Lactation for Infant Feeding Expertise (LIFE) Abstract book

LIFE Introduction for Scientists.

What is LIFE?

LIFE (Lactation for Infant Feeding Expertise) is a dynamic research initiative focused on characterization of human milk composition. LIFE also explores the association between human milk and its components with maternal health as well as nutritional status, growth and other developmental outcomes in infants. LIFE involves numerous external collaborators and key opinion leaders in the field of human milk science.

Human Milk Composition and Analysis

Quantification of Vitamins A, E, and K and Carotenoids in Submilliliter Volumes of Human Milk

Background: Human milk is the optimal nutrition for all newborns in the first 6 months of life. In order to assess the nutritional needs of the breastfed infant, human milk is often characterized for multiple nutrients. Objective: To ensure that we minimize the volume of milk dedicated for research and optimize the number of nutrients characterized, we developed analytical methodologies for the determination of vitamins A (retinol), E (alpha and gamma tocopherol), K (phylloquinone and menaquinone4), and five carotenoids (β-carotene, lycopene, βcryptoxanthin, lutein, and zeaxanthin) using <1 mL human milk. Method: Vitamins E and K and carotenoids are simultaneously isolated from 750 μL milk by liquidliquid extraction (LLE). Tocopherols and carotenoids are determined by normal-phase LC with fluorescence and ultraviolet detection respectively. Vitamin K is analyzed on the same extracts after resuspension and clean-up by reversed phase liquid chromatography coupled to tandem MS.
The analysis of vitamin A involves saponification of 200 μL milk followed by LLE and determination by normal- phase LC with UV detection. Results: Full single-laboratory validation at four different concentration levels is presented. Recovery rates were within 90-105% in all except one case (retinol at 1.9 μg/mL, 88% recovery), with RSDs of repeatability and intermediate reproducibility below 10 and 15%, respectively for all the compounds. Conclusions and Highlights: To the best of best knowledge, this is the first report that allows for the characterization and quantification of vitamins A, E, and K and five carotenoids in <1 mL human milk.

Comparison of Macronutrient Content in Human Milk Measured by Mid-Infrared Human Milk Analyzer and Reference Methods

OBJECTIVE: The study aims at evaluating mid-infrared human milk analyzer (HMA) accuracy and precision, in human milk (HM). STUDY DESIGN: Röse-Gottlieb, highperformance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD), Kjeldahl and amino acid analysis (AA) were selected as references for total fat, lactose and total protein determination. RESULTS: No significant difference was observed in lactose content between HMA and HPAEC-PAD. Significant differences were observed in fat and protein content between HMA and reference methods. However, the difference in fat content was lower than 12%, and therefore within the variability declared by supplier.
For protein determination, the BCA protein assay was selected. No significant differences were observed in total protein content measured by BCA assay, Kjeldahl and AA methods. CONCLUSIONS: HMA was reliable for the quantification of total fat and lactose content, but not for total protein one. The latter was measured by BCA assay, which yielded comparable results to Kjeldahl and AA methods.

Quantification of Total Cholesterol in Human Milk by Gas Chromatography

Human milk provides the key nutrients necessary for infant growth and development. The objective of this study was to develop and validate a method to analyze the cholesterol content in liquid human milk samples along lactation. Direct saponification of the sample using ethanolic potassium hydroxide solution under cold conditions was applied and unsaponifiable matter was separated by centrifugation. Cholesterol was converted into its trimethylsilyl ether and the derivative analyzed by gas chromatography coupled with a flame ionization detector. Cholesterol was quantified using epicoprostanol as internal standard. The method is suitable for the determination of cholesterol in only 0.3 g of human milk.

It has been validated showing good repeatability (CV(r) < 15%) and intermediate reproducibility (CV(iR) < 15%). The method was used to analyze human milk obtained from five mothers collected at day 30(±3), 60 (±3) and 120 (±3) after delivery. The cholesterol content in human milk slightly decreased from 13.1 mg/100 g at 1 month to 11.3 mg/100 g 120 days after delivery. The method can also be used to determine desmosterol, an intermediate in cholesterol synthesis. 

Quantitative Determination of Non-Lactose Milk Oligosaccharides 

A method for the determination of non-lactose oligosaccharides (NLO) in milk using liquid chromatography has been developed. Oligosaccharides were labelled with a fluorescent tag, 2-aminobenzamide (2AB), and were identified by comparison of their retention times to those of oligosaccharide standards, their mass (as measured by mass spectrometry) and their fragmentation patterns in the mass spectrometer. The concentrations of the NLO in milk have been determined using 2 different approaches: (1) by preparing a calibration curve using genuine standards of each oligosaccharide. (2) by preparing a calibration curve using maltotriose as a universal standard for all NLO, and assuming all 2AB labelled oligosaccharides give an equimolar response in the detector.
The accuracy of the method was assessed by spike-recovery experiments. Using genuine NLO standards for calibration, recoveries were in the range 96-114%. Using maltotriose as a universal calibrant, recoveries were in the range 86- 120%. Method precision was assessed by determining the relative standard deviation of the results under repeatability (RSD(r)) and intermediate reproducibility (RSD(iR)) conditions. In most cases RSD(r) and RSD(iR) were below 5% irrespective of calibration method, but increased when NLO levels were close to LoQ.

Quantification of Glycerophospholipids and Sphingomyelin in Human Milk and Infant Formula by High Performance Liquid Chromatography Coupled with Mass Spectrometer Detector

Phospholipids and sphingomyelin have a central role in infant nutrition, phospholipid acting as a nutrient carrier of long chain polyunsaturated fatty acids and sphingomyelin having an important role in cognitive function. However, analytical methods to precisely characterize and quantify these compounds in maternal milk are needed. Phospholipids and sphingomyelin were extracted using chloroform and methanol and separated on Polaris 3 Si column 250×2.0mm from Varian and analyzed by high performance liquid chromatography (HPLC) coupled with mass spectrometer detector (MS). The analytical method was validated and repeatability, intermediate reproducibility, and recovery values were calculated. The relative standard deviation of repeatability (CV(r)) and intermediate reproducibility (CV(iR)) values ranged between 2.3 and 7.2% and 9.5 and 17.8%, respectively and the recovery values between 96 and 109%. Finally, the validated method was tested on human milk samples and on infant formula which were analysed also by HPLC coupled with evaporative light scattering detector (ELSD). In human milk, sphingomyelin (9.28mg100mL-1) was the most abundant compound, followed by phosphatidylcholine (5.39mg100mL-1), phosphatidylethanolamine (2.85mg100mL-1) and phosphatidylinositol (1.82mg100mL-1).

