P-053 Torin 2 as a Potential Enhancer of Human Sperm Motility: A Dose- and Time- Dependent Analysis

Abstract Study question Can Torin 2, an mTOR inhibitor, enhance human sperm motility in vitro without compromising viability? Summary answer Torin 2 can enhance human sperm motility in a dose- and time-dependent manner, with minimal impact on sperm viability at optimal concentrations. What is known already Globally, male factor infertility accounts for 30-50% of infertility cases with impaired sperm motility being a leading cause. Enhancing motility is crucial for improving natural conception and assisted reproduction outcomes. Various group of compounds, including phosphodiesterase inhibitors, xanthine derivates and lysophosphatidic acid, have been explored to enhance sperm motility, but often lack specificity and raise concerns about embryo toxicity. Torin 2, a potent ATP-competitive mTOR inhibitor with established anticancer, cell proliferation and anti-inflammatory properties, presents a novel but underexplored approach for sperm motility enhancement. However, its effects on sperm function and safety profile in reproductive applications remain unknown. Study design, size, duration Samples underwent basic semen analysis per WHO6, followed by cryopreservation and thawing, then were divided into three groups: Control (DMSO only), Group 2 (0.25 µM Torin 2), and Group 3 (0.5 µM Torin 2). Motility and vitality were analysed at baseline, and at 15-, 30-, and 60-minutes post-treatment using using computer-assisted semen analysis (CASA) and eosin-nigrosin staining. All groups were tested simultaneously, and experiments were repeated per donor. Ethical approval was obtained (EoSRES REC 1). Participants/materials, setting, methods Eighteen male (18–40 years) recruited per HFEA Code of Practice provided semen samples. After semen analysis, normozoospermia samples were assessed for motility and vitality using CASA and eosin-nigrosin staining. Following cryopreservation and thawing, assessments were repeated at 15-, 30-, and 60-minutes post-treatment. Motility was categorized as progressive, non-progressive, or immotile, while vitality was classified as alive or dead. Paired t-tests analysed motility changes post-Torin 2. Two-way ANOVA assessed treatment-time interactions on motility and vitality. Main results and the role of chance After sperm preparation (before freezing), the observed progressive (A+B), non-progressive and immotile sperm cells were 59.58 ± 20.49%, 4.44 ± 2.67 % and 35.89 ± 20.96 % respectively. Cryopreservation reduced motility, increasing immotile sperm to 91.78 ± 6.76%. Vitality assessment showed that 51.15 ± 17.07% of the frozen-thawed sperm were alive. Motility assessments, at 15 minutes (T1), 30 minutes (T2), and 60 minutes (T3) post-Torin 2 addition, showed statistically significant increase in progressive motility for both Torin 2 treatment groups compared to controls. Group 2 with lower dose of Torin 2 (0.25µM) showed the highest progressive motility at all time points (19.70 ± 18.30% at T1, 20.72 ± 17.99% at T2, 17.88 ± 15.66% at T3, p < 0.01). Non-progressive motility increased slightly, but only T1 was significant (p = 0.04). Immotile category remained unchanged across all groups and all time points (p > 0.05). Sperm vitality analysis showed no significant reductions at T1 and T2 (p > 0.05), but a decrease occurred at T3 in Groups 2 (p < 0.01) and Group 3 (p < 0.05), while Group 1 remained unchanged (p > 0.05). Despite this, effect size was minimal (Partial Eta Squared = 0.035), indicating limited biological impact. Limitations, reasons for caution While this study pioneers the investigation of Torin 2 in sperm motility enhancement, additional research is needed to determine its long-term effects and optimize dosing. Future studies should explore concentrations below 0.25 µM to assess whether lower doses achieve similar benefits while further minimizing potential impact on sperm viability. Wider implications of the findings Our findings highlight the potential of Torin 2, as a novel agent for enhancing sperm motility, particularly at an optimal concentration of 0.25 µM. This study provides a foundation for future research on the mechanistic pathways involved and the feasibility of incorporating Torin 2 into assisted reproductive technologies. Trial registration number No