Probiotics to prevent necrotising enterocolitis in very preterm or very low birth weight infants

Abstract

Background

Intestinal dysbiosis may contribute to the pathogenesis of necrotising enterocolitis (NEC) in very preterm or very low birth weight (VLBW) infants. Dietary supplementation with probiotics to modulate the intestinal microbiome has been proposed as a strategy to reduce the risk of NEC and associated mortality and morbidity in very preterm or VLBW infants.

Objectives

To determine the effect of supplemental probiotics on the risk of NEC and associated mortality and morbidity in very preterm or very low birth weight infants.

Search methods

We searched CENTRAL, MEDLINE, Embase, the Maternity and Infant Care database, and CINAHL from inception to July 2022. We searched clinical trials databases and conference proceedings, and examined the reference lists of retrieved articles.

Selection criteria

We included randomised controlled trials (RCTs) and quasi‐RCTs comparing probiotics with placebo or no probiotics in very preterm infants (born before 32 weeks' gestation) and VLBW infants (weighing less than 1500 g at birth).

Data collection and analysis

Two review authors independently evaluated risk of bias of the trials, extracted data, and synthesised effect estimates using risk ratios (RRs), risk differences (RDs), and mean differences (MDs), with associated 95% confidence intervals (CIs). The primary outcomes were NEC and all‐cause mortality; secondary outcome measures were late‐onset invasive infection (more than 48 hours after birth), duration of hospitalisation from birth, and neurodevelopmental impairment. We used the GRADE approach to assess the certainty of the evidence.

Main results

We included 60 trials with 11,156 infants. Most trials were small (median sample size 145 infants). The main potential sources of bias were unclear reporting of methods for concealing allocation and masking caregivers or investigators in about half of the trials. The formulation of the probiotics varied across trials. The most common preparations contained Bifidobacterium spp., Lactobacillus spp., Saccharomyces spp., andStreptococcus spp., alone or in combination.

Very preterm or very low birth weight infants

Probiotics may reduce the risk of NEC (RR 0.54, 95% CI 0.46 to 0.65; I² = 17%; 57 trials, 10,918 infants; low certainty). The number needed to treat for an additional beneficial outcome (NNTB) was 33 (95% CI 25 to 50). Probiotics probably reduce mortality slightly (RR 0.77, 95% CI 0.66 to 0.90; I² = 0%; 54 trials, 10,484 infants; moderate certainty); the NNTB was 50 (95% CI 50 to 100). Probiotics probably have little or no effect on the risk of late‐onset invasive infection (RR 0.89, 95% CI 0.82 to 0.97; I² = 22%; 49 trials, 9876 infants; moderate certainty). Probiotics may have little or no effect on neurodevelopmental impairment (RR 1.03, 95% CI 0.84 to 1.26; I² = 0%; 5 trials, 1518 infants; low certainty).

Extremely preterm or extremely low birth weight infants

Few data were available for extremely preterm or extremely low birth weight (ELBW) infants. In this population, probiotics may have little or no effect on NEC (RR 0.92, 95% CI 0.69 to 1.22, I² = 0%; 10 trials, 1836 infants; low certainty), all‐cause mortality (RR 0.92, 95% CI 0.72 to 1.18; I² = 0%; 7 trials, 1723 infants; low certainty), or late‐onset invasive infection (RR 0.93, 95% CI 0.78 to 1.09; I² = 0%; 7 trials, 1533 infants; low certainty). No trials provided data for measures of neurodevelopmental impairment in extremely preterm or ELBW infants.

Authors' conclusions

Given the low to moderate certainty of evidence for the effects of probiotic supplements on the risk of NEC and associated morbidity and mortality for very preterm or VLBW infants, and particularly for extremely preterm or ELBW infants, there is a need for further large, high‐quality trials to provide evidence of sufficient validity and applicability to inform policy and practice.

Plain language summary

Probiotics for prevention of necrotising enterocolitis in very preterm or very low birth weight infants

Review question
Does giving very preterm or very low birth weight infants probiotics prevent necrotising enterocolitis?

Background

Very preterm infants (those born more than eight weeks early) and very low birth weight infants (those weighing less than 1.5 kg at birth) are at risk of developing necrotising enterocolitis, a severe condition where tissues in the lining of the infant's bowel become inflamed and start to die. This condition can lead to death, serious infection, long‐term disability, and developmental problems.

What did we want to find out?

One way to help prevent necrotising enterocolitis may be to add probiotics (dietary supplements containing potentially beneficial bacteria or yeasts) to milk feeds. We wanted to find out whether probiotic supplementation might benefit very preterm and very low birth weight infants. Specifically, we wanted to know if probiotic supplementation was better than placebo (dummy treatment) or no treatment for improving:

• necrotising enterocolitis;
• death from any cause;
• serious infection;
• duration of hospitalisation from birth; and
• neurodevelopmental disability.

What did we do?

We searched several important databases to identify randomised controlled trials (trials that assign participants to one of two or more treatment groups at random) that investigated the use of probiotics for preventing necrotising enterocolitis in very preterm and very low birth weight infants. We compared and summarised the results of the studies and rated our confidence in the evidence based on factors such as study methods and sizes.

What did we find?

We found 60 trials with 11,156 infants. Most trials were small and had design flaws that might have biased their findings.

Main results

Giving very preterm and very low birth weight infants probiotics, compared with giving them placebo or no treatment, may reduce their risk of necrotising enterocolitis, and probably reduces their risk of death. Probiotics probably have little or no effect on serious infection and may have little or no effect on disability or developmental outcomes. Probiotics compared with placebo or no treatment may have little or no effect on necrotising enterocolitis, death, or serious infection in extremely preterm infants (those born more than 12 weeks early) or extremely low birth weight infants (those weighing less than 1.0 kg at birth).

What are the limitations of this evidence?

The methods used in the included trials may have exaggerated the benefits of giving probiotics to very preterm and very low birth weight infants. Furthermore, the effect could have been biased by small trials with unreliable methods.

Because we have little confidence or moderate confidence in the evidence for the effects of probiotic supplements in very preterm or very low birth weight infants, there is a need for additional large, high‐quality trials to provide evidence of sufficient validity and applicability to inform policy and practice.

How up to date is this evidence?

The evidence is up to date to July 2022.

Summary of findings

Summary of findings 1. Probiotics compared to control in very preterm or very low birth weight infants.

Probiotics compared to placebo or no probiotics in very preterm or very low birth weight infants
Patient or population: very preterm or very low birth weight infants Setting: neonatal care centres worldwide Intervention: probiotics Comparison: placebo or no probiotics
Outcomes	Anticipated absolute effects* (95% CI)		Risk ratio (95% CI)	№ of participants (trials)	Certainty of the evidence (GRADE)
	Risk with control	Risk with probiotics
Necrotising enterocolitis (before hospital discharge)	60 per 1000	33 per 1000 (28 to 39)	0.54 (0.46 to 0.65)	10,918 (57)	⊕⊕⊝⊝ Lowa,b
All‐cause mortality (before hospital discharge)	63 per 1000	48 per 1000 (42 to 57)	0.77 (0.66 to 0.90)	10,484 (54)	⊕⊕⊕⊝ Moderatea
Late‐onset invasive infection (before hospital discharge)	173 per 1000	154 per 1000 (142 to 168)	0.89 (0.82 to 0.97)	9876 (49)	⊕⊕⊕⊝ Moderatea
Neurodevelopmental impairment (18 months to 3 years)	194 per 1000	200 per 1000 (163 to 245)	1.03 (0.84 to 1.26)	1518 (5)	⊕⊕⊝⊝ Lowa,c
*The risk in the intervention group (and its 95% CI) is based on the assumed risk in the comparison group and the relative effect of the intervention (and its 95% CI). CI: confidence interval.
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.

