BREASTFEEDING MEDICINE

Volume 15, Number 9, 2020 Clinical Research

ª Mary Ann Liebert, Inc.
DOI: 10.1089/bfm.2020.0195

Factors Affecting the Composition

of Expressed Fresh Human Milk

Quyen Pham,1 Pinkal Patel,1 Babak Baban,2 Jack Yu,3 and Jatinder Bhatia1

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
Downloaded by 201.230.169.118 from www.liebertpub.com at 12/12/20. For personal use only.

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.

Keywords: breast milk expression, breast milk handling, breast milk storage, HMO

Introduction functional state only during lactation.5 The first phase of

lactation, defined as secretory differentiation, starts a,bout 20

B reast milk is a highly variable and complex biologi-

cal fluid that contains more than 200 identified com-
ponents.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 (ca-
sein micelles), membranes, membrane-bound globules, and
live cells.3 Its constituents can be broadly divided into cate-
gories, 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
weeks of pregnancy. During this phase, the breast develops
the capacity to synthesize milk products, marked by matu-
ration of lactocytes. This phase requires the action of ma-
ternal progesterone, prolactin, and human placental lactogen.
The second phase, defined as secretory activation, is trig-
gered 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.
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 lacta-
tion. Macronutrient components of breast milk vary within
mothers and across lactation, but are conserved across pop-
ulations 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

1
Department of Pediatrics, Medical College of Georgia, Augusta University, Augusta, Georgia, USA.
2
Department of Oral Biology and Diagnostic Services, Dental College of Georgia, Augusta University, Augusta, Georgia, USA.
3
Department of Plastic and Reconstructive Surgery, Medical College of Georgia, Augusta University, Augusta, Georgia, USA.

551
 552 PHAM ET AL.

Table 1. Breast Milk Components

Macronutrients Micronutrients
Nonprotein
Carbohydrate Protein nitrogen Fat (emulsion) Vitamins Minerals
Lactose, Casein, whey, Urea, creatinine, Triacylglycerides, Pantothenic acid, Calcium,
oligosaccharides mucins, nucleotides, diacylglycerides, riboflavin, magnesium,
a-lectalbumin, free amino monoacylglycerides, thiamin, phosphorus,
lactoferrin, acids, peptides free fatty acids, ascorbic acid, electrolytes
secretory IgA, phospholipids, folate, vitamin (sodium,
lysozyme cholesterol B6, 12, K, D potassium,
and E, biotin, chloride), trace
niacin, retinol, metals (iron,
carotenoids zinc, copper,
manganese,
selenium, iodine,
fluoride)
Adapted from Kleinman and Greer.8
Downloaded by 201.230.169.118 from www.liebertpub.com at 12/12/20. For personal use only.

IgA, immunoglobulin A.

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 menstrua-
tion, 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 concentra-
tions 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 re-
moval of breast milk from a mother’s breast without an in-
fant’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 en-
gorgement, 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 con-
tains high amounts of secretory immunoglobulin A (IgA),
lactoferrin, leukocytes, as well as developmental factors such
as epidermal growth factor.14–16 Colostrum contains rela-
tively low lactose, indicating its primary function to be im-
munologic 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 concen-
trations 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 compo-
sition 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 co-
lostrum 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, compo-
sition 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 immu-
nity 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 pasteuriza-
tion on these cells and subsequent effects need further study.
Maternal genetic status. Genetic factors in lactating
women have been shown to influence breast milk composi-
tion, 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 larg-
est amount of carbohydrate after lactose in breast milk.
HMOs are indigestible by the gut, their primary function is
 COMPOSITION OF EXPRESSED FRESH HUMAN MILK 553

MUC1; MUC4
immunological (prebiotics), and they influence intestinal col-
onization. HMOs act as a prebiotic, provide a carbon source

Mucins
for commensal bacteria, such as Bifidobacteria spp., Bac-
teriodes spp., and Lactobacillus spp,25–27 which prevent col-
onization 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 oli-

Oligosacc and glycans

glycosaminoglycans
gosaccharides in human milk.29 The production of HMO is

HMOs, gangliosides,
genetically determined. Different HMO profiles occur as a
result of specific transferase enzymes expressed in lacto-
cytes.30 For example: human milk fucosylated oligosaccha-
ride 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
leptin, ghrelin

mothers (lack FUT2 enzyme) represent about 30% of women

Metabolic
hormones
Adiponectin,

worldwide. They produce milk lacking in alpha 1-2-fucosylated

oligosaccharides. Infants consuming the milk lacking these
Downloaded by 201.230.169.118 from www.liebertpub.com at 12/12/20. For personal use only.

