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.

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.

SARS-CoV-2 specific antibody trajectories in mothers and infants over two months following maternal infection.

Infants exposed to caregivers infected with SARS-CoV-2 may have heightened infection risks relative to older children due to their more intensive care and feeding needs. However, there has been limited research on COVID-19 outcomes in exposed infants beyond the neonatal period. Between June 2020 - March 2021, we conducted interviews and collected capillary dried blood spots from 46 SARS-CoV-2 infected mothers and their infants (aged 1-36 months) for up to two months following maternal infection onset (COVID+ group, 87% breastfeeding). Comparative data were also collected from 26 breastfeeding mothers with no known SARS-CoV-2 infection or exposures (breastfeeding control group), and 11 mothers who tested SARS-CoV-2 negative after experiencing symptoms or close contact exposure (COVID- group, 73% breastfeeding). Dried blood spots were assayed for anti-SARS-CoV-2 S-RBD IgG and IgA positivity and anti-SARS-CoV-2 S1 + S2 IgG concentrations. Within the COVID+ group, the mean probability of seropositivity among infant samples was lower than that of corresponding maternal samples (0.54 and 0.87, respectively, for IgG; 0.33 and 0.85, respectively, for IgA), with likelihood of infant infection positively associated with the number of maternal symptoms and other household infections reported. COVID+ mothers reported a lower incidence of COVID-19 symptoms among their infants as compared to themselves and other household adults, and infants had similar PCR positivity rates as other household children. No samples returned by COVID- mothers or their infants tested antibody positive. Among the breastfeeding control group, 44% of mothers but none of their infants tested antibody positive in at least one sample. Results support previous research demonstrating minimal risks to infants following maternal COVID-19 infection, including for breastfeeding infants.

Brief Research Report: Estimation of the Protein Digestibility-Corrected Amino Acid Score of Defatted Walnuts.

Introduction: Walnuts are considered a good source of essential fatty acids, which is unique among tree nuts. Walnuts are also composed of about 10-15% protein, but the quality of this protein has not been evaluated. Pistachios and almonds have been evaluated for their protein content using a protein digestibility-corrected amino acid score (PDCAAS), but it is unclear how the quality of protein in walnuts relates to that in other commonly consumed tree nuts. The objective of this study was to substantiate the protein quality of walnuts by determining their PDCAAS. Methods: A small, 10-day dietary intervention trial was conducted using male Sprague-Dawley rats (n = 8, 4 per group) with two diets: a nitrogen-free diet and a diet containing protein exclusively from defatted walnuts. Feed intake and fecal output of nitrogen were measured to estimate the true protein digestibility, and the amino acid compositions of walnuts compared to child and adult populations were used to calculate amino acid scores (AAS) and PDCAAS. Results: The true protein digestibility score of raw walnuts was calculated to be 86.22%. Raw walnuts contained 15.6 g protein/g walnut with AAS of 0.45 and 0.63 for children aged 6 months to 3 years and 3-10 years, respectively. For each population, a PDCAAS of 39 and 46% was calculated, respectively, using a protein conversion constant of 5.30. Using a protein constant of 6.25, a PDCAAS of 39% (6 months - 3 years) or 46% (3-10 years) was calculated. Conclusions: This is the first known assessment of the PDCAAS of walnuts. Like almonds, they appear to have a low-to-moderate score, indicating they are not a quality source of protein.

Variation in Human Milk Composition Is Related to Differences in Milk and Infant Fecal Microbial Communities.

Previously published data from our group and others demonstrate that human milk oligosaccharide (HMOs), as well as milk and infant fecal microbial profiles, vary by geography. However, little is known about the geographical variation of other milk-borne factors, such as lactose and protein, as well as the associations among these factors and microbial community structures in milk and infant feces. Here, we characterized and contrasted concentrations of milk-borne lactose, protein, and HMOs, and examined their associations with milk and infant fecal microbiomes in samples collected in 11 geographically diverse sites. Although geographical site was strongly associated with milk and infant fecal microbiomes, both sample types assorted into a smaller number of community state types based on shared microbial profiles. Similar to HMOs, concentrations of lactose and protein also varied by geography. Concentrations of HMOs, lactose, and protein were associated with differences in the microbial community structures of milk and infant feces and in the abundance of specific taxa. Taken together, these data suggest that the composition of human milk, even when produced by relatively healthy women, differs based on geographical boundaries and that concentrations of HMOs, lactose, and protein in milk are related to variation in milk and infant fecal microbial communities.