Quantification of 1,3-olein-2-palmitin (OPO) and Palmitic Acid in sn2 Position of Triacylglycerols in Human Milk by Liquid Chromatography Coupled with Mass Spectrometry

This study describes the identification and quantification of fatty acids in the sn-2 position of triacylglycerols (TAG) and of the most abundant TAG regioisomers in human milk by liquid chromatography coupled with highresolution mass spectrometry (HPLC-HRMS). Over 300 individual TAG species were observed and 1,3- olein-2- palmitin (OPO) was identified as the most abundant TAG regioisomer. Validation of the HPLC-HRMS method showed repeatability and intermediate reproducibility values ranging from 3.1 to 16.6% and 4.0 to 20.7%, respectively, and accuracy ranging from 75 to 97%. Results obtained by the HPLC-HRMS method were comparable to results from the ISO 6800 method for the quantification of palmitic acid in the sn-2 position of TAG (81.4 and 81.8 g 100 g-1 total palmitic acid, respectively). Processing the data obtained with the HPLC- HRMS method is extremely time consuming and, therefore, a targeted method suitable for the quantification of OPO in human milk samples by ultra-performance (UP) LC coupled with triple quadrupole (QQQ) MS was developed and validated.
OPO identification and quantification by UPLC-QQQ were based on nominal mass and a fragmentation pattern obtained by multiple reaction monitoring experiments. The method was validated in terms of accuracy and precision by analyzing different aliquots of the same human milk sample over time and comparing the results with values obtained by HPLCHRMS. Intermediate reproducibility was <15% and trueness comparable to HPLC-HRMS. Quantification of OPO in human milk samples collected at 30, 60 and 120 days postpartum showed that OPO content varies between 333 ± 11.8 and 383 ± 18.0 mg 100mL-1.

Regional, socioeconomic, and dietary factors influencing B-vitamins in human milk of urban Chinese lactating women at different lactation stages

Background: Adequate B-vitamins concentrations in human milk are considered to be a prerequisite for healthy development of infants in early life. This study aims to determine the concentrations of B-vitamins in human milk from Chinese women and the relationships between their concentrations and different geographical origin, lactation stages, socioeconomic characteristics, and dietary intake.

Methods: Human milk was obtained from 443 healthy lactating women from Beijing (n = 150), Suzhou (n = 146), and Guangzhou (n = 147) cities. Thiamine, riboflavin, vitamin B3 (nicotinamide and nicotinic acid), and vitamin B6 (pyridoxal, pyridoxine, and pyridoxamine) in human milk were analyzed by high performance liquid chromatography-tandem mass spectrometry. Pantothenic acid, biotin, and folates in human milk were analyzed by microbiological assay. The information from one 24-h dietary recall and socioeconomic characteristics were collected by interview and structured questionnaire, respectively.

Results: B-vitamins concentrations in human milk varied greatly among individuals. The median concentrations of B-vitamins of postpartum 5–11 d, 12–30 d, 31–60 d, 61– 120 d, and 121–240 d were respectively as follows: thiamine 3.13, 5.07, 4.28, 5.65, 6.28 (μg/100 g); riboflavin 20.8, 20.2, 11.9, 13.6, 15.6 (μg/100 g); vitamin B3 194.0, 300.0, 261.0, 212.5, 218.0 (μg/100 g); pantothenic acid 236.5, 291.0, 254.0, 179.0, 189.0 (μg/100 g); vitamin B6 6.34, 7.58, 8.60, 9.34, 10.20 (μg/100 g); biotin 0.462, 0.834, 0.606, 0.523, 0.464 (μg/100 g); folates 0.730, 2.390, 2.440, 2.420, 2.330 (μg/100 g). The levels of B-vitamins presented regional differences and varied significantly among different lactation stages. The inversely associations of thiamine, vitamin B6, and folates with maternal BMI were found in multivariate analyses (p < 0.05), as well as higher pantothenic acid, folates, and biotin concentrations in lactating women with supplement intake when compared with those without (p < 0.05). Riboflavin concentrations associated with regular exercise was found in multivariate analyses (p < 0.05).

Conclusions: The present study indicated regional and socioeconomic factors, lactation stage, and supplement intake may influence B-vitamins concentrations of human milk in healthy Chinese mothers. Further studies on accurate and complete analysis of all vitamin forms are crucial for giving a more comprehensive understanding of vitamin status in human milk.

Concentrations of Carotenoids and Tocopherols in Breast Milk from Urban Chinese Mothers and Their Associations with Maternal Characteristics: A Cross-Sectional Study

Milk composition remains the best estimate of infant requirements. The aims of this study were to quantify carotenoids and tocopherols in human milk from healthy Chinese mothers, and to explore their associations with lactation stage, region, socio-economic and obstetric characteristics, and dietary intake. Human milk was obtained from 509 healthy mothers, and concentrations of carotenoids and tocopherols were analyzed by Ultra High Performance Liquid Chromatography. The mothers' socio-economic and obstetric characteristics and dietary intake through a single 24-h dietary recall were evaluated. The median concentrations (μg/100 mL) of each component of 0-4 days, 5-11 days, 12-30 days, 31- 60 days, 61-120 days, and 121-240 days postpartum were respectively as follows: β-carotene 8.0, 2.8, 2.1, 1.7, 1.9, 1.8; β-cryptoxanthin 6.2, 3.4, 2.4, 1.7, 1.8, 2.1; lutein 5.7, 7.0, 2.2, 2.9, 2.8, 3.7; lycopene 6.3, 2.5, 1.8, 1.4, 1.4, 1.5; zeaxanthin 1.0, 1.4, 0.8, 0.8, 1.0, 1.1; α-tocopherol 645, 382, 239, 206, 212, 211; γ-tocopherol 68, 63, 70, 73, 68, 88.
The levels of those components varied significantly among different lactation stages and presented regional differences. Associations of carotenoid contents with maternal education, delivery mode, and present body mass index were found in multivariate analyses. These results suggested that lactation stage, region, and socio-economic and obstetric factors were associated with human milk concentrations of carotenoids and tocopherols in healthy Chinese mothers.

