Human milk microbiome: associations with maternal diet and infant growth.

Introduction: Ingestion of human milk (HM) is identified as a significant factor associated with early infant gut microbial colonization, which has been associated with infant health and development. Maternal diet has been associated with the HM microbiome (HMM). However, a few studies have explored the associations among maternal diet, HMM, and infant growth during the first 6 months of lactation. Methods: For this cross-sectional study, Mam-Mayan mother-infant dyads (n = 64) were recruited from 8 rural communities in the Western Highlands of Guatemala at two stages of lactation: early (6-46 days postpartum, n = 29) or late (109-184 days postpartum, n = 35). Recruited mothers had vaginally delivered singleton births, had no subclinical mastitis or antibiotic treatments, and breastfed their infants. Data collected at both stages of lactation included two 24-h recalls, milk samples, and infant growth status indicators: head-circumference-for-age-z-score (HCAZ), length-for-age-z-score (LAZ), and weight-for-age-z-score (WAZ). Infants were divided into subgroups: normal weight (WAZ ≥ -1SD) and mildly underweight (WAZ < -1SD), non-stunted (LAZ ≥ -1.5SD) and mildly stunted (LAZ < -1.5SD), and normal head-circumference (HCAZ ≥ -1SD) and smaller head-circumference (HCAZ < -1SD). HMM was identified using 16S rRNA gene sequencing; amplicon analysis was performed with the high-resolution ANCHOR pipeline, and DESeq2 identified the differentially abundant (DA) HMM at the species-level between infant growth groups (FDR < 0.05) in both early and late lactation. Results: Using both cluster and univariate analyses, we identified (a) positive correlations between infant growth clusters and maternal dietary clusters, (b) both positive and negative associations among maternal macronutrient and micronutrient intakes with the HMM at the species level and (c) distinct correlations between HMM DA taxa with maternal nutrient intakes and infant z-scores that differed between breast-fed infants experiencing growth faltering and normal growth in early and late lactation. Conclusion: Collectively, these findings provide important evidence of the potential influence of maternal diet on the early-life growth of breastfed infants via modulation of the HMM.

Preschool child growth attainment and velocity during an agriculture intervention in rural Panama may be diminished by soil-transmitted helminths.

Background: Agricultural interventions are often recommended to address undernutrition in subsistence farming communities. However, intensified agriculture exposure can increase soil transmitted helminth (STH) infections, which are linked with poor child growth. This study examined impacts of the VERASAN public health and agricultural intervention program on preschool child growth attainment (HAZ and WAZ) and relative growth velocity over 7 months [change in height-for-age (∆HAD) and weight-for-age (∆WAD) difference]. Methods: VERASAN was initiated in 15 subsistence farming communities in rural Panama experiencing chronic undernutrition. Activities targeted improved household food security, preschool child diets and growth by intensifying and diversifying household agriculture. Our objectives were to explore the relationship between VERASAN and preschool child growth attainment (HAZ and WAZ) and velocity (∆HAD and ∆WAD) during one agricultural cycle in 238 households. We compared those new to VERASAN with those involved for 1 or 5 years, and identified if agricultural practices, food security, diet diversity and treatment of pre-existing STH infection were associated with growth attainment or velocity. Results: Prior participation in VERASAN did not directly influence WAZ, HAZ or ΔHAD but VERASAN-related benefits had an indirect influence. ΔHAD was positively associated with VERASAN-associated improvements in diet diversity and food security. HAZ and WAZ during land preparation were positively associated with diet diversity and HAZ with food security during harvest. HAZ was negatively associated with children visiting the agricultural plot, consuming leafy green vegetables and pre-existing hookworm infections. Both agricultural season and STH influenced ΔWAD. Children in VERASAN for 1 or 5 years experienced growth faltering between land preparation and growing season, but not those new to VERASAN. In contrast, between growing and harvest, ∆WAD declined in children new to VERASAN compared to children in VERASAN for longer. ΔWAD from land preparation to harvest was higher with pre-existing Ascaris infection whereas it was lower between growing season and harvest for pre-existing hookworm infection. Conclusion: In a context of preschool child growth faltering, malnutrition and STH infections, improved food security, agricultural production and diet diversity associated with VERASAN were associated with improved growth. In contrast, STH infections were negatively associated with some, but not all, growth outcomes.

Human milk microbial species are associated with infant head-circumference during early and late lactation in Guatemalan mother-infant dyads.

Human milk contains abundant commensal bacteria that colonize and establish the infant's gut microbiome but the association between the milk microbiome and head circumference during infancy has not been explored. For this cross-sectional study, head-circumference-for-age-z-scores (HCAZ) of vaginally delivered breastfed infants were collected from 62 unrelated Mam-Mayan mothers living in eight remote rural communities in the Western Highlands of Guatemala during two stages of lactation, 'early' (6-46 days postpartum, n = 29) or 'late' (109-184 days postpartum, n = 33). At each stage of lactation, infants were divided into HCAZ ≥ -1 SD (early: n = 18; late: n = 14) and HCAZ < -1 SD (early: n = 11; late: n = 19). Milk microbiome communities were assessed using 16S ribosomal RNA gene sequencing and DESeq2 was used to compare the differential abundance (DA) of human milk microbiota with infant HCAZ subgroups at both stages of lactations. A total of 503 ESVs annotated 256 putative species across the 64 human milk samples. Alpha-diversity using Chao index uncovered a difference in microbial community richness between HCAZ ≥ -1 SD and HCAZ < -1 SD groups at late lactation (p = 0.045) but not at early lactation. In contrast, Canonical Analysis of Principal Coordinates identified significant differences between HCAZ ≥ -1 SD and HCAZ < -1 SD at both stages of lactation (p = 0.003); moreover, 26 milk microbial taxa differed in relative abundance (FDR < 0.05) between HCAZ ≥ -1 SD and HCAZ < -1 SD, with 13 differentially abundant at each lactation stage. Most species in the HCAZ ≥ -1 SD group were Streptococcus species from the Firmicutes phylum which are considered human colonizers associated with human milk whereas the HCAZ < -1 SD group at late lactation had more differentially abundant taxa associated with environmentally and 'potentially opportunistic' species belonging to the Actinobacteria genus. These findings suggest possible associations between brain growth of breastfed infants and the milk microbiome during lactation. Importantly, these data provide the first evidence of cross talk between the human milk microbiome and the infant brain that requires further investigation.