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.

Comparison of Two Approaches for the Metataxonomic Analysis of the Human Milk Microbiome.

Recent work has demonstrated the existence of large inter-individual and inter-population variability in the microbiota of human milk from healthy women living across variable geographical and socio-cultural settings. However, no studies have evaluated the impact that variable sequencing approaches targeting different 16S rRNA variable regions may have on the human milk microbiota profiling results. This hampers our ability to make meaningful comparisons across studies. In this context, the main purpose of the present study was to re-process and re-sequence the microbiome in a large set of human milk samples (n = 412) collected from healthy women living at diverse international sites (Spain, Sweden, Peru, United States, Ethiopia, Gambia, Ghana and Kenya), by targeting a different 16S rRNA variable region and reaching a larger sequencing depth. Despite some differences between the results obtained from both sequencing approaches were notable (especially regarding alpha and beta diversities and Proteobacteria representation), results indicate that both sequencing approaches revealed a relatively consistent microbiota configurations in the studied cohorts. Our data expand upon the milk microbiota results we previously reported from the INSPIRE cohort and provide, for the first time across globally diverse populations, evidence of the impact that different DNA processing and sequencing approaches have on the microbiota profiles obtained for human milk samples. Overall, our results corroborate some similarities regarding the microbial communities previously reported for the INSPIRE cohort, but some differences were also detected. Understanding the impact of different sequencing approaches on human milk microbiota profiles is essential to enable meaningful comparisons across studies. Clinical Trial Registration: www.clinicaltrials.gov, identifier NCT02670278.

Characterization of SARS-CoV-2 RNA, Antibodies, and Neutralizing Capacity in Milk Produced by Women with COVID-19.

Whether mother-to-infant SARS-CoV-2 transmission can occur during breastfeeding and, if so, whether the benefits of breastfeeding outweigh this risk during maternal COVID-19 illness remain important questions. Using RT-qPCR, we did not detect SARS-CoV-2 RNA in any milk sample (n = 37) collected from 18 women following COVID-19 diagnosis. Although we detected evidence of viral RNA on 8 out of 70 breast skin swabs, only one was considered a conclusive positive result. In contrast, 76% of the milk samples collected from women with COVID-19 contained SARS-CoV-2-specific IgA, and 80% had SARS-CoV-2-specific IgG. In addition, 62% of the milk samples were able to neutralize SARS-CoV-2 infectivity in vitro, whereas milk samples collected prior to the COVID-19 pandemic were unable to do so. Taken together, our data do not support mother-to-infant transmission of SARS-CoV-2 via milk. Importantly, milk produced by infected mothers is a beneficial source of anti-SARS-CoV-2 IgA and IgG and neutralizes SARS-CoV-2 activity. These results support recommendations to continue breastfeeding during mild-to-moderate maternal COVID-19 illness.IMPORTANCE Results from prior studies assaying human milk for the presence of SARS-CoV-2, the causative virus of COVID-19, have suggested milk may act as a potential vehicle for mother-to-child transmission. Most previous studies are limited because they followed only a few participants, were cross-sectional, and/or failed to report how milk was collected and/or analyzed. As such, considerable uncertainty remains regarding whether human milk is capable of transmitting SARS-CoV-2 from mother to child. Here, we report that repeated milk samples collected from 18 women following COVID-19 diagnosis did not contain SARS-CoV-2 RNA; however, risk of transmission via breast skin should be further evaluated. Importantly, we found that milk produced by infected mothers is a source of anti-SARS-CoV-2 IgA and IgG and neutralizes SARS-CoV-2 activity. These results support recommendations to continue breastfeeding during mild-to-moderate maternal COVID-19 illness as milk likely provides specific immunologic benefits to infants.

Milk From Women Diagnosed With COVID-19 Does Not Contain SARS-CoV-2 RNA but Has Persistent Levels of SARS-CoV-2-Specific IgA Antibodies.

