Comparative Metabolomics and Microbiome Analysis of Ethanol versus OMNImet/gene•GUT Fecal Stabilization.

Metabolites from feces provide important insights into the functionality of the gut microbiome. As immediate freezing is not always feasible in gut microbiome studies, there is a need for sampling protocols that provide the stability of the fecal metabolome and microbiome at room temperature (RT). Here, we investigated the stability of various metabolites and the microbiome (16S rRNA) in feces collected in 95% ethanol (EtOH) and commercially available sample collection kits with specific preservatives OMNImet•GUT/OMNIgene•GUT. To simulate field-collection scenarios, the samples were stored at different temperatures at varying durations (24 h + 4 °C, 24 h RT, 36 h RT, 48 h RT, and 7 days RT) and compared to aliquots immediately frozen at -80 °C. We applied several targeted and untargeted metabolomics platforms to measure lipids, polar metabolites, endocannabinoids, short-chain fatty acids (SCFAs), and bile acids (BAs). We found that SCFAs in the nonstabilized samples increased over time, while a stable profile was recorded in sample aliquots stored in 95% EtOH and OMNImet•GUT. When comparing the metabolite levels between aliquots stored at room temperature and at +4 °C, we detected several changes in microbial metabolites, including multiple BAs and SCFAs. Taken together, we found that storing samples at RT and stabilizing them in 95% EtOH yielded metabolomic results comparable to those from flash freezing. We also found that the overall composition of the microbiome did not vary significantly between different storage types. However, notable differences were observed in the α diversity. Altogether, the stability of the metabolome and microbiome in 95% EtOH provided results similar to those of the validated commercial collection kits OMNImet•GUT and OMNIgene•GUT, respectively.

Human milk: From complex tailored nutrition to bioactive impact on child cognition and behavior.

Human milk is a highly complex liquid food tailor-made to match an infant's needs. Beyond documented positive effects of breastfeeding on infant and maternal health, there is increasing evidence that milk constituents also impact child neurodevelopment. Non-nutrient milk bioactives would contribute to the (long-term) development of child cognition and behavior, a process termed 'Lactocrine Programming'. In this review we discuss the current state of the field on human milk composition and its links with child cognitive and behavioral development. To promote state-of-the-art methodologies and designs that facilitate data pooling and meta-analytic endeavors, we present detailed recommendations and best practices for future studies. Finally, we determine important scientific gaps that need to be filled to advance the field, and discuss innovative directions for future research. Unveiling the mechanisms underlying the links between human milk and child cognition and behavior will deepen our understanding of the broad functions of this complex liquid food, as well as provide necessary information for designing future interventions.

Infant gut microbiota and negative and fear reactivity.

BACKGROUND: Studies indicate that gut microbiota is related to neurodevelopmental and behavioral outcomes. Accordingly, early gut microbiota composition (GMC) has been linked to child temperament, but research is still scarce. The aim of this study was to examine how early GMC at 2.5 months is associated with child negative and fear reactivity at 8 and 12 months since they are potentially important intermediate phenotypes of later child psychiatric disorders. METHODS: Our study population was 330 infants enrolled in the longitudinal FinnBrain Birth Cohort Study. Gut microbiota composition was analyzed using stool sample 16s rRNA sequencing. Negative and fear reactivity were assessed using the Laboratory Temperament Assessment Battery (Lab-TAB) at child's age of 8 months (n =150) and the Infant Behavior Questionnaire-Revised Short Form (IBQ-R SF) at child's age of 12 months (n = 276). CONCLUSIONS: We found a positive association between alpha diversity and reported fear reactivity and differing microbial community composition based on negative reactivity for boys. Isobutyric acid correlated with observed negative reactivity, however, this association attenuated in the linear model. Several genera were associated with the selected infant temperament traits. This study adds to the growing literature on links between infant gut microbiota and temperament informing future mechanistic studies.

Gut microbiota diversity but not composition is related to saliva cortisol stress response at the age of 2.5 months.

