Review nature publishing group Protective effects of amniotic fluid in the setting of necrotizing enterocolitis Soham Dasgupta1 and Sunil Kumar Jain1 Necrotizing enterocolitis (NEC) is the most common life cost of taking care of medical NEC was $74,004 and surgical threatening condition affecting preterm infants. NEC occurs in NEC was $198,040 per infant, respectively (8). 1–5% of all neonatal intensive care admissions and 5–10% of After more than half a century of NEC research in animal very low birth weight infants. The protective role of human models and in vitro research, we could not find a “magic breast milk (BM) has been well established. It has also been bullet” to prevent NEC. However, in last decade the most shown that amniotic fluid (AF) and BM have many similarities effective tools to decrease burden of NEC have been NEC in terms of presence of growth and other immune- prevention initiatives like breast milk (BM) feeding, blood modulatory factors. This finding led to the initial hypothesis transfusion guidelines, and antibiotics stewardship in PI that AF may exert similar protective effects against the (3,9,10). During fetal life, fetal intestine gets exposed to high development of NEC, as does BM. Multiple studies have concentrations of many trophic factors by swallowing AF. But elucidated the presence of growth factors in AF and the preterm birth interrupts AF swallowing, which deprives the protective effect of AF against NEC. Studies have also intestine of PI of these trophic factors. In this review, we will described possible mechanisms how AF protects against review the role of various trophic factors and cytokines in the NEC. At present, research in this particular area is extremely development of intestine and role of decreasing/preventing active and robust. This review summarizes the various studies NEC by providing trophic factors via supplementing AF. looking at the protective effects of AF against the develop- ment of NEC. It also provides an insight into future directions, PATHOGENESIS the vast potential of AF as a readily available biologic medium, The pathogenesis of NEC is complex and multifactorial. and the ethical barriers that must be overcome before Prematurity, bacterial colonization of the gut, and formula using AF. feeding are the three main factors responsible in the pathogenesis of NEC. Prematurity leads to impaired peristal- sis, deficiencies in components of the mucus layer, and disruption of the integrity of the intestinal tract epithelial tight ecrotizing enterocolitis (NEC) is a gastrointestinal junctions (11). Ninflammatory disease, which affects predominantly preterm infants. Bell-staging criteria (1) is most commonly Role of Toll-Like Receptor 4 in Pathogenesis of NEC used to define the severity of NEC. NEC is the most common Toll-like receptor 4 (TLR4) is an innate immune receptor that life threatening condition affecting preterm infants (PI) (2). recognizes lipopolysaccharide (LPS) found in Gram-negative NEC occurs in 1–5% of all neonatal intensive care admissions bacteria. TLR4 is expressed at higher levels in the premature and 5–10% of all very low birth weight (o1,500 g) infants (3) than the full-term intestine in human infants, rodents, and leading to high morbidity and mortality. Patel et al. (4) other species (12). TLR4 is expressed at high levels in showed that NEC has emerged as the leading cause of developing fetal intestine. After preterm birth, intestinal TLR4 mortality in extremely low birth weight infants. The study levels remain high, gut bacterial colonization during postnatal found that in extremely PI, overall mortality related to period leads to exaggerated activation of TLR4 signaling. This pulmonary, immaturity, infection, and central nervous system exaggerated activation of TLR4 leads to development of NEC (2000–2011) has declined. However, mortality secondary to in premature infants by increased enterocyte apoptosis, NEC has increased during the same time period. Intestinal impaired mucosal healing, and enhanced pro-inflammatory perforation, short gut syndrome, and TPN-associated choles- cytokine release (13). The intestine of PI have increased tasis are the common causes of morbidity and mortality bacterial colonization, altered microcirculatory perfusion, and related to NEC (5,6). Long-term adverse outcomes include the immaturity of the immune system compared with term stricture formation, development of recurrent NEC, and infants (12,13). These factors work together to develop development of short bowel syndrome (7). In 2011, overall NEC in PI. 1Department of Pediatrics, University of Texas Medical Branch, Galveston, Texas. Correspondence: Sunil Kumar Jain ([email protected]) Received 6 October 2016; accepted 3 May 2017; advance online publication 