Background: Limited data are available regarding the balance of risks and benefits from human milk and/or breastfeeding during and following maternal infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Objective: To investigate whether SARS-CoV-2 can be detected in milk and on the breast after maternal coronavirus disease 2019 (COVID-19) diagnosis; and characterize concentrations of milk immunoglobulin (Ig) A specific to the SARS-CoV-2 spike glycoprotein receptor binding domain (RBD) during the 2 months after onset of symptoms or positive diagnostic test. Methods: Using a longitudinal study design, we collected milk and breast skin swabs one to seven times from 64 lactating women with COVID-19 over a 2-month period, beginning as early as the week of diagnosis. Milk and breast swabs were analyzed for SARS-CoV-2 RNA, and milk was tested for anti-RBD IgA. Results: SARS-CoV-2 was not detected in any milk sample or on 71% of breast swabs. Twenty-seven out of 29 (93%) breast swabs collected after breast washing tested negative for SARS-CoV-2. Detection of SARS-CoV-2 on the breast was associated with maternal coughing and other household COVID-19. Most (75%; 95% CI, 70-79%; n=316) milk samples contained anti-RBD IgA, and concentrations increased (P=.02) during the first two weeks following onset of COVID-19 symptoms or positive test. Milk-borne anti-RBD IgA persisted for at least two months in 77% of women. Conclusion: Milk produced by women with COVID-19 does not contain SARS-CoV-2 and is likely a lasting source of passive immunity via anti-RBD IgA. These results support recommendations encouraging lactating women to continue breastfeeding during and after COVID-19 illness.

COVID-19 and human milk: SARS-CoV-2, antibodies, and neutralizing capacity.

Background: It is not known whether SARS-CoV-2 can be transmitted from mother to infant during breastfeeding, and if so whether the benefits of breastfeeding outweigh this risk. This study was designed to evaluate 1) if SARS-CoV-2 RNA can be detected in milk and on the breast of infected women, 2) concentrations of milk-borne anti-SARS-CoV-2 antibodies, and 3) the capacity of milk to neutralize SARS-CoV-2 infectivity. Methods: We collected 37 milk samples and 70 breast swabs (before and after breast washing) from 18 women recently diagnosed with COVID-19. Samples were analyzed for SARS-CoV-2 RNA using RT-qPCR. Milk was also analyzed for IgA and IgG specific for the nucleocapsid protein, receptor binding domain (RBD), S2 subunit of the spike protein of SARS-CoV-2, as well as 2 seasonal coronaviruses using ELISA; and for its ability to neutralize SARS-CoV-2. Results: We did not detect SARS-CoV-2 RNA in any milk sample. In contrast, SARS-CoV-2 RNA was detected on several breast swabs, although only one was considered conclusive. All milk contained SARS-CoV-2-specific IgA and IgG, and levels of anti-RBD IgA correlated with SARS-CoV-2 neutralization. Strong correlations between levels of IgA and IgG to SARS-CoV-2 and seasonal coronaviruses were noted. Conclusions: Our data do not support maternal-to-child transmission of SARS-CoV-2 via milk; however, risk of transmission via breast skin should be further evaluated. Importantly, milk produced by infected mothers is a source of anti-SARS-CoV-2 IgA and IgG and neutralizes SARS-CoV-2 activity. These results support recommendations to continue breastfeeding during mild-to-moderate maternal COVID-19 illness.

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

The novel coronavirus SARS-CoV-2 has emerged as one of the most compelling 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 have rapidly engaged to find answers and solutions. One area of active inquiry is understanding the mode(s) of SARS-CoV-2 transmission. While respiratory droplets are a known mechanism of transmission, other mechanisms are possible. 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 coronavirus (including SARS-CoV-2) via human milk and/or breastfeeding. There is 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. There are 9 case studies of human milk tested for SARS-CoV-2; none detected the virus. Importantly, none of the published studies on coronaviruses and human milk report validation of their 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.

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.

Corrigendum: What's Normal? Microbiomes in Human Milk and Infant Feces Are Related to Each Other but Vary Geographically: The INSPIRE Study.

[This corrects the article DOI: 10.3389/fnut.2019.00045.].

What's Normal? Microbiomes in Human Milk and Infant Feces Are Related to Each Other but Vary Geographically: The INSPIRE Study.

