Effects of neonatal nutrition interventions on neonatal mortality and child health and development outcomes: A systematic review

Aamer Imdad; Faseeha Rehman; Evans Davis; Deepika Ranjit; Gamael S S Surin; Suzanna L Attia; Sarah Lawler; Abigail A Smith; Zulfiqar A Bhutta

doi:10.1002/cl2.1141

. 2021 Mar 5;17(1):e1141. doi: 10.1002/cl2.1141

Effects of neonatal nutrition interventions on neonatal mortality and child health and development outcomes: A systematic review

Aamer Imdad ^1,^✉, Faseeha Rehman ², Evans Davis ³, Deepika Ranjit ⁴, Gamael S S Surin ⁴, Suzanna L Attia ⁵, Sarah Lawler ⁶, Abigail A Smith ⁷, Zulfiqar A Bhutta ⁸

PMCID: PMC8356300 PMID: 37133295

Abstract

Background

The last two decades have seen a significant decrease in mortality for children <5 years of age in low and middle‐income countries (LMICs); however, neonatal (age, 0–28 days) mortality has not decreased at the same rate. We assessed three neonatal nutritional interventions that have the potential of reducing morbidity and mortality during infancy in LMICs.

Objectives

To determine the efficacy and effectiveness of synthetic vitamin A, dextrose oral gel, and probiotic supplementation during the neonatal period.

Search Methods

We conducted electronic searches for relevant studies on the following databases: PubMed, CINAHL, LILACS, SCOPUS, and CENTRAL, Cochrane Central Register for Controlled Trials, up to November 27, 2019.

Selection Criteria

We aimed to include randomized and quasi‐experimental studies. The target population was neonates in LMICs. The interventions included synthetic vitamin A supplementation, oral dextrose gel supplementation, and probiotic supplementation during the neonatal period. We included studies from the community and hospital settings irrespective of the gestational age or birth weight of the neonate.

Data Collection and Analysis

Two authors screened the titles and extracted the data from selected studies. The risk of bias (ROB) in the included studies was assessed according to the Cochrane Handbook of Systematic Reviews. The primary outcome was all‐cause mortality. The secondary outcomes were neonatal sepsis, necrotizing enterocolitis (NEC), prevention and treatment of neonatal hypoglycaemia, adverse events, and neurodevelopmental outcomes. Data were meta‐analyzed by random effect models to obtain relative risk (RR) and 95% confidence interval (CI) for dichotomous outcomes and mean difference with 95% CI for continuous outcomes. The overall rating of evidence was determined by the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach.

Main Results

Sixteen randomized studies (total participants 169,366) assessed the effect of vitamin A supplementation during the neonatal period. All studies were conducted in low‐ and middle‐income (LMIC) countries. Thirteen studies were conducted in the community setting and three studies were conducted in the hospital setting, specifically in neonatal intensive care units. Studies were conducted in 10 different countries including India (four studies), Guinea‐Bissau (three studies), Bangladesh (two studies), and one study each in China, Ghana, Indonesia, Nepal, Pakistan, Tanzania, and Zimbabwe. The overall ROB was low in most of the included studies for neonatal vitamin A supplementation. The pooled results from the community based randomized studies showed that there was no significant difference in all‐cause mortality in the vitamin A (intervention) group compared to controls at 1 month (RR, 0.99; 95% CI, 0.90–1.08; six studies with 126,548 participants, statistical heterogeneity I ² 0%, funnel plot symmetrical, grade rating high), 6 months (RR, 0.98; 95% CI, 0.89–1.07; 12 studies with 154,940 participants, statistical heterogeneity I ² 43%, funnel plot symmetrical, GRADE quality high) and 12 months of age (RR, 1.04; 95% CI, 0.94–1.14; eight studies with 118,376 participants, statistical heterogeneity I ² 46%, funnel plot symmetrical, GRADE quality high). Neonatal vitamin A supplementation increased the incidence of bulging fontanelle by 53% compared to control (RR, 1.53; 95% CI, 1.12–2.09; six studies with 100,256 participants, statistical heterogeneity I ² 65%, funnel plot symmetrical, GRADE quality high). We did not identify any experimental study that addressed the use of dextrose gel for the prevention and/or treatment of neonatal hypoglycaemia in LMIC. Thirty‐three studies assessed the effect of probiotic supplementation during the neonatal period (total participants 11,595; probiotics: 5854 and controls: 5741). All of the included studies were conducted in LMIC and were randomized. Most of the studies were done in the hospital setting and included participants who were preterm (born < 37 weeks gestation) and/or low birth weight (<2500 g birth weight). Studies were conducted in 13 different countries with 10 studies conducted in India, six studies in Turkey, three studies each in China and Iran, two each in Mexico and South Africa, and one each in Bangladesh, Brazil, Colombia, Indonesia, Nepal, Pakistan, and Thailand. Three studies were at high ROB due to lack of appropriate randomization sequence or allocation concealment. Combined data from 25 studies showed that probiotic supplementation reduced all‐cause mortality by 20% compared to controls (RR, 0.80; 95% CI, 0.66–0.96; total number of participants 10,998, number needed to treat 100, statistical heterogeneity I ² 0%, funnel plot symmetrical, GRADE quality high). Twenty‐nine studies reported the effect of probiotics on the incidence of NEC, and the combined results showed a relative reduction of 54% in the intervention group compared to controls (RR, 0.46; 95% CI, 0.35–0.59; total number of participants 5574, number needed to treat 17, statistical heterogeneity I ² 24%, funnel plot symmetrical, GRADE quality high). Twenty‐one studies assessed the effect of probiotic supplementation during the neonatal period on neonatal sepsis, and the combined results showed a relative reduction of 22% in the intervention group compared to controls (RR, 0.78; 95% CI, 0.70–0.86; total number of participants 9105, number needed to treat 14, statistical heterogeneity I ² 23%, funnel plot symmetrical, GRADE quality high).

Authors' Conclusions

Vitamin A supplementation during the neonatal period does not reduce all‐cause neonatal or infant mortality in LMICs in the community setting. However, neonatal vitamin A supplementation increases the risk of Bulging Fontanelle. No experimental or quasi‐experimental studies were available from LMICs to assess the effect of dextrose gel supplementation for the prevention or treatment of neonatal hypoglycaemia. Probiotic supplementation during the neonatal period seems to reduce all‐cause mortality, NEC, and sepsis in babies born with low birth weight and/or preterm in the hospital setting. There was clinical heterogeneity in the use of probiotics, and we could not recommend any single strain of probiotics for wider use based on these results. There was a lack of studies on probiotic supplementation in the community setting. More research is needed to assess the effect of probiotics administered to neonates in‐home/community setting in LMICs.

1. PLAIN LANGUAGE SUMMARY

1.1. Neonatal probiotic supplementation can improve infant illness and reduce death, but vitamin A does not, and may have adverse effects

Nutritional support during the 1st month of life is vital for the short‐ and long‐term survival of the newborn. Neonatal nutrition interventions have the potential to decrease death and illness in young infants in LMICs.

1.1.1. What is this review about?

This review assesses the efficacy of synthetic vitamin A, dextrose, and probiotic supplementation during the neonatal period. These interventions were assessed separately and not in combination with each other.

What is the aim of this review?

This Campbell systematic review assesses the efficacy of three neonatal nutritional interventions that have the potential of reducing morbidity and mortality during infancy in LMICs: synthetic vitamin A, dextrose, and probiotic supplementation.

1.1.2. What studies are included?

Sixteen studies that assessed the effect of vitamin A supplementation during the neonatal period were included. Thirteen of these studies were conducted in the community setting and three studies were conducted in the hospital setting. All the included studies on neonatal vitamin A supplementation were conducted in LMICs. Most of the studies had a low ROB.

No experimental studies were found that evaluated the use of dextrose for the prevention or treatment of low blood sugar during the neonatal period.

Thirty‐three studies assessed the use of probiotics during the 1st month of life. All included studies on probiotic supplementation were randomized and conducted in LMICs. Most of the included studies had a low ROB. The probiotics studies mainly included babies born early and/or with low birth weight, and these studies were mostly conducted in hospital settings.

1.1.3. Key results

Combined results from thirteen vitamin A studies conducted in the community settings showed that there was no significant effect of vitamin A supplementation for reduction of death in young infants at 1, 6, or 12 months of age. Neonatal vitamin A supplementation increases the risk of bulging fontanelle by 53%. The pooled data from probiotics studies showed that this intervention reduced the risk of death by 20% compared to controls. Further analysis showed that compared to controls, probiotic supplementation reduced the risk of a severe form of gastrointestinal illness in neonates called NEC by 54%. Probiotic supplementation also reduced the risk of blood infection called sepsis by 22% compared to controls. The quality grade ratings for these outcomes were “high.”

1.1.4. What are the main findings of the review?

The pooled data from probiotics studies showed that this intervention reduced the risk of death by 20% compared to controls. Further analysis showed that compared to controls, probiotic supplementation reduced the risk of a severe form of gastrointestinal illness in neonates called NEC by 54%. Probiotic supplementation also reduced the risk of blood infection called sepsis by 22% compared to controls. The quality grade ratings for these outcomes were “high.”

1.1.5. What do the findings of this review mean?

Vitamin A supplementation during the 1st month of life does not reduce the risk of death during the 1st year of life in LMICs. However, neonatal vitamin A supplementation increases the risk of bulging fontanelle, which may cause damage to the brain.

We did not find any experimental studies from LMICs that assessed the use of dextrose gel supplementation during the 1st month of life for the prevention or treatment of low blood sugar.

Probiotic supplementation during the 1st month of life to babies born preterm and/or low birthweight can reduce the risk of death, blood infection and bowel sickness (NEC).

There was clinical heterogeneity in the use of probiotics and we could not recommend any single strain or combination of probiotics for wider use based of these results.

There is a lack of studies on probiotic supplementation in the 1st month of life in community settings. More research is needed to assess the effect of probiotics administered to neonates in home/community settings in LMICs.

1.1.6. How up‐to‐date is this review?

The review authors searched for studies published up to November 2019.

