Recent Advances in Necrotizing Enterocolitis Research Strategies for Implementation in Clinical Practice

INTRODUCTION

Our Current Understanding of Necrotizing Enterocolitis as a Disease Entity

Necrotizing enterocolitis (NEC) is an idiopathic, inflammatory bowel necrosis of premature infants that involves the small and the large intestine (Box 1). Despite decades of research, the etiopathologic hypotheses remain incomplete and still focused on disparate elements such as immaturity of the gut mucosal barrier, mucosal injury, ischemia, inflammation, and microbial dysbiosis, which may eventually allow luminal bacteria to translocate into the bowel wall to cause cellular necrosis and unregulated inflammation. In the clinical setting, about half of all infants with NEC respond to bowel rest, antibiotics, and supportive medical measures, but others develop progressive bowel disease or require surgical intervention; a third of these patients eventually die.¹ There is an unmet need for novel research strategies for prevention, early diagnosis, clinical monitoring, and treatment of NEC.

Box 1.

• Necrotizing enterocolitis (NEC) is an inflammatory necrosis of the bowel wall, and is a leading cause of morbidity and mortality of premature infants.

• NEC is often preceded by bacterial overgrowth. Lesions are marked by necrosis, inflammation, hemorrhages, and pneumatosis.

• Associations: prematurity, bacterial overgrowth, anemia/red blood cell transfusions, splanchnic compromise, and antibiotic use.

• Biomarkers may be useful in early diagnosis of NEC and further monitoring.

• Maternal milk feeding may be superior in various quantities to any other human milk substitute.

• Probiotics are likely to be useful in prevention of NEC.

• Antibiotic stewardship may protect against NEC through effects on bacterial flora.

• Protective immunomodulators may include lactoferrin; Toll-like receptor antagonists; amniotic fluid; growth factors; and specific chemicals, such as oligosaccharide 2′-fucosyllactose and peptidoglycans.

• Intestinal stem cells may have a role in prevention of NEC.

Most patients with NEC show disease evolution over a period of 24 to 48 hours, although a third may progress rapidly over just a few hours. The onset of NEC at a postnatal age of 2 to 5 weeks typically shows an inverse relationship with the gestational age at birth. At presentation, the systemic clinical signs are usually nonspecific and may include tachycardia with increased lethargy, apnea, and temperature instability; gastrointestinal signs may include feeding intolerance, delayed gastric emptying, abdominal distention, tenderness, and ileus with decreased bowel sounds. Grossly bloody stools are seen in approximately 25%. The severity of illness is often staged by using the modified Bell criteria, which define disease progression with an early stage of nonspecific systemic/gastrointestinal inflammatory response, a more definite stage of gastrointestinal disease and localized peritonitis, and eventually an advanced stage with diffuse peritonitis and systemic inflammatory response syndrome.

On histology, NEC lesions in the bowel wall are characterized by coagulative necrosis; inflammatory changes; bacterial overgrowth; pneumatosis intestinalis (gaseous cysts in the bowel wall); and, depending on the time elapsed since disease onset, reparative changes in the bowel wall.^2,3 The leukocyte infiltrates in NEC lesions show numerous newly recruited blood monocytes and macrophages (12.8 ± 1.1 cells/high-power field [HPF] in control tissue vs 128.6 ± 9.4 cells/HPF in NEC lesions).⁴ There is also a modest increase in neutrophils (7.7 ± 1.7 cells/HPF in control vs 37.9 ± 5.8 cells/HPF in NEC). The increase in lymphocytes is less prominent, although there may be important changes in lymphocyte subsets.⁵ This article summarizes the research strategies currently underway and others on the horizon, hoping that a multipronged approach will eventually improve the outcomes of preterm infants at risk of NEC.

Hematological and Inflammatory Changes During Necrotizing Enterocolitis

Anemia and red blood cell (RBC) transfusions have been associated with NEC. Numerous retrospective clinical studies have shown that 25% to 40% of all patients with NEC develop intestinal injury within 48 hours of receiving an RBC transfusion.^6–12 Compared with patients who develop NEC and do not have a history of a recent RBC transfusion, neonates with transfusion-associated NEC are often born at an earlier gestation, have lower birth weights, and have a delayed NEC onset at 3 to 5 weeks of postnatal age. In a recent study, MohanKumar and colleagues¹³ showed that mouse pups rendered anemic by timed phlebotomy on postnatal days 2 to 10 and then given RBC transfusions 24 hours later developed NEC-like intestinal injury within 12 to 24 hours. These mice showed prominent bowel necrosis, inflammation, and submucosal edema/separation of the lamina propria in the ileocecal region and colon. The anemic intestine showed extensive infiltration with inflammatory macrophages, which were activated by subsequent RBC transfusions via a lipopolysaccharide receptor (Toll-like receptor-4 [TLR4])–mediated mechanisms to cause bowel injury. TLR4 expression is known to be higher in the premature intestine than in term infants. Chelation of RBC degradation products with haptoglobin, absence of TLR4, macrophage depletion, and inhibition of macrophage activation were protective.¹³ Intestinal injury worsened with increasing severity and duration of anemia before transfusion, indicating a need for reevaluation of the current transfusion guidelines for premature infants.

Patients with NEC are also usually thrombocytopenic and show platelet counts less than 150 × 10⁹/L within 24 to 72 hours of onset of disease.^14–17 The severity of this thrombocytopenia usually correlates with the Bell clinical stage of NEC, and a rapid decrease in platelet counts to less than 100 × 10⁹/L is a sensitive, although not specific, predictor of bowel gangrene and/or the need for surgical intervention.^14,17 Although most patients with NEC and thrombocytopenia do not show signs of disseminated intravascular coagulation, the primary mechanism for thrombocytopenia is widely thought to be increased platelet destruction. In a recent study, Namachivayam and colleagues¹⁸ used their murine model of NEC to investigate the role of platelets in the pathogenesis of NEC. Ten-day-old mouse pups develop thrombocytopenia at 12 to 15 hours and develop an acute necrotizing ileocolitis resembling human NEC within 24 hours. Neonatal intestinal macrophages released tissue factor as early as 3 hours after the initiation of mucosal injury, which led to the activation of circulating thrombin and then platelets, causing widespread activation of inflammatory cascades and gut mucosal injury. Consistent with the murine data, there were increased levels of circulating tissue factor and thrombin-antithrombin complexes in patients with NEC.

TLR4-mediated lymphocyte influx may also play an important role in the development of NEC. Egan and colleagues⁵ showed that human and murine NEC is rich in lymphocytes that are required for NEC development. They showed that recombination activating gene 1–deficient (Rag1^−/−) mice were protected from NEC and the transfer of intestinal lymphocytes from NEC mice into naive mice induced intestinal inflammation. Similar to the findings in anemia and RBC transfusion–related NEC, intestinal TLR4 expression was required for CCR9/CCL25 signaling and consequent lymphocyte influx. TLR4 also mediated a STAT3-mediated lymphocyte polarization toward increased proinflammatory cluster of differentiation (CD) 3⁺, CD4⁺, interleukin (IL)-17⁺, and reduced tolerogenic Foxp3⁺ T-regulatory lymphocytes (Tregs). T-helper (Th)-17 lymphocytes were required for NEC development, because inhibition of STAT3 or IL-17 receptor signaling attenuated NEC in mice, whereas IL-17 release impaired enterocyte tight junctions, increased enterocyte apoptosis, and reduced enterocyte proliferation, leading to NEC.

Vascular Remodeling and Microcirculation

Development of intestinal vasculature and intestinal vasoregulation are prime targets for NEC research.^19–21 In premature infants, splanchnic blood flow limitations may arise from limited production of endogenous vasodilators such as nitric oxide (NO) from dysfunctional endothelial cells²² or excessive production of vasoconstrictors such as catecholamines and endothelins.²³ Blood flow disruptions related to maternal preeclampsia and congenital heart disease are also recognized as risk factors for NEC.^24,25 Endothelial TLR4 activation can also contribute to NEC pathogenesis by diminishing endothelial NO synthase expression and NO production.²² Clearly, optimizing splanchnic vasoregulation is a strategic area of research to decrease intestinal injury and inflammation.

Increasing evidence indicates a role of gut microvasculature maldevelopment in NEC pathogenesis.²¹ In mice, the intestinal microvasculature rapidly expands within the perinatal period from a thin, rudimentary matrix into a much more complex and organized network of blood vessels.²⁶ In our murine NEC model, impaired postnatal development of the gut microvasculature may predispose to histologic intestinal injury.²⁷ Near term, the fetal intestine shows strong expression of proangiogenic vascular endothelial cell factor (VEGF) and its cognate receptor VEGFR2, but this maturational increase is impaired by prenatal inflammation. Murine pups treated with a VEGFR2 inhibitor before experimental NEC show increased intestinal injury and mortality,²⁷ whereas promotion of intestinal VEGF production may be protective.²⁰ These experimental findings are consistent with decreased VEGF expression in healthy margins of human intestinal tissue resected for NEC compared with controls.²⁸ Therapeutic approaches to preserve intestinal microvasculature development and local VEGF and VEGFR2 expression may help prevent NEC in high-risk premature infants.²⁷

Microbiome and Biotics Research

Microbial dysbiosis preceding NEC is characterized by a proteobacterial bloom and decrease in other phyla, namely Firmicutes and Bacteroidetes.²⁹ Microbiome optimization to offset dysbiosis is a promising research strategy. Manipulating gut microbiota composition by administering targeted antibiotics; supplementing specific strains of beneficial bacteria (probiotics), microbial products, and paraprobiotics (inactivated probiotics); or by modulating the TLR4 response to dysbiosis are promising research strategies.

The traditional microbial optimization method has been the use of probiotics, both single species and combinations of species of bacteria such as bifidobacteria and lactobacilli, and nonpathogenic fungi such as Saccharomyces. The World Health Organization (WHO) defines probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host.”³⁰ The mechanisms by which probiotics may induce beneficial effects include gut barrier enhancement, improved epithelial survival, immune response modulation (eg, reduced responses from the TLR4 receptor, modulation of the effects of inflammatory cytokines), and competitive inhibition of gut colonization by pathogens.^31–33 Systematic reviews on probiotics, including the Cochrane Review, show that probiotics decrease NEC, late-onset sepsis, and all-cause mortality.^34–38 However, questions regarding the ideal combination of probiotics, duration of use, and dosage remain. Probiotic-related sepsis remains rare, but reports of sepsis³⁹ caused by contamination of probiotic products call for continued clinical caution in the selection of recipients.^40–42 Novel probiotic delivery systems such as biofilms, which may be more efficacious, are also being researched.⁴³

The term paraprobiotics is used to define nonviable microbial cells (intact or broken) or crude cell extracts (ie, with a complex chemical composition), which, when administered (orally or topically) in adequate amounts, confer a benefit on the human or animal recipient.⁴⁴ The live cells (probiotics) are inactivated by heat, chemicals (eg, formalin), gamma or ultraviolet rays, or sonication.^44,45 Each of these inactivation methods affects the cells differently and, therefore, induces variable effects on immunomodulation.

Prebiotics are indigestible substances that stay unabsorbed and serve as substrates for growth of probiotic organisms. In a meta-analysis of 7 trials (417 infants), prebiotics did not decrease late-onset sepsis, NEC, or feed tolerance but increased the growth of Bifidobacterium in the stools.⁴⁶ Synbiotics, a combination of prebiotics and probiotics, has been investigated in few trials with varying results, and more research is needed.⁴⁷

Human Milk Oligosaccharides and Glycans

Human milk oligosaccharides (HMOs) have gained increased awareness because of their luminal effects as prebiotics, which enhanced growth of beneficial bacteria (such as bifidobacteria), and as decoys in preventing pathogen colonization, and intestinal effects via promotion of the gut epithelial barrier and through maturation and enhancement of the leukocyte barrier in the lamina propria.^48,49 There may be some inconsistency in the HMO content not only by gestation (preterm vs term human milk) but also among mothers because of their secretor or genetic status. Lower amounts of HMO in their mothers’ milk may predispose some infants to late-onset sepsis or NEC. Supplementation of preterm formula with HMOs may have beneficial effects on intestinal injury and inflammation.⁵⁰ Although HMO supplementation has been reported to be beneficial in animal models of intestinal injury,^51–53 human trials are yet to be conducted.

