Cellular and transcriptional diversity over the course of human lactation.

Human breast milk (hBM) is a dynamic fluid that contains millions of cells, but their identities and phenotypic properties are poorly understood. We generated and analyzed single-cell RNA-sequencing (scRNA-seq) data to characterize the transcriptomes of cells from hBM across lactational time from 3 to 632 d postpartum in 15 donors. We found that the majority of cells in hBM are lactocytes, a specialized epithelial subset, and that cell-type frequencies shift over the course of lactation, yielding greater epithelial diversity at later points. Analysis of lactocytes reveals a continuum of cell states characterized by transcriptional changes in hormone-, growth factor-, and milk production-related pathways. Generalized additive models suggest that one subcluster, LC1 epithelial cells, increases as a function of time postpartum, daycare attendance, and the use of hormonal birth control. We identify several subclusters of macrophages in hBM that are enriched for tolerogenic functions, possibly playing a role in protecting the mammary gland during lactation. Our description of the cellular components of breast milk, their association with maternal­infant dyad metadata, and our quantification of alterations at the gene and pathway levels provide a detailed longitudinal picture of hBM cells across lactational time. This work paves the way for future investigations of how a potential division of cellular labor and differential hormone regulation might be leveraged therapeutically to support healthy lactation and potentially aid in milk production.

Milk glycan metabolism by intestinal bifidobacteria: insights from comparative genomics.

Bifidobacteria are early colonizers of the human neonatal gut and provide multiple health benefits to the infant, including inhibiting the growth of enteropathogens and modulating the immune system. Certain Bifidobacterium species prevail in the gut of breastfed infants due to the ability of these microorganisms to selectively forage glycans present in human milk, specifically human milk oligosaccharides (HMOs) and N-linked glycans. Therefore, these carbohydrates serve as promising prebiotic dietary supplements to stimulate the growth of bifidobacteria in the guts of children suffering from impaired gut microbiota development. However, the rational formulation of milk glycan-based prebiotics requires a detailed understanding of how bifidobacteria metabolize these carbohydrates. Accumulating biochemical and genomic data suggest that HMO and N-glycan assimilation abilities vary remarkably within the Bifidobacterium genus, both at the species and strain levels. This review focuses on the delineation and genome-based comparative analysis of differences in respective biochemical pathways, transport systems, and associated transcriptional regulatory networks, providing a foundation for genomics-based projection of milk glycan utilization capabilities across a rapidly growing number of sequenced bifidobacterial genomes and metagenomic datasets. This analysis also highlights remaining knowledge gaps and suggests directions for future studies to optimize the formulation of milk-glycan-based prebiotics that target bifidobacteria.

Lactational transfer of sulforaphane-N-acetylcysteine in vivo and in human breast milk.

Sulforaphane (SFN) is a bioactive phytonutrient found in cruciferous vegetables. There is a lack of detailed information on the lactational transfer of SFN and SFN metabolites, and potential pharmacological effects on breastfeeding infants. We carried out two maternal supplementation studies in a mouse model, wherein lactating dams received either vehicle, 300 or 600 ppm SFN from postnatal day (PND) 1 to 5, or in a second experiment, vehicle or 600 ppm SFN from PND 1 to 14. The parent compound was only detectable in milk and plasma from dams receiving 600 ppm SFN for five days. The predominant metabolite SFN-N-acetylcysteine (SFN-NAC) was readily detected in milk from dams receiving 300 and 600 ppm SFN for five days or 600 ppm for 14 days. Maternal SFN-NAC plasma levels were elevated in both 600 ppm groups. Maternal hepatic and pulmonary expression of NRF2-related genes, Nqo1, Gsta2, Gstm1, and Gstp1, were significantly increased, generally following a dose-response; however, offspring induction varied. PND5 neonates in the 600-ppm group exhibited significantly elevated expression of Nqo1, Gsta2, and Gstp1 in liver, and Gstm1 and Gstp1 in lung. Findings support maternal dietary supplementation with SFN induces NRF2-related gene expression in neonates via lactational transfer of SFN-NAC. However, NQO1 enzyme activity was not significantly elevated, highlighting the need to optimize dosing strategy. Additionally, in a pilot investigation of lactating women consuming a typical diet, without any purified SFN supplementation, 7 out of 8 breast milk samples showed SFN-NAC above the limit of quantification (LOQ). Notably, the one sample below the LOQ was collected from the only participant who reported no consumption of cruciferous vegetables in the past 24 h. The parent compound was not detected in any of the human breast milk samples. Overall, these data indicate lactational transfer of SFN-NAC at dietary relevant levels. Future studies are needed to evaluate pharmacokinetics and pharmacodynamics of lactational transfer for potential preventive or therapeutic effects in breastfeeding children.