^aDowngraded one level for high risk of bias due to uncertainty about methods used to generate random sequence, conceal allocation, and mask outcome assessment.
^bDowngraded one level for publication bias (funnel plot asymmetry and statistical evidence consistent with trial size; trials favouring controls missing).
^cDowngraded one level for serious imprecision of effect estimate (95% CI around estimate consistent with substantial harm or benefit).

Summary of findings 2. Probiotics compared to control in extremely preterm or extremely low birth weight infants.

Probiotics compared to placebo or no probiotics in extremely preterm or extremely low birth weight infants
Patient or population: extremely preterm or extremely low birth weight infants Setting: neonatal care centres globally Intervention: probiotics Comparison: placebo or no probiotics
Outcomes	Anticipated absolute effects* (95% CI)		Risk ratio (95% CI)	№ of participants (trials)	Certainty of the evidence (GRADE)
	Risk with control (extremely preterm or ELBW)	Risk with Probiotics
Necrotising enterocolitis (before hospital discharge)	94 per 1000	87 per 1000 (65 to 114)	0.92 (0.69 to 1.22)	1836 (10)	⊕⊕⊝⊝ Lowa
All‐cause mortality (before hospital discharge)	132 per 1000	121 per 1000 (95 to 156)	0.92 (0.72 to 1.18)	1723 (7)	⊕⊕⊝⊝ Lowa
Late‐onset invasive infection (before hospital discharge)	274 per 1000	255 per 1000 (214 to 299)	0.93 (0.78 to 1.09)	1533 (7)	⊕⊕⊝⊝ Lowa
Neurodevelopmental impairment (18 months to 3 years)	No trials provided subgroup data for analysis.
*The risk in the intervention group (and its 95% CI) is based on the assumed risk in the comparison group and the relative effect of the intervention (and its 95% CI). CI: confidence interval.
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.

^aDowngraded one level for high risk of bias (uncertainty about methods used to generate random sequence, conceal allocation, and mask assessments) in many trials, and one level for serious imprecision of effect estimate (95% CI around estimate consistent with substantial harm or benefit).

Background

The intestinal microbiome may play an important role in the pathogenesis of necrotising enterocolitis (NEC; Embleton 2017). Probiotics are microorganisms that benefit the host by modulating the intestinal microbiome and promoting mucosal barrier functions and resistance to pathogens. Dietary supplementation with probiotics has been proposed as a strategy to reduce the risk of NEC and associated morbidity and mortality in very preterm or very low birth weight (VLBW) infants.

Description of the condition

NEC, a syndrome of acute intestinal necrosis of unknown aetiology, affects around 5% of very preterm or VLBW infants (Horbar 2012). The major risk predictors for NEC include extremely preterm birth or extremely low birth weight (ELBW), and evidence of intrauterine growth restriction or absent or reversed end‐diastolic flow velocities in Doppler studies of the foetal aorta or umbilical artery (Samuels 2017). Infants who develop NEC experience more infections, have lower levels of nutrient intake, grow more slowly, and have longer stays in intensive care and hospital than gestation‐comparable infants without NEC (Battersby 2018; Berrington 2012). The associated mortality rate is around 20%, and infants who develop NEC, especially if associated with bloodstream infections, have a higher risk of neurodevelopmental problems and disability compared with their peers (Hickey 2018; Martin 2010).

The pathogenesis of NEC remains unclear but is thought to involve intestinal dysbiosis, infection, and inflammation (Eaton 2017; Mara 2018; Morgan 2011). Emerging evidence supports the theory that the intestinal microbiome affects the risk of developing NEC (Masi 2019; Olm 2019; Stewart 2012; Warner 2016). Most very preterm or VLBW infants who develop NEC have received enteral milk feeds; feeding with human milk rather than cow's milk formula reduces the risk of NEC (Quigley M 2019). One putative explanation for this protective effect is that prebiotic substances in human milk promote the growth of non‐pathogenic probiotic microorganisms, predominantly lactobacilli and bifidobacteria, that modulate the intestinal microbiome and promote mucosal barrier functions (Embleton 2017; Granger 2020; Walsh 2019). However, compared with human milk‐fed term infants, very preterm or VLBW infants typically harbour fewer probiotic microorganisms and more potential pathogens such as enterococci and Enterobacteriaceae, which might be due to dysbiotic effects of enteral fasting and antibiotic exposure (Stewart 2017).

Given the putative role of probiotics in maintaining the structure, integrity, and function of the intestinal barrier, the possibility that supplemental probiotics might be effective in preventing NEC is of considerable research interest (Berrington 2019; Patel 2018).

Description of the intervention

The probiotic preparations most commonly used as enteral supplements contain one or more strains of bacteria (typically bifidobacteria or lactobacilli) or the fungus Saccharomyces boulardii (Thomas 2010). Other bacteria with probiotic properties include Bacillus clausii, Enterococcus faecium, and Streptococcus thermophilus. Exogenous probiotics can colonise the mucosal surface of the human gastrointestinal tract (Abdulkadir 2016; Zmora 2018). A range of probiotic supplements are available commercially as single‐ or multiple‐strain preparations and have been used to prevent and treat infectious or inflammatory gastrointestinal conditions in adults. However, despite biological plausibility and underpinning preclinical studies, the evidence is of low certainty for most conditions (Bron 2017; Koretz 2018; Kunk 2019; Lerner 2019; Suez 2019). Furthermore, there are reports of serious unexpected adverse events and outcomes of probiotic supplementation in critically ill adults (Besselink 2008; Boyle 2006).

Probiotics for very preterm infants

Policies and practices for the use of probiotic supplements to prevent NEC in very preterm or VLBW infants vary within and between countries (Duffield 2019; Poindexter 2021; Viswanathan 2016). Parents have expressed willingness to consider probiotics for their very preterm or VLBW infants if evidence of benefit and safety exists (Sesham 2014). Enteral administration of commercially available supplements of lyophilised probiotic microorganisms (usually multispecies preparations containing lactobacilli, bifidobacteria, or both) is established in some settings (Robertson 2020). However, routine use outwith trials remains limited because of uncertainty about the optimal constitution of preparations (strains of microorganisms and dosing strategies), quality control and safety issues (including contamination of products with potential pathogens), and national licencing processes and regulatory requirements (Berrington 2019; Fleming 2019; Pell 2019; Poindexter 2021; van den Akker 2020; Vermeulen 2020). Although probiotic supplementation in immunocompetent adults is considered safe, research suggests that exogenous probiotic microorganisms have caused bacteraemia or fungaemia in very preterm or VLBW infants (Bertelli 2015; Esaiassen 2016; Jenke 2012; Sakurai 2021; Zbinden 2015).

How the intervention might work

Intestinal probiotic microorganisms are thought to exert their beneficial effects via several mechanisms. Probiotics may out‐compete pathogens for nutrients and limit pathogen growth via production of inhibitory organic acids (postbiotics) and antimicrobial compounds (Embleton 2017; Patel 2015). Infants supplemented with probiotics harbour fewer potential pathogens in the intestine (Alcon‐Giner 2020). Other putative actions include stimulating differentiation and proliferation of enterocytes, enhancing expression of intestinal digestive enzymes, and improving intestinal mucosal barrier integrity (Bron 2017; Johnson‐Henry 2016; Sanders 2019).

Why it is important to do this review

NEC and associated complications, particularly infections, are the commonest causes of mortality and serious morbidity beyond the early neonatal period in very preterm or VLBW infants (Berrington 2012). Since probiotic supplementation might reduce the risk of NEC, appraising and synthesising the trial evidence about the effectiveness and safety of probiotic supplementation could inform practice, policy, and research (Embleton 2016; Quigley E 2019). Current international policy statements that exist to guide practice do not make unconditional recommendations for use of any probiotic combination for very preterm or VLBW infants (Marchand 2012; van den Akker 2020).

