The effects of chronic unpredicted mild stress on maternal negative emotions and gut microbiota and metabolites in pregnant rats

{"Background": "Maternal exposure to psychosocial stress during pregnancy increases risk for adverse pregnancy outcomes and mood disturbances. The gut-brain axis, particularly the gut microbiota and its metabolites, has been implicated in stress-related behavioral changes, but the specific relationship between maternal chronic stress, gut microbial composition, and fecal metabolites during pregnancy was unclear. This study aimed to establish a rat model of chronic stress during pregnancy and use 16S rRNA sequencing and GC-MS metabolomics to characterize gut microbiota and metabolite changes, linking them to maternal emotional behavior.", "Methods": "Sixteen female Sprague-Dawley rats were randomly assigned to a stress model group or control group (n=8 each). The stress group was exposed to 21 consecutive days of CUMS, with one of nine randomly assigned stressors daily (food/water deprivation, crowding, humidity, warm water swimming, shaking, restraint, heat, tail clamping). Mating occurred on day 3 of CUMS. Plasma corticosterone was measured via radioimmunoassay at baseline and days 1, 7, 14, and 21. Behavioral assessments included the sucrose preference test (SPT) and open-field test (OFT) at the same time points. Fecal samples from 6 rats per group were analyzed using 16S rRNA V3-V4 sequencing (Illumina HiSeq) and GC-MS metabolomics. Statistical analyses used repeated-measures ANOVA, Student's t-test, PLS-DA/OPLS-DA, and Spearman correlation.", "Key Results": "CUMS significantly elevated plasma corticosterone in stressed dams compared with controls, peaking at day 14 (t=5.414, P=0.001). Stressed rats exhibited depression-like behaviors: reduced sucrose preference at days 14 and 21 (t=2.557, 2.897; P=0.029, 0.016), reduced horizontal movement at day 7 (t=2.950, P=0.013), and reduced vertical movement at day 4 (t=3.257, P=0.005). Alpha diversity indices (Sobs, Chao, Ace, Shannon) were significantly lower in stressed rats, while Simpson index was higher (all P<0.05). At the family level, Ruminococcaceae decreased from 32.67% to 23.45% and Prevotellaceae increased from 0.03% to 10.45% (P<0.05). PLS-DA and OPLS-DA showed complete separation of metabolic profiles between groups. Seven differential metabolites were identified (VIP>1, P<0.05): trigonelline, 3,4-dihydroxymandelic acid, and phenylalanine were decreased, while pantothenic acid, 2,3-diphosphoglycerate, 3-hydroxypyridine, and 4-hydroxybenzyl cyanide were increased. KEGG pathway analysis identified 11 significantly altered metabolic pathways (all P<0.05), including nicotinate/nicotinamide metabolism (P=0.045), aminoacyl-tRNA biosynthesis (P=0.0426), and phenylalanine/tyrosine/tryptophan biosynthesis (P=0.0289). Spearman correlation revealed vertical movement and sucrose preference were negatively correlated with Prevotellaceae (r=-0.90, P<0.05). Plasma corticosterone was negatively correlated with Leuconostocaceae (r=-0.89, P<0.05) and Aerococcaceae (r=-0.90, P<0.05). Ruminococcaceae was negatively correlated with 2,3-diphosphoglycerate (r=-0.89, P<0.05).", "Clinical Implications": "These findings support the gut-brain axis hypothesis that chronic stress during pregnancy induces gut dysbiosis and metabolite perturbations that may underlie maternal negative emotions. The differential metabolites identified represent potential biomarkers for stress-related maternal harm and candidate targets for intervention. The strong correlations between Prevotellaceae and behavioral measures, and between specific bacterial families and metabolites, suggest avenues for probiotic or metabolite-based therapies to mitigate the adverse effects of prenatal stress on maternal mental health. Further research is needed to establish causal mechanisms and to translate these findings to pregnant women."}