Comparing Extracellular and Intracellular Antioxidants in Human Sperm Rapid Freezing: Hypotaurine Versus Melatonin.

Abstract

Introduction: Even with the significant advancements in sperm cryopreservation, the addition of intracellular or extracellular antioxidants in preparation and freezing media remains an understudied topic. Objective: We examined the effects of hypotaurine and melatonin on the routine and functional tests of sperm and the expression of HspA2 and Caspase9 during the human sperm rapid freezing process. Methods: Following the collection of 34 normospermia semen samples, each sample was split into four experimental groups: fresh (F), freezing control (C) (human tubal fluid medium and 0.5M sucrose), and two freezing groups with the inclusion of 2 mM melatonin (MEL) and 50 mM hypotaurine (HYP). A straw held 100 μL of the sample, which was then cryopreserved in liquid nitrogen to accomplish rapid freezing. Before and after rapid freezing-thawing, the sperm classical parameters and the expression levels of HspA2 and Caspase9 were assessed. Results: The HYP group exhibited higher normal morphology (p < 0.001), viability (p < 0.001), and higher acrosome integrity (p < 0.001) and lower DNA fragmentation index (DFI) (p < 0.001) than the C and MEL groups. No significant difference was observed in the total and progressive motility percentage among the antioxidant and frozen control groups. The MEL group had a significantly higher level of HspA2 mRNA compared with F and C groups (p < 0.05). The expression of Caspase9 was unaffected by including MEL and HYP in all experimental groups. Conclusion: Hypotaurine, as an extracellular antioxidant, is more effective than melatonin as an intracellular antioxidant in reducing deleterious cryoinjuries on morphology, viability, acrosome reaction, and DFI.