抗氧化剂改善冻融人精子氧化应激性DNA损伤的研究

目的 探讨抗坏血酸盐、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)对冻融人精子氧化应激性DNA损伤的可能保护作用.方法 30份健康可生育男性精液分别添加改良人精子冷冻保护液,依所添加的抗氧化剂及终浓度分6组;检测各组冻融前后常规精子参数,精子核DNA完整性以及冻融后活性氧(ROS)水平.结果 (1)冻融后抗坏血酸盐300 μmol/L组与CAT 200 U、400 U组a+b级精子下降少于对照组(均P＜0.05),精子活率复苏率、ROS水平则分别高于和低于对照组(67%±14%、68%±14%、69%±15%vs 59%±10%,均P＜0.05;30±13、30±11、30±11 vs 37±17,均P＜0.05).(2)添加抗坏血酸盐300 μmol/L组,CAT 200 U、400 U组的彗星细胞尾部DNA百分比及Olive尾矩与冷冻前相比,差异无统计学意义(P＞0.05);但分别低于对照组(41%±4%、40%±7%、40%±6%vs 46%±6%,均P＜0.01;7.7±1.2、7.5±1.6、7.8±1.9 vs 10.1±3.1,均P＜0.01).其余试验组上述指标则明显高于冷冻前(均P＜0.01),而与对照组差异无统计学意义(P＞0.05).(3)冻融后各实验组a+b级精子与ROS水平有负相关性(P＜0.05或P＜0.01).除抗坏血酸盐600 μmol/L组外,其余试验组尾部DNA%、Olive尾矩则分别与其对应组ROS有显著正相关性(P＜0.05、P＜0.01).结论 在精液冷冻保护剂中添加一定浓度的抗坏血酸盐、CAT可减少冷冻产生的过量ROS,进而减轻后者对精子核DNA的氧化应激性损伤,从而提高冻融人精子质量.

Reducing oxidative DNA damage by adding antioxidants in human semen samples undergoing cryopreservation procedure

OBJECTIVE: To evaluate the protective effects of oxidative DNA damage by adding antioxidants: ascorbate, catalase (CAT), and superoxide dismutase (SOD) in human semen samples undergoing cryopreservation procedure. METHODS: Semen sample form 30 fertile men were mixed with modified cryoprotectant and divided into six groups according to the category and concentration of antioxidants: ascorbate 300 micromol/L, ascorbate 600 micromol/L, CAT 200 U/ml, CAT 400 U/ml, SOD 200 U/ml, and SOD 400 U/ml. Comet assay was conducted to measure the percentage of comet cells, and the nuclear DNA damaged parameters: tail DNA percentage (TD%) and Olive tail moment (OTM). Flow cytometry was used to detect the reactive oxidative species (ROS). The motility (a + b grade), viable recovery rate, nuclear DNA integrity and reactive oxidative species (ROS) of all groups were analyzed before and/or after freeze-thawing. RESULTS: (After cryopreservation, compared with the control group, the a + b grade sperm rates of the ascorbate 300 micromol/L, CAT 200 U, and CAT 400 U groups were all higher than that of the control group (all P < 0.05), however, the levels of reactive oxygen species (ROS) of the ascorbate 300 micromol/L, CAT 200 U, and CAT 400 U groups were 30 +/- 13, 30 +/- 11, and 30 +/- 11 respectively, all significantly lower than that of the control group (37 +/- 17 , all P < 0.05). The viable recovery rates of the ascorbate 300 micromol/L , CAT 200 U, and CAT 400 U groups were 67% +/- 14%, 68% +/- 14%, and 69% -/+ 15% respectively, all significantly higher than that of the control group (59% +/- 10%, all P < 0.05). (2) The TD% levels of the ascorbate 300 micromol/L, CAT 200 U, and CAT 400 U groups were 41% +/- 4%, 40% +/- 7%, 40% +/- 6%, all similar to that of the raw semen (all P > 0.05), but significantly lower than that of the control group (46% +/- 6%, all P < 0.01). The OTM levels of the ascorbate 300 micromol/ L, CAT 200 U, and CAT 400 U groups were 7.7 +/- 1.2, 7.5 +/- 1.6, and 7.8 +/- 1.9, all similar to that of the raw semen (all P > 0.05), but significantly lower than that of the control group (10.1 +/- 3.1, all P < 0.01) too. The TD% and OTM levels of the other groups were all significantly higher than that of the raw semen (all P < 0.01), but not significantly different from those of the control group (all P > 0.05). (3) ROS was significantly negatively correlated with the motility in all groups (P < 0.05 or P < 0.01). Apart from the ascorbate 600 micromol/L group, the TD% and OTM of the other groups were all significantly positively correlated with the ROS (P < 0.05 or P < 0.01). CONCLUSION: Supplementation of ascorbate or CAT reduces the level of ROS that induces sperm nuclear DNA damage, and improves the human sperm quality in the process of freeze-thawing.