Factors Affecting the Composition of Expressed Fresh Human Milk

Abstract

Breast milk is considered the ideal and preferred feeding for all infants through the first 4–6 months of life. It provides many short and long-term benefits to the infant and mother. In the absence of breastfeeding, expressed breast milk is the best way to provide nutrition. In the United States, the majority of breastfeeding mothers express milk at some point during the course of lactation. Breast milk is a dynamic fluid and its content changes with duration of lactation and varies between and among women. Many factors such as maternal diet and medications affect the constituents of breast milk. In addition, method of breast milk expression, handling, and storage can also influence its contents.

Introduction

Breast milk is a highly variable and complex biological fluid that contains more than 200 identified components.¹ Over time, as analytical techniques improved, the number of recognized components has increased. The American Academy of Pediatrics recommends breast milk as the only nutrient for healthy, term infants for approximately the first 6 months of life and supports continued breastfeeding for at least the first year of life.² Breast milk consists of several compartments, including true solutions, colloids (casein micelles), membranes, membrane-bound globules, and live cells.³ Its constituents can be broadly divided into categories, such as aqueous and lipid fractions or nutritive and nonnutritive constituents. The concentration of contents varies among women, stages of lactation, and over the course of the day. There are environmental and maternal factors that can affect human milk composition as well as how the milk is stored and handled.

Normal Lactation and Lactation Cycle

Normal human lactation should be comfortable for both the mother and infant and should be providing adequate milk for the infant's optimal growth and development.⁴ Lactation begins with conception and pregnancy induces changes in the mammary gland such as ductal proliferation and subsequent alveolar development. The mammary gland reaches a mature functional state only during lactation.⁵ The first phase of lactation, defined as secretory differentiation, starts a,bout 20 weeks of pregnancy. During this phase, the breast develops the capacity to synthesize milk products, marked by maturation of lactocytes. This phase requires the action of maternal progesterone, prolactin, and human placental lactogen. The second phase, defined as secretory activation, is triggered by delivery of the placenta and begins around 60 hours after birth (range of 24–72 hours).^6,7 This phase is marked by copious breast milk secretion. The initial product from the mammary gland following birth is colostrum, which is available to the infant for the first 60 hours (range of 24–72 hours).^6,7 Tables 1 and 2 list the various components of breast milk. The nutritional components of milk are derived from three sources, by maternal stores, maternal diet and synthesis in the lactocytes.

Table 1.

Breast Milk Components

Macronutrients				Micronutrients
Carbohydrate	Protein	Nonprotein nitrogen	Fat (emulsion)	Vitamins	Minerals
Lactose, oligosaccharides	Casein, whey, mucins, α-lectalbumin, lactoferrin, secretory IgA, lysozyme	Urea, creatinine, nucleotides, free amino acids, peptides	Triacylglycerides, diacylglycerides, monoacylglycerides, free fatty acids, phospholipids, cholesterol	Pantothenic acid, riboflavin, thiamin, ascorbic acid, folate, vitamin B6, 12, K, D and E, biotin, niacin, retinol, carotenoids	Calcium, magnesium, phosphorus, electrolytes (sodium, potassium, chloride), trace metals (iron, zinc, copper, manganese, selenium, iodine, fluoride)

Adapted from Kleinman and Greer.⁸

IgA, immunoglobulin A.

Table 2.

Bioactive Factors in Human Milk

Cells	Igs	Cytokines	Chemokines	Growth factors	Hormones	Metabolic hormones	Oligosacc and glycans	Mucins
Macrophage; stem cells	IgA/sIgA; IgG, IgM	IL-6, 7, 8, 10; IFN-γ, TGF-ß, TNF-α	G-CSF, MIF	EGF; HB-EGF, VEGF, NGF, IGF, erythropoeitin	Calcitonin; somatostatins	Adiponectin, leptin, ghrelin	HMOs, gangliosides, glycosaminoglycans	MUC1; MUC4

HMOs, human milk oligosaccharides; MIF, macrophage migratory inhibitory factor.

Breast Milk—A Dynamic Biological Fluid

The major changes of human milk content are observed in the first month of life, and then remain relatively stable, although subtle changes do occur over the course of lactation. Macronutrient components of breast milk vary within mothers and across lactation, but are conserved across populations despite variations in maternal nutritional status.⁹ Overall, the nutritional quality of breast milk is conserved but some vitamins (vitamin A, C, B-6, and B-12) and fatty acid (FA) composition depend on maternal diet. There are other factors besides maternal diet, which may influence breast milk content. Nommsen et al. found that after 4 months postpartum, the macronutrient concentrations of human milk are related to one or more of the following factors: maternal body mass index (BMI), protein intake, return of menstruation, and nursing frequency. They also found that mothers who produce higher quantities of milk tend to have lower milk concentrations of fat and protein but higher concentrations of lactose.¹⁰ Breast milk content is also influenced by milk expression and storage. For example, the fat content decreases following storage, freezing, and thawing process. Ideally, breast feeding is the optimal way to preserve all the components. However, if direct breastfeeding is not possible, the alternative is milk expression either manually or by a manual/electric pump. Milk expression is defined as the removal of breast milk from a mother's breast without an infant's mouth at her nipple. Survey data indicate that 85% of breastfeeding mothers express milk at some point before 4 months of age.¹¹ Reasons for milk expression by mothers include employed mothers returning to work, storing milk for unexpected maternal/infant separation, to relieve breast engorgement, or birth of a premature infant.^11,12

Factors known to influence human milk composition are discussed below.

