Prenatal dietary supplements influence the infant airway microbiota in a randomized factorial clinical trial

Asthma and chronic inflammatory diseases impose a large burden, and early-life exposures, including during gestation, shape later disease risk. Prior randomized trials demonstrated that prenatal supplementation with n-3 long-chain polyunsaturated fatty acids (LCPUFA) and high-dose vitamin D reduces wheeze/asthma in offspring. These nutrients modulate immune development (e.g., cytokine regulation, T-cell maturation, antimicrobial peptide production, epithelial barrier function) and have been linked to microbial changes. Early-life microbiota, especially gut composition and specific airway genera, has been associated with later asthma risk. This study asks whether maternal n-3 LCPUFA and vitamin D supplementation alters maternal vaginal, infant gut, and infant airway microbiota, and whether microbiota changes mediate the supplements’ protective effects on childhood asthma.

Evidence indicates prenatal and early-life environments are critical for asthma pathogenesis. Trials have shown fish oil–derived n-3 LCPUFA and high-dose vitamin D in pregnancy reduce childhood wheeze/asthma. Mechanistically, n-3 LCPUFA compete with arachidonic acid pathways and modulate cytokines/Th cell maturation; vitamin D/VDR influences Tregs, antimicrobial peptides, and epithelial junctions. Observational and animal studies link vitamin D and n-3 intake to gut microbiome alterations. Early-life gut microbiome maturation and reduced diversity have been associated with allergic disease risk, and early airway colonization with specific bacteria (e.g., Streptococcus) has been linked to later asthma and wheeze. These data motivate testing whether prenatal nutrient supplementation modifies early microbiota and contributes to clinical benefits.

Design: Nested, two-by-two factorial, double-blind, placebo-controlled randomized clinical trial within the population-based COPSAC2010 cohort. Pregnant women were randomized 1:1 to receive n-3 LCPUFA or placebo and independently 1:1 to receive vitamin D3 or placebo from gestational week 24 to 1 week postpartum, yielding four groups (n-3/vitamin D; n-3/placebo; placebo/vitamin D; placebo/placebo). Primary clinical endpoint for the parent cohort was persistent wheeze/asthma diagnosed by a validated algorithm. Ethics approvals and informed consent obtained; trials registered (NCT00798226, NCT00856947). Interventions: n-3 LCPUFA 2.4 g/day (55% EPA, 37% DHA; triacylglycerol form) vs placebo (olive oil). Vitamin D3 2400 IU/day vs placebo, on top of recommended 400 IU/day (dose comparison 2800 vs 400 IU/day). Participants: Of 736 women in COPSAC2010, 693 women and 695 children contributed at least one microbiota sample in the n-3 trial; 580 women and 581 children in the vitamin D trial. Randomization produced comparable baseline characteristics. Sampling: Maternal vaginal swabs at gestational weeks 24 and 36. Infant airway samples (hypopharyngeal aspirates) at 1 week, 1 month, and 3 months. Infant fecal samples at 1 week, 1 month, and 1 year. Samples stored at −80 °C. Airway immunology: At 1 month, nasal mucosal lining fluid collected by nasosorption; 20 cytokines/chemokines quantified by high-sensitivity electrochemiluminescence multiplex assays. Laboratory procedures: DNA extracted (Mobio PowerMAG; epMotion 5075). 16S rRNA gene V4 region amplified (primers 515F/806R) using two-step PCR; libraries pooled and sequenced on Illumina MiSeq (2×250 bp). Bioinformatics: Demultiplexing via MiSeq software; trimming (biopieces); chimera checking (usearch); OTU clustering (UPARSE); taxonomic classification (mothur). Samples with <2000 reads removed. Sequencing summary: 4991 samples; mean depth 54,682 reads; 6846 unique OTUs detected. Statistical analysis: Conducted in R v3.3.0. Alpha diversity: Shannon index. Beta diversity: weighted UniFrac; PERMANOVA (10,000 permutations) for group differences; PCoA for visualization. Differential abundance (DA): Wilcoxon rank-sum at phylum level; metagenomeSeq feature model at genus and OTU levels after filtering (≥10% presence, ≥0.01% mean relative abundance). Multiple testing controlled by FDR (Benjamini–Hochberg), with exploratory significance threshold FDR q<0.1. Additive effects: Spearman correlation of number of interventions (0,1,2) with taxa and PCoA features. Microbiota maturation: Microbiota-by-age z-score (MAZ) via random forest predicting microbiota age; 10-fold CV; metrics: microbial maturity (predicted – median microbiota age) and MAZ (standardized). Mediation analysis: Parametric survival regression models for time to persistent wheeze/asthma onset; mediator = PCo1 of 1-month airway microbiota; estimates of average direct effect (ADE), average causal mediated effect (ACME), and proportion mediated (R packages survival and mediation). Covariates: For microbiota analyses, effects tested with/without adjustment for delivery mode, siblings, antibiotics in pregnancy/first month, birth season, pets at birth, and sex.

