Supplementary Figure 3: The maternal vaginal microbiome does not impact hypothalamic–pituitary–adrenal stress axis responsivity in female offspring.
(a) Production of corticosterone following a 15 min restraint stress in vaginally delivered and cross-fostered control and prenatal stress-exposed female offspring is different across time but not by treatment or its interaction (two-way ANOVA, main effect of time, F3,33 = 63.35, P = 0.000000000000088). N = 6 vaginally delivered control females; N = 7 vaginally delivered prenatal stress-exposed females. Data is representative of two experimental replicates. Two-way ANOVA with time as a repeated measure. Data are presented as mean ± range with bars representing min and max. (b) Production of corticosterone following a 15 min restraint stress in C→C, C→S, S→C, and S→S females is different across time but not by treatment or its interaction (two-way ANOVA, main effect of time, F3,24 = 130.7, P = 0.000000000000005). N = 3 C→C females; N = 3 C→S females; N = 3 S→C females; N = 3 S→S females. Data is representative of two experimental replicates. Two-way ANOVA with time as a repeated measure. Data are presented as mean ± range with bars representing min and max. (c) Total Area Under the Curve (AUC) production of corticosterone in females following a 15 min restrain stress. (Left) Total AUC corticosterone levels did not differ between vaginally delivered and cross-fostered control females and prenatal stress-exposed females (Unpaired two-sided t-Test, t11 = 1.013, P = 0.33). (Right) Total AUC corticosterone levels did not differ between C→C, C→S, S→C, and S→S females (one-way ANOVA, F3,8 = 2.346, P = 0.1490). N = 6 vaginally delivered control females; N = 7 vaginally delivered prenatal stress-exposed females; N = 3 C→C females; N = 3 C→S females; N = 3 S→C females; N = 3 S→S females. Data is representative of two experimental replicates. Unpaired two-sided t-Test comparing vaginally delivered and cross-fostered offspring; One-way ANOVA compared cesarean delivered and inoculated offspring. Data are presented as mean ± SEM with individual data points overlaid.
