Lactation in the human

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In this article, the development of the mammary gland in the human fetus is described followed by a comparison of the situation and anatomy of the mammary glands in the human with the glands in rodents and dairy animals. The hormones that bring about further development at puberty and in pregnancy are described along with the changes in the ductal structure of the gland. We consider the changes in milk composition that take place during secretory activation, the onset of milk secretion that occurs postpartum in women. The structure of milk is outlined emphasizing the various milk components obtained by differential centrifugation including whey, milk fat globules, casein micelles, extracellular vesicles, milk cells, and bacteria. Finally, we consider the minimal data addressing breast involution after weaning in the human. In a study of fetal mammary glands of 72 human fetuses, less than 2 mo gestational age, Gusterson and colleagues (Anbazhagan et al., 1991) found the ductal system was variably developed but contained both luminal and basal epithelial cells. The fetal glands showed a chronological developmental pattern with secretory-type changes likely brought about by maternal lactogenic hormones accessing the fetus. There was evidence of milk secretion soon after birth. Involutional changes occurred by the time the infant was 2 mo of age leaving a rudimentary nipple and duct system similar in both males and females. More detailed recent studies (Gusterson and Stein, 2012) show that breast development starts about the fifth week of intrauterine life with appearance of a 2- to 4-cell-thick “milk streak” in the abdominal ectoderm extending from the chest to the groin. The thoracic area in this streak begins to thicken in the sixth or seventh week of gestation and the rest of the streak disappears. Next indentations begin to appear in this thickened region followed by the down-growth of epithelial tubes that become mammary ducts with a lumen. They appear to be lined by cuboidal epithelial cells and surrounded by mesenchymal cells (Figure 1). By the second trimester, a layer of myoepithelial cells can be discerned below the epithelial layer. In addition, adipose tissue begins to accumulate around the ductal structures. At birth, the breast tissue is similar in males and females and continues this way until puberty when hormones in the female bring about differentiation. Embryonic mammary bud in a 16-wk male fetus. The epithelial cells in the lumen of the bud (L) are surrounded by a condensed layer of mesenchymal cells (M) (Howard and Gusterson, 2000). Used by permission. The molecular underpinnings of these developmental stages are known primarily from studies in mice (Hens and Wysolmerski, 2005) and include signaling by members of both the wingless-related integration site (Wnt) and fibroblast growth factor (FGF) families. In the milk streak these appear to interact with T-box family transcription factors TBX2 and TBX3. As the steak develops into placodes the members of these family appear to be joined by ectodysplasin (Eda), a member of the tumor necrosis factor ligand family. Eda is present in the mesenchymal compartment of the mouse placode and its receptor Edar is found in the epithelium. As the placode develops into the milk bud, a large number of additional proteins including insulin-like growth factor 1 (IGF-1), hedgehog (Hh), parathyroid hormone-related protein (PTHrP), neuregulin (NRG3), and their associated receptors appear to join the Wnt and FGF families in specifying developmental processes (Biswas et al., 2022). Limited information about these pathways and factors is available for the human mammary gland, which is difficult to study because of the relative unavailability of tissues. Two or three years prior to the menarche, very low levels of circulating estrogen from the ovaries stimulate ductal development, acting on estrogen receptor alpha (ERα) in mammary epithelial cells. Insulin-like growth factor-1 (IGF-1), secreted by the liver or locally by stromal cells under the direction of growth hormone (GH), may foster branching of the ductal system (Macias and Hinck, 2012). As puberty progresses, small protrusions branch off the primary ducts and expand into lobular structures called terminal duct lobular units (TDLU) that become more complex as time goes by (Figure 2). These complex ductile units extend to the periphery of the gland by the end of puberty. While progesterone is added to the plasma hormones during the luteal phase of the menstrual cycle, there is conflicting evidence as to whether it stimulates TDLU growth (Lee and Kelleher, 2016). In pig mammary glands, which have significant similarity to the human mammary gland, progesterone has no effect on proliferation in the presence of estrogen and prolactin; in the absence of these hormones, it actually inhibits proliferation (Horigan et al., 2009). In the mouse, progesterone receptor knockout models show that progesterone is essential for side branching at puberty and alveolar development during pregnancy (Ismail et al., 2003; Neville et al., 2023). Whole mount preparation of the human breast from a 15-yr-old human female. Note the ducts with lobular structures branching from their sides and termini. Copious