Mitochondrial DNA analysis in a cohort of stillbirths with brainstem and cardiac conduction system abnormalities

Stillbirth (SB) accounts for over 60% of perinatal deaths in high-income countries, with a significant portion of cases remaining unexplained following thorough anatomopathological investigation. Mitochondrial DNA (mtDNA) alterations were analyzed in 42 SB cases with brainstem and cardiac conduction system (CCS) anomalies and in 32 control fetuses without these anomalies. DNA extracted from brainstem tissues was analyzed in both groups. In addition, unaffected tissues from the SB cases were examined for intra-individual comparison. The analysis included mtDNA sequencing, haplogroup determination, copy number (CN) quantification, and evaluation of displacement loop (D-loop) instability and methylation. Across the entire SB cohort, a total of 158 variants were identified, with a significant enrichment of variants observed in cases without CCS anomalies (p = 0.024). Affected brainstem tissues exhibited significantly higher mtDNA-CN compared with both control brainstem tissues (p < 0.0001) and unaffected tissues (p = 0.005), with levels higher in mild lesions than in severe lesions (p = 0.02). D-loop instability was identified in 37% of cases, and D-loop methylation levels were consistently higher in affected brainstem tissues compared with both control brainstem tissues (p < 0.0001) and unaffected tissues (p = 0.0001). These findings support mitochondrial dysfunction as a key contributor to fetal demise and mtDNA-CN as a potential biomarker for SB.