Objectives

To determine the effect of supplemental probiotics on the risk of NEC and associated mortality and morbidity in very preterm or VLBW infants.

Methods

Criteria for considering studies for this review

Types of studies

We included randomised controlled trials (RCTs) and quasi‐RCTs.

Types of participants

Eligible trials enrolled very preterm infants (born before 32 weeks' gestation) or VLBW infants (weighing less than 1500 g at birth). We prespecified analyses for extremely preterm infants (born before 28 weeks' gestation) or ELBW infants (weighing less than 1000 g at birth).

Types of interventions

We included enteral administration of any probiotic or probiotic combination for at least one week compared with placebo or no treatment.

We categorised probiotic preparations at the genus level (Bifidobacterium spp., Lactobacillus spp., Sacchromyces spp., Streptococcal spp., others, and combinations thereof).

Types of outcome measures

We focused on assessing effects on infant‐ and family‐important outcomes, principally neonatal morbidities that plausibly affect rates of mortality or neurodisability. We did not include surrogate outcomes such as stool colonisation patterns.

Primary outcomes

• NEC before hospital discharge, confirmed at surgery or autopsy or using the following standardised clinical and radiological criteria (VON 2020):

  • at least one of: bilious gastric aspirate or emesis, abdominal distention, or blood in stool; and

  • at least one of: abdominal radiograph showing pneumatosis intestinalis, gas in the portal venous system, or free air in the abdomen.

• All‐cause mortality before hospital discharge

Secondary outcomes

• Late‐onset invasive infection, as determined by culture of bacteria or fungus from blood or cerebrospinal fluid or from a normally sterile body space (from 48 hours after birth until discharge from hospital)

• Late‐onset infection with the supplemented probiotic microorganism before discharge from hospital

• Duration of hospitalisation from birth (days)

• Neurodevelopmental impairment assessed by a validated test after 12 months' post‐term (neurological evaluations, developmental scores, and classifications of disability, including cerebral palsy and auditory and visual impairment).

Search methods for identification of studies

We used the criteria and standard methods of Cochrane Neonatal.

Electronic searches

We searched the following electronic databases using a combination of text words and MeSH terms (Appendix 1).

• Cochrane Central Register of Controlled Trials (CENTRAL; 2022, Issue 7), in the Cochrane Library

• MEDLINE via Ovid (1946 to July 2022)

• Embase via Ovid (1974 to July 2022)

• Maternity & Infant Care Database via Ovid (1971 to June 2022)

• Cumulative Index to Nursing and Allied Health Literature (CINAHL; 1982 to July 2022)

We limited the search outputs with filters for clinical trials as recommended in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2020). We applied no language restrictions.

We searched the following trials registries for ongoing or recently completed trials.

• The US National Institutes of Health Ongoing Trials Registry ClinicalTrials.gov (clinicaltrials.gov)

• The World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP; trialsearch.who.int)

• The ISRCTN Registry (www.isrctn.com)

Searching other resources

We examined the reference lists of all included articles for any other potentially eligible trials.

Data collection and analysis

We used the standard methods of Cochrane Neonatal for data collection and analysis.

Selection of studies

Two review authors (SS and WM) independently screened the titles and abstracts of all studies and eliminated those that were clearly ineligible. The same two review authors then read the full‐text articles of all potentially eligible trials and excluded those that did not meet our inclusion criteria, recording reasons for exclusion. We discussed disagreements until reaching a consensus, consulting a third review author (SO) for a final decision as necessary.

Data extraction and management

Two review authors (SS, SO, or WM) extracted data independently, using a piloted data extraction form. Specifically, we collected data related to the design, methodology, participants, interventions, outcomes, and treatment effects of included studies. We discussed disagreements until reaching a consensus. If the trial reports provided insufficient data, we contacted the trial authors for further information.

Assessment of risk of bias in included studies

Two review authors (SS, SO, or WM) independently assessed the risk of bias (low, high, or unclear) of all included trials using the Cochrane risk of bias tool (RoB 1), which covers the following domains (Higgins 2011).

• Random sequence generation (selection bias)

• Allocation concealment (selection bias)

• Blinding (masking) of participants, personnel, and outcome assessors (performance and detection bias)

• Incomplete outcome data (attrition bias)

• Selective reporting (reporting bias)

• Other potential sources of bias

Had any disagreements occurred, we would have resolved them through discussion or by involving the third assessor. See Appendix 2 for a description of risk of bias for each domain. 

For cluster‐randomised trials, which randomise groups of individuals (rather than individuals) to the different interventions, we additionally planned to assess bias arising from prior knowledge of cluster‐allocation (identification/recruitment bias, suggested by baseline imbalances in characteristics of participants rather than of clusters) and bias arising from the timing of identification and recruitment of participants (Higgins 2020).

Measures of treatment effect

We analysed the treatment effects in the individual trials by calculating risk ratios (RRs) and risk differences (RDs) for dichotomous data and mean differences (MDs) for continuous data, with their respective 95% confidence intervals (CIs). We also determined the number needed to treat for an additional beneficial outcome (NNTB) or an additional harmful outcome (NNTH) for analyses with a statistically significant RD.

Unit of analysis issues

The unit of analysis was the participating infant in individually randomised trials. For cluster‐randomised trials, we undertook analyses at the level of the individual while accounting for intra‐cluster correlations in the data using methods recommended in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2020). Cross‐over studies were not eligible for inclusion.

Dealing with missing data

We requested additional data from trial authors when data on important outcomes were missing or unclear. If unavailable, we planned to undertake sensitivity analyses to assess the potential impact of missing outcome data.

Assessment of heterogeneity

We examined treatment effects in individual trials and heterogeneity between trial results by inspecting the forest plots if more than one trial was included in a meta‐analysis. We calculated the I² statistic for each analysis to quantify inconsistency across studies and to describe the percentage of variability in effect estimates that may be due to heterogeneity rather than to sampling error. If we detected moderate heterogeneity (I² > 50%) or high heterogeneity (I² > 75%), we planned to explore possible causes (differences in study design, participants, interventions, or outcome assessments).

Assessment of reporting biases

For meta‐analyses with data from 10 or more trials, we created funnel plots and assessed funnel plot asymmetry visually and with Egger's test (for continuous outcomes) or Harbord's modification of Egger's test (for dichotomous outcomes; Harbord 2006).

Data synthesis

We used a fixed‐effect inverse‐variance meta‐analysis for combining data where trials examined the same intervention, and the populations and methods of the trials were sufficiently similar.

Subgroup analysis and investigation of heterogeneity

When we detected high statistical heterogeneity (I² > 75%), we planned to examine the potential causes in the following subgroup analyses for all outcomes.

• Genus of probiotics or combinations (Bifidobacterium spp., Lactobacillus spp., Sacchromyces spp., Streptococcal spp., others, or combinations thereof)

• Type of enteral feeding permitted for participating infants (human milk, formula, or mixed)

Sensitivity analysis

We planned sensitivity analyses to determine how estimates were affected by including only studies at low risk of bias in all domains.

Summary of findings and assessment of the certainty of the evidence

Two review authors (SS, SO, or WM) used the GRADE approach to assess the certainty of the evidence for the outcomes NEC, all‐cause mortality, late‐onset invasive infection, and neurodevelopmental impairment (Schünemann 2013; Walsh 1986).

We considered evidence from RCTs as high certainty to begin with, downgrading by one level for serious (or two levels for very serious) limitations based on the following domains.

• Design (study limitations)

• Inconsistency across studies

• Indirectness of the evidence

• Imprecision of estimates

• Presence of publication bias.

We used GRADEpro GDT to create a summary of findings table for each comparison and to report the certainty of the evidence. The GRADE approach classifies the certainty of a body of evidence as one of the following four grades.

• 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.

