Identification of potential microRNA markers in plasma vesicles for giant panda pregnancy diagnosis

Although extracellular vesicles microRNAs have potential as pregnancy biomarkers in humans and important economic animals, they are largely uncharacterized in wildlife like the giant panda, a species with considerable difficulty in pregnancy diagnosis. This study reports that microRNAs present in plasma vesicles can serve as effective biomarkers for diagnosing true pregnancy in giant pandas. Abstract: Extracellular vesicle microRNAs play direct and indirect roles in pregnancy across humans and animal models, making them potential biomarkers for pregnancy detection. Here, a total of 15 blood samples were collected to profile extracellular vesicle microRNAs across three experimental groups representing different gestational stages (early, mid, and late pregnancy), with pseudopregnancy (nonparturient) and estrus samples serving as controls. Eight microRNAs (miR-126a-3p, miR-599-5p, miR-3925-3p, miR-365a-3p, miR-362-5p, miR-143-3p, miR-501b-3p, and miR-324a-3p) were found to be coexpressed in both mid and late pregnancy but were absent in the pseudopregnancy (nonparturient) group. Gene Ontology analysis revealed that the predicted target genes of seven out of the eight microRNAs were enriched in several common immune-related terms, suggesting that these microRNAs may contribute to pregnancy maintenance through immune regulation in the giant panda. Protein-protein interaction network analysis further showed that the seven microRNAs target 20 key immune-related genes, thereby providing essential foundational data for future investigations of miRNA-mRNA interactions during pregnancy in giant pandas. Additionally, the expression patterns of miR-3925-3p and miR-599-5p were validated by real-time fluorescence quantitative PCR using an additional 20 blood samples. Collectively, this study identifies several promising diagnostic biomarkers for true pregnancy and provides foundational insights into the role of microRNAs in immune regulation during gestation in the giant panda.