抗氧化剂保护精子离心过程氧化应激损伤的实验研究

发布时间：2018-06-02 17:38

本文选题：活性氧 + 精液质量　；参考：《广西医科大学》2014年硕士论文

【摘要】：目的：研究通过测定精液活性氧浓度与精液常规参数及与体外受精结局的关系，分析活性氧含量与精液质量及体外受精是否存在相关性，评估精液活性氧在男性不育诊断及治疗中的应用价值。方法：资料来自2012年10至2013年1月因输卵管因素不孕在本中心初次接受IVF-ET诊疗的72对夫妇，运用Diff-Quik精子形态学分析法进行严格精子形态学分析，用苯胺蓝染色法进行精子核成熟度检测，化学发光法检测精液活性氧含量，于体外受精第一天观察受精情况，第三天观察受精卵卵裂及优质胚胎情况。Pearson相关分析法分析精液活性氧含量与精液质量及IVF结局的关系。结果:72例患者精液活性氧含量(72.19±3.56)RLU/s。Pearson相关分析显示精液活性氧含量与精子活率、正常形态率、受精率、正常受精率均呈显著负相关(r=-0.799、-0.827、-0.768、-0.792，P=0.012、0.032、0.000、0.003，均0.05)，与精子密度、不成熟精子率呈正相关(r=0.317、0.794，P=0.008、0.001，均0.05)，与男方年龄、精液量及IVF卵裂率、优胚率无相关性(P均0.05)。结论：精液活性氧含量与精液质量及体外受精结局有相关性。适量活性氧在精子的发生、成熟和获能具有一定的作用，然而，过量活性氧可致精子活力下降，，受精率降低；但对IVF卵裂率和优质胚胎率无显著影响。目的:研究体外离心对正常精子与少弱精子的氧化应激损伤；探讨抗氧化剂谷胱甘肽、左卡尼汀在精子体外离心过程中抗氧化应激损伤的作用效果。方法:按WHO标准选取35份正常精子样本和38份少弱精子样本，每份样本各取1800ul，分为空白对照组、离心对照组、谷胱甘肽离心组和左卡尼汀离心组，各450ul。对照组：仅加入EBSS平衡液不离心；离心对照组：加入EBSS平衡液并离心；谷胱甘肽离心组：加入含有一定浓度谷胱甘肽的EBSS平衡液并离心；左卡尼汀离心组：加入含有一定浓度左卡尼汀的EBSS平衡液并离心，检测各组活性氧（ROS）、丙二醛（MDA）及精子DNA断裂指数（DFI）。统计检验采用随机区组设计方差分析并做组间两两比较。结果:1.四组组间比较均有统计学意义。正常精子组：FROS=280.10、P=0.000；FMDA=15.98，P=0.001；FDFI=74.84，P=0.002，P均0.05；少弱精子组：FROS=9.45、P=0.000；FMDA=15.79，P=0.000；FDFI=13.56，P=0.000，P均0.05。2.两两比较示正常精子组和少弱精子组的离心对照组ROS、MDA及DFI水平较空白对照组均明显升高(P均0.05)；谷胱甘肽离心组与左卡尼汀离心组的ROS、MDA及DFI水平均较离心对照组间明显降低，差异有统计学意义(P均0.05)；而谷胱甘肽离心组及左卡尼汀离心组间的比较均无统计学意义(P均0.05))。结论:正常精子和少弱精子在离心处理过程中均会产生过量活性氧导致过氧化损伤;在精液离心前添加一定浓度的左卡尼汀或谷胱甘肽可减少离心过程中产生的过量活性氧对精子的氧化应激性损伤，从而提高精子质量。
[Abstract]:Objective: to study the relationship between the concentration of reactive oxygen species (Ros) in semen and semen routine parameters and the outcome of in vitro fertilization (IVF), and to analyze the correlation between Ros content and semen quality and in vitro fertilization (IVF). To evaluate the value of semen reactive oxygen species (Ros) in the diagnosis and treatment of male infertility. Methods: from October 2012 to January 2013, 72 couples who were first treated with IVF-ET because of fallopian tube infertility were analyzed by Diff-Quik sperm morphology analysis. The sperm nuclear maturity was detected by aniline blue staining, the active oxygen species in semen was detected by chemiluminescence, and the fertilization was observed on the first day of in vitro fertilization. On the third day, the cleavage of fertilized eggs and high quality embryos were observed. Pearson correlation analysis was used to analyze the relationship between the content of Ros and the quality of semen and the outcome of IVF. Results the results of 72.19 卤3.56)RLU/s.Pearson correlation analysis showed that there was a significant negative correlation between semen Ros content and sperm motility, normal morphology, fertilization rate, normal fertilization rate, and normal fertilization rate. There was a significant negative correlation between Ros content and sperm density in 72 patients with Ros 0.012, 0. 032, 0. 003, 0. 05, 0. 05, and 0. 779 9, 0. 827, 0. 768 and 0. 792%, 0. 032, 0. 000, 0. 003, 0. 05, 0. 05, 0. 05, 0. 05, 0. 