Development of a Highly Sensitive pH Probe for Spermatozoa and Its Application in the Etiological Diagnosis and Treatment of Asthenospermia

Declining fertility rates, mainly due to reduced sperm quality, challenge the health of mankind. Thus, there is an urgent need for advanced male infertility diagnostics. Despite the known importance of sperm intracellular pH (pHi) in sperm activity during fertilization, the absence of tools to detect subtle changes in spermatozoa limits the knowledge and clinical application of sperm pHi. Here, we developed RCPH, a novel dual-color fluorescent probe, demonstrating high precision (ΔpHi < 0.1) in monitoring spermatozoa pHi with spatial–temporal response. In a cohort of 79 healthy individuals and 35 asthenospermia patients, RCPH detection accurately reflects clinical parameters. With an unprecedented resolution, RCPH revealed that the sperm pHi in asthenospermia patients was 0.1 units lower compared to healthy controls. RCPH also proved effective in drug screenings that enhance pHi, thereby improving sperm motility. RCPH not only advances our knowledge of sperm pHi but also offers diagnostic potential to predict fertility outcomes and enhance in vitro fertilization (IVF) success for reproductive medicine and etiology-based interventions.