2. BACKGROUND

The decline in rates of neonatal (age, 0–28 days) mortality has been slower than the decline in child mortality between 1990 and 2016 (Alkema et al., 2014; Bhutta et al., 2015). Neonatal mortality accounted for 46% of child mortality in 2016 compared to 40% of all under‐five mortality rates in 1990 (WHO, 2017a). Globally, the percentage of neonatal mortality is the highest in South Asia and Sub‐Saharan Africa (Alkema et al., 2014). Optimal nutritional support during the neonatal period is vital to the short and long term survival of the newborn (Bhutta et al., 2013; WHO, 2017b). Poor nutritional status of neonates is a major cause of illness and can lead to poor growth, increased risk of infection, bleeding, and neonatal death (Bhutta et al., 2013; WHO, 2017b). The risk of morbidity and mortality during the neonatal period is higher in LMICs where many births happen at home and the prevalence of maternal malnutrition and incidence of low birth weight (birth weight <2500 g) and preterm birth (gestational age <37 weeks) is high (Bhutta et al., 2013; Lee et al., 2017; WHO, 2017b). This review focused on three nutritional interventions during neonatal periods that have the potential to reduce illness and death during infancy in LMIC.

2.1. Description of the condition

The approach to nutritional management of newborn depends on maternal nutritional status, comorbidities during pregnancy (such as gestational diabetes), pregnancy duration (term vs. preterm birth), events at birth (such as birth asphyxia), birth weight (low birth weight vs. normal birth weight) and available resources for postpartum care of the mother and the baby (such as skilled birth attendant, home vs. facility birth, availability of neonatal intensive care) (Bhutta et al., 2013; WHO, 2015, 2017a, 2017b). The most important nutritional intervention after birth is breastfeeding, which is covered in a separate Campbell review of this series. There are a number of other nutritional interventions that have been proposed in addition to breastfeeding. It is beyond the scope of this review to comprehensively evaluate all the possible nutritional interventions during the neonatal period. We limited our review to the following three interventions: neonatal synthetic vitamin A supplementation, oral dextrose gel supplementation, and probiotic supplementation during the neonatal period in LMIC. Below in this section and in the rest of the introduction, we describe the rationale and importance of reviewing these interventions.

2.1.1. Neonatal vitamin A deficiency (VAD)

Globally, about 190 million children and 19.1 million pregnant women are vitamin A deficient based on serum retinol levels (i.e., serum retinol <0.70 μmol/L) (WHO, 2009a). VAD is most prevalent in South Asia and Africa (Stevens et al., 2015). VAD is associated with increased risk of blindness, infections, and mortality (Imdad et al., 2017). Most of the newborns are vitamin A deficient and rely on supplementation from maternal breast milk (Haider et al., 2017). High prevalence of maternal VAD in LMICs increases the risk of neonatal VAD. There has been interest in vitamin A supplementation during neonatal period to assess if it reduces risk of illness and death (Haider et al., 2017; WHO, 2009b), as it has been shown to reduce morbidity and mortality in children 6–59 months of age (Imdad et al., 2017).

2.1.2. Hypoglycemia during the neonatal period

Hypoglycemia (low blood sugar) is common during the immediate neonatal period. The definition of neonatal hypoglycaemia varies. The American Academy of Pediatrics defines neonatal hypoglycaemia as blood glucose below 47 mg/dl (2.61 mmol/L); however, other societies such as the Pediatric Endocrine Society define neonatal hypoglycaemia as blood glucose <50 mg/dl (2.77 mmol/L; Thompson‐Branch & Havranek, 2017; Thornton et al., 2015). Recurrent, severe, and/or persistent hypoglycaemia can lead to complications such as death; there is limited evidence to show that blood sugars below a certain level leads to long‐term brain damage (Kaiser et al., 2015; McKinlay et al., 2017; Thornton et al., 2015). About 10–15% of otherwise healthy newborns have low blood sugar, and the rate is much higher among infants with additional risk factors such as large for gestational age, small for gestational age, low birth weight, preterm birth, infant of diabetic mother, and newborns with perinatal asphyxia (Thompson‐Branch & Havranek, 2017). Additional risk factors for neonatal hypoglycaemia include neonatal sepsis, prolonged labor, and maternal medication use such as use of β‐agonists and β‐blockers (Thompson‐Branch & Havranek, 2017). The recommended initial intervention to treat neonatal hypoglycaemia is to offer feeding in the form of breastfeeding followed by formula feeding if breastfeeding is unsuccessful. Persistent hypoglycaemia may require IV dextrose supplementation and admission to a neonatal intensive care unit (Thompson‐Branch & Havranek, 2017; Thornton et al., 2015). In LMIC, where a significant proportion of births happen at home and incidence of low birth weight and preterm birth is high, prevention and treatment of hypoglycaemia encounters additional challenges (Singhal et al., 1991, 1992; WHO, 2017b; Williams, 1997). The instruments to test blood sugar might not be available in low‐resource settings; In addition, formula, IV dextrose, and intensive care units might not be available to treat persistent and/or severe hypoglycaemia. Recent studies have tested simple interventions such as oral dextrose gel to treat neonatal hypoglycaemia and to prevent hypoglycaemia in high‐risk newborns (Hegarty et al., 2016; Weston et al., 2016).

2.1.3. Neonatal sepsis and NEC

Neonatal sepsis and NEC are neonatal morbidities that can be fatal (Oza et al., 2015; WHO, 2017b). Neonatal sepsis is the presence of an infection leading to systemic illness. Bacterial sepsis is common in LMIC and is a significant risk factor of morbidity and mortality in these countries (WHO, 2017a). NEC is a condition that occurs in newborns and can lead to intestinal injury and death. The extent of injury may vary from mucosal injury to full thickness intestinal wall injury. NEC happens most commonly in preterm babies and especially in extremely preterm babies (<28 weeks gestational age; AlFaleh & Anabrees, 2014; Patel & Denning, 2015). Multiple factors lead to the development of NEC in preterm infants including altered bacterial gut flora affecting the protective intestinal barrier, decreased intestinal motility, and the increased susceptibility of preterm infants to inflammation and infections (Patel & Denning, 2015). Recent studies have shown that an imbalance between commensal bacteria and pathogenic bacteria (intestinal dysbiosis) makes babies vulnerable to pathogenic bacterial growth in the intestine which then causes inflammation that may contribute to neonatal sepsis and/or NEC (Arrieta et al., 2014; Deshmukh et al., 2014; Gewolb et al., 1999; Panigrahi et al., 2017). There is an increasing interest in correction of intestinal dysbiosis by probiotics to prevent NEC and neonatal sepsis. Data from early studies on probiotic use in neonates from LMIC is encouraging (AlFaleh & Anabrees, 2014; Rao et al., 2016).

2.2. Description of the intervention

2.2.1. Neonatal vitamin A supplementation

Vitamin A is a term used for a subclass of the family of fat soluble compounds named retinoic acids. Vitamin A is found in nature in two forms, provitamin A carotenoids and preformed vitamin A, which is essential to human bodily function. Plant‐based foods are the main source of provitamin A carotenoids, of which β‐carotene is the most commonly known. Animal‐based foods are the main source of preformed vitamin A (Bates, 1995; Haider & Bhutta, 2011). Vitamin A from animal sources (retinol, retinal, retinoic acid, and retinyl esters) is the most active form, and synthetic vitamin A retinol has been used in most intervention trials in the past (Haider & Bhutta, 2011; Imdad et al., 2017). Plant‐based foods may not be an adequate source of vitamin A, as the gastrointestinal conversion ratio from carotenoid‐to‐retinol varies from 6:1 to 26:1 (US Institute of Medicine, Food and Nutrition Board). VAD may, therefore, exist in areas even when there is high consumption of plant‐based foods such as in South Asia and Africa (Imdad et al., 2017; Stevens et al., 2015).

2.2.2. Oral dextrose gel supplementation during neonatal period

Dextrose gel is a thickened aqueous solution that contains the concentrated simple carbohydrate dextrose. It can be administered by direct application to oral, buccal, or sublingual mucosa and can increase blood sugars rapidly by absorption through the highly vascularized and thin mucus membranes of the oral mucosa (Hegarty et al., 2016). Detxrose gel is a low cost nonproprietary intervention, and the gel can be prepared in hospital pharmacies. The typical ingredients include water, glucose, a gelling agent, and preservatives (Hegarty et al., 2016). The decision to use dextrose gel in a neonate should be taken on individual basis and should be avoided in neonates with compromised neurological or respiratory status (Hegarty et al., 2016; Weston et al., 2016).

2.2.3. Probiotic supplementation during neonatal period

Prebiotics are supplements that promote the growth of commensal bacteria (AlFaleh & Anabrees, 2014; Panigrahi et al., 2017). Probiotics contain live bacteria that enrich pools of commensal bacteria (AlFaleh & Anabrees, 2014; Millar et al., 2003; Panigrahi et al., 2017). Synbiotics are a combination of prebiotics and probiotics and might have synergistic effect (Johnson‐Henry et al., 2016, Nandhini et al., 2016; Panigrahi et al., 2017). These supplements are meant to optimise gut health and their hypothesized mechanisms of actions include enhanced gut barrier function, inhibition of gut colonization with pathogenic bacteria, improvement in colonization with healthy commensal bacteria that protect the infant from enteropathogenic infection through production of acetate, enhanced innate immunity, and increased maturation of the enteric nervous system (Rao et al., 2016). Recent data have shown that probiotic supplements can prevent the incidence of NEC in preterm babies (AlFaleh & Anabrees, 2014; Millar et al., 2003; Patel & Denning, 2015; van den Akker et al., 2018). There is also promising data on use of probiotocs/synbiotics for prevention of neonatal sepsis (Rao et al., 2016; Panigrahi et al., 2017). The most commonly used strains of probiotics include Lactobacillus and Bifidobacterium (Rao et al., 2016).

2.3. How the intervention might work

2.3.1. Neonatal vitamin A supplementation

Vitamin A has an effect on cell differentiation and helps maintain normal functioning of epithelial cells (Bates, 1995; Bhutta et al., 2013; Haider & Bhutta, 2011). It is considered anti‐infective because it helps to maintain the protective epithelial barrier of the skin and mucosa, which protects the body from infections. Vitamin A helps in the regeneration of the epithelium and therefore maintains the integrity of the body's first line of defence. These mechanisms may help prevent infections in newborns (McCullough et al., 1999; Wolbach, 1933). Synthetic vitamin A supplementation has been shown to reduce morbidity and mortality in children 6–59 months of age (Imdad et al., 2017). The potential side effects of synthetic vitamin A supplementation include vomiting and bulging fontanelle (Imdad et al., 2016, 2017; Haider & Bhutta, 2011; Haider et al., 2017). Excess vitamin A supplementation can cause toxicity that presents in the form of a bulging fontanelle in children under 1 year, headaches, vomiting, diarrhea, loss of appetite, and irritability (Haider et al., 2017; Imdad et al., 2017).