Human brain and intestinal microbes reportedly maintain a constant bidirectional connection through diverse neural, endocrine, immune, and metabolic pathways. Increasing evidence indicates that this communication system, referred to as microbiota-gut-brain axis, enables the gut microbes to influence several aspects of brain function and behavior, including hypothalamic-pituitary-adrenal (HPA) axis stress responses, and on the other hand, stress can affect gut microbiota. However, the role of gut microbiota in the HPA axis functioning in humans remains to be specified especially in early life. This study aimed at identifying the potential link between the cortisol stress response and the gut microbiota at the age of 2.5 months. Fecal microbiota profiles were acquired by 16S rRNA gene sequencing, while salivary cortisol responses after an exposure to a mild acute stressor represented the HPA axis reactivity. We observed that a blunted cortisol stress response was weakly associated with a diverse gut microbiota diversity at the age of 2.5 months. Gut microbiota composition was not associated with cortisol stress responsiveness, but rather with covariates, i.e. factors that influence gut microbiota composition and colonization.LAY SUMMARYThis exploratory study aimed at identifying possible links between cortisol stress responses and fecal microbiota composition in early infancy. In a well-characterized study population of 2.5-month-old infants, we observed that an attenuated cortisol stress responsiveness after a mild stressor was weakly associated with a diverse fecal microbiota. Our results suggest that the gut microbiota composition is associated with environmental factors, such as delivery mode and number of siblings, rather than with cortisol stress responsiveness, in this age group.

Associations between maternal socioeconomic, psychosocial and seasonal factors, infant characteristics and human milk cortisol concentrations.

OBJECTIVES: Glucocorticoids are one component of human milk (HM) potentially affecting offspring development. Previous studies have identified various maternal, obstetric and socioeconomic characteristics that are associated with HM cortisol concentration but the literature is still scarce concerning these determinants in human populations. We aimed to identify which factors are linked with HM cortisol concentration at 2 months postpartum. METHODS: We analyzed data from 340 lactating Finnish mothers using ordinary least squares regression with log-transformed HM cortisol concentration as the dependent variable. Potential predictors included obstetric and maternal factors (maternal age, parity status, delivery mode, gestational age, pre-pregnancy obesity, and smoking in pregnancy), socioeconomic status (education and socioeconomic class), subjective economic well-being, maternal psychosocial factors (postpartum depression and anxiety symptoms), infant sex and age, and HM sample characteristics (time of the day and season of the year at sample collection). RESULTS: The strongest and most robust predictors were season of the year of sample collection and parity status. HM cortisol concentration was significantly higher for primiparas than multiparas. HM samples collected in summer showed significantly higher cortisol concentrations than those collected in winter, spring or autumn. CONCLUSIONS: The findings suggest that parity and season of the year at sample collection may be important factors to control for when examining HM cortisol. The strongest and most robust associations were related to maternal and sample characteristics and not to socioeconomic and psychosocial distress. This may be related to the fact that the study was conducted in a low-risk population.

Gut microbiota composition is associated with temperament traits in infants.

BACKGROUND: One of the key behavioral phenotypes in infancy are different temperament traits, and certain early life temperament traits have been shown to precede later mental health problems. Differences in the gut microbiota composition (GMC) have been suggested to link with neurodevelopment. For example, toddler temperament traits have been found to associate with differences in GMC; however, studies in infants are lacking although infancy is a rapid period of neurodevelopment as well as GM development. Thus, we aimed to investigate association between infant GMC and temperament. METHODS: The study population (n = 301, 53% boys) was drawn from the FinnBrain Birth Cohort Study. Stool samples were collected from the 2.5-month-old infants and sequenced with 16S Illumina MiSeq platform. GMC taxonomic composition (at Genus and OTU level), observed sample clusters, diversity and richness were investigated in relation to the maternal reports of Infant Behavior Questionnaire -Revised (IBQ-R) at the age of 6 months. RESULTS: Three sample clusters (Bifidobacterium/Enterobacteriaceae, Bacteroides, V. Dispar) based on GMC were identified, of which Bifidobacterium/Enterobacteriaceae-cluster presented with higher scores on the IBQ-R main dimension regulation and its subscale duration of orienting compared to Bacteroides-cluster. The clusters associated with temperament in a sex-dependent manner. The IBQ-R main dimension surgency (positive emotionality) was associated positively both with genus Bifidobacterium and Streptococcus. Alpha diversity had a negative association with negative emotionality and fear reactivity. CONCLUSION: This is the first study demonstrating associations, but not causal connections, between GMC and temperament in young infants in a prospective design.

Human milk cortisol concentration predicts experimentally induced infant fear reactivity: moderation by infant sex.

