Maternal Bacterial Engraftment in Multiple Body Sites of Cesarean Section Born Neonates after Vaginal Seeding-a Randomized Controlled Trial

Abstract

Children delivered by elective, prelabor Cesarean section (C-section) are not exposed to the birth canal microbiota and, in relation to vaginally delivered children, show altered microbiota development. Perturbed microbial colonization during critical early-life windows of development alters metabolic and immune programming and is associated with an increased risk of immune and metabolic diseases. In nonrandomized studies, vaginal seeding of C-section-born neonates partially restores their microbiota colonization to that of their vaginally delivered counterparts, but without randomization, confounding factors cannot be excluded. In a double-blind, randomized, placebo-controlled trial, we determined the effect of vaginal seeding versus placebo seeding (control arm) on the skin and stool microbiota of elective, prelabor C-section-born neonates (n = 20) at 1 day and 1 month after birth. We also examined whether there were between-arm differences in engraftment of maternal microbes in the neonatal microbiota. In relation to the control arm, vaginal seeding increased mother-to-neonate microbiota transmission and caused compositional changes and a reduction in alpha diversity (Shannon Index) of the skin and stool microbiota. The neonatal skin and stool microbiota alpha diversity when maternal vaginal microbiota is provided is intriguing and highlights the need of larger randomized studies to determine the ecological mechanisms and effects of vaginal seeding on clinical outcomes. IMPORTANCE Children delivered by elective C-section are not exposed to the birth canal and show altered microbiota development. Impairing microbial colonization during early life alters metabolic and immune programming and is associated with an increased risk of immune and metabolic diseases. In a double-blind, randomized, placebo-controlled trial, we determined the effect of vaginal seeding on the skin and stool microbiota of elective C-section born neonates and found that vaginal seeding increased mother-to-neonate microbiota transmission and caused compositional changes and a reduction in the skin and stool microbiota diversity. The reduction of neonatal skin and stool microbiota diversity when maternal vaginal microbiota is provided is intriguing and highlights the need of larger randomized studies to determine the ecological mechanisms and effects of vaginal seeding on clinical outcomes.

Keywords: Cesarean section; microbiome; microbiota; neonate; randomized controlled trial; vaginal microbiome transfer; vaginal seeding.

FIG 1

Differences in bacterial DNA load and composition between treatment groups at differing body sites, collection types, and times. (A) Bacterial DNA load in maternal vaginal swabs (both inoculated with vaginal fluids), gauze (control gauze not inoculated with vaginal fluids), infant skin (forearm), and infant stool. (B) Bacterial load of the 20 most abundant genera in gauzes from the two groups. Samples and taxa are ordered by unsupervised hierarchical clustering based on bacteria load. Bacterial load was estimated by 16S rRNA gene copy number based on qPCR using 16S rRNA gene universal primers. Wilcoxon signed-rank tests were performed for intergroup comparisons, with statistical significance indicated as follows: *, P < 0.05; ***, P < 0.001.

FIG 2

Shannon diversity index by treatment group at differing body sites, collection types, and times. Shannon diversity index values were estimated using the R package DivNet and assessed for significance using the function “betta” from the R package breakaway. Intergroup comparisons were considered statistically significant and were indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3

Log₂ fold differences of ASV relative abundance by treatment group in transitional stool (A), day 30 stool (B), and day 1 skin (C). The dots represent the mean log₂ fold change of relative abundance compared with the control group; the bars indicate the standard error of log₂ fold change. All taxa were detected as significantly different between treatment group by ANCOM with default parameters and with a threshold of 0.9 (i.e., >90% comparisons indicate that the significant taxa have different relative abundance between groups). (D) Change in the relative abundance of top 15 taxa at the genus level between infant transitional stool and day 30 stool by treatment group.

FIG 4

Proportion of maternal bacterial sources in infant and maternal body sites. (A) Contribution of maternal bacterial sources from different body sites to the microbiota of the infant skin (i), infant transitional stool (ii), and infant day 30 stool (iii). (B) Contribution of maternal vaginal bacteria (from vaginal swab) to the microbiota of the infant skin (i), infant transitional stool (ii), and infant day 30 stool (iii). (C) Bacterial sharing between vagina and other maternal body sites. Wilcoxon signed-rank tests were performed to compare source differences between groups, with statistical significance indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.