Maternal factors

Stages of lactation

Human milk goes through three major phases of change: colostrum, transitional milk, and mature milk; however, these are not distinct classes of milk, but refer to the gradual alteration in the content of milk throughout the lactation.¹³ The first fluid produced by mother after delivery is colostrum. It is produced in small quantities and considered the “first vaccine” of the neonate and contains high amounts of secretory immunoglobulin A (IgA), lactoferrin, leukocytes, as well as developmental factors such as epidermal growth factor.^14–16 Colostrum contains relatively low lactose, indicating its primary function to be immunologic and gut priming (trophic) rather than nutritional. Transitional milk produced from 7 to 14 days postpartum is marked by accelerated milk production and is characterized by a decrease in immunoglobulin and total protein concentrations and an increase in lactose, fat, and total energy. Transitional milk supports the nutritional needs of the infant during the period of returning to birth weight. Breast milk after about 2 weeks of delivery is considered mature milk which supports the healthy term infant through the first 4–6 months of life. Usually, no dramatic changes in the composition of the mature milk occurs; however, subtle changes occur in the milk content over the course of lactation. Total protein and lipids show a gradual decline during the first 6 months of lactation, whereas lactose is initially low in colostrum and transitional milk, but then increases in mature milk and remains at the same level for up to 6 months.¹⁷ Protein levels decrease over the first 4–6 weeks regardless of timing of delivery.¹⁸ In addition to protein content, composition also changes throughout lactation. In early lactation, ratio of whey to casein is ∼80:20 while it changes to ∼50:50 in late lactation.¹⁹

Human milk contains 32 soluble factors and 5 cell types. The proinflammatory and anti-inflammatory milk cytokines continue to be described.²⁰ Innate lymphoid cells (ILCs), a new class of lineage negative lymphoid cells, appear to be important to the intestinal microbiome and adaptive immunity of the infant.²¹ ILCs are important in inflammation, immunity, and tissue homeostasis. In a recent study, the presence of ILCs in fresh human milk, with high ILC1s, followed by ILC3s and ILC2s were described.^22,23 These milk ILCs may impart innate immunity in newborns; the effect of milk expression, storage, handling, and pasteurization on these cells and subsequent effects need further study.

Maternal genetic status

Genetic factors in lactating women have been shown to influence breast milk composition, which explains the variations in its content among women. The factors such as hormones, which have influence on biosynthetic processes of the mammary gland can also modify the milk composition.²⁴ The prime example is human milk oligosaccharides (HMOs), which are the second largest amount of carbohydrate after lactose in breast milk. HMOs are indigestible by the gut, their primary function is immunological (prebiotics), and they influence intestinal colonization. HMOs act as a prebiotic, provide a carbon source for commensal bacteria, such as Bifidobacteria spp., Bacteriodes spp., and Lactobacillus spp,^25–27 which prevent colonization by pathogenic bacteria. For breast feeding infants, HMOs play an important role in preventing respiratory tract and diarrheal diseases.^20,28 There are over 200 different oligosaccharides in human milk.²⁹ The production of HMO is genetically determined. Different HMO profiles occur as a result of specific transferase enzymes expressed in lactocytes.³⁰ For example: human milk fucosylated oligosaccharide synthesis is controlled by the same fucosyltransferase genes (FUT2 and FUT3) that control secretor and Lewis blood group types.³¹ FUT2 (alpha-1-2 fucosyltransferase) gene is codified by secretor gene and allows classification of Se⁺ (secretor) and Se⁻ (nonsecretor) mothers. Nonsecretor mothers (lack FUT2 enzyme) represent about 30% of women worldwide. They produce milk lacking in alpha 1-2-fucosylated oligosaccharides. Infants consuming the milk lacking these compounds exhibit delayed colonization of bifidobacteria, higher abundance of streptococcus taxa, and are at higher risk of diarrheal diseases.^32–35 FUT3 (alpha-1-3-4-fucosyltransferase) gene is codified by Lewis gene and allows classification of Le⁺ and Le⁻. A nonsecretor mother secretes alpha 1, 3 fucosylated oligosaccharides in two to fivefold higher concentrations than secretor mothers.³⁶ Maternal phenotypes can be divided as Se⁺/Le⁺, Se⁺/Le⁻, Se⁻/Le⁺, and Se⁻/Le⁻. The breast milk from these different maternal genotypes has shown significant differences in HMO profiles, and may protect their infants against certain infections to a greater or lesser extent depending on the presence of specific HMOs.^{25,34,37–39}

Infant factors

There is an association between infant birth weight and volume of milk produced.