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)
Table 2. Bioactive Factors in Human Milk

gene is codified by Lewis gene and allows classification of

somatostatins

Le+ and Le-. A nonsecretor mother secretes alpha 1, 3 fuco-

Hormones

sylated oligosaccharides in two to fivefold higher concentra-

Calcitonin;

tions 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 sig-
nificant differences in HMO profiles, and may protect their
VEGF, NGF, IGF,

infants against certain infections to a greater or lesser extent

depending on the presence of specific HMOs.25,34,37–39
Growth factors

erythropoeitin
IL-6, 7, 8, 10; IFN-c, G-CSF, MIF EGF; HB-EGF,

Infant factors. There is an association between infant

birth weight and volume of milk produced.
HMOs, human milk oligosaccharides; MIF, macrophage migratory inhibitory factor.

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.
Chemokines

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 pre-
TGF-ß, TNF-a

term than term milk.18

Cytokines

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 af-
fected by maternal diet, but increases with maternal BMI, and
IgG, IgM

decreases in mothers producing higher amount of milk.10 Fat

Macrophage; IgA/sIgA;

in human milk is a highly variable component. Maternal diet

Igs

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
stem cells

profile varies with maternal diet. Also, the nature of the fat
consumed by the mother influences the FA composition.
Cells

Dietary FAs are transferred rapidly to breast milk, and within

2–3 days, breast milk changes to mimic that of dietary fat.45
 554
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 Pana-
gos 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 polyunsatu-
rated 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
Downloaded by 201.230.169.118 from www.liebertpub.com at 12/12/20. For personal use only.
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. re-
ported 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 wo-
men 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 unaf-
fected 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 de-
fined as hindmilk. Hindmilk contains higher fat concentra-
tion, energy density, and concentrations of vitamins A and
E.55–58 There is not much difference in protein content be-
tween foremilk and hindmilk.59 Prematurity also affects
volume of the foremilk and hindmilk. Degree of prematurity
is significantly related to the relative proportion of fore-
milk/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
PHAM ET AL.
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 stim-
ulates 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 gesta-
tion). 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 breast-
feeding 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 al-
cohol 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 psycho-
active substance in the world. There is some evidence that
caffeine intake may reduce breast milk production.63 Caf-
feine may cause irritability and sleep disruption in breast-
feeding 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
 COMPOSITION OF EXPRESSED FRESH HUMAN MILK
breast milk displays circadian variation with higher concen-
tration 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 effec-
tive 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 simu-
lating 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
Downloaded by 201.230.169.118 from www.liebertpub.com at 12/12/20. For personal use only.
contamination negligible. There are several different con-
tainers available for breast milk storage such as, glass,
polyethylene, polypropylene, polycarbonate, polyether sul-
fone, 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. Poly-
propylene 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 proper-
ties of the milk.
Room temperature. Fresh expressed human milk can be
stored safely at room temperature (10–29C, 50–85F). The
optimal time for room temperature storage depends on
cleanliness of the expression technique and actual tempera-
ture. For example: warmer ambient temperatures are asso-
ciated with increased bacterial counts. Four hours may be a
reasonable limit to store fresh milk in room temperature
(*25C).82–84 Breast milk should not be stored at body
temperature, as a study has shown that there was a 40% in-
crease 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
555
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 (4C) 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 co-
lostrum, such as IgA, cytokines, and growth factors are not
diminished by refrigeration for 48 hours.88
Freezing. Freezing human milk (-4C to -20C) 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 ongo-
ing lipase activity.90 Lactoferrin levels and bioactivity are
significantly lower in human milk frozen at -20C for 3
months.91–93 Certain cytokines, IgA and growth factors from
colostrum are stable for at least 6 months at -20C.76,88
Frozen breast milk should be thawed before using. There are
several methods to thaw the breast milk:
o Place the container in the refrigerator
o Place in a container of warm or lukewarm water
o 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 40C). 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, how-
ever 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 nu-
trition 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, hu-
