Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in preterm infants and often appears after enteral feeding. In addition, the risk of developing NEC is higher (6 to 10-fold) in formula-fed preterm infants than those fed exclusively breast milk (1). This intestinal inflammatory disease is characterized by an injury of the gut (barrier disruption and necrosis) that may lead to its perforation and followed by multisystem organ failure and death (2).
Increased Gut Permeability
Higher intestinal permeability is observed in premature infants fed formula than those fed breast milk (3). Increased intestinal permeability is associated with necrosis in NEC and intestinal dysbiosis. Moreover, Penn et al. (4) demonstrated that numerous digested formulas are cytotoxic on intestinal epithelial cells, endothelial cells, and neutrophils, whereas breast milk is not. These results suggest that high concentrations of free fatty acids (FFAs) in the formula may contribute to the pathogenesis of NEC.
Increased Endotoxin Levels
Increased enteral endotoxin level is associated with increasing dysbiosis that could lead to the development of NEC. Kaufman et al. (5) have recently demonstrated that preterm formulas contain endotoxin levels (from 4.5 to 47 EU/mL, depending on the brand and formula product). Detection of endotoxins in infant formulas could be related to the presence of lipopolysaccharides (LPS), which is a component of Gram-negative bacterial cell walls. LPS can activate TLR4 signaling, and their interaction (binding) induces gut inflammation and enterocyte apoptosis, which result in a reduction of mucosal repair. Thus, endotoxin exposure via enteral formulas may contribute to neonatal gut dysbiosis, microbial translocation, and NEC. The FDA has an endotoxin limit of 120 EU/kg/day for intravenous products. Kaufman et al. (5) reported that most preterm formulas had between 396 to 5688 EU/Kg/day of endotoxins. Pasteurization and sterilization destroy microbes, but they do not eliminate LPS or endotoxin. Therefore, the preterm/infant formula may promote NEC development due to the stimulation of pro-inflammatory responses in their immature gut.
Breast Milk Proteins Against NEC
One major factor influencing the protective effect of breast milk against the development of NEC is the presence of bioactive proteins, including immunoglobulins and transforming growth factor-beta (TGF-b). Maheshwari et al. (10) found that TGF-b supplementation decreased the risk of NEC in a neonatal mouse model. They demonstrated that TGF-b2 suppressed macrophage cytokine production and mucosal inflammatory responses in the developing intestine (10). Secretory IgA (sIgA), IgG, IGF-1, and EGF in breast milk could promote intestinal growth and enhance the gut barrier against pathogenic microbes and toxins (11). The transfer of antibodies and growth factors from mother’s milk to her preterm infant is critical because the preterm immune system is more vulnerable to microbial infections due to the decreased production of IgA, IgM, IgG and defensins, and upregulation proinflammatory TLRs and cytokines (9).
Alteration of Gut Microbiota by Infant Formula
Preterm/infant formula could impair the microbiome in preterm infants and promote NEC. Bacteria isolated in blood and stool samples from premature infants with NEC are often related to Klebsiella, Escherichia coli, Enterobacter, and other Gram-negative bacteria from the Enterobacteriaceae family (6). The abundance of Enterobacteriaceae, Staphylococcaceae (include S. aureus), and Clostridiaceae in infant stools were higher in formula-preterm infants than in those fed breast milk or donor breast milk (7). In contrast, breast milk and donor breast milk enhances commensal bacterial colonization (especially Bifidobacteria), which reduces pathogenic bacteria's ability to colonize the infant's gut (7). Histopathologic signs of NEC were observed in 57% of pig fed formula and in 5% feds colostrum (8). Formula-induced mucosal atrophy and dysfunction that predispose to NEC and bacterial overgrowth (9).
HMOS on Infant Microbiome
Human milk oligosaccharides (HMO) enhance the proliferation of Bifidobacterium and Lactobacillus and their colonization into the gut. The colonization of these beneficial bacteria (also named probiotics) inhibits and prevents the attachment of pathogenic bacteria in the immature intestine, which reduces the risk of NEC (9). Wejryd et al. (12) demonstrated that low diversity of HMOs was associated with NEC development. Breast milk contains a large diversity of HMOs, whereas bovine or synthetic HMO added into infant formula contains a low diversity. The low diversity (or absence) of HMOs in infant formula may select and promote the overgrowth of pathogenic microbiota (Enterobacteriaceae) that contribute to the development of NEC (9).
Donor Breast Milk vs Infant Formula
One meta-analysis demonstrated that formula-fed preterm infants are associated with a higher risk of developing NEC than donor breast milk-fed preterm infants (13). Another study (14) found that premature infants fed donor breast milk reduced the incidence of surgical NEC compared to premature infants fed bovine-based preterm formula. This study was a multicenter, blinded, randomized controlled trial where preterm-delivering mothers did not intend to provide breast milk to their infant (14). However, fresh breast milk has greater protective properties against NEC than donor breast milk due to its higher levels of immunoglobulins and other bioactive proteins. These anti-infective proteins are partially destroyed during milk pasteurization and freeze-thawing steps.
While the strategies for preventing NEC in preterm infants are still incompletely understood, it is well established that the risk of NEC is increased by the ingestion of infant formula and decreased by the ingestion of breast milk. More investigations are needed to develop donor milk products simulating colostrum or amniotic fluid and enhance the gut development in preterm infants who don’t have access to breast milk.
References:
1. Lucas A, Cole TJ. Breast milk and neonatal necrotising enterocolitis. The Lancet. 1990;336:1519-23.
2. Good M, Sodhi CP, Hackam DJ. Evidence-based feeding strategies before and after the development of necrotizing enterocolitis. Expert review of clinical immunology. 2014;10:875-84.
3. Taylor SN, Basile LA, Ebeling M, Wagner CL. Intestinal permeability in preterm infants by feeding type: mother's milk versus formula. Breastfeeding medicine. 2009;4:11-5.
4. Penn AH, et al. Digested formula but not digested fresh human milk causes death of intestinal cells in vitro: implications for necrotizing enterocolitis. Pediatric research. 2012;2:560-7.
5. Kaufman D, Perks P, Greenberg R. Endotoxin content of enteral formulas and fortifiers used in the NICU: A potential unrecognized source of bowel injury. PAS 2021, Virtual Pediatric Academic Societies 2021.
6. Duffy LC, et al. Bacterial toxins and enteral feeding of premature infants at risk for necrotizing enterocolitis. In Bioactive Components of Human Milk 2001 (pp. 519-527). Springer, Boston, MA.
7. Parra-Llorca A, et al. Preterm gut microbiome depending on feeding type: significance of donor human milk. Frontiers in microbiology. 2018;9:1376.
8. Sangild PT et al. Diet-and colonization-dependent intestinal dysfunction predisposes to necrotizing enterocolitis in preterm pigs. Gastroenterology. 2006;130:1776-92.
9. Sangild PT, et al. Diet-and colonization-dependent intestinal dysfunction predisposes to necrotizing enterocolitis in preterm pigs. Gastroenterology. 2006 May 1;130(6):1776-92.
10. Maheshwari A, et al. TGF-β2 suppresses macrophage cytokine production and mucosal inflammatory responses in the developing intestine. Gastroenterology. 2011;140:242-53.
11. Eibl MM, Wolf HM, Fürnkranz H, Rosenkranz A. Prophylaxis of necrotizing enterocolitis by oral IgA-IgG: review of a clinical study in low birth weight infants and discussion of the pathogenic role of infection. Journal of clinical immunology. 1990;10:72S-9S.
12. Wejryd E, et al. Low diversity of human milk oligosaccharides is associated with necrotising enterocolitis in extremely low birth weight infants. Nutrients. 2018;10:1556.
13. Quigley M, Embleton ND, McGuire W. Formula versus donor breast milk for feeding preterm or low birth weight infants. Cochrane Database of systematic reviews. 2019.
14. Cristofalo EA, et al. Randomized trial of exclusive human milk versus preterm formula diets in extremely premature infants. The Journal of pediatrics. 2013;163:1592-5.
About the Author
Dr. Demers is a Scientist with an expertise in Neonatal Immunology and Microbiology. Her long-term goal is to improve the immunity and growth development of premature infants. Her objective is to identify how antibodies, cytokines, and growth factors in human milk influence the function of the infant’s guts to protect against infection and pediatric diseases in premature infants. She also wants to understand how maternal factors influence the levels of these bioactive proteins in human milk and impact infant health outcomes.