Multiple Indicators of Undernutrition, Infection, and Inflammation in Lactating Women Are Associated with Maternal Iron Status and Infant Anthropometry in Panama: The MINDI Cohort.

Maternal infections, nutrient deficiencies, and inflammation (MINDI) co-exist in lactating indigenous women in Panama, but their impact on maternal iron status and infant growth is unknown. For this secondary analysis of cross-sectional data of lactating mothers from our MINDI cohort, we investigated associations of MINDI variables with maternal anemia, elevated serum transferrin receptor (sTfR), low serum iron, hepcidin, ferritin, and infant weight-for-age (WAZ), length-for-age (LAZ), and head-circumference-for-age (HCAZ) Z-scores in 99 mother-infant dyads. A bootstrapping resampling procedure preselected covariates for inclusion in multivariable regressions models from chronic maternal infections and nutritional status [folate, vitamins A, D, retinol-binding protein (RBP), insulin-growth factor-1 (IGF-1)] and inflammation [C-reactive protein (CRP), cytokines, platelet indices] indicators. Anemia was prevalent (53.5%) but underestimated due to widespread low plasma volume (<2.2 L, 79.9%) and was associated with indicators of malnutrition [lower IGF-1, body mass index (BMI), vitamin D, and intake of green/leafy vegetables], but not inflammation. Higher CRP was associated with lower serum iron, and higher hepcidin and ferritin, whereas maternal platelets were associated with lower HCAZ (ß = −0.22), WAZ (ß = −0.17), and LAZ (ß = −0.17). Higher LAZ was also associated with maternal serum vitamin D (ß = 0.23), whereas maternal iron supplementation lowered LAZ (ß = −0.22). Assessment of iron status in this MINDI cohort is complex and supplementation strategies must consider consequences for both the mother and the infant.

Human milk microbiome is shaped by breastfeeding practices.

There is evidence that breastfeeding practices may impact the milk microbiota diversity and differential abundance at the genera level; however, the possibility that distinct feeding practices, such as exclusive (EBF) and non-exclusive breastfeeding (non-EBF), might alter the milk microbiome at the species level has not been explored. This cross-sectional study analyzed the milk microbiome of 64 Mam-Mayan indigenous mothers from San Juan Ostuncalco in Guatemala. Two breastfeeding practices [exclusive (EBF) vs non-exclusive (non-EBF)] were analyzed at two stages of lactation [early (5-46 days post-partum) vs late (109-184 days post-partum)]. EBF was defined as offering only human milk and non-EBF was defined as feeding the infant herbal teas (agüitas) and/or complementary foods while continuing to breastfeed. Results identified four clusters with distinct microbial communities that segregated bacterial species by both breastfeeding practices and stage of lactation. Comparison among these clusters identified several notable patterns. First, during EBF, the microbiome differed by stage of lactation where there was a shift in differential abundance from Actinobacteria and Firmicutes in early to Bacteroidetes and Proteobacteria species in late lactation. Second, a similar comparison between non-EBF mothers by stage of lactation also identified a higher differential abundance of Actinobacteria and Firmicutes species in early lactation, but only Proteobacteria and not Bacteroidetes in late lactation, indicating a further shift in the milk microbial ecosystem with fewer oral bacteria present in late lactation. Third, comparisons between EBF and non-EBF mothers at both early and late lactation showed that mothers who exclusively breastfed had more differentially abundant species in early (11 vs 1) and late (13 vs 2) lactation. Fourth, EBF at early and late lactation had more commensal and lactic acid bacteria, including Lactobacillus gasseri, Granulicatella elegans, Streptococcus mitis, and Streptococcus parasanguinis, compared to those who did not exclusively breastfeed. Collectively, these results show that EBF has more differentially abundant bacteria, including commensal and lactic acid bacteria, and that the addition of agüitas (herbal teas) and/or complementary foods modify the milk microbiome composition by reducing the oral bacteria and introducing more environmentally sourced bacteria to the ecosystem.

Maternal nematode infection upregulates expression of Th2/Treg and diapedesis related genes in the neonatal brain.

