Gestational Diabetes Mellitus Placentas Exhibit Mitonuclear Transcriptional Imbalance Co-occurring with m6A Regulatory Gene Dysregulation: Insights from Bulk RNA Sequencing

Background Gestational diabetes mellitus (GDM) affects approximately 14% of pregnancies globally and is associated with placental dysfunction and adverse perinatal outcomes. While mitochondrial dysfunction in GDM placentas is well-documented, the coordinated relationship between mitochondrial DNA (mtDNA)-encoded and nuclear genome-encoded oxidative phosphorylation (OXPHOS) gene expression — termed mitonuclear transcriptional imbalance, has not previously been described in this context. Methods We performed transcriptomic analysis of bulk RNA-seq data from 14 GDM (GDMA1, n = 5; GDMA2, n = 9) and 11 normoglycemic control placental samples from the publicly available dataset GSE249311. Differential expression analysis was performed using DESeq2. Gene Set Enrichment Analysis (GSEA) was performed using the Hallmark gene set collection. A rank-based Mitonuclear Index was computed using ssGSEA with the predefined Hallmark OXIDATIVE_PHOSPHORYLATION gene set (MSigDB, n = 200 genes) to avoid circularity with differential expression results. Robustness was assessed by leave-one-out analysis and comparison across three scoring algorithms (ssGSEA, GSVA, Z-score module score). Directional consistency was assessed in an independent microarray cohort (GSE70493, n = 63). Results All 13 mtDNA-encoded respiratory chain genes were significantly downregulated in GDM placentas (log₂FC ≈ − 1.4, adjusted P < 10⁻⁹), while nuclear-encoded OXPHOS genes were the most significantly upregulated pathway by GSEA (NES = 2.15, adjusted P = 1.19 × 10⁻⁸). The Mitonuclear Index was significantly elevated in GDM versus controls (Wilcoxon P = 8.75 × 10⁻⁵, rank-biserial r = 0.72) and was consistently observed across GDMA1, GDMA2, and T2DM in pregnancy subtypes. Leave-one-out analysis confirmed robustness across all 25 samples (all iterations P < 0.001). The signal was directionally concordant across alternative scoring approaches, though statistical support varied by method (ssGSEA P < 0.001; Z-score P = 0.044; GSVA P = 0.066). Six m6A regulatory genes were significantly dysregulated, including upregulation of METTL14, WTAP, and RBM15, co-occurring with the mitonuclear transcriptional state. Conclusions These findings suggest mitonuclear transcriptional imbalance as a candidate placental transcriptional phenotype associated with maternal hyperglycemia. Future mechanistic studies are warranted to determine whether impaired mtDNA transcription, retrograde mitochondrial signaling, and m6A epitranscriptomic remodeling jointly contribute to placental adaptation in GDM.