• No significant effects of either prenatal n-3 LCPUFA or vitamin D on maternal vaginal microbiota (alpha or beta diversity) at week 36, despite 12 weeks of supplementation (PERMANOVA p>0.2).
• No significant effects on infant fecal microbiota at 1 week, 1 month, or 1 year (alpha and beta diversity, all p>0.2–0.9).
• Infant airway microbiota: At 1 month, both interventions significantly altered beta diversity (weighted UniFrac PERMANOVA; n-3 LCPUFA F=3.74, R2≈0.007, p=0.005; vitamin D F=4.228, R2≈0.007, p=0.004). No significant interaction between interventions; effects remained after adjustment for covariates. No effects at 1 week or 3 months.
• Differential abundance at 1 month in airways: Both interventions associated with decreased Firmicutes and increased Proteobacteria. n-3 LCPUFA significantly decreased Gemella (p=0.005) and Veillonella (p=0.002) at genus level. Trends suggested decreases in Streptococcus and Staphylococcus and increases in Moraxella and Haemophilus, though individual genus effects mostly not FDR-significant. OTU-level exploratory results: n-3 increased 4 OTUs and decreased 6 OTUs (including three Veillonella); vitamin D increased OTUs in Neisseria and Haemophilus and decreased seven Streptococcus OTUs (five putative S. pneumoniae, others S. mitis/oralis); none remained significant after multiple-testing correction (FDR q≥0.1).
• Additive effects: PCo1 of 1-month airway microbiota shifted stepwise with number of interventions (0,1,2) (Wilcoxon p<0.001), with corresponding stepwise decrease in Firmicutes and increase in Proteobacteria; Streptococcus decreased and Moraxella increased across 0→2 interventions (uncorrected p<0.05; FDR not significant for genera).
• Microbiota maturation (MAZ): n-3 LCPUFA decreased airway MAZ at 1 week (p=0.004) and increased at 1 month (p=0.01); vitamin D showed similar, weaker trends. Additive per-intervention effects: −0.2 MAZ at 1 week (p=0.007) and +0.1 at 1 month (p<0.05). No intervention effects on gut MAZ or airway MAZ at 3 months.
• Airway immune mediators at 1 month associated with intervention-driven microbiota variation (PCo1): positive associations with CCL4, TNF-α, CXCL8, IL-1β; negative with CCL2 and CCL17 (linear models, n=585).
• Mediation analysis: Estimated proportion of asthma risk reduction mediated by 1-month airway microbiota was minor and not statistically significant: 9% for n-3 LCPUFA (p=0.11) through age 5 years; 5.5% for vitamin D (p=0.39) for persistent wheeze through age 3 years.
• Mode of delivery did not modify the intervention effects on 1-month airway microbiota (no interaction in beta diversity or PCo1 shift).

The study demonstrates that prenatal supplementation with n-3 LCPUFA and high-dose vitamin D selectively modulates the infant airway microbiota at 1 month of age, without detectable effects on maternal vaginal or infant gut microbiota. Both interventions produced similar and additive community shifts (reduced Firmicutes, increased Proteobacteria; decreases in Streptococcus, Staphylococcus, Gemella, Veillonella; increases in Moraxella and Haemophilus), suggesting shared or convergent pathways influencing early airway colonization, potentially via immune modulation. The intervention-associated microbiota variation correlated with a distinct airway immune mediator profile (elevated CCL4, TNF-α, CXCL8, IL-1β; reduced CCL2, CCL17), supporting an interaction between microbial colonization and mucosal immunity during a critical early-life window. Despite affecting genera previously linked to asthma risk (e.g., reductions in Streptococcus, Gemella, Veillonella), mediation analysis indicates that airway microbiota alterations account for only a minor, statistically non-significant fraction of the clinical protection against asthma/wheeze, implying that other mechanisms (immune maturation, eicosanoid pathways, epigenetic modulation, lung development) likely contribute substantially. The effects were time-specific (evident at 1 month but not at 1 week or 3 months), consistent with dynamic airway microbial maturation and a potential window of opportunity. Delivery mode did not alter effects, arguing against mediation via altered perinatal transfer from maternal gut. Overall, the findings address the hypothesis that prenatal nutrients can shape early airway microbiota and suggest limited mediation of clinical benefits through these microbial changes, while highlighting immune–microbiota interactions in early infancy.

Prenatal n-3 LCPUFA and high-dose vitamin D supplementation in a randomized factorial trial significantly and additively modified the infant airway microbiota at 1 month of age, without affecting maternal vaginal or infant gut microbiota. The interventions shifted overall community structure and relative abundances of key genera and were associated with distinct airway immune mediator profiles. However, mediation analysis suggests that these microbiota changes explain only a minor, non-significant proportion of the reductions in asthma/persistent wheeze risk. The study provides causal evidence that prenatal dietary supplements can influence early airway colonization and immune milieu, underscoring a time-sensitive window in early life. Future research should include denser longitudinal sampling around the first month, maternal gut and immediate postnatal infant sampling to assess transfer pathways, functional metagenomics/transcriptomics to capture microbial function and non-bacterial components (fungi, viruses), and mechanistic studies integrating immune profiling and epigenetics to delineate causal pathways.

• Sampling scope: No maternal gut microbiota sampling before/after intervention and no immediate post-birth infant sampling, limiting assessment of maternal–infant microbial transfer.
• Temporal resolution: Airway sampling intervals may have missed the precise window of maximal effect; no effect observed at 3 months when the microbiota is more stable.
• Methodological scope: 16S rRNA amplicon sequencing limits resolution to bacteria and lacks functional insights; no assessment of fungi, parasites, or viruses.
• Statistical power: Mediation effects were small and non-significant; splitting overall clinical effects into mediated and direct pathways reduces power.
• Multiple testing: Many OTU-level differences did not survive FDR correction, reflecting exploratory nature and modest effect sizes.
• Generalizability: Cohort largely Caucasian with specific regional context; findings may not generalize to other populations.