Intestinal nematode infections common during pregnancy have recently been shown to have impacts that extend to their uninfected offspring including altered brain gene expression. If maternal immune signals reach the neonatal brain, they might alter neuroimmune development. We explored expression of genes associated with four distinct types of T cells (Th1, Th2, Th17, Treg) and with leukocyte transendothelial migration and endocytosis transport across the blood-brain barrier (BBB) in the postnatal brain of offspring of nematode-infected mice, through secondary analysis of a whole brain gene expression database. Th1/Th17 expression was lowered by maternal infection as evidenced by down-regulated expression of IL1ß, Th1 receptors and related proteins, and of IL22 and several Th17 genes associated with immunopathology. In contrast, Th2/Treg related pathways were upregulated as shown by higher expression of IL4 and TGF-ß family genes. Maternal infection also upregulated expression of pathways and integrin genes involved in transport of leukocytes in between endothelial cells but downregulated endosome vesicle formation related genes that are necessary for endocytosis of immunoglobulins across the BBB. Taken together, pup brain gene expression indicates that maternal nematode infection enhanced movement of leukocytes across the neonatal BBB and promoted a Th2/Treg environment that presumably minimizes the proinflammatory Th1 response in the pup brain.

Infant Anthropometry and Growth Velocity Before 6 Months are Associated with Breastfeeding Practices and the Presence of Subclinical Mastitis and Maternal Intestinal Protozoa in Indigenous Communities in Guatemala.

BACKGROUND: The possibility that maternal health status and breastfeeding practices contribute to growth faltering before 6 mo is underexplored. OBJECTIVES: This longitudinal study investigated whether indicators of subclinical mastitis (SCM) and breast inflammation, maternal fecal-oral contamination, and/or breastfeeding practices were associated with infant anthropometry or growth velocity before 6 mo. METHODS: Indigenous Mam-Mayan mother-infant dyads (n = 140) were recruited. Breast milk was collected at early (<6 wk) and established (4-6 mo) lactation when maternal and infant anthropometry were measured. Milk Na:K ratio as an indicator of SCM and concentrations of 4 proinflammatory cytokines were measured. Maternal stool samples were examined for the presence of intestinal parasites including nonpathogenic protozoa (Endolimax nana, Iodamoeba bütschlii, Entamoeba coli, Blastocystis hominis). Questionnaires characterized breastfeeding and hygiene practices. Multiple linear regression identified factors associated with infant growth attainment [weight-for-age z-score (WAZ), length-for-age z-score (LAZ), and head circumference-for-age z-score (HCAZ)] and growth velocity (expressed as change per day from early to established lactation). Multiple logistic regression identified factors associated with increased odds of underweight, stunting, and low head circumference at both lactation stages. RESULTS: A higher Na:K ratio, individual nonpathogenic protozoa, and specific breastfeeding and hygiene practices were associated with impaired growth before 6 wk and at 4-6 mo in exclusively breastfed infants. Growth velocity for weight was inversely associated with Entamoeba coli but cranial growth was associated positively with Iodamoeba bütschlii whereas feeding colostrum in early lactation was protective and decreased the odds of an HCAZ < -2 SD. Finally, the presence of SCM in early lactation increased the likelihood of both WAZ < -2 SD and LAZ < -2 SD by 6 wk. CONCLUSIONS: Prevention of SCM can improve early infant weight, but measures that promote the feeding of colostrum and reduce exposure to fecal-oral contamination might be required to minimize infant growth faltering in breastfed infants.

Maternal and cord blood parameters are associated with placental and newborn outcomes in indigenous mothers: A case study in the MINDI cohort / Parámetros maternos y de sangre de cordón asociados con medidas placentarias y del recién nacido: Un estudio de casos en madres indígenas de la cohorte MINDI

Abstract Background: Multiple infections, nutrient deficiencies and inflammation (MINDI) occur in indigenous communities, but their associations with perinatal outcomes have not been described. Objective: To assess maternal and cord blood micronutrient and inflammation status in peripartum mothers from the Ngäbe-Buglé comarca in Panama, and their associations with placental and infant outcomes. Methods: In 34 mother-newborn dyads, placental weight and diameter were measured, and maternal and cord blood were processed for complete cell counts, serum C-reactive protein, ferritin, serum transferrin receptor (sTfR), vitamins A and D. Blood volumes were calculated using Nadler's formula. Results: Mothers had low plasma volume (<2.8 L, 96%), vitamin A (52.9%), vitamin D (29.4%), iron (58.8%) and hemoglobin (23.5%), but high hematocrit (>40%, 17.6%) and inflammation (C-reactive protein >8.1 mg/L, 85.3%). Birthweights were normal, but low placental weight (35.3%), low head circumference Z-scores (17.6%), and low cord hemoglobin (5.9%), iron (79.4%), vitamin A (14.7%) and vitamin D (82.3%) were identified. Maternal and cord vitamin D were highly correlated. Higher maternal plasma volume was associated with heavier placentae (β= 0.57), and higher cord D (β= 0.43) and eosinophils (β= 0.43) with larger placentae. Hemoconcentration (higher cord hematocrit) was associated with lower newborn weight (β= -0.48) and head circumference (β= -0.56). Inflammation [higher maternal neutrophils (β= -0.50), and cord platelets (β= -0.32)] was associated with lower newborn length and head circumference. Conclusion: Maternal-newborn hemoconcentration, subclinical inflammation and multiple nutrient deficiencies, particularly neonatal vitamin D deficiency, were identified as potential targets for interventions to improve pregnancy outcomes in vulnerable communities.