adipose tissue is generally present as well. A terminal duct lobular unit (TDLU) is indicated by the green circle. From Howard and Gusterson (2000). Used by permission. In early pregnancy, secretion of estrogen and progesterone from the ovary is stimulated by chorionic gonadotropin secreted by trophoblasts of the developing placenta. By the second trimester, estrogen and placental lactogen are secreted by the syncytiotrophoblasts in the developing placenta. These hormones along with placental lactogen stimulate growth and differentiation of milk secreting structures (Lee and Kelleher, 2016). The epithelial bilayer structure is maintained throughout development including both the luminal cells, which will become the milk secreting cells, and the basal cells, which will become the myoepithelium responsible for milk ejection. The syncytiotrophoblasts in the placenta also secrete human placental lactogen, a GH-like molecule that promotes secretory differentiation probably along with prolactin (PRL) from the pituitary whose secretion increases throughout pregnancy. PRL secretion is maintained at high levels in the early period after birth. While a colostral-like secretion can be detected in the alveolar lumens, full secretion of milk is inhibited by the high concentration of progesterone present in late pregnancy (Kuhn, 1969). The anatomy of the primate breast has been well-documented in macaques and appears to be similar to the anatomy of the human breast (Cline, 2007). It differs from that of dairy animals where ducts from the milk-secreting alveoli converge on an udder which can have 2 to 4 teats (Cunningham et al., 2005) and from rodents in which multiple ducts converge below the nipple (Cole, 1933). In mice, the ducts develop lobular structures only in pregnancy, one of the many differences between the mouse and human mammary gland. The porcine mammary gland appears to be a good model for the development of the human mammary gland (Horigan et al., 2009). In humans and primates, in general, multiple ductile structures terminate on the nipple as shown in Figure 3A, a famous early image of the structure of the lactating breast obtained by A.P. Copper, a surgeon in London who injected colored wax into each of the orifices terminating on the nipple (Cooper, 1840). After dissection, it could be seen that each of the ducts extends far into the breast terminating in alveoli (Figure 3B), where the milk is synthesized and held until suckling brings about oxytocin secretion. Oxytocin, in turn, causes contraction of the myoepithelial cells surrounding the alveoli and ejection of milk. Figure 3B comes from the same analysis by A.P. Cooper and magnifies the expanded alveolar structures of the lactating breast. The lactating human breast. A. The ductile system of the lactating human breast. The breast of a fully lactating woman was injected with wax through the mammary ducts at the nipple by a 19th century surgeon, A.P. Cooper. B. Magnified structure of the human breast. Images by A.P. Cooper, published 1840 (Cooper, 1840). Milk is synthesized by the luminal alveolar cells and stored in the acini shown in Figure 3B. Milk synthesis and secretion can be altered by changes in the placental factors that govern mammary development, retained placental fragments after birth of the infant, and obesity, all of which may hinder mammary development into a fully secreting gland (Neville et al., 2023). Copious milk production is initiated in women in the first few days after birth during secretory activation brought about by a fall in progesterone and maintained prolactin (PRL; Neville et al., 2001). In dairy and other mammals, the fall in plasma progesterone occurs prior to parturition initiating lactation prior to birth. In women, however, the fall in progesterone takes place over 2 to 3 d postpartum so that small amounts of colostrum are secreted during the first few days postpartum followed by secretion of transitional milk. The first changes in composition of the breast secretion after birth are a fall in sodium and chloride and a rise in lactose concentrations reflecting closure of the tight junctions between the luminal epithelial cells (Figure 4, top). Milk volume rises more slowly representing activation of the metabolic pathways that underlie synthesis the major components of milk including casein. Concomitant with the rise in milk volume there is a fall in the concentrations of two of the major protective components of human milk, lactoferrin and secretory IgA (sIgA; Figure 4, bottom). Under normal circumstances milk volume plateaus between 500 and 600 ml/day by day 6 postpartum and increases to 700–800 ml/day by the first month, a rate that is maintained up to 6 mo of exclusive breastfeeding (Cox et al., 1996). These milk volumes appear to be characteristic of healthy breastfeeding parents throughout the world. Secretory activation in women. A. Changes in milk volume, lactose, sodium, and chloride concentrations in the first 8 days after birth. B. Changes in concentrations of lactoferrin and sIgA. Inset: changes in the rate of secretion of these two milk proteins. From Neville M.C. et al. (2001), used by permission. The volume of the mammary secretion in the first 2 d after parturition (colostrum) is low, ~100 ml/day (Figure 4top). It increases rapidly up