Development is a continuous cascade that does not stop at the moment of birth but progresses along a continuum. Although in utero and ex utero life have unique aspects, one progresses from the other. Amniotic fluid is the physical buffer and barrier to the outside world and is the fluid that the fetus swallows, bathing first the rudimentary primitive gut that later becomes the gastrointestinal tract. Although amniotic fluid is not the nutritional mainstay of the fetus, it contributes up to 15% of fetal nutritional requirements (1,2) and plays a significant role in gut development and maturation (3). In contrast, human milk becomes the foodstuff essential for the neonate's survival. Human milk also is more than its nutritional component, because it can stimulate cell growth and repair and can provide immune protection and enhance immunocompetence (4–6). Amniotic fluid and human milk share bioactivity, the capacity to stimulate cells to grow or undergo reparative processes.
The concept that amniotic fluid and human milk share certain qualities along a continuum of fetal and neonatal growth is not new (4,7–9). However, the findings of Hirai et al. (10) in this issue present a new perspective to an old topic. In a unique, in vitro, human fetal small intestinal cell model, the authors demonstrated the trophic properties of amniotic fluid and human milk. They then compared the trophic effect of individual growth factors previously identified in amniotic fluid and human milk: EGF, insulinlike growth factor 1, transforming growth factor α, hepatocyte growth factor, fibroblast growth factor, and vascular endothelial growth factor. Monoclonal antibodies partially reversed the trophic effects of these growth factors, supporting the premise of their role in gut development. The authors also present exciting new data about the mechanism of action of these bioactive factors in amniotic fluid and human milk. Through the use of genistein, a tyrosine kinase inhibitor, they reversed the effect of these growth factors and that of amniotic fluid and human milk.
In this cell model, human milk's ability to stimulate FHs-74 cells was significantly greater than that of amniotic fluid: a 5% solution of human milk was comparable to a 40% solution of amniotic fluid in terms of growth-promoting activities, as measured by 3H thymidine incorporation. Although the gut's own ability to generate bioactive factors for its growth and regeneration is well documented (11–13), this ability comes with maturation, which is often lacking in neonates, particularly premature infants. The neonatal gut's need to undergo further differentiation rapidly is clear because prolonged gut maturation increases the risk of nutritional malabsorption (from decreased surface area of crypts and villi), infection, and systemic access of the outside world (1,3,6). Human milk's provision of growth factors and gut peptides facilitates this developmental process.
Although Hirai, et al. (10) found that EGF concentration in amniotic fluid increased as a function of advancing gestational age, they found no differences in the proliferation effect of amniotic fluid from earlier gestations compared with later gestations (see Fig. 4). Why did this occur? Again the single growth factor approach to any cell system is simplistic; other growth factors in conjunction with EGF likely account for the equivalent bioactivity of amniotic fluid in this in vitro model. Wagner and Forsythe (14), also using FHs-74 cells in vitro, showed a synergistic relationship between EGF and transforming growth factor α that was greater than the combined effect of EGF and insulinlike growth factor 1 or of all three factors combined. Both groups report a narrow physiologic range at which the growth factors tested maximally stimulated fetal gut epithelial cells.
Booth, et al. (11) used a developing rat epithelium model that also supports the interactional effect of bioactive substances on gut epithelial cells. In their model, no single growth factor promoted exclusive proliferation of the epithelial cells, but when insulinlike growth factor 1, EGF, transforming growth factor α, and PDGF were combined, epithelial cell numbers increased. In vitro studies only suggest how these growth factors and gut peptides act, whether in concert, serially, or separately, on the recipient gut epithelia. The interplay of these factors in vivo remains largely unknown, but through the work of Hirai et al. and others, we have a glimpse of their interaction and effect on gastrointestinal epithelial cells of the developing fetus and neonate. Strategies to assess the interactional effect of gut peptides and growth factors collectively on gut epithelial development and maturation become essential in developing new therapies for gastrointestinal diseases (3).
In vivo, many of our findings about gut development and maturation come from the preterm infant (15). The observation that when fed human milk, the preterm infant, who is closest to the fetal state, has enhanced gut maturation, less feeding intolerance, decreased incidence of necrotizing enterocolitis, and a decreased incidence of infection when compared with formula-fed infants speaks to breast milk bioactivity (16). The finding that human milk also is involved in ongoing gut reparative processes comes from both in vitro and in vivo studies. Rao et al. (17) demonstrated in a Caco-2 cell xanthine oxidase injury model that a dilution of human milk was protective against injury, which was lacking in cells incubated with proprietary infant formulas. Whether amniotic fluid provides an avenue for reparative processes in the fetal gut is unclear, but the work of Mulvilli et al. (2,18) in their fetal rabbit model, and of Hirai et al. (10) suggest that it does.
Another unique difference between human milk and amniotic fluid is their physical structures. Human milk contains aqueous, fat, and cellular compartments, whereas amniotic fluid contains an aqueous compartment in which cells are suspended (19,20). The aqueous or whey fraction of human milk is juxtaposed with fat that remains in solution as an emulsified component. The mammary epithelial cell is responsible for enveloping fat (that consists mainly of triglycerides) with its apical membrane, creating the milk fat globule. The milk fat molecule membrane has cell surface receptors and transmembrane growth factors on its surface that likely contribute to milk's bioactivity. Similarly, the cellular component of human milk is not trivial, with cells—a combination of macrophages, lymphocytes, neutrophils, and rare mammary epithelial cells—all numbering 1 × 105/mL in colostrum and 1 × 103/mL in mature milk. In contrast, unless chorioamnionitis occurs, amniotic fluid's cell count is less than that of colostrum, with the upper limit of normal being 1.2 to 1.3 × 104 cells/mL.
Few experiments that study gut epithelial cell interactions have been performed with fat or milk leukocytes. In Hirai et al., human milk's bioactivity and stimulation of FHs-74 cell growth is only partially measured by its aqueous components, with little known about the interaction of the milk fat globule, its membranes, or milk's cellular compartment on the gut epithelium. The work of Hirai et al. is a good beginning to the story of how amniotic fluid prepares the fetal gut for in utero existence, and of how human milk continues the journey of gut development in the neonate. We look to the future for more detailed experiments to elaborate more completely the function and interaction of all milk compartments with the gut. Who knows, we may discover a whole new signaling system between mother and neonate.
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