Results

Description of studies

Results of the search

After removing duplicates from the search results, we screened 9998 (900 new) titles and abstracts. We reviewed 96 full‐text articles or trial registration records. We included 60 studies (4 new; 56 from previous version of review), excluded 26 studies, listed five studies as awaiting classification, and identified five ongoing studies. For details, see Figure 1.

1.

Study flow diagram: review update 2022.

Included studies

See the Characteristics of included studies table.

We included 60 trials with 11,156 infants. This update added four new trials with 344 participants (Alshaikh 2022; Matin 2022; Moreno‐Sanz 2022; Sowden 2022).

Four trials were conducted before 2000, the rest after 2000. Geographical spread was wide, though most trials took place in Europe (23 trials) or Asia (23 trials). Only one trial took place in sub‐Saharan Africa (Zeber‐Lubecka 2016).

Nine trials were multicentre (Al‐Hosni 2012; Costeloe 2015; Dani 2002; Dilli 2015; Hays 2015; Jacobs 2013; Lin 2008; Manzoni 2009; Totsu 2014), and the rest were single‐centre.

One trial, based in 19 neonatal units in Japan, used a cluster design, with the unit of randomisation being the neonatal unit (Totsu 2014). The remaining trials randomised individual infants to intervention or control groups.

Population

There were fewer than 100 infants enrolled in 25 trials, between 100 and 199 in 20 trials, between 200 and 499 in 13 trials, and 500 or more in three trials: Costeloe 2015 (n = 1310), Dani 2002 (n = 585), and Jacobs 2013 (n = 1099).

Most trials enrolled only very preterm or VLBW infants, with average birth weights of around 1000 g to 1200 g, and average gestational ages at birth of 28 to 32 weeks. Eight trials enrolled infants with a gestational age of up to 34 weeks or a birth weight of up to 1800 g (Chandrashekar 2018; Dashti 2014; Fujii 2006; Hernandez‐Enriquez 2016; Mohan 2006; Ren 2010; Strus 2018; Tewari 2015). We included these trials because the average gestation at birth was under 32 weeks or the average birth weight was under 1500 g.

Three trials included only ELBW infants (Al‐Hosni 2012; Alshaikh 2022; Wejryd 2019). Four trials excluded infants with a birth weight below the 10th percentile for the reference population (i.e. who were small for their gestational age; Al‐Hosni 2012; Hays 2015; Indrio 2017; Kitajima 1997). No trials specified exclusion of infants with evidence of absent or reversed end‐diastolic flow velocities on antenatal Doppler studies of the foetal aorta or umbilical artery.

In most trials, infants could be fed either human milk or formula. Eight trials excluded formula‐fed infants (Alshaikh 2022; Roy 2014; Samanta 2009; Shadkam 2015; Shashidhar 2017; Tewari 2015; Van Niekerk 2014; Wejryd 2019), and five trials excluded human milk‐fed infants (Costalos 2003; Chrzanowska‐Liszewska 2012; Indrio 2017; Reuman 1986; Stratiki 2007).

Interventions and comparisons

The probiotic preparations varied across trials: 34 used single‐genus probiotics (most commonly Bifidobacterium spp. or Lactobacillus spp.), and 26 used multigenus combinations (most commonly Bifidobacterium spp. plus Lactobacillus spp.). Most preparations were commercially available products supplied by the manufacturer for use in the trial.

The specific preparations in each trial were as follows.

• Bifidobacterium spp. (14 trials)

  • B. breve (Costeloe 2015; Fujii 2006; Hikaru 2010; Kitajima 1997; Li 2019; Patole 2014; Wang 2007)

  • B. lactis (Dilli 2015; Mihatsch 2010; Mohan 2006; Stratiki 2007)

  • B. bifidum (Totsu 2014)

  • B. adolescentis (Huang 2009)

  • B. lactis, B. longum, or both (three intervention groups; Hays 2015)

• Lactobacillus spp. (nine trials)

  • L. rhamnosus (Agarwal 2003; Chrzanowska‐Liszewska 2012; Dani 2002; Manzoni 2006; Manzoni 2009; Millar 1993)

  • L. reuteri (Oncel 2014)

  • L. acidophilus (Reuman 1986)

  • L. paracasei (Matin 2022)

• Sacchromyces spp. (four trials)

  • Sacchromyces boulardii (Costalos 2003; Demirel 2013; Serce 2013; Zeber‐Lubecka 2016).

• Bacillus spp. (two trials):

  • Bacillus clausii (Tewari 2015);

  • Bacillus coagulans (Lactobacillus sporogenes in report; Sari 2011).

• Bifidobacterium spp. plus Lactobacillus spp. (11 trials)

  • B. breve, B. bifidum, B. infantis, B. longum, and L. rhamnosus (Alshaikh 2022)

  • B. breve and L. casei (Braga 2011)

  • B. bifidum, B. longum, B. infantis, L. rhamnosus, L. paracasei,L. casei, L. acidophilus, and L. latis (Chowdhury 2016)

  • B. bifidum and L. acidophilus (Lin 2005; Lin 2008; Saengtawesin 2014)

  • B. longum and L. rhamnosus (Rougé 2009)

  • B. longum, B. bifidum, B. lactis, and L. acidophilus (Roy 2014)

  • B. longum, B. bifidum, B.infantis, and L. acidophilus (Samanta 2009)

  • B. longum and L. salivarius (Moreno‐Sanz 2022)

  • B. bifidum, B. infantis, and L. acidophilus (Sowden 2022)

• Bifidobacterium spp. plus Streptococcus spp. (two trials)

  • B. infantis, B. lactis, and S. thermophilus (Jacobs 2013)

  • B. infantis, B. bifidum (Lactobacillus bifidus in report), and S. thermophilus (Bin‐Nun 2005)

• Bifidobacterium spp. plus Lactobacillus spp. plus Sacchromyces spp. (four trials)

  • B. infantis, L. rhamnosus, L. casei, L. plantarum, L. acidophilus, and S. boulardii (Dutta 2015)

  • B. bifidum, L acidophilus, and S. boulardii (Hariharan 2016)

  • B. longum, L.acidophilus, L. rhamnosus, and S. boulardii (Chandrashekar 2018; Shashidhar 2017)

• Bifidobacterium spp. plus Lactobacillus spp. plus Streptococcus spp. (five trials)

  • B longum, B. breve, L. acidophilus, L. rhamnosus, L. bulgaricus, L. casei, and S. thermophilus (Dashti 2014)

  • B. infantis, L. rhamnosus, L. casei, L. plantarum, L acidophilus, and S. thermophilus (Fernández‐Carrocera 2013)

  • B. infantis, L acidophilus, and Enterococcus faecium (Kanic 2015)

  • B. infantis, L. acidophilus, Enterococcus faecium, and Bacillus cereus (Ren 2010)

  • Bifidobacterium spp. (not specified), L. acidophilus, L. delbrueckii, and S. thermophilus (Rehman 2018)

Half (30) of the trials gave control infants milk feeds that were not supplemented with probiotics. The others used active placebos, most commonly maltodextrin or corn starch powder (20 trials).

Most trials initiated probiotic administration (and placebo if used) during the first week after birth, typically with the first enteral feed. The lyophilised probiotics were reconstituted in water or milk, and administered to supply 10⁸ to 10⁹ colony forming units per dose, once or twice daily via a gastric feeding tube. In most trials, the intervention period was at least six weeks, typically until 34 to 36 weeks' postmenstrual age, or until discharge from hospital. Thirteen of the trials administered the intervention for a shorter period, between seven and 30 days (Braga 2011; Costalos 2003; Dutta 2015; Huang 2009; Kitajima 1997; Matin 2022; Millar 1993; Mohan 2006; Ren 2010; Reuman 1986; Shadkam 2015; Sowden 2022; Van Niekerk 2014). One trial continued the intervention until the infant reached 2000 g bodyweight (Totsu 2014).

