Associations of Plasma Gut Microbiota-Derived TMAO and Precursors in Early Pregnancy with Gestational Diabetes Mellitus Risk: A Nested Case-Control Study

<b>Objectives:</b> Gut microbiota-derived metabolites-trimethylamine N-oxide (TMAO) and its precursors choline, betaine, and carnitine-have been linked to various health outcomes. However, their role in gestational diabetes mellitus (GDM) remains unclear due to inconsistent findings. This study aims to investigate the associations between maternal plasma concentrations of these metabolites during early pregnancy and the risk of GDM. <b>Methods:</b> A nested case-control study was performed in the Shanghai Birth Cohort. GDM cases and non-GDM controls were matched according to maternal age at a ratio of 1:4. Three hundred twenty-one identified GDM cases and 1284 controls were included. Maternal plasma concentrations of TMAO and its precursors were measured between 12 and 16 weeks of gestation in early pregnancy using high-performance liquid chromatography-tandem mass spectrometry. Conditional logistic regression models were applied to assess associations between metabolite levels and GDM risk and to calculate odds ratios (ORs) and their 95% confidence intervals (CIs). Multivariate linear regressions evaluated relationships between metabolite concentrations and glycemic indicators. Stratified and sensitivity analyses were conducted to ensure robustness. <b>Results:</b> Maternal plasma levels of TMAO, choline, betaine, and carnitine in early pregnancy were 1.95 μmol/L (IQR, 1.16-3.20), 9.25 μmol/L (IQR, 7.31-11.98), 20.51 μmol/L (IQR, 16.92-24.79), and 17.13 μmol/L (IQR, 13.33-21.16), respectively. Betaine and carnitine were significantly higher in GDM cases (<i>p</i> = 0.002 and <i>p</i> = 0.042, respectively). No significant associations were identified between TMAO levels and GDM risk and glycemic indicators. Each SD increase in choline was associated with a 16% higher GDM risk (OR = 1.16, 95% CI: 1.01, 1.34, <i>p</i> = 0.039), while increased betaine and carnitine levels were linked to a 19% (OR = 0.81, 95% CI: 0.70, 0.95; <i>p</i> = 0.010) and 20% (OR = 0.80, 95% CI: 0.69, 0.94; <i>p</i> = 0.007) lower risk, respectively. Restricted cubic spline models showed no evidence of non-linear relationships (<i>p</i>_{for non-linearity} > 0.05). Interaction analyses indicated that the protective effect of betaine may be more pronounced in parous women. <b>Conclusions:</b> Higher early pregnancy levels of betaine and carnitine were associated with a reduced GDM risk, while elevated choline levels increased the risk. The protective association between betaine and GDM was more pronounced in parous women. No significant relationship was found between TMAO and GDM. The roles of choline, betaine, and carnitine in glucose metabolism warrant further investigation.