Comparing Top-Down Proteoform Identification: Deconvolution, PrSM Overlap, and PTM Detection.

Generating top-down tandem mass spectra (MS/MS) from complex mixtures of proteoforms benefits from improvements in fractionation, separation, fragmentation, and mass analysis. The algorithms to match MS/MS to sequences have undergone a parallel evolution, with both spectral alignment and match-counting approaches producing high-quality proteoform-spectrum matches (PrSMs). This study assesses state-of-the-art algorithms for top-down identification (ProSight PD, TopPIC, MSPathFinderT, and pTop) in their yield of PrSMs while controlling false discovery rate. We evaluated deconvolution engines (ThermoFisher Xtract, Bruker AutoMSn, Matrix Science Mascot Distiller, TopFD, and FLASHDeconv) in both ThermoFisher Orbitrap-class and Bruker maXis Q-TOF data (PXD033208) to produce consistent precursor charges and mass determinations. Finally, we sought post-translational modifications (PTMs) in proteoforms from bovine milk (PXD031744) and human ovarian tissue. Contemporary identification workflows produce excellent PrSM yields, although approximately half of all identified proteoforms from these four pipelines were specific to only one workflow. Deconvolution algorithms disagree on precursor masses and charges, contributing to identification variability. Detection of PTMs is inconsistent among algorithms. In bovine milk, 18% of PrSMs produced by pTop and TopMG were singly phosphorylated, but this percentage fell to 1% for one algorithm. Applying multiple search engines produces more comprehensive assessments of experiments. Top-down algorithms would benefit from greater interoperability.

Human milk immune factors, maternal nutritional status, and infant sex: The INSPIRE study.

OBJECTIVES: Breastfeeding is an energetically costly and intense form of human parental investment, providing sole-source nutrition in early infancy and bioactive components, including immune factors. Given the energetic cost of lactation, milk factors may be subject to tradeoffs, and variation in concentrations have been explored utilizing the Trivers-Willard hypothesis. As human milk immune factors are critical to developing immune system and protect infants against pathogens, we tested whether concentrations of milk immune factors (IgA, IgM, IgG, EGF, TGFß2, and IL-10) vary in response to infant sex and maternal condition (proxied by maternal diet diversity [DD] and body mass index [BMI]) as posited in the Trivers-Willard hypothesis and consider the application of the hypothesis to milk composition. METHODS: We analyzed concentrations of immune factors in 358 milk samples collected from women residing in 10 international sites using linear mixed-effects models to test for an interaction between maternal condition, including population as a random effect and infant age and maternal age as fixed effects. RESULTS: IgG concentrations were significantly lower in milk produced by women consuming diets with low diversity with male infants than those with female infants. No other significant associations were identified. CONCLUSIONS: IgG concentrations were related to infant sex and maternal diet diversity, providing minimal support for the hypothesis. Given the lack of associations across other select immune factors, results suggest that the Trivers-Willard hypothesis may not be broadly applied to human milk immune factors as a measure of maternal investment, which are likely buffered against perturbations in maternal condition.

Breastfeeding Beyond 12 Months: Is There Evidence for Health Impacts?

Because breastfeeding provides optimal nutrition and other benefits for infants (e.g., lower risk of infectious disease) and benefits for mothers (e.g., less postpartum bleeding), many organizations recommend that healthy infants be exclusively breastfed for 4 to 6 months in the United States and 6 months internationally. Recommendations related to how long breastfeeding should continue, however, are inconsistent. The objective of this article is to review the literature related to evidence for benefits of breastfeeding beyond 1 year for mothers and infants. In summary, human milk represents a good source of nutrients and immune components beyond 1 year. Some studies point toward lower infant mortality in undernourished children breastfed for >1 year, and prolonged breastfeeding increases interbirth intervals. Data on other outcomes (e.g., growth, diarrhea, obesity, and maternal weight loss) are inconsistent, often lacking sufficient control for confounding variables. There is a substantial need for rigorous, prospective, mixed-methods, cross-cultural research on this topic.

Key genetic variants associated with variation of milk oligosaccharides from diverse human populations.