Resumen Antecedentes: Las Múltiples Infecciones, Nutrición Deficiente e Inflamación (MINDI), son frecuentes en comunidades indígenas, sin embargo, sus asociaciones con resultados de salud perinatales no han sido descritos. Objetivo: Evaluar la inflamación y los micronutrientes en sangre materna y de cordón de madres en trabajo de parto en la comarca Ngäbe-Buglé en Panamá, así como sus asociaciones con medidas placentarias y del recién nacido. Métodos: En 34 pares madre-recién nacido, se midieron peso y diámetro placentario, y se analizaron muestras de sangre materna y de cordón umbilical para hemograma completo, proteína-C reactiva (PCR), ferritina, receptor sérico de transferrina (RsTf), vitaminas A y D. Se usó la fórmula de Nadler para calcular volúmenes sanguíneos. Resultados: Las madres presentaron volumen plasmático (<2.8 L, 96%), vitamina A (52.9%), vitamina D (29.4%), hierro (58.8%) y hemoglobina (23.5%) bajos, pero el 17.6% presentaron hematocrito >40% y 85.3% presentaron inflamación (PCR >8.1 mg/L). Los pesos al nacer fueron normales, pero se identificó bajo peso placentario (35.3%), bajo puntaje-z de circunferencia cefálica neonatal, y en sangre de cordón, bajos hemoglobina (5.9%), hierro (79.4%), vitamina A (14.7%) y vitamina D (82.3%). Se encontró una fuerte correlación positiva entre la vitamina D materna y de sangre de cordón. Un mayor volumen plasmático materno se asoció con placentas de mayor peso (β= 0.57), en tanto que concentraciones más altas de vitamina D (β= 0.43) y mayor número de eosinófilos (β= 0.43) se asociaron con mayor diámetro placentario. Una mayor hemoconcentración (hematocrito en cordón más alto) se asoció con menores peso al nacer (β= -0.48) y circunferencia cefálica (β= -0.56). La inflamación [mayor número de neutrófilos maternos (β= -0.50) y plaquetas en sangre de cordón (β= -0.32)] se asoció con menor talla y circunferencia cefálica neonatales. Conclusión: La hemoconcentración materna y del recién nacido, la inflamación subclínica y las múltiples deficiencias en micronutrientes, particularmente la deficiencia de vitamina D neonatal, se identificaron como potenciales áreas de intervención para mejorar los resultados de salud del embarazo en comunidades vulnerables.

A gastrointestinal nematode in pregnant and lactating mice alters maternal and neonatal microbiomes.

The maternal microbiome is understood to be the principal source of the neonatal microbiome but the consequences of intestinal nematodes on pregnant and lactating mothers and implications for the neonatal microbiome are unknown. Using pregnant CD1 mice infected with Heligmosomoides bakeri, we investigated the microbiomes in maternal tissues (intestine, vagina, and milk) and in the neonatal stomach using MiSeq sequencing of bacterial 16S rRNA genes. Our first hypothesis was that maternal nematode infection altered the maternal intestinal, vaginal, and milk microbiomes and associated metabolic pathways. Maternal nematode infection was associated with increased beta-diversity and abundance of fermenting bacteria as well as Lactobacillus in the maternal caecum 2 days after parturition, together with down-regulated carbohydrate, amino acid and vitamin biosynthesis pathways. Maternal nematode infection did not alter the vaginal or milk microbiomes. Our second hypothesis was that maternal infection would shape colonization of the neonatal microbiome. Although the pup stomach microbiome was similar to that of the maternal vaginal microbiome, pups of infected dams had higher beta-diversity at day 2, and a dramatic expansion in the abundance of Lactobacillus between days 2 and 7 compared with pups nursing uninfected dams. Our third hypothesis that maternal nematode infection altered the composition of neonatal microbiomes was confirmed as we observed up-regulation of several putatively beneficial microbial pathways associated with synthesis of essential and branched-chain amino acids, vitamins, and short-chain fatty acids. We believe this is the first study to show that a nematode living in the maternal intestine is associated with altered composition and function of the neonatal microbiome.

INTERGROWTH-21 Identifies High Prevalence of Low Symphysis-Fundal Height in Indigenous Pregnant Women Experiencing Multiple Infections, Nutrient Deficiencies, and Inflammation: The Maternal Infections, Nutrient Deficiencies, and Inflammation (MINDI) Cohort.

BACKGROUND: In the absence of ultrasound, symphysis-fundal height (SFH) can assess maternal-fetal well-being as it is associated with gestational age, fetal weight, and amniotic fluid volume. However, other modifiers of SFH, including maternal infections, nutrient deficiencies, and inflammation (MINDI), have not been widely explored. OBJECTIVES: Our objectives were 2-fold: 1) to assess prevalence of low SFH in indigenous Panamanian women using both Pan-American Health Organization (PAHO) and INTERGROWTH-21 standards and 2) to explore associations of SFH with maternal health indicators: infections (oral, skin, urogenital, nematode infections), nutrient deficiencies [protein and iron indicators (ferritin, serum iron, serum transferrin receptor, hepcidin), folate, and vitamins A, D, and B-12], and inflammation [leukocytes, C-reactive protein (CRP), cytokines]. METHODS: For this cross-sectional study, low-SFH-for-gestational-age was assessed using PAHO and INTERGROWTH <10th centile in 174 women at ≥16 weeks of gestation. Bootstrapping selected MINDI variables for inclusion in multivariable fractional polynomial (MFP) logistic regressions for low SFH. Associations of MINDI variables with hepcidin were also investigated. RESULTS: Prevalence of low SFH was 8% using PAHO, but using INTERGROWTH, 50.6% had SFH <10th centile, including 37.9% <3rd centile. Both PAHO-SFH <10th centile and INTERGROWTH-SFH <3rd centile were associated with higher hepcidin (OR = 1.12, P = 0.008, and OR = 3.04, P = 0.001, respectively) and with lower TNF-α (OR = 0.73, P = 0.012, and OR = 0.93, P = 0.015, respectively). Wood-smoke exposure increased the odds of PAHO-SFH <10th centile (OR = 1.19, P = 0.009), whereas higher BMI decreased the odds of INTERGROWTH-SFH <3rd centile (OR = 0.87, P = 0.012). Lower pulse pressure (OR = 0.90, P = 0.009) and lower inflammatory responses [lower lymphocytes (OR = 0.21, P = 0.026), IL-17 (OR = 0.89, P = 0.011)] distinguished SFH <3rd centile from SFH ≥3rd to <10th centiles using INTERGROWTH-21 standards. The MFP regression for hepcidin controlling for SFH (adjusted R 2 = 0.40, P = 0.001) revealed associations with indicators of inflammation (CRP, P < 0.0001; IL-17, P = 0.012), acidic urinary pH (P = 0.008), and higher intake of supplements (P = 0.035). CONCLUSIONS: Associations of low SFH with MINDI variables, including hepcidin, highlight its potential for early detection of multicausal in utero growth faltering.

