[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).


CONCLUSIONS
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.
Li
Zhi-ling
ZL
Reproductive Center, First Affiliated Hospital of Medical College, Shantou University, Shantou, China.
Lin
Qiong-lin
QL
Liu
Rong-ju
RJ
Xie
Wen-yan
WY
Xiao
Wan-fen
WF
D000328:Adult; D000975:Antioxidants / Q000494:pharmacology*; D001205:Ascorbic Acid / Q000494:pharmacology; D002374:Catalase / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects*; D006801:Humans; D008297:Male; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D012661:Semen / Q000187:drug effects* / Q000378:metabolism; D012662:Semen Preservation / Q000379:methods*; D013482:Superoxide Dismutase / Q000494:pharmacology
D000975:Antioxidants; D017382:Reactive Oxygen Species; D002374:Catalase; D013482:Superoxide Dismutase; D001205:Ascorbic Acid