Plasma and Brain-Derived Extracellular Vesicle Biomarkers Following a Randomized Controlled Trial of Choline for Neurodevelopment in Fetal Alcohol Spectrum Disorder: A Pilot Analysis

Background: Postnatal choline supplementation improves memory, non-verbal IQ, executive function, and white-matter microstructure in children with fetal alcohol spectrum disorder (FASD) across randomized controlled trials; however, peripheral and brain biomarkers of these effects are uncharacterized. Methods: We measured eight immune and neurotrophic biomarkers (TNFα, transferrin receptor 1 [TfR1], BDNF, ferritin, MCP-1, RANTES, CRP, and Eotaxin) in plasma and brain-derived extracellular vesicles (BDEVs) from 24 children with FASD randomized to choline (n = 13; 7 male, 6 female) or placebo (n = 11; 7 male, 4 female), before and approximately nine months after treatment (23 with paired pre/post samples). Values were log10-transformed to correct for non-normal distribution. We modeled Group × Time effects with linear mixed models, adjusted for age, sex, and sample storage duration, cross-checked with cluster-robust ordinary least squares, and tested plasma-to-BDEV concordance for each analyte with linear regression adjusted for age and sex. Multiplicity was corrected by Benjamini–Hochberg FDR. Results: Plasma MCP-1 showed a nominally significant Group × Time interaction (p = 0.035, q = 0.28), rising over time in the placebo but not the choline group. Ferritin and CRP showed similar patterns that did not reach significance (interaction p = 0.099 and p = 0.118; q = 0.31 for both). RANTES and BDNF rose significantly in BDEVs over time, without evidence of a differential change by treatment group, and survived FDR correction (both q ≤ 0.02); ferritin also rose significantly (p = 0.021) but its Time effect did not survive FDR correction (q = 0.06). BDEV TfR1 was nominally lower in the choline group than placebo at both timepoints (p = 0.023; q = 0.13). Plasma levels were associated with BDEV levels for five markers (ferritin, TfR1, TNFα, Eotaxin, RANTES; all p < 0.05), independent of age, sex, time, and group; all five associations survived FDR correction (q ≤ 0.01). The plasma-BDEV association for MCP-1 became non-significant when adjusted for time and group (p = 0.134, q = 0.13). CRP and BDNF showed a between-person, but not within-person, plasma-BDEV association. Conclusions: In this small, exploratory sample of children with FASD, a condition characterized by brain iron deficiency and inflammation, choline was associated with an attenuated rise in pro-inflammatory plasma MCP-1 relative to placebo, generating a new hypothesized candidate biomarker for further study. Most BDEV markers changed over time (Pre-to-Post), not with treatment. Plasma and BDEV BDNF were not significantly associated overall, although a between-person association was present without a corresponding within-person signal, suggesting that inferring central nervous system function from plasma BDNF should proceed with caution. These hypothesis-generating findings support further, larger-scale investigation of BDEV content analysis as a candidate complement to this trial program’s cognitive and neuroimaging outcomes. Replication and expansion of the BDEV panel of analytes in a larger sample are needed. Trial registration: ClinicalTrials.Gov [NCT02735473]; registered 2 April 2016.