5 July 2017. doi:10.1038/pr.2017.144 584 Pediatric RESEARCH Volume 82 | Number 4 | October 2017 Copyright © 2017 International Pediatric Research Foundation, Inc. Amniotic fluid in the setting of NEC | Review Gut Bacterial Colonization of Premature Infants and enterocyte abnormalities. Neonates born with congenital Gut colonization of pathological Gram-negative bacteria in PI intestinal obstruction also showed villus blunting and poorly leads to an alteration of the normal gut flora. Colonization of organized crypts distal to the site of obstruction (23). In the the gut in the early neonatal period happens in two waves sheep model, the effects of interrupted AF swallowing on the (14). The first wave, which is similar in both term and PI, intestinal mucosa were gradually reversed following predominantly depends on the mode of childbirth. The the removal of esophageal ligatures and restitution of AF second wave of colonization in term infants is determined by ingestion, but not with infusion of lactated Ringer’s solution. feeding type. Breastfed infants mainly have Bifidobacteria/ These findings indicated that the trophic effects of AF were Bacteroides and formula-fed infants predominantly have secondary to the bioactive molecules present in AF rather Streptococci/Staphylococci/Lactobacilli (15). In the case of PI, than due to the flow of fluid through the gut (23). There are the second wave of colonization is influenced less by the type many trophic factors in AF like insulin-like growth factors I of feeding, and is characterized by high numbers of and II (IGF I and II), epidermal growth factor (EGF), Clostridiaceae and Enterobacteriaceae and very low relative hepatocyte growth factor (HGF), and others that modify numbers of Bifidobacteria and Bacteroides. Several investiga- intestinal nutrient absorption and development, which are tors have shown a link between this abnormal gut microbiota reviewed in Drozdowski and Thomson (24). in PI and the development of NEC. PI are also exposed to longer courses and duration of antibiotic therapy, which may EFFECT OF CYTOKINES ON THE DEVELOPMENT OF THE GIT lead to significant alteration of the flora of the normal The development, maturation, and maintenance of the gastrointestinal tract (GIT) (3). complex functions of the GIT require interaction with Even though TLR4 has significant role in the pathogenesis multiple environmental exposures. Maheshwari (25) of NEC, other pathways have been shown to be important as described cytokines in AF and their role in the development well. Increased expression of platelet-activating factor in gut of GIT. In addition to the cytokines and growth factors mucosal injury and intestinal epithelial barrier function is delivered to the intestinal mucosa via AF, the fetal GIT also associated with NEC (16). constitutively expresses many cytokines and growth factors at Pro-inflammatory cytokines like LPS cause epithelial injury high levels. Intestinal epithelial cells express receptors for leading to epithelial barrier injury and development of NEC most of these cytokines, and evidence from in vitro and via nitric oxide. Ford et al. compared iNOS mRNA in the animal models suggest that many of these agents can increase intestinal tissue from infants with and without NEC. iNOS enterocyte proliferation, migration, and differentiation, pre- mRNA levels were significantly higher in the intestinal vent apoptosis, and promote mucosal restitution, all of which samples from infants who had NEC compared with controls. protect against NEC (19,26). Further, immunochemistry and in situ hybridization con- The cytokines described by Maheshwari et al. and their role firmed that the predominant source of iNOS activity was in the development of the GIT are described in Table 1. enterocytes in the intestinal samples from infants with NEC (17,18). Understanding the exact pathogenesis of NEC is crucial to ROLE OF ANTIMICROBIAL PEPTIDES IN AMNIOTIC FLUID implementing strategies to prevent it. Human BM and Antimicrobial proteins and peptides (APP) serve as a broad amniotic fluid (AF) have been shown to contain numerous spectrum anti-infective molecules. AF is a good source of bio-factors that may be protective against the development of many APP like neutrophil defensins, bactericidal permeability NEC in PI. This is discussed further in this review article. increasing proteins, calprotectin, and human B defensin-2 (HBD-2) among others (27,28). These APP account for the FETAL SWALLOWING OF AF antimicrobial activity of the AF, and their concentrations The fact that NEC is not seen in utero raised the possibility of increase under conditions where there is microbial invasion of the presence of protective factors in AF against the the amniotic cavity. APPs are believed