Human Milk Microbiota in an Indigenous Population Is Associated with Maternal Factors, Stage of Lactation, and Breastfeeding Practices.

BACKGROUND: Human milk contains a diverse community of bacteria that are modified by maternal factors, but whether these or other factors are similar in developing countries has not been explored. Our objective was to determine whether the milk microbiota was modified by maternal age, BMI, parity, lactation stage, subclinical mastitis (SCM), and breastfeeding practices in the first 6 mo of lactation in an indigenous population from Guatemala. METHODS: For this cross-sectional study, Mam-Mayan indigenous mothers nursing infants aged <6 mo were recruited. Unilateral human milk samples were collected (n = 86) and processed for 16S rRNA sequencing at the genus level. Microbial diversity and relative abundance were compared with maternal factors [age, BMI, parity, stage of lactation, SCM, and 3 breastfeeding practices (exclusive, predominant, mixed)] obtained through questionnaires. RESULTS: Streptococcus was the most abundant genus (33.8%), followed by Pseudomonas (18.7%) and Sphingobium (10.7%) but relative abundance was associated with maternal factors. First, Lactobacillus and Streptococcus were more abundant in early lactation whereas the common oral (Leptotrichia) and environmental (Comamonas) bacteria were more abundant in established lactation. Second, Streptococcus,Lactobacillus,Lactococcus,Leuconostoc, and Micrococcus had a higher abundance in multiparous mothers compared with primiparous mothers. Third, a more diverse microbiota characterized by a higher abundance of lactic acid bacteria (Lactobacillus,Leuconostoc, and Lactococcus), Leucobacter, and Micrococcus was found in mothers with a healthy BMI. Finally, distinct microbial communities differed by stage of lactation and by exclusive, predominant, or mixed breastfeeding practices. CONCLUSION: Milk bacterial communities in an indigenous community were associated with maternal factors. Higher microbial diversity was supported by having a healthy BMI, the absence of SCM, and by breastfeeding. Interestingly, breastfeeding practices when assessed by lactation stage were associated with distinct microbiota profiles.

Distinct Changes Occur in the Human Breast Milk Microbiome Between Early and Established Lactation in Breastfeeding Guatemalan Mothers.

Human breast milk contains a diverse community of bacteria, but as breast milk microbiome studies have largely focused on mothers from high income countries where few women breastfeed to 6 months, the temporal changes in the breast milk microbiome that occur during later lactation stages have not been explored. For this cross-sectional study, microbiota from breast milk samples of Mam-Mayan mothers living in eight remote rural communities in the Western Highlands of Guatemala were analyzed. All mothers delivered vaginally and breastfed their infants for 6 months. Breast milk from 76 unrelated mothers was used to compare two lactation stages, either "early" (6-46 days post-partum, n = 33) or "late" (109-184 days post-partum, n = 43). Breast milk microbial communities were assessed using 16S ribosomal RNA gene sequencing and lactation stages were compared using DESeq2 differential abundance analysis. A total of 1,505 OTUs were identified, including 287 which could be annotated as putative species. Among several maternal factors, lactation stage explained microbiome variance and inertia in ordination with the most significance (p < 0.001). Differential abundance analysis identified 137 OTUs as significantly higher in either early or late lactation. These included a general shift from Staphylococcus and Streptococcus species in early lactation to Sphingobium and Pseudomonas species in late lactation. Species enriched in early lactation included putative commensal bacteria known to colonize the infant oral and intestinal tracts whereas species enriched in late lactation had a uniform functional trait associated with aromatic compound degradation. Differentially abundant species also included several species which have not previously been reported within breast milk, such as Janthinobacterium agaricidamnosum, Novosphingobium clariflavum, Ottowia beijingensis, and Flavobacterium cucumis. These discoveries describe temporal changes to the breast milk microbiome of healthy Guatemalan mothers from early to late lactation. Collectively, these findings illustrate how studying under-represented human populations might advance our understanding of factors that modulate the human milk microbiome in low and middle income countries (LMIC).

Emerging frontiers in human milk microbiome research and suggested primers for 16S rRNA gene analysis.

Human milk is the ideal food for infants due to its unique nutritional and immune properties, and more recently human milk has also been recognized as an important source of bacteria for infants. However, a substantial amount of fundamental human milk microbiome information remains unclear, such as the origin, composition and function of the community and its members. There is emerging evidence to suggest that the diversity and composition of the milk microbiome might differ between lactation stages, due to maternal factors and diet, agrarian and urban lifestyles, and geographical location. The evolution of the methods used for studying milk microbiota, transitioning from culture dependent-approaches to include culture-independent approaches, has had an impact on findings and, in particular, primer selection within 16S rRNA gene barcoding studies have led to discrepancies in observed microbiota communities. Here, the current state-of-the-art is reviewed and emerging frontiers essential to improving our understanding of the human milk microbiome are considered.

