Physiology of Lactation

Six Factors of Mammary Organization

Box 3.2 ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ presents six well-documented factors that help explain the organization of mammary growth. Much of this work has resulted since the availability of “knockout” studies in mice and associated techniques.

The coordination of epithelial and stromal activity in the mammary gland is complex. Hepatocyte growth and scatter factor has been associated with the process during puberty. ²⁰ Another growth factor, heregulin, a member of the epidermal growth factor (EGF) family, has been identified in the stroma of mammary ducts during pregnancy.

Neville ¹⁵ has diagrammed the regulation of mammary development ( Fig. 3.2 ). She notes that the concentrations of estrogen, progesterone, and lactogenic hormone in the form of prolactin or placental lactogen (PL) greatly increase, enhance alveolar development, and result in the differentiation of alveolar cells. Although many investigators have contributed pieces to the puzzle of mammogenesis, Neville succeeded in creating the current visualization. ¹⁵

Prepubertal Growth

Mammogenesis occurs in two phases as the gland responds to the hormones of puberty and later of pregnancy. ³ During the prepubertal phase, the primary and secondary ducts that develop in the fetus in utero continue to grow in both boys and girls in proportion to growth in general. Shortly before puberty, a more rapid expansion of the duct system begins in girls. The growth of the duct system seems to depend predominantly on estrogen and does not occur in the absence of ovaries. The complete growth of the alveoli requires stimulation by progesterone as well.

Studies of hypophysectomized animals have shown the failure of full mammary growth even with adequate estrogen and progesterone. The secretion of prolactin and somatotropin by the pituitary gland results in mammary growth. Adrenocorticotropic hormone (ACTH) and thyroid-stimulating hormone (TSH) acting on the adrenal and thyroid glands also play a minor role in the growth of the mammary gland.

Growth and development during organogenesis involve the interaction of cells with extracellular matrices and neighboring cells. ²¹ Necropsy breast specimens from six male and eight female infants ranging in age from 1 day to 9 months were studied to determine the process of organogenesis in humans. ²² Integrins were expressed in a pattern that correlates with morphologic and functional differentiation of the normal mammary gland. Integrins are transmembrane glycoproteins that form receptors for extracellular matrix proteins, such as fibronectin, laminin, and collagen. Integrins are widely expressed in normal tissue and are considered critical to the control of cell growth and differentiation. This suggests integrin’s involvement in the functional characterization of the adhesion molecules in the breast.

Pubertal Growth

When the hypophyseal–ovarian–uterine cycle is established, a new phase of mammary growth, which includes extensive branching of the system of ducts and proliferation and canalization of the lobuloalveolar units at the distal tips of the branches, begins. ²¹ The organization of the stromal connective tissue forms the interlobular septa. The ducts, ductules (terminal intralobular ducts), and alveolar structures are formed by double layers of cells. One layer, the epithelial cells, circumscribes the lumen. The second layer, the myoepithelial cells, surrounds the inner epithelial cells and is bordered by a basement lamina.

Menstrual Cycle Growth

The cyclic changes of the adult mammary gland can be associated with the menstrual cycle and the hormonal changes that control that cycle. Estrogens stimulate parenchymal proliferation, with the formation of epithelial sprouts. This hyperplasia continues into the secretory phase of the cycle. Anatomically, when the corpus luteum provides increased amounts of estrogens and progesterone, there is lobular edema, thickening of the epithelial basal membrane, and secretory material in the alveolar lumen. ²¹ Lymphoid and plasma cells infiltrate the stroma. Clinically, mammary blood flow increases in this luteal phase. This increased flow is experienced by women as fullness, heaviness, and turgescence. The breast may become nodular because of interlobular edema and ductular-acinar growth.

After the onset of menstruation and the reduction of sex steroid levels, milk-secretory prolactin action is limited. Postmenstrual changes occur rapidly, with degeneration of glandular cells and proliferation tissue, loss of edema, and a decrease in breast size. The ovulatory cycle actually enhances mammary growth in the early years of menstruation (until about age 30 to 35 years) because the postmenstrual regression of the glandular-alveolar growth after each cycle is not complete. ²¹ These changes of ductal and lobular proliferation, which occur during the follicular phase before ovulation, continue in the luteal phase and regress after the menstrual phase, exemplifying the sensitivity of this target organ to variations in the balance of hormones.

