭＤＡ）和精浆ＳＯＤ测定，抗精子抗体与精浆Ｔ细胞ＣＤ４／ＣＤ８比例测定等。结果提示：１．ＣＢＰ可通过多方面机制影响精液质量和男性生育力；２．ＣＢＰ患者精液中白细胞及其活性产物在精子膜过氧化机制中发挥重要     

腺苷脱氨酶在人睾丸及附睾的分布

应用本实验室前期制备的特异性的抗人精子膜结合腺苷脱氨酶（ＡＤＡ）同功酶的单克隆抗体（ＭｃＡｂ）和免疫组化技术，我们分析了该同功酶在正常人睾丸和附睾中的分布。实验结果显示：（１）ＡＤＡ在正常人睾丸中无分布，而在附睾头部，附睾管腔上皮细胞中开始分布；（２）ＡＤＡ在正常人附睾管腔上皮细胞中的分布，由附睾头部至尾部，其密度递增。这一研究结果提示：（１）ＡＤＡ分布具有附睾特异性；（２）人精子膜结合ＡＤＡ可能来自附睾管腔上皮细胞。     

精液白细胞与精子功能间关系的研究

为探讨精液中白细胞与精液质量的关系，对１１例生育男性、１８例生殖道感染性不育及１６例特发性男性不育患者，分别进行精液常规分析、精液白细胞计数、ＭＤＡ测定、精子活力测定及精子细胞膜完整性的测定。结果显示：（１）生育组白细胞密度为７３．２±２９万／ｍｌ，显著低于感染组（１４６．１±９２．２万／ｍｌ）及弱精子症组（１１１．３±５３．４万／ｍｌ），同时精子活动率、ＳＶＴ、精子头部膜完整率、尾部膜完整率均显著高于其它二组；生育组精液中ＭＤＡ值为４．５９１±２．５２１ｎｍ／ｍｌ，显著低于不育组。死亡率显著低于其它二组。（２）相关分析显示ＭＤＡ与白细胞密度成正相关，与精子活动率、ＳＶＴ、膜完整率成负相关。研究结果显示：（１）精液白细胞可通过膜脂质过氧化反应对精子功能造成损伤；（２）膜脂质过氧化反应可伴随白细胞的增加而增加，但同时又受精液中ＳＯＤ的抑制。结论：在病理情况下，白细胞增多可通过ＭＤＡ导致精子运动力、活动率下降，精子膜完整性受损，从而使精子受精能力下降。     

大鼠附睾精子成熟过程中膜流动性变化的研究

精子在附睾内的运行过程中，结构和功能发生变化，最终达到成熟，并贮存于附睾尾部。本文用５－Ｄｏｘｙｌ－ｓｔｅａｒｉｃａｃｉｄ（５ＤＳＡ）和１６－Ｄｏｘｙｌ－ｓｔｅａｒｉｃａｃｉｄ（１６ＤＳＡ）为标记，在电子自旋共振（ＥＳＲ）波谱仪上研究成年ＳＤ雄鼠附睾头、体、尾各段的精子膜流动性的变化，并用低渗膨胀（ＨＯＳ）试验测定精子膜对渗透压的反应能力。结果表明，从附睾头部到尾部，精子膜膜脂的流动性逐步降低，特别是从体部到尾部更为明显（Ｐ＜０．０１），而精子膜对渗透压的反应能力也逐渐降低，主要表现在从头部移行到体部的精子（Ｐ＜０．０１）。精子成熟过程中的脂质过氧化反应，可能是降低膜脂流动性的原因之一。     

冻精损伤和精子膜脂质过氧化反应的关系

为研究冻精损伤与精子膜脂质过氧化反应的绎１０例正常自愿者精液作冷冻处理，检测冷冻前后精质过氧化物的水平变化及在冷冻剂中加入抗氧化剂ＶｉｔＣ，三冻精活动率，存活率是否改善。     

体外添加黄芪注射液对人精子膜拮抗脂质过氧化的作用

目的 :观察体外添加黄芪注射液对弱精子症患者精子膜脂质过氧化的拮抗作用。方法 :应用计算机辅助精子分析仪 (CASA)和丙二醛 (MDA)及超氧化物歧化酶 (SOD)检测试剂盒 ,分别分析体外添加黄芪注射液 (A组 )对 30例弱精子症患者精子运动参数的影响 ,检测精子悬液MDA含量及总SOD(TSOD)力 ,并进行相关分析 ,同时设立空白对照组进行对比观察。结果 :A组的精子活率 (Mot)、前向运动精子百分率、曲线速度 (VCL)、平均路径速度 (VAP)和精子头摆幅度 (ALH)较空白对照组显著提高 (P <0 .0 5或P <0 .0 1) ;A组精子悬液的MDA含量显著降低 (P <0 .0 5 ) ,TSOD活力与对照组比较差异无统计学意义 (P >0 .0 5 )。A组MDA含量与Mot、前向运动精子百分率、VAP和ALH间均有显著性负相关 (P <0 .0 5或P <0 .0 1)。结论 :体外添加黄芪注射液对弱精子症患者精子具有拮抗脂质过氧化作用 ,为临床应用黄芪减少递质对精子的损害提供理论基础。     