This association appears to be related to greater sucking strength, frequency, or feeding duration among larger infants, all of which could increase milk volume.^40,41 Milk content differs from mothers who deliver preterm than at term. Bauer and Gerss compared the composition of breast milk of mothers who delivered premature infants <28 weeks over the first 8 weeks of lactation with term milk. They found that the carbohydrate, fat, and energy contents were significantly higher in preterm milk than term. The protein content of both preterm and term milk decreased with duration of lactation demonstrating significantly higher values in extremely preterm than term milk.¹⁸

Maternal diet

Maternal diet has been shown to have little effect on total protein, carbohydrate, and certain minerals, but affects FAs, certain vitamins, zinc, calcium, selenium, iodine, and fluoride.^42–44 Breast milk protein concentration is not affected by maternal diet, but increases with maternal BMI, and decreases in mothers producing higher amount of milk.¹⁰ Fat in human milk is a highly variable component. Maternal diet intake acts as an important contributing factor for determining the variation in composition of polyunsaturated FAs in milk. Insull et al. showed that diet composition affects breast milk fat synthesis.⁴⁵ Particularly, long-chain polyunsaturated FAs profile varies with maternal diet. Also, the nature of the fat consumed by the mother influences the FA composition. Dietary FAs are transferred rapidly to breast milk, and within 2–3 days, breast milk changes to mimic that of dietary fat.⁴⁵

Besides fat content, the vitamin content of breast milk is influenced by the mother's vitamin status. If maternal vitamin intake is chronically low, its level in milk is also low.⁴⁶

Maternal health status

One of the common health problems today is obesity. The prepregnancy obesity rate has increased from 17.6% to 20.5% from 2003 to 2009.⁴⁷ Panagos et al. collected breast milk from lean (BMI 18–25 kg/m²) and obese (BMI >30 kg/m²) mothers between 6 and 10 weeks postpartum and found that the total protein, lactose, fat, and energy content of breast milk were similar between obese and lean mothers, but obese mothers had increased saturated FA, trans FA, and decreased monosaturated FA and polyunsaturated FA content compared with milk of lean mothers.⁴⁸ Maternal diabetes, cystic fibrosis, hypoproteinemia, allergic diseases, and type I hyperlipoproteinemia also affect fat composition of breast milk.⁴⁹

Milk volume

Studies conducted across the world indicate that the average milk production is ∼750 to 800 mL/day in women with widely varying dietary habits and with varying nutritional status.⁴⁶ During the early postpartum period, there is a positive association between nursing frequency and milk production when milk supply is being established.^50–53 Hopkinson et al. studied 32 mothers of preterm infants and found sufficient milk production was achieved when milk was pumped five or more times per day during the first month postpartum.⁵² The milk volume affects certain components of breast milk such as protein, and lactose. Nommsen et al. reported milk protein concentration was negatively related to milk volume at 6 and 9 months and positively related to nursing frequency at 6 months and ideal body weight at 9 months. They also found milk lactose concentration to be positively related to milk volume at 6 and 9 months.¹⁰ It is assumed that high fluid intake is needed to produce enough breast milk. However, evidence suggests that lactating women can tolerate a considerable amount of water restriction and supplemental fluid has little effect on milk volume. Prentice et al. found mother's milk volume remained unaffected despite losing 7.6% of their body water during fasting (from 5:00 am to 7:30 pm) over several days; however, changes in milk composition (lower lactose concentration, increased osmolality) indicated changes in mammary cell permeability.⁵⁴

Each milk expression

Composition of milk differs from the beginning to the end of a feeding or milk expression. At the initiation of the expression or feeding, the milk (let down) is defined as foremilk. The remainder of the milk obtained until complete breast emptying or feeding is finished is defined as hindmilk. Hindmilk contains higher fat concentration, energy density, and concentrations of vitamins A and E.^55–58 There is not much difference in protein content between foremilk and hindmilk.⁵⁹ Prematurity also affects volume of the foremilk and hindmilk. Degree of prematurity is significantly related to the relative proportion of foremilk/hindmilk volume. Foremilk volume increases while hindmilk decreases with the degree of prematurity.⁵⁷

Substance use

Maternal substances usage may affect both milk volume and its composition. Authors suggest readers should refer to LactMed.⁶⁰ Following are some of the commonly used substances and their effects on breast milk.

