Alterations in the Vaginal Microbiome by Maternal Stress Are Associated With Metabolic Reprogramming of the Offspring Gut and Brain

Abstract

The neonate is exposed to the maternal vaginal microbiota during parturition, providing the primary source for normal gut colonization, host immune maturation, and metabolism. These early interactions between the host and microbiota occur during a critical window of neurodevelopment, suggesting early life as an important period of cross talk between the developing gut and brain. Because perturbations in the prenatal environment such as maternal stress increase neurodevelopmental disease risk, disruptions to the vaginal ecosystem could be a contributing factor in significant and long-term consequences for the offspring. Therefore, to examine the hypothesis that changes in the vaginal microbiome are associated with effects on the offspring gut microbiota and on the developing brain, we used genomic, proteomic and metabolomic technologies to examine outcomes in our mouse model of early prenatal stress. Multivariate modeling identified broad proteomic changes to the maternal vaginal environment that influence offspring microbiota composition and metabolic processes essential for normal neurodevelopment. Maternal stress altered proteins related to vaginal immunity and abundance of Lactobacillus, the prominent taxa in the maternal vagina. Loss of maternal vaginal Lactobacillus resulted in decreased transmission of this bacterium to offspring. Further, altered microbiota composition in the neonate gut corresponded with changes in metabolite profiles involved in energy balance, and with region- and sex-specific disruptions of amino acid profiles in the developing brain. Taken together, these results identify the vaginal microbiota as a novel factor by which maternal stress may contribute to reprogramming of the developing brain that may predispose individuals to neurodevelopmental disorders.

Figure 1.

Early pregnancy stress altered protein content of the maternal vaginal mucosal environment measured by proteomics assessment. A, Heat map of vaginal proteins with significantly different abundance levels between control and stress-exposed females at E7.5 and PN2, demonstrating changes in protein profiles across pregnancy and exposure to stress. B, OPLS-DA score plots showing significant separation of vaginal protein profiles at E7.5 (n = 10) vs PN2 (n = 11), indicating that the vaginal proteome was significantly different at these 2 time points. C, Volcano plot of vaginal proteins demonstrating the contribution that each protein made to the differences between time points. Highlighted in red are proteins with VIP scores above 1 and considered to be strong contributors to group separation, showing that most proteins are down-regulated at PN2 relative to E7.5. VIP, Variable Importance in the Projection. D, Differentially abundant proteins identified by volcano plots grouped into functional annotation categories using DAVID functional clustering tools, indicating that differentially abundant proteins cluster to pathways related to cytoskeletal reorganization. E, OPLS-DA scores plot showing clear separation between control (n = 6) and stress (n = 5) groups at PN2, suggesting that stress drives distinct vaginal protein profiles at this time. F, Volcano plot of vaginal proteins demonstrating the contribution that each protein makes to the differences between control and stress dams. Highlighted in red are proteins with VIP scores above 1 and considered to be strong contributors to group separation, showing 45 proteins differentially regulated by stress at PN2. G, Proteins differentially regulated by stress were grouped into functional annotation categories using the DAVID clustering tool. Negative DAVID enrichment scores correspond to significantly enriched pathways by proteins decreased by stress, whereas positive DAVID enrichment scores correspond to significantly enriched pathways by proteins increased by stress.

Figure 2.

Stress early in pregnancy altered maternal Lactobacillus transmission to offspring and more broadly disrupted neonate gut microbiota composition. A, Vaginal Lactobacillus abundance at PN2 was significantly decreased in dams exposed to EPS (n = 8) compared with control dams (n = 13). B, No difference in overall PN2 vaginal microbial census was noted. C, Comparison of Lactobacillus abundance in the PN2 neonate gut from control (n = 13) and offspring exposed to EPS (n = 8). Asterisks indicate a main effect of treatment by univariate analysis. D, No difference in PN2 neonate gut microbial census was noted. E, Maternal vaginal Lactobacillus levels were significantly positively correlated with neonate distal gut Lactobacillus levels. Solid line signifies the best-fit line from a linear regression, and the dotted lines signify the 95% confidence interval (CI) for that best fit line. F, No relationship was noted between the maternal fecal Lactobacillus and offspring gut Lactobacillus levels. G, Disrupting transmission of maternal vaginal microbiota by cesarean delivery significantly reduced total bacterial count in the PN2 neonate gut, whereas seeding cesarean delivered offspring with maternal vaginal fluid recovered total bacterial count compared with VD offspring. VD, vaginally delivered; CS, non-inoculated cesarean delivered; CSI, inoculated CS (n = 4–5 offspring/mode of delivery). Asterisks indicate a significant difference between CS offspring and all other groups determined by univariate analysis. H, Effect of EPS on selected bacterial genera in the PN2 neonate gut. In control offspring, a clear sex difference in microbiota composition was noted, whereas EPS disrupted microbiota composition disrupted this sex difference with male EPS offspring showing increased colonization by strict anaerobes and a microbiota profile more similar to control females (n = 8 per sex and treatment). *, P < .05.

Figure 3.

Stress alterations in neonate microbiota correlate with changes in colon and plasma metabolites as measured by metabolomics assessment. A, OPLS-DA plots showed clear separation between control (n = 10) and EPS (n = 11) offspring, indicating that EPS exposures resulted in distinct colon metabolite profiles already at PN2. B, Colon metabolites that were significantly increased or decreased in EPS offspring relative to controls were clustered into metabolic pathways identified by MSEA. Dotted lines signify mean value of control offspring. C, OPLS-DA plots showed clear separation between control (n = 14) and EPS (n = 12) offspring, indicating that EPS exposure resulted in distinct plasma metabolite profiles already at PN2. D, Plasma metabolites that were significantly increased or decreased in EPS offspring relative to controls clustered into metabolic pathways identified by MSEA.

Figure 4.

In accordance with changes in gut and plasma metabolites, EPS altered free AA levels in the PN2 brain in a region- and sex-specific manner. A, OPLS-DA scores plots showed significant brain region-specific differences in AA profiles of the hippocampus (Hpc), PvThal, and the PVN at PN2. B and C, AA differences with Log2 fold change in the PVN demonstrating that EPS altered the PVN AA profile in a sex-dependent manner. Stressed males (B) displayed down-regulation compared with control males, whereas stressed females (C) were more likely to display up-regulation of AAs compared with control females.