Outcomes

Fifty‐seven trials reported the number of infants who developed NEC, and 54 trials reported mortality prior to hospital discharge. For 49 trials, we obtained published or unpublished data for the number of infants with at least one episode of culture‐confirmed infection. Other in‐hospital outcomes included time to establish full enteral feeding, rate of weight gain, and duration of hospital stay (23 trials). Five trials reported neurodevelopmental or cognitive outcomes (Jacobs 2013; Lin 2005; Oncel 2014; Sari 2011; Totsu 2014). One trial assessed cognition in participants aged three to five years (Patole 2014). Three trials reported no outcomes of interest for this review (Agarwal 2003; Li 2019; Moreno‐Sanz 2022).

Excluded studies

We excluded 26 studies (see the Characteristics of excluded studies table). The most common reasons for exclusion were ineligible population (most participants not very preterm or VLBW), ineligible intervention (prebiotics or synbiotics), and ineligible design (not randomised). Four screened articles were secondary reports for included trials.

Ongoing studies

See the Characteristics of ongoing studies table. We identified five ongoing studies (Marißen 2019; NCT00977912; NCT01181791; NCT01375309; NCT04541771). Three of these studies were completed before 2014. We received no responses from the investigators regarding current status or availability of outcomes data (NCT00977912; NCT01181791; NCT01375309).

Awaiting classification

See the Characteristics of studies awaiting classification table. Five studies are awaiting classification (Coleta 2013; Kaban 2019; Niazi 2021; Punnahitananda 2006; Reznikov 2022). We received no responses from the investigators regarding study design or other characteristics to determine eligibility for this review.

Risk of bias in included studies

Methodological quality varied across the included trials (Figure 2).

2.

Risk of bias summary: review authors' judgements about each risk of bias item for each included study.

Allocation

We considered 27 trials at low risk of selection bias because they employed adequate methods to generate the random sequence (typically computer‐generated randomisation) and to conceal allocation (typically central or pharmacy allocation or storage of allocation codes in sealed envelopes; we did not consider it necessary for reports to describe the envelopes as opaque). There was insufficient information on randomisation and allocation concealment methods in 26 trials (unclear risk of bias). Five quasi‐RCTs used alternate allocation (high risk of bias).

Blinding

We judged 28 trials at low risk of performance bias and detection bias. These were placebo‐controlled (usually maltodextrin), or the report or trial authors indicated that the people responsible for preparing the intervention (mixing the probiotic in milk) were not directly involved in other caregiving duties or outcome assessment (e.g. pharmacy staff). In 13 trials, control infants received milk feeds without probiotic supplements, but it was unclear whether staff were aware of the group allocation (unclear risk of bias). There were no masking measures in 19 trials (high risk of bias).

Incomplete outcome data

Attrition bias did not appear to be an issue in most trials (outcome data reported for more than 80% of randomised cohorts).

Selective reporting

Most reports did not mention a trial protocol. However, reporting bias was unlikely to be an issue in trials that reported our primary outcome and our infant‐important outcomes (low risk of bias). In trials that assessed surrogate outcomes such as stool colonisation or intestinal permeability, clinical outcome data were generally available from the trial authors.

Other potential sources of bias

We found no evidence of between‐group baseline differences in participant characteristics or demographics (particularly average birth weight, gestational age at birth, and exposure to antenatal corticosteroids) in most trials. Two trials did not report baseline demographic characteristics so were at unclear risk of bias (Fernández‐Carrocera 2013; Ren 2010). We judged three trials at high risk of other bias because the average birth weight was higher in the intervention group than in the control group (Dilli 2015; Sadowska‐Krawczenko 2012), or because antenatal corticosteroid exposure was higher in the intervention group than in the control group (Totsu 2014).

Effects of interventions

See: Table 1; Table 2

Comparison 1. Probiotics versus placebo or no probiotics in very preterm or very low birth weight infants

See Table 1.

Primary outcomes

Necrotising enterocolitis

Meta‐analysis of data from 57 trials (10,918 infants) produced the following results (Analysis 1.1; Figure 3)

1.1. Analysis.

Comparison 1: Probiotics versus placebo or no probiotics (very preterm or very low birth weight infants), Outcome 1: Necrotising enterocolitis

3.

Forest plot of comparison: 1 Probiotics versus placebo or no probiotics, outcome: 1.1 Necrotising enterocolitis.

• RR 0.54, 95% CI 0.46 to 0.65; I² = 17%

• RD −0.03, 95% CI −0.04 to −0.02

• NNTB 33, 95% CI 25 to 50

There was evidence of funnel plot asymmetry consistent with publication bias favouring the treatment effect (Harbord's modified test for bias −0.71, 95% CI −1.40 to −0.01; P = 0.05; Figure 4).

4.

Funnel plot of comparison: 1 Probiotics versus placebo or no probiotics, outcome: 1.1 Necrotising enterocolitis.

We assessed the certainty of evidence as low using the GRADE approach.

Probiotics compared with placebo or no probiotics may reduce NEC before hospital discharge in very preterm or VLBW infants.

All‐cause mortality

Meta‐analysis of data from 54 trials (10,484 infants) produced the following results (Analysis 1.2; Figure 5).

1.2. Analysis.

Comparison 1: Probiotics versus placebo or no probiotics (very preterm or very low birth weight infants), Outcome 2: All‐cause mortality

5.

Forest plot of comparison: 1 Probiotics versus placebo or no probiotics, outcome: 1.2 Mortality.

• RR 0.77, 95% CI 0.66 to 0.90; I² = 0%

• RD −0.02, 95% CI −0.02 to −0.01

• NNTB 50, 95% CI 50 to 100

There was no evidence of funnel plot asymmetry (Harbord's modified test for bias −0.37, 95% CI −0.99 to 0.24; P = 0.24; Figure 6).

6.

Funnel plot of comparison: 1 Probiotics versus placebo or no probiotics, outcome: 1.2 Mortality.

We assessed the certainty of evidence as moderate using the GRADE approach.

Probiotics compared with placebo or no probiotics probably reduce all‐cause mortality before hospital discharge in very preterm or VLBW infants.

Secondary outcomes

Late‐onset invasive infection

Meta‐analysis of data from 49 trials (9876 infants) produced the following results (Analysis 1.3).

1.3. Analysis.

Comparison 1: Probiotics versus placebo or no probiotics (very preterm or very low birth weight infants), Outcome 3: Late‐onset invasive infection

• RR 0.89, 95% CI 0.82 to 0.97; I² = 22%

• RD −0.02, 95% CI −0.03 to 0.00

There was no evidence of funnel plot asymmetry (Harbord's modified test for bias −0.06, 95% CI −0.45 to 0.33; P = 0.87)

We assessed the certainty of evidence as moderate using the GRADE approach.

Probiotics compared with placebo or no probiotics probably have little or no effect on late‐onset invasive infection before hospital discharge in very preterm or VLBW infants.

Late‐onset infection with the supplemented probiotic microorganism

No studies reported invasive infection caused by the supplemented probiotic microorganisms.

Duration of hospitalisation from birth

Meta‐analysis of data from 24 trials (5572 infants) suggested a reduction in duration of hospitalisation with probiotics (MD −1.68 days, 95% CI −3.08 to −0.28; I² = 30%; Analysis 1.4).

1.4. Analysis.

Comparison 1: Probiotics versus placebo or no probiotics (very preterm or very low birth weight infants), Outcome 4: Duration of hospitalisation from birth (days)

Two other trials reported data that we were unable to meta‐analyse: Oncel 2014 reported shorter median duration of hospitalisation (38 versus 46 days), and Tewari 2015 reported no difference in duration of hospitalisation (data not provided).

