The development of new, non-hormonal contraceptive technologies is essential for meeting global reproductive health and family planning needs. Copper (Cu2+) is an active pharmaceutical ingredient (API) in popular intrauterine devices as well as non-hormonal multipurpose prevention technologies being developed to provide contraception and protect against sexually-transmitted infections (STIs) and bacterial vaginosis. Historically, the standard for evaluating effective contraceptive API concentrations has been rapid and complete inhibition of motility. However, this method neglects the long-term effects that APIs might have on other vital structures such as the plasma membrane, and aspects of sperm physiology, such as capacitation. A more integrated approach to assessing mechanisms of action and impacts on multiple sperm function endpoints may uncover that lower API concentrations, rather than those needed for full motility inhibition, are effective. For example, sperm may remain motile after leaving the vagina but die in the uterus due to a compromised plasma membrane undergoing premature exocytosis. Such results could potentially save compounds previously discarded for toxicity reasons. Our comprehensive methodology for evaluating the effects of APIs on multiple aspects of sperm physiology beyond motility has revealed that Cu2+ can pathologically increase sperm plasma membrane fluidity leading to premature acrosome exocytosis and early cell death. Based on literature showing Cu2+ produces reactive oxygen species and preliminary data indicating Cu2+-treated sperm have increased membrane lipid peroxidation, we will conduct four assays to determine if Cu2+ impacts the membrane via lipid peroxidation (Aim 1). These assays will involve quantifying byproducts of lipid peroxidation, spatially and temporally visualizing peroxidation in live sperm, and ascertaining whether antioxidant treatment or inhibition of phospholipase A2 can reduce Cu2+'s effects on membrane dynamics. In Aim 2, we will expand our range of non-hormonal contraceptive ingredients by testing promising APIs using our holistic, multi-endpoint approach, examining effects on motility, viability, membrane fluidity, capacitation (the functional maturation of sperm within the female reproductive tract), acrosome exocytosis, and synergy with Cu2+. We will then determine their mechanisms of action and conduct preliminary in-vitro toxicological assessments. Together, these experiments will have both scientific and clinical significance. Understanding the molecular mechanisms of Cu2+ and other APIs on sperm structure and function will help us target synergistic combinations that induce peroxidation while inhibiting sperm's oxidative stress defense pathways. Such synergistic combinations could improve efficacy and reduce toxicity. Notably, such combinations are also ideal to reduce the development of antimicrobial resistance, which is critical for multipurpose prevention technologies that must protect against STIs.