Comparative profiling of extracellular vesicles and miRNA cargo from in vivo- and in vitro-derived bovine embryos during blastulation and hatching

Background: Extracellular vesicles (EVs) play a central role in embryo–maternal communication, transporting bioactive molecules that reflect the embryo's physiological state and developmental competence. This study examined how embryonic origin and developmental stage shape the characteristics and molecular content of EVs secreted by bovine embryos. Results: IVV embryos exhibited higher developmental competence and secreted larger EVs whose concentrations remained stable across developmental windows. In contrast, IVP embryos released smaller and more abundant vesicles, particularly during hatching, indicating origin- and stage-specific regulation of EV output. Distinct miRNA profiles clearly separated both embryo types. EVs from IVV embryos were enriched in miRNAs associated with implantation and lineage specification (e.g., miR-124, miR-125, miR-181), whereas EVs from IVP embryos contained higher levels of miRNAs linked to stress response, apoptosis, and the unfolded protein response (e.g., miR-23b, miR-92a, miR-409). Consistently, functional enrichment analyses revealed that IVV-derived miRNAs targeted pathways related to immune modulation and purinergic signaling, while IVP-derived miRNAs were associated with calcium transport and endoplasmic reticulum stress pathways. Together, these differences point to divergent regulatory programs shaped simultaneously by embryonic origin and developmental progression. Conclusions: Embryonic origin and developmental stage modulate both the biophysical properties and molecular signatures of embryo-secreted EVs. The distinct miRNA landscapes identified in IVV and IVP embryos reflect contrasting developmental trajectories and support the potential of EV-derived miRNAs as non-invasive biomarkers of embryo quality and developmental competence.