Fig. 3. Modeling compounding maternal risk factors results in an adverse response to birth-associated microbial exposure.

A Schematic of experimental timeline for the induction of pregestational excessive weight gain, glucose intolerance, and microbiota alterations through the consumption of a high-fat low-fiber diet (HFt-LFb). Day 0 refers to the beginning of the dietary treatment and does not reflect chronological age. Created with BioRender.com. B Consumption of a high-fat low-fiber diet accelerates body weight gain in females compared with females consuming a low-fat high-fiber diet (two-way ANOVA, main effect of time, F_{5, 120} = 94.17, P < 0.0001; main effect of diet, F_{1, 24} = 30.70, P < 0.0001; time*diet interaction, F_{5, 120} = 36.61, P < 0.0001). N = 12 LFt-HFb females, 20 HFt-LFb females per timepoint. Data represented as mean ± SEM. Time (in weeks) is measured from time of diet switch. Data are representative of two independent experiments. **P < 0.01, ***P < 0.001. C Principal coordinates analysis demonstrating temporal dynamics of diet on the gut microbiota, whereby 1wk consumption of a high-fat low-fiber diet resulted in separate clustering from females consuming a low-fat high-fiber diet that failed to recover during the treatment window. N = 12 LFt-HFb females, 20 HFt-LFb females per timepoint, total of 68 samples that passed quality filtering. D Mean relative abundance of top ten taxa showing rapid changes to the fecal microbiota following transition to a high-fat low-fat diet, characterized by a loss of Clostridiales. N = 12 LFt-HFb females, 20 HFt-LFb females per timepoint, total of 68 samples that passed quality filtering. E Expression of HIF-1a is significantly decreased in the colon following 6-week consumption of a high-fat low-fiber diet relative to females consuming a low-fat high-fiber diet (two-sided t-test, t₄ = 4.9, P = 0.008), indicating possible disruption in hypoxia homeostasis in the colon. N = 3 LFt-HFb females, 3 HFt-LFb females. Data represented as mean ± SEM with individual data points overlaid. **P < 0.01. F Left, Plasma glucose levels during a glucose tolerance test in females consuming either a high-fat low-fiber or low-fat high-fiber diet. Females consuming a high-fat low-fiber diet showed significant delay in glucose clearance (two-way ANOVA, main effect of time, F_{4, 108} = 106.33, P < 0.0001; main effect of diet, F_{1, 27} = 15.86, P = 0.0005; time*diet interaction, F_{4, 108} = 15.44, P < 0.0001). Right, AUC of total plasma glucose levels showing increase glucose levels in females consuming a high-fat low-fiber diet (two-tailed t-test, t₂₄ = 4.055, P = 0.005). N = 9 LFt-HFb females, 17 HFt-LFb females Data represented as mean ± SD with individual data points overlaid. ***P < 0.001. G Schematic of experimental design to determine the impact of prenatal exposure to compounding maternal adversities, such as diet and presence of a common member of CST IV, during pregnancy on offspring outcomes. We induced the pregestational phenotype and colonized pregnant females to G. vaginalis 11E4 (Gv) gestational day 13.5 and 15.5. At gestational day 18.5, offspring from all treatment groups were colonized with the nonoptimal CST IV human vaginal microbiota. Created with BioRender.com. H Survival of offspring from dams that experience a single or multiple compounding adversities. All pups were C-section delivered and gavaged with human CST IV inoculant, showing the highest offspring mortality risk in HFt-LFb+Gv offspring that were exposed to CST IV at birth. LFt-HFb = low-fat high-fiber; HFt-LFb = high-fat low-fiber; Gv = G. vaginalis 11E4. Kaplan–Meier survival analysis. N = 20 offspring per treatment condition. I Compounding effects of maternal diet, maternal vaginal colonization by G. vaginalis (Gv), and exposure to CST IV on absolute number of neutrophils in the circulation of postnatal day 1 male pups. Triple-hit male pups show significant increase in neutrophils compared with vaginally delivered and CST IV inoculated offspring (One-way ANOVA, main effect of treatment, F_{3, 11} = 8.452, P = 0.0034; VD vs. Triple Hit, P = 0.0188; CST IV vs. Triple Hit, P = 0.0026). N = 3 vaginally delivered males, 5 CST IV inoculated LFt-HFb males, 4 CST IV inoculated HFt-LFb males, 3 CST IV inoculated HFt-LFb+Gv males. Data represented as mean ± SEM with individual data points overlaid. *P < 0.05, **P < 0.01.