Detection of Development-Specific MicroRNAs in Rabbit Embryos and Culture Media: A Potential Biomarker Approach for...

MicroRNAs (miRNAs) are short, non-coding RNA molecules that play a crucial role in regulating various biological processes by influencing post-transcriptional gene expression and gene silencing. Background/Objectives: In this study, rabbit embryos were utilised as a model system to investigate potential biomarkers relevant to human embryo development. Seven microRNAs (miRNAs) identified in the embryo culture medium were evaluated as biomarkers by analysing the correlation between their expression levels and the developmental quality of rabbit embryos at days 4 and 6. Methods: We analysed the expression of seven development-specific miRNAs (miR-24-3p, miR-28-3p, miR-103a-3p, miR-181a-5p, miR-191-5p, miR-320a-3p, miR-378a-3p) in 4-day-old and 6-day-old rabbit embryos, along with their culture media. Results: Our findings revealed significant differences in the expression levels of these miRNAs between the 4-day-old and 6-day-old embryos. On the other hand, the expression patterns observed in the culture medium samples showed less variation between the two age groups. Nonetheless, analysis of miRNA expression profiles in the spent culture medium from individually cultured embryos enabled the identification of lower-quality embryos, characterised by smaller size and impaired or delayed development. Conclusions: The detection of these miRNAs in embryo culture medium may serve as a reliable indicator of successful progression to the blastocyst stage. Our experimental results identified specific miRNAs whose expression profiles differ according to embryonic stage and quality, thereby reflecting key developmental milestones. Notably, the detectability of these miRNAs in the medium-without prior RNA isolation-indicates their active secretion into the extracellular environment. By synthesising our findings with the existing literature, we refined a panel of miRNAs essential for the development of implantation-competent embryos in both rabbits and humans. Consequently, we developed a non-invasive assay for predicting implantation and pregnancy outcomes, which may have significant applications in human reproductive medicine.