Fowler et al. ²³ measured cyclic changes in the composition and volume of the breast during the menstrual cycle using nuclear magnetic resonance T1-weighted imaging. The T1 relaxation time (spin-lattice T1 relaxation) is a measure of the rate of energy loss from tissues after T1 excitation. This energy loss depends on the biophysical environment of the excited protons. A short T1, therefore, indicates the presence of lipids and organic structures that bind water tightly. A longer T1 occurs with greater hydration and with the greatest amount of cellular water. This study revealed that the lowest total breast volume and parenchymal volume of T1 and water content occurred between days 6 and 15 of the cycle. Between days 16 and 28, T1 rose sharply, and it peaked on the 25th day. The rise in parenchymal volume in the second half of the cycle resulted from not only increased tissue water but also from growth and increased tissue fluid, according to Fowler et al. ²³

Growth During Pregnancy

Hormonal influences on the breast cause profound changes during pregnancy ( Figs. 3.3 and 3.4 ). Early in pregnancy, a marked increase in ductular sprouting, branching, and lobular formation is evoked by luteal and placental hormones. ²¹ PL, prolactin, and chorionic gonadotropin have been identified as contributors to the accelerated growth (see Fig. 3.4 ). The dichorionic ductular sprouting has been attributed to estrogen, and lobular formation to progesterone.

Prolactin is essential for complete lobular-alveolar development of the gland. Growth of the mammary lobular-alveolar system requires the “lactogenic hormone complex” of estrogen, progesterone, prolactin, and certain metabolic hormones. ²⁴ Prolactin, as with other protein hormones, exerts its effect through receptors for the initiation of milk secretion located on the alveolar cell surfaces. The induction of milk synthesis requires insulin-induced cell division and the presence of cortisol.

From the third month of gestation, secretory material that resembles colostrum appears in the acini. Prolactin from the anterior pituitary gland stimulates the glandular production of colostrum. By the second trimester, PL begins to stimulate the secretion of colostrum. A mother who delivers after 16 weeks’ gestation will secrete colostrum, even though she has had a nonviable infant. This demonstrates the effectiveness of hormonal stimulation on lactation.

An estrogen-mediated increase in prolactin secretion in pregnancy may produce as much as a 10- to 20-fold increase in plasma prolactin. This effect may be partially controlled by lactogen from the placenta, which inhibits the production of prolactin. ³ Hormonal regulation of the growth and proliferation of the mammary gland cells has been carefully studied in many species.

Studies of mice in which receptors for each of the hormones have been ablated demonstrate that progesterone and prolactin (or possibly placenta lactogen) are key to alveolar development in pregnancy. The major inhibitor of milk production during pregnancy has been shown to be progesterone, whereas estrogen also contributes to secretion inhibition. ²¹

Hormonal Preparation of Breast for Lactation

A complex sequence of events, governed by hormonal action, prepares the breast for lactation (see Fig. 3.3 ). During pregnancy, 17β-estradiol stimulates the ductal system of epithelial cells to elongate. In contrast to puberty, however, when estrogens appear to directly and indirectly stimulate breast development, estrogens have no indispensable role in mammary development during pregnancy except as a prolactin potentiator: according to Neville, ¹² when estrogen levels are low in pregnancy, the breast still develops. Estrogen levels are normally high in pregnancy, but not for mammogenesis. Induced lactation in the cow is dependably reproduced with 7 days of estrogen and progesterone treatment. Progesterone, in turn, induces the specific epithelial cells of the tubular invaginations to produce distinct ducts, which branch from the main tubules. ³ , ²¹

The end result of the combined actions of estrogen and progesterone is a richly branched arborization of the gland. Highly differentiated secretory alveolar cells develop at the ends of these ducts under the influence of prolactin ( Fig. 3.5 ).

Serum growth factor, which is present in normal human serum, and insulin can stimulate the stem cells of the gland to proliferate. These dividing cells are further directed to the formation of alveoli by corticosteroid hormones. At least two types of cells are identified in the epithelial layer of the gland: stem cells and secretory alveolar cells. At this point in the pregnancy, prolactin influences the production of the constituents of milk.

Transforming growth factor-β (TGF-β) influences pattern formation in the developing mammary gland and may negatively regulate ductal growth as well. ¹⁸ The pattern of mammary ductal development varies widely among species and is a function of both genotype and hormonal status. Normal human breast cells secrete TGF-β and are themselves inhibited by it, suggesting an autoregulatory feedback circuit that may be modulated by estradiol. Growth and patterning of the ductal tree are regulated in part by TGF-β operating through an autocrine feedback mechanism and by paracrine circuits associated with epithelial-stromal interactions. ²⁴

Stages of Lactogenesis

Stage I

Stage I lactogenesis starts approximately 12 weeks before parturition and is heralded by significant increases in lactose, total proteins, and immunoglobulin; decreases in sodium and chloride; and the gathering of substrate for milk production. The composition of prepartum secretion is fairly constant until delivery, as monitored by the milk protein α-lactalbumin.

