Effect of vitamin D on apoptotic marker, reactive oxygen species and human sperm parameters during the process of cryopreservation

Sperm cryopreservation plays an undeniable role in assisted reproductive technology. However, this process significantly reduces the motility, viability, morphology and nuclear integrity of sperm. Reasons of these changes were oxidative stress and apoptosis. The aim of this study was to evaluate the influence of vitamin D on the survival and integrity of fertile sperm after cryopreservation. Semen sample of 18 males with normal parameters was used. After swimming up, each sample was divided into two parts. 20 µmol vitamin D was added to one part as experimental group and the other part was left untreated as control group. The samples in all groups were frozen for 14 days. Post-thawing, the groups were evaluated for sperm motility, and viability using eosin staining, morphology using the Diff-Quick staining and apoptosis by TUNEL, Annexin-V and caspase-3 activity assay. By using nitrobluetetraxolium test and thiobarbituric acid, the reactive oxygen species (ROS) and lipid peroxidation of sperms were measured, respectively. In comparison with control groups, motile and viable sperm concentration was substantially higher in treated groups (P-value<0.05); however, morphological analysis did not show any remarkable changes. Also, ROS and lipid peroxidation values were dramatically reduced by vitamin D (P-value<0.05). TUNEL and Annexin assay for apoptosis were considerably lower in treated groups (P-value<0.05), but caspase activity assay revealed no significant difference between groups. The results have shown that the addition of vitamin D to a freezing medium leads to higher quality and function of human sperm.