to day 4 and is classified as colostrum up to day 6. Transitional milk is often designated as milk secreted from day 10 to 14 and mature milk from day 15 on (Nyquist et al., 2022). Because humans have transplacental immunity, meaning that immunoglobulins are transmitted to the fetus across the placenta, unlike ungulate species like cattle, human colostrum is low in IgG and IgM (Bigler et al., 2022). It is, however, high in IgA which is thought to protect the gastrointestinal system from unwanted pathogens. Human colostrum also contains high levels of the protective proteins lactoferrin and lysozyme as well as larger concentrations of fat-soluble vitamins (A, E, and K) than term milk. Whether the small volume of colostrum in the first 2 d postpartum is important to the infant or whether it is mainly protective of the nonsuckled breast is a question that has not been seriously addressed. However, suckling by the infant shortly after birth results in the secretion of oxytocin, which may help parental-infant bonding by interacting with the pleasure receptors in the hypothalamus (see below; Quintana et al., 2019). High levels of PRL are present in the plasma of women at birth averaging between 100 and 300 µg/L (Figure 5). Suckling induces a significant rise in plasma PRL (Cox et al., 1996). However, plasma PRL falls with duration of breastfeeding. For example, at 6 mo in the same women basal prolactin declined about 60 µg/L rising to 90 µg/L after suckling. However, the rate of milk production did not change significantly. A predominant role of prolactin in the maintenance of human lactation has been called into question by studies showing only small effects of galactagogues such as domperidone on milk volume secretion (Grzeskowiak et al., 2019). Plasma prolactin levels before (solid bars) and after (open bars) feeding as well as milk volume production (filled circles) in 11 fully breastfeeding Australian women. From Cox et al. (1996), used by permission. Suckling or other infant signals such as crying lead to oxytocin secretion by the posterior pituitary. This oxytocin travels through the blood stream to the alveoli in the breast, interacting with the myoepithelial cells whose subsequent contraction leads to milk ejection. The oxytocin also migrates to the hypothalamus where it apparently stimulates the pleasure reward centers helping bring about maternal–infant bonding (Quintana et al., 2019). The major corticosteroid in human plasma is cortisol. The hormone has a natural diurnal rhythm and is elevated in response to stress. Cortisol appears to be necessary to establish the functionality of human mammary cells in culture. It is thought to be necessary for secretory activation although there is data from human hair levels of cortisol indicating that stress levels of cortisol are associated with delayed secretory activation (Caparros-Gonzalez et al., 2019). The level of cortisol during lactation has been shown to be associated with changes in the lipid composition of milk (Linderborg et al., 2020; Ziomkiewicz et al., 2021). This hormone has been shown to be important for mammary differentiation in the pregnant mouse (Neville et al., 2013). Its role in mammary differentiation in humans has not been elucidated. However, recent studies have shown that insulin plays an essential role in secretory differentiation, secretory activation, and mature milk production. There may also be an association between glucose intolerance and lactation problems in women. Milk is a complex ecosystem with many different components that benefit the infant in a myriad of different ways. The “whey” fraction obtained after centrifugation to remove precipitable components (see below) contains soluble proteins as well as the major carbohydrate fractions consisting of lactose (~71 g/L) and a large number of oligosaccharides (~12 g/L; Newburg and Neubauer, 1995) with diverse structures. Structural components of human milk separated by various centrifugation methods include milk fat globules, casein micelles, milk cells, exosomes, and bacteria often referred to as microbiota. We will start by discussing nutrients present in whey. Lactose and oligosaccharides are the most predominant nutrients in human milk, lactose being present at about 75 g/L compared to cow’s milk, which has only about 50 g/L (Ventrella et al., 2021). Oligosaccharides are present at about 15 g/L in mature milk and can have up to 150 different structures with about 20 structures accounting for about 90% of the total oligosaccharides (Smilowitz et al., 2023). Lactose is easily digested in the infant gut by lactase whereas oligosaccharides are poorly digested by the infant gut enzymes but are often a source of calories for the microbial population. Because oligosaccharides can attach to some microbial recognition sites on enterocytes, they also provide protection against bacterial infection. In addition, many of the milk proteins including lactoferrin, lysozyme and sIgA are heavily glycosylated with the glycosylation patterns differing over the course of lactation. The most abundant soluble or whey proteins in human milk are alpha-lactalbumin (~3.3 g/L), lactoferrin (~2.0 g/L), sIgA (~1.0 g/L), lysozyme (29 to 96 µg/ml; Jaeser et al., 2022), bile salt stimulated lipase, and serum albumin (0.4 g/L). There are many others