Lactogenesis is initiated in the postpartum period by a fall in plasma progesterone, but prolactin levels remain high ( Fig. 3.6 ). The initiation of the process does not depend on suckling by the infant until the third or fourth day, when the secretion declines if milk is not removed from the breast. ²⁷

Stage II

Stage II lactogenesis includes an increase in blood flow and oxygen and glucose uptake as well as a sharp increase in the citrate concentration, considered a reliable marker for lactogenesis stage II. Stage II at 2 to 3 days postpartum begins clinically when the secretion of milk is copious and biochemically when plasma α-lactalbumin levels peak (paralleling the period when “the milk comes in”). The major changes in milk composition continue for 10 days, when “mature milk” is established. The establishment of the mature milk supply, once called galactopoiesis , is now referred to as stage III of lactogenesis.

The profound changes in milk composition have been established for the period of transition to mature milk in relation to an increase in milk volume. Detailed studies of successfully lactating women were performed by Neville et al., ²⁸ who report that a significant fall in sodium, chloride, and protein and a rise in lactose precede the major increase in milk volume during early lactogenesis. At 46 to 96 hours postpartum, copious milk production is accompanied by an increase in citrate, glucose, free phosphate, and calcium concentrations and a decrease in pH.

The breast, one of the most complex endocrine target organs, has been prepared during pregnancy and responds to the release of prolactin by producing the constituents of milk (see Fig. 3.5 ). The lactogenic effects of prolactin are modulated by the complex interplay of pituitary, ovarian, thyroid, adrenal, and pancreatic hormones ( Fig. 3.7 ).

The Science of Suckling

The ability to lactate is characteristic of all mammals, from the most primitive to the most advanced, and the instinct to feed through suckling occurs spontaneously in infants postpartum. Normal, alert newborns have been observed to “crawl” to the nipple and latch on unassisted when placed on the maternal abdomen following a normal delivery and the clamping and severing of the umbilical cord. ²⁹ The divergence of suckling patterns, however, makes it urgent that human patterns be studied specifically. Some aquatic mammals, such as whales, nurse under water; others, such as the seal and sea lion, nurse on land. Various erect or recumbent postures are assumed by different terrestrial mammals. ³⁰ Nursing may be continuous, as in the joey attached to a marsupial teat, or at widely different intervals characteristic of the species and parallel to the nutrient concentrations of the milk. The intervals may be a half hour for dolphins, an hour for pigs, a day for rabbits, 2 days for tree shrews, or a week for northern fur seals.

New anatomy research, gathered for the first time in 160 years since the brilliant work with dissections by Sir Ashley Cooper, has been generated in the laboratory of Peter Hartmann in Australia and his eclectic team of scientists. They have had access to the latest digital technology and have shown that the milk ducts of the breast are small ( Fig. 3.8 ), compressible, superficial, and closely intertwined. ³¹ There are no “dilated sinuses” that store large amounts of milk. The amount of adipose tissue in the breast is very variable and not a measure of the amount of glandular tissue; there is twice as much glandular tissue as fat. ³² Magnetic resonance imaging has identified some central ducts in the breasts of lactating women. The anatomy of the lactating breast was redefined with ultrasound imaging in Hartmann’s laboratory. ³³ Ducts were found to number four to eight, and branches drain glandular tissue directly beneath the nipple and merge into a collecting duct very close to the nipple. They do increase in diameter during milk ejection. Milk production is not dependent on neural stimulation but is hormonal. Milk ejection is critical to successful lactation. A failure to remove milk results in decreased milk production. Multiple milk ejections occur during breastfeeding, even though a woman usually only senses the first milk ejection.

Although many anatomic distinctions exist as well, the principal mechanism of milk removal common to all mammals is the contractile response of the mammary myoepithelium under the hormonal influence of oxytocin released from the neurohypophysis. ³⁴

The key function in all species is effective control of milk delivery to the young in the right amount and at the appropriate intervals, which requires a production system, exit channels, a prehensile appendage, an expulsion mechanism, and a retention mechanism. The primary, secondary, and tertiary ducts form an uninterrupted channel for the passage of milk from the milk-producing alveoli to the prehensile appendage. A process of erection of the areolar region facilitates prehension by the young during suckling. The principal object of the suction produced by the facial musculature of the young is to draw the nipple into the mouth and retain it there. Positive pressure is used to expel milk from the gland by the contractile changes in the mammary gland provided by the myoepithelial cells. The sympathetic nervous stimuli can oppose milk ejection by increasing vasoconstrictor tone, thereby reducing the access of circulating oxytocin to the mammary myoepithelium. Sympathetic activity also can occur during conditions of apprehension or muscular exertion. The milk-ejection reflex can be blocked by emotional disturbance or reflex excitation of the neurohypophysis. The central nervous system control of milk ejection indeed suggests that restraining mechanisms exist to ensure that milk ejection can only occur under circumstances wholly conducive to the effective removal of milk by the suckling young.