A Novel Methodology for the Quantification of B-Vitamers in Breast Milk

With this report we present the development, validation and application of an analytical methodology for the quantification of 18 water soluble vitamers and secreted or biological forms in breast milk. On a relatively low amount of breast milk (200 μL), we applied isotope dilution-based sample preparation based on a combination of enzymatic treatment and protein precipitation using acidic methanol enriched with stable isotope labelled internal standards. Compounds separation was achieved by reversed-phase liquid chromatography and detection performed by tandem mass spectrometry in positive electrospray ionization mode. To perform the quantification of 18 water-soluble vitamers, procured pooled breast milk was used to build matrix-matched calibration curves, as labelled internal standards were not available for each vitamer. The analytical approach has been validated according to the EMA guidelines. The overall performance of the method was considered adequate, with 0.3-28.3% and 0.9-32.6% intra and inter-day precision respectively and averaged accuracy reaching 92.2-107.5%. In addition, performed freeze/thaw stability studies showed the potential degradation of some vitamers. We therefore recommend particular attention in sample collection with rather having dedicated aliquots with small volumes. The feasibility of this analytical approach has been evaluated by quantifying various breast milk samples that were procured from an external supplier. The main forms found in breast milk were thiamine monophosphate for B1, flavin adenine dinucleotide for B2, nicotinamide for B3, pyridoxal for B6 and 5-methyl tetrahydrofolic acid for B9. In addition, we newly reported nudifloramide as B3 form present in breast milk. With this analytical approach, it will give more confidence to provide a comprehensive assessment of the presence of watersoluble vitamins in breast milk. This will enable the accurate evaluation of the nutritional requirements of infants.

Amino Acid Composition of Breast Milk from Urban Chinese Mothers

Human breast milk (BM) amino acid (AA) composition may be impacted by lactation stage or factors related to geographical location. The present cross-sectional study is aimed at assessing the temporal changes of BMAA over lactation stages in a large cohort of urban mothers in China. Four hundred fifty BM samples, collected in three Chinese cities covering eight months of lactation were analyzed for free (FAA) and total (TAA) AA by ophthalaldehyde/fluorenylmethylchloroformate (OPA/FMOC) derivatization. Concentrations and changes over lactation were aligned with previous reports. Both the sum and the individual TAA values significantly decreased during the first periods of lactation and then generally leveled off. Leucine and methionine were respectively the most and the least abundant indispensable amino acids across all the lactation stages, whereas glutamic acid + glutamine (Glx) was the most and cystine the least abundant dispensable AA. The contribution of FAA to TAA levels was less than 2%, except for free Glx, which was the most abundant FAA. In conclusion, the AA composition of the milk from our cohort of urban Chinese mothers was comparable to previous studies conducted in other parts of the world, suggesting that this is an evolutionary conserved trait largely independent of geographical, ethnic, or dietary factors.

Nutritive and Bioactive Proteins in Breastmilk

Protein ingested with breast milk provides indispensable amino acids which are necessary for new protein synthesis for growth and replacement of losses via urine, feces, and the skin. Protein gain in the body of an infant is highest during the first months when protein concentrations in breast milk are higher than during later stages of lactation. Low-birth-weight infants have higher protein needs than term infants and need protein supplements during feeding with breastmilk. Based on our better understanding of protein evolution in breastmilk during the stages of lactation, new infant formulas with lower protein concentration but better protein quality have been created, successfully tested, and are now available in many countries. Besides providing indispensable amino acids, bioactive protein in breast milk can be broadly classified into 4 major functions, that is, providing protection from microbial insults and immune protection, aiding in digestive functions, gut development, and being carriers for other nutrients. Individual proteins and their proposed bioactivities are summarized in this paper in brief. Indeed, some proteins like lactoferrin and sIgA have been extensively studied for their biological functions, whereas others may require more data in support to further validate their proposed functions.

Mineral compositions in breast milk of healthy Chinese lactating women in urban areas and its associated factors

BACKGROUND: Optimal mineral intakes are important for infant growth and development. However, data on mineral compositions of breast milk in Chinese women are scarce, and most were acquired before 1990. The objectives of this study were three-fold: (1) to investigate the mineral compositions of Chinese healthy mothers' breast milk in different lactation stages; (2) to explore correlations among mineral concentrations in breast milk; and (3) to explore the associated factors affecting mineral compositions in breast milk.

METHODS: The inductively coupled plasma mass spectrometry (ICP-MS) was used to analyze mineral concentrations in breast-milk of 444 healthy lactating women from three cities in China. A questionnaire was used to survey socio-demographic characteristics and pregnancy history. Food intakes by lactating women were measured using both food frequency questionnaire and one cycle of 24-hour dietary recall.

RESULTS: Mineral compositions of breast milk varied in different regions. Concentrations of most minerals were higher in the first one or two months of lactation, and then decreased with time, except for magnesium and iron. Inter-mineral correlations existed among several minerals. The calcium-to-phosphorus ratio was above 2:1 in each lactation stage. Women with caesarean section had higher concentration of iodine in the transitional milk (349.9 µg/kg) compared to women with natural delivery (237.5 µg/kg, P < 0.001). Dietary mineral intakes, supplements, food intake frequencies in the recent 6 months, maternal age and maternal BMI did not show significant correlations with concentrations of milk minerals (all P > 0.05).

CONCLUSIONS: Milk minerals decreased with time, and changed most rapidly in the first one or two months of lactation. Caesarean section might affect the iodine level in transitional milk.

Direct Quantification of Fatty Acids in Human Milk by Gas Chromatography

Human milk provides the key nutrients necessary for the infants' growth and development. The fatty acid composition of human milk has been extensively studied over xuthe last 20 years and the results obtained by analyzing the fatty acid profile followed by lipid extraction and expressing data as g per 100g of fatty acids. The main drawback is that normalizing data set does not give any information on the amount of fatty acid mother's milk and therefore the level of intake by the infant. The objective of the present study was to develop and validate a direct method to analyze the fatty acid content in liquid human milk samples. Hydrochloric acid in a solution of methanol was selected as the catalyst and methyl undecanoate (11:0) as the internal standard together with tritridecanoin (13:0 TAG) to monitor transesterification performance. The separation of fatty acid methyl esters (FAME) was performed using a 100 m highly polar capillary column and a certified calibration mixture used to calculate experimental response factors. The method is suitable to quantify fatty acids in human milk from a 250 μL sample and allow expression of the data in mg of fatty acids per deciliter of human milk as well as weight % of fatty acids. The method has been validated and show a good repeatability [CV(r)<15% and CV(r)<20% for the concentrations close to the LOQ] and a good intermediate reproducibility [CV(iR)<15% and CV(iR)<20% for the concentrations close to the LOQ]. The method was applied to analyze human milk samples obtained from 50 mothers 4 weeks post partum and the data are provided in absolute and relative quantity. These results show that the inter-individual variability of the fatty acid content in human milk is of prime importance and such information cannot be captured with normalized data sets.

