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Background: Gestational diabetes mellitus (GDM) poses significant health risks for both mothers and children, contributing to long-term complications such as type 2 diabetes and cardiovascular disease. This study explores the potential of microRNAs (miRNAs) as biomarkers for GDM by analyzing peripheral blood samples from GDM patients. Method: Ten samples, including peripheral blood from 5 GDM patients and 5 controls, were collected to perform the RNA sequencing analysis. Differentially expressed miRNAs were further validated by quantitative real-time polymerase chain reaction. Results: A total of 2287 miRNAs were identified, 229 of which showed differential expression. Validation by qRT-PCR confirmed significant up-regulation of miR-5193, miR-5003-3p, miR-3127-5p, novel-miR-96, miR-6734-5p, and miR-122-5p, while miR-10395-3p was down-regulated. Bioinformatics analyses revealed the involvement of these miRNAs in pathways associated with herpes simplex virus 1 infection. Conclusion: This study provides insights into the differential expression of miRNAs in GDM patients and their potential roles in disease pathogenesis. It suggests that the differentially expressed miRNAs could serve as potential biomarkers for GDM, shedding light on the complex molecular mechanisms involved. [ABSTRACT FROM AUTHOR]