Little consideration has been given to the possibility of human infant development being shaped via lactocrine programming, and by breast milk cortisol levels specifically. Despite animal models indicating that glucocorticoid (GC) exposure via lactation might modify brain development and behavior, only one study has reported that milk cortisol levels were positively associated with infant negative affectivity, especially fearfulness and sadness-early emerging risk factors for internalizing difficulties such as anxiety. The aim of the current study was to investigate whether human milk cortisol is associated with mother-reported fearfulness and experimentally induced infant fear reactivity. Mother-infant dyads (n = 65) enrolled in the FinnBrain Cohort Study participated. Breast milk samples were obtained 2.5 months postpartum, and milk cortisol concentrations were ascertained using validated luminescence immunoassay methodology. Infant fear reactivity was assessed using maternal reports 6 months postpartum and in a laboratory 8 months postpartum. There was a significant interaction between infant sex and milk cortisol such that higher milk cortisol was related to higher infant fear reactivity in a laboratory setting in girls (ß = 0.36, p = .04) but not in boys (ß = -0.15, p = .40). Milk cortisol was not associated with mother-reported infant fearfulness. Results suggest that higher human milk cortisol concentrations are associated with elevated experimentally induced fear in infancy. Findings support lactocrine programming, and suggest that mothers may "communicate" vital information about stressful environments via cortisol contained in breast milk, shaping girls' early emotional reactivity.

Infant fecal microbiota composition and attention to emotional faces.

The gut microbiota has been suggested to influence neurodevelopment in rodents. Preliminary human studies have associated fecal microbiota composition with features of emotional and cognitive development as well as differences in thalamus-amygdala connectivity. Currently, microbiota-gut-brain axis studies cover heterogenous set of infant and child brain developmental phenotypes, while microbiota associations with more fine-grained aspects of brain development remain largely unknown. Here (N = 122, 53% boys), we investigated the associations between infant fecal microbiota composition and infant attention to emotional faces, as bias for faces is strong in infancy and deviations in early processing of emotional facial expressions may influence the trajectories of social-emotional development. The fecal microbiota composition was assessed at 2.5 months of age and analyzed with 16S rRNA gene sequencing. Attention to emotional faces was assessed with an age-appropriate face-distractor paradigm, using neutral, happy, fearful, and scrambled faces and salient distractors, at 8 months of age. We observed an association between a lower abundance of Bifidobacterium and a higher abundance of Clostridium with an increased "fear bias," that is, attention toward fearful versus happy/neutral faces. This data suggests an association between early microbiota and later fear bias, a well-established infant phenotype of emotionally directed attention. However, the clinical significance or causality of our findings remains to be assessed. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Human milk metabolome is associated with symptoms of maternal psychological distress and milk cortisol.

The composition of human milk is subject to considerable variation, but the effects of maternal stress are largely unknown. We studied differences in human milk metabolome between Finnish mothers (n = 120, secretors) with symptoms of prenatal symptoms of psychological distress and milk cortisol concentrations. Human milk samples acquired at 2.5 months postpartum were analyzed using targeted 1H NMR metabolomics. Self-reported scores for depression (EPDS), overall anxiety (SCL-90), and pregnancy-related anxiety (PRAQ) were used to evaluate psychological distress. Prenatal psychological distress was positively associated with concentrations of short-chain fatty acids, caprate, and hypoxanthine (q < 0.0012). Milk cortisol was positively associated with lactate concentration (q < 0.05). Changes in the human milk metabolome were shown to be associated with maternal psychological distress and concentration of milk cortisol in a dissimilarly, suggesting alterations in bacterial and energy metabolism of the mother, respectively.

Interactions between cortisol and lipids in human milk.

BACKGROUND: Human breast milk is one of the key early postnatal biological exposures for the developing child. It includes bioactive compounds, such as cortisol and fatty acids, which may be linked via the mother's lipid metabolism. METHODS: This study investigated the associations between cortisol and lipids in human milk at the infant age of 2.5 months. Human milk cortisol concentrations were measured using luminescence immunoassay, and two groups of milks (n = 50 each) were formed based on either high (> 10 nmol/L) or low (< 3 nmol/L) cortisol levels. Lipids, as fatty acid content and composition of neutral (triacylglycerol-rich) and polar (phospholipid-rich) lipids, were measured with gas chromatography. The samples originated from the FinnBrain Birth Cohort Study. RESULTS: The percentage of phospholipid-rich lipids of total lipids was 33.08% ± 1.33%. In triacylglycerol-rich lipids, high cortisol level in milk was associated with higher lauric (12:0, mass % and mg/mL), myristic (14:0, mass % and mg/mL), eicosenoic (20:1n - 9, mass %), docosenoic (22:1n - 9, mass %, and mg/mL) acids, and to lower palmitic acid (16:0, mass %) compared with low cortisol levels in milk. In phospholipid-rich lipids, high cortisol level was associated with higher myristic (14:0, mass %) and docosenoic (22:1n - 9, mass %) acids. After adjusting for pre-pregnancy BMI and sampling time by linear regression, the milk cortisol remained a significant predictor for lauric and myristic acids in triacylglycerol-rich lipids, and myristic and docosenoic acid in phospholipid-rich lipids (ß = 0.23 to 0.38 and p < 0.05 for each). CONCLUSIONS: This study revealed certain significant associations between milk cortisol and the fatty acid composition of human milk, indicating that cortisol might be one of the factors affecting the origin of the lipids in human milk.