Neurodevelopmental impairment

Five trials reported severe neurodevelopmental impairment (either motor, sensory, or cognitive) in surviving children. Three assessed children using Bayley Scales of Infant Development II (BSID‐II) at 18 to 24 months (Oncel 2014; Sari 2011), or three years (Lin 2005) post‐term. One trial assessed Bayley‐III composite scales, Movement Assessment Battery for Children, and Wechsler Preschool and Primary Scale of Intelligence Full Scale Intelligence Quotient at two to five years (Jacobs 2013). One trial, undertaken in Japan, used the Kyoto Scale of Psychological Development 2001 (similar to the Bayley III scales) and physical examination to assess neurodevelopmental status at 18 months post‐term (Totsu 2014).

Completeness of neurodevelopmental follow‐up assessment was balanced between groups in all trials but varied across trials, as follows.

• Lin 2005: 90%

• Sari 2011: 84%

• Totsu 2014: 73%

• Oncel 2014: 68%

• Jacobs 2013: 48%

No individual trials showed an effect of probiotics on neurodevelopmental impairment. A meta‐analysis of data from five trials (1518 infants) produced the following results (Analysis 1.5).

1.5. Analysis.

Comparison 1: Probiotics versus placebo or no probiotics (very preterm or very low birth weight infants), Outcome 5: Neurodevelopmental impairment

• RR 1.03, 95% CI 0.84 to 1.26; I² = 0%

• RD 0.01, 95% CI −0.02 to 0.03

We assessed the certainty of evidence as low using the GRADE approach.

Probiotics compared with placebo or no probiotics may have little or no effect on neurodevelopmental impairment at 18 months to three years in very preterm or VLBW infants.

Cerebral palsy

No individual trials showed an effect of probiotics on cerebral palsy. A meta‐analysis of data from five trials (1512 infants) suggested little or no effect (RR 1.13, 95% CI 0.74 to 1.72; I² = 18%; Analysis 1.6).

1.6. Analysis.

Comparison 1: Probiotics versus placebo or no probiotics (very preterm or very low birth weight infants), Outcome 6: Cerebral palsy

Visual impairment

No individual trials showed an effect of probiotics on visual impairment. A meta‐analysis of data from four trials (1356 infants) suggested little or no effect (RR 0.50, 95% CI 0.14 to 1.80; I² = 0%; Analysis 1.7).

1.7. Analysis.

Comparison 1: Probiotics versus placebo or no probiotics (very preterm or very low birth weight infants), Outcome 7: Visual impairment

Hearing impairment

No individual trials showed an effect of probiotics on hearing impairment. A meta‐analysis of data from four trials (1356 infants) suggested little or no effect (RR 0.46, 95% CI 0.18 to 1.17; I² = 32%; Analysis 1.8)

1.8. Analysis.

Comparison 1: Probiotics versus placebo or no probiotics (very preterm or very low birth weight infants), Outcome 8: Hearing impairment

Cognitive performance

Patole 2014 assessed 42% of eligible participants aged three to five years using the Mullen's Scale of Early Learning tool. Analysis showed little or no effect of probiotics on the "early learning composite score" (RR −1.00, 95% CI −6.38, 4.38; Analysis 1.9)

1.9. Analysis.

Comparison 1: Probiotics versus placebo or no probiotics (very preterm or very low birth weight infants), Outcome 9: Cognitive performance

Comparison 2. Probiotics versus control in extremely preterm or ELBW infants

See Table 2.

Three trials restricted participation to ELBW infants (Al‐Hosni 2012; Alshaikh 2022; Wejryd 2019). Seven trials reported subgroup data for extremely preterm or ELBW infants (Costeloe 2015; Jacobs 2013; Oncel 2014; Roy 2014; Sowden 2022; Tewari 2015; Wang 2007).

Primary outcomes

Necrotising enterocolitis

Meta‐analysis of data from 10 trials (1836 infants) produced the following results (Analysis 2.1).

2.1. Analysis.

Comparison 2: Probiotics versus placebo or no probiotics (extremely preterm or extremely low birth weight infants), Outcome 1: Necrotising enterocolitis

• RR 0.92, 95% CI 0.69 to 1.22; I² = 0%

• RD −0.01, 95% CI −0.03 to 0.02

We assessed the certainty of evidence as low using the GRADE approach.

Probiotics compared with placebo or no probiotics may have little or no effect on NEC before hospital discharge in extremely preterm or ELBW infants.

All‐cause mortality

Meta‐analysis of data from seven trials (1723 infants) produced the following results (Analysis 2.2).

2.2. Analysis.

Comparison 2: Probiotics versus placebo or no probiotics (extremely preterm or extremely low birth weight infants), Outcome 2: All‐cause mortality

• RR 0.92, 95% CI 0.72 to 1.18; I² = 0%

• RD −0.01, 5% CI −0.04 to 0.02

We assessed the certainty of evidence as low using the GRADE approach.

Probiotics compared with placebo or no probiotics may have little or no effect on all‐cause mortality before hospital discharge in extremely preterm or ELBW infants.

Secondary outcomes

Late‐onset invasive infection

Meta‐analysis of data from seven trials (1533 infants) produced the following results (Analysis 2.3).

2.3. Analysis.

Comparison 2: Probiotics versus placebo or no probiotics (extremely preterm or extremely low birth weight infants), Outcome 3: Late‐onset invasive infection

• RR 0.93, 95% CI 0.78 to 1.09; I² = 0%

• RD −0.02, 95% CI −0.06 to 0.02

We assessed the certainty of evidence as low using the GRADE approach.

Probiotics compared with placebo or no probiotics may have little or no effect on late‐onset invasive infection before hospital discharge in extremely preterm or ELBW infants.

Late‐onset infection with the supplemented probiotic microorganism

No trials reported invasive infection caused by the supplemented probiotic microorganisms.

Duration of hospitalisation from birth

Analysis of data from two trials (84 infants) suggested little or no effect of probiotics of duration of hospitalisation (MD 2.18 days, 95% CI −13.8 to 18.21; I² = 77%; Analysis 2.4)

2.4. Analysis.

Comparison 2: Probiotics versus placebo or no probiotics (extremely preterm or extremely low birth weight infants), Outcome 4: Duration of hospitalisation from birth (days)

Neurodevelopmental impairment

No trials provided subgroup data on neurodevelopmental outcomes for meta‐analysis. Three trials stated that probiotics had no effect on the rate of severe neurodevelopmental impairment in the extremely preterm or ELBW subgroup (Jacobs 2013; Sari 2011; Totsu 2014).

Subgroup comparison by genus of probiotics

Primary outcomes

Necrotising enterocolitis

There was no evidence of subgroup differences depending on genus of probiotics for NEC (Chi² = 10.44, df = 7 (P = 0.16), I² = 33.0%; Analysis 1.1; Figure 3).

All‐cause mortality

There was no evidence of subgroup differences depending on genus of probiotics for all‐cause mortality (Chi² = 3.38, df = 7 (P = 0.85), I² = 0%; Analysis 1.2; Figure 5).

Secondary outcomes

Late‐onset invasive infection

There was no evidence of subgroup differences depending on genus of probiotics for late‐onset invasive infection (Chi² = 2.73, df = 7 (P = 0.91), I² = 0%; Analysis 1.3).

Duration of hospitalisation from birth

There was no evidence of subgroup differences depending on genus of probiotics for duration of hospitalisation (Chi² = 5.99, df = 6 (P = 0.42), I² = 0%; Analysis 1.4).

Neurodevelopmental impairment

There was no evidence of subgroup differences depending on genus of probiotics for neurodevelopmental impairment (Chi² = 1.48, df = 4 (P = 0.83), I² = 0%; Analysis 1.5).

Cerebral palsy

There was no evidence of subgroup differences depending on genus of probiotics for cerebral palsy (Chi² = 4.86, df = 4 (P = 0.30), I² = 17.7%; Analysis 1.6).

