Identification of potential MicroRNA Markers in Plasma Vesicles for Giant Panda Pregnancy Diagnosis

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 pseudo-pregnancy(non-parturient) 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 co-expressed in both mid- and late pregnancy but were absent in the pseudo-pregnancy (non-parturient) 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 RT-qPCR 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.