Multiomic Strategies and Integration of Microbiome, Proteome, Metabolome, and Epigenome Data

Advancing technology, including next-generation sequencing, proteomics, metabolomics, transcriptomics, and epigenomics, has provided better and holistic understanding of perinatal pathophysiology.⁵⁴ There is an ever-increasing need for integrative analysis of the human microbiome, with increasing information on genomics and the proteome, metabolome, and the epigenome, with integrative analysis, and correlation of these to the clinical outcomes. The host genome provides the backdrop for additional and multiple system interactions. The host and these microbes also produce large quantities of metabolites, adding another layer of complexity. Metabolomics is the science of detecting small molecules, the result of metabolic pathways from biological specimens such as plasma, serum, urine, and tissues, and is the latest of the omics technologies.^55–58 This testing detects the products of the metabolic pathways in an organism, which may be useful in diagnosis, prediction, prognosis, or assigning disease status (biomarker detection). Metabolomics allows identification of distinct patterns of small molecules generated during both host and microbial cellular metabolism and may be useful in searching for biomarkers of microbiome patterns and dysbiosis. Metabolite patterns are dynamic, changing with gestational age, time, or disease process, and evaluation at a time point provides a snapshot of the metabolic milieu of the organism. The complexity and the numerous metabolites that need to be measured need sophisticated analytical techniques. Nuclear magnetic resonance spectroscopy and mass spectrometry are the most common techniques used. The metabolites produced by microbes and/or the host may also regulate transcriptional and translational events that can be evaluated using transcriptomics and proteomics.⁵⁹

There is a need for integration, analysis, and interpretation of information collected from different platforms related to the microbiome, transcription, proteomics, metabolomics, and the immune function to have a comprehensive view of these biological processes. This need brings up a significant bioinformatics challenge given the complexity of biological systems, the technological limits, the large number of biological variables, and limitations in the number of biological samples. Many network-based and graphical models (bayesian and nonbayesian) are being used to integrate these data. Bioinformatic techniques and expertise in dealing with these multiomics data are often the key in multiomic studies.^60,61 Integration of multiomics data may give a holistic view of pathophysiologic processes that lead to perinatal diseases and may inform novel preventive and therapeutic approaches.

Liquid biopsy of stools

A novel method of assessing microbiome and host responses is to salvage intestinal cells from stools, which can then be tested for microbiome, gene expression, and epigenetic profiles all in the same stool specimen, which can be followed longitudinally.⁶²

Immunomodulation

Intestinal injury and inflammation and necrosis are important features of NEC, and excessive inflammation has been proposed as a causal factor in NEC.⁶³ Hence the interest in immunomodulators, which can decrease inflammation and intestinal injury.

Lactoferrin

Lactoferrin is a multifunctional molecule that has iron binding and immunomodulatory properties. It is a component of the innate immune system and may decrease inflammation. A meta-analysis of 12 studies, including small trials done worldwide, found that enteral supplementation of lactoferrin decreases length of stay but not mortality or NEC.⁶⁴ Two large recent trials of lactoferrin (ELFIN in the United Kingdom and LIFT in Australia and New Zealand) failed to show a decrease in sepsis, mortality. Or NEC.^65,66

Toll-like receptor antagonists

TLR4 signaling is an important signaling pathway that enhances intestinal inflammation via the lipopolysaccharide of gram-negative bacteria.⁶⁷ Therefore, TLR4 antagonists may be helpful in decreasing intestinal inflammation and injury. A novel family of TLR4 inhibitors was described by Hackam and colleagues,^68,69 including a lead TLR4 antagonist, C34, which is available for clinical use. C34 is an oligosaccharide, binds to the synthetic lipid A analogue eritoran (E5564), and thus inhibits TLR4. C34 inhibits TLR4 in vitro in intestinal epithelial cells and macrophages, and reduces TLR4-mediated inflammation in mouse models of endotoxemia and NEC.^68,69 Phase 1 clinical trials of C34 are being planned.

Other natural products, such as amniotic fluid,^70,71 breast milk,⁷² and human milk oligosaccharide 2′-fucosyllactose,⁷³ have been shown to attenuate TLR4 signaling. Reciprocal expression of TLR4 and TLR9 signaling is seen in NEC, and TLR9 attenuates inflammation by antagonizing TLR4 mechanisms.⁷⁴ Peptidoglycan of gram-positive organisms may trigger TLR2 signaling, which has an antiinflammatory (via IL-10) and mucosal protective effect.⁷⁵ Variants of TLR2 have been associated with more severe inflammation. Novel strategies that use TLR2 ligands may decrease inflammation and disease severity in NEC.⁷⁵ TLR9 (CPG-DNA is a TLR9 ligand) inhibits TLR4 signaling and is one of the mechanisms of action of probiotics.⁷⁴

Growth Factors in the Prevention of Necrotizing Enterocolitis: Role for Amniotic Fluid and Breast Milk

Growth factors are essential for development, growth, and health of the gastrointestinal tract, and understanding the ontogeny may help clinicians understand the timing of NEC onset.⁷⁶ The amniotic fluid that bathes the fetal intestines and breast milk that is given after birth contain growth factors for the optimum development and function of the intestine. It can be hypothesized that decreased or absent growth factors during critical stages during fetal or postnatal development may increase the risk of NEC. Growth factors mediate cellular activities, namely cellular proliferation, migration, differentiation, and survival. This research has focused on enterocyte growth and trophic factors such as the epidermal growth factor (EGF) and heparin binding– EGF, but similar beneficial roles have been identified for glucagonlike peptide 2, insulinlike growth factor 1, erythropoietin, growth hormone, hepatocyte growth factor, and even for inflammatory mediators such as CXC chemokines such as IL-8.

The role of amniotic fluid in the prevention of NEC, which contains growth and trophic factors, is being researched.⁷⁰ By urination and swallowing, the fetus contributes to the volume and composition of the amniotic fluid mostly in the second half of pregnancy. Interruption of amniotic fluid flow in the intestines causes mucosal and villus atrophy, which can be reversed by restoring flow of amniotic fluid but not by Ringer lactate.^77,78 This finding shows that trophic factors (insulinlike growth factors I and II, EGF, hepatocyte growth factor) in the amniotic fluid are more essential to fetal gut development and maturation than the volume of the fluid.⁷⁹ In addition, the amniotic fluid also contains cytokines and stem cells (SCs).⁸⁰ Animal studies have shown that amniotic fluid administration, postnatally in mice, decreases TLR4 signaling and inflammation, and enhances repair of the intestine.^71,81–83 However, questions remain regarding collection of amniotic fluid; when, how, what gestation, and when to administer to neonates; sterilization practices; donor amniotic fluid; and potential adverse effects of this intervention.

Human Stem Cells and Their Role in Prevention of Necrotizing Enterocolitis

The use of SCs, which have the potential to help in intestinal restitution from injury, is a promising avenue of research in NEC prevention. SCs produce an array of cytokines, growth factors, microRNAs, and extracellular vesicles that may decrease intestinal injury in animal models of NEC.⁸⁴ Research from the Besner laboratory has reported that different types of SC reduce the incidence and severity of NEC and preserve intestinal barrier function during NEC.^85,86 SCs from the amniotic fluid also protect the intestines from injury and facilitate repair, and hence amniotic fluid therapy holds great promise for prevention of NEC.⁸⁷ Once NEC has set in, rescue therapy is also possible with enteric neural SCs. Administered SCs engraft at low rates and do not explain the beneficial effects of SC administration, and, therefore, SC-secreted factors may be more important. SC-derived exosomes as a paracrine secretion that is responsible for the beneficial effects of SCs is currently being researched.^85,86

BIOMARKER DISCOVERY FOR EARLY DIAGNOSIS AND MONITORING

The signs and symptoms in the early phases of NEC are nonspecific and can be confused with other diseases (spontaneous intestinal perforation [SIP]), ischemic bowel caused by cardiovascular diseases, or food protein–sensitive enteritis. Pneumoperitoneum on the radiograph may also be caused by SIP without intestinal necrosis, and pneumatosis intestinalis may be confused with air bubbles mixed with stool. Ultrasonography examination of the abdomen may be useful but can be subjective, needs expertise to perform and interpret, and becomes more specific in the later stages of the disease. Biomarkers that can diagnose NEC in the early stages of the disease are necessary to differentiate it from other diagnoses. None of the currently available biomarkers have adequate sensitivity or specificity for use in diagnostic tests. Development of a disease-specific screening tool may not only help in the early diagnosis of the disease but also improve the understanding of the pathogenesis and aid targeted treatment.

Biomarkers, end points, and other tools (BEST) defines a biomarker as “a defined characteristic that is, measured as an indicator of normal biological processes, pathogenic processes, or response to an exposure or intervention.”⁸⁸ Molecular, histologic, radiographic, or physiologic characteristics are types of biomarkers. A biomarker would be highly useful in the following clinical scenarios⁸⁹:

1. Risk assessment of likelihood or probability of NEC in all very low birth weight infants.

2. Early NEC diagnosis, before progression of disease to irreversible intestinal injury.

3. Excluding NEC in infants with symptoms suspicious for NEC (ie, high negative predictive value and specificity).

4. Early NEC prognostication, determining which newborns are likely to have disease progression.

CATEGORIES OF BIOMARKERS

1. Biomarkers associated with systemic inflammation: C-reactive protein (CRP),white cell count, thrombocytopenia, procalcitonin, and other cytokines. Increased CRP and procalcitonin levels have been reported to be highly specific for NEC diagnosis in some studies, although these studies do not include a control group of patients with sepsis alone without NEC, leading to misclassification bias.