Zuranolone Concentrations in the Breast Milk of Healthy, Lactating Individuals: Results From a Phase 1 Open-Label Study.

PURPOSE/BACKGROUND: Zuranolone is a positive allosteric modulator of both synaptic and extrasynaptic γ-aminobutyric acid type A receptors and a neuroactive steroid approved as an oral, once-daily, 14-day treatment course for adults with postpartum depression in the United States. This study assessed zuranolone transfer into breast milk. METHODS/PROCEDURES: Healthy, nonpregnant, lactating adult female participants received once-daily 30 mg zuranolone from day (D)1 through D5 in this phase 1 open-label study. The relative infant dose (RID; weight-adjusted proportion of the maternal dose in breast milk over 24 hours) for 30 mg zuranolone was assessed at D5. An RID for 50 mg zuranolone was estimated using a simulation approach across a range of infant ages and weights. FINDINGS/RESULTS: Of 15 enrolled participants (mean age, 30.1 years), 14 completed the study. The mean RID for 30 mg zuranolone at D5 was 0.357%; the mean steady-state milk volume over D3 to D5 decreased from baseline by 8.3%. Overall unbound zuranolone in plasma was low (≤0.49%). Plasma concentrations peaked at D5 before decreasing in a biexponential manner. There was strong concordance between the temporal profiles of zuranolone concentrations in plasma and breast milk. The estimated mean RID for 50 mg zuranolone based on a milk intake of 200 mL/kg per day was 0.984%. All treatment-emergent adverse events reported by participants were mild, the most common being dizziness (n = 3). IMPLICATIONS/CONCLUSIONS: Zuranolone transfer into the breast milk of healthy, nonpregnant, lactating adult female participants was low; the estimated RID for 50 mg zuranolone was <1%, well below the <10% threshold generally considered compatible with breastfeeding.

Human colostrum in vitro protein digestion: peptidomics by liquid chromatography-Orbitrap-high-resolution MS and prospection for bioactive peptides via bioinformatics.

Breast milk is known to contain bioactive peptides that are released during digestion, being a major source of bioactive peptides to the new-born, some of which act against invading pathogens. However, the formation of bioactive peptides during digestion of human colostrum remains largely uninvestigated. This study aimed to investigate the formation of peptides during simulated digestion of human colostrum from adult women and to prospect antimicrobial peptides. For this purpose, we used high-resolution MS to monitor the release of peptides during in vitro digestion. Bioinformatics was used for the prospection of antimicrobial activity of peptides. During simulated digestion (oral, gastric and duodenal phases), 2318 peptide sequences derived from 112 precursor proteins were identified. At the end of simulated digestion, casein-derived peptide sequences were the most frequently observed. Among precursors, some proteins were seen for the first time in this study. The resulting peptides were rich in proline, glutamine, valine and leucine residues, providing characteristic traits of antimicrobial peptides. From bioinformatics analysis, seven peptides showed potentially high antimicrobial activity towards bacteria, viruses and fungi, from which the latter was the most prominent predicted activity. Antimicrobial peptides released during digestion may provide a defence platform with controlled release for the new-born.

Polyphenol exposure of mothers and infants assessed by LC-MS/MS based biomonitoring in breast milk.