Visual impairment

There was no evidence of subgroup differences depending on genus of probiotics for visual impairment (Chi² = 1.75, df = 2 (P = 0.42), I² = 0%; Analysis 1.7).

Hearing impairment

There was no evidence of subgroup differences depending on genus of probiotics for hearing impairment (Chi² = 4.35, df = 3 (P = 0.23), I² = 31.1%; Analysis 1.8).

Subgroup comparison by type of enteral feed (human milk versus formula versus mixed)

Primary outcomes

Necrotising enterocolitis

There was no evidence of subgroup differences depending on the type of enteral feed for NEC (Chi² = 3.92, df = 2 (P = 0.14), I² = 49.0%; Analysis 3.1).

3.1. Analysis.

Comparison 3: Subgroup analysis by type of feeding, Outcome 1: Necrotising enterocolitis

All‐cause mortality

There was no evidence of subgroup differences depending on the type of enteral feed for all‐cause mortality (Chi² = 2.97, df = 2 (P = 0.23), I² = 32.8%; Analysis 3.2).

3.2. Analysis.

Comparison 3: Subgroup analysis by type of feeding, Outcome 2: All‐cause mortality

Secondary outcomes

Late‐onset invasive infection

There was no evidence of subgroup differences depending on the type of enteral feed for late‐onset invasive infection (Chi² = 4.33, df = 2 (P = 0.11), I² = 53.8%; Analysis 3.3).

3.3. Analysis.

Comparison 3: Subgroup analysis by type of feeding, Outcome 3: Late‐onset invasive infection

Duration of hospitalisation from birth

There was some evidence of subgroup differences depending on the type of enteral feed for duration of hospitalisation, with effect size larger in formula than human milk, and larger in human milk than in the mixed group (Chi² = 6.01, df = 2 (P = 0.05), I² = 66.7%; Analysis 3.4).

3.4. Analysis.

Comparison 3: Subgroup analysis by type of feeding, Outcome 4: Duration of hospitalisation from birth (days)

Neurodevelopmental impairment

In all trials that reported neurodevelopmental impairment, the infants may have received human milk, formula, or both.

Sensitivity meta‐analyses of trials at low risk of bias across all domains

Primary outcomes

Necrotising enterocolitis

Sensitivity meta‐analysis of 17 trials (4649 infants) at low risk of bias produced the following results, suggesting a reduced risk of NEC with probiotics (Analysis 4.1).

4.1. Analysis.

Comparison 4: Sensitivity analyses: trials at low risk of bias, Outcome 1: Necrotising enterocolitis

• RR 0.70, 95% CI 0.55 to 0.89; I² = 25%

• RD −0.02, 95% CI −0.03 to −0.01

• NNTB 50, 95% CI 33 to 100

All‐cause mortality

Sensitivity meta‐analysis of 17 trials (4649 infants) at low risk of bias produced the following results, suggesting little or no effect of probiotics on all‐cause mortality (Analysis 4.2).

4.2. Analysis.

Comparison 4: Sensitivity analyses: trials at low risk of bias, Outcome 2: All‐cause mortality

• RR 0.86, 95% CI 0.69 to 1.07; I² = 0%

• RD −0.01, 95% CI −0.02 to 0.00

Late‐onset invasive infection

Sensitivity meta‐analysis of 17 trials (4649 infants) at low risk of bias produced the following results, suggesting little or no effect of probiotics on late‐onset invasive infection (Analysis 4.3).

4.3. Analysis.

Comparison 4: Sensitivity analyses: trials at low risk of bias, Outcome 3: Late‐onset invasive infection

• RR 0.89, 95% CI 0.78 to 1.01; I² = 0%

• RD −0.02, 95% CI −0.04 to 0.00

Duration of hospitalisation from birth

Sensitivity meta‐analysis of seven trials (2838 infants) at low risk of bias suggested little or no effect of probiotics on duration of hospitalisation from birth (MD −2.45 days, 95% CI −5.45 to 0.56; I² = 44%; Analysis 4.4).

4.4. Analysis.

Comparison 4: Sensitivity analyses: trials at low risk of bias, Outcome 4: Duration of hospitalisation from birth (days)

Neurodevelopmental impairment

Sensitivity meta‐analysis of two trials (913 infants) at low risk of bias produced the following results, suggesting little or no effect of probiotics on neurodevelopmental impairment (Analysis 4.5).

4.5. Analysis.

Comparison 4: Sensitivity analyses: trials at low risk of bias, Outcome 5: Neurodevelopmental impairment

• RR 0.99, 95% CI 0.76 to 1.27; I² = 0%

• RD 0.00, 95% CI −0.05 to 0.05

Cerebral palsy

Sensitivity meta‐analysis of two trials (913 infants) at low risk of bias produced the following results, suggesting little or no effect of probiotics on cerebral palsy (Analysis 4.6).

4.6. Analysis.

Comparison 4: Sensitivity analyses: trials at low risk of bias, Outcome 6: Cerebral palsy

• RR 1.14, 95% CI 0.68 to 1.92; I² = 0%

• RD 0.01, 95% CI −0.02 to 0.04

Visual impairment

Sensitivity meta‐analysis of two trials (913 infants) at low risk of bias produced the following results, suggesting little or no effect of probiotics on visual impairment (Analysis 4.7).

4.7. Analysis.

Comparison 4: Sensitivity analyses: trials at low risk of bias, Outcome 7: Visual impairment

• RR 2.91, 95% CI 0.12 to 71.21; I² not applicable

• RD 0.00, 95% CI −0.01 to 0.01

Hearing impairment

Sensitivity meta‐analysis of two trials (913 infants) at low risk of bias produced the following results, suggesting little or no effect of probiotics on hearing impairment (Analysis 4.8).

4.8. Analysis.

Comparison 4: Sensitivity analyses: trials at low risk of bias, Outcome 8: Hearing impairment

• RR 0.30, 95% CI 0.09 to 0.98; I² = 60%

• RD −0.02, 95% CI −0.03 to −0.00

Discussion

Summary of main results

Meta‐analysis of data from more than 50 trials, with more than 10,000 participants in total, showed that enteral supplementation with probiotics may reduce the risk of NEC. Probiotics probably reduce mortality, though sensitivity meta‐analysis of trials at low risk of bias did not show a clear effect. Probiotics probably have little or no effect on late‐onset invasive infection. No trials reported instances of invasive infection caused by the probiotic organisms being tested. Meta‐analysis of data from five trials suggested that probiotics may have little or no effect on neurodevelopmental impairment. According to GRADE assessment, the certainty of the evidence in this review is low to moderate.

Overall completeness and applicability of evidence

Most of the trials were undertaken after the year 2000 in healthcare facilities worldwide, though predominantly in Europe and Asia. The findings should be applicable to current care practices for very preterm or VLBW infants, including infants who are small for gestation at birth (only four trials excluded such infants, and none defined evidence of abnormal end‐diastolic flow velocities in foetal Doppler studies as an exclusion criterion). The average event rate for NEC in the control group was 6%, consistent with estimates from prevalence studies in very preterm of VLBW infants in high‐income countries (Battersby 2018). We prespecified a comparison including only data for extremely preterm or ELBW infants. Three trials restricted participation to this population, and a further six trials reported subgroup data. Meta‐analyses suggested little or no effect of probiotics on NEC, all‐cause mortality, late‐onset invasive infection, or duration of hospitalisation from birth. However, these estimates are imprecise, and the CIs around the point estimates do not rule out important benefits or harms in this subpopulation, consistent with the effects seen in the meta‐analyses including the entire very preterm or VLBW population.