In all species that have been studied, a rise in intramammary pressure and flow of milk occurs as a reflex event in suckling. The excitation of the neurohypophysis results in the release of oxytocin, which is conveyed via the bloodstream to mammary capillaries, where it evokes contraction of the myoepithelium. The successive ejection-pressure peaks, demonstrated in lactating women, can be duplicated more accurately by a series of separate oxytocin injections than by the same total dose as a single injection or by a continuous infusion of the hormone. This strongly suggests that oxytocin is released from the neurohypophysis in spurts. The study of suckling patterns in all species shows a high degree of ritualization, which in turn suggests a close neural connection between cognitive or behavioral and hormonal responses.

Attention has focused on the mechanisms that control suckling behavior, on its incidence, on events that precipitate and terminate it, on the effects of stress, and on how development modifies it. Suckling is characteristic of each species and is vital for survival. Suckling means to take nourishment at the breast and specifically refers to “breastfeeding” in all species. Sucking , however, means to draw into the mouth by means of a partial vacuum, which is the process employed when bottle-feeding. Sucking also means to consume by licking.

Although suckling has been studied in the young and mothers in other species, a large portion of human data has been collected using a rubber nipple and bottle. Other mammals suckle only in the nutritive mode, whether receiving milk from the nipple or not. Human infants were noted to have two distinct patterns with rubber nipples: a nutritive mode and a nonnutritive mode. ³³ , ³⁵ When this work was repeated using the breastfeeding model, no difference between nutritive and nonnutritive suckling rates was seen but, rather, a continuous variation of the suckling rate in response to the milk-flow rate. ³⁶ Suckling rates in other species correlate with milk composition and species-specific feeding schedules (one suck per second in great apes and four to five sucks per second in sheep and goats).

In further experiments, an inverse linear relationship was found between milk flow and the suckling rate. Thus, the higher the milk flow, the lower the suckling rate. In human infants younger than 12 weeks of age, suckling will terminate with sleep and be reinstated on awakening, a pattern that is well described in other species. In infants older than 12 weeks, suckling is not always terminated by sleep. At 12 to 24 weeks, infants will play with the nipple, explore the mother, and not always elicit nipple attachment. Continuous measurement of milk intake during a given feeding from one breast showed a progressive reduction in intake volume per suck and an increase in the proportion of time spent pausing between bursts of sucking.

Using the miniature Doppler ³⁷ ultrasound flow transducer, Woolridge and Baum ³⁸ studied 32 normal mother–baby pairs from 5 to 9 days postpartum. Intakes during trials averaged 34.2 g (±3.7 g) on the first breast and 26.2 g (±3.5 g) on the second breast. At the start of feeds, the average suck volume was approximately 0.14 mL/suck, which decreased to approximately 0.10 mL/suck or less. The mean latency for the release of milk was 2.2 minutes after the infant began to suckle. The researchers also noted that on the first breast, the flow increased and stabilized after 2 minutes, with concomitant slowing and stabilizing of the sucking pattern during the remainder of the feed. On the second breast, the suck volume fell off dramatically toward the end of the feed (50% reduction from peak to end of feed; Fig. 3.9 ).

These observations support the theory that infants become satiated at the breast, and milk remains unconsumed in the breast. During the first month of life, infants consume a given amount of fluid with decreasing investment of time. ³⁷ The amount of fluid per suck increases over time. The control of intake appears to come under intrinsic control of the infant during the first month of life. ³⁹

A cineradiographic study of breastfeeding was done by Ardran et al. ⁴⁰ in 1957 and compared with a similar study of bottle-feeding. ⁴¹ The nipples and areolae of 41 breastfeeding mothers were coated with a paste of barium sulfate in lanolin, and cineradiographic films were taken with the infant at the breast. These were then reviewed meticulously. Box 3.3 lists the authors’ conclusions in their original description. These observations are of historical interest, but newer techniques in imagery have more accurately described the understanding of human suckling.