Exposure to polyphenols is relevant throughout critical windows of infant development, including the breastfeeding phase. However, the quantitative assessment of polyphenols in human breast milk has received limited attention so far, though polyphenols may positively influence infant health. Therefore, a targeted LC-MS/MS assay was developed to investigate 86 analytes representing different polyphenol classes in human breast milk. The sample preparation consisted of liquid extraction, salting out, freeze-out, and a dilution step. Overall, nearly 70% of the chemically diverse polyphenols fulfilled all strict validation criteria for full quantitative assessment. The remaining analytes did not fulfill all criteria at every concentration level, but can still provide useful semi-quantitative insights into nutritional and biomedical research questions. The limits of detection for all analyzed polyphenols were in the range of 0.0041-87 ng*mL-1, with a median of 0.17 ng*mL-1. Moreover, the mean recovery was determined to be 82% and the mean signal suppression and enhancement effect was 117%. The developed assay was applied in a proof-of-principle study to investigate polyphenols in breast milk samples provided by twelve Nigerian mothers at three distinct time points post-delivery. In total, 50 polyphenol analytes were detected with almost half being phenolic acids. Phase II metabolites, including genistein-7-ß-D-glucuronide, genistein-7-sulfate, and daidzein-7-ß-D-glucuronide, were also detected in several samples. In conclusion, the developed method was demonstrated to be fit-for-purpose to simultaneously (semi-) quantify a wide variety of polyphenols in breast milk. It also demonstrated that various polyphenols including their biotransformation products were present in breast milk and therefore likely transferred to infants where they might impact microbiome development and infant health.

Human milk-specific fat components enhance the secretion of ghrelin by MGN3-1 cells.

Triacylglycerols (TAGs) are a major fat component in human milk. Since gastric lipase produces 1,2-diacylglycerol from TAGs, we focused on the bioactivity of human milk-derived diacylglycerols in stomach cells. Ghrelin is produced in the stomach and acts as an important regulator of growth hormone secretion and energy homeostasis. In this study, we showed that 1-oleoyl-2-palmitoylglycerol (OP) increased ghrelin secretion, whereas 1,3-dioleoyl-2-palmitoylglycerol (OPO), a major component of human milk TAGs, did not increase ghrelin secretion in the ghrelin-secreting cell line, MGN3-1. Therefore, diacylglycerol OP may directly contribute to the regulation of ghrelin secretion. We also found that 2-palmitoylglycerol and 1- and 2-oleoylglycerol increased ghrelin secretion. Finally, we demonstrated that intracellular cAMP levels and preproghrelin and ghrelin O-acyl transferase expression levels were enhanced by OP treatment in MGN3-1 cells. This may represent an example of a novel mother-infant interaction mediated by fat components derived from human breast milk.

The impact of thawing duration on leptin and adiponectin levels in frozen human milk samples.

BACKGROUND: Measurements of hormone concentrations in human milk from understudied populations across the world is of growing scientific interest. Due to limited access to suitable laboratory facilities at many research sites, human milk samples are often shipped frozen to distant laboratories for analysis. Shipping and handling exposes samples to the risk of thawing and degradation as the results of delays, mishandling, or other unforeseen circumstances. Similar degradation risk factors are well known in serum samples. However, the vulnerability of hormone degradation in human milk to thawing during transportation is largely unexplored. Leptin and adiponectin are of particular importance due to their roles in feeding behavior and metabolism in infants, hence our focus on these two hormones. In addition, leptin and adiponectin have been shown to be vulnerable to pasteurization temperature degradation in human milk samples. AIMS: We tested the degradation of human milk metabolic hormones - leptin and adiponectin - in a controlled environment in response to a variety of freeze/thaw conditions that are reflective of potential temperature fluctuations during transportation. MATERIALS & METHODS: Human milk samples were experimentally subjected to thaw durations ranging from 4 to 16 h and assayed for hormonal levels to observe changes from baseline. Samples were also subjected to two additional experimental conditions, re-freezing after an extended period outside freezers or remaining thawed with additional ice packs, to investigate the impact of common transportation conditions. RESULTS: We found the assayed levels of leptin were not significantly impacted by an extended thaw cycle of 16 h. However, leptin levels were impacted by the extended period of 40 h outside of the freezer. Adiponectin showed a decrease in concentration percentage after the initial 16-h thaw period, but the increased degradation between 0-16 h and 16-40 h was not as severe as that seen in the leptin samples. DISCUSSION & CONCLUSION: The results of this experiment can be used to inform hormone measurement consistency given different thawing lengths or freezing conditions during transportation. Additionally, this research informs decisions regarding transportation, storage/handling, and data analysis in human milk research.

Time intervals between pumping did not affect breastmilk protein produced by mothers of preterm infants.

