Dual-Parameter Hypoxia Sensing in Spermatozoa: A Transformative Nitroreductase/Viscosity Fluorescent Probe Unravels Metabolic Dysregulation Biomarkers for Andrological Diagnostics

Sperm energy metabolism, including oxidative phosphorylation and glycolysis, is critical for sperm function. Environmental stressors like hypoxia can disrupt metabolic activities, potentially leading to fertilization failure. As an NADH-dependent flavin reductase, altered nitroreductase (NTR) levels may reflect hypoxic metabolic abnormalities. However, no studies have reported the detection of hypoxia conditions in spermatozoa to date. To address this diagnostic gap, we first performed metabolomic analysis on normal and clinically infertile spermatozoa. Hypoxia-mediated metabolic dysregulation in spermatozoa is unveiled as a pivotal mechanism underlying idiopathic male infertility, with clinical metabolomics revealing impaired anaerobic glycolysis (50% lactate reduction, <i>p</i> < 0.0001; 1174-fold PEP accumulation, <i>p</i> < 0.0001 vs controls). Subsequently, to address the critical need for monitoring the hypoxia microenvironment of spermatozoa, we developed <b>PPy</b>, a dual-response fluorescent probe with NTR and viscosity. This first-in-spermatozoa probe demonstrates viscosity-driven near-infrared enhancement at 706 nm (32-fold, Stokes shift >220 nm) and NTR-activated green emission around 562 nm, enabling real-time hypoxia mapping in live systems. Confocal validation in pathological models (H₂O₂-stressed/infertile spermatozoa) quantified obvious NTR activity loss and diminished capacity for viscosity regulation and successfully established NTR as a diagnostic biomarker (AUC = 0.975, ΔMFI < 12.39% with 100% sensitivity and 90% specificity). Thus, our study introduces innovative tools and actionable biomarkers for clinical andrology settings, which uncover profound hypoxia-induced impairments in spermatozoa of infertile patients, thereby advancing both therapeutic strategies for infertility and mechanistic investigations into fertilization disorders.