Smoking

Smoking may reduce milk volume through an inhibitory effect on prolactin. Prolactin is essential for normal lactogenesis and it promotes milk synthesis. Oxytocin stimulates contraction of the myoepithelial cells, which leads to milk ejection. Hopkinson et al. compared milk volume in mothers who smoked cigarettes versus those who did not after delivery of their preterm infants (28–32 weeks gestation). Milk production was significantly less among those who smoked, with or without adjusting for age, race, parity, gravidity, infant's birth weight, and pumping frequency. Total protein nitrogen, lactose, calcium, and phosphorus content did not differ in milks from mother who smoked versus who did not smoke, while fat concentrations were lower in the milk from mothers who smoked.⁶¹

Alcohol

The literature on alcohol usage during lactation and pregnancy is scarce. The potential effects of maternal alcohol ingestion on infants consuming breast milk depends on occasional use versus chronic abuse. Alcohol is excreted into breast milk in concentrations similar to those in maternal blood. The effect of occasional alcohol consumption on milk production is small, temporary, and unlikely to be of clinical relevance. The moderate alcohol consumption by a breastfeeding mother (up to 1 standard drink per day, defined as 12 ounces of 5% beer, 8 ounces of 7% malt liquor, 5 ounces 12% wine, or 1.5 ounces of 40% liquor) is not known to be harmful to the infant, especially if the mother waits at least 2 hours before nursing. Expressing or pumping milk after drinking alcohol, and then discarding it (“pumping and dumping”) does not reduce the amount of alcohol present in the milk. Breast milk continues to contain alcohol as long as alcohol is present in the mother's blood stream.⁶² Refraining from alcohol drinking during breastfeeding is the safest option (CDC), but if alcohol is used, lactating woman should limit their intake to no more than 0.5 g of alcohol/kg of body weight, as intake over this level may impair the milk ejection reflex.

Caffeine

Caffeine is the most widely consumed psychoactive substance in the world. There is some evidence that caffeine intake may reduce breast milk production.⁶³ Caffeine may cause irritability and sleep disruption in breastfeeding infants whose mothers consume 10 or more cups of coffee (∼1 g caffeine) per day, but findings are equivocal.⁶⁴

Maternal medication use

The detailed and up-to-date source of information regarding the safety of maternal medications when the mother is breastfeeding is LactMed.⁶⁰ It is an Internet-accessed source published by the National Library of Medicine/National Institutes of Health. Generally, breastfeeding/breast milk is not recommended when mothers are receiving medications from the following classes of drugs: amphetamines, chemotherapy agents, ergotamines, and statins. To cover the effects of all medications on breast milk is beyond the scope of this article.

Circadian variation

Breast milk is a dynamic biological fluid, contents of which not only change over the course of lactation but also over the course of a day. Lipid content of breast milk displays circadian variation with higher concentration found in evening samples.^65,66 Circadian variations in protein and carbohydrate content is less pronounced than fat, except higher levels of tryptophan are found in the evening sample.⁶⁷ Studies have shown diurnal variation in calcium, phosphorus, magnesium, iron, nucleotides, cortisol, and melatonin.^68–75

Other factors affecting breast milk

How the milk is expressed and collected

There are various methods of breast milk expression. The most effective and safe is breastfeeding by the infant. Hand expression is the other method to collect breast milk. The ideal artificial collection method is by an electric pump. An electric pump cycles the negative pressure with a rhythmic action simulating suckling, which would provide good fat content as compared with hand expression. The tubing and breast flange must be cleansed thoroughly (in dishwasher) to keep bacterial contamination negligible. There are several different containers available for breast milk storage such as, glass, polyethylene, polypropylene, polycarbonate, polyether sulfone, or bags.⁷⁶ The storage container can alter the cellular component of the milk, as the cells adhere to the walls of glass containers but not to polyethylene or polypropylene containers.⁷⁷ Water-soluble constituents and IgA remain stable in both glass and polypropylene containers. Polypropylene containers are easier to handle than glass.

Glass or polypropylene containers appear similar in their effects on adherence of lipid-soluble nutrients to the container surface, the concentration of IgA, and the number of viable white blood cells in the stored milk.⁷⁸

Polyethylene containers use cause a decrease of up to 60% in IgA and bactericidal effects of milk when compared with the use of Pyrex container (Pyrex is a type of tempered glass).

Storage containers should be thoroughly washed before use and breast pump kits must be completely dismantled and thoroughly rinsed and washed. They should always be air dried or dried with paper towels.^79,80 Chemical disinfectant is not ideal and if soap is not available, then boiling water is preferable.⁸¹

Temperature

Human milk is sensitive to the effect of temperature. Heating above the physiological temperature significantly impacts nutritional and immunological properties of the milk.

Room temperature

Fresh expressed human milk can be stored safely at room temperature (10–29°C, 50–85°F). The optimal time for room temperature storage depends on cleanliness of the expression technique and actual temperature. For example: warmer ambient temperatures are associated with increased bacterial counts. Four hours may be a reasonable limit to store fresh milk in room temperature (∼25°C).^82–84 Breast milk should not be stored at body temperature, as a study has shown that there was a 40% increase in proteolytic products above the baseline and 440–710% increase in free FA concentration than in expressed fresh milk.⁸³ This degree of free FA concentration is not negligible since free FAs are cytotoxic and may lead to cellular damage.⁸⁵

Refrigeration

Freshly expressed breast milk can be stored safely at refrigerator temperature (4°C) for up to 4 days without changing its integrity, such as pH, albumin, total protein, lactose, and lipid content.^81,86 Bertino et al. found that lipid composition and lipase activity remained stable up to 4 days in the refrigerator.⁸⁷ Immunological factors in colostrum, such as IgA, cytokines, and growth factors are not diminished by refrigeration for 48 hours.⁸⁸

