Background Prolonged exposure of human spermatozoa to seminal plasma (SP) causes pronounced deterioration of sperm motility. The effect varies by donor and is consequential for both fertility and diagnostic applications. Whether this is a direct effect on motility, mitochondrial function, or sperm vitality, and whether sperm-independent ageing of seminal plasma plays a role, is unclear. We sought to investigate the cause of this deterioration and underlying mechanism.
Methods Percoll-isolated spermatozoa (min n=6 donors) were incubated in BWW (control) or SP for up to 24 h at 37°C. Enzymes (trypsin, PSA), calcium chelation (BAPTA-AM) and aldehyde scavengers (N-acetylcysteine, L-carnosine) probed inhibitory mechanisms; SP fractionation by size, pre-incubation of SP without spermatozoa (24 h), and media exchange by washing or electrophoresis interrogated the responsible factor and reversibility. Endpoints were motility (CASA), mitochondrial membrane potential (MMP; JC-1) and vitality (far-red LIVE/DEAD).
Results Whole SP and its >100 kDa fraction caused pronounced motility loss by 24 h. The >100 kDa fraction reduced total motility from 66.7±7.3% to 16.4±15.8% (p=0.006), while MMP (52.6±11.6% of live cells) and vitality (72.0±10.1%) were largely retained. SP also reduced MMP (high-MMP from 78.3% vs 43.4%, p=0.013), but motility loss did not correlate with MMP loss. Motility loss was not reversed by washing, nor prevented by proteases, aldehyde scavenging, calcium chelation, or electrophoretic isolation. Aged SP (pre-incubated 24 h) exacerbated motility loss at 3 h versus fresh SP (total 41.5±4.3% vs 54.1±4.5%, p=0.009; progressive 6.6±1.3% vs 12.9±2.2%, p=0.004), without affecting vitality.
Conclusion Seminal plasma undergoes sperm-independent ageing that accelerates motility loss independently of sperm vitality and mitochondrial potential. A soluble, high-molecular-weight activity may underlie the persistent suppression of sperm motility. Sperm-independent SP ageing is thus a previously unrecognised contributor to motility loss, with implications for fertility treatment and diagnostics.