Supplementation of freezing and thawing media with brain-derived neurotrophic factor protects human sperm from freeze-thaw-induced damage

Objective To investigate the effects of brain-derived neurotrophic factor (BDNF) supplementation to freezing and thawing media on frozen-thawed human sperm parameters. Design Laboratory study. Setting University hospital. Patient(s) Semen samples from 21 healthy fertile men. Intervention(s) We measured reactive oxygen species (ROS) by flow cytometry using the probes dichlorofluorescin diacetate for intracellular hydrogen peroxide (H 2 O 2 ) and dihydroethidium for intracellular superoxide anion (O 2 –• ), sperm plasma membrane integrity by flow cytometry, caspase-3 activity using ELISA, and AKT phosphorylation status using Western blot in sperm that was cryopreserved and thawed in media either supplemented with BDNF or without BDNF supplementation (control). Main Outcome Measure(s) Sperm motility, viability, ROS levels, caspase-3 activity and AKT phosphorylation. Result(s) The percentage of motile and viable sperm cells was significantly higher in BDNF-supplemented groups as compared with the nonsupplemented (control) group. There was a significant difference in AKT phosphorylation status between BDNF-supplemented groups and the control group. Moreover, the levels of intracellular H 2 O 2 and caspase-3 activity were significantly lower in the sperm cells that were frozen and thawed in media supplemented with BDNF compared with in the control group. Conclusion(s) BDNF supplementation to sperm freezing or thawing media has protective effects against oxidative stress and apoptosis in frozen-thawed human spermatozoa and could improve sperm function, probably through the activation of AKT.