05 respectively. There was a positive correlation between immature sperm rate and age, semen volume and cleavage rate of IVF, and there was no correlation between the rate of superior embryo and the age of male, sperm volume and cleavage rate of IVF, and there was no correlation between the rate of superior embryo and the age of male side, the quantity of spermatozoa and the rate of cleavage of IVF. Conclusion: the content of Ros in semen is correlated with the quality of semen and the outcome of in vitro fertilization. Proper amount of Ros had certain effect on spermatogenesis, maturation and capacitation. However, excessive Ros could decrease sperm motility and fertilization rate, but had no significant effect on IVF cleavage rate and high quality embryo rate. Aim: to investigate the effects of antioxidant glutathione and levacarnitine on oxidative stress injury of normal sperm and oligozoospermia during centrifugation in vitro. Methods: according to WHO standard, 35 normal sperm samples and 38 oligozoospermia samples were divided into three groups: blank control group, centrifugal control group, glutathione centrifugation group and levacarnitine centrifugation group, each with 450 ululins. The control group only added EBSS equilibrium solution without centrifugation, the centrifugal control group added EBSS equilibrium solution and centrifugation, the glutathione centrifugation group added EBSS equilibrium solution containing a certain concentration of glutathione and centrifugation. Levacarnitine centrifugation group: the EBSS equilibrium solution containing a certain concentration of L-carnitine was added and centrifuged. The reactive oxygen species (Ros), malondialdehyde (MDA) and sperm DNA breakage index (DNA) were detected in each group. Random block design ANOVA was used to test the statistical results. The result is 1: 1. There were significant differences among the four groups. Normal spermatozoa group: normal spermatozoa group (280.10) P0. 000% FMDAA 15.98% P0. 001 FDFI (74.84) (P = 0.002) P, P = 0. 05; in oligozoospermia group, it is 9. 45% P0. 000 FROS0. 000% FROSU 15. 79P0. 000FDFI 13.56P0. 000 (P = 0. 05.2p = 0. 05.2P = 0. 05.2P = 0. 05.2P = 0. 05.2P = 0. 05.2P = 0.05.2respectively). The results showed that the levels of rosid MDA and DFI in normal sperm group and oligozoospermia group were significantly higher than those in blank control group, while the levels of rosid MDA and DFI in glutathione centrifugation group and leucarnitine centrifugation group were significantly lower than those in control group. The difference was statistically significant (P < 0.05), but there was no significant difference between glutathione centrifugation group and L-carnitine centrifugation group (P < 0.05). Conclusion: both normal spermatozoa and oligozoospermia can produce excessive reactive oxygen species (Ros) and lead to peroxidation damage during centrifugation. Adding a certain concentration of leucarnitine or glutathione to semen before centrifugation can reduce the oxidative stress damage of spermatozoa produced by excessive reactive oxygen species during centrifugation and improve the quality of spermatozoa.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.8

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