2.3.2. Oral dextrose gel supplementation during neonatal period

The absorption of dextrose gel through the oral mucosa leads to entry of glucose into lingual veins and into the internal jugular vein. This pathway provides almost immediate delivery of glucose to the systemic circulation and bypasses first pass liver metabolism through the portal circulation. If proven effective in preventing and treating hypoglycaemia, dextrose gel can avoid the need of intravenous glucose and reduce separation of baby from mother (Hegarty et al., 2016; Weston et al., 2016). The intervention is simple enough that it does not require special skills (such as IV placement) and can be administered by community, lay health workers, or the caregiver herself. Potential adverse effects include vomiting, choking, gagging, respiratory distress, and delay of treatment for severe hypoglycaemia (Hegarty et al., 2016; Weston et al., 2016).

2.4. Probiotics supplementation during neonatal period

Newborn, and especially preterm, babies have immature intestines free of normal commensal bacteria that would normally protect them from developing NEC and sepsis by inhibiting the growth of pathogenic bacteria in the intestines (AlFaleh & Anabrees, 2014; Patel & Denning, 2015; Rao et al., 2016). Probiotics are used to proactively colonize the intestines with beneficial bacteria such as Lactobacillus species (Millar et al., 2003; Patel & Denning, 2015). Probiotics therefore reduce the growth of pathogenic bacteria which would otherwise increase the risk of NEC and sepsis. Also probiotics promote gut immunity by increasing IgA levels and contributing to improved mucosal barrier function (Patel & Denning, 2015). These protective mechanisms reduce intestinal permeability by producing a protective mucosal barrier against bacteria and increase the production of anti‐inflammatory cytokines (Deshpande et al., 2017; Millar et al., 2003). Probiotics are especially protective in preterm babies with immature intestinal microbiomes and neonates on antibiotics; antibiotics may reduce bacterial diversity in the intestine and thus also dispose to colonization by pathogenic bacteria causing NEC. Prebiotics and probiotics can be given together in the form of a synbiotic to improve the gut flora and can potentially reduce all‐cause neonatal mortality (Johnson‐Henry et al., 2016; Panigrahi et al., 2017). Probiotics are considered safe; however, there are concerns regarding probiotic supplementation in extremely premature or immunocompromised neonates. A few cases of neonatal sepsis have been reported that were thought to be caused by probiotics (Dani et al., 2016).

2.5. Why it is important to do this review

2.5.1. Neonatal vitamin A supplementation

Randomized trials on neonatal vitamin A supplementation have produced conflicting results with some studies (mostly from South Asia) showing a mortality benefit while no major benefit in other studies (mostly from Africa) (Haider et al., 2017) and some studies showing even an increased risk of infant mortality in certain populations (Smith et al., 2016).The exact reason for this difference in results is not clear. Previous reviews (Haider et al., 2017; Gogia & Sachdev, 2009) and a WHO technical consultation (WHO, 2009b) have hypothesized on what factors may explain these varied results. Our group has previously published a Cochrane review on the evidence on neonatal vitamin A supplementation (Haider & Bhutta, 2011), and we wanted to update the previous review. The previous review included studies conducted in the community setting. In this review, we considered studies conducted in both the community and the hospital setting in LMIC. We also included neurodevelopment outcomes for this review that were not covered in the previous Cochrane review.

2.5.2. Oral dextrose gel supplementation during neonatal period

Oral dextrose gel has been studied in the prevention and treatment of neonatal hypoglycaemia in high‐income countries (Hegarty et al., 2017; Weston et al., 2016); however, it was not clear if similar studies were available from LMICs. Our objective was to consider both randomized and nonrandomized observational studies with a control arm. We hypothesized that the use of dextrose may be more beneficial in LMIC than in high‐income countries, as the incidence of neonatal hypoglycaemia might be higher in these countries due to an increased rate of preterm and low birth weight birth.

2.5.3. Probiotics supplementation during neonatal period

The effect of probiotic supplementation for the prevention of NEC and neonatal sepsis has been assessed in previous reviews (AlFaleh & Anabrees, 2014; Rao et al., 2016; van den Akker et al., 2018). Most of these reviews included studies from both high‐ and LMICs. Deshpande et al. (2017) reviewed studies from LMIC where neonates were supplemented with probiotics. More studies (Amini et al., 2017; Chowdhury et al., 2016; Guney‐Varal et al., 2017; Hernández‐Enríquez et al., 2016; Hussain et al., 2016) have been published since the publication of Deshpande et al.'s (2017) review. Overall, our objective was to assess the current evidence for the effect of probiotic supplementation during the neonatal period in the hospital and community setting in LMIC.

3. OBJECTIVES

3.1. Primary objectives

To determine the efficacy of the following interventions on neonatal morbidity and mortality:

1.
Synthetic vitamin A supplementation,
2.
Oral dextrose gel supplementation, and
3.
Oral probiotic supplementation.

A detailed description of background and methods for this review was published in the form of a protocol as Imdad et al. (2019).

4. METHODS

4.1. Criteria for considering studies for this review

4.1.1. Types of studies

We included the following study designs:

Randomized controlled trials (RCTs), where participants were randomly assigned either individually or in clusters to intervention and comparison groups. Cross‐over designs were also eligible for inclusion.
Quasi‐experimental designs, which include:

a.
Natural experiments: studies where nonrandom assignment was determined by factors that were out of the control of the investigator. One common type includes allocation based on exogenous geographical variation.
b.
Controlled before‐after studies (CBA), in which measures were taken of an experimental group and a comparable control group both before and after the intervention. We also require that appropriate methods were used to control for confounding, such as statistical matching (e.g., propensity score matching or covariate matching) or regression adjustment (e.g., difference‐in‐differences, instrumental variables).
c.
Regression discontinuity designs; here, allocation to intervention/control was based upon a cut‐off score.
d.
Interrupted time series studies, in which outcomes were measured in the intervention group at at least three time points before the intervention and after the intervention.

4.1.2. Types of participants

Participants for this review included neonates (aged 0–28 days) from LMICs. We included neonates regardless of their health status. This includes low birth weight and preterm babies. However, studies that focused on neonates with congenital anomalies were excluded. We considered studies that included older age population groups in addition to neonates only if we could disaggregate relevant data for the neonatal population. For example, a study might include infants up to 6 months of age. We included such a study if the disaggregated data were available for neonates (0–28 days). Even though we planned to assess later childhood outcomes, we did not plan to include studies that recruited participants after the neonatal period.

4.1.3. Types of interventions

The following interventions were included in the review:

1.
Neonatal vitamin A supplementation compared to no supplementation or placebo: we considered only oral synthetic vitamin A supplementation. There was no restriction on the dosage and frequency of the medicine. The comparison group could include a placebo or standard of care.
2.
Oral dextrose gel supplementation during the neonatal period compared to no supplementation: we placed no limits on the dose or frequency of the dextrose supplementation. We only considered dextrose gel as the intervention and excluded dextrose given in other forms such as intravenous, nasogastric tube, or mixed with infant formula. The reason to exclude forms other than dextrose gel was that administration of dextrose in those forms may require special circumstances (like trained staff to place an IV) or special delivery vehicles, such as formula, that may not be available in LMIC. The comparison group included placebo or standard of care.
3.
Neonatal oral probiotics/synbiotics compared to no probiotic supplementation: probiotics are live microbial organisms that are given to promote the growth of commensal gut bacteria and prevent the growth of pathogenic bacteria. Prebiotics are dietary supplements that promote the growth of commensal bacteria. Synbiotics are a combination of prebiotics and probiotics (Millar et al., 2003; Patel & Denning, 2015). We placed no limits on the dose or frequency of probiotics. We included studies that used probiotics and synbiotics supplementation and excluded studies that used only prebiotics. Comparison groups included placebo or standard of care.

Each of the above interventions (i.e., vitamin A, dextrose, or probiotics) was summarized separately, and the interventions were not compared to each other directly or indirectly.

4.1.4. Types of outcome measures

Primary outcomes

The primary outcomes were:

1.
All‐cause neonatal mortality (death between 0 and 28 days of life)
2.
All cause infant mortality at 6 months (death between 0 days and 6 months of life)
3.
All‐cause infant mortality at 12 months (death between 0 days and 12 months life).

We anticipated that studies might not report the outcomes in the follow‐up period mentioned above for the primary outcomes. If a study did not report mortality outcomes at day 28, 6 months, or 12 months, we contacted authors for data for the same. If segregated data were not available from authors, we included mortality data as follows: mortality in the first 6 weeks of life was included as neonatal mortality at day 28; between 3 and 6 months was included as 6 months, and between 9 and 12 months was included as 12 months. If the follow up was not clear, we included the mortality data at the longest follow‐up.

Secondary outcomes

The secondary outcomes included:

1.
Sepsis‐specific mortality measured between 0 and 28 days, 0 days and 6 months, and 0 days and 12 months of life
2.
Neonatal sepsis (as defined by authors) in the first 6 weeks of life
3.
NEC as defined by authors
4.
VAD
5.
Prevention of Hypoglycemia (as defined by authors) during the neonatal period
6.
Treatment of Hypoglycemia (recurrence of hypoglycaemia after the episode treated)
7.
Any adverse reactions during the intervention period
8.
Serious adverse events
9.
Neurodevelopmental outcomes at 12 and 24 months and at the longest follow‐up.

The term neurodevelopment is a composite term that refers to cognitive, neurologic, and/or sensory outcomes. The term neurodevelopment may include intellectual disability as measured on the Mental Developmental Index of the Bayley Scales of Infant Development; gross motor delay measured on Gross Motor Function Classification System, and hearing and vision loss requiring amplification devices.

In order to be eligible for inclusion in the review, a study should have reported at least one of the primary or secondary outcomes. This was assessed at the full‐text review stage.

Duration of follow‐up

We included all participants in eligible studies that had outcomes of interest measured. There were no restrictions based on the duration of exposure, duration of follow‐up, or timing of the outcome measurement. If the duration of treatment exceeded the neonatal period (i.e., 28 days), we considered another 2 weeks maximum but did not include studies in which the treatment went beyond 6 weeks of supplementation. We included mortality outcomes measured at 28 days, 6 months, 12 months of life, and at the longest follow‐up as reported by authors.

Type of settings

We included studies conducted in LMIC. Low‐income countries were defined as those with a gross national income (GNI) per capita of USD 1005 or less in 2016, and middle‐income economies were those with a GNI per capita between USD 1006 and 3955 in 2016 (World Bank, 2017).

4.2. Search methods for identification of studies

The identification of studies included various methods, such as electronic and other sources. We did not exclude any based on the outcome at the screening stages.

4.2.1. Electronic searches

The electronic search for relevant studies was done in the following databases: PubMed, CINAHL, LILACS, SCOPUS, and CENTRAL (Cochrane Central Register for Controlled Trials).

Appendix 1 gives the search strategy for PubMed, CINAHL, LILACS, SCOPUS, and CENTRAL. It includes keywords and MeSH terms as appropriate. This approach includes a search strategy for the population (neonates) and interventions of interest. We planned to run searches for each intervention separately. We first ran the search for the population, which is the same for each intervention. Then we ran the search for each intervention. We then combined both searches by using “AND” and kept the searches in a separate EndNote file.

An example of a search strategy for vitamin A for PubMed was as follows:

(((((“Vitamin A”[Mesh]) OR (Vitamin A[tiab] OR Aquasol A[tiab] OR Retinol[tiab] OR All Trans Retinol[tiab] OR All‐Trans‐Retinol[tiab] OR Vitamin A1[tiab] OR Vitamin A 1[tiab] OR 11‐cis‐Retinol[tiab] OR 11 cis Retinol[tiab] OR Tretinoin[tiab])AND Supplement*[tiab]))AND ((“Infant”[Mesh] OR “Premature Birth”[Mesh]) OR (Neonat*[tiab] OR neo nat*[tiab]) OR (newborn* OR new Born*[tiab] OR newly born*[tiab]) OR (preterm[tiab] OR preterms[tiab] OR pre term[tiab] OR pre terms[tiab]) OR (premature*[tiab] AND (birth*[tiab] OR born[tiab] OR deliver*[tiab])) OR (low[tiab] AND (birthweight*[tiab] OR birth weight*[tiab])) OR (lbw[tiab] OR vlbw[tiab] OR elbw[tiab]) OR infant*[tiab] OR (baby[tiab] OR babies[tiab])))) NOT (“Animals”[Mesh] NOT (“Animals”[Mesh] AND “Humans”[Mesh]))

We applied restriction of “humans” to searches. We did not apply any restrictions on searches based on outcomes, study design, or language. There was no restriction on date of publication.

The searches were conducted for vitamin A on December 10, 2018 (updated on November 13, 2019); probiotics on February 8, 2019 (updated on November 27, 2019); and dextrose on April 25, 2019 (updated on November 26, 2019).

4.2.2. Searching other resources

Other resources included the search for ongoing trials at www.clinicaltrials.gov and WHO's ICTRP trials database. We also searched websites of international agencies such as WHO (including WHO's Reproductive Health Library), UNICEF, Global Alliance for Improved Nutrition (GAIN), International Food Policy Research Institute, International Initiative for Impact Evaluation (3ie), Nutrition International (NI), World Bank, USAID and USAID affiliates (e.g., FANTA, SPRING), and the World Food Programme.

Grey literature search sources included NI, GAIN, International Food Policy and Research Institute (IFPRI), and the WHO library database (WHOLIS).

We searched the reference lists of all included studies. We did citation searches of included studies in Google Scholar and Web of Science. We also searched the reference sections of previously published systematic reviews and the latest published studies. We contacted the experts and authors of the newest published studies to ask about any additional studies. Duplicates were removed.

4.3. Data collection and analysis

4.3.1. Selection of studies

Two authors independently screened titles/abstracts using prespecified inclusion/exclusion criteria. A full text was reviewed for the studies selected in the initial screening, and the same inclusion/exclusion criteria were applied. If there was a conflict about the inclusion of a study between the two reviewers, a third reviewer (ZAB) was consulted. We used a web‐based software “Covidence” (Covidence, 2019) to do both title/abstract and full‐text screening. This software allows simultaneous independent screening of studies, and inter‐reviewer reliability can be assessed by checking the number of conflicts in the resolved conflict page following each stage of screening.

Description of methods used in primary research

We expected that the majority of the included studies would be randomized or cluster‐randomized. We extracted the information on study design explicitly and made a careful differentiation between experimental and observational studies. We aimed to analyze randomized and nonrandomized studies separately.

Criteria for determination of independent findings

We anticipated that authors might report the results of a study in multiple publications. We coded such trials as a single study to avoid double counting of the data and included all the relevant outcomes decided a priori for this review. If a pilot study was done before the larger study, we included the two studies separately unless the data from the pilot study was included in the main trial. When a clinical trial registration number was available for a study, we searched that number on PubMed to locate all the published studies linked to that trial number.

4.3.2. Data extraction and management

Details of study coding categories

The data from included studies were abstracted into a standardized data abstraction form by two authors. We extracted data in duplicates, and any discrepancies were resolved by discussion firs. A third reviewer (ZAB) was consulted if the conflict existed after the initial discussion.

The data extraction sheet had the following information.

General study information: authors, publication year, study design
Study setting: World Bank region, country, World Bank income level, city/town, urban/urban slum/rural/mixed setting, duration of data collection, date of data collection
Study population: sample size recruited, sample size analysed, female (%), description of participants (i.e., inclusion/exclusion criteria applied to recruitment)
Intervention characteristics: type of intervention, duration of intervention, unit of randomization (where applicable), dose, frequency of provision, duration of follow up, attrition rate
Quality assessment

Each quantitative outcome sheet contained the following:

Subgroup (if applicable)
Subgroup sample size
Outcome type
Outcome units
Outcomes

a.
Outcome measure treatment group
b.
Outcome measure comparison group
c.
Standard deviation

Effect size:

a.
Effect measure
b.
95% CI

4.3.3. Assessment of ROB in included studies

We used the Cochrane ROB tool (Higgins & Green, 2011) for randomized studies. The Cochrane ROB tool includes the following items:

Selection bias: random sequence generation and allocation concealment
Performance bias: blinding of participants and personnel
Detection bias: blinding of outcome assessment
Attrition bias: incomplete outcome data
Reporting bias: selective reporting
Other sources of bias

Two authors independently performed the ROB assessments for each study. A third reviewer was involved to resolve any disagreements (ZAB). An overall score was not provided.

4.3.4. Measures of treatment effect

We performed a meta‐analysis for the synthesis of quantitative data when the included studies had comparable participants, interventions, and outcomes. We did not assess the effect on outcome across the interventions, such as is done in network meta‐analysis. Each intervention was analysed separately. We analysed continuous and dichotomous data separately. For dichotomous outcomes, results were presented as summary risk ratios with 95% CI. We combined risk ratios (events per child) and rate ratios (events per child year) for incidence data because of their similar interpretation and scale. For continuous outcomes, we presented the summary results as the mean difference with 95% CI when data were available on the same scale across the studies. We used the standardized mean difference with 95% CI when data were presented in different scales across the studies.

To avoid reviewer bias, we planned to predetermine the preference for specific data for certain outcomes. For example, for mortality outcomes, we gave preference to denominators in the following order: number with the definite outcome known, number randomized, and child‐years. For morbidity data such as neonatal sepsis where both survivors and nonsurvivors might have contributed data, we gave preference to child years, number with the definite outcome known, and number randomized. For randomized trials, we gave preference to data that required the least manipulation by authors or inference by reviewers. We extracted the raw values (e.g., means and SDs) and built the intention‐to‐treat (ITT) analysis where applicable.

We anticipated that cause‐specific morbidity or mortality data might not be readily available, as febrile illness due to respiratory, urinary, or central nervous system infection during the neonatal period are often categorized under a broader term of neonatal sepsis (WHO, 2017b).

4.3.5. Unit of analysis issues

As we planned to include multiple interventions, all interventions and outcomes within those interventions, were meta‐analyzed separately.

For randomized trials, we meta‐analyzed individual and cluster‐randomized trials in the same analysis. We assessed analyses in the cluster‐randomized trials to ensure that clustering was appropriately accounted for within the analysis of the primary study, such that study precision was not over or under‐estimated within our analysis. If the authors adjusted for cluster randomization, no further adjustment was made. In case a cluster‐randomized study was not adjusted by primary authors, we adjusted effect estimates by using the mean cluster size (M) and the intra‐cluster correlation coefficient (ICC) to calculate the design effect as follows: design effect = 1 + (M − 1) ICC. We then used the design effect to adjust the study data such that a trial was reduced to its effective sample size or standard error of the summary estimate was inflated. We used the ICC given in the published studies. If the ICC was not available from the published study, we contacted the authors for the same. If the ICC was not available from the authors, we used ICC from the similar studies done in the similar region and on a similar population or took it from the previously published reviews (Haider et al., 2017).

Multiple‐arm trials

We included studies with multiple intervention arms, but we only included the arms that were eligible for the review. We selected one pair (with appropriate intervention and control group) that satisfied the inclusion criteria of the review and excluded the rest. In case there were more than two groups eligible for inclusion, we combined these groups into a single pair‐wise comparison. In multiple‐arm trials using two different doses of the same intervention, we combined the two groups to avoid double counting the participants in the control group.

4.3.6. Dealing with missing data

Any missing data were noted including loss to follow‐up and dropouts. The reasons for the missing data were taken from the studies, and if it was not mentioned in the studies, the authors were contacted for the same. If the authors reported the adjusted values for missing data, we used the adjusted values.

4.3.7. Assessment of heterogeneity

Statistical heterogeneity was assessed using τ ², I ², and significance of the χ ² test. We also assessed statistical heterogeneity by visually inspecting the forest plots.

4.3.8. Assessment of reporting biases

A funnel plot and its symmetry were used to assess publication bias if the number of included studies for intervention was more than 10. If the funnel plot was suggestive of publication bias, we further investigated the publication bias with the use of Egger's test (Higgins & Green, 2011).

4.3.9. Data synthesis

Synthesis procedures and statistical analysis

We used the software Review Manager 5.3 (Review Manager, 2019) to conduct the statistical analysis. For randomized trials, we followed the ITT analysis. If ITT was not available, and the author reported the analyses as specified in the protocol, we reconstructed the data to create an ITT analysis.

We used a random‐effect model to account for expected heterogeneity in the intervention, comparisons, or setting within studies included in a given synthesis. We used the generic inverse variance method of meta‐analysis for fixed effect models and random effect models. This method of meta‐analysis gives weight to studies based on their variance in a way that a study with low variance gets a high weight and vice versa.

We interpreted the results of the meta‐analysis based on p value at the 95% confidence level (a value <0.05 was considered statistically significant) and reported both significant and nonsignificant results. For subgroup analysis, we used an interaction test to determine if there was a relevant difference in effect across subgroups.

We assessed the quality of overall evidence using the GRADE approach. This method of quality assessment considers study type, within‐study ROB (methodological quality), directness of evidence, heterogeneity, precision of effect estimates, and risk of publication bias (Guyatt et al., 2011). We rated the quality of the body of evidence for each key outcome as “high,” “moderate,” “low,” or “very low.”

4.3.10. Subgroup analysis and investigation of heterogeneity

Neonatal vitamin A supplementation

Although we had planned a number of subgroup analyses for neonatal vitamin A supplementation; however, a recent IPD analysis (West et al., 2019) covered both individual and study level subgroup analyses, so we did not perform any subgroup analysis for vitamin A supplementation at this stage

Neonatal probiotic supplementation

1.
Gestational age: term and preterm
2.
Strains used in probiotics: single strain versus multiple strain and of type of strain used in each probiotic
3.
Strains used in probiotics: contains Lactobacillus versus Bifidobacterium versus both
4.
Settings: community‐based versus hospital setting
5.
Type of feedings: breastmilk versus formula milk versus mixed.

Oral dextrose gel supplementation

1.
Gestational age: term and postterm versus late preterm (35–36 weeks) versus moderately preterm (30–34 weeks) versus extremely preterm (<30 weeks)
2.
Dose: equal or <200 mg/kg versus >200 mg/kg
3.
Frequency: one versus more than one dose
4.
Time of administration: ≤1 h of age versus after 1 h of age versus after 2 h of age.

4.3.11. Sensitivity analysis

1.
High quality studies versus low quality studies. The quality of study was subjectively based on the ROB assessment. Even though we considered all the domains included in the Cochrane ROB tool, we gave higher importance to sequence generation and allocation concealment, as most of the outcomes for this review were objective, and it was less likely that the results of the included studies would have been biased by a lack of blinding.
2.
Random versus fixed effect models. We chose this sensitivity analysis to assess if the summary estimates will change significantly based on use of random versus fixed effect model. There is no exact criterion to choose between the two models, and we wanted to make sure that estimates were not significantly different between the two models.

Treatment of qualitative research

We did not plan to include qualitative research.

4.3.12. Summary of findings and assessment of the certainty of the evidence

Summary of findings' tables

We constructed “Summary of findings” tables for all of the primary outcomes using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) criteria (GRADEpro GDT 2015). These covered consideration of within‐study ROB (methodological quality), directness of evidence, heterogeneity, precision of effect estimates and risk of publication bias. We rated the certainty of evidence for each key outcome as “high,” “moderate,” “low,” or “very low.” The GRADE evidence is described in Table 1. Nonrandomised studies were initially rated as “low” quality. If there were no serious methodological flaws, we upgraded the evidence for studies with a large magnitude of effect; presence of a dose response relationship; and effect of plausible residual confounding.

Table 1.

Effect of probiotic supplementation during neonatal period: Subgroup analysis

Outcome or subgroup	No. of studies	Effect estimate: relative risk	Test for subgroup difference
All‐cause mortality: subgroup analysis: settings
Hospital based	22	0.78 [0.65, 0.94]	p = .31
Community based	3	1.25 [0.51, 3.05]	I ² = 0%
All‐cause mortality: subgroup analysis: type of probiotics
Preparation contain a single strain of probiotics	9	0.80 [0.61, 1.05]	p = .95
Preparation contained multiple strains of probiotics	12	0.80 [0.58, 1.09]	I ² = 0%
Preparation contained synbiotics (prebiotics + probiotics)	5	0.69 [0.29, 1.61]
All‐cause mortality: subgroup analysis: type of participants
Study include preterm/low birth weight babies	24	0.79 [0.65, 0.95]	p = .47
Study included term infants only	1	1.38 [0.31, 6.08]	I ² = 0%
All‐cause mortality: subgroup analysis: type of feedings
Baby received breastmilk only	14	0.81 [0.62, 1.05]	p = .44
Baby received formula milk only	1	1.38 [0.31, 6.08]	p = .44
Baby received both both breastmilk and formula milk	8	0.69 [0.48, 0.99]	I ² = 0%
Type of feeding was unclear	3	1.33 [0.63, 2.81]	I ² = 0%
All‐cause mortality: subgroup analysis: probiotics preparation
Preparation contained Lactobacillus	10	0.82 [0.63, 1.05]	p = .47
Preparation contained Bifidobacterium	1	0.43 [0.17, 1.09]	p = .47
Preparation contained both Lactobacillus and Bifidobacterium	13	0.71 [0.47, 1.08]	I ² = 0%
Preparation contained Saccharomyces boulardii only	2	1.12 [0.46, 2.71]	I ² = 0%
Necrotizing enterocolitis: subgroup analysis: probiotic preparation
Preparation contained Lactobacillus	13	0.39 [0.25, 0.61]	p = .05
Preparation contained Bifidobacterium	1	0.20 [0.09, 0.47]
Preparation contained both Lactobacillus and Bifidobacterium	14	0.49 [0.36, 0.68]
	14	0.49 [0.36, 0.68]	I ² = 60.5%
Preparation contained S. boulardii only	2	0.94 [0.45, 1.95]	I ² = 60.5%
Necrotizing enterocolitis: subgroup analysis: type of feeding
Baby received breastmilk only	13	0.43 [0.31, 0.59]	p = .74
Baby received formula only	1	0.21 [0.03, 1.76]	p = .74
Baby received both breastmilk and formula milk	9	0.55 [0.33, 0.92]	I ² = 0%
Type of feeding was unclear	7	0.41 [0.17, 1.00]	I ² = 0%
Necrotizing enterocolitis: subgroup analysis: type of probiotics
Preparation contained a single strain of probiotics	12	0.48 [0.30, 0.76]	p= .50
Preparation contained multiple strains of probiotics	15	0.48 [0.35, 0.67]	I ² = 0%
Preparation contained synbiotics (prebiotics + probiotics)	3	0.28 [0.12, 0.67]
Neonatal sepsis: subgroup analysis: probiotic preparation
Preparation contained Lactobacillus	11	0.74 [0.62, 0.87]	p = .79
Preparation contained Bifidobacterium	1	0.81 [0.60, 1.09]	p = .79
Preparation contained both Bifidobacterium and Lactobacillus	6	0.83 [0.68, 1.02]	I ² = 0%
Preparation contained S. boulardii only	3	0.73 [0.57, 0.94]	I ² = 0%
Neonatal sepsis: subgroup analysis: type of feeding
Baby received breastmilk only	8	0.71 [0.61, 0.83]	p = .04
Baby received formula milk only	2	0.59 [0.22, 1.56]	p = .04
Baby received both formula and breastmilk only	6	0.77 [0.65, 0.90]	I ² = 65%
Type of feeding was unclear	4	0.95 [0.82, 1.09]	I ² = 65%
Neonatal sepsis: type of probiotics
Preparation contained single strain of probiotics	8	0.84 [0.74, 0.96]	p = .21
Preparation contained multiple strains of probiotics	9	0.81 [0.68, 0.97]	p = .21
Preparation contained multiple strains of probiotics	9	0.81 [0.68, 0.97]	I ² = 35%
Preparation contained synbiotics (prebiotics + probiotics)	4	0.67 [0.54, 0.83]	I ² = 35%
Neonatal sepsis: subgroup analysis: settings
Hospital based	19	0.83 [0.76, 0.91]	p = .19
Community based	2	0.67 [0.49, 0.91]	I ² = 42%

Vitamin A compared to placebo for neonatal health
Patient or population: neonates (0‐28 days)
Setting: low and middle income countries
Intervention: vitamin A
Comparison: placebo
Outcomes	Relative effect (95% CI)	No. of participants (studies)	Certainty of the evidence (GRADE)
All‐cause neonatal mortality	RR, 0.99 (0.90–1.08)	126,548 (6 RCTs)	⊕⊕⊕⊕ HIGH
All‐cause mortality at 6 months of age	RR, 0.98 (0.89–1.07)	154,940 (12 RCTs)	⊕⊕⊕⊕ HIGH
All‐cause mortality at 12 months of age	RR, 1.04 (0.94–1.14)	118,376 (8 RCTs)	⊕⊕⊕⊕ HIGH
Adverse Events: Bulging Fontanelle 48–72 h	RR, 1.53 (1.12–2.09)	100,562 (6 RCTs)	⊕⊕⊕⊕ HIGH
*The risk in the intervention group (and its 95% confidence interval) is based on the assumed risk in the comparison group and the relative effect of the intervention (and its 95% CI).
GRADE Working Group grades of evidence
High certainty: We are very confident that the true effect lies close to that of the estimate of the effect
Moderate certainty: We are moderately confident in the effect estimate: The true effect is likely to be close to the estimate of the effect, but there is a possibility that it is substantially different
Low certainty: Our confidence in the effect estimate is limited: The true effect may be substantially different from the estimate of the effect
Very low certainty: We have very little confidence in the effect estimate: The true effect is likely to be substantially different from the estimate of effect

Probiotics supplementation compared to control during neonatal period
Patient or population: neonates (Mmost of the included studies had preterm and low birth weight neonates)
Setting: low and middle income countries
Intervention: probiotics/synbiotics
Comparison: control
Outcomes	No. of participants (studies) Follow up	Certainty of the evidence (GRADE)	Relative effect (95% CI)	Anticipated absolute effects* (95% CI)
Outcomes	No. of participants (studies) Follow up	Certainty of the evidence (GRADE)	Relative effect (95% CI)	Risk with control	Risk difference with probiotics (intervention)
All‐cause mortality	10904 (25 RCTs)	⊕⊕⊕⊕ HIGH^a, ^b, ^c, ^d	RR, 0.80 (0.66–0.96)	Study population
All‐cause mortality	10904 (25 RCTs)	⊕⊕⊕⊕ HIGH^a, ^b, ^c, ^d	RR, 0.80 (0.66–0.96)	47 per 1000	9 fewer per 1000 (15 fewer to 1 fewer)
Neonatal sepsis	8918 (21 RCTs)	⊕⊕⊕⊕ HIGH^a, ^d, ^e	RR, 0.78 (0.70–0.86)	Study population
Neonatal sepsis	8918 (21 RCTs)	⊕⊕⊕⊕ HIGH^a, ^d, ^e	RR, 0.78 (0.70–0.86)	205 per 1000	45 fewer per 1000 (62 fewer to 29 fewer)
Necrotizing enterocolitis	55574 (29 RCTs)	⊕⊕⊕⊕ HIGH^a, ^d, ^f	RR, 0.46 (0.35–0.61)	Study population
Necrotizing enterocolitis	55574 (29 RCTs)	⊕⊕⊕⊕ HIGH^a, ^d, ^f	RR, 0.46 (0.35–0.61)	101 per 1000	55 fewer per 1000 (66 fewer to 41 fewer)
*The risk in the intervention group (and its 95% confidence interval) is based on the assumed risk in the comparison group and the relative effect of the intervention (and its 95% CI).
CI: Confidence interval; RR: Risk ratio; OR: Odds ratio;
GRADE Working Group grades of evidence
High certainty: We are very confident that the true effect lies close to that of the estimate of the effect
Moderate certainty: We are moderately confident in the effect estimate: The true effect is likely to be close to the estimate of the effect, but there is a possibility that it is substantially different
Low certainty: Our confidence in the effect estimate is limited: The true effect may be substantially different from the estimate of the effect
Very low certainty: We have very little confidence in the effect estimate: The true effect is likely to be substantially different from the estimate of effect

Methods	A block‐randomized, double‐masked, placebo‐controlled intervention trial conducted in Bangladesh
Participants	Inclusion criteria: "consent of the mother and willingness to have their infant participate; singleton birth at MCHTI clinic and eligible for vaccination according to the national and MCHTI clinic policy".
	Exclusion criteria: "planned at home delivery because of the low likelihood of vaccination at MCHTI within 48 h of birth, (2) congenital disease or a serious infection showing that the infant was not healthy; infant with birth weight <1500 g and inability to enrol within 48 h of birth due to lack of timely notification or other exceptional circumstances".
Interventions	Intervention group: 50,000 IU vitamin A (retinyl palmitate)
	Comparison: Placebo (unfortified soya based oil)
	The intervention was delivered within 48 h of birth
Outcomes	Neonatal mortality, adverse events, microbiome changes, thymus size
Notes	Data on mortality and bulging fontanelle were taken from publication (J Nutr 2019;00:1–8). Data on mortality was reported at 15 weeks. We included the data with “all‐cause mortality at 6 months".

Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Quote: "The randomization lists of vitamin A and placebo within each group were generated by WHO, using Stata, v11"
Allocation concealment (selection bias)	Low risk	Quote: "Preplanned statistical analyses using arbitrary group identifiers…"
		Comment: Most likely done
Blinding of participants and personnel (performance bias)	Low risk	Quote: "dose of VA in oil or an identical placebo (PL) within 48…"
		Comment: Most likely done
Blinding of outcome assessment (detection bias)	Low risk	Quote: "Preplanned statistical analyses using arbitrary group identifiers (group 1, group 2) were completed on 4 April 2014 before unblinding…"
Incomplete outcome data (attrition bias)	Low risk	Attrition rate: 5.2%
Selective reporting (reporting bias)	Low risk	Author prespecified the outcomes. Trial was registered as ClinicalTrials.gov: NCT01583972.
Other bias	Low risk	No other risk of bias was noted

Methods	Prospective randomized control trial conducted in Iran
Participants	Inclusion Criteria: "All premature newborns (n = 115) weighting 750–1500 g or <32 weeks' gestation who received antibiotics and total parenteral nutrition in NICU of Vali Asr Hospital were included"
	Exclusion Criteria: "Premature babies <750 and more than 1500 g and neonates with congenital heart disease, congenital malformations, and immune system deficiency, even in their family members, were excluded from the study."
Interventions	Intervention: Multistrain powder probiotic infant formula containing Streptococcus thermophilus, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacterium infantis, Lactobacillus casei. The dose was 0.8–1 g per day in 8–10 doses given for 13 days
	Comparison: Enteral feed without probiotic
Outcomes	NEC
Notes	Data were taken from table 3

Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Quote: "In this double blind randomized clinical trial (RCT), block randomization was used and 60 cases were randomly divided into 2 groups."
Allocation concealment (selection bias)	Unclear risk	No clear information was available about allocation concealment
Blinding of participants and personnel (performance bias)	Unclear risk	No clear information available
Blinding of outcome assessment (detection bias)	Unclear risk	No clear information available
Incomplete outcome data (attrition bias)	Low risk	Minimal loss to follow up
Selective reporting (reporting bias)	Low risk	Authors seem to report all the outcomes irrespective of their statistical significance
Other bias	Low risk	No other risk of bias was noted

Methods	A randomized double‐blind placebo‐controlled trial India
Participants	Inclusion Criteria: Inborn, VLBW (birth weight (BW) < 1500 g) neonates admitted in NICU and requiring respiratory support in the form of oxygen inhalation through nasal prongs or head box, continuous positive airway pressure (CPAP), high flow nasal cannula (HFNC), or mechanical ventilation (MV) at the age of 24 h, were included.
	Exclusion Criteria: Neonates with major congenital malformation, any life‐threatening condition such as reversal of umbilical artery end‐diastolic blood flow on antenatal Doppler, perinatal asphyxia with moderate to severe hypoxic ischemic encephalopathy, shock with escalating doses of vasopressors, recurrent seizures, and suspected inborn errors of metabolism
Interventions	Intervention: 10,000 IU of retinol/dose, alternate days, 28 days or until discharge
	Compairson: Placebo
Outcomes	All‐cause mortality, sepsis, NEC
Notes	Study conducted in very low birth weight babies

Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Quote: "Randomization into vitamin A or placebo group was done using random permuted blocks of 4, 6, and 8, prepared by an independent statistician not involved in the study."
Allocation concealment (selection bias)	Low risk	Quote: "Allocation into vitamin A or placebo group was done using serially numbered opaque and sealed envelopes by on‐duty residents who were appropriately trained for the process beforehand. Allocation concealment was maintained throughout the study."
Blinding of participants and personnel (performance bias)	Low risk	Quote: "Vitamin A and placebo oral solutions were supplied in identical bottles of 20 mL with dropper marked at 1 mL…."
Blinding of outcome assessment (detection bias)	Low risk	Quote: "Treating physicians, nursing staffs, and the parents were unaware about the composition of the bottles."
Incomplete outcome data (attrition bias)	Low risk	Three patients from the intervention and two patients from the placebo group left against medical advice
Selective reporting (reporting bias)	Low risk	Authors seem to report all the relevant outcomes
Other bias	Low risk	No other risk of bias was noted

Methods	Randomized placebo controlled trial conducted in Guinea‐Bissau
Participants	Inclusion Criteria: Weight at least 2500 g at presentation and no signs of overt illness or malformations
	Exclusion Criteria: Weight <2500 g at presentation and/or signs of overt illness and/or malformations. Also, infants who died in the maternity ward before the vaccination team could arrive
	Total number randomized to the intervention group: 2145
	Total number randomized to the control group: 2200
Interventions	Intervention: 50,000 IU vitamin A intradermally
	Control: 0.5 ml vegetable oil intradermally
	Common intervention given to all groups: 10 IU vitamin E intradermally
Outcomes	Primary outcomes: Overall mortality
	Other outcomes: Bulging fontanelles, vomiting, irritability, infections, fever, skin problems, and healthcare contacts
Notes	Vitamin A supplementation appeared to benefit boys but was harmful to girls

Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Quote: "The mother drew a lot from an envelope prepared by the study supervisor. Each envelope contained 100 lots—50 marked “1” and 50 marked “2”—indicating from which of two numbered bottles, “1” or “2,” the child should receive the supplement"
		Comment: Most likely done
Allocation concealment (selection bias)	Low risk	Quote: "The lots were folded, making it impossible to tell what was written on them before they were opened"
		Comment: Most likely done
Blinding of participants and personnel (performance bias)	Low risk	Quote: "When asked, none of the three assistants who were responsible for the randomisation procedures at the hospital and at the heath centres had any idea which bottles contained vitamin A and which placebo. We concluded that the blinding of mothers and assistants was successful"
		Comment: Most likely done
Blinding of outcome assessment (detection bias)	Unclear risk	Quote: "Accumulating evidence for sex differential effects of vitamin A supplementation during the trial made us hypothesise before we started the analyses that supplementation would be particularly beneficial for boys"
		Comment: Most likely done
Incomplete outcome data (attrition bias)	Low risk	Total number of loss to follow up: 70 (1.6%)
		The loss to follow up was not balanced
Selective reporting (reporting bias)	Low risk	Authors seem to report all the outcomes irrespective of their statistical significance
Other bias	Low risk	No other risk of bias was noted

Methods	Randomized placebo controlled two by two factorial trial conducted in Guinea‐Bissau
Participants	Inclusion criteria: Weight <2500 g at presentation
	Exclusion criteria: Weight >2500 g at presentation
	Total number randomized to the intervention group: 864
	Total number randomized to the control group: 872
Interventions	Intervention: 25,000 IU vitamin A intradermally
	Control: 0.5 ml vegetable oil intradermally
	Common intervention given to all groups: 10 IU vitamin E intradermally
Outcomes	Primary outcomes: Infant mortality
	Other outcomes: Fever, septicaemia, malaria, malnutrition, and respiratory infections
Notes

Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Quote: "Once consent was provided, the mother drew an envelope from a bag. Each bag was prepared by the study supervisor and contained 48 envelopes; each envelope contained a lot name. Within each bag were 12 envelopes with lots marked “BCG 6,” 12 marked “BCG 7,” 12 marked “no BCG 6,” and 12 marked “no BCG 7.” The numbers “6” and “7” indicated from which of two numbered bottles, “6” or “7,” the child should receive treatment (that is, either 25,000 IU vitamin A or placebo)"
		Comment: Most likely done
Allocation concealment (selection bias)	Low risk	Quote: "The envelopes were closed and non‐transparent, making it impossible to identify the allocation before the envelopes were opened"
		Comment: Most likely done
Blinding of participants and personnel (performance bias)	Unclear risk	Quote: "Once consent was provided, the mother drew an envelope from a bag"
		"Each bag was prepared by the study supervisor and contained 48 envelopes; each envelope contained a lot name"
		Comment: Most likely done
Blinding of outcome assessment (detection bias)	Low risk	Quote: "Follow‐up was performed by assistants who were unaware of the allocated treatment"
		Comment: Most likely done
Incomplete outcome data (attrition bias)	Unclear risk	Total number of loss to follow up: 145 (8.4%)
		The loss to follow up was not balanced
Selective reporting (reporting bias)	Low risk	Authors seem to report all the outcomes irrespective of their statistical significance
Other bias	Low risk	No other risk of bias was noted

Methods	Double‐blind, placebo‐controlled randomized trial conducted in Guinea‐Bissau
Participants	Inclusion criteria: Normal birth‐weight neonates who were healthy and due for BCG vaccination
	Exclusion criteria: Birth weight <2500 g at presentation or overt illness and/or malformations
	Total number randomized to the intervention group 1 (50,000 IU vitamin A): 2015
	Total number randomized to the intervention group 25 (25,000 IU vitamin A): 2011
	Total number randomized to the control group: 2022
Interventions	Intervention: 50,000 IU vitamin A intradermally or 25,000 IU vitamin A intradermally
	Control: 0.5 ml Vegetable oil intradermally
	Common intervention given to all groups: 10 IU vitamin E intradermally
Outcomes	Primary outcome: Infant mortality
	Other outcomes: None measured
Notes

Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Quote. "Each envelope was prepared by the data manager, who did not take part in the enrolment procedures, and contained 48 folded lots indicating from which of 3 numbered bottles—“3,” “4,” or “5”—the child should receive his or her supplement"
		Comment: Most likely done
Allocation concealment (selection bias)	Low risk	Quote. "…48 folded lots indicating from which of 3 numbered bottles—“3,” “4,” or “5”—the child should receive his or her supplement"
		Comment: Most likely done
Blinding of participants and personnel (performance bias)	Low risk	Quote. "At each inclusion site, the randomization procedure was carried out by 1 carefully trained assistant every day except during short vacations. After providing consent, the mother drew a lot from an envelope. Each envelope was prepared by the data manager, who did not take part in the enrolment procedures…"
		Comment: Most likely done
Blinding of outcome assessment (detection bias)	Low risk	Quote. "The registration system assistants and the special team were unaware of the allocated treatment, because they were not present during enrolment, and the information was not transferred to the children's vaccination card or follow‐up forms"
		Comment: Most likely done
Incomplete outcome data (attrition bias)	Unclear risk	Quote: “Of 6053 children invited to participate, 6048 were randomly allocated to each of the 3 groups (50,000 IU vitamin A, 25,000 IU vitamin A, or placebo) (Figure 1). The 3 randomly assigned groups were similar in terms of their background characteristics (Table 1). A total of 176 deaths occurred; 2 of these were due to accidents and were censored. Fourteen deaths occurred after the child had been eligible for a national vitamin A campaign. Hence, censoring for accidents and subsequent VAS, the cohort had 160 deaths during 4125 person‐years of risk, corresponding to an MR of 39 per 1000 person‐years”
Selective reporting (reporting bias)	Low risk	Authors seem to report all the outcomes irrespective of their statistical significance
Other bias	Low risk	No other risk of bias was noted

Methods	Randomized, double blind control study conducted in Brazil
Participants	Inclusion criteria: All infants included in this study were born locally and admitted to the Neonatal Intensive Care Unit (NICU) with a birth weight from 750 to 1499 g, and had no major congenital malformations, life threatening chromosomal alterations, or congenital infections
Interventions	Intervention: Probiotic supplementation: Bifidobacterium breve and Lactobacillus casei: The intervention was started on the second day of life and was maintained until 30 d of life, a diagnosis of NEC, discharge from the hospital, or death, whichever occurred first. The dose was 3 ml human milk from the bank milk to which L. casei and B. breve had been added providing 3.5 × 10⁷ to 3.5 × 10⁹ CFU
	Comparison: The control group received the same volume of human milk without probiotics
Outcomes	Mortality, NEC and sepsis
Notes	Authors did not do intention to treat analysis. We created the intention to treat analysis by taking the number randomized as denominators. The data on outcomes was taken from table 2 of the main manuscript

Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Randomization was carried out in blocks of 10, and the list of random numbers was generated by the subprogram Epitable from Epi‐Info 6.04
Allocation concealment (selection bias)	Low risk	A sealed envelope with the identification number in ascending order, containing information about which group they belonged to, was provided for each infant and sent to the hospital's nutritional centre
Blinding of participants and personnel (performance bias)	Low risk	Neither the medical and nursing staff responsible for monitoring the infants nor the researchers were aware of which group the infants were allocated to
Blinding of outcome assessment (detection bias)	Low risk	Neither the medical and nursing staff responsible for monitoring the infants nor the researchers were aware of which group the infants were allocated to
Incomplete outcome data (attrition bias)	Low risk	Attrition in intervention group was 2% and 7% in the control group
Selective reporting (reporting bias)	Low risk	Most of the outcomes were reported
Other bias	Low risk	No other bias was noted

Methods	A randomized controlled trial conducted in Bangladesh
Participants	Inclusion crietria: Preterm (<33 woks), VLBW (<1500 g) infants who are able to tolerate oral feeds and survive beyond 48 h
	Exclusion criteria: Babies with suspicion of clinical sepsis, presence of prenatal asphyxia, major congenital anomaly and babies who expired due to other neonatal illness were excluded
Interventions	Intervention: Probiotic supplementation: Bifidobacterium breve and Lactobacillus casei: The dose was 3 ml once daily of solution containing Bifidobacterium breve and Lactobacillus casei 10⁶ CFU. The intervention was continued for at least 10 days
	Comparison: No probiotics
Outcomes	NEC, all‐cause mortality
Notes	Authors did not perform intention to treat analysis, however, we created the intention to treat analysis from Figure 1. The data for mortality was taken from Figure 1 and the data for NEC was taken from table 2

Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Coding to group 1 and 2 was done by a faculty of another department not related to this study. First case was selected to 1 group by lottery method and subsequent group was continued accordingly
Allocation concealment (selection bias)	Low risk	Participants and investigators did not know group allocation
Blinding of participants and personnel (performance bias)	Low risk	Probiotics were added to breast milk by registrar or assistant registrar of the corresponding unit before feeding
Blinding of outcome assessment (detection bias)	Low risk	Participants and investigators did not know group allocation
Incomplete outcome data (attrition bias)	Low risk	15% attrition. Reasons for loss to follow up reported.
Selective reporting (reporting bias)	Low risk	All expected outcomes were reported
Other bias	Low risk	No concerns for other risk of bias

Methods	A randomized double‐blind controlled trial conducted in South Africa in community settings
Participants	Inclusion criteria: "healthy", full term (37–42 weeks), born to HIV + formula feeding mothers, ≤3 days old, 2500–4500 g, singleton birth
	Exclusion criteria: Congenital illness or malformation affecting growth; significant perinatal disease, antibiotics in 1st 3 days of life, caregivers could not comply, or in another trial
Interventions	Intervention: Probiotics: Formula containing prebiotic (bovine milk‐derived oligosaccharides) and probiotic ((B. lactis strain CNCM‐I‐3446 with 1 × 10⁷ cfu/g. of powder formula). The duration of intervention was 6 months
	Comparison: Formula without prebiotic and probiotic
Outcomes	All‐cause mortality
Notes	The data was taken from the last paragraph of the result section. We note that the intervention group received both prebiotics and probiotics and the control group did not receive any prebiotics or probiotics. Some of the study participants were HIV positive

Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Quote: "The randomization was performed using the in‐house TrialSys software".
Allocation concealment (selection bias)	Low risk	Formulas labelled similarly
Blinding of participants and personnel (performance bias)	Low risk	Parents (caregivers), investigators, study support staff, and the clinical project managers were blinded to the identity of the products.
Blinding of outcome assessment (detection bias)	Low risk	Likely same care‐team
Incomplete outcome data (attrition bias)	Low risk	Total loss to follow up was 1%
Selective reporting (reporting bias)	Low risk	Authors do not seem to selectively report outcomes
Other bias	Low risk	No other risk of bias was noted.

PERMALINK

Effects of neonatal nutrition interventions on neonatal mortality and child health and development outcomes: A systematic review

Aamer Imdad

Faseeha Rehman

Evans Davis

Deepika Ranjit

Gamael S S Surin

Suzanna L Attia

Sarah Lawler

Abigail A Smith

Zulfiqar A Bhutta

Abstract

Background

Objectives

Search Methods

Selection Criteria

Data Collection and Analysis

Main Results

Authors' Conclusions

1. PLAIN LANGUAGE SUMMARY

1.1. Neonatal probiotic supplementation can improve infant illness and reduce death, but vitamin A does not, and may have adverse effects

1.1.1. What is this review about?

1.1.2. What studies are included?

1.1.3. Key results

1.1.4. What are the main findings of the review?

1.1.5. What do the findings of this review mean?

1.1.6. How up‐to‐date is this review?

2. BACKGROUND

2.1. Description of the condition

2.1.1. Neonatal vitamin A deficiency (VAD)

2.1.2. Hypoglycemia during the neonatal period

2.1.3. Neonatal sepsis and NEC

2.2. Description of the intervention

2.2.1. Neonatal vitamin A supplementation

2.2.2. Oral dextrose gel supplementation during neonatal period

2.2.3. Probiotic supplementation during neonatal period

2.3. How the intervention might work

2.3.1. Neonatal vitamin A supplementation

2.3.2. Oral dextrose gel supplementation during neonatal period

2.4. Probiotics supplementation during neonatal period

2.5. Why it is important to do this review

2.5.1. Neonatal vitamin A supplementation

2.5.2. Oral dextrose gel supplementation during neonatal period

2.5.3. Probiotics supplementation during neonatal period

3. OBJECTIVES

3.1. Primary objectives

4. METHODS

4.1. Criteria for considering studies for this review

4.1.1. Types of studies

4.1.2. Types of participants

4.1.3. Types of interventions

4.1.4. Types of outcome measures

Primary outcomes

Secondary outcomes

Duration of follow‐up

Type of settings

4.2. Search methods for identification of studies

4.2.1. Electronic searches

4.2.2. Searching other resources

4.3. Data collection and analysis

4.3.1. Selection of studies

Description of methods used in primary research

Criteria for determination of independent findings

4.3.2. Data extraction and management

Details of study coding categories

4.3.3. Assessment of ROB in included studies

4.3.4. Measures of treatment effect

4.3.5. Unit of analysis issues

Multiple‐arm trials

4.3.6. Dealing with missing data

4.3.7. Assessment of heterogeneity

4.3.8. Assessment of reporting biases

4.3.9. Data synthesis

Synthesis procedures and statistical analysis

4.3.10. Subgroup analysis and investigation of heterogeneity

Neonatal vitamin A supplementation

Neonatal probiotic supplementation

Oral dextrose gel supplementation

4.3.11. Sensitivity analysis

Treatment of qualitative research

Methods	A prospective, double‐blinded randomized study conducted in China
Participants	Inclusion criteria: Formula‐fed preterm infants, gestational age ≥30 and <37 weeks; birthweight ≥1500 g and ≤ 2000 g with vital sign and hemodynamic parameters stable
	Exclusion criteria: Congenital diseases, expected hospitalisations <2 weeks and maternal or neonatal antibiotics or other probiotics before admission
Interventions	Intervention: Lactobacillus reuteri DSM 17938, five drops daily for minim of 7 days. Each drop had 1×10⁸ colony‐forming units
	Comparison: Placebo
Outcomes	Sepsis, NEC, growth
Notes	Data were taken from table 2

Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Quote: "Randomization was conducted according to a random computer‐determined allocation order considering gestational age"
		Comment: Most likely done
Allocation concealment (selection bias)	Unclear risk	No clear information available for allocation concealment
Blinding of participants and personnel (performance bias)	Low risk	Quote: "Blinding was possible because the nurses who administered L. reuteri to the infants were not involved in the daily care and the attending neonatal team was unaware of the randomization assignments"
Blinding of outcome assessment (detection bias)	Unclear risk	It was not clear if the families of the participating neonates were aware of the treatment assignments
Incomplete outcome data (attrition bias)	Low risk	About 18% attrition reported that was balanced in two groups
Selective reporting (reporting bias)	Low risk	Authors seem to report all the relevant outcomes
Other bias	Low risk	No other risk of bias was noted

Methods	Prospective triple‐blinded, interventional, randomized clinical trial
Participants	Inclusion criteria: Birth weight of 700‐1800 g, stable hemodynamic, be able to have enteral feeding, and written parental consent.
	Exclusion criteria: Evidence or suspicion of congenital intestinal obstruction or perforation, prenatal or postnatal diagnosis of gastroschisis, large omphalocele, or congenital diaphragmatic hernia, and major congenital anomalies."
Interventions	Intervention: Protexin (probiotics). Protexin (Restore): 1 × 109 CFU (colony forming unit), 1 g (one sachet) contains: Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus bulgaricus, Lactobacillus casei, Streptococcus thermophilus, Bifidobacterium breve, and Bifidobacterium
	The dose was as follows
	– Neonates weighing <1000 g were fed with a half of sachet once daily (5×10⁸ CFU of probiotics), – Neonates weighing 1001–1500 g were fed with 3/4 of a sachet once daily (7.5×10⁸ CFU of probiotics) – Neonates weighing more than 1500 g were fed with a full sachet once daily (1×10⁹ CFU of probiotics).
	Control: "placebo that was physically indistinguishable from the probiotic powder"
Outcomes	NEC, mortality and sepsis
Notes	The data for NEC, mortality and sepsis was taken from table 2. We assumed that group A was the intervention group and group B was the control. Authors did not mention clearly in the paper which group is the intervention group and which one is the control group.The duration of intervention was not clearly stated

Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	No clear data is available to support the assessment however the two groups were comparable after randomizations and allocation seems to be concealed. So less likely that randomizations was not done properly
Allocation concealment (selection bias)	Low risk	Quote: "To blind the trial the probiotic and placebo sachets were set in similar indistinguishable packages"
Blinding of participants and personnel (performance bias)	Low risk	Quote: "The control group was fed with milk and a placebo that was physically indistinguishable from the probiotic powder"
Blinding of outcome assessment (detection bias)	Low risk	Quote: "After starting the feeding, infants were observed continuously by a chart containing basic information like daily weight, feeding volume, abdominal girth, appearance of erythema of abdominal wall, loose stools with blood, vomiting, and orogastric tube suction volume. The amount of feeding was advanced slowly, if tolerated, with no more than a 20 ml/kg/d"
Incomplete outcome data (attrition bias)	Low risk	Quote: "Feeding was discontinued if there was any sign of feeding intolerance (defined as the presence of gastric aspirate in the amount that was more than a half of the previous feeding or abdominal distension)"
Selective reporting (reporting bias)	Low risk	Authors seem to report all the relevant outcomes
Other bias	Low risk	No other risk of bias was noted

Methods	Prospective, blinded, randomized control trial conducted in Turkey
Participants	Inclusion criteria: Neonates born ≤32 weeks and birthweight ≤1500 g who survived to start enteral feedings.
	Exclusion criteria: Major congenital anomalies, lack of parental consent, death in first seven days after study start
Interventions	Intervention: Probiotic supplementation: S. boulardii: Dose was 250 mg (5 billion cfu), added to breastmilk or formula, frequency was once daily and supplementation continued till discharge
	Comparison: Placebo
Outcomes	NEC, sepsis, mortality
Notes	Data was not analysed as intention to treat analysis. We created the intention to treat analysis by using the number randomized and the outcome numbers given in table 3

Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Quote, "Randomisation was simple and unadjusted and was performed using sequential numbers generated at the computer centre of the NICU"
Allocation concealment (selection bias)	Low risk	Quote, "The allocations were sealed in opaque, sequentially numbered envelopes"
Blinding of participants and personnel (performance bias)	Low risk	Quote, "The supplements were prepared by personnel on the breast milk team following the instructions in the sealed envelope. These individuals were the only personnel who were aware of the group assignments, and they were not involved in the care of the infants"
Blinding of outcome assessment (detection bias)	Low risk	Quote, "The supplements were prepared by personnel on the breast milk team following the instructions in the sealed envelope. These individuals were the only personnel who were aware of the group assignments, and they were not involved in the care of the infants"
Incomplete outcome data (attrition bias)	Low risk	7/278 dropped out. The intervention and the control group were fairly similar
Selective reporting (reporting bias)	Low risk	Authors seems to report all the outcomes mentioned in the analysis plan
Other bias	Low risk	No other bias was noted

Methods	Prospective, randomized, controlled trial conducted in TurkeyQuote
Participants	Inclsuin criteria:: "VLBW infants with a gestational age of <32 weeks and a birth weight of <1500 g, born at or transferred to the NICU within the 1st week of life and fed enterally before inclusion, were eligible
	Exclusion criteria: Infants with any disease other than those linked to prematurity or congenital anomalies of the intestinal tract, not fed enterally or who died before the seventh day after birth, whose mothers had taken nondietary probiotic supplements, and whose parents refused to participate were excluded"
Interventions	Intervention: Probitics/Prebiotics
	Multiple Arm trial
	1) Probiotic (Bifiidobacterium lactis, 5×10⁹ colony‐forming units) 2) Prebiotic (inulin, 900 mg) 3) Synbiotic (Bifidobacterium lactis, 5×10⁹ colony‐forming units, 30 mg plus inulin, 900 mg 4) The control group: maltodextrin powder as a placebo
	The dose was 1 sachet per day of pre/probiotics with breast milk or formula until discharge or death, for a maximum of 8 weeks, whichever comes first
Outcomes	NEC, sepsis and mortality
Notes	The data were taken from table 3. We included data as probiotics+synbiotics vs. prebiotics+placebo

Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Quote: "Infants were randomized by balanced blocks using sealed envelopes"
Allocation concealment (selection bias)	Low risk	Quote: "Infants were randomized by balanced blocks using sealed envelopes"
Blinding of participants and personnel (performance bias)	Low risk	Quote: " In feeding units, sachets were opened and mixed with 1 ml of sterile water or breastmilk immediately before administration to infants who were receiving enteral feeding on the day of the supplementation. The feeding team was not involved in the care of the infant and followed directions on the sealed envelopes"
Blinding of outcome assessment (detection bias)	Low risk	Quote: "The only personnel who knew of the infants group assignments were the investigators". As the outcomes were mostly objective, it is less likely that study had significant detection bias
Incomplete outcome data (attrition bias)	Low risk	Minimal loss to follow up reasons reported
Selective reporting (reporting bias)	Low risk	Authors seem to report all the relevant outcomes
Other bias	Low risk	No other sources of bias were noted

Methods	A randomized, placebo‐controlled trial conducted in India
Participants	Inclusion criteria:
	(1) Neonates born at 27–33 weeks gestation in our hospital (2) aged <96 h of life (3) who were likely to either remain admitted in hospital or reside within 30 km of the hospital for the next 28 days (4) who were tolerating at least 15 ml/kg/day of milk feeds
	Exclusion criteria:
	(1) a gastro‐intestinal malformation (2) prior NEC or sepsis (3) any life‐threatening malformation that limited estimated life expectancy to less than a month
Interventions	Intervention group: Probiotic Lactobacillus acidophilus (662.5 million), Lactobacillus rhamnosus (362.5 million), Bifidobacterium longum (87.5 million), and Saccharomyces boulardii (137.5 million)
	Four groups
	A. High‐dose long course 10¹⁰ cells 12 hourly for 21 days B. High‐dose short course 10¹⁰ cells 12 hourly for days 1–14; followed by placebo from days 15–21 C. Low‐dose long course 10⁹ cells 12 hourly for 21 days D. Control group: Placebo for 21 days
Outcomes	NEC, mortality and sepsis
Notes	We combined all the probiotics groups (group A + B + C) to avoid the double counting of the placebo.

Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Quote: "A block randomized sequence was generated online by an investigator who was not involved in the recruitment of subjects"
Allocation concealment (selection bias)	Low risk	Quote: "A block randomized sequence was generated online by an investigator who was not involved in the recruitment of subjects"
Blinding of participants and personnel (performance bias)	Low risk	Quote: "The external appearance and the contents of the sachets of high dose, low dose, and placebo were identical looking.
Blinding of outcome assessment (detection bias)	Low risk	As the intervention was concealed properly, less likely that outcomes assessors were aware of the allocation
Incomplete outcome data (attrition bias)	Low risk	Minimal loss to follow up
Selective reporting (reporting bias)	Low risk	Authors seems to report all the relevant outcomes
Other bias	Low risk	No other risk of bias was noted