Human milk oligosaccharides (HMO), the third most abundant component of human milk, are thought to be important contributors to infant health. Studies have provided evidence that geography, stage of lactation, and Lewis and secretor blood groups are associated with HMO profile. However, little is known about how variation across the genome may influence HMO composition among women in various populations. In this study, we performed genome-wide association analyses of 395 women from 8 countries to identify genetic regions associated with 19 different HMO. Our data support FUT2 as the most significantly associated (P < 4.23-9 to P < 4.5-70) gene with seven HMO and provide evidence of balancing selection for FUT2. Although polymorphisms in FUT3 were also associated with variation in lacto-N-fucopentaose II and difucosyllacto-N-tetrose, we found little evidence of selection on FUT3. To our knowledge, this is the first report of the use of genome-wide association analyses on HMO.

Microbiomes and Childhood Malnutrition: What Is the Evidence?

Both undernutrition and overnutrition continue to represent enduring global health crises, and with the growing implications of both forms of malnutrition occurring simultaneously in individuals and populations (referred to as the double burden of malnutrition), understanding their biological and environmental causes is a primary research and humanitarian necessity. There is growing evidence of a bidirectional association between variation in the gastrointestinal (GI) microbiome and risk of/resilience to malnutrition during early life. For example, studies of siblings who discordantly do or do not develop severe malnutrition show clear differences in the diversity and composition of fecal microbiomes. These differences are transiently lessened during refeeding but re-emerge thereafter. These findings have been somewhat recapitulated using animal models, but small sample sizes and limited range complicate interpretation of results and applicability to humans. Mechanisms driving these differences are currently unknown but likely involve a combination of inflammatory pathways (and perhaps antioxidant status of the host) and effects on nutrient availability, requirements, and utilization by both host and microbe. A less robust literature also suggests that variation in GI microbiome is associated with risk for obesity during childhood. The putative impact of GI microbiomes on malnutrition is likely modified by a variety of important variables such as genetics (likely driven, in part, by evolution), environmental pathogen exposure and its timing, dietary factors, and cultural/societal pattern (e.g., use of antibiotics). Given the growing double burden of malnutrition, this topic demands a focused interdisciplinary approach that expands from merely characterizing differences and longitudinal changes in fecal microbes to examining their functionality during early life. Understanding the complex composition of human milk and how its components impact establishment and maintenance of the recipient infant's GI microbiome will also undoubtedly shed important light on this topic.

SARS-CoV-2 and human milk: What is the evidence?

The novel coronavirus SARS-CoV-2 has emerged as one of the most compelling and concerning public health challenges of our time. To address the myriad issues generated by this pandemic, an interdisciplinary breadth of research, clinical and public health communities has rapidly engaged to collectively find answers and solutions. One area of active inquiry is understanding the mode(s) of SARS-CoV-2 transmission. Although respiratory droplets are a known mechanism of transmission, other mechanisms are likely. Of particular importance to global health is the possibility of vertical transmission from infected mothers to infants through breastfeeding or consumption of human milk. However, there is limited published literature related to vertical transmission of any human coronaviruses (including SARS-CoV-2) via human milk and/or breastfeeding. Results of the literature search reported here (finalized on 17 April 2020) revealed a single study providing some evidence of vertical transmission of human coronavirus 229E; a single study evaluating presence of SARS-CoV in human milk (it was negative); and no published data on MERS-CoV and human milk. We identified 13 studies reporting human milk tested for SARS-CoV-2; one study (a non-peer-reviewed preprint) detected the virus in one milk sample, and another study detected SARS-CoV-2 specific IgG in milk. Importantly, none of the studies on coronaviruses and human milk report validation of their collection and analytical methods for use in human milk. These reports are evaluated here, and their implications related to the possibility of vertical transmission of coronaviruses (in particular, SARS-CoV-2) during breastfeeding are discussed.

Strong Multivariate Relations Exist Among Milk, Oral, and Fecal Microbiomes in Mother-Infant Dyads During the First Six Months Postpartum.

