The sperm survival switch: circRNA-mediated regulatory loops control apoptotic signalling in human spermatozoa

Abstract Male reproductive health is declining worldwide, partly due to lifestyle factors including obesity, which is linked to increased sperm apoptosis and impaired assisted reproductive technologies (ARTs) outcomes. This growing issue underscores the urgent need for novel molecular biomarkers capable of improving sperm quality assessment. In this context, circular RNAs (circRNAs) are emerging as regulators of germ cell functions and apoptosis, but their role as early apoptotic markers in human sperm under lifestyle-related stress remains poorly explored. In this study, by using the obesity as experimental condition of sperm apoptosis-induction, we performed a comprehensive circRNA profiling in High-quality spermatozoa (A-SPZ) collected from Normal-weight and Obese men. Obese sperm exhibit a pro-apoptotic phenotype associated with a cargo of differentially expressed (DE-) circRNAs enriched in pro-apoptotic regulatory networks. Mechanistically, circRNAs arise both from the trigger of FUS-regulated endogenous backsplicing and via extracellular vesicles (EVs) mediated delivery, thereby establishing a functional pro-apoptotic circRNA regulatory circuit. Furthermore, Obese men exhibit a dysregulated sphingosine 1-phosphate (S1P) signaling axis, along with an imbalanced S1P–to–ceramide rheostat within EVs and increased levels of pro-apoptotic ceramide. Finally, the in vitro sperm apoptosis induction confirmed a functional circRNA–apoptosis axis, identifying a subset of circRNAs responsive to pro-apoptotic stimuli that engage in a self-reinforcing regulatory loop involving pro-CASPASE3 activation. Our findings elucidate a pro-apoptotic propensity in Obese A-SPZ positioning circRNAs as putative molecular biomarkers candidate for early apoptosis. These spermatic circRNAs, which are significantly up-regulated under apoptotic conditions, exert a dual functional role: (i) serving as diagnostic markers for early-stage apoptosis and (ii) acting as active molecular adapters to enhance apoptosis activation. These circRNAs could be suggested as early sperm apoptotic biomarkers enhancing the precision of sperm quality assessment and, in turn, the success of ART procedures.