**Background:** Choline is an essential micronutrient crucial for brain development, particularly the hippocampus, and may influence growth. However, few studies have examined postnatal choline status and child outcomes in low- and middle-income countries (LMICs). This secondary analysis of the Mazira Project—a randomized trial providing one egg daily for 6 months to Malawian children aged 6–9 months—aimed to assess cross-sectional and predictive associations between plasma choline and measures of growth and neurodevelopment during the complementary feeding period (6–15 months). The trial previously found no effect of eggs on growth or development, and no improvement in plasma choline, so this analysis sought to understand those null results.
**Methods:** Data came from 400 children (200 per trial arm) who provided adequate blood samples at baseline and 6-month follow-up. Plasma choline was measured semi-quantitatively via UPLC-MS/MS (relative intensity units) in all 400, and quantitatively via LC-MS/MS in a subsample of 60. Growth outcomes included length-for-age (LAZ), weight-for-age (WAZ), weight-for-length (WLZ), head circumference-for-age (HCAZ) z-scores, and dichotomous stunting, underweight, and low head circumference. Conditional growth measures (residuals from regression on prior anthropometry) were also calculated. Neurodevelopment was assessed using the Malawi Developmental Assessment Tool (MDAT; fine motor, gross motor, language, personal-social), visual paired comparison (VPC) eye-tracking (novelty preference and peak look length), Infant Orienting with Attention (IOWA) eye-tracking (response time), and an elicited imitation task (actions recalled). Statistical analyses used linear and logistic regression with robust standard errors, adjusting for time point, group assignment, and pre-specified covariates (e.g., age, sex, maternal education, household assets, inflammation markers). Exploratory analyses examined choline metabolites (betaine, dimethylglycine [DMG], trimethylamine N-oxide [TMAO]) and effect modifiers.
**Key Results:** Mean plasma choline decreased from 17.1 µmol/L at baseline (age 6–9 months) to 14.6 µmol/L at 6-month follow-up (age 12–15 months). Stunting prevalence was 13.8% at baseline and 19.5% at follow-up. In cross-sectional analyses, plasma choline was not significantly associated with most growth outcomes in fully adjusted models, except for a weak negative association with LAZ (estimate −0.09 per 1 SD, 95% CI −0.17 to −0.01, p<0.05). For neurodevelopment, cross-sectional fully adjusted models showed a significant negative association with VPC peak look length (−183.0 ms per 1 SD, 95% CI −348.1 to −17.9) and a significant positive association with IOWA response time (8.84 ms per 1 SD, 95% CI 1.66–16.03), indicating faster processing but slower orienting with higher choline. In predictive models (baseline choline predicting 6-month outcomes), higher baseline choline was significantly associated with lower MDAT fine motor z-scores (−0.13 per 1 SD, 95% CI −0.22 to −0.04). No other predictive associations were significant. Exploratory analyses of betaine, DMG, and TMAO showed mostly null associations, except baseline TMAO was positively associated with peak look length (231.7 ms per 1 SD, 95% CI 100.4–363.0) and elicited imitation actions recalled (0.23 per 1 SD, 95% CI 0.03–0.43) at follow-up. Effect modification analyses yielded no clear patterns, with significant results near the expected 5% by chance.
**Clinical Implications:** This study found that plasma choline was not consistently associated with growth or neurodevelopment in young Malawian children, despite very low choline intake (mean estimated intake 102 mg/day vs. Adequate Intake of 150 mg/day). The few significant associations were small and in inconsistent directions, with three of four suggesting poorer outcomes with higher choline. These null findings help explain why the egg intervention did not improve growth or development in the Mazira Project, as both the link from eggs to choline status and from choline to outcomes were absent. The results suggest that in settings with severe undernutrition, high inflammation, and multiple environmental constraints, choline may not be a limiting factor for growth and development, or plasma choline may be an inadequate biomarker. The authors recommend that adequate choline intake should still be encouraged, but future research requires validated biomarkers and rigorous designs to clarify choline's role in diverse contexts.