BACKGROUND: Neonatal gastrointestinal (GI) bacterial community structure may be related to bacterial communities of the mother, including those of her milk. However, very little is known about the diversity in and relationships among complex bacterial communities in mother-infant dyads. OBJECTIVE: Our primary objective was to assess whether microbiomes of milk are associated with those of oral and fecal samples of healthy lactating women and their infants. METHODS: Samples were collected 9 times from day 2 to 6 mo postpartum from 21 healthy lactating women and their infants. Milk was collected via complete breast expression, oral samples via swabs, and fecal samples from tissue (mothers) and diapers (infants). Microbiomes were characterized using high-throughput sequencing of the 16S ribosomal RNA (rRNA) gene. Alpha and beta diversity indices were used to compare microbiomes across time and sample types. Membership and composition of microbiomes were analyzed using nonmetric multidimensional scaling and canonical correlation analysis (CCA). The contribution of various bacterial communities of the mother-infant dyad to both milk and infant fecal bacterial communities were estimated using SourceTracker2. RESULTS: Bacterial community structures were relatively unique to each sample type. The most abundant genus in milk and maternal and infant oral samples was Streptococcus (47.1% ± 2.3%, 53.9% ± 1.3%, and 69.1% ± 1.8%, respectively), whereas Bacteroides were predominant in maternal and infant fecal microbiomes (22.9% ± 1.3% and 21.4% ± 2.4%, respectively). The milk microbiome was more similar to the infant oral microbiome than the infant fecal microbiome. However, CCA suggested strong associations between the complex microbial communities of milk and those of all other sample types collected. CONCLUSIONS: These findings suggest complex microbial interactions between breastfeeding mothers and their infants and support the hypothesis that variation in the milk microbiome may influence the infant GI microbiome.

Household composition and the infant fecal microbiome: The INSPIRE study.

OBJECTIVES: Establishment and development of the infant gastrointestinal microbiome (GIM) varies cross-culturally and is thought to be influenced by factors such as gestational age, birth mode, diet, and antibiotic exposure. However, there is little data as to how the composition of infants' households may play a role, particularly from a cross-cultural perspective. Here, we examined relationships between infant fecal microbiome (IFM) diversity/composition and infants' household size, number of siblings, and number of other household members. MATERIALS AND METHODS: We analyzed 377 fecal samples from healthy, breastfeeding infants across 11 sites in eight different countries (Ethiopia, The Gambia, Ghana, Kenya, Peru, Spain, Sweden, and the United States). Fecal microbial community structure was determined by amplifying, sequencing, and classifying (to the genus level) the V1-V3 region of the bacterial 16S rRNA gene. Surveys administered to infants' mothers identified household members and composition. RESULTS: Our results indicated that household composition (represented by the number of cohabitating siblings and other household members) did not have a measurable impact on the bacterial diversity, evenness, or richness of the IFM. However, we observed that variation in household composition categories did correspond to differential relative abundances of specific taxa, namely: Lactobacillus, Clostridium, Enterobacter, and Klebsiella. DISCUSSION: This study, to our knowledge, is the largest cross-cultural study to date examining the association between household composition and the IFM. Our results indicate that the social environment of infants (represented here by the proxy of household composition) may influence the bacterial composition of the infant GIM, although the mechanism is unknown. A higher number and diversity of cohabitants and potential caregivers may facilitate social transmission of beneficial bacteria to the infant gastrointestinal tract, by way of shared environment or through direct physical and social contact between the maternal-infant dyad and other household members. These findings contribute to the discussion concerning ways by which infants are influenced by their social environments and add further dimensionality to the ongoing exploration of social transmission of gut microbiota and the "old friends" hypothesis.

Social networks, cooperative breeding, and the human milk microbiome.

