Human Milk and Lactation

Mammogenesis

The breast begins to develop in utero, undergoing the first of many developmental changes necessary for proper breastfeeding to occur. A bulb-shaped mammary bud can be discerned in the fetus at 18-19 weeks' gestation. Inside the bud, a rudimentary mammary ductal system is formed, which is present at birth. After birth, growth of the gland parallels that of the child until puberty. The normal anatomy of the mammary gland following pubertal development is shown in the images below.

Schematic diagram of the breast.

Frontal view of lactating breast.

The basic unit of the mammary gland is the alveolus or acinus cell that connects to a ductule. Each ductule was believed to independently drain to a duct that, in turn, emptied into lactiferous sinuses. These lactiferous sinuses drain to 15-25 openings in the nipple, allowing milk to flow to the recipient infant.

More recently, researchers such as Ramsay et al (2005) have questioned the existence of lactiferous sinuses. ^[5] Extensive real-time ultrasonography of 21 fully lactating women provided better understanding of the anatomy of the lactating human breast, as shown below.

On ultrasonography, milk ducts appear as superficial, hypoechoic tubular structures with echogenic walls whose milk-fat globules appear as echoes. The ducts are easily compressed, do not display typical sinuses, and anatomically appear to transport the milk rather than store it.

The ducts can be traced from the base of the nipple back into the parenchyma. The mean number of main ducts greater than 0.55 mm diameter at the base of the nipple was approximately 10 for the left breast and 9 for the right breast. Although the duct diameter was increased at multiple branch points, the typical saclike appearance of lactiferous sinuses under the areola was not observed during scanning. The mean number of ducts and the diameter of the main ducts were not related to nipple diameter, areola radius, or milk production for individual breasts. Adipose and glandular tissue distribution differed widely among women but not between breasts. In addition, the proportion of glandular and fat tissue and the number and size of ducts were not related to milk production.

At puberty, released estrogen stimulates breast tissue to enlarge through growth of mammary ducts into the preexisting mammary fat pad. Progesterone, secreted in the second half of the menstrual cycle, causes limited lobuloalveolar development. The effects of estrogen and progesterone facilitate the formation of the characteristic structure of the adult breast, which is the terminal duct lobular unit. However, full alveolar development and maturation of epithelium requires the hormones of pregnancy.

Lactogenesis

In lactogenesis, the mammary gland develops the capacity to secrete milk. Lactogenesis includes all processes necessary to transform the mammary gland from its undifferentiated state in early pregnancy to its fully differentiated state sometime after pregnancy. This fully differentiated state allows full lactation. The 2 stages of lactogenesis are discussed below.

Stage 1 occurs by mid pregnancy. In stage 1, the mammary gland becomes competent to secrete milk. Lactose, total protein, and immunoglobulin concentrations increase within the secreted glandular fluid, whereas sodium and chloride concentrations decrease. The gland is now sufficiently differentiated to secrete milk, as evidenced by the fact that women often describe drops of colostrum on their nipples in the second or third trimester. However, high circulating levels of progesterone and estrogen hold the secretion of milk in check.

Stage 2 of lactogenesis occurs around the time of delivery. It is defined as the onset of copious milk secretion. In stage 2, blood flow, oxygen, and glucose uptake increase, and citrate concentration increases sharply. Increased milk citrate is considered a reliable marker for the second stage of lactogenesis. Progesterone plays a key role in this stage. Removal of the placenta (ie, the source of progesterone during pregnancy) is necessary for the initiation of milk secretion; however, the placenta does not inhibit established lactation. Work by Haslam and Shyamala reveals that progesterone receptors are lost in lactating mammary tissues, thus decreasing the inhibitory effect of circulating progesterone. ^{[6, 7]} In addition, maternal secretion of insulin, growth hormone (GH), cortisol, and parathyroid hormone (PTH) facilitates the mobilization of nutrients and minerals that are required for lactation.