most of whose functions in milk are not currently clear (Smilowitz et al., 2023). α-lactalbumin is a component of lactose synthase, the enzyme which combines glucose and galactose to make lactose. It is the most abundant whey protein and is present in the Golgi compartment of the secreting mammary alveolar cell where it interacts with the major disaccharide synthesizing enzyme, galactosyl transferase, permitting this enzyme to utilize glucose as a substrate. Lactoferrin, present in high concentrations in colostrum (Figure 4), is a glycoprotein with significant antibacterial, antiviral, and even antitumor effects. It binds two ferric ions with high affinity, to its effects by the many bacteria to A of lactoferrin, called can be from both and human lactoferrin and may have significant in the infant gut as well as in the mammary gland. is present in human but not in cow’s milk. It has been shown to the bacterial composition of porcine milk to a more healthy et al., 2013). salt stimulated is a glycoprotein that is very in the found in milk after they the and The serum albumin in human milk is from the plasma by across the luminal milk secreting Its concentration is about of its serum concentration and its in milk is not at this Human milk also contains a large of growth and many of which may have functions in the infant, but are to (Smilowitz et al., 2023). A and protein in milk is casein. are into a structure called the casein These complex structures are to in in human milk and up to 600 in milk. In human milk, there are two of and milk has called of which there are two and is and about the molecular of human casein is to its glycosylation in the and The casein binds about of the lysozyme in human milk et al., however, the of the lysozyme was not different from that of the The in milk are contained in structures called milk fat they a surrounded by a up of and milk fat proteins. The milk fat a large of to the infant and the lipid is on lactation and of soluble signaling like as well as and are also present in the milk fat that to and their to receptors on the (see oligosaccharides The milk fat also contains a of as the is being secreted from the alveolar this is a source of that can into the molecular of human milk secretion et al., 2013). A recent study has that human milk fat different proteins compared to milk in which different proteins et al., has that human milk contains a and of (Smilowitz et al., 2023). The causes of the microbial may include maternal time feeding and many Milk bacteria may help to the It was thought that most cells in human milk cells. However, recent detailed studies of the of cells by centrifugation et al., et al., have shown that the of cells in healthy human milk are the milk secreting cells. of cells have also been with about of the cell population. of these cell in various associated with milk secretion have the to provide into the molecular of milk secretion in the human. Because of human breast tissue has been difficult in the into the of human milk secretion are likely in the from studies of milk cells. in human milk include three to 150 in to in and et al., 2022), by the of of small which bud off the into many small The with the of the luminal epithelial cells their to be into the milk from of the the into the milk to in are from cells. The include not only from luminal epithelial cells but from the cells in milk. It is that small extracellular from cells other than milk cells may across the mammary into milk. They may be in the milk or be to milk cells or milk fat The of are from the of the secreting cells and include and which may to the They have a composition which may be altered by and evidence that gut and et al., 2022). They can across the infant gut and many developmental processes in the such as the of cells and other developmental They may The composition of is by maternal obesity, birth, and a postpartum between and has been described in in the mouse mammary because breast are in the human during involution many studies However, showing changes in by et al. on cells from human milk obtained and 14 d after of breast feeding et al., 2016). The cell present to cells from colostrum in early about as and cells. and with transcription factors during involution in the mouse gland including and as well as and These that involution is after of milk in women. The of the human mammary gland is very different from that of which have been the source of of about the of milk secretion. studies of the cells in human milk have shown that the large of cells in human milk are that many of milk secreting cells and an for the study of the molecular of human lactation. Human breast milk has very different from most available including milk from and other dairy animals. In human milk is in protective proteins including lactoferrin, and lysozyme as well as a of oligosaccharides which may bacterial in the infant Neville has been on the of the mammary gland the and is an in this published first on in the mammary gland, in and has over 100 and in the the on in and the at that in was a of the of and a that is currently in its of was the on the of the from its in until the over to in from as of and and of and in In continues and in the of mammary gland and In of the in for on factors that human mammary development, milk and milk of