man breast milk plays a crucial role in establishing infantile
microbiome by providing both the microorganisms as well as
nutrition, thus a multidirectional and symbiotic relation-
ship between milk and microbiota is created, and leads
to improvement and maintenance of homeostasis and tis-
sue integrity.98 Hence, while milk is composed of cells,
nutrients, and proteins beneficial to neonates, it is un-
doubtedly important for milk to be handled and upheld
appropriately so that all components are functional, intact,
and viable.
Disclosure Statement
No competing financial interests exist.
 556
Funding Information
No funding to declare for this article.
References
1. Blanc B. Biochemical aspects of human milk—Comparison
with bovine milk. World Rev Nutr Diet 1981;36:1–89.
2. Breastfeeding and the use of human milk. Pediatrics 2012;
129:e827–e841.
3. Ruegg M, Blanc B. Structure and properties of the partic-
ulate constituents of human milk. A review. J Food Struct
1982;1:4.
4. Boss, M, Gardner H, Hartmann P. Normal Human Lacta-
tion: Closing the gap. F1000Res 2018;7:F1000 Faculty
Rev-801.
5. Hassiotou F, Geddes D. Anatomy of the human mammary
gland: Current status of knowledge. Clin Anat 2013;26:
29–48.
6. Kent JC. How breastfeeding works. J Midwifery Womens
Health 2007;52:564–570.
Downloaded by 201.230.169.118 from www.liebertpub.com at 12/12/20. For personal use only.
7. Dewey KG, Nommsen-Rivers LA, Heinig MJ, et al. Risk
factors for suboptimal infant breastfeeding behavior, de-
layed onset of lactation, and excess neonatal weight loss.
Pediatrics 2003;112(Pt 1):607–619.
8. Kleinman RE, Greer FR. Pediatric Nutrition, 7th ed. American
Academy of Pediatrics Committee on Nutrition, Elk Grove
Village, IL: American Academy of Pediatrics, 2013.
9. Prentice A. Regional variations in the composition of hu-
man milk. In: Handbook of Milk Composition, Jensen RG,
ed. San Diego, CA: Academic Press, Inc., 1995, p. 919.
10. Nommsen LA, Lovelady CA, Heinig MJ, et al. Determi-
nants 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 J, Fein S, Shealy K, et al. Prevalence of
breast milk expression and associated factors. Pediatrics
2008;122:S63.
12. Clemons SN, Amir LH. Breastfeeding women’s experience
of expressing: A descriptive study. J Hum Lact 2010;26:
258–265.
13. Jenness R. The composition of human milk. Semin Peri-
natol 1979;3:225–239.
14. Castellote C, Casillas R, Ramirez-Santana C, et al. Pre-
mature delivery influences the immunological composi-
tion of colostrum and transitional and mature human milk.
J Nutr 2011;141:1181–1187.
15. Pang WW, Hartmann PE. Initiation of human lactation:
Secretory differentiation and secretory activation. J Mam-
mary Gland Biol Neoplasia 2007;12:211–221.
16. Kulski JK, Hartmann PE. Changes in human milk com-
position during the initiation of lactation. Aust J Exp Biol
Med Sci 1981;59:101–114.
17. Koletzko B. Human milk lipids. Ann Nutr Metab 2016;
69(Suppl 2):28–40.
18. Bauer J, Gerss J. Longitudinal analysis of macronutrients
and minerals in human milk produced by mothers of pre-
term infants. Clin Nutr 2011;30:215–220.
19. Lonnerdal B. Nutritional and physiologic significance of
human milk proteins. Am J Clin Nutr 2003;77:1537S–1543S.
20. Newburg DS, Walker WA. Protection of the neonate by the
innate immune system of developing gut and of human
milk. Pediatr Res 2007;61:2–8.
21. Artis D, Spits H. The biology of innate lymphoid cells.
Nature 2015;517:293–301.
PHAM ET AL.
22. Baban B, Malik A, Bhatia J, et al. Presence and profile of
innate lymphoid cells in human breast milk. JAMA Pediatr
2018;172:594–596.
23. Yu JC, Khodadadi H, Malik A, 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 L. Human milk oligosaccharides: Every baby needs a
sugar mama. Glycobiology 2012;22:1147–1162.
26. Bashiardes S, Thaiss CA, Elinav E. It’s in the milk: Feeding
the microbiome to promote infant growth. Cell Metab
2016;23:393–394.
27. Timmis K, Jebok F. Microbiome yarns: Human milk oli-
gosaccharides, Bifidobacterium and immunopowergames.
Microb Biotechnol 2018;11:437–441.
28. Morrow AL, Ruiz-Palacios GM, Altaye M, 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 JB, Freeman SL, Lebrilla CB, 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 N, Kampmann B, Mehring Le-Doare K. Human
breast milk: A review on its composition and bioactivity.
Early Hum Dev 2015;91:629–635.
31. Davidson B, Meinzen-Derr JK, Wagner CL, et al. Fuco-
sylated oligosaccharides in human milk in relation to ges-
tational age and stage of lactation. Adv Exp Med Biol 2004;
554:427–430.
32. Smilowitz JT, Lebrilla CB, Mills DA, et al. Breast milk
oligosaccharides: Structure-function relationships in the
neonate. Annu Rev Nutr 2014;34:143–169.
33. Kunz C, Meyer C, Collado MC, et al. Influence of gesta-
tional age, secretor and Lewis blood group status on the
oligosaccharide content of human milk. J Pediatr Gastro-
enterol Nutr 2017;64:789–798.
34. Lewis ZT, Totten SM, Smilowitz JT, et al. Maternal fu-
cosyltransferase 2 status affects the gut bifidobacterial
communities of breastfed infants. Microbiome 2015;3:13.
35. Newburg DS, Ruiz-Palacios GM, Altaye M, et al. Innate
protection conferred by fucosylated oligosaccharides of
human milk against diarrhea in breastfed infants. Glyco-
biology 2004;14:253–263.
36. Pratico G, Capuani G, Tomassini A, et al. Exploring human
breast milk composition by NMR-based metabolomics. Nat
Prod Res 2014;28:95–101.
37. Fanos V. 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 F, Fanos V, Reali A. ‘‘Omics’’ in human co-
