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

Maternal dietary interventions during pregnancy with fish oil and high dose vitamin D have been shown to reduce the incidence of asthma and wheeze in offspring, potentially through microbial effects in pregnancy or early childhood. Here we analyze the bacterial compositions in longitudinal samples from 695 pregnant women and their children according to intervention group in a nested, factorial, double-blind, placebo-controlled, randomized trial of n-3 long-chain fatty acids and vitamin D supplementation. The dietary interventions affect the infant airways, but not the infant fecal or maternal vaginal microbiota. Changes in overall beta diversity are observed, which in turn associates with a change in immune mediator profile. In addition, airway microbial maturation and the relative abundance of specific bacterial genera are altered. Furthermore, mediation analysis reveals the changed airway microbiota to be a minor and non-significant mediator of the protective effect of the dietary interventions on risk of asthma. Our results demonstrate the potential of prenatal dietary supplements as manipulators of the early airway bacterial colonization.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection

Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability

Hans Bisgaard
Nov 27, 2019 Data was collected during the visits to the clinical research unit and stored in a dedicated custom online database. The data was doublechecked against source data and subsequently locked. An audit trail was run routinely.
Fastq-files demultiplexed by the MiSeq Controller Software were trimmed for amplification primers, diversity spacers and sequencing adapters (biopieces), mate-paired and quality filtered (USEARCH v7.0.1090, parameter: -maxee 0.5). UPARSE was used for OTU clustering (>97% identity) as recommended, in particular removing singletons after de-replication. Chimera checking was performed with USEARCH against the Genomes OnLine Database as recommended. Representative sequences were classified (Mothur v1.25.0, using the wang function at 0.8 confidence threshold) against the Mothur formatted version of the Ribosomal Database Project v9. A phylogenetic tree was constructed from an alignment of representative sequences made with Mothur's align_seqs function against the Greengenes database (may 2013 version). Alignments were then input to Fasttree in nucleotide mode. All data analyses were conducted using the statistical software R v3.5.2, using the packages phyloseq, lme4, lmerTest, vegan, mixOmics, DMwR, mediation and ggplot2.
Summary and feature-level data underlying Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative. samples with a 16S rRNA amplicon sequencing depth of below 2000 reads were excluded as they were deemed unreliable and defined as failed.
To replicate the findings a new clinical trial would have to be done, including regular microbial sampling. We are working on financing such a study, but as of yet we are the only cohort with this experimental setup.
The mothers were randomly allocated to either n-3 LCPUFA/placebo and Vitamin D/placebo. Due to the high number of mothers we were successful in creating two groups with similar baseline characteristics, and no significant allocation differences were observed (see Table 1) .
Both investigators and study participants (Cohort mothers and children) were blinded during the study period.