Oral administration of maternal vaginal microbes at birth to restore gut microbiome development in infants born by caesarean section: A pilot randomised placebo-controlled trial

Abstract

Background: Birth by caesarean section (CS) is associated with aberrant gut microbiome development and greater disease susceptibility later in life. We investigated whether oral administration of maternal vaginal microbiota to infants born by CS could restore their gut microbiome development in a pilot single-blinded, randomised placebo-controlled trial (Australian New Zealand Clinical Trials Registry, ACTRN12618000339257).

Methods: Pregnant women scheduled for a CS underwent comprehensive antenatal pathogen screening. At birth, healthy neonates were randomised to receive a 3 ml solution of either maternal vaginal microbes (CS-seeded, n = 12) or sterile water (CS-placebo, n = 13). Vaginally-born neonates were used as the reference control (VB, n = 22). Clinical assessments occurred within the first 2 h of birth, and at 1 month and 3 months of age. Infant stool samples and maternal vaginal extracts from CS women underwent shotgun metagenomic sequencing. The primary outcome was gut microbiome composition at 1 month of age. Secondary outcomes included maternal strain engraftment, functional potential of the gut microbiome, anthropometry, body composition, and adverse events.

Findings: Despite the presence of viable microbial cells within transplant solutions, there were no observed differences in gut microbiome composition or functional potential between CS-seeded and CS-placebo infants at 1 month or 3 months of age. Both CS groups displayed the characteristic signature of low Bacteroides abundance, which contributed to a number of biosynthesis pathways being underrepresented when compared with VB microbiomes. Maternal vaginal strain engraftment was rare. Vaginal seeding had no observed effects on anthropometry or body composition. There were no serious adverse events associated with treatment.

Interpretation: Our pilot findings question the value of vaginal seeding given that oral administration of maternal vaginal microbiota did not alter early gut microbiome development in CS-born infants. The limited colonisation of maternal vaginal strains suggest that other maternal sources, such as the perianal area, may play a larger role in seeding the neonatal gut microbiome.

Funding: Health Research Council of New Zealand, A Better Start - National Science Challenge.

Keywords: Birth; Caesarean section; Gut microbiome; Infant development; Maternal strain transmission; Metagenomics; Vaginal seeding.

Fig. 1

CONSORT diagram showing the flow of participants through the ECOBABe trial, including infants born by caesarean section (CS) or vaginally (VB).

Fig. 2

Comparisons of microbiome composition and diversity in infants born by caesarean section who received vaginal seeding (CS-seeded) or placebo (CS-placebo), and infants born vaginally (VB). Non-metric multi-dimensional scaling plots based on genus-level Bray Curtis dissimilarities showing the variation in microbiome composition in: (a) maternal vaginal and infant faecal samples at 1 month and 3 months of age; and (b) faecal samples from infants in the three study groups at 1 month and 3 months of age. Significant differences in infant microbiome composition based on birth mode, intervention group, feeding mode, and sex was assessed by PERMANOVA (see Supplementary Table 3). (c) Shannon diversity index and (d) gene richness, normalised by sequencing depth, for infant faecal microbiomes at 1 month and 3 months of age; each box represents the median and inter-quartile range (IQR), and whiskers the range of the data (expanding up to 1.5 x IQR). Group differences assessed by Kruskal–Wallis test.

Fig. 3

Taxonomic differences in infant faecal microbiomes at 1 and 3 months of age in infants born by caesarean section who received vaginal seeding (CS-seeded) or placebo (CS-placebo), and infants born vaginally (VB). (a) Relative abundance of bacterial families in infant faecal microbiomes. Bacterial families whose relative abundances were <1% are categorised as “Other”. (b) Relative abundances of Bacteroides, Bifidobacterium, and Lactobacillus genera in infant faecal microbiomes. Each box represents the median and inter-quartile range (IQR), and whiskers the range of the data (expanding up to 1.5 x IQR). ***p < 0.001 for an overall difference in relative abundances among the three study groups, assessed by a Kruskal–Wallis test.

Fig. 4

Microbial metabolic pathways that were more abundant in the faecal microbiomes of infants born vaginally (VB) in comparison to infants born by caesarean section who received vaginal seeding (CS-seeded) or placebo (CS-placebo). Differences in MetaCyc pathway abundances were assessed using general linearised models, as implemented in MaAsLin2, and were adjusted for feeding mode and sex. Cells represent the mean pathway abundance expressed in copies per million (CPM) for each infant group at 1 month (1m) and 3 months (3m) of age. Broad-level functions were categorised based on MetaCyc “superclasses”.

Fig. 5

Maternal vaginal strains detected at 1 month (1m) and 3 months (3m) of age, in faecal samples of infants born by caesarean section who received vaginal seeding (CS-seeded) or placebo (CS-placebo). (a) The five maternal vaginal strains that were detected in the faecal microbiomes of CS infants. (b) Phylogenetic tree of different Bifidobacterium breve strains from infant faecal samples and vaginal samples from CS mothers. Scale bar signifies difference in sequence similarity between strains as determined by single nucleotide polymorphism (SNP)-based haplotyping. Strains from reference genomes and infants born vaginally (VB) are included for context. An example of a probable maternal strain transmission event is highlighted in the grey box.