Maternal prenatal psychological distress and hair cortisol levels associate with infant fecal microbiota composition at 2.5 months of age.

BACKGROUND: Maternal prenatal stress associates with infant developmental outcomes, but the mechanisms underlying this association are not fully understood. Alterations in the composition and function of infant intestinal microbiota may mediate some of the observed health effects, a viewpoint that is supported by animal studies along with a small human study showing that exposure to prenatal stress modifies the offspring's intestinal microbiota. In the current study, we aim to investigate the associations between maternal prenatal psychological distress (PPD) and hair cortisol concentration (HCC) with infant fecal microbiota composition in a large prospective human cohort. METHODS: The study population was drawn from FinnBrain Birth Cohort Study. Maternal PPD was measured with standardized questionnaires (EPDS, SCL, PRAQ-R2, Daily Hassles) three times during pregnancy (n = 398). A measure addressing the chronicity of PPD was composed separately for each questionnaire. HCC was measured from a five cm segment at gestational week 24 (n = 115), thus covering the early and mid-pregnancy. Infant fecal samples were collected at the age of 2.5 months and analyzed with 16S rRNA amplicon sequencing. RESULTS: Maternal chronic PPD (all symptom measures) showed positive associations (FDR < 0.01) with bacterial genera from phylum Proteobacteria, with potential pathogens, in infants. Further, chronic PPD (SCL, PRAQ-R2, and Daily Hassles negative scale) associated negatively with Akkermansia. HCC associated negatively with Lactobacillus. Neither maternal chronic PPD nor HCC associated with infant fecal microbiota diversity. CONCLUSION: Chronic maternal PPD symptoms and elevated HCC associate with alterations in infant intestinal microbiota composition. In keeping with the earlier literature, maternal PPD symptoms were associated with increases in genera fromProteobacteria phylum. Further research is needed to understand how these microbiota changes are linked with later child health outcomes.

Gut Microbiota Composition in Mid-Pregnancy Is Associated with Gestational Weight Gain but Not Prepregnancy Body Mass Index.

BACKGROUND: Pregnancy is a time of numerous hormonal, metabolic, and immunological changes for both the mother and the fetus. Furthermore, maternal gut microbiota composition (GMC) is altered during pregnancy. One major factor affecting GMC in pregnant and nonpregnant populations is obesity. The aim was to analyze associations between maternal overweight/obesity, as well as gestational weight gain (GWG) and GMC. Moreover, the modifying effect of depression and anxiety symptom scores on weight and GMC were investigated. METHODS: Study included 46 women from the FinnBrain Birth Cohort study, of which 36 were normal weight, and 11 overweight or obese according to their prepregnancy body mass index (BMI). Stool samples were collected in gestational week 24, and the GMC was sequenced with Illumina MiSeq approach. Hierarchical clustering was executed to illuminate group formation according to the GMC. The population was divided according to Firmicutes and Bacteroidetes dominance. Symptoms of depression, general anxiety, and pregnancy-related anxiety were measured by using standardized questionnaires. RESULTS: Excessive GWG was associated with distinct GMC in mid-pregnancy as measured by hierarchical clustering and grouping according to Firmicutes or Bacteroidetes dominance, with Bacteroidetes being prominent and Firmicutes being less prominent in the GMC among those with increased GWG. Reduced alpha diversity was observed among the Bacteroidetes-dominated subjects. There were no zero-order effects between the abundances of bacterial genera or phyla, alpha or beta diversity, and prepregnancy BMI or GWG. CONCLUSION: Bacteroidetes-dominated GMC in mid-pregnancy is associated with increased GWG and reduced alpha diversity.