Direct RNA sequencing reveals PBMC transcriptome alterations and epitranscriptomic regulations in gestational diabetes mellitus

Gestational diabetes mellitus (GDM) is a common metabolic complication of pregnancy, with chronic low-grade inflammation playing an important role in its pathogenesis. However, most transcriptomic studies of GDM rely on short-read sequencing, which limits the detection of full-length transcripts, complex isoforms, and RNA modifications. This pilot study aimed to characterize transcriptomic and RNA modification profiles in peripheral blood mononuclear cells (PBMCs) from pregnancies with normal glucose tolerance and those with GDM using long-read direct RNA (dRNA) sequencing. PBMC samples were collected from three women with GDM and three gestational age-matched controls without maternal complications. Bulk RNA was extracted and sequenced using the Oxford Nanopore Technologies platform. Transcript quantification and discovery were performed with IsoQuant, and isoform classification with SQANTI3. Differential gene expression (DGE), enrichment analysis, differential transcript usage (DTU), and RNA modification analyses were conducted. DGE analysis identified 439 candidate differentially expressed genes in GDM, including MYOM2 , OLFM1 , and HBG2 . Gene Ontology analysis indicated downregulation of blood-related processes such as erythrocyte development, blood coagulation, and oxygen transport. KEGG pathway analysis suggested enrichment of the PI3K-Akt signaling pathway. Thirteen genes showed significant DTU, with FBXO7 displaying the most pronounced isoform changes. Additionally, seven genes exhibited DTU events with at least one novel isoform. Differential RNA modification analysis highlighted SND1 , HLA-DPB1 , ZNF701 , UNK , and CNST as prominently modified transcripts. These findings reveal GDM-associated alterations in transcriptomic and RNA modification profiles, suggesting a potential role of epitranscriptomic regulation in immune-related molecular dysregulation in GDM.