OBJECTIVES: We present the first available data on the human milk microbiome (HMM) from small-scale societies (hunter-gatherers and horticulturalists in the Central African Republic [CAR]) and explore relationships among subsistence type and seasonality on HMM diversity and composition. Additionally, as humans are cooperative breeders and, throughout our evolutionary history and today, we rear offspring within social networks, we examine associations between the social environment and the HMM. Childrearing and breastfeeding exist in a biosocial nexus, which we hypothesize influences the HMM. METHODS: Milk samples from hunter-gatherer and horticultural mothers (n = 41) collected over two seasons, were analyzed for their microbial composition. A subsample of these women's infants (n = 33) also participated in detailed naturalistic behavioral observations which identified the breadth of infants' social and caregiving networks and the frequency of contact they had with caregivers. RESULTS: Analyses of milk produced by CAR women indicated that HMM diversity and community composition were related to the size of the mother-infant dyad's social network and frequency of care that infants receive. The abundance of some microbial taxa also varied significantly across populations and seasons. Alpha diversity, however, was not related to subsistence type or seasonality. CONCLUSION: While the origins of the HMM are not fully understood, our results provide evidence regarding possible feedback loops among the infant, the mother, and the mother's social network that might influence HMM composition.

Human Milk Microbial Community Structure Is Relatively Stable and Related to Variations in Macronutrient and Micronutrient Intakes in Healthy Lactating Women.

Background: The human milk microbiome has been somewhat characterized, but little is known about changes over time and relations with maternal factors such as nutrient intake.Objective: We sought to characterize the human milk microbiome and described associations with maternal nutrient intake, time postpartum, delivery mode, and body mass index (BMI; in kg/m2).Methods: Milk samples (n = 104) and 24-h diet recalls were collected 9 times from 21 healthy lactating women from day 2 to 6 mo postpartum. Women were classified by BMI as healthy weight (<25) or overweight or obese (≥25). Bacterial taxa were characterized with the use of the high-throughput sequencing of the 16S ribosomal RNA gene.Results: The milk microbiome was relatively constant over time, although there were small changes in some of the lesser-abundant genera. Relative abundances of several taxa were associated with BMI, delivery mode, and infant sex. For instance, overweight and obese mothers produced milk with a higher relative abundance of Granulicatella than did healthy-weight women (1.8% ± 0.6% compared with 0.4% ± 0.2%, respectively; P < 0.05). Relative abundances of several bacterial taxa were also associated with variations in maternal dietary intake. For example, intakes of saturated fatty acids (rs = -0.59; P = 0.005) and monounsaturated fatty acids (rs = -0.46; P = 0.036) were inversely associated with the relative abundance of Corynebacterium; total carbohydrates (rs = -0.54; P = 0.011), disaccharides (rs = -0.47; P = 0.031), and lactose (rs = -0.51; P = 0.018) were negatively associated with Firmicutes; and protein consumption was positively correlated with the relative abundance of Gemella (rs = 0.46; P = 0.037).Conclusions: Factors associated with variations in the human milk microbiome are complex and may include maternal nutrient intake, maternal BMI, delivery mode, and infant sex. Future studies designed to investigate the relation between maternal nutrient intake and the milk microbiome should strive to also evaluate dietary supplement usage and analyze the collected milk for its nutrient content.

Validation of reliable and selective methods for direct determination of glyphosate and aminomethylphosphonic acid in milk and urine using LC-MS/MS.

Simple high-throughput procedures were developed for the direct analysis of glyphosate [N-(phosphonomethyl)glycine] and aminomethylphosphonic acid (AMPA) in human and bovine milk and human urine matrices. Samples were extracted with an acidified aqueous solution on a high-speed shaker. Stable isotope labeled internal standards were added with the extraction solvent to ensure accurate tracking and quantitation. An additional cleanup procedure using partitioning with methylene chloride was required for milk matrices to minimize the presence of matrix components that can impact the longevity of the analytical column. Both analytes were analyzed directly, without derivatization, by liquid chromatography tandem mass spectrometry using two separate precursor-to-product transitions that ensure and confirm the accuracy of the measured results. Method performance was evaluated during validation through a series of assessments that included linearity, accuracy, precision, selectivity, ionization effects and carryover. Limits of quantitation (LOQ) were determined to be 0.1 and 10 µg/L (ppb) for urine and milk, respectively, for both glyphosate and AMPA. Mean recoveries for all matrices were within 89-107% at three separate fortification levels including the LOQ. Precision for replicates was ≤ 7.4% relative standard deviation (RSD) for milk and ≤ 11.4% RSD for urine across all fortification levels. All human and bovine milk samples used for selectivity and ionization effects assessments were free of any detectable levels of glyphosate and AMPA. Some of the human urine samples contained trace levels of glyphosate and AMPA, which were background subtracted for accuracy assessments. Ionization effects testing showed no significant biases from the matrix. A successful independent external validation was conducted using the more complicated milk matrices to demonstrate method transferability.

Fecal Microbial Community Structure Is Stable over Time and Related to Variation in Macronutrient and Micronutrient Intakes in Lactating Women.

BACKGROUND: The fecal microbiota has been characterized in some adult populations, but little is known about its community structure during lactation. OBJECTIVES: We characterized the maternal fecal microbiome during lactation and explored possible mediating factors such as nutrition. METHODS: Fecal samples were collected from 20 lactating women from 2 d to 6 mo postpartum, and bacterial taxa were characterized with the use of high-throughput sequencing. Bacterial community structure (at each taxonomic level) and relations between bacterial taxa and environmental and dietary variables were visualized and analyzed with the use of stacked bar charts, principal component analysis, and multivariate analyses such as nonmetric multidimensional scaling and canonical correlation analysis. RESULTS: Complex bacterial community structure was somewhat similar to those previously published for other adult populations (although there were some notable differences), and there were no clear associations with time postpartum or anthropometric or environmental variables. However, Spearman rank correlations suggested that increased intake of pantothenic acid, riboflavin, vitamin B-6, and vitamin B-12 were related to increased relative abundance of Prevotella (r = 0.45, 0.39, 0.34, and 0.24, respectively; P ≤ 0.01) and decreased relative abundance of Bacteroides (r = -0.55, -0.46, -0.32, and -0.35, respectively; P ≤ 0.01). Intakes of copper, magnesium, manganese, and molybdenum were positively associated with Firmicutes (r = 0.33, 0.38, 0.44, and 0.51, respectively; P ≤ 0.01) and negatively associated with Bacteroidetes (r = -0.38, -0.44, -0.48, and -0.53, respectively; P ≤ 0.01). Overall, data consistently suggest that increased consumption of a more nutrient- and calorie-rich diet was positively associated with relative abundance of Firmicutes. CONCLUSIONS: The fecal microbiome of lactating women is relatively stable in the postpartum period and somewhat similar to that of other adult populations. Variation in dietary constituents may be related to that of relative abundance of individual bacterial taxa. Controlled dietary intervention studies will be required to determine whether these associations are causal in nature.

Total Nitrite and Nitrate Concentration in Human Milk and Saliva during the First 60 Days Postpartum-A Pilot Study.

Nitric oxide (NO) in human milk may have important functions in lactation and infant health. This longitudinal pilot cohort study investigated the total nitrite and nitrate (NOx) concentration in human milk and maternal saliva during the first 60 days postpartum. Additionally, we explored the association between selected breastfeeding variables and milk and saliva NOx concentration. Human milk and maternal saliva samples were collected on days 2, 5, 14, 30, and 60 postpartum and analyzed for NOx concentration. Breastfeeding data were collected through self-assessed questions. Data analyses were performed using mixed models. The concentration of NOx in milk was significantly higher during the first 30 days compared to day 60, and there was a positive association between milk and saliva NOx concentrations throughout the entire study period. In absolute numbers, partially breastfeeding mothers had a lower concentration of NOx in milk on day 2 compared to exclusively breastfeeding mothers (8 vs. 15.1 µM, respectively). Partially breastfeeding mothers reported a later start of secretory activation and fewer mothers in this group started breastfeeding within the first hour after birth. Due to the small numbers, these differences could not be statistically evaluated. Further research is warranted to elucidate the role of NO in lactation success and breastfeeding outcomes.

Maternal Cannabis Use during Lactation and Potential Effects on Human Milk Composition and Production: A Narrative Review.

Cannabis use has increased sharply in the last 20 y among adults, including reproductive-aged women. Its recent widespread legalization is associated with a decrease in risk perception of cannabis use during breastfeeding. However, the effect of cannabis use (if any) on milk production and milk composition is not known. This narrative review summarizes current knowledge related to maternal cannabis use during breastfeeding and provides an overview of possible pathways whereby cannabis might affect milk composition and production. Several studies have demonstrated that cannabinoids and their metabolites are detectable in human milk produced by mothers who use cannabis. Due to their physicochemical properties, cannabinoids are stored in adipose tissue, can easily reach the mammary gland, and can be secreted in milk. Moreover, cannabinoid receptors are present in adipocytes and mammary epithelial cells. The activation of these receptors directly modulates fatty acid metabolism, potentially causing changes in milk fatty acid profiles. Additionally, the endocannabinoid system is intimately connected to the endocrine system. As such, it is probable that interactions of exogenous cannabinoids with the endocannabinoid system might modify release of critical hormones (e.g., prolactin and dopamine) that regulate milk production and secretion. Nonetheless, few studies have investigated effects of cannabis use (including on milk production and composition) in lactating women. Additional research utilizing robust methodologies are needed to elucidate whether and how cannabis use affects human milk production and composition.

Cannabis use, decision making, and perceptions of risk among breastfeeding individuals: the Lactation and Cannabis (LAC) Study.

OBJECTIVE: Our primary objective was to understand breastfeeding individuals' decisions to use cannabis. Specifically, we investigated reasons for cannabis use, experiences with healthcare providers regarding use, and potential concerns about cannabis use. METHODS: We collected survey data from twenty breastfeeding participants from Washington and Oregon who used cannabis at least once weekly. We documented individuals' cannabis use and analyzed factors associated with their decisions to use cannabis during lactation. Qualitative description was used to assess responses to an open-ended question about potential concerns. RESULTS: Fifty-five percent of participants (n = 11) reported using cannabis to treat or manage health conditions, mostly related to mental health. Eighty percent of participants (n = 16) reported very few or no concerns about using cannabis while breastfeeding, although participants who used cannabis for medical purposes had significantly more concerns. Most participants (n = 18, 90%) reported receiving either no or unhelpful advice from healthcare providers. Four themes arose through qualitative analysis, indicating that breastfeeding individuals are: 1) identifying research gaps and collecting evidence; 2) monitoring their child's health and development; 3) monitoring and titrating their cannabis use; and 4) comparing risks between cannabis and other controlled substances. CONCLUSIONS: Breastfeeding individuals reported cannabis for medical and non-medical reasons and few had concerns about cannabis use during breastfeeding. Breastfeeding individuals reported using a variety of strategies and resources in their assessment of risk or lack thereof when deciding to use cannabis. Most participants reported receiving no helpful guidance from healthcare providers.

Human Milk Cannabinoid Concentrations and Associations with Maternal Factors: The Lactation and Cannabis (LAC) Study.

Background and Objectives: As cannabis use increases among reproductive-aged women, there is a growing need to better understand the presence of cannabinoids in milk produced by women using cannabis. It is unclear how concentrations of cannabinoids such as delta-9-tetrahydrocannabinol (Δ9-THC) persist in milk after cannabis use and what factors contribute to variation in milk Δ9-THC concentrations. Our objectives were to measure cannabinoids in human milk following cannabis abstention, after single and repeated instances of cannabis use, and identify factors contributing to concentration variation. Methods: The Lactation and Cannabis (LAC) Study prospectively observed 20 breastfeeding participants who frequently used cannabis (≥1/week), had enrolled <6 months postpartum, were feeding their infant their milk ≥5 times/day, and were not using any illicit drugs. Participants collected a baseline milk sample after ≥12 hours of abstaining from cannabis and five milk samples at set intervals over 8-12 hours after initial cannabis use. Participants completed surveys and recorded self-directed cannabis use during the study period. Results: Δ9-THC peaked 120 minutes after a single instance of cannabis use (median, n = 9). More instances of cannabis use during the study period were associated with greater Δ9-THC area-under-the-curve concentrations (ρ = 0.65, p = 0.002), indicating Δ9-THC bioaccumulation in most participants. Baseline Δ9-THC logged concentration was positively associated with self-reported frequency of cannabis use (b = 0.57, p = 0.01). Conclusions: Cannabinoids are measurable in human milk following cannabis use, and concentrations remain elevated with repeated cannabis use over a day. Substantial variation in Δ9-THC milk concentrations reflects individual differences in characteristics and behavior, including average postpartum frequency of cannabis use.

Prevalence and duration of SARS-CoV-2 fecal shedding in breastfeeding dyads following maternal COVID-19 diagnosis.

Background: There is a paucity of data on the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in feces of lactating women with coronavirus disease 2019 (COVID-19) and their breastfed infants as well as associations between fecal shedding and symptomatology. Objective: We examined whether and to what extent SARS-CoV-2 is detectable in the feces of lactating women and their breastfed infants following maternal COVID-19 diagnosis. Methods: This was a longitudinal study carried out from April 2020 to December 2021 involving 57 breastfeeding maternal-infant dyads: 33 dyads were enrolled within 7 d of maternal COVID-19 diagnosis, and 24 healthy dyads served as controls. Maternal/infant fecal samples were collected by participants, and surveys were administered via telephone over an 8-wk period. Feces were analyzed for SARS-CoV-2 RNA. Results: Signs/symptoms related to ears, eyes, nose, and throat (EENT); general fatigue/malaise; and cardiopulmonary signs/symptoms were commonly reported among mothers with COVID-19. In infants of mothers with COVID-19, EENT, immunologic, and cardiopulmonary signs/symptoms were most common, but prevalence did not differ from that of infants of control mothers. SARS-CoV-2 RNA was detected in feces of 7 (25%) women with COVID-19 and 10 (30%) of their infants. Duration of fecal shedding ranged from 1-4 wk for both mothers and infants. SARS-CoV-2 RNA was sparsely detected in feces of healthy dyads, with only one mother's and two infants' fecal samples testing positive. There was no relationship between frequencies of maternal and infant SARS-CoV-2 fecal shedding (P=0.36), although presence of maternal or infant fever was related to increased likelihood (7-9 times greater, P≤0.04) of fecal shedding in infants of mothers with COVID-19.

Breastfeeding patterns are associated with human milk microbiome composition: The Mother-Infant Microbiomes, Behavior, and Ecology Study (MIMBES).

The human milk microbiome (HMM) is hypothesized to be seeded by multiple factors, including the infant oral microbiome during breastfeeding. However, it is not known whether breastfeeding patterns (e.g., frequency or total time) impact the composition of the HMM. As part of the Mother-Infant Microbiomes, Behavior, and Ecology Study (MIMBES), we analyzed data from naturalistic observations of 46 mother-infant dyads living in the US Pacific Northwest and analyzed milk produced by the mothers for its bacterial diversity and composition. DNA was extracted from milk and the V1-V3 region of the 16S rRNA gene was amplified and sequenced. We hypothesized that number of breastfeeding bouts (breastfeeding sessions separated by >30 seconds) and total time breastfeeding would be associated with HMM α-diversity (richness, diversity, or evenness) and differential abundance of HMM bacterial genera. Multiple linear regression was used to examine associations between HMM α-diversity and the number of breastfeeding bouts or total time breastfeeding and selected covariates (infant age, maternal work outside the home, frequency of allomother physical contact with the infant, non-household caregiving network). HMM richness was inversely associated with number of breastfeeding bouts and frequency of allomother physical contact, but not total time breastfeeding. Infants' non-household caregiving network was positively associated with HMM evenness. In two ANCOM-BC analyses, abundances of 5 of the 35 most abundant genera were differentially associated with frequency of breastfeeding bouts (Bifidobacterium, Micrococcus, Pedobacter, Acidocella, Achromobacter); 5 genera (Bifidobacterium, Agreia, Pedobacter, Rugamonas, Stenotrophomonas) were associated with total time breastfeeding. These results indicate that breastfeeding patterns and infant caregiving ecology may play a role in influencing HMM composition. Future research is needed to identify whether these relationships are consistent in other populations and if they are associated with variation in the infant's gastrointestinal (including oral) microbiome.