lostrum and mature milk: Looking to old data with new
eyes. Nutrients 2017;9:843.
39. Urbaniak C, McMillan A, Angelini M, et al. Effect of
chemotherapy on the microbiota and metabolome of human
milk, a case report. Microbiome 2014;2:24.
40. Prentice A. The effect of maternal parity on lactational
performance in a rural African community. In: Human
Lactation 2: Maternal and Environmental Factors, Hamosh
M, Goldman AS, eds. New York: Plenum Press, 1986,
pp. 165–173.
 COMPOSITION OF EXPRESSED FRESH HUMAN MILK
41. Dewey KG, Lönnerdal B. Infant self-regulation of breast
milk intake. Acta Paediatr Scand 1986;75:893–898.
42. Bravi F, Wiens F, Decarli A, et al. Impact of maternal
nutrition on breast-milk composition: A systemic review.
Am J Clin Nutr 2016;104:646–662.
43. Prentice A. Constituents of human milk. Food Nutr Bull
1996;17:305–312.
44. Keikha M, Bahreynian M, Saleki M, et al. Macro- and
micronutrients of human milk composition: Are they re-
lated to maternal diet? A comprehensive systematic review.
Breastfeed Med 2017;12:517–527.
45. Insull W, Jr., Hirsch J, James T, 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 SC, Kim SY, Sharma AJ, et al. Is obesity still in-
Downloaded by 201.230.169.118 from www.liebertpub.com at 12/12/20. For personal use only.
creasing among pregnant women? Prepregnancy obesity
trends in 20 states, 2003–2009. Prev Med 2013;56:372–
378.
48. Panagos PG, Vishwanathan R, Penfield-Cyr A, et al. Breast
milk from obese mothers has proinflammatory properties
and decreased neuroprotective factors. J Perinatol 2016;36:
284–290.
49. Hamosh M. Human milk in disease. Lipids 1992;27:848–
857.
50. de Carvalho M, Robertson S, Friedman A, et al. Effect of
frequent breastfeeding on early milk production and infant
weight gain. Pediatrics 1983;72:307–311.
51. de Carvalho M, Anderson DM, Giangreco A, et al. Fre-
quency of milk expression and milk production by mothers
of nonnursing premature neonates. Am J Dis Child 1985;
139:483–485.
52. Hopkinson JM, Schanler RJ, Garza C. Milk production by
mothers of premature infants. Pediatrics 1988;81:815–820.
53. Salariya EM, Easton PM, Cater JI. Human Milk in the
Modern World. Oxford: Oxford University Press, 1978.
54. Prentice AM, Lamb WH, Prentice A, et al. The effect of
water abstention on milk synthesis in lactating women. Clin
Sci 1984;66:291–298.
55. Valentine CJ, Nancy H, Richard S. Hindmilk improves
weight gain in low birth weight infants fed human milk.
J Pediatr Gastroenterol Nutr 1994;18:474–477.
56. Bishara R, Dunn MS, Merko SE, et al. Nutrient composi-
tion 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 MS, Merko SE, 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 AA, William O, Fidelia B. Hindmilk and weight
gain in preterm very low birthweight infants. Pediatr Int
2007;49:156–160.
59. Saarela T, Kokkonen J, Koivisto M. 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).
557
61. Hopkinson J, Schanler RJ, Fraley JK, 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 J. 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 M, Stanier JE. Dirunal variation in the fat content
of breast-milk. Lancet 1949;2:235–237.
66. Lubetzky R, Mimouni FB, Dollberg S, et al. Consistent
circadian variations in creamatocrit over the first 7 weeks of
lactation. Breastfeed Med 2007;2:15–18.
67. Cubero J, Valero V, Sanchez J, 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 RK, Nakra VK, Pandey HN, et al. Studies on cir-
cadian periodicity of plasma, breast milk and urinary cal-
cium in lactating Indian women. Trop Georg Med 1984;36:
345–349.
69. Pundir S, Wall CR, Mitchell CJ, et al. Variation of human
milk glucocorticoids over 24hrs period. J Mammary Gland
Biol Neoplasia 2017;22:85–92.
70. van der Voorn B, de Waard M, van Goudoever JB, 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 MV, Kirksey A. Variation in zinc, calcium, and
magnesium concentrations of human milk within a 24-hour
period from 1 to 6 months of lactation. J Pediatr Gastro-
enterol Nutr 1988;7:100–106.
72. Barkova EN, Nazarenko EV, Zhdanova EV. Diurnal vari-
ations in qualitative composition of breast milk in women
with iron deficiency. Bull Exp Biol Med 2005;140:394–396.
73. Sanchez CL, Cubero J, Sanchez J, et al. The possible role of
human milk nucleotides as sleep inducers. Nutr Neurosci
2009;12:2–8.
74. Illnerova H, Buresova M, Presl J. Melatonin rhythm in
human milk. J Clin Endocrinol Metab 1993;77:838–841.
75. Katzer D, Pauli L, Mueller A, et al. Melatonin concentra-
tions and antioxidative capacity of human breast milk ac-
cording to gestational age and the time of day. J Hum Lact
2016;32:NP105–NP110.
76. Chang YC, Chen CH, Lin MC. The macronutrients in hu-
man milk changes after storage in various containers. Pe-
diatr Neonatol 2012;53:205–209.
77. Goldblum RM, Garzaab C, Johnson CA, et al. Human milk
banking I. Effects of container upon immunologic factors in
mature milk. Nutr Res 1981;1:449–459.
78. Manohar A, Williamson M, Koppikar G. Effect of storage
of colostrum in various containers. Indian Pediatr 1997;34:
293–295.
79. Pittard WB, 3rd, Geddes K, Brown S, et al. Bacterial
contamination of human milk: Container type and method
of expression. Am J Perinatol 1991;8:25–27.
80. Price E, Weaver G, Hoffman P, et al. Decontamination of
breast pump milk collection kits and related items at home
and in hospital: Guidance from a Joint Working Group of
 558 PHAM ET AL.