Quantification of Phospholipids Classes in Human Milk

Phospholipids are integral constituents of the milk fat globule membranes and they play a central role in infants' immune and inflammatory responses. A methodology employing liquid chromatography coupled with evaporative light scattering detector has been optimized and validated to quantify the major phospholipids classes in human milk. Phospholipids were extracted using chloroform and methanol and separated on C18 column. Repeatability, intermediate reproducibility, and recovery values were calculated and a large sample set of human milk analyzed. In human milk, phospholipid classes were quantified at concentrations of 0.6 mg/100 g for phosphatidylinositol; 4.2 mg/100 g for phosphatidylethanolamine, 0.4 mg/100 g for phosphatidylserine, 2.8 mg/100 g for phosphatidylcholine, and 4.6 mg/100 g for sphingomyelin. Their relative standard deviation of repeatability and intermediate reproducibility values ranging between 0.8 and 13.4 % and between 2.4 and 25.7 %, respectively. The recovery values ranged between 67 and 112 %. Finally, the validated method was used to quantify phospholipid classes in human milk collected from 50 volunteers 4 weeks postpartum providing absolute content of these lipids in a relatively large cohort. The average content of total phospholipids was 23.8 mg/100 g that corresponds to an estimated mean intake of 140 mg phospholipids/day in a 4-week old infant when exclusively breast-fed.

N-Linked Glycan Profiling of Mature Human Milk by HighPerformance Microfluidic Chip Liquid Chromatography Time-ofFlight Tandem Mass Spectrometry

N-Linked glycans of skim human milk proteins were determined for three mothers. N-Linked glycans are linked to immune defense, cell growth, and cell-cell adhesion, but their functions in human milk are undetermined. Protein-bound N-linked glycans were released with peptidyl N-glycosidase F (PNGase F), enriched by graphitized carbon chromatography, and analyzed with Chip-TOF MS. To be defined as N- glycans, compounds were required, in all three procedural replicates, to match, within 6 ppm, against a theoretical human N-glycan library and be at least 2-fold higher in abundance in PNGase F-treated than in control samples. Fifty-two N-linked glycan compositions were identified, and 24 were confirmed via tandem mass spectra analysis. Twenty-seven compositions have been found previously in human milk, and 25 are novel compositions. By abundance, 84% of N-glycans were fucosylated and 47% were sialylated. The majority (70%) of total N-glycan abundance was composed of N-glycans found in all three milk samples.

OMICS-Rooted Studies of Milk Proteins, Oligosaccharides and Lipids

Milk has co-evolved with mammals and mankind to nourish their offspring and is a biological fluid of unique complexity and richness. It contains all necessary nutrients for the growth and development of the newborn. Structure and function of biomolecules in milk such as the macronutrients (glyco-) proteins, lipids, and oligosaccharides are central topics in nutritional research. Omics disciplines such as proteomics, glycomics, glycoproteomics, and lipidomics enable comprehensive analysis of these biomolecule components in food science and industry. Mass spectrometry has largely expanded our knowledge on these milk bioactives as it enables identification, quantification and characterization of milk proteins, carbohydrates, and lipids. In this article, we describe the biological importance of milk macronutrients and review the application of proteomics, glycomics, glycoproteomics, and lipidomics to the analysis of milk. Proteomics is a central platform among the Omics tools that have more recently been adapted and applied to nutrition and health research in order to deliver biomarkers for health and comfort as well as to discover beneficial food bioactives.

Modulation of Neonatal Microbial Recognition: TLR-Mediated Innate Immune Responses Are Specifically and Differentially Modulated by Human Milk

The mechanisms controlling innate microbial recognition in the neonatal gut are still to be fully understood. We have sought specific regulatory mechanisms operating in human breast milk relating to TLR-mediated microbial recognition. In this study, we report a specific and differential modulatory effect of early samples (days 1-5) of breast milk on ligand-induced cell stimulation via TLRs. Although a negative modulation was exerted on TLR2 and TLR3-mediated responses, those via TLR4 and TLR5 were enhanced. This effect was observed in human adult and fetal intestinal epithelial cell lines, monocytes, dendritic cells, and PBMC as well as neonatal blood. In the latter case, milk compensated for the low capacity of neonatal plasma to support responses to LPS. Cell stimulation via the IL-1R or TNFR was not modulated by milk. This, together with the differential effect on TLR activation, suggested that the primary effect of milk is exerted upstream of signaling proximal to TLR ligand recognition. The analysis of TLR4-mediated gene expression, used as a model system, showed that milk modulated TLR-related genes differently, including those coding for signal intermediates and regulators. A proteinaceous milk component of > or =80 kDa was found to be responsible for the effect on TLR4. Notably, infant milk formulations did not reproduce the modulatory activity of breast milk. Together, these findings reveal an unrecognized function of human milk, namely, its capacity to influence neonatal microbial recognition by modulating TLR-mediated responses specifically and differentially. This in turn suggests the existence of novel mechanisms regulating TLR activation.

Soluble Forms of Toll-Like Receptor (TLR)2 Capable of Modulating TLR2 Signaling Are Present in Human Plasma and Breast Milk

Dysregulation of the initial, innate immune response to bacterial infection may lead to septic shock and death. Toll-like receptors (TLRs) play a crucial role in this innate immune response, and yet the regulatory mechanisms controlling microbial-induced TLR triggering are still to be fully understood. We have therefore sought specific regulatory mechanisms that may modulate TLR signaling. In this study, we tested for the possible existence of a functionally active soluble form of TLR2. We demonstrated the existence of natural soluble forms of TLR2 (sTLR2), which we show to be capable of modulating cell activation. We found that blood monocytes released sTLR2 constitutively and that the kinetics of sTLR2 release increased upon cell activation. Analysis of cells expressing the human TLR2 cDNA or its c-myc-tagged version indicated that sTLR2 resulted from the posttranslational modification of the TLR2 protein in an intracellular compartment. Moreover, an intracellular pool of sTLR2 is maintained. sTLR2 was found naturally expressed in breast milk and plasma. Milk sTLR2 levels mirrored those of the TLR coreceptor soluble CD14. Depletion of sTLR2 from serum resulted in an increased cellular response to bacterial lipopeptide. Notably, serum sTLR2 was lower in tuberculosis patients. Coimmunoprecipitation experiments and computational molecular docking studies showed an interaction between sTLR2 and soluble CD14 in plasma and milk. These findings suggest the existence of a novel and specific innate immune mechanism regulating microbialinduced TLR triggering, and may lead to new therapeutics for the prevention and/or treatment of severe infectious diseases.

Innate Recognition of Bacteria in Human Milk is Mediated by a MilkDerived Highly Expressed Pattern Recognition Receptor, Soluble CD14

Little is known about innate immunity to bacteria after birth in the hitherto sterile fetal intestine. Breast- feeding has long been associated with a lower incidence of gastrointestinal infections and inflammatory and allergic diseases. We found in human breast milk a 48-kD polypeptide, which we confirmed by mass spectrometry and sequencing to be a soluble form of the bacterial pattern recognition receptor CD14 (sCD14). Milk sCD14 (m-sCD14) concentrations were up to 20-fold higher than serum sCD14 from nonpregnant, pregnant, or lactating women. In contrast, lipopolysaccharide (LPS)-binding protein was at very low levels. Mammary epithelial cells produced 48-kD sCD14. m-sCD14 mediated activation by LPS and whole bacteria of CD14 negative cells, including intestinal epithelial cells, resulting in release of innate immune response molecules. m-sCD14 was undetectable in the infant formulas and commercial (cows') milk tested, although it was present in bovine colostrum. These findings indicate a sentinel role for sCD14 in human milk during bacterial colonization of the gut, and suggest that m-sCD14 may be involved in modulating local innate and adaptive immune responses, thus controlling homeostasis in the neonatal intestine.

Dynamics of Human Milk Composition over the Lactation Period

Longitudinal Changes of Mineral Concentrations in Preterm and Term Human Milk from Lactating Swiss Women

An adequate mineral supply to preterm infants is essential for normal growth and development. This study aimed to compare the mineral contents of human milk (HM) from healthy mothers of preterm (28-32 weeks) and full term (>37 weeks) infants. Samples were collected weekly for eight weeks for the term group (n = 34) and, biweekly up to 16 weeks for the preterm group (n = 27). Iron, zinc, selenium, copper, iodine, calcium, magnesium, phosphorus, potassium, and sodium were quantitatively analyzed by Inductively Coupled PlasmaMass Spectrometry. The mineral contents of both HM showed parallel compositional changes over the period of lactation, with occasional significant differences when compared at the same postpartum age. However, when the comparisons were performed at an equivalent postmenstrual age, preterm HM contained less zinc and copper from week 39 to 48 (p < 0.002) and less selenium from week 39 to 44 (p < 0.002) than term HM. This translates into ranges of differences (min-max) of 53% to 78%, 30% to 72%, and 11% to 33% lower for zinc, copper, and selenium, respectively. These data provide comprehensive information on the temporal changes of ten minerals in preterm HM and may help to increase the accuracy of the mineral fortification of milk for preterm consumption.

Longitudinal Analysis of Macronutrient Composition in Preterm and Term Human Milk: A Prospective Cohort Study

BACKGROUND: Mother's own milk is the optimal source of nutrients and provides numerous health advantages for mothers and infants. As they have supplementary nutritional needs, very preterm infants may require fortification of human milk (HM). Addressing HM composition and variations is essential to optimize HM fortification strategies for these vulnerable infants.

AIMS: To analyze and compare macronutrient composition in HM of mothers lactating very preterm (PT) (28 0/7 to 32 6/7 weeks of gestational age, GA) and term (T) infants (37 0/7 to 41 6/7 weeks of GA) over time, both at similar postnatal and postmenstrual ages, and to investigate other potential factors of variations.

METHODS: Milk samples from 27 mothers of the PT infants and 34 mothers of the T infants were collected longitudinally at 12 points in time during four months for the PT HM and eight points in time during two months for the T HM. Macronutrient composition (proteins, fat, and lactose) and energy were measured using a midinfrared milk analyzer, corrected by bicinchoninic acid (BCA) assay for total protein content.

RESULTS: Analysis of 500 HM samples revealed large inter- and intra-subject variations in both groups. Proteins decreased from birth to four months in the PT and the T HM without significant differences at any postnatal time point, while it was lower around term equivalent age in PT HM. Lactose content remained stable and comparable over time. The PT HM contained significantly more fat and tended to be more caloric in the first two weeks of lactation, while the T HM revealed higher fat and higher energy content later during lactation (three to eight weeks). In both groups, male gender was associated with more fat and energy content. The gender association was stronger in the PT group, and it remained significant after adjustments.

CONCLUSION: Longitudinal measurements of macronutrients compositions of the PT and the T HM showed only small differences at similar postnatal stages in our population. However, numerous differences exist at similar postmenstrual ages. Male gender seems to be associated with a higher content in fat, especially in the PT HM. This study provides original information on macronutrient composition and variations of HM, which is important to consider for the optimization of nutrition and growth of PT infants.

Longitudinal Analysis of Macronutrient Composition in Preterm and Term Human Milk: A Prospective Cohort Study

BACKGROUND: Mother's own milk is the optimal source of nutrients and provides numerous health advantages for mothers and infants. As they have supplementary nutritional needs, very preterm infants may require fortification of human milk (HM). Addressing HM composition and variations is essential to optimize HM fortification strategies for these vulnerable infants. AIMS: To analyze and compare macronutrient composition in HM of mothers lactating very preterm (PT) (28 0/7 to 32 6/7 weeks of gestational age, GA) and term (T) infants (37 0/7 to 41 6/7 weeks of GA) over time, both at similar postnatal and postmenstrual ages, and to investigate other potential factors of variations.

METHODS: Milk samples from 27 mothers of the PT infants and 34 mothers of the T infants were collected longitudinally at 12 points in time during four months for the PT HM and eight points in time during two months for the T HM. Macronutrient composition (proteins, fat, and lactose) and energy were measured using a midinfrared milk analyzer, corrected by bicinchoninic acid (BCA) assay for total protein content.

RESULTS: Analysis of 500 HM samples revealed large inter- and intra-subject variations in both groups. Proteins decreased from birth to four months in the PT and the T HM without significant differences at any postnatal time point, while it was lower around term equivalent age in PT HM. Lactose content remained stable and comparable over time. The PT HM contained significantly more fat and tended to be more caloric in the first two weeks of lactation, while the T HM revealed higher fat and higher energy content later during lactation (three to eight weeks). In both groups, male gender was associated with more fat and energy content. The gender association was stronger in the PT group, and it remained significant after adjustments.

CONCLUSION: Longitudinal measurements of macronutrients compositions of the PT and the T HM showed only small differences at similar postnatal stages in our population. However, numerous differences exist at similar postmenstrual ages. Male gender seems to be associated with a higher content in fat, especially in the PT HM. This study provides original information on macronutrient composition and variations of HM, which is important to consider for the optimization of nutrition and growth of PT infants.

