Human semen cryopreservation has been widely used as a method of preserving fertility. For this, several methodologies has been used, such as: low freezing and vitrification. In order to reduce freezing damages, several studies used antioxidant and stimulant substances, however, their efficiency as well as their role in the functional control of spermatozoa have not been established in literature. In previous study conduced by our group, we observed that melatonin, an antioxidant, and caffeine, a stimulant, showed benefits in sperm quality when added on normozoospermic seminal samples cryopreserved by low freezing. Thus, the objective of this study is to evaluate the effect of melatonin and caffeine supplementation on sperm quality of post-thaw spermatozoa by slow freezing and vitrification. For this, this prospective study used 30 normozoospermic seminal samples from patients aged from 19 to 45 years in the routine of the Androscience Laboratory and HCFMUSP. Then, the samples were cryopreserved by low freezing and vitrification methods without supplement or with 2mM of melatonin. After thawing, the samples were analyzed or supplemented with 2 mM of caffeine. At the end of the experiments, we obtained 4 groups: Post-thaw samples without supplementation or supplemented with melatonin and caffeine, by low freezing or vitrification methods. Seminal parameters for count and motility, analyzed by WHO criteria, mitochondrial activity, by diaminobenzidine staining, evaluation of DNA fragmentation rate, by the SCSA® method, and dosage of radical oxygen species (ROS) levels, by the luminescence method, pre-cryopreservation and post-thaw were carried out with or without supplementation. The data were analyzed by the paired Student's t-test and by one-way analysis of variance with repeated measurements, followed by the Holm-Sidak post-test, adopting an alpha of 5%.
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