the Healthcare Infection Society and Infection Prevention
Society. J Hosp Infect 2016;92:213–221.
81. Eglash A, Simon L; Academy of Breastfeeding Medicine.
ABM Clinical Protocol #8: Human milk storage infor-
mation for home use for full term infants, revised 2017.
Breastfeed Med 2017;12:390–395.
82. Eteng M, Ebong P, Eyong E, 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 M, Ellis L, Pollock D, et al. Breastfeeding and the
working mother: Effect of time and temperature of short-
term storage on proteolysis, lipolysis, and bacterial growth
in milk. Pediatrics 1996;97:492–498.
84. Nwankwo M, Offor E, Okolo A, et al. Bacterial growth
in expressed breast milk. Ann Trop Paediatr 1988;8:
92–95.
85. Penn AH, Altshuler AE, Small JW, et al. Digested formula
but not digested fresh human milk causes death of intestinal
cells in vitro: Implications for necrotizing enterocolitis.
Downloaded by 201.230.169.118 from www.liebertpub.com at 12/12/20. For personal use only.
92. Rollo DE, Radmacher PG, Turcu RM, et al. Stability of lac-
toferrin in stored human milk. J Perinatol 2014;34:284–286.
93. Takci S, Gulmez D, Yigit S, et al. Effects of freezing on
the bactericidal activity of human milk. J Pediatr Gastro-
enterol Nutr 2012;55:146–149.
94. Ovesen L, Jakobsen J, Leth T, et al. The effect of micro-
wave heating on vitamins B1 and E, and linoleic and li-
nolenic acids, and immunoglobulins in human milk. Int J
Food Sci Nutr 1996;47:427–436.
95. Quan R, Yang C, Rubinstein S, et al. Effects of microwave
radiation on anti-infective factors in human milk. Pedia-
trics 1992;89:667–669.
96. Sigman M, Burke K, Swarner O, et al. Effects of micro-
waving human milk: Changes in IgA content and bacterial
count. J Am Diet Assoc 1989;89:690–692.
97. Witkowska-Zimny M, Kaminska-El-Hassan E. Cells of
human breast milk. Cell Mol Biol Lett 2017;22:11.
98. Kaingade P, Somasundaram I, Nikam A, et al. Breast milk
cell components and its beneficial effects on neonates:
Need for breast milk cell banking. J Pediatr Neonatal In-

