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

Abstract

Background: Extracellular vesicles (EVs) represent an important component of embryo-maternal communication by conveying molecular signals that reflect the embryo's physiological state and developmental competence. However, the combined impact of embryonic origin and developmental stage on EV molecular composition has remained largely unexplored. In this study, a comparison is presented for the first time between EVs secreted by bovine embryos produced in vivo (IVV) and in vitro (IVP) during the blastulation and hatching stages, providing evidence of how these factors shape their biological profiles.

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 influence the biophysical properties and miRNA composition of embryo-secreted EVs, reflecting distinct developmental trajectories between IVV and IVP embryos. This study provides the first direct evidence that embryonic origin significantly modulates EV physicochemical features and miRNA cargo, highlighting their role in embryo-maternal communication and supporting the use of EV-derived miRNAs as novel non-invasive biomarkers of embryo quality and developmental competence.