The development of real-time ultrasound improved the definition of images. Several studies have been published using this noninvasive technique to observe the action of the infant’s tongue and buccal mucosa and the maternal nipple areola. Using a video recorder in the 1980s that allowed frame-by-frame analysis and recorded simultaneous respiration, the pattern of suck, swallow, and breathing was documented during a period of active suckling at the breast. A suck was defined by Weber et al. ⁴² as the beginning of one indentation of the nipple by the tongue to the beginning of the next. Weber et al. had examined six breastfed and six bottle-fed infants between 1 and 6 days of life. Not all sucks were associated with a swallow. Box 3.4 summarizes the process.

Observations of suckling using improved techniques from 2 to 26 weeks showed that suckling starts with a series of fast sucking movements and then stabilizes. In a 2-week-old breastfeeding infant, sucking and breathing pattern proportions alternated smoothly at about two sucks to one breath, with swallowing occurring with every suck. Bottle-feeding patterns were variable and sometimes asynchronous with sucking and breathing.

The process of suckling has been described as a pulsating process similar to peristalsis along the rest of the gastrointestinal (GI) tract. This undulating motion, as described by cineradiography, did not involve stroking or friction, as was clearly pointed out by Woolridge. ³⁷ The nipple should not move in and out of the infant’s mouth if the breast is positioned correctly. The tip of the tongue does not move along the nipple. The positive pressure of the tongue against the teat (areola and nipple), coupled with ejection of the milk from increased intraductal pressure, evacuates the milk, not suction. The negative pressure created in the mouth holds the nipple and breast in place and reduces the “work” to refill the ducts. Visual observations and videotapes made in our laboratory to study suckling show the undulating motion of the external buccal surfaces even in newborns. Ultrasound confirms the molding of the buccal mucosa and tongue around the teat, leaving no space.

In breastfeeding, the tongue action is a “rolling,” or peristaltic, action from the tip of the tongue to the base, not side to side. In bottle-feeding, the tongue action is more piston-like or squeezing. When the infant rests between sucks, the human nipple is indented by the tongue, and the latex teat is expanded in bottle-feeding ( Figs. 3.10 and 3.11 ).

The change in nipple dimensions during suckling is detailed by Smith et al., ⁴³ who also used ultrasound and examined 16 term infants ages 60 to 120 days and their mothers. They demonstrated that human nipples are highly elastic and elongate during active feeding, including approximately 2 cm of areola, to form a teat approximately twice its resting length. They also showed that infants’ cheeks (buccal membranes), with their thick layer of fatty tissue, known as sucking fat pads , act to make a passive seal to create a vacuum (as opposed to the concept that the cheeks are sucked in by the negative pressure). Milk ejection was noted to occur after maximal compression of the nipple.

Multifunctional Nature of Prolactin

Prolactin, the lactogenic hormone, is essential for glucocorticoid stimulation of the milk-protein genes. ³

Synthesis and secretion are not restricted to the anterior pituitary gland but include multiple sites in the brain (cerebral cortex, hippocampus, amygdala, cerebellum, brainstem, and spinal cord). It is also produced in the placenta, amnion, decidua, and uterus. Evidence suggests that lymphocytes from the immune system, thymus, and spleen release bioactive prolactin. Prolactin is found in the epithelial cells of the lactating mammary gland and the milk itself. Prolactin reaches the milk by crossing the mammary epithelial cell basement membrane, attaches to a specific prolactin-binding protein, and ultimately moves by exostosis through the apical membranes into the alveolar lumen. Prolactin mRNA in milk contains more prolactin variants than serum. Milk prolactin participates in the maturation of the neuroendocrine and immune systems.

The information generated by the use of knockout mice with prolactin knockouts or prolactin-receptor knockouts has refined the understanding of mammary morphogenesis and subsequent lactogenesis. ¹⁰ It has been confirmed that prolactin does not operate alone but depends on estrogen, progesterone, and glucocorticoids, as well as insulin, thyroid hormone, parathyroid hormone, and even oxytocin (see Fig. 3.7 ). Prolactin also stimulates the uptake of some amino acids, the uptake of glucose, and the synthesis of milk sugar and milk fats. ¹

Plasma prolactin varies in relation to psychosocial stress. Utilizing four different real-life stress studies in a longitudinal design, Theorell ⁵⁶ found that changing situations associated with passive coping are accompanied by increased plasma prolactin levels. Changing situations associated with active coping are associated with unchanged or even lowered prolactin levels. The regulation of plasma prolactin is part of a dopaminergic system (see the list of pharmacologic suppressors in the next section).