Pediatr Res 2012;72:560–567. divid Med 2017;6:e060115.

86. Goshal B, Lahiri S, Kar K, et al. Changes in biochemical
contents of expressed breast milk on refrigerator storage. Address correspondence to:
Indian Pediatr 2012;49:836–837. Pinkal Patel, MD
87. Bertino E, Giribaldi M, Baro C, et al. Effect of prolonged Department of Pediatrics
refrigeration on the lipid profile, lipase activity, and oxi- Medical College of Georgia
dative status of human milk. J Pediatr Gastroenterol Nutr Augusta University
2013;56:390–396. 1120 15th Street, BI-6033
88. Ramı́rez-Santana C, Pérez-Cano FJ, Audı́ C, et al. Effects Augusta, GA 30912
of cooling and freezing storage on the stability of bioac- USA
tive factors in human colostrum. J Dairy Sci 2012;95:2319–
2325. E-mail: pipatel@augusta.edu
89. Garcı́a-Lara NR, Escuder-Vieco D, Garcı́a-Algar O, et al.
Effect of freezing time on macronutrients and energy
content of breastmilk. Breastfeed Med 2012;7:295–301. Jatinder Bhatia, MD
90. Vázquez-Román S, Escuder-Vieco D, Garcı́a-Lara NR, Department of Pediatrics
et al. Impact of freezing time on dornic acidity in three Medical College of Georgia
types of milk: Raw donor milk, mother’s own milk, and Augusta University
pasteurized donor milk. Breastfeed Med 2016;11:91–93. Augusta, GA 30912
91. Raoof NA, Adamkin DH, Radmacher PG, et al. Compar- USA
ison of lactoferrin activity in fresh and stored human milk.
J Perinatol 2016;36:207–209. E-mail: jatindeb@augusta.edu