Maternal and cord blood parameters are associated with placental and newborn outcomes in indigenous mothers: A case study in the MINDI cohort.

Background: Multiple infections, nutrient deficiencies and inflammation (MINDI) occur in indigenous communities, but their associations with perinatal outcomes have not been described. Objective: To assess maternal and cord blood micronutrient and inflammation status in peripartum mothers from the Ngäbe-Buglé comarca in Panama, and their associations with placental and infant outcomes. Methods: In 34 mother-newborn dyads, placental weight and diameter were measured, and maternal and cord blood were processed for complete cell counts, serum C-reactive protein, ferritin, serum transferrin receptor (sTfR), vitamins A and D. Blood volumes were calculated using Nadler's formula. Results: Mothers had low plasma volume (<2.8 L, 96%), vitamin A (52.9%), vitamin D (29.4%), iron (58.8%) and hemoglobin (23.5%), but high hematocrit (>40%, 17.6%) and inflammation (C-reactive protein >8.1 mg/L, 85.3%). Birthweights were normal, but low placental weight (35.3%), low head circumference Z-scores (17.6%), and low cord hemoglobin (5.9%), iron (79.4%), vitamin A (14.7%) and vitamin D (82.3%) were identified. Maternal and cord vitamin D were highly correlated. Higher maternal plasma volume was associated with heavier placentae (ß= 0.57), and higher cord D (ß= 0.43) and eosinophils (ß= 0.43) with larger placentae. Hemoconcentration (higher cord hematocrit) was associated with lower newborn weight (ß= -0.48) and head circumference (ß= -0.56). Inflammation [higher maternal neutrophils (ß= -0.50), and cord platelets (ß= -0.32)] was associated with lower newborn length and head circumference. Conclusion: Maternal-newborn hemoconcentration, subclinical inflammation and multiple nutrient deficiencies, particularly neonatal vitamin D deficiency, were identified as potential targets for interventions to improve pregnancy outcomes in vulnerable communities.

Antecedentes: Las Múltiples Infecciones, Nutrición Deficiente e Inflamación (MINDI), son frecuentes en comunidades indígenas, sin embargo, sus asociaciones con resultados de salud perinatales no han sido descritos. Objetivo: Evaluar la inflamación y los micronutrientes en sangre materna y de cordón de madres en trabajo de parto en la comarca Ngäbe-Buglé en Panamá, así como sus asociaciones con medidas placentarias y del recién nacido. Métodos: En 34 pares madre-recién nacido, se midieron peso y diámetro placentario, y se analizaron muestras de sangre materna y de cordón umbilical para hemograma completo, proteína-C reactiva (PCR), ferritina, receptor sérico de transferrina (RsTf), vitaminas A y D. Se usó la fórmula de Nadler para calcular volúmenes sanguíneos. Resultados: Las madres presentaron volumen plasmático (<2.8 L, 96%), vitamina A (52.9%), vitamina D (29.4%), hierro (58.8%) y hemoglobina (23.5%) bajos, pero el 17.6% presentaron hematocrito >40% y 85.3% presentaron inflamación (PCR >8.1 mg/L). Los pesos al nacer fueron normales, pero se identificó bajo peso placentario (35.3%), bajo puntaje-z de circunferencia cefálica neonatal, y en sangre de cordón, bajos hemoglobina (5.9%), hierro (79.4%), vitamina A (14.7%) y vitamina D (82.3%). Se encontró una fuerte correlación positiva entre la vitamina D materna y de sangre de cordón. Un mayor volumen plasmático materno se asoció con placentas de mayor peso (ß= 0.57), en tanto que concentraciones más altas de vitamina D (ß= 0.43) y mayor número de eosinófilos (ß= 0.43) se asociaron con mayor diámetro placentario. Una mayor hemoconcentración (hematocrito en cordón más alto) se asoció con menores peso al nacer (ß= -0.48) y circunferencia cefálica (ß= -0.56). La inflamación [mayor número de neutrófilos maternos (ß= -0.50) y plaquetas en sangre de cordón (ß= -0.32)] se asoció con menor talla y circunferencia cefálica neonatales. Conclusión: La hemoconcentración materna y del recién nacido, la inflamación subclínica y las múltiples deficiencias en micronutrientes, particularmente la deficiencia de vitamina D neonatal, se identificaron como potenciales áreas de intervención para mejorar los resultados de salud del embarazo en comunidades vulnerables.

Demonstration gardens improve agricultural production, food security and preschool child diets in subsistence farming communities in Panama.