The stages of lactation can be summarized as follows (adapted from Riordan and Auerbach, 1998): ^[8]

• Mammogenesis: Mammary (breast) growth occurs. The size and weight of the breast increase.
• Lactogenesis
• Stage 1 (late pregnancy): Alveolar cells are differentiated from secretory cells.
• Stage 2 (day 2 or 3 to day 8 after birth): The tight junction in the alveolar cell closes. Copious milk secretion begins. Breasts are full and warm. Endocrine control switches to autocrine (supply-demand) control.
• Galactopoiesis (later than 9 d after birth to beginning of involution): Established secretion is maintained. Autocrine system control continues.
• Involution (average 40 d after last breastfeeding): Regular supplementation is added. Milk secretion decreases from the buildup of inhibiting peptides.

Lactation

See the list below:

• Two essential hormones (prolactin and oxytocin)
• During the second stage of lactogenesis, the breast becomes capable of milk production. For the ongoing synthesis and secretion of human milk, the mammary gland must receive hormonal signals. These signals, which are in direct response to stimulation of the nipple and areola (mammae), are then relayed to the central nervous system. This cyclical process of milk synthesis and secretion is termed lactation. Lactation occurs with the help of 2 hormones, prolactin (PRL) and oxytocin. Although PRL and oxytocin act independently on different cellular receptors, their combined actions are essential for successful lactation.
• Prolactin
• Milk synthesis occurs in the mammary gland epithelial cells in response to PRL activation of epithelial cell PRL receptors. PRL, a polypeptide hormone synthesized by lactotrophic cells in the anterior pituitary, is structurally similar to GH and placental lactogen (PL), which appear to have cytokine functions. The secretion of PRL appears to be both positively and negatively regulated; however, its main locus of control comes from hypothalamic inhibitory factors, the most important of which is dopamine, acting through the D2 subclass of dopamine receptors present in lactotrophs. PRL stimulates mammary glandular ductal growth and epithelial cell proliferation and induces milk protein synthesis.
• Research during the past several decades has led to a deeper understanding of PRL's role in the body. PRL-related knockout models support PRL's pivotal role in lactation and reproduction, which suggests that most of PRL's target tissues are modulated rather than dependent on PRL.
• The significance of PRL can be seen in the inhibition of lactogenesis using bromocriptine and other dopamine analogues, which are PRL inhibitors.
• Oxytocin
• The other important hormone involved in the milk ejection or letdown reflex is oxytocin. When the neonate is placed at the breast and begins suckling, oxytocin is released. The suckling infant stimulates the touch receptors that are densely located around the nipple and areola. The tactile sensations create impulses that, in turn, activate the dorsal root ganglia via the intercostals nerves (4, 5, 6). These impulses ascend the spinal cord, creating an afferent neuronal pathway to both the paraventricular nuclei of the hypothalamus where oxytocin is synthesized and secreted by the pituitary gland. The stimulation of the nuclei causes the release of oxytocin down the pituitary stalk and into the posterior pituitary gland, where oxytocin is stored.
• The infant's suckling creates afferent impulses that stimulate the posterior pituitary gland. This releases oxytocin in a pulsatile fashion to adjacent capillaries, traveling to the mammary myoepithelial cell receptors that, in turn, stimulate the cells to contract. Oxytocin causes the contraction of the myoepithelial cells that line the ducts of the breast. These smooth muscle–like cells, when stimulated, expel milk from alveoli into ducts and subareolar sinuses that empty through a nipple pore.
• Milk secretion directly correlates with synthesis
• The regulation of milk synthesis is quite efficient. Milk synthesis remains remarkably constant at approximately 800 mL/d. However, the actual volume of milk secreted may be adjusted to the requirement of the infant by feedback inhibitor of lactation, a local factor secreted into the milk; therefore, the rate of milk synthesis is related to the degree of breast emptiness or fullness. The emptier breast produces milk faster than the fuller one.
• Milk production is responsive to maternal states of well-being. Thus, stress and fatigue adversely affect a woman's milk supply. The mechanism for this effect is the down-regulation of milk synthesis with increased levels of dopamine, norepinephrine, or both, which inhibit PRL synthesis. Relaxation is key for successful lactation.