Freezing

Freezing human milk (−4°C to −20°C) is safe for at least 3 months. Freezing human milk beyond 90 days has shown significant decreases in fat and energy content.⁸⁹ There is a significant increase in acidity of frozen human milk by 3 months mainly due to increase in free FAs from ongoing lipase activity.⁹⁰ Lactoferrin levels and bioactivity are significantly lower in human milk frozen at −20°C for 3 months.^91–93 Certain cytokines, IgA and growth factors from colostrum are stable for at least 6 months at −20°C.^76,88 Frozen breast milk should be thawed before using. There are several methods to thaw the breast milk:

○ Place the container in the refrigerator

○ Place in a container of warm or lukewarm water

○ Place the container under lukewarm running water.

Warming thawed breast milk to room temperature should be done over a period of 20 minutes in lukewarm water (at 40°C). Breast milk should not be thawed or heated in a microwave since uneven heating of the milk occurs and creates hot spots, which may not be safe for the baby.⁹⁴ Microwave effectively decreases bacteria in the milk, however it significantly decreases the activity of immunological factors.^95,96 CDC does not recommend thawing breast milk in microwave and recommends using breast milk within 24 hours of thawing in the refrigerator. Breast milk should be used within 2 hours if it is brought to room temperature or warmed after refrigeration or frozen.

Conclusions

Breast milk is a natural product, providing the basic nutrition and partial immunity for the newborn during several months of early life. Human breast milk is a complex and dynamic fluid with a heterogeneous matrix. In addition to nutrient factors, milk is composed of immune cells, stem cells, epithelial cells, and bacteria.⁹⁷ Most importantly, human breast milk plays a crucial role in establishing infantile microbiome by providing both the microorganisms as well as nutrition, thus a multidirectional and symbiotic relationship between milk and microbiota is created, and leads to improvement and maintenance of homeostasis and tissue integrity.⁹⁸ Hence, while milk is composed of cells, nutrients, and proteins beneficial to neonates, it is undoubtedly important for milk to be handled and upheld appropriately so that all components are functional, intact, and viable.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

No funding to declare for this article.

References

Blanc

Biochemical aspects of human milk—Comparison with bovine milk. World Rev Nutr Diet, 1981; 36:1–89.

Breastfeeding and the use of human milk. Pediatrics, 2012; 129:e827–e841.

Ruegg

, Blanc

. Structure and properties of the particulate constituents of human milk. A review. J Food Struct, 1982; 1:4.

Boss

, Gardner

, Hartmann

Normal Human Lactation: Closing the gap. F1000Res, 2018; 7:F1000 Faculty Rev-801.

Hassiotou

, Geddes

. Anatomy of the human mammary gland: Current status of knowledge. Clin Anat, 2013; 26:29–48.

Kent

JC.

How breastfeeding works. J Midwifery Womens Health, 2007; 52:564–570.

Dewey

, Nommsen-Rivers

, Heinig

, et al. Risk factors for suboptimal infant breastfeeding behavior, delayed onset of lactation, and excess neonatal weight loss. Pediatrics, 2003; 112(Pt 1):607–619.

Kleinman

, Greer

. Pediatric Nutrition, 7th ed. American Academy of Pediatrics Committee on Nutrition, Elk Grove Village, IL: American Academy of Pediatrics, 2013.

Prentice

Regional variations in the composition of human milk. In: Handbook of Milk Composition, Jensen

, ed. San Diego, CA: Academic Press, Inc., 1995, p. 919.

10.

Nommsen

, Lovelady

, Heinig

, et al. Determinants of energy, protein, lipid and lactose concentrations in human milk during the first 12 mo of lactation: The DARLING Study. Am J Clin Nutr, 1991; 53:457–465.

11.

Labinar-Wolfe

, Fein

, Shealy

, et al. Prevalence of breast milk expression and associated factors. Pediatrics, 2008; 122:S63.

12.

Clemons

, Amir

. Breastfeeding women's experience of expressing: A descriptive study. J Hum Lact, 2010; 26:258–265.

13.

Jenness

The composition of human milk. Semin Perinatol, 1979; 3:225–239.

14.

Castellote

, Casillas

, Ramirez-Santana

, et al. Premature delivery influences the immunological composition of colostrum and transitional and mature human milk. J Nutr, 2011; 141:1181–1187.

15.

Pang

, Hartmann

. Initiation of human lactation: Secretory differentiation and secretory activation. J Mammary Gland Biol Neoplasia, 2007; 12:211–221.

16.

Kulski

, Hartmann

. Changes in human milk composition during the initiation of lactation. Aust J Exp Biol Med Sci, 1981; 59:101–114.

17.

Koletzko

Human milk lipids. Ann Nutr Metab, 2016; 69(Suppl 2):28–40.

18.

Bauer

, Gerss

. Longitudinal analysis of macronutrients and minerals in human milk produced by mothers of preterm infants. Clin Nutr, 2011; 30:215–220.