Antioxidative effect of melatonin on human spermatozoa

The ability of melatonin to suppress experimentally induced lipid peroxidation (LPO) in sperm membrane was investigated in 41 samples of infertile men. Iron/ascorbate (0.04/0.2 mmol)-induced LPO was measured by the formation of malondialdehyde (MDA) using the thiobarbituric acid method. Sperm incubated in the presence of melatonin (2-6 mmol) exhibited a concentration-dependent decrease of MDA generated from hydroperoxide of the sperm plasma membrane in the presence of promoter system. Addition of 6 mmol of melatonin significantly reduced the rate of lipid peroxidation in sperm of unselected donors (mean +/- SE in control samples = 26.4 +/- 2.9 vs. 6.5 +/- 1.1 nmol MDA/10(8) sperm in melatonin-treated samples: n = 16, p < .005). Inhibitory effect of melatonin was also significant in the presence of 0.015 mmol of ferrous ions (20.5 +/- 1.7 vs. 7.9 +/- 1.6 nmol MDA/10(8) sperm in melatonin-treated samples: n = 7, p < .02) and (.005 mmol of ferrous ions (20.2 +/- 2.8 vs. 9.9 +/- 2.4 nmol MDA/ 10(8) sperm: n = 6, p < .05). Comparing the effect of melatonin with that of Trolox, an analog of vitamin E. a similar effect at concentration of 0.1-0.2 mmol of Trolox was found (25.2 +/- 2.9 vs. 11.8 +/- 1.2 nmol MDA/10(8) sperm in Trolox-treated samples: n = 7, p < .005). The obtained data of in vitro experiments show that melatonin is 40-fold less efficient than Trolox in achieving the 50% reduction in LPO (4 vs. 0.1 mmol). Since the physiological concentration of melatonin in human semen is at the nanomolar level, its antioxidative role in vivo is probably of minor importance.     

Total antioxidant status and lipid peroxidation with and without in vitro zinc supplementation in infertile men

The aim of this study was to assess the total antioxidant capacity (TAC) and malondialdehyde (MDA) level in infertile men with asthenozoospermia and asthenoteratozoospermia compared to fertile donors, and to examine the effect of zinc on sperm lipid peroxidation and antioxidant status in infertile and fertile men. Semen samples provided by infertile men (n = 38) and fertile donors (controls; n = 12) were exposed to 6 mmol/L of zinc for 2 hr at 37°C. After semen analysis, lipid peroxidation was detected by MDA assay and seminal TAC was assessed by colorimetric method using TAS (total antioxidant status) Kit. TAC was significantly lower in infertile group compared to controls (p = .037). However, lipid peroxidation did not alter in infertile patients compared to controls (p > .05). After in vitro incubation of samples with zinc, a significant increase in TAC level was found only in infertile men (p < .001). Meanwhile, zinc had no effect on sperm lipid peroxidation in both fertile and infertile men (p > .05). Our data indicate that antioxidant treatment based on zinc in vitro supplementation may be helpful to enhance the rate of seminal antioxidant status in infertile men; however, it does not prevent sperm lipid peroxidation.     

Interleukin-6 (IL-6) in seminal plasma of infertile men, and lipid peroxidation of their sperm

Concentrations of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in seminal fluid, as well as levels of sperm lipid membrane peroxidation, were investigated in fertile and infertile men. Semen samples, obtained by masturbation from 37 infertile and 14 fertile men, were examined for the presence of TNF-alpha and IL-6. The level of lipid peroxidation of the sperm membrane was measured by determining malondialdehyde (MDA) formation. The correlation between the IL-6 and the TNF-alpha concentrations in seminal plasma with the levels of lipid peroxidation of the sperm membranes was statistically evaluated. The IL-6 concentration in seminal plasma of infertile men was significantly higher than that of fertile men (p < .05). Similarly, the level of membrane lipid peroxidation was higher for the semen of infertile men than that of fertile men (p < .001). A significant positive correlation was found between IL-6 levels in seminal plasma and membrane sperm lipid peroxidation (p < .002), but not between this parameter and TNF-alpha levels in seminal plasma. These findings suggest a possible association between IL-6 seminal plasma levels and lipid peroxidation of sperm membrane. Stimulation of reactive species production by human sperm and leucocytes, induced by the high levels of IL-6, could explain these results.     