Temporal Changes of Major Protein Concentrations in Preterm and Term Human Milk. A Prospective Cohort Study

BACKGROUND: Proteins are major contributors to the beneficial effects of human milk (HM) on preterm infant health and development. Alpha-lactalbumin, lactoferrin, serum albumin and caseins represent approximately 85% of the total HM protein. The temporal changes of these proteins in preterm (PT) HM and its comparison with term (T) HM is poorly characterized.

AIMS: To quantify and compare the temporal changes of the major proteins in PT HM and T HM. METHODS: HM was collected for 4 months postpartum at 12 time points for PT HM (gestational age 28 0/7-32 6/7 weeks; 280 samples) and for 2 months postpartum at 8 time points for T HM (gestational age 37 0/7-41 6/7 weeks; 220 samples). Proteins were measured with a micro-fluidic LabChip system.

RESULTS: Casein, alpha-lactalbumin and lactoferrin decreased with advancing stages of lactation in PT and T HM, whereas serum albumin remained stable. Only marginal differences between PT and T HM were observed for alpha-lactalbumin during postpartum weeks 3-5 and for serum albumin at the first week. However, a comparison of HM provided to preterm and term infants at the same postmenstrual ages revealed that alphalactalbumin contents were significantly lower in PT HM than in T HM during the 39-48 postmenstrual weeks.

CONCLUSIONS: This study provides comprehensive information of the longitudinal changes of major proteins in PT and T HM, and suggests limited availability of alphalactalbumin, a nutritionally important protein, in breastfed PT infants after reaching the term corrected age. This information may be important to optimize HM protein fortification, although its biological relevance needs to be confirmed by intervention studies.

Temporal Progression of Fatty Acids in Preterm and Term Human Milk of Mothers from Switzerland

We longitudinally compared fatty acids (FA) from human milk (HM) of mothers delivering term and preterm infants. HM was collected for 4 months postpartum at 12 time points for preterm and for 2 months postpartum at 8 time points for term group. Samples were collected from the first feed of the morning, and single breast was fully expressed. FA were analyzed by gas chromatography coupled with flame ionization detector. Oleic, palmitic and linoleic acids were the most abundant FA across lactation and in both groups. Preterm colostrum contained significantly (p < 0.05) higher 8:0, 10:0, 12:0, sum medium chain fatty acids (MCFA), 18:3 n3 FA compared to term counterparts. Preterm mature milk contained significantly higher 12:0, 14:0, 18:2 n-6, sum saturated fatty acids (SFA), and sum MCFA. We did not observe any significant differences between the preterm and term groups for docosahexaenoic acid, arachidonic acid and eicosapentaenoic acid at any stage of lactation. Overall, preterm milk was higher for SFA with a major contribution from MCFA and higher in 18:2 n-6. These observational differences needs to be studied further for their implications on preterm developmental outcomes and on fortification strategies of either mothers' own milk or donor human milk.

Longitudinal Evolution of True Protein, Amino Acids and Bioactive Proteins in Breast Milk: A Developmental Perspective

The protein content of breast milk provides a foundation for estimating protein requirements of infants. Because it serves as a guideline for regulatory agencies issuing regulations for infant formula composition, it is critical that information on the protein content of breast milk is reliable. We have therefore carried out a meta-analysis of the protein and amino acid contents of breast milk and how they evolve during lactation. As several bioactive proteins are not completely digested in the infant and therefore represent "non- utilizable" protein, we evaluated the quantity, mechanism of action and digestive fate of several major breast milk proteins. A better knowledge of the development of the protein contents of breast milk and to what extent protein utilization changes with age of the infant will help improve understanding of protein needs in infancy. It is also essential when designing the composition of infant formulas, particularly when the formula uses a "staging" approach in which the composition of the formula is modified in stages to reflect changes in breast milk and changing requirements as the infant ages.

Temporal Changes of Human Breast Milk Lipids of Chinese Mothers

Fatty acids (FA), phospholipids (PL), and gangliosides (GD) play a central role in infant growth, immune and inflammatory responses. The aim of this study was to determine FA, PL, and GD compositional changes in human milk (HM) during lactation in a large group of Chinese lactating mothers (540 volunteers) residing in Beijing, Guangzhou, and Suzhou. HM samples were collected after full expression from one breast and while the baby was fed on the other breast. FA were assessed by direct methylation followed by gas chromatography (GC) analysis. PL and GD were extracted using chloroform and methanol. A methodology employing liquid chromatography coupled with an evaporative light scattering detector (ELSD) and with time of flight (TOF) mass spectrometry was used to quantify PL and GD classes in HM, respectively. Saturated FA (SFA), monounsaturated FA (MUFA), and PL content decreased during lactation, while polyunsaturated FA (PUFA) and GD content increased. Among different cities, over the lactation time, HM from Beijing showed the highest SFA content, HM from Guangzhou the highest MUFA content and HM from Suzhou the highest n-3PUFA content. The highest total PL and GD contents were observed in HM from Suzhou. In order to investigate the influence of the diet on maternal milk composition, a careful analyses of dietary habits of these population needs to be performed in the future. 

Temporal Changes of Protein Composition in Breast Milk of Chinese Urban Mothers and Impact of Caesarean Section Delivery

Human breast milk (BM) protein composition may be impacted by lactation stage or factors related to geographical location. The present study aimed at assessing the temporal changes of BM major proteins over lactation stages and the impact of mode of delivery on immune factors, in a large cohort of urban mothers in China. 450 BM samples, collected in three Chinese cities, covering 8 months of lactation were analyzed for αlactalbumin, lactoferrin, serum albumin, total caseins, immunoglobulins (IgA, IgM and IgG) and transforming growth factor (TGF) β1 and β2 content by microfluidic chip- or ELISA-based quantitative methods. Concentrations and changes over lactation were aligned with previous reports. α-lactalbumin, lactoferrin, IgA, IgM and TGF-β1 contents followed similar variations characterized by highest concentrations in early lactation that rapidly decreased before remaining stable up to end of lactation. TGF-β2 content displayed same early dynamics before increasing again. Total caseins followed a different pattern, showing initial increase before decreasing back to starting values. Serum albumin and IgG levels appeared stable throughout lactation. In conclusion, BM content in major proteins of urban mothers in China was comparable with previous studies carried out in other parts of the world and C-section delivery had only very limited impact on BM immune factors. 