19.

Lonnerdal

Nutritional and physiologic significance of human milk proteins. Am J Clin Nutr, 2003; 77:1537S–1543S.

20.

Newburg

, Walker

. Protection of the neonate by the innate immune system of developing gut and of human milk. Pediatr Res, 2007; 61:2–8.

21.

Artis

, Spits

. The biology of innate lymphoid cells. Nature, 2015; 517:293–301.

22.

Baban

, Malik

, Bhatia

, et al. Presence and profile of innate lymphoid cells in human breast milk. JAMA Pediatr, 2018; 172:594–596.

23.

, Khodadadi

, Malik

, et al. Innate immunity of neonates and infants. Front Immunol, 2018; 9:1759.

24.

Institute of Medicine (US). Milk volume. In: Nutrition During Lactation. Washington, DC: National Academies Press, 1991, pp. 80–112.

25.

Bode

Human milk oligosaccharides: Every baby needs a sugar mama. Glycobiology, 2012; 22:1147–1162.

26.

Bashiardes

, Thaiss

, Elinav

. It's in the milk: Feeding the microbiome to promote infant growth. Cell Metab, 2016; 23:393–394.

27.

Timmis

, Jebok

. Microbiome yarns: Human milk oligosaccharides, Bifidobacterium and immunopowergames. Microb Biotechnol, 2018; 11:437–441.

28.

Morrow

, Ruiz-Palacios

, Altaye

, et al. Human milk oligosaccherides blood group epitopes and innate immune protection against campylobacter and calcivirus diarrhea in breastfed infants. Adv Exp Med Biol, 2004; 554:443–446.

29.

German

, Freeman

, Lebrilla

, et al. Human milk oligosaccharides: Evolution, structures and bioselectivity as substrates for intestinal bacteria. Nestle Nutr Workshop Ser Pediatr Program, 2008; 62:205–222.

30.

Andreas

, Kampmann

, Mehring Le-Doare

. Human breast milk: A review on its composition and bioactivity. Early Hum Dev, 2015; 91:629–635.

31.

Davidson

, Meinzen-Derr

, Wagner

, et al. Fucosylated oligosaccharides in human milk in relation to gestational age and stage of lactation. Adv Exp Med Biol, 2004; 554:427–430.

32.

Smilowitz

, Lebrilla

, Mills

, et al. Breast milk oligosaccharides: Structure-function relationships in the neonate. Annu Rev Nutr, 2014; 34:143–169.

33.

Kunz

, Meyer

, Collado

, et al. Influence of gestational age, secretor and Lewis blood group status on the oligosaccharide content of human milk. J Pediatr Gastroenterol Nutr, 2017; 64:789–798.

34.

Lewis

, Totten

, Smilowitz

, et al. Maternal fucosyltransferase 2 status affects the gut bifidobacterial communities of breastfed infants. Microbiome, 2015; 3:13.

35.

Newburg

, Ruiz-Palacios

, Altaye

, et al. Innate protection conferred by fucosylated oligosaccharides of human milk against diarrhea in breastfed infants. Glycobiology, 2004; 14:253–263.

36.

Pratico

, Capuani

, Tomassini

, et al. Exploring human breast milk composition by NMR-based metabolomics. Nat Prod Res, 2014; 28:95–101.

37.

Fanos

Metabolomics, milk-oriented microbiota (MOM) and multipotent stem cells: The future of research on breast milk. J Pediatr Neonat Individual Med, 2015; 4:e040115.

38.

Bardanzellu

, Fanos

, Reali

. “Omics” in human colostrum and mature milk: Looking to old data with new eyes. Nutrients, 2017; 9:843.

39.

Urbaniak

, McMillan

, Angelini

, et al. Effect of chemotherapy on the microbiota and metabolome of human milk, a case report. Microbiome, 2014; 2:24.

40.

Prentice

The effect of maternal parity on lactational performance in a rural African community. In: Human Lactation 2: Maternal and Environmental Factors, Hamosh

, Goldman

, eds. New York: Plenum Press, 1986, pp. 165–173.

41.

Dewey

, Lönnerdal

. Infant self-regulation of breast milk intake. Acta Paediatr Scand, 1986; 75:893–898.

42.

Bravi

, Wiens

, Decarli

, et al. Impact of maternal nutrition on breast-milk composition: A systemic review. Am J Clin Nutr, 2016; 104:646–662.

43.

Prentice

Constituents of human milk. Food Nutr Bull, 1996; 17:305–312.

44.

Keikha

, Bahreynian

, Saleki

, et al. Macro- and micronutrients of human milk composition: Are they related to maternal diet? A comprehensive systematic review. Breastfeed Med, 2017; 12:517–527.

45.

Insull

Jr. , Hirsch

, James

, et al. The fatty acids of human milk. II. Alterations produced by manipulation of caloric balance and exchange of dietary fats. J Clin Invest, 1959; 38:443–450.

46.

Institute of Medicine Committee on Nutritional Status During Pregnancy and Lactation. Milk composition. In: Nutrition During Lactation. Washington, DC: National Academies Press, 1991, pp. 113–152.