Sperm oxidative stress and the effect of an oral vitamin E and selenium supplement on semen quality in infertile men

Numerous studies have reported beneficial effects of antioxidant drugs on semen quality, but there is no well-defined therapeutical protocol in male infertility. This study aimed to test the effects of vitamin E and selenium supplementation on lipid peroxidation and on sperm parameters. The study included 54 voluntary and infertile men who produced semen samples for spermiogram and for spectrophotometric measurement of a lipid peroxidation marker, the malondialdehyde (MDA), and produced blood samples for high-performance liquid chromatography assessment of serum vitamin E level. The trial was randomized and open. Twenty-eight men were supplemented daily by vitamin E (400 mg) and selenium (225 microg), during 3 months. The remaining 26 patients received vitamin B (4,5 g/day) for the same duration. Only 20 patients achieved their treatment and returned for control analysis. MDA concentrations in sperm were much less than in seminal plasma and motility and viability were inversely correlated with semen MDA levels. In contrast to vitamin B supplementation, vitamin E and selenium supplementation produced a significant decrease in MDA concentrations and an improvement of sperm motility. The results confirm the protective and beneficial effects of vitamin E and selenium on semen quality and advocate their use in male infertility treatment.     

Human sperm motility and lipid peroxidation in different ascorbic acid concentrations: an in vitro analysis

Summary.  Human spermatozoal motility, viability and lipid peroxidation (LPO) have been assessed in Ringer-Tyrode supplemented with different concentrations of ascorbic acid (AA) ranging from 50 to 4000 μ m . Ascorbic acid in concentrations below 1000 μ m protects spermatozoa from free radical damage as evidenced from improvement in their motility and viability. Concomitantly, there is also witnessed depletion of malondialdehyde generation (an end product of LPO) following AA treatment. Ascorbic acid at 1000 μ m concentration and above, however, is not protective, as evidenced by abrupt fall in sperm motility and viability and concomitant increase in LPO.     

Free radical scavenger effect of rebamipide in sperm processing and cryopreservation

Aim: To study the effect of rebamipide added to semen samples and cryoprotectant on reactive oxygen species (ROS) production. Methods: Semen samples from 30 fertile and healthy volunteers were collected by masturbation after 2 days - 3 days of abstinence. After liquefaction, the specimens were diluted with sperm wash media to a uniform density of 20×106/mL. Rebamipide was added to semen samples and cryoprotectant to a final concentration of 10 μmol/L, 30 μmol/L, 100 μmol/L or 300 μmol/L. Specimens were incubated at 37 ℃ in a 0.5 % CO2 incubator for 1 h or cryopreserved at -196 ℃ LN2 for 3 days. The sperm motility and viability and the levels of ROS and lipid peroxidation of sperm membrance were assessed before and after incubation and cryopreservation by means of computer assisted semen analyzer, eosin-nigrosin stain, chemiluminescence and thiobarbituric acid assay, respectively. Results: The sperm motility was significantly increased after incubation with 100 μmol/L and 300 μmol/L rebamipide (P     

Chlamydia trachomatis and sperm lipid peroxidation in infertile men

Aim: To relate the presence of anti-Chlamydial trachomatis IgA in semen with sperm lipid membrane peroxidation and changes in seminal parameters. Methods: Semen samples of the male partners of 52 couples assessed for undiagnosed infertility were examined for the presence of IgA antibody against C. trachomatis. The level of sperm membrane lipid peroxidation was estimated by determining the malondialdehyde (MDA) formation. Results: Sperm membrane of infertile males with positive IgA antibodies against C. trachomatis showed a higher level of lipid peroxidation than that of infertile males with negative IgA antibody (P<0.05). There was a positive correlation (P< 0.01) between the level of C. trachomatis antibody and the magnitude of sperm membrane lipid peroxidation. All the other tested semen parameters were found to be similar in the two groups. Conclusion: The activation of immune system by C. trachomatis may promote lipid peroxidation of the sperm membrane. This could be the way by which C. trachomatis a     

Lipid peroxidation in human spermatozoa and maintenance of progressive sperm motility

Washed and deep frozen spermatozoa of 46 patients from an infertility clinic were separated into 3 different groups depending on their progressive motility (expressed as the sperm motile efficiency index according to Ishii et al ., 1977), determined 0 and 3  h after liquefaction, and were examined for their lipid peroxidation (LPO) potential by means of the thiobarbituric acid assay. Spontaneous and iron-catalysed generation (after 15, 30 and 60  min incubation) of thiobarbituric acid-reactive substances (TBARS) was measured spectrophotometrically. Spontaneous LPO revealed the highest generation of TBARS in the group of spermatozoa with initially normal progressive motility and decreased maintenance of progressive motility after 3  h of aerobic incubation. Iron-catalysed LPO generally revealed the highest amounts of TBARS after 60  min, especially in the aforementioned group with decreased motility maintenance. The differences between this group and the two other groups were highly significant. Consequently, spermatozoa with initially normal progressive motility but decreased maintenance of motility, generated higher amounts of stable LPO products than others, which suggests that loss of motility under aerobic incubation seems to be the consequence of enhanced LPO processes.