The review findings are likely to be broadly applicable to infants fed enterally with human milk, formula, or both. Formula feeding increases risk of NEC, and the risk‐benefit balance of probiotic supplementation could differ between human milk‐ and formula‐fed very preterm or VLBW infants (Quigley M 2019). Prespecified subgroup analyses showed no difference in effects on the primary outcomes between trials that included only human milk‐fed infants versus trials that included infants fed with formula, either alone or with human milk. However, the reported data in trials that permitted human milk or formula feeding were insufficient to analyse subgroup effects at an infant level by type of enteral feeds received.

The main challenge in applying the findings of this review is the heterogeneity of the interventions tested. Data from the only two large (more than 1000 infants) high‐quality trials illustrate this concern (Costeloe 2015; Jacobs 2013). The largest trial of probiotic supplementation (n = 1310) showed that a single‐strain preparation of B. breve is probably ineffective in reducing NEC (Costeloe 2015). Conversely, the combination of B. infantis, S. thermophilus, and B. lactis used in the other large trial (n = 1099) is probably effective in reducing the risk of NEC, though not mortality or infection (Jacobs 2013). However, prespecified subgroup analyses showed no evidence of differences in effect sizes depending on the genus of the probiotics used, broadly consistent with recent network analyses of different probiotic combinations (Bi 2019; Morgan 2020; van den Akker 2018). These findings should be interpreted with cation, as indirect comparisons are not randomised. Differences in effect between trials or groups of trials could be due to many factors, including methodological quality, types of participants, setting, and other practices and policies such as feeding protocols and antibiotic stewardship. Effect estimates may be confounded by species and strain level differences that affect how probiotic organisms interact with each other and endogenous microorganisms in the intestine of immature infants (Millar 2012). Consequently, the optimal probiotic composition or combination is unlikely to be determined reliably by analyses of the existing trial data.

Quality of the evidence

The certainty of evidence assessed with the GRADE approach was low or moderate for the prespecified outcomes (Table 1; Table 2). About half of the trials had methodological weaknesses, including in methods used to conceal random allocation and to mask clinicians, parents, and caregivers to the intervention (Figure 2). Knowledge of the intervention group could have affected caregivers' or assessors' subjective perceptions of outcomes; for example, it may have influenced decisions on whether to investigate or diagnose NEC or invasive infection.

Most of the included trials were small (median n = 149). The asymmetry evident in the funnel plot for the meta‐analysis of the effect on NEC was consistent with small‐study bias (Figure 4). One explanation is publication bias, which is the tendency for articles that report statistically significant effects to be submitted and accepted for publication (Gale 2020). Publication bias, as well as other sources of small‐study bias, has become increasingly evident as an important contributor to exaggerated effect size estimates in meta‐analyses of interventions to improve outcomes in very preterm or VLBW infants (Ohlsson 2020; Pammi 2020). Another concern is that in many of the trials that assessed the effect of probiotics on clinical outcomes, it was unclear how investigators had defined the sample size, and whether trial 'stopping rules' existed. If trial investigators were able to monitor accumulating outcome data until an effect on an outcome was detected, this may have resulted in a tendency to detect spurious effects that inflate the pooled estimate of effect sizes.

Attrition bias due to loss of outcome data from randomised participants was not a concern for the in‐hospital outcomes (NEC, death, infection) assessed in this review. However, completeness of long‐term neurodevelopmental outcome data ranged from 48% to 90% across the trials that reported such assessments. The degree of incomplete follow‐up assessment was balanced across the intervention and control groups in each trial. Although this is reassuring with regard to the impact of attrition bias on effect estimates, some concern remains that the assessed population may not be representative of the entire cohort (Tin 1998). For this reason, the evidence suggesting that probiotics have little or no effect on neurodevelopmental outcomes is of low certainty.

Potential biases in the review process

The main concern with meta‐analysis of the effect on NEC is the possibility that the findings are subject to small‐study biases, including publication bias. Trials that reported statistically significant or potentially important effects may have contributed more data to the meta‐analysis than other trials (Hopewell 2009). We attempted to minimise this threat by searching the proceedings of major international perinatal conferences to identify trial reports that were not published in full form in journals. However, it is possible that some trials were undertaken but not reported; such trials are less likely than published trials to have detected statistically significant or clinically important effects.

We contacted trial authors for unpublished data (Young 2011). In several cases, authors of 'proof of concept' or exploratory trials that aimed primarily to assess whether probiotic administration affected intestinal (stool) colonisation patterns or permeability or immune function were able to provide unpublished clinical outcome data for inclusion in meta‐analyses.

We identified no potential risk of bias due to the funding sources of the included trials (where reported). In related contexts, such as manufacturers of breast milk substitutes funding infant feeding trials, this conflict is important to note (Cleminson 2015), but we did not consider it a substantial risk of bias in the case of this review. Manufacturers of probiotic products supported some of the trials by supplying the intervention at no or low cost (noted in the Characteristics of included studies table), but we considered they were unlikely to have a conflict of interest in the trial outcome for this relatively niche indication.

Agreements and disagreements with other studies or reviews

Our findings are broadly consistent with other systematic reviews of probiotics for preterm infants (summarised in Jarrett 2019). Our review differs from others in the following key respects.

• We restricted the population of interest to very preterm and VLBW infants to enhance applicability to those infants at high risk of developing NEC and associated complications.

• We included trials that assessed probiotics only and excluded trials that assessed prebiotics or synbiotics.

• We conducted genus‐level subgroup analyses to explore differences in effect sizes depending on the probiotic or combination of probiotics assessed.

• We included formal statistical evaluation to assess the risk of small‐study bias for the major outcomes.

• We prespecified sensitivity analyses to determine how trial methodological quality affected effect sizes.

• We included a formal GRADE assessment of the certainty of the evidence at outcomes level to help inform policy, practice, and research (Gephart 2020).

Authors' conclusions

Implications for practice.

Despite the quantity of trial data, we found low‐ to moderate‐certainty evidence on the effects of probiotic supplements on the risk of necrosing enterocolitis (NEC) and associated morbidity and mortality in very preterm or very low birth weight (VLBW) infants. The evidence for all assessed outcomes in extremely preterm or extremely low birth weight (ELBW) infants was of low certainty.

As well as concerns that effect size estimates are inflated by biases in the existing trials (including publication bias), a major barrier to implementing the findings is that existing analyses cannot determine the optimal constitution of probiotics (strains, doses, timing of introduction, duration of use) for routine prophylactic use (Fleming 2022). A variety of commercially available probiotic preparations are in use in a minority of neonatal units internationally, but widespread use appears to be limited by availability and by regulatory and licencing issues. Although the data from the included trials are reassuring with regard to safety, there have been reports of probiotic bacteraemia or fungaemia and other adverse effects in preterm infants (Bertelli 2015; Esaiassen 2016; Jenke 2012; Zbinden 2015). It remains unclear whether different strains or combinations have different safety profiles.

Implications for research.

Given the uncertainty about whether (and which) probiotics affect important outcomes in very preterm or VLBW infants, further large, high‐quality trials are needed to provide evidence of sufficient validity and applicability to inform policy and practice. Before starting such a trial, it is essential for investigators to determine whether families and clinicians would support it.

Any planned trials should attempt to ensure that caregivers and assessors are masked to the intervention, as investigation and diagnosis of important outcomes such as NEC, invasive infection, and neurodevelopmental impairment can be subjective. While it may be appropriate to be broadly inclusive of very preterm and VLBW infants, trials should ensure sufficient power to assess effects in extremely preterm or ELBW infants, and to explore interactions with the type of enteral feed given the potential of human milk components to moderate any probiotic effects.

A key concern in planning any trial is choosing the appropriate intervention to assess. Two options appear favourable. First, a confirmatory trial of the probiotic combination Bifidobacterium infantis, Streptococcus thermophilus and Bifidobacterium lactis, already shown to be likely to reduce the risk of NEC in one high‐quality trial in Australasia (Jacobs 2013). Alternatively, investigators may consider a pragmatic choice based on multistrain products in established use in their regions; this would provide some availability and quