Single-particle analysis of small extracellular vesicles from human follicular fluid unveils immunomodulatory PD-L1 ⁺ subpopulations and potentially fertility biomarkers

In certain cell systems, small extracellular vesicles bearing PD-L1 (PD-L1⁺ sEVs) have been shown to suppress T-cell immunity. We investigated whether a distinct profile of PD-L1⁺ sEVs exists in human follicular fluid (FF), a microenvironment where immune tolerance is crucial for proper follicular development. We characterized the expression and colocalization of CD63, CD81, CD9, and PD-L1 in sEVs derived from FF of women undergoing fertility treatments (<i>n</i> = 10), utilizing single-particle interferometric reflectance imaging sensing combined with single-particle antibody capture and immunofluorescence labeling. Additionally, sEV size distribution was analysed <i>via</i> atomic force microscopy. These integrated techniques revealed that the majority of tetraspanin-expressing EVs in human FF are smaller than 50 nm. Statistical analysis revealed a significant difference in PD-L1 co-expression across CD63, CD81, and CD9, confirming a preferential association of PD-L1 with CD9⁺ sEVs. Coefficients of variation across the cohort further indicated that PD-L1/CD9 co-expression was the most consistent among patients, suggesting a stable and distinct sEV subpopulation. These findings underscore the potential of PD-L1⁺ sEVs as biomarkers for immune regulation in reproductive treatments. The discovery of distinct PD-L1⁺ sEV subpopulations suggests a role in modulating immune responses within the follicular microenvironment. Further studies are warranted to investigate the functional relevance of these vesicles in predicting fertility outcome, promoting local immune tolerance, and facilitating follicular development.