47.

Fisher

, Kim

, Sharma

, et al. Is obesity still increasing among pregnant women? Prepregnancy obesity trends in 20 states, 2003–2009. Prev Med, 2013; 56:372–378.

48.

Panagos

, Vishwanathan

, Penfield-Cyr

, et al. Breast milk from obese mothers has proinflammatory properties and decreased neuroprotective factors. J Perinatol, 2016; 36:284–290.

49.

Hamosh

Human milk in disease. Lipids, 1992; 27:848–857.

50.

de Carvalho

, Robertson

, Friedman

, et al. Effect of frequent breastfeeding on early milk production and infant weight gain. Pediatrics, 1983; 72:307–311.

51.

de Carvalho

, Anderson

, Giangreco

, et al. Frequency of milk expression and milk production by mothers of nonnursing premature neonates. Am J Dis Child, 1985; 139:483–485.

52.

Hopkinson

, Schanler

, Garza

. Milk production by mothers of premature infants. Pediatrics, 1988; 81:815–820.

53.

Salariya

, Easton

, Cater

. Human Milk in the Modern World. Oxford: Oxford University Press, 1978.

54.

Prentice

, Lamb

, Prentice

, et al. The effect of water abstention on milk synthesis in lactating women. Clin Sci, 1984; 66:291–298.

55.

Valentine

, Nancy

, Richard

. Hindmilk improves weight gain in low birth weight infants fed human milk. J Pediatr Gastroenterol Nutr, 1994; 18:474–477.

56.

Bishara

, Dunn

, Merko

, et al. Nutrient composition of hindmik produced by mothers of very low birth weight infants born less than 28 weeks gestation. J Hum Lact, 2008; 24:159–167.

57.

Bishara, Dunn

, Merko

, et al. Volume of foremilk, hindmilk, and total milk produced by mothers of very preterm infants born at less than 28 weeks gestation. J Hum Lact, 2009; 25:272–279.

58.

Ogechi

, William

, Fidelia

. Hindmilk and weight gain in preterm very low birthweight infants. Pediatr Int, 2007; 49:156–160.

59.

Saarela

, Kokkonen

, Koivisto

. Macronutrient and energy contents of human milk fractions during the first six months of lactation. Acta Pediatr, 2005; 94:1176–1181.

60.

Drugs and Lactation Database (LactMed). National Library of Medicine (NJM). September 16, 2016. Available at https://toxnet.nlm.nih.gov/newtoxnet/lactmed.htm (accessed September 12, 2018).

61.

Hopkinson

, Schanler

, Fraley

, et al. Milk production by mothers of premature infants: Influence of cigarette smoking. Pediatrics, 1992; 90:934–938.

62.

Centers of Disease Control and Prevention. Fetal alcohol spectrum disorders (FASDs). Centers of Disease Control and Prevention, March 27, 2018. Available at www.cdc.gov/ncbddd/fasd/alcohol-use.html (accessed September 12, 2018).

63.

Liston

Breastfeeding and the use of recreational drugs—Alcohol, caffeine, nicotine and marijuana. Breastfeed Rev, 1998; 6:27–30.

64.

Ryu

JE.

Caffeine in human milk and in serum of breast-fed infants. Dev Pharmacol Ther, 1985; 8:329–337.

65.

Gunther

, Stanier

. Dirunal variation in the fat content of breast-milk. Lancet, 1949; 2:235–237.

66.

Lubetzky

, Mimouni

, Dollberg

, et al. Consistent circadian variations in creamatocrit over the first 7 weeks of lactation. Breastfeed Med, 2007; 2:15–18.

67.

Cubero

, Valero

, Sanchez

, et al. The circadian rhythm of tryptophan in breast milk affects the rhythms of 6-sulfamethoxymelatonin and sleep in the newborn. Neuro Endocrinol Lett, 2005; 26:657–661.

68.

Singh

, Nakra

, Pandey

, et al. Studies on circadian periodicity of plasma, breast milk and urinary calcium in lactating Indian women. Trop Georg Med, 1984; 36:345–349.

69.

Pundir

, Wall

, Mitchell

, et al. Variation of human milk glucocorticoids over 24hrs period. J Mammary Gland Biol Neoplasia, 2017; 22:85–92.

70.

van der Voorn

, de Waard

, van Goudoever

, et al. Breast-milk cortisol and cortisone concentrations follow the diurnal rhythm of maternal hypothalamus-pituitary-adrenal axis activity. J Nutr, 2016; 146:2174–2179.

71.

Karra

, Kirksey

. Variation in zinc, calcium, and magnesium concentrations of human milk within a 24-hour period from 1 to 6 months of lactation. J Pediatr Gastroenterol Nutr, 1988; 7:100–106.

72.

Barkova

, Nazarenko

, Zhdanova

. Diurnal variations in qualitative composition of breast milk in women with iron deficiency. Bull Exp Biol Med, 2005; 140:394–396.

73.

