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

Even with the significant advancements in sperm cryopreservation, the addition of intracellular or extracellular antioxidants in preparation and freezing media remains an understudied topic.

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.

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.

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.

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.