Embryo-derived extracellular vesicle miRNA cargo reflects developmental competence in porcine in vitro-produced...

Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication during embryo development and represent a promising source of non-invasive biomarkers of embryo developmental competence. This study characterized for the first time EVs released by porcine in vitro-produced embryos and compared the microRNA (miRNA) cargo of EVs derived from blastocysts (BL) and non-blastocysts (N-BL) to identify molecular signatures associated with developmental competence. Porcine embryos were produced by standard in vitro maturation, fertilization, and culture procedures. On day 7 of in vitro culture, embryos were classified as BL and N-BL, washed and cultured in groups of 50 for 6 h. Conditioned media were subjected to EV isolation by size-exclusion chromatography and characterized by dynamic light scattering, transmission electron microscopy, and flow cytometry. Total RNA was extracted and subjected to small RNA sequencing. No significant differences were detected in EV concentration or mean particle size between groups. A total of 123 miRNAs were identified, including 105 shared, 15 BL-exclusive, and 3 N-BL-exclusive. Among the shared miRNAs, ssc-miR-148a, ssc-miR-99a, ssc-miR-122, and ssc-miR-151a were upregulated, whereas ssc-miR-181b was downregulated in BL-derived EVs. Functional enrichment analysis revealed biological processes associated with developmental signaling, cellular organization and vesicle trafficking in BL-derived EVs, whereas N-BL-derived EVs were associated with apoptosis, cytokine-mediated signaling, developmental arrest and epigenetic regulation. Collectively, these findings demonstrate that the miRNA cargo of embryo-derived EVs reflects embryo developmental competence and identify EV-associated miRNAs as promising non-invasive biomarkers and candidate regulators of early embryonic development in porcine in vitro production systems.