Microbiota in Breast Milk of Chinese Lactating Mothers

The microbiota of breast milk from Chinese lactating mothers at different stages of lactation was examined in the framework of a Maternal Infant Nutrition Growth (MING) study investigating the dietary habits and breast milk composition in Chinese urban mothers. We used microbiota profiling based on the sequencing of fragments of 16S rRNA gene and specific qPCR for bifidobacteria, lactobacilli and total bacteria to study microbiota of the entire breast milk collected using standard protocol without aseptic cleansing (n = 60), and the microbiota of the milk collected aseptically (n = 30). We have also investigated the impact of the delivery mode and the stage of lactation on the microbiota composition. The microbiota of breast milk was dominated by streptococci and staphylococci for both collection protocols and, in the case of standard collection protocol, Acinetobacter sp. While the predominance of streptococci and staphylococci was consistently reported previously for other populations, the abundance of Acinetobacter sp. was reported only once before in a study where milk collection was done without aseptic cleansing of the breast and rejection of foremilk. Higher bacterial counts were found in the milk collected using standard protocol. Bifidobacteria and lactobacilli were present in few samples with low abundance. We observed no effect of the stage of lactation or the delivery mode on microbiota composition. Methodological and geographical differences likely explain the variability in microbiota composition reported to date.

Protein Evolution of Human Milk 

Given the documented short- and long-term advantages of breastfeeding, human milk (HM) as a sole source of nutrition for the first few months of newborn life is considered a normative standard. Each macroconstituent of HM plays a crucial role in the growth and development of the baby. Lipids are largely responsible for providing more than 50% of the energy as well as providing essential fatty acids and minor lipids that are integral to all cell membranes. Carbohydrates can be broadly divided into lactose and oligosaccharides, which are a readily digestible source of glucose and indigestible nonnutritive components, respectively. Proteins in HM provide essential amino acids indispensable for the growth of infants. What is more interesting is that protein concentration profoundly changes from colostrum to mature milk. In this report, we share data from an observatory, single-center, longitudinal trial assessing the constituents of HM collected 30, 60 and 120 days postpartum from 50 mothers (singleton deliveries: 25 male and 25 female infants). The protein content decreased with evolving stages of lactation from an average of 1.45 to 1.38 g/100 ml. The data did not show any gender differences as it was reported for lipid content at 120 days postpartum by our group. Additionally, we also share consolidated literature data on protein evolution of HM during the first year of lactation.

Longitudinal Evolution of the Concentration of Gangliosides GM3 and GD3 in Human Milk

It has been reported that dietary gangliosides may have an important role in preventing infections and in brain development during early infancy. However, data related to the evolution of their concentration over the different stages of lactation are scarce. Liquid chromatography coupled with electrospray ionization high resolution mass spectrometer (LC/ESI-HR-MS) has been optimized to quantify the two major ganglioside classes, i.e., aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer (GD3) and aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer (GM3) in human milk. Gangliosides were extracted using chloroform and methanol, further purified by solid-phase extraction and separated by reversed-phase liquid chromatography. Repeatability, intermediate reproducibility, and recovery values were assessed to validate the method. In human milk, GD3 and GM3 could be quantified at the level of 0.1 and 0.2 μg/mL, respectively, with relative standard deviation of repeatability [CV(r)] and intermediate reproducibility [CV(iR)] values ranging from 1.9 to 15.0 % and 1.9 to 22.5 %, respectively. The described method was used to quantify GD3 and GM3 in human milk samples collected from 450 volunteers between 0 and 11 days and at 30, 60 and 120 days postpartum, providing for the first time the concentration of these minor lipids in a large cohort. The content of total gangliosides ranged from 8.1 and 10.7 μg/mL and the mean intake of gangliosides in infants 30, 60 and 120 days postpartum could be estimated at about 5.5, 7.0 and 8.6 mg of total gangliosides per day, respectively, when infants were exclusively breastfed.

Dynamics of Human Milk Nutrient Composition of Women from Singapore with A Special Focus on Lipids

BACKGROUND: A recent report suggested that human milk (HM) composition not only changes with lactation stages but also vary according to gender of the offspring. In spite of available literature, the dynamic changes of HM composition still remain to be completely explored and characterized. Progress in analytical technologies together with quantitative sampling of HM allows for a better quantification of HM nutrients and thereby providing a deeper understanding of the dynamics of HM secretion.

OBJECTIVE: To characterize and quantify HM nutrients based on appropriate for analyses sampling procedures and advanced analytical methodologies.

CLINICAL STUDY DESIGN: We conducted an observatory, single center, longitudinal trial with HM collection at 30, 60, and 120 days postpartum from 50 mothers (singleton-deliveries of 25 male and 25 female infants). HM samples were analyzed for lipid, lactose, energy density, fatty acids, phospholipids, and gangliosides. Longitudinal analyses of the datasets have been carried out using linear mixed models. RESULTS: HM for male infants compared to females at 120 days, were higher for energy content and lipids by 24 and 39%, respectively. Similarly, other bioactive lipids such as linoleic acid, phospholipids and gangliosides were also significantly different based on the gender of the infant. Significant stage-based differences were observed for total lipids, energy density, phospholipids, and gangliosides. Such difference in HM composition may stem from different energy needs to cope up for individual growth and development.

CONCLUSION: Collectively, the current observations affirm that HM secretion, especially the lipid composition, is a very dynamic and personalized biological process.

Human Milk Oligosaccharides

Impact of Maternal Characteristics on Human Milk Oligosaccharide Composition over the First 4 Months of Lactation in a Cohort of Healthy European Mothers

Human milk oligosaccharide (HMO) composition varies among lactating mothers and changes during the course of lactation period. Interindividual variation is largely driven by fucosyltransferase (FUT2 and FUT3) polymorphisms resulting in 4 distinct milk groups. Little is known regarding whether maternal physiological status contributes to HMO variability. We characterized the trajectories of 20 major HMOs and explored whether maternal pre-pregnancy body mass index (ppBMI), mode of delivery, or parity may affect milk HMO composition. Using longitudinal breastmilk samples from healthy mothers (n = 290) across 7 European countries, we characterized HMO composion and employed mixed linear models to explore associations of maternal characteristics with individual HMOs. We observed HMOspecific temporal trajectories and milk group dependencies. We observed relatively small but significant differences in HMO concentrations based on maternal ppBMI, mode of delivery and parity. Our findings suggest that HMO composition to be regulated time-dependently by an enzyme as well as substrate availability and that ppBMI, mode of delivery, and parity may influence maternal physiology to affect glycosylation marginally within the initital period of lactation. Our observational study is the largest European standardized and longitudinal (up to 4 months) milk collection study assessing HMO concentrations and basic maternal characteristics. Time of lactation and milk groups had the biggest impact on HMO variation. Future studies need to elucidate these observations and assess the physiological significance for the breastfed infant. 