AIM: Few studies investigate factors that might influence the content of expressed breastmilk. This study aims to investigate the influence of the intervals between breastmilk pumping and the time of the day on protein and fat concentration in breastmilk. METHODS: Mothers of very preterm infants in a neonatal ward who expressed more than 400 mL per day were included. Expressed breastmilk was obtained from each mother over 30 h who were pumping at strictly planned and varying intervals: 2, 3, 4 and 6 h. All samples were analysed using infrared transmission spectroscopy. RESULTS: Ten mothers participated at a median of 22 days postpartum. A total of 176 milk samples were analysed, and the average protein and fat concentrations in g/100 mL were 1.1 ± 0.23 and 4.2 ± 1.3, respectively. The time intervals between breast pumping sessions did not impact protein content, but fat content decreased by longer intervals (p < 0.01). The time of the day for milk pumping did not influence the protein or fat content. CONCLUSION: A single milk sample collected after any 2-6 h interval, at any time during the day, represents the protein content in the breastmilk, but not the fat content which decreased with longer intervals.

Peptidome comparison on the immune regulation effects of different casein fractions in a cyclophosphamide mouse model.

The protein composition of human milk plays a crucial role in infant formula milk powder formulation. Notably, significant differences exist between bovine casein and human milk casein. Previous studies have shown that casein hydrolysates could enhance immune function; however, gastrointestinal dyspepsia in infants affects the type and function of peptides. Therefore, the present study used peptidomics to sequence and analyze hydrolyzed peptides from different casein fractions. Additionally, animal experiments were conducted to assess the functionality of these casein fractions and elucidate their differences. The results revealed variations in peptide composition among the different casein fractions of formula milk powder. Interestingly, milk powder formulated with both ß- and κ-casein (BK) exhibited significant enrichment of peptides related to the immune system. Moreover, the BK group significantly alleviated immune organ damage in cyclophosphamide-treated mice and regulated serum levels of pro-inflammatory and anti-inflammatory factors. Furthermore, feeding different casein fractions influenced the intestinal microflora of cyclophosphamide-treated mice, with the BK group mitigating the changes caused by cyclophosphamide. In conclusion, the findings suggest that BK formula in milk powder has the potential to positively enhance immunity. This study provides a robust theoretical basis for human-emulsified formula milk powder development.

Maternal-Infant Factors in Relation to Extracellular Vesicle and Particle miRNA in Prenatal Plasma and in Postpartum Human Milk.

MicroRNAs (miRNA) in extracellular vesicles and particles (EVPs) in maternal circulation during pregnancy and in human milk postpartum are hypothesized to facilitate maternal-offspring communication via epigenetic regulation. However, factors influencing maternal EVP miRNA profiles during these two critical developmental windows remain largely unknown. In a pilot study of 54 mother-child dyads in the New Hampshire Birth Cohort Study, we profiled 798 EVP miRNAs, using the NanoString nCounter platform, in paired maternal second-trimester plasma and mature (6-week) milk samples. In adjusted models, total EVP miRNA counts were lower for plasma samples collected in the afternoon compared with the morning (p = 0.024). Infant age at sample collection was inversely associated with total miRNA counts in human milk EVPs (p = 0.040). Milk EVP miRNA counts were also lower among participants who were multiparous after delivery (p = 0.047), had a pre-pregnancy BMI > 25 kg/m2 (p = 0.037), or delivered their baby via cesarean section (p = 0.021). In post hoc analyses, we also identified 22 specific EVP miRNA that were lower among participants who delivered their baby via cesarean section (Q < 0.05). Target genes of delivery mode-associated miRNAs were over-represented in pathways related to satiety signaling in infants (e.g., CCKR signaling) and mammary gland development and lactation (e.g., FGF signaling, EGF receptor signaling). In conclusion, we identified several key factors that may influence maternal EVP miRNA composition during two critical developmental windows, which should be considered in future studies investigating EVP miRNA roles in maternal and child health.

Human milk oligosaccharides reduce necrotizing enterocolitis-induced neuroinflammation and cognitive impairment in mice.

