Background/ObjectivesObesity in pregnancy associates with changes in the glucose-insulin axis. We hypothesized that these changes affect the maternal metabolome already in the first trimester of human pregnancy and, thus, aimed to identify these metabolites.Patients/MethodsWe performed untargeted metabolomics (HPLC-MS/MS) on maternal serum (n= 181, gestational weeks 4+0–11+6). For further analysis, we included only non-smoking women as assessed by serum cotinine levels (ELISA) (n= 111). In addition to body mass index (BMI) and leptin as measures of obesity and adiposity, we metabolically phenotyped women by their fasting glucose, C-peptide and insulin sensitivity (ISHOMAindex). To identify metabolites (outcome) associated with BMI, leptin, glucose, C-peptide and/or ISHOMA(exposures), we used a combination of univariable and multivariable regression analyses with multiple confounders and machine learning methods (Partial Least Squares Discriminant Analysis, Random Forest and Support Vector Machine). Additional statistical tests confirmed robustness of results. Furthermore, we performed network analyses (MoDentify package) to identify sets of correlating metabolites that are coordinately regulated by the exposures.ResultsWe detected 2449 serum features of which 277 were annotated. After stringent analysis, 15 metabolites associated with at least one exposure (BMI, leptin, glucose, C-peptide, ISHOMA). Among these, palmitoleoyl ethanolamine (POEA), an endocannabinoid-like lipid endogenously synthesized from palmitoleic acid, and N-acetyl-L-alanine were consistently associated with C-peptide in all the analyses (95% CI: 0.10–0.34; effect size: 21%;p< 0.001; 95% CI: 0.04–0.10; effect size: 7%;p< 0.001). In network analysis, most features correlating with palmitoleoyl ethanolamide and N-acetyl-L-alanine and associated with C-peptide, were amino acids or dipeptides (n= 9, 35%), followed by lipids (n= 7, 27%).ConclusionsWe conclude that the metabolome of pregnant women with overweight/obesity is already altered early in pregnancy because of associated changes of C-peptide. Changes of palmitoleoyl ethanolamide concentration in pregnant women with obesity-associated hyperinsulinemia may reflect dysfunctional endocannabinoid-like signalling.