Background: Microbial communities in human milk and those in feces from breastfed infants vary within and across populations. However, few researchers have conducted cross-cultural comparisons between populations, and little is known about whether certain "core" taxa occur normally within or between populations and whether variation in milk microbiome is related to variation in infant fecal microbiome. The purpose of this study was to describe microbiomes of milk produced by relatively healthy women living at diverse international sites and compare these to the fecal microbiomes of their relatively healthy infants. Methods: We analyzed milk (n = 394) and infant feces (n = 377) collected from mother/infant dyads living in 11 international sites (2 each in Ethiopia, The Gambia, and the US; 1 each in Ghana, Kenya, Peru, Spain, and Sweden). The V1-V3 region of the bacterial 16S rRNA gene was sequenced to characterize and compare microbial communities within and among cohorts. Results: Core genera in feces were Streptococcus, Escherichia/Shigella, and Veillonella, and in milk were Streptococcus and Staphylococcus, although substantial variability existed within and across cohorts. For instance, relative abundance of Lactobacillus was highest in feces from rural Ethiopia and The Gambia, and lowest in feces from Peru, Spain, Sweden, and the US; Rhizobium was relatively more abundant in milk produced by women in rural Ethiopia than all other cohorts. Bacterial diversity also varied among cohorts. For example, Shannon diversity was higher in feces from Kenya than Ghana and US-California, and higher in rural Ethiopian than Ghana, Peru, Spain, Sweden, and US-California. There were limited associations between individual genera in milk and feces, but community-level analyses suggest strong, positive associations between the complex communities in these sample types. Conclusions: Our data provide additional evidence of within- and among-population differences in milk and infant fecal bacterial community membership and diversity and support for a relationship between the bacterial communities in milk and those of the recipient infant's feces. Additional research is needed to understand environmental, behavioral, and genetic factors driving this variation and association, as well as its significance for acute and chronic maternal and infant health.

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.

Comparison of commercially-available preservatives for maintaining the integrity of bacterial DNA in human milk.

BACKGROUND: Inhibiting changes to bacteria in human milk between sample collection and analysis is necessary for unbiased characterization of the milk microbiome. Although cold storage is considered optimal, alternative preservation is sometimes necessary. RESEARCH AIM/QUESTION: The objective of this study was to compare the effectiveness of several commercially-available preservatives with regard to maintaining bacterial DNA in human milk for delayed microbiome analysis. Specifically, we compared Life Technologies' RNAlater® stabilization solution, Biomatrica's DNAgard® Saliva, Advanced Instruments' Broad Spectrum Microtabs II™, and Norgen Biotek Corporation's Milk DNA Preservation and Isolation Kit. METHODS: Aliquots of 8 pools of human milk were treated with each preservative. DNA was extracted immediately and at 1, 2, 4, and 6wk, during which time milk was held at 37°C. The V1-V3 region of the bacterial 16S rRNA gene was amplified and sequenced. Changes in bacterial community structure and diversity over time were evaluated. RESULTS: Comparable to other studies, the most abundant genera were Streptococcus (33.3%), Staphylococcus (14.0%), Dyella (6.3%), Pseudomonas (3.0%), Veillonella (2.5%), Hafnia (2.0%), Prevotella (1.7%), Rhodococcus (1.6%), and Granulicatella (1.4%). Overall, use of Norgen's Milk DNA Preservation and Isolation Kit best maintained the consistency of the bacterial community structure. Total DNA, diversity, and evenness metrics were also highest in samples preserved with this method. CONCLUSIONS: When collecting human milk for bacterial community analysis in field conditions where cold storage is not available, our results suggest that Norgen's Milk DNA Preservation and Isolation Kit may be a useful method, at least for a period of 2weeks.

What's Normal? Immune Profiling of Human Milk from Healthy Women Living in Different Geographical and Socioeconomic Settings.

Human milk provides a very wide range of nutrients and bioactive components, including immune factors, human milk oligosaccharides, and a commensal microbiota. These factors are essential for interconnected processes including immunity programming and the development of a normal infant gastrointestinal microbiome. Newborn immune protection mostly relies on maternal immune factors provided through milk. However, studies dealing with an in-depth profiling of the different immune compounds present in human milk and with the assessment of their natural variation in healthy women from different populations are scarce. In this context, the objective of this work was the detection and quantification of a wide array of immune compounds, including innate immunity factors (IL1ß, IL6, IL12, INFγ, TNFα), acquired immunity factors (IL2, IL4, IL10, IL13, IL17), chemokines (IL8, Groα, MCP1, MIP1ß), growth factors [IL5, IL7, epidermal growth factor (EGF), granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, TGFß2], and immunoglobulins (IgA, IgG, IgM), in milk produced by healthy women of different ethnicities living in different geographic, dietary, socioeconomic, and environmental settings. Among the analyzed factors, IgA, IgG, IgM, EGF, TGFß2, IL7, IL8, Groα, and MIP1ß were detected in all or most of the samples collected in each population and, therefore, this specific set of compounds might be considered as the "core" soluble immune factors in milk produced by healthy women worldwide. This approach may help define which immune factors are (or are not) common in milk produced by women living in various conditions, and to identify host, lifestyle, and environmental factors that affect the immunological composition of this complex biological fluid. Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT02670278.

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.