Necrotizing enterocolitis (NEC) is the leading cause of morbidity and mortality in premature infants. One of the most devastating complications of NEC is the development of NEC-induced brain injury, which manifests as impaired cognition that persists beyond infancy and which represents a proinflammatory activation of the gut-brain axis. Given that oral administration of the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'-FL) and 6'-sialyslactose (6'-SL) significantly reduced intestinal inflammation in mice, we hypothesized that oral administration of these HMOs would reduce NEC-induced brain injury and sought to determine the mechanisms involved. We now show that the administration of either 2'-FL or 6'-SL significantly attenuated NEC-induced brain injury, reversed myelin loss in the corpus callosum and midbrain of newborn mice, and prevented the impaired cognition observed in mice with NEC-induced brain injury. In seeking to define the mechanisms involved, 2'-FL or 6'-SL administration resulted in a restoration of the blood-brain barrier in newborn mice and also had a direct anti-inflammatory effect on the brain as revealed through the study of brain organoids. Metabolites of 2'-FL were detected in the infant mouse brain by nuclear magnetic resonance (NMR), whereas intact 2'-FL was not. Strikingly, the beneficial effects of 2'-FL or 6'-SL against NEC-induced brain injury required the release of the neurotrophic factor brain-derived neurotrophic factor (BDNF), as mice lacking BDNF were not protected by these HMOs from the development of NEC-induced brain injury. Taken in aggregate, these findings reveal that the HMOs 2'-FL and 6'-SL interrupt the gut-brain inflammatory axis and reduce the risk of NEC-induced brain injury.NEW & NOTEWORTHY This study reveals that the administration of human milk oligosaccharides, which are present in human breast milk, can interfere with the proinflammatory gut-brain axis and prevent neuroinflammation in the setting of necrotizing enterocolitis, a major intestinal disorder seen in premature infants.

Streptococcus lactarius MB622 and Streptococcus salivarius MB620 isolated from human milk reduce chemokine IL-8 production in response to TNF-α in Caco-2 cell line, an exploratory study.

Streptococci are a predominant genera of the human milk microbiome. Among different lactic acid bacteria (LAB) a few Streptococcal strains are also considered as probiotics. Probiotic bacteria are reported to modulate immunity when consumed in adequate amount and bacterial hydrophobicity can be considered as a preliminary experiment for the adhesive capability of probiotic bacteria to the epithelial cells. The present study aimed to investigate the probiotic, hydrophobic and immune modulation property of Streptococcus lactarius MB622 and Streptococcus salivarius MB620, isolated from human milk. S. lactarius MB622 and S. salivarius MB620 displayed higher hydrophobicity (78 % and 59 % respectively) in addition to intrinsic probiotic properties such as gram positive classification, catalase negative activity, resistance to artificially stimulated gastric juice and gastrointestinal bile salt concentration. In conclusion Streptococcus lactarius MB622 and Streptococcus salivarius MB620 isolated from human milk when administered in sufficient amount and for certain duration could be used to reduce inflammation inside the colon by reducing the production of inflammatory booster (IL-8) in diseased state.

Regulation of adipogenesis by exosomal milk miRNA.

Milk is a rich source of miRNA packaged in exosomes. Evidence for the systemic uptake and tissue distribution of milk exosomes was reported in newborn and adult humans and animals. Breastfeeding in infants was associated with a reduced risk of obesity. Numerous adipogenesis-related miRNAs have been detected in human milk exosomes. It has been demonstrated that ingested exosomal milk miRNAs may alter gene expression in offspring to regulate their metabolism and growth. In humans, consumption of other species' milk, such as cows and goats, is continued through adulthood. Since miRNAs are conserved, the concern of cross-species transfer of adipogenic miRNA has been raised in recent years, and the increase in obesity worldwide was attributed partially to dairy milk consumption by humans. However, evidence is still weak. Research emphasizes the need for an adequate number of exosomal milk's miRNAs to reach the target cell for biological action to be achieved. It was reported that obese women's milk had less miRNA-148a and miRNA-30b, which may affect the fat acquisition of their babies. Some exosomal milk miRNAs, such as miRNA-29, miRNA-148, miRNA-30b and miRNA-125b, may have epigenetic effects on milk recipients. Moreover, the ability of milk exosomes to cross the gastrointestinal barrier makes them a promising oral drug delivery tool. Yet, exosomes may also be tagged with specific ligands which target certain tissues. Thus, milk exosomes can be engineered and loaded with certain miRNAs responsible for adipocyte differentiation, conversion, or browning. Modifications in the miRNA cargo of exosomes can benefit human health and be an alternative to traditional drugs.

Direct Use of Plasma or Milk Vitamin A Specific Activity Data to Model Retinol Kinetics and Predict Vitamin A Stores in Theoretical Lactating Women.