OBJECTIVES: To explore impacts of a demonstration garden-based agricultural intervention on agricultural knowledge, practices and production, food security and preschool child diet diversity of subsistence farming households. DESIGN: Observational study of households new to the intervention or participating for 1 or 5 years. Variables measured were agricultural techniques learned from the intervention and used, agricultural production, household food insecurity (FIS) and child diet diversity (DDS), over one agricultural cycle (during land preparation, growing and harvest months). SETTING: Fifteen rural subsistence farming communities in Panama. PARTICIPANTS: Households participating in intervention (n 237) with minimum one preschool child. RESULTS: After 1 year, participants had more learned and applied techniques, more staple crops produced and lower FIS and higher DDS during land preparation and growing months compared with those new to the intervention. After 5 years, participants grew more maize, chickens and types of crops and had higher DDS during growing months and, where demonstration gardens persisted, used more learned techniques and children ate more vitamin A-rich foods. Variables associated with DDS varied seasonally: during land preparation, higher DDS was associated with higher household durable asset-based wealth; during growing months, with greater diversity of vegetables planted and lower FIS; during harvest, with older caregivers, caregivers working less in agriculture, more diverse crops and receiving food from demonstration gardens. CONCLUSIONS: The intervention improved food production, food security and diets. Sustained demonstration gardens were important for continued use of new agricultural techniques and improved diets.

A Multi-Sectoral Approach Improves Early Child Development in a Disadvantaged Community in Peru: Role of Community Gardens, Nutrition Workshops and Enhanced Caregiver-Child Interaction: Project "Wawa Illari".

Background: Multi-dimensional monitoring evaluation and learning strategies are needed to address the complex set of factors that affect early child development in marginalized populations, but few studies have explored their effectiveness. Objective: To compare improvement of health and development of children 0-3 years between intervention communities (IC) and control communities (CC) from peripheral settlements of Lima. Sequential interventions included: (1) home and community gardens, (2) conscious nutrition, and (3) parenting workshops following the International Child Development Program (ICDP). Methods: Interventions were delivered by community health promoters (CHPs) using a "step-by-step" learning system. Both IC and CC were monitored before the interventions began, at 8 and 12 months (n = 113 IC and 127 CC children). Data were collected on household characteristics, diet, food security, health indicators (history of diarrhea and respiratory infections, hemoglobin, intestinal parasites, anthropometry), caregiver-child interactions and stress, and achievement of Pan-American Health Organization age-specific developmental milestones. Stepwise multiple logistic regressions were used to determine if the interventions affected food insecurity, as well as motor, social/cognitive and language delays. Results: At baseline, 2.6% were categorized as "suspected developmental delay" and 14.2% were on "alert for development delay." Food insecurity, diarrhea and respiratory infections were lowered following the interventions. Through the "step-by-step" approach, caregivers in IC gained skills in gardening, conscious nutrition and parenting that reduced the risk of food insecurity [Adjusted Risk Ratio = 0.20 (95% CI: 0.08-0.51)] and language delay [0.39 (0.19-0.82)] but not motor or social/cognitive delay. Use of a multiple micronutrient supplement decreased the risk of motor delay [0.12 (0.03-0.56)], but more pets were associated with higher risk of motor [3.24 (1.47-7.14)] and social/cognitive delay [2.72 (1.33-5.55)], and of food insecurity [1.73 (1.13-2.66)]. Conclusion: The combined interventions delivered by CHPs helped to mitigate the impact of adversity on food insecurity and language delay. Additional improvements may have been detected if the interventions had continued for a longer time. Our results indicate that control of infections and pets may be needed to achieve measurable results for motor and social/cognitive development. Continuous monitoring facilitated adjusting implementation strategies and achieving positive developmental outcomes.

Associations of History of Displacement, Food Insecurity, and Stress With Maternal-Fetal Health in a Conflict Zone: A Case Study.

Background: In populations with a history of conflict, early identification of pregnant women who are at risk of adverse pregnancy outcomes is challenging, especially if sonography is not available. We evaluated the performance of symphysis-fundal height (SFH) for identification of high-risk pregnancies and investigated if food security and diet quality, clinical biomarkers, and stress were associated with SFH and two known indicators of maternal-fetal well-being, sonography-estimated fetal weight and amniotic fluid index (AFI). Methods: For this cross-sectional study, 61 women with high-risk pregnancies were recruited after referral to the obstetrics and gynecology unit at San José Hospital in Popayán, Colombia. Multiple stepwise linear and ordered logistic regressions were used to identify associations of SFH, sonography-estimated fetal weight and AFI classification with history of displacement, food insecurity, post-traumatic stress symptoms as well as biopsychosocial risk evaluated through the Colombian risk scale. Results: History of displacement was associated with lower SFH Z-scores, but higher hemoglobin, taking iron supplements and a higher diastolic blood pressure were associated with higher SFH Z-scores. SFH was also associated with AFI but not with sonography-estimated fetal weight. Stress indicators were associated with a higher AFI. In contrast family support, an element of the Colombian biopsychosocial risk assessment, was associated with a higher sonography-estimated fetal weight, whereas more hours of sleep/day were associated with lower sonography-estimated fetal weight. Conclusion: SFH was not only associated with biological factors known to affect maternal/fetal health but also with history of displacement, thus validating its use in conflict areas for pregnancy assessment. Associations of biopsychosocial stressors with maternal-fetal outcomes highlight the need for a systematic assessment of stress in pregnant women from conflict zones.

Identification of High-Risk Pregnancies in a Remote Setting Using Ambulatory Blood Pressure: The MINDI Cohort.

