Efeito do resveratrol e do ácido ascórbico na criopreservação de sêmen humano

Abstract

Cryopreservation of human semen is very important in reproductive medicine, but it may cause damages to the spermatozoa, probably due to the production of reactive oxygen species (ROS), which may cause structural damage to the biomolecules. Resveratrol and ascorbic acid are important antioxidants, however, until now, there are only few studies about its use in cryopreservation process. Therefore, the aim of this study was to analyze the oxidative stress markers and seminal standard parameters before and after the cryopreservation of human semen with and without these antioxidants. Ejaculated semen from 20 infertile men and 10 fertile donors, with proven fertility, were examined. Individuals with azoospermia, leukocytospermia or under antioxidant supplementation were excluded from the study. Oxidative stress markers including the determination of the levels of thiobarbituric acid reactive species (TBARS) and the activity of the antioxidant enzymes superoxide dismutase (Sod) and catalase (Cat). DNA damages were quantified by comet assay. After the evaluation of standard seminal assays, semen samples were divided into 5 aliquots, which were treated as follows: (a) without antioxidants; (b) with 0.1 mM of resveratrol; (c) with 1.0 mM of resveratrol; (d) with 10.0 mM of resveratrol and (e) with 10.0 mM of ascorbic acid. Samples were incubated for 1 hour and, then, they were cryopreserved in liquid nitrogen at -196°C. The TEST-Yolk buffer was used as cryoprotector agent in all the samples. Besides, it was evaluated, also, the seric values of TBARS, Sod, Cat and uric acid both in infertile and fertile men. The results showed that the use of ascorbic acid or resveratrol did not change the concentration and morphology of the spermatozoids. However, none of the antioxidants were able to prevent the decrease in the mobility induced by the cryopreservation process. Cryopreservation causes a decrease in Sod activity in infertile men and an increase of TBARS levels, both in infertile and fertile men. It was observed a negative correlation between spermatozoa motility and TBARS levels in infertile men, both before (-0, 868, p<0,05) and after (-0, 897, p<0,05) the cryopreservation process. Resveratrol was able to avoid the decrease in Sod activity in infertile men and the increase in TBARS levels both in infertile and fertile men samples. Ascorbic acid was able to reduce the oxidative lipid damages induced by cryopreservation only in infertile men. It was also observed that the cryopreservation process induced a significant increase of DNA damages (mainly types 2, 3 and 4) in infertile and fertile men. The addition of ascorbic acid reduces DNA damages only in infertile men. On the other hand, resveratrol was capable to reduce DNA damages both in infertile and fertile men. In fact, the use of 10.0mM of resveratrol was able to avoid all the DNA damages induced by the cryopreservation in infertile men. Infertile men showed high seric levels of TBARS, uric acid and Sod activity than the fertile men. Although other studies are necessary, the data presented in this work suggests the possibility to use resveratrol and ascorbic acid in human cryopreservation procedures, both for fertile and infertile men, at least, for ICSI (Intracytoplasmic Sperm Injection), as it does not need motile sperm for achieving pregnancy.