Evaluation of prolonged in vitro incubation of human sperm at room temperature

The longevity in vitro of viability and motility of spermatozoa from a single ejaculate is seldom studied. The physiological decay of human sperm under room temperature in vitro remains a critical yet under-explored area. While standard clinical procedures typically involve short-term sperm handling, the development of novel techniques demands a deeper understanding of 'shelf-life' of sperm. This pilot study aimed to characterise human sperm longevity at room temperature by longitudinally assessing motility, viability and DNA fragmentation, and to evaluate whether culture medium replacement could extend in vitro sperm motility. Twenty-six semen samples with sperm concentration ≥ 15 million/ml and motility ≥ 40% were collected and motile sperm were concentrated using discontinuous gradient centrifugation. A subset of 20 samples was evaluated to determine the longitudinal dynamics of motility and viability. To establish the temporal profile of DNA damage, processed samples were collected using the swim-up method at Days 0, 3, 6, 9 and 12. The remaining 6 samples were used for motility parameter observations using computer-assisted semen analysis both with and without culture medium replacement. Following initial processing, the samples exhibited a baseline motility of 91.2 ± 3.4% and viability of 94.6 ± 3.5%. During 20-day incubation, sperm motility and viability declined in a time-dependent manner, with motility decreasing more rapidly than viability. Motility persisted for an average of 13.4 days. The 20 samples showed viability of 13.4 ± 4.4% at D20. In 10 samples that were fully observed for viability, the longevity range being 35-50 days. The D3 (2.4 ± 1.4%) and D6 (5.7 ± 5.8%) DNA fragmentation levels of progressively motile sperm showed no differences with D0 levels (3.9 ± 2.8%), but at later time points, progressively motile sperm showed increased DNA fragmentation, reaching 9% at D9 (p = 0.003) and 11.2% at D12 (p = 0.01). Additionally, processed sperm exposed to regular culture medium replacement showed prolonged longevity in regard to motility. In this pilot study, after routine processing in vitro and left at room temperature, human sperm had a motility longevity of between 8 and 18 days and a viability longevity of between 35 and 50 days, although after D9 progressively motile sperm displayed significantly increased DNA damage. Regular replacement of culture medium could support and prolong sperm motility longevity.