Maternal Metabolomic Signatures of Spontaneous Preterm Birth in Bangladesh: Evidence from the AMANHI Bangladesh Cohort

Spontaneous preterm birth (sPTB), defined as natural labor onset before 37 weeks of gestation, is a major contributor to neonatal morbidity and mortality, especially in low- and middle-income countries. Despite its global impact, the biological mechanisms underlying sPTB remain poorly understood. This study employed untargeted metabolomics and a nested case-control study design to investigate maternal serum metabolites that are differentially expressed in sPTB and the associated biological pathways. Cases (sPTB, n=267) and controls (healthy term births, n=864) were drawn from a prospective pregnancy cohort of 3,000 women enrolled in the Alliance for Maternal and Newborn Health Improvement (AMANHI) study in Bangladesh. Study participants were selected in early pregnancy from two rural subdistricts of the Sylhet district in Bangladesh, with data collected between 2014 and 2018. Maternal blood samples were obtained at two time points during pregnancy: once in early pregnancy (before 20 weeks) and in late pregnancy (at or after 20 weeks). Serum metabolites were profiled using an untargeted metabolomics approach based on high-throughput untargeted liquid chromatography-mass spectrometry (LC-MS), yielding 55,541 metabolites, for which 650 were annotated. Statistical analyses revealed significant associations (FDR < 0.1) between sPTB and 268 metabolites in early pregnancy and 617 in late pregnancy. In cases of sPTB, triglyceride levels were markedly reduced in early pregnancy. In contrast, phosphocholines, sphingomyelins, and very long-chain dicarboxylic acids exhibited differential expression in late pregnancy, suggesting disruptions in lipid metabolism. The rate of change in hydroxyisovaleric acid levels across paired samples was significantly higher in sPTB cases, indicating its potential as a dynamic biomarker. Metabolite set enrichment analyses highlighted key differences in triglyceride and dicarboxylic acid pathways, implicating roles in energy metabolism, inflammation, and placental function. Dihydrothymine was elevated across both time-points, indicating its role in pyrimidine metabolism. These findings provide valuable insights into the biological basis of sPTB and highlight the potential of metabolomic biomarkers for risk assessment and targeted intervention.