Follicular fluid-derived small extracellular vesicles during individual in vitro maturation improve blastocyst development.

INTRODUCTION: We evaluated the impact of follicular fluid-derived small extracellular vesicles (FF-sEVs) supplementation during oocyte maturation in vitro on bovine embryo outcomes, comparing group and individual culture systems. METHODS: Follicular fluid was aspirated from dominant follicles of four nulliparous Holstein heifers at 4.5 days post-ovulation. Small extracellular vesicles were isolated, characterized, and pooled to ensure balanced donor contribution. To confirm uptake, FF-sEVs were fluorescently labelled and co-cultured with cumulus-oocyte complexes (COCs) during in vitro maturation. Fluorescent labelling confirmed FF-sEVs internalization by oocytes and granulosa cells. Next, COCs were matured in vitro with FF-sEVs at varying concentrations (group system: 0, 5, 10, 25, 50 μg/mL; individual system: 0, 6.5, 12.5, 25 μg/mL), fertilized, and cultured. Blastocyst quality was assessed via differential-apoptotic staining. RESULTS: In group culture, the control group exhibited higher day 8 blastocyst rates compared to 10, 25, and 50 μg/mL FF-sEVs groups, while 5 μg/mL FF-sEVs showed no difference. Blastocysts developed from oocytes matured in 25 and 50 μg/mL groups had reduced total cell numbers versus controls and groups matured in lower FF-sEVs concentrations. Conversely, individual maturation with 6.5 μg/mL FF-sEVs enhanced day 8 blastocyst rate, total cell counts, inner cell mass, and reduced apoptotic ratios compared to all other groups. DISCUSSION AND CONCLUSION: We propose that intercellular communication in group cultures, potentially mediated by endogenous embryotropins (including sEVs), may mask FF-sEVs benefits. In individual systems, where such interactions are absent (or minimal), FF-sEVs significantly improved embryo competence. These findings underscore FF-sEVs as a promising tool to refine assisted reproductive technologies, contingent on culture conditions.