Background: Ambulatory blood pressure is a potential tool for early detection of complications during pregnancy, but its utility in impoverished settings has not been assessed. This cross-sectional study aimed to determine whether maternal infections, nutrient deficiencies and inflammation (MINDI) were associated with four measures of maternal blood pressure (BP) and to determine their association with symphysis-fundal-height (SFH). Methods: Environmental and dietary factors, intake of iron and a multiple-nutrient supplement (MNS), markers of inflammation, protein, anemia, folate, vitamins B12, A and D status, and urogenital, skin, oral and intestinal nematode infections were measured in indigenous pregnant Panamanian women. Stepwise multiple linear and logistic regression models explored determinants of systolic and diastolic blood pressure (SBP, DBP), hypotension (SBP < 100 and DBP < 60), mean arterial pressure (MAP), elevated MAP (eMAP), and pulse pressure (PP). Associations of BP with intestinal nematodes and with SFH Z scores (≥16 wk) were also explored. Results: Despite absence of high SBP or DBP, 11.2% of women had eMAP. Furthermore, 24.1% had hypotension. Linear regression showed that hookworm infection was associated with higher SBP (P = 0.049), DBP (P = 0.046), and MAP (P = 0.016), whereas Ascaris was associated with lower DBP (P = 0.018) and MAP (P = 0.028). Trichomonas was also associated with lower SBP (P < 0.0001) and MAP (P = 0.009). The presence of Trichuris (OR: 6.7, 95% CI 1.0-44.5) and folic acid deficiency (OR: 6.9, 95% CI 1.4-33.8) were associated with increased odds of eMAP. The odds of low BP was higher in the presence of Ascaris (OR: 3.63 ± 2.28, P = 0.040), but odds were lowered by MNS (OR: 0.35 ± 0.11, P = 0.001), more intake of animal-source foods/wk (OR: 0.7, 95% CI 0.5-0.9) and by higher concentrations of IL-17 (OR: 0.87 ± 0.05, P = 0.016). Conclusion: MINDI were bi-directionally associated with blood pressure indicators. In this MINDI cohort, infections, nutrients and cytokines both raised, and lowered BP indices. The presence of eMAP identified pregnant women at risk of hypertension whereas low PP was associated with lower SFH. Therefore, MAP and PP may help in detecting women at risk of adverse pregnancy outcomes in settings with limited access to technology.

Anthropometry before Day 46 and Growth Velocity before 6 Months of Guatemalan Breastfed Infants Are Associated with Subclinical Mastitis and Milk Cytokines, Minerals, and Trace Elements.

BACKGROUND: Subclinical mastitis (SCM) is an inflammatory condition of the mammary gland, but its association with human milk composition and infant growth is not well described. OBJECTIVES: We investigated whether SCM, milk cytokines, and/or estimated intakes of milk minerals and trace elements were associated with infant anthropometry or growth velocity before 6 mo. METHODS: Breast milk was collected from Mam-Mayan mothers (n = 114) at both early (2-46 d) and established (4-6 mo) lactation. Concentrations of 9 elements (sodium, potassium, calcium, magnesium, copper, iron, manganese, selenium, zinc) analyzed by inductively-coupled plasma mass spectrometry were used to estimate infants' daily intakes. Concentrations of 4 cytokines were measured. Milk concentrations and infants' estimated elemental intakes and anthropometric measurements during early and established lactation were compared by SCM status. Multiple regression was used to identify factors associated with infant growth attainment (<46 d) for infant weight- (WAZ), length- (LAZ), and head circumference-for-age (HCAZ) and weight-for-length (WLZ) z scores and with growth velocity (expressed as Δ/d) from early to established lactation. RESULTS: SCM prevalence was higher in early (30%) than established (10%) lactation. Breast milk of SCM mothers had higher cytokine concentrations and higher magnesium in early and higher selenium concentrations in both early and established lactation (Padj < 0.0121). At day 46, regression models showed inverse associations of SCM with WLZ and IL-1ß with LAZ (Padj < 0.0150). In contrast, linear growth velocity was positively associated with IL-1ß measured in early lactation (Padj < 0.0124), whereas cranial growth velocity was positively associated with IL-8 measured during established lactation ( Padj < 0.0124). CONCLUSIONS: Despite a high prevalence of inadequate intakes, only infants' intake of milk magnesium during early lactation was associated with linear growth velocity in breastfed infants <6 mo. Evidence shows that SCM, breast-milk cytokines, and infants' estimated intakes of select elements are independently associated with growth attainment and growth velocity during lactation.

Household food insecurity in Panamanian subsistence farming communities is associated with indicators of household wealth and constraints on food production.

OBJECTIVE: To determine if constraints on agricultural production were a novel construct in the Panama Food Security Questionnaire (FSQ) and to characterize agricultural and economic determinants of food insecurity during the planting, growing and harvesting time periods in subsistence farming communities. DESIGN: This longitudinal study followed households during land preparation, growing and harvest periods in one agricultural cycle. Agricultural production and economic variables were recorded and the Panama FSQ was administered. Exploratory factor analysis was used to verify construct validity of the FSQ. A food insecurity score (FIS), ranging from 0 to 42, was derived. Multiple regression analyses of FIS were conducted for each agricultural period. SETTING: Fifteen rural villages in Panama. PARTICIPANTS: Subsistence farming households (n 237). RESULTS: The FSQ contained four constructs: (i) ability to buy food; (ii) decreased amount/number of meals; (iii) feeling hungry; and (iv) lower agricultural production because of weather or lack of resources. Although most households were mildly food insecure in all time periods, determinants of food insecurity differed in each. Higher FIS was associated during land preparation with less rice and legumes planted and lower asset-based wealth; during growing months with less rice, more maize and pigeon peas planted and not selling produce; and during harvest with less rice planted, fewer chickens and lower income. CONCLUSIONS: Constraints on agriculture was a novel construct of the Panama FSQ. Different income-related variables emerged in each agricultural period. Planting staple foods and raising chickens were associated with food security, but some crop choices were associated with food insecurity.