Human Milk Oligosaccharides in the Milk of Mothers Delivering Term versus Preterm Infants

Human milk oligosaccharides (HMOs) are a major component of human milk, and play an important role in protecting the infant from infections. Preterm infants are particularly vulnerable, but have improved outcomes if fed with human milk. This study aimed to determine if the HMO composition of preterm milk differed from that of term milk at equivalent stage of lactation and equivalent postmenstrual age. In all, 22 HMOs were analyzed in 500 samples of milk from 25 mothers breastfeeding very preterm infants (< 32 weeks of gestational age, < 1500g of birthweight) and 28 mothers breastfeeding term infants. The concentrations of most HMOs were comparable at equivalent postpartum age. However, HMOs containing α-1,2-linked fucose were reduced in concentration in preterm milk during the first month of lactation. The concentrations of a number of sialylated oligosaccharides were also different in preterm milk, in particular 3'-sialyllactose concentrations were elevated. At equivalent postmenstrual age, the concentrations of a number of HMOs were significantly different in preterm compared to term milk. The largest differences manifest around 40 weeks of postmenstrual age, when the milk of term infants contains the highest concentrations of HMOs. The observed differences warrant further investigation in view of their potential clinical impact.

Human Milk Oligosaccharides: Factors Affecting Their Composition and Their Physiological Significance

Human milk oligosaccharides (HMOs) are elongations of the milk sugar lactose by galactose, Nacetylglucosamine, fucose; and sialic acid. The HMO composition of breast milk is strongly influenced by polymorphisms of the maternal fucosyltransferases, FUT2 and FUT3, and by the stage of lactation. Clinical observational studies with breastfed infant-mother dyads associate specific HMOs with infant gut microbiota, morbidity, infectious diarrhea, and allergies. Observational and basic research data suggest that HMOs influence the establishment of early-life microbiota and mucosal immunity and inhibit pathogens, thereby contributing to protection from infections. Clinical intervention trials with infant formula supplemented with the single HMO, 2'-fucosyllactose (2'FL), or with 2 HMOs, 2'FL and lacto-N-neotetraose (LNnT), demonstrated that they allow for ageappropriate growth and are well tolerated. A priori defined exploratory outcomes related feeding an infant formula with 2 HMOs to fewer reported illnesses of the lower respiratory tract and reduced need for antibiotics during the first year of life compared to feeding a control formula. In parallel, early-life microbiota composition shifted towards that of breastfed infants. Together, HMOs likely contribute to immune protection in part through their effect on early-life gut microbiota, findings that warrant further clinical research to improve our understanding of HMO biology and significance for infant nutrition.

Longitudinal Change of Selected Human Milk Oligosaccharides and Association to Infants' Growth, an Observatory, Single Center, Longitudinal Cohort Study

BACKGROUND: Human milk is the recommended and sole nutrient source for newborns. One of the largest components of human milk is oligosaccharides (HMOs) with major constituents determined by the mother genotype for the fucosyltransferase 2 (FUT2, secretor) gene. HMO variation has been related with infant microbiota establishment, diarrhea incidence, morbidity and mortality, IgE associated eczema and body composition.

OBJECTIVES: We investigated the (i) dependence of several major representative HMOs on the FUT2 status assessed through breast milk 2'Fucosyllactose (2'FL) and (ii) the relation of the 2'FL status with infant growth up to 4 months of life. DESIGN: From an open observatory, single center, longitudinal cohort study with quantitative human milk collection at 30, 60, and 120 days postpartum from 50 mothers, who gave birth to 25 female and 25 male singleton infants, we collected a representative sample of human milk. We quantified the following 5 representative HMOs: 2'FL, Lacto-N-tetraose (LNT), Lacto-N-neotetraose (LNnT), 3'Sialyllactose (3'SL) and 6'Sialyllactose (6'SL). We grouped the milk samples and corresponding infants according to the measured milk 2'FL concentrations at 30 days of lactation, which clustered around low concentrations (95% CI of mean 12-42 mg/L) and high concentrations (95% CI of mean 1880-2460 mg/L) with the former likely representing secretor negative mothers. Infant anthropometric measures were recorded at birth, 1, 2 and 4 months of age. Relations among the quantified HMOs and the relation of the high and low 2'FL HMOs groups with infant growth parameters were investigated via linear mixed models. RESULTS: The milk samples with low 2'FL concentration had higher LNT and lower LNnT concentrations compared to the samples with high 2'FL. The milk 3'- and 6'SL concentrations were independent of 2'FL. Over lactation time we observed a drop in the concentration of 2'FL, LNT, LNnT and 6'SL, especially from 1 to 2 months, while 3'SL remained at relatively constant concentration from 1 month onwards. Up to 4 months of age, we did not observe significant differences in body weight, body length, body mass index and head circumference of the infants who consumed breast milk with low or high FUT2 associated HMO concentrations and composition. CONCLUSIONS: Our findings on HMO concentrations over time of lactation and clusters based on 2'FL concentrations confirm previous observations and suggest that LNnT and LNT are 'co-regulated' with the FUT2 dependent 2'FL concentration, with LNnT showing a positive and LNT a negative relation. Further, our findings also suggest that the relatively substantial variation in HMOs between the high and low 2'FL clusters do not impact infant growth of either sex up to 4 months of age.

Temporal Change of the Content of 10 Oligosaccharides in the Milk of Chinese Urban Mothers

Breastfed infants tend to be less prone to infections and may have improved cognitive benefits compared to formula-fed infants. Human milk oligosaccharides (HMO) are the third most abundant component of human milk, but are absent from formulae. They may be partially responsible for the benefits of breastfeeding. In this cross-sectional observational study, the HMO composition of milk from Chinese mothers was studied to determine the impact of stage of lactation, mode of delivery and geographical location. The content of 10 HMO was measured by HPLC in 446 milk samples from mothers living in three different cities in China. Around 21% of the samples contained levels of 2'-fucosyllactose (2'-FL) below the limit of quantification, which is similar to the frequency of fucosyltransferase-2 non-secretors in other populations, but 2'-FL was detected in all samples. Levels of most of the HMO studied decreased during the course of lactation, but the level of 3-fucosyllactose increased. Levels of 2'-FL and 3-fucosyllactose seem to be strongly correlated, suggesting some sort of mechanism for co-regulation. Levels of 6'-sialyllactose were higher than those of 3'- sialyllactose at early stages of lactation, but beyond 2-4 months, 3'-sialyllactose was predominant. Neither mode of delivery nor geographical location had any impact on HMO composition.