Significance: Hatching from the zona pellucida is a prerequisite for embryo implantation and is less likely to occur in vitro for reasons unknown. Extracellular vesicles (EVs) are secreted by the embryo into the culture medium. Yet the role that embryonic EVs and their cargo microRNAs (miRNAs) play in blastocyst hatching has not been elucidated, partially due to the difficulties of isolating them from low amounts of culture medium. Here, we optimized EV-miRNA isolation from medium conditioned by individually cultured bovine embryos and subsequently showed that miR-378a-3p, which was up-regulated in EVs secreted by blastocysts, plays a crucial role in promoting blastocyst hatching. This demonstrates the regulatory effect of miR-378-3p on hatching, which is an established embryo quality parameter linked with implantation.

Biomarkers are biomolecules used to identify or predict the presence of a specific disease or condition. They play an important role in early diagnosis and may be crucial for treatment. MicroRNAs (miRNAs), a group of small non-coding RNAs, are more and more regarded as promising biomarkers for several reasons. Dysregulation of miRNAs has been linked with development of several diseases, including many different types of cancer, and abnormal levels can be present in early stages of tumor development. Because miRNAs are stable molecules secreted and freely circulating in blood and urine, they can be sampled with little or no invasion. Here, we present an overview of the current literature, focusing on the types of cancers for which dysregulation of miR-665 has been associated with disease progression, recurrence, and/or prognosis. It needs to be emphasized that the role of miR-665 sometimes seems ambiguous, in the sense that it can be upregulated in one cancer type and downregulated in another and can even change during the progression of the same cancer. Caution is thus needed before using miR-665 as a biomarker, and extrapolation between different cancer types is not advisable. Moreover, more detailed understanding of the different roles of miR-665 will help in determining its potential as a diagnostic and prognostic biomarker.

Extracellular vesicles (EVs) contain microRNAs (miRNAs), which are important regulators of embryonic development. Nevertheless, little is known about the precise molecular processes controlling blastocyst development and quality. In a previous study, we identified bta-miR-665 as one of the miRNAs more abundantly present in extracellular vesicles of embryo-conditioned culture media of blastocysts compared to degenerate ones. Here, we investigated the effect and regulatory roles of bta-miR-665 in blastocyst development by supplementation of bta-miR-665 mimics or inhibitors to the culture media. Supplementation of bta-miR-665 mimics improved cleavage and blastocyst rate (P < 0.01), and blastocyst quality as indicated by increased inner cell mass rates and reduced apoptotic cell ratios (P < 0.01). Furthermore, supplementation of bta-miR-665 inhibitors had the opposite effect on these phenotypes. Low input transcriptome analysis and RT-qPCR revealed that bta-miR-665 acts on genes linked to microtubule formation and apoptosis/cell proliferation. These insights not only elucidate the important role of bta-miR-665 in embryo development, but also underscore its potential in improving reproductive efficiency in bovine embryo culture.