Biochemistry of human milk

Human milk is a unique, species-specific, complex nutritive fluid with immunologic and growth-promoting properties. This unique fluid actually evolves to meet the changing needs of the baby during growth and maturation. Milk synthesis and secretion by the mammary gland involve numerous cellular pathways and processes (summarized in the table below).

The pathways for milk secretion and synthesis by the mammary epithelial cell. I: Exocytosis of milk protein, lactose, and other components of the aqueous phase in Golgi-derived secretory vesicles. II: Milk fat secretion via the milk fat globule. III: Direct movement of monovalent ions, water, and glucose across the apical membrane of the cell. IV: Transcytosis of components of the interstitial space. V: The paracellular pathway for plasma components and leukocytes. Pathway V is open only during pregnancy, involution, and in inflammatory states such as mastitis. SV = Secretory vesicle; RER = Rough endoplasmic reticulum; BM = Basement membrane; MFG = Milk fat globule; CLD = Cytoplasmic lipid droplet; N = Nucleus; PC = Plasma cell; FDA = Fat-depleted adipocyte; TJ = Tight junction; GJ = Gap junction; D = Desmosome; ME = Myoepithelial cell.

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The processing and packaging of nutrients within human milk changes over time as the recipient infant matures. For example, early milk or colostrum has lower concentrations of fat than mature milk but higher concentrations of protein and minerals (see the image below). This relationship reverses as the infant matures.

Lactose, protein, and total lipid concentrations in human milk.

Important biochemical points are discussed below.

Fore and hind milk (important differences)

In addition to the changes from colostrum to mature milk that mirror the needs of the developing neonate, variation exists within a given breastfeeding session. The milk first ingested by the infant (fore milk) has a lower fat content. As the infant continues to breastfeed over the next several minutes, the fat content increases. This hind milk is thought to facilitate satiety in the infant. Finally, the diurnal variations in breast milk reflect maternal diet and daily hormonal fluctuations.

Specific enzymes to aid neonatal digestion

Human milk contains various enzymes; some are specific for the biosynthesis of milk in the mammary gland (eg, lactose synthetase, fatty acid synthetase, thioesterase), whereas others are specific for the digestion of proteins, fats, and carbohydrates that facilitate the infant's ability to break down food and to absorb human milk. Certain enzymes also serve as transport moieties for other substances, such as zinc, selenium, and magnesium.

Three-dimensional structure of human milk

Under a microscope, the appearance of human milk is truly amazing. Although it is a fluid, human milk has substantial structure in the form of compartmentation. Nutrients and bioactive substances are sequestered within the various compartments of human milk. The most elegant example of this structure involves lipids. Lipids are enveloped at the time of secretion from the apical mammary epithelial cell within its plasma membrane, becoming the milk-fat globule. Certain proteins, growth factors, and vitamins also become sequestered within this milk-fat globule and are embedded within the membrane itself.

The membrane acts as a stabilizing interface between the aqueous milk components and compartmentalized fat. This interface allows controlled release of the lipolysis products and transfer of polar materials into milk serum (aqueous phase). The bipolar characteristics of the membrane are also necessary for the emulsion stability of the globules themselves; thus, the structure of human milk provides readily available fatty acids and cholesterol for micellar absorption in the small intestine.

Proteins, carbohydrates, and designer fats for optimal brain development

Human milk provides appropriate amounts of proteins (primarily alpha-lactalbumin and whey), carbohydrates (lactose), minerals, vitamins, and fats for the growing term infant. The fats are composed of cholesterol, triglycerides, short-chain fatty acids, and long-chain polyunsaturated (LCP) fatty acids. The LCP fatty acids (18- to 22-carbon length) are needed for brain and retinal development. Large amounts of omega-6 and omega-3 LCP fatty acids, predominately the 20-carbon arachidonic acid (AA) and the 22-carbon docosahexaenoic acids (DHAs), are deposited in the developing brain and retina during prenatal and early postnatal growth.

