Association of the Infant Gut Microbiome With Early Childhood Neurodevelopmental Outcomes

This ancillary cohort study of the Vitamin D Antenatal Asthma Reduction Trial (VDAART) examines the association of the gut microbiome in infancy with development of motor skills, problem solving, communication, and personal and social skills at preschool age.

Pregnant women (who reported or whose partner reported allergies/asthma) were randomized (n=880) during the first trimester of pregnancy (10-18 weeks) to one of two treatment arms of a clinical trial: 4000 IU vitamin D + prenatal vitamins or 400 IU vitamin D + prenatal vitamins.
Seventy infants were excluded, due to fetal or neonatal deaths (N=29), loss to follow-up during the prenatal period (N=27) or loss to follow-up after delivery (N=3). Of the 810 infants remaining, 333 infants donated stool samples.

Stool sample Collection and Sequencing of Bacterial 16S rRNA gene
Mothers were asked to collect ½ teaspoon of their infant's stool from a dirty diaper using a tongue depressor and to store the sample in their home freezer. Frozen stool samples were then brought to the clinical center by the infant's mother within 1-2 days or picked up at participants' homes within 24 hours of collection and stored at -80 o C. Stool samples were not collected if the infant had taken antibiotics within the past 7 days. A total of 333 infants had stool samples (age 3-6 months) collected. DNA extractions were performed on stool samples, and the bacterial rRNA 16S gene (V3 to V5 hyper-variable regions) was amplified. Pyrosequencing data (Roche 454 Titanium) were produced by the Genome Center (TGI) at Washington University in St.

Bioinformatics Pipeline for Processing of Bacterial 16S Sequences
Quality filtering, trimming, and chimera checking were performed. The quality filtering protocol used allows one mismatch in the primer and zero mismatches in the barcode. Chimera slayer software was used to remove chimeric reads. All samples that passed quality control screening were classified from phylum to genus level at a confidence threshold of 0.5 using the Ribosomal Database Project Naive Bayesian Classifier version 2.2, training set 6. The reads whose taxonomic assignments were lower than a 0.5 confidence threshold were assigned to the unclassified group. All samples were scaled to 1000 reads before analysis.

Covariates (Additional information about Race classification)
Infant's racial/ethnic group was derived from initial enrollment data on race/ethnicity of each biological parent. The 'African American' race/ethnicity category included infants of African American ancestry only, as well as those with mixed African and Hispanic ancestry. Infants of Native American or Asian descent were included in the 'Other' race category.

Multivariate Associations
MaAsLin analytic software performs boosted, additive general linear models to detect associations between one group of data (metadata/predictors, including ASQ-3 binary outcomes for typical development vs. potential delayed development) and another group (microbial abundance of specific taxa). In this multivariate approach, all measured taxa are considered as outcomes simultaneously.

. MaAsLin Analysis Results: Association of Gut Microbiome Taxa (Those Present in at Least 10 Subjects) in Infants (Ages 3-6 Months) with Typical Personal/Social Development on the ASQ-3 at Age 3 Years, N=307
* Nominally significant features (P < 0.05) are reported. Analysis adjusted for gestational age, race, gender, breast feeding in 1 st 6 months of life, c-section, age at ASQ, maternal education, maternal marital status, maternal age, low income (<$30,000), antibiotics in the first days of life, treatment group and clinical site **Number of participants with detectable feature 0.517589 *Nominally significant features (P < 0.05) are reported. Analysis adjusted for gestational age, race, gender, breast feeding in 1 st 6 months of life, c-section, age at ASQ, maternal education, maternal marital status, maternal age, low income (<$30,000), antibiotics in the first days of life, treatment group and clinical site **Number of participants with detectable feature