Mitochondrial uncoupler BAM15 attenuates cryopreservation-induced damage in human sperm by stabilizing mitochondrial...

Human sperm cryopreservation is essential for sperm banking and assisted reproduction, yet freeze-thaw stress promotes oxidative injury that reduces motility and damages the acrosome and nuclear DNA. Here, we tested whether the mitochondrial uncoupler BAM15 improves post-thaw human sperm quality and examined mechanisms linked to mitochondrial homeostasis. Ejaculates were cryopreserved using a standard protocol supplemented with graded concentrations of BAM15. After thawing, total and progressive motility and viability were assessed. Flow cytometry quantified the DNA fragmentation index and the proportion of high DNA stainability cells. Mitochondrial membrane potential, intracellular reactive oxygen species, and lipid peroxidation were measured to evaluate mitochondrial function and oxidative status. Ultrastructural preservation of the acrosome, plasma membrane, midpiece mitochondria, and flagellar axoneme was examined by transmission electron microscopy. Compared with untreated controls, BAM15 increased total and progressive motility and improved viability. BAM15 reduced DNA fragmentation and decreased high DNA stainability, indicating enhanced genomic integrity. Consistently, BAM15 improved mitochondrial membrane potential while suppressing intracellular reactive oxygen species and lipid peroxidation, supporting attenuation of freeze-thaw oxidative damage. Transmission electron microscopy further revealed more continuous acrosomal and plasma membranes, fewer swollen or vacuolated midpiece mitochondria, and improved preservation of axonemal architecture. Collectively, these findings identify BAM15 as a promising cryopreservation supplement that stabilizes mitochondrial homeostasis and improves the functional and structural quality of human sperm after thawing.