BACKGROUND: Many applications of the Simulation, Analysis and Modeling software use data on the fraction of an orally administered tracer dose (FD) in plasma; thus, researchers must scale-up measured analyte concentration to the total plasma pool. For studies in lactating women, estimating breast milk pool size is challenging. OBJECTIVES: The objectives were to determine whether the standard vitamin A modeling approach using FD data could be modified to use vitamin A specific activity in milk (SAm) and/or plasma (SAp) for compartmental analysis of vitamin A kinetics and status in theoretical lactating women. METHODS: Using 12 previously studied theoretical subjects with a wide range of assigned values for vitamin A total body stores (TBS) and the coefficient ("FaS") needed to predict TBS using a retinol isotope dilution equation, we simulated data for SAp and SAm for 49 d after oral administration of labeled vitamin A. Then we modeled datasets for SAp and SAm, as well as only SAp or SAm, incorporating a linear scaling factor to automatically convert SA to FD and including several physiologically reasonable constraints as input data. As outcomes, we compared model-predicted TBS and FaS to assigned values. RESULTS: Scaling factors effectively adjusted SA data to adequately predict vitamin A mass in plasma and breast milk pools. Data for SAp and SAm provided model predictions of TBS that were comparable to assigned values (range: 85-107%); using only SAp, ratios ranged from 92% to 108% and for SAm from 85% to 108%. Parallel results were obtained for simulated FaS. CONCLUSIONS: Results show that SA data from plasma and/or milk can be used directly for modeling vitamin A during lactation in theoretical subjects, providing accurate estimates of TBS and FaS. Results suggest that, in free-living lactating women, researchers might measure only SAp or only SAm and adequately describe whole-body vitamin A metabolism and status.

Preterm Pigs Fed Donor Human Milk Have Greater Liver ß-Carotene Concentrations than Pigs Fed Infant Formula.

BACKGROUND: Milk carotenoids may support preterm infant health and neurodevelopment. Infants fed human milk often have higher blood and tissue carotenoid concentrations than infants fed carotenoid-containing infant formula (IF). Donor human milk (DHM) is a supplement to mother's own milk, used to support preterm infant nutrition. OBJECTIVES: We tested whether tissue and plasma ß-carotene concentrations would be higher in preterm pigs fed pasteurized DHM versus premature IF. METHODS: This is a secondary analysis of samples collected from a study of the effects of enteral diet composition on necrotizing enterocolitis incidence. Preterm pigs received partial enteral feeding of either DHM (n = 7) or premature IF (n = 7) from 2 to 7 d of age. The diets provided similar ß-carotene (32 nM), but DHM had higher lutein, zeaxanthin, and lycopene, whereas IF had higher total vitamin A. Plasma, liver, and jejunum carotenoid and vitamin A concentrations were measured by HPLC-PDA. Jejunal expression of 12 genes associated with carotenoid and lipid metabolism were measured. RESULTS: Liver ß-carotene concentrations were higher in DHM- than IF-fed piglets (23 ± 4 compared with 16 ± 2 µg/g, respectively, P = 0.0024), whereas plasma and jejunal ß-carotene concentrations were similar between diets. Liver vitamin A stores were higher in piglets fed IF than DHM (50.6 ± 10.1 compared with 30.9 ± 7.2 µg/g, respectively, P=0.0013); however, plasma vitamin A was similar between groups. Plasma, liver, and jejunum concentrations of lutein, zeaxanthin, and lycopene were higher with DHM than IF feeding. Relative to piglets fed DHM, jejunal low density lipoprotein receptor (Ldlr) expression was higher (61%, P = 0.018) and cluster determinant 36 (Cd36) expression (-27%, P = 0.034) was lower in IF-fed piglets. CONCLUSIONS: Preterm pigs fed DHM accumulate more liver ß-carotene than IF-fed pigs. Future studies should further investigate infant carotenoid bioactivity and bioavailability.

Acute decrease in mothers' cortisol following nursing and milk expression.