2. Biomarkers specific for gut injury: calprotectin, intestinal fatty acid binding protein, claudins, and interalpha inhibitor protein.

3. Microbial metabolites including fecal volatile organic compounds (VOCs).

4. Composite of multiple biomarkers; for example, combination of inflammatory, gut injury specific and imaging.

Metabolomics allow identification of distinct patterns of small molecules generated during host and microbial cellular metabolism, which may detect microbiome and metabolomic signatures of dysbiosis.^90–94 Morrow and colleagues⁹⁵ evaluated urinary metabolome patterns in association with gut dysbiosis preceding NEC. Alanine was directly correlated with the relative abundance of Firmicutes, and inversely correlated with the relative abundance of both Proteobacteria and Propionibacterium.⁹⁵ Ratio of alanine to histidine was positively associated with overall NEC and inversely associated with the relative abundance of Propionibacterium. Other studies show differences in metabolites related to carbohydrate, steroid hormone biosynthesis, gluconic acid, leukotriene and prostaglandin metabolism, lineolate metabolism, lipid metabolism, and intracellular signaling between infants with NEC and control infants.^96–98 There is no unifying hypothesis related to altered metabolism in NEC (host or microbial) and integration of all omics platforms, including metabolomics, is necessary to understand the pathophysiology in NEC.

Fecal VOCs are considered to reflect not only gut microbiota composition but also their metabolic activity and concurrent interaction with the host.⁹⁹ A NEC-specific or a sepsis-specific microbial or metabolic signature has not yet been determined. Identification of disease-specific VOCs and microbiota composition may increase understanding on pathophysiologic mechanisms in NEC. To date, only 2 studies report on fecal VOCs in NEC.^99–101 Garner and colleagues¹⁰¹ observed that the number of VOCs increases significantly with age in infants without NEC (n = 7), whereas a reduction was observed in infants with NEC (n = 6) days before diagnosis. The NEC cases did not show 4 specific esters: hexadecenoic acid ethyl ester, 2-ethylhexyl acetic ester, decanoic acid ethyl ester, and dodecanoic acid ethyl ester, which were present in control infants. de Meij and colleagues¹⁰⁰ reported that fecal VOC profiles allowed discrimination between cases (n = 13) and controls (n = 14) up to 3 days before clinical onset of NEC, with increasing accuracy closer to the day of diagnosis.

Currently available biomarkers lack utility⁸⁹ because they have not been rigorously assessed in appropriate samples of preterm infants. They lack validity testing with reproducibility of results; for example, IAIP, hydrogen excretion, VOCs, fecal microbiota analysis, and proteomics. There is an urgent need to evaluate these biomarkers in prospective multicenter studies to assess analytical validity (reproducibility), clinical validity (discrimination and calibration), and ability to generalize.

SUMMARY

This article summarizes the multipronged research being pursued in the domain of NEC. Advances in technology and multiomic platforms of the microbiome, transcriptomics, proteomics, and metabolomics have provided an opportunity to have a holistic view on biological processes in NEC, which can then be targeted to improve clinical outcomes. Multiomics can also provide host-related or microbiome-related biomarkers for early diagnosis and monitoring. However, most of this cutting-edge research needs further evaluation in experimental situations, and then for reproducibility and the ability to generalize in wider clinical applications.

Best Practices.

Best Practices

What is the current practice for [enter disease/conditions]

Best practice/guideline/care path objectives

What changes in current practice are likely to improve outcomes?

Is there a clinical algorithm? [author: either create your own, use from your article, or search for one in an Elsevier publication]

Major recommendations

Describe strength of the evidence

Bibliographic sources [author: this is important to list current sources relevant to evidence]

Summary statement

KEY POINTS.

• Evaluation of microbiome optimization, immunomodulation, growth factors, and human milk glycans, and integration of multiomics data from various platforms as research strategies for prevention of necrotizing enterocolitis (NEC).

• Validation of novel biomarkers for early diagnosis of disease and monitoring in multicentre clinical trials.