An infant, particularly a preterm infant, may have a limited ability to synthesize optimal levels of AA and DHA from linoleic and linolenic acid. Therefore, these 2 fatty acids may be considered essential fatty acids. Many infant formulas in the United States have added AA, DHA, or both. The amount of AA and DHA in breast milk varies with the maternal diet. ^{[1, 9]} The unique blend of fatty acids in the breast milk has been linked to the development of innate and adaptive immune regulation.

Prior to routine fortification of formulas with DHA and AA, infants who received breast milk demonstrated better visual acuity at age 4 months than formula-fed infants, as well as slightly enhanced cognitive development. This has not been a universal finding, however, and some have continued to doubt the benefits of DHA and ARA.

However, in a study of children at age 5 years who were breastfed and whose mothers were given a modest DHA supplement until 4 months postpartum, there was a significant improvement in sustained attention when compared to children whose mothers were not given DHA. ^[10]

A study compared growth and bone mineralization in very low birth weight infants fed preterm formula with those who received term formula; the conclusion was that preterm formula better aided in growth and development. ^[11]

One study examined maternal dietary manipulation of fatty acid concentration and neurodevelopmental differences in human milk. ^[12] Despite higher levels of AA and DHA in the heavily supplemented maternal groups, no differences were observed in the neurodevelopmental outcomes of the 3 groups. This finding supports a more global effect of human milk as opposed to a single agent that renders developmental differences.

Thus, whether healthy term infants benefit from the addition of DHA and AA to formula remains unclear because they are able to convert very LCP fatty acids to DHA and AA. Ill term infants and those born prematurely are most likely to benefit from formulas enriched with DHA, AA, or both.

Rather than producing better vision or greater intelligence, breast milk may somehow protect the developing neonatal brain from injury or less optimal development by providing necessary building materials and growth factors that act synergistically rather than in isolation.

A study by Dallas et al indicated that milk produced by women who deliver preterm demonstrates a high level of protein breakdown by endogenous proteases, with the investigators suggesting that such breakdown may reduce difficulties associated with the immature digestive systems of preterm infants. The study, which looked at a total of 32 term and 28 preterm milk samples (from eight mothers and 14 mothers, respectively), found preterm milk to have a significantly higher peptide count than term milk. Cleavage-site analysis suggested that the protease plasmin is more active in preterm milk and that cytosol aminopeptidase and carboxypeptidase B2 also break down milk proteins. ^[13]

Immunologic properties of human milk

Through the years, knowledge about the immune properties and effects of human milk has grown. A recommended comprehensive review by one of the pioneers in the field, Dr. Armand Goldman, appeared in Breastfeeding Medicine (2007). ^[14] Below are the highlights of just some of many known immune properties and functions of human milk.

• Human milk immunoglobulins
• Human milk contains all of the different antibodies (M, A, D, G, E), but secretory immunoglobulin A (sIgA) is the most abundant. Milk-derived sIgA is a significant source of passively acquired immunity for the infant during the weeks before the endogenous production of sIgA occurs. During this time of reduced neonatal gut immune function, the infant has limited defense against ingested pathogens. Therefore, sIgA is an important protective factor against infection.
• Assuming that the mother and her infant, who are closely associated, share common flora, the antigenic specificity of the mother's sIgA in her milk is directed against the same antigens in the neonate. Maternal immunoglobulin A (IgA) antibodies derived from the gut and respiratory immune surveillance systems are transported via blood and lymphatic circulations to the mammary gland, ultimately to be extruded into her milk as sIgA. The packaging of IgA with a secretory component unique to the mammary gland protects the sIgA from stomach acids, allowing it to reach the small intestine intact.
• Other immunologic properties of human milk
• In addition to antibodies, human milk has numerous factors that can affect the intestinal microflora of the baby. These factors enhance the colonization of some bacteria while inhibiting the colonization by others. The immunologic components include lactoferrin, which binds to iron, thus making it unavailable to pathogenic bacteria; lysozyme, which enhances sIgA bactericidal activity against gram-negative organisms; oligosaccharides, which intercept bacteria and form harmless compounds that the baby excretes; milk lipids, which damage membranes of enveloped viruses; and mucins, which are present on the milk-fat globule membrane. Mucins adhere to bacteria and viruses and help eliminate them from the body. Interferon and fibronectin have antiviral activities and enhance lytic properties of milk leukocytes.
• Our understanding of the interactional effect of these bioactive constituents, the impact of microbiota on gut function, and development (and role of human milk in that development) is just beginning to be understood. ^{[15, 16]} These constituents clearly have profound effects of the health status of individuals throughout life, particularly during infancy.
• Human milk leukocytes
• Macrophages comprise 40-60% of the cells in colostrum, with the remainder of cells primarily consisting of lymphocytes and polymorphonucleocytes. Extruded into the milk are rare mammary epithelial cells and the plasma membrane-bound lipid droplets referred to as milk-fat globules. By 7-10 days postpartum, with the transition from colostrum to mature milk, the percentage of macrophages then increases to 80-90% at a concentration of 10⁴ -10⁵ human milk macrophages per milliliter of milk. Milk leukocytes can tolerate extremes in pH, temperature, and osmolality. They have been shown to survive for as long as a week in baboons and lambs.
• Passive immunity from mother to recipient breastfeeding infant
• While awaiting endogenous maturation of the baby's own immunologic systems, various immunologic and bioactive milk components act synergistically to provide a passive immunologic support system from the mother to her infant in the first days to months after birth. Ingested milk passively immunizes the neonate. Numerous studies have clearly documented this scenario and its clinical benefit, demonstrating decreased risk for gastrointestinal and respiratory infections, particularly during the first year of life.
• Evidence is increasing that these immune and bioactive substances prime the neonatal GI and immune systems in their selective recognition of antigens and development of cellular signaling. This may explain the decreased risk of intestinal and respiratory allergy in children who have been breastfed and the lower-than-predicted risk of autoimmune diseases in the breastfed population. Direct effects are difficult to prove given the multifactorial nature of such diseases; however, when taken together, the data support the beneficial nature of human milk for the developing infant.

Bioactive properties of human milk

Human milk also contains growth modulators, such as epidermal growth factor (EGF), nerve growth factor (NGF), insulinlike growth factors (IGFs), and interleukins. Transforming growth factor (TGF)–alpha, TGF-beta, and granulocyte colony-stimulating factor (G-CSF) are also identified in human milk. These growth modulators are produced either by the epithelial cells of the mammary gland or by activated macrophages, lymphocytes (mainly T cells), or neutrophils in the milk. EGF and TGF-alpha were found at higher concentrations in the milk of mothers who delivered prematurely compared with those who delivered at term. EGF, TGF-alpha, and human milk stimulate fetal small intestinal cell proliferation in vitro, with the greatest increase in cell proliferation seen following exposure to human milk.

Certain bioactive substances and live cells in milk appear to influence neonatal gut maturation and growth through their transfer of developmental information to the newborn. Although most of these biosubstances have been identified in mother's milk in quantities that exceed maternal serum levels, their exact role in human newborns is uncertain; most current information is from animal models whose development may significantly differ.

Conclusion

Human milk, in addition to its numerous nutrients that make it an ideal food source for the growing term infant, is a bioactive fluid that evolves from colostrum to mature milk as the infant matures. This bioactive fluid contains numerous factors and live cells that, in concert, promote the growth and well-being of the breastfeeding infant. Oliver Wendell Holmes said it best when he stated, "A pair of substantial mammary glands has the advantage over the two hemispheres of the most learned professor's brain, in the art of compounding a nutritious fluid for infants." With the ever-expanding knowledge resulting from current research, commercial formula clearly cannot replicate all of the valuable properties that are inherent in human milk.