The glucocorticoid hormone cortisol is an integral component of signaling pathways related to stress reactivity, energy balance, immune function, and other processes. In animal models, lactation is robustly associated with alterations in glucocorticoid signaling, and limited data suggest that similar changes may occur across human lactation. We asked whether milk letdown/secretion in breastfeeding mothers was associated with changes in cortisol, and whether such effects required presence of an infant. We measured changes in maternal salivary cortisol concentrations before and after nursing, the expression of breastmilk with an electric pump, or control activities. Participants conducted pre-session and post-session sampling (at 30 min) for all conditions, and provided a sample of pumped milk from one session. Both nursing and mechanical expression of breastmilk but not control were associated with equivalent declines in maternal cortisol concentration from pre-session values, indicating an effect of milk letdown on circulating cortisol independent of infant contact. Pre-session maternal salivary cortisol concentration was strongly and positively correlated with cortisol concentration in pumped milk samples, indicating that cortisol ingested by offspring provides a signal of maternal cortisol levels. Self-reported maternal stress was associated with higher pre-session cortisol concentrations, as well as with a larger drop in cortisol following nursing or pumping. These findings demonstrate that milk release-in the presence or absence of a suckling infant-regulates cortisol in mothers, and supports the potential for maternal signaling through breastmilk.

Human milk-derived extracellular vesicles alleviate high fat diet-induced non-alcoholic fatty liver disease in mice.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD), characterized by excessive hepatic lipid accumulation, imposes serious challenges on public health worldwide. Breastfeeding has been reported to reduce the risk of NAFLD. Extracellular vesicles (EVs) are bilayer membrane vesicles released from various cells into the extracellular space, participating in multiple life processes. Whether EVs from human milk exert metabolic benefits against NAFLD is worth investigating. METHODS AND RESULTS: In this study, the EVs were isolated from human milk collected from healthy mothers and quantified. Functional analyses were performed using the NAFLD mouse model and free fatty acid (FFA)-stimulated mouse primary hepatocytes. The results showed that human milk-derived EVs could effectively alleviate high fat diet-induced hepatic steatosis and insulin resistance in mice with NAFLD via inhibiting lipogenesis and increasing lipolysis. The FFA-induced lipid accumulation was also inhibited in hepatocytes after treatment with human milk-derived EVs. Mechanistically, the human milk derived-EVs cargo (proteins and miRNAs), which linked to lipid metabolism, may be responsible for these beneficial effects. CONCLUSION: The findings of this study highlighted the therapeutic benefits of human milk-derived EVs and provided a new strategy for NAFLD treatment.

Human Milk Exosomes Induce ZO-1 Expression via Inhibition of REDD1 Expression in Human Intestinal Epithelial Cells.

Human milk exosomes (HMEs) enhance intestinal barrier function and contribute to an improvement in inflammation and mucosal injury, such as necrotizing enteritis (NEC), in infants. Here, we aimed to elucidate the intracellular factors involved in HME-induced expression of zonula occludens-1 (ZO-1), a tight junction protein, in Caco-2 human intestinal epithelial cells. HME treatment for 72 h significantly increased transepithelial electrical resistance in these cells. The mean ZO-1 protein levels in cells treated with HME for 72 h were significantly higher than those in the control cells. The mRNA and protein levels of regulated in development and DNA damage response 1 (REDD1) in HME-treated cells were significantly lower than those in the control cells. Although HME treatment did not increase the mechanistic target of rapamycin (mTOR) level in Caco-2 cells, it significantly increased the phosphorylated mTOR (p-mTOR) level and p-mTOR/mTOR ratio. The ZO-1 protein levels in cells treated with an inducer of REDD1, cobalt chloride (CoCl2) alone were significantly lower than those in the control cells. However, ZO-1 protein levels in cells co-treated with HME and CoCl2 were significantly higher than those in cells treated with CoCl2 alone. Additionally, REDD1 protein levels in cells treated with CoCl2 alone were significantly higher than those in the control cells. However, REDD1 protein levels in cells co-treated with HME and CoCl2 were significantly lower than those in cells treated with CoCl2 alone. This HME-mediated effect may contribute to the development of barrier function in the infant intestine and protect infants from diseases.

Metabolomics can provide new insights into perinatal nutrition.

Perinatal nutrition is a key factor related to the Developmental Origin of Health and Disease hypothesis, which states that each and every event that happens during the periconceptional period and pregnancy can affect the health status of an individual. Metabolomics can be a very useful tool for gathering information about the effect of perinatal nutrition on both mothers and newborn infants. This non-systematic review focuses on the main metabolites detected by this technique, with regard to gestational diabetes, intrauterine growth restriction and breast milk. Conclusion. Nutrition, metabolome and microbiome interactions are gaining interest in the scientific community.