Maternal mental illnesses, such as depression and anxiety, are increasingly prevalent, significantly impacting children's development. Preventing these illnesses requires identifying resilience factors in addition to risk factors. The gut-brain axis, the bidirectional communication between the gut microbiome and the central nervous system, has emerged as a crucial area of research in mental health. The gut microbiome influences the brain through various pathways including the autonomic nervous system, neuropeptides, hormones, and the immune system. Studies have linked gut dysbiosis (imbalance in gut microbiota) to psychological disorders and poor physical health, characterized by reduced SCFA-producing bacteria (like butyrate-producing bacteria). Physical health is also intrinsically linked to mental well-being; poor physical health often accompanies mental illnesses. Parenting stress, a significant risk factor for maternal mental illness, has been largely unexplored in relation to gut microbiota in postpartum mothers. This study aimed to comprehensively evaluate the relationships between gut microbiota, physical health, parenting stress, and psychological resilience in Japanese mothers. Two studies were conducted: Study 1, a large-scale analysis of mothers with infants and toddlers, investigated the association between parenting stress and gut microbiota; and Study 2, focused on primiparous mothers in the early postpartum period, explored the relationship between gut microbiota, physical and physiological function (autonomic nervous system, oxytocin), and psychological resilience. This is the first study to explore these complex interrelationships in non-clinical postpartum mothers.

Existing research highlights the growing prevalence of maternal mental illness and its detrimental effects on children's well-being. The gut-brain axis is increasingly recognized as a key player in mental health, with studies linking gut dysbiosis (imbalance of gut microbiota) to depression, anxiety, and other psychological disorders. These studies often reveal reduced levels of SCFA-producing bacteria, particularly butyrate-producing bacteria, in individuals with these conditions. The importance of physical health in mental well-being is also established, with physical symptoms commonly associated with mental illness. Parenting stress, a major risk factor for maternal mental illness, is typically measured using the Parenting Stress Index (PSI). However, research investigating the link between parenting stress and the gut microbiome in postpartum mothers is limited, particularly studies using the PSI. Previous research during pregnancy has explored the relationship between perceived stress and the gut microbiome, but postpartum studies using parenting stress measures like the PSI are lacking. This gap underscores the need for research exploring these crucial interconnections, especially the role of the gut microbiome as a potential mediator of stress and resilience in postpartum mothers.

The study comprised two parts: Study 1 and Study 2. **Study 1:** This study involved 339 Japanese mothers raising children aged 0-4 years. Participants were excluded if they were under medical treatment, had taken antibiotics in the past three months, were taking psychiatric medication, or had incomplete questionnaire responses. Parenting stress was assessed using the Parenting Stress Index (PSI), with participants exceeding predefined cutoffs classified as high-risk. Physical health was assessed using the Multidimensional Physical Scale (MDPS), sleep duration and quality were self-reported. Dietary and lifestyle habits were assessed using the Mykinso Pro questionnaire. Stool samples were collected and analyzed for gut microbiome composition and diversity (alpha and beta diversity using Shannon index and UniFrac distances). **Study 2:** This study focused on 27 primiparous Japanese mothers within 3-6 months postpartum. Data was collected at two visits. At the first visit, body composition (BMI, body fat percentage, SMI, ECW/TBW), grip strength, two-step test, and gait speed were measured. Questionnaires included the Resilience Scale (RS25), the Japan Resilience Scale (J-RS), and the Center for Epidemiological Studies Depression Scale (CESD). At the second visit, saliva samples were collected for oxytocin measurement, a 3-minute resting electrocardiogram was recorded for autonomic nervous system assessment (cardiac vagal and sympathetic indices using Lorenz plot analysis), and the PSI was administered. Stool samples were collected for microbiome analysis using 16S rRNA gene sequencing and metagenomic shotgun sequencing. Statistical analyses included Mann-Whitney U-tests, PERMANOVA, Pearson correlations, and regression analyses.

**Study 1:** Mothers at high risk of parenting stress exhibited lower alpha and beta diversity of gut microbiota compared to the healthy group. Significant differences were found in the abundance of several bacterial genera, including reduced SCFA-producing bacteria (*Lachnospira*, *Veillonella*, *Alistipes*, and *Phascolarctobacterium*), fewer immunity-related bacteria (*Odoribacter* and *Sutterella*), and increased inflammatory bacteria (*Escherichia-Shigella*). High-risk mothers also reported poorer sleep quality and worse MDPS scores (indicating digestive dysfunction, physical depression, and impaired microcirculation). These findings remained significant even after controlling for age and education. **Study 2:** A positive correlation was observed between gut microbiota diversity (Shannon α-diversity) and vagal nerve activity, which was also positively correlated with psychological resilience (J-RS). Regression analysis revealed associations between specific bacterial genera and both psychological resilience and physical function. *Blautia* SC05B48 showed a positive association with resilience and physical function. *Clostridium* SY8519 was positively associated with resilience but negatively with oxytocin. *Collinsella aerofaciens* showed a positive association with resilience and a negative association with gait speed. *Eggerthella lenta* was negatively associated with resilience and positively associated with parenting stress and grip strength. *Faecalibacterium prausnitzii* was positively associated with sympathetic nerve activity. Approximately 41% of primiparous mothers in this study met criteria for sarcopenia (low skeletal muscle mass index [SMI]), indicating poor muscle mass and motor function.

The findings provide compelling evidence for the intricate relationship between the gut microbiome, physical and physiological factors, and maternal mental health. Study 1 demonstrates the association of gut dysbiosis and reduced SCFA-producing bacteria with higher parenting stress risk. This suggests that an altered gut microbiome might contribute to increased stress vulnerability. Study 2's findings highlight the role of vagal nerve activity and specific bacterial genera in psychological resilience. The positive correlation between gut microbiota diversity and vagal activity suggests a possible pathway linking the gut to brain function and emotional regulation. The identified bacterial genera (*Blautia*, *Clostridium*, *Collinsella*, and *Eggerthella*) offer potential targets for interventions aimed at improving maternal mental health. These results provide valuable insights into the complex interplay of factors influencing maternal resilience and suggest potential avenues for personalized interventions.

This study reveals crucial associations between the gut microbiome, physical health, and psychological resilience in mothers. Reduced microbiome diversity and specific bacterial imbalances are linked to increased parenting stress risk. Vagal nerve activity plays a significant role in resilience, and certain bacterial genera show promising associations with both resilience and physical function. These findings suggest potential avenues for personalized interventions targeting the gut microbiome to improve maternal mental health, but further longitudinal studies with larger sample sizes are needed to confirm these findings and explore causal relationships. Dietary interventions may also be beneficial, given the potential impact of the Japanese diet on specific bacterial genera.

Several limitations should be considered. The cross-sectional design of both studies prevents the establishment of causal relationships. Study 2's small sample size limits the statistical power and generalizability of the findings, also hindering the inclusion of covariates in regression analyses. The study primarily focuses on Japanese mothers, and the findings may not be generalizable to other populations. Future research should address these limitations by conducting longitudinal studies with larger and more diverse samples to clarify the causal relationships and generalizability of the findings.