Sanchez

, Cubero

, Sanchez

, et al. The possible role of human milk nucleotides as sleep inducers. Nutr Neurosci, 2009; 12:2–8.

74.

Illnerova

, Buresova

, Presl

. Melatonin rhythm in human milk. J Clin Endocrinol Metab, 1993; 77:838–841.

75.

Katzer

, Pauli

, Mueller

, et al. Melatonin concentrations and antioxidative capacity of human breast milk according to gestational age and the time of day. J Hum Lact, 2016; 32:NP105–NP110.

76.

Chang

, Chen

, Lin

. The macronutrients in human milk changes after storage in various containers. Pediatr Neonatol, 2012; 53:205–209.

77.

Goldblum

, Garzaab

, Johnson

, et al. Human milk banking I. Effects of container upon immunologic factors in mature milk. Nutr Res, 1981; 1:449–459.

78.

Manohar

, Williamson

, Koppikar

. Effect of storage of colostrum in various containers. Indian Pediatr, 1997; 34:293–295.

79.

Pittard

, 3rd, Geddes

, Brown

, et al. Bacterial contamination of human milk: Container type and method of expression. Am J Perinatol, 1991; 8:25–27.

80.

Price

, Weaver

, Hoffman

, et al. Decontamination of breast pump milk collection kits and related items at home and in hospital: Guidance from a Joint Working Group of the Healthcare Infection Society and Infection Prevention Society. J Hosp Infect, 2016; 92:213–221.

81.

Eglash

, Simon L; Academy of Breastfeeding

Medicine

. ABM Clinical Protocol #8: Human milk storage information for home use for full term infants, revised 2017. Breastfeed Med, 2017; 12:390–395.

82.

Eteng

, Ebong

, Eyong

, et al. Storage beyond three hours at ambient temperature alters the biochemical and nutritional qualities of breastmilk. Afr J Reprod Health, 2001; 5:130–134.

83.

Hamosh

, Ellis

, Pollock

, et al. Breastfeeding and the working mother: Effect of time and temperature of shortterm storage on proteolysis, lipolysis, and bacterial growth in milk. Pediatrics, 1996; 97:492–498.

84.

Nwankwo

, Offor

, Okolo

, et al. Bacterial growth in expressed breast milk. Ann Trop Paediatr, 1988; 8:92–95.

85.

Penn

, Altshuler

, Small

, et al. Digested formula but not digested fresh human milk causes death of intestinal cells in vitro: Implications for necrotizing enterocolitis. Pediatr Res, 2012; 72:560–567.

86.

Goshal

, Lahiri

, Kar

, et al. Changes in biochemical contents of expressed breast milk on refrigerator storage. Indian Pediatr, 2012; 49:836–837.

87.

Bertino

, Giribaldi

, Baro

, et al. Effect of prolonged refrigeration on the lipid profile, lipase activity, and oxidative status of human milk. J Pediatr Gastroenterol Nutr, 2013; 56:390–396.

88.

Ramírez-Santana

, Pérez-Cano

, Audí

, et al. Effects of cooling and freezing storage on the stability of bioactive factors in human colostrum. J Dairy Sci, 2012; 95:2319–2325.

89.

García-Lara

, Escuder-Vieco

, García-Algar

, et al. Effect of freezing time on macronutrients and energy content of breastmilk. Breastfeed Med, 2012; 7:295–301.

90.

Vázquez-Román

, Escuder-Vieco

, García-Lara

, et al. Impact of freezing time on dornic acidity in three types of milk: Raw donor milk, mother's own milk, and pasteurized donor milk. Breastfeed Med, 2016; 11:91–93.

91.

Raoof

, Adamkin

, Radmacher

, et al. Comparison of lactoferrin activity in fresh and stored human milk. J Perinatol, 2016; 36:207–209.

92.

Rollo

, Radmacher

, Turcu

, et al. Stability of lactoferrin in stored human milk. J Perinatol, 2014; 34:284–286.

93.

Takci

, Gulmez

, Yigit

, et al. Effects of freezing on the bactericidal activity of human milk. J Pediatr Gastroenterol Nutr, 2012; 55:146–149.

94.

Ovesen

, Jakobsen

, Leth

, et al. The effect of microwave heating on vitamins B1 and E, and linoleic and linolenic acids, and immunoglobulins in human milk. Int J Food Sci Nutr, 1996; 47:427–436.

95.

Quan

, Yang

, Rubinstein

, et al. Effects of microwave radiation on anti-infective factors in human milk. Pediatrics, 1992; 89:667–669.

96.

Sigman

, Burke

, Swarner

, et al. Effects of microwaving human milk: Changes in IgA content and bacterial count. J Am Diet Assoc, 1989; 89:690–692.

97.

Witkowska-Zimny

, Kaminska-El-Hassan

. Cells of human breast milk. Cell Mol Biol Lett, 2017; 22:11.

98.

Kaingade

, Somasundaram

, Nikam

, et al. Breast milk cell components and its beneficial effects on neonates: Need for breast milk cell banking. J Pediatr Neonatal Individ Med, 2017; 6:e060115.