Development of a cAMP biosensor-based assay for measuring serum bioactive concentrations of FSH

Follicle-stimulating hormone (FSH) plays a pivotal role in reproductive physiology and is increasingly recognized for its involvement in the extragonadal system. The biological activity of FSH, which is modulated by glycosylation, varies with age, reproductive status, and pathological conditions. Although the serum FSH immunoreactive concentrations are measured routinely for patients with reproductive disorders, the significance of assessing the biological activity of FSH remains unclear. In this study, we aimed to develop a novel, rapid, and animal-free assay to quantify FSH bioactive concentrations using HEK293 cells co-expressing the human FSH receptor (FSHR) and a cyclic adenosine monophosphate biosensor. The assay demonstrated high specificity, reproducibility, and linearity, with minimal cross-reactivity with structurally related hormones. Application to clinical serum samples from postmenopausal women revealed a strong correlation between FSH immunoreactive and bioactive concentrations. Notably, estrogen replacement therapy resulted in a significant reduction in both FSH immunoreactive and bioactive concentrations, as well as in the FSH bioactive -to-immunoreactive concentration ratio, suggesting that FSH glycosylation patterns may have been altered, leading to a decrease in its bioactive concentrations. The findings collectively suggested that assessing FSH bioactive concentrations, in addition to immunoreactive concentrations, may provide further insights into hormonal regulation and its relevance to therapeutic evaluation. The biosensor-based assay could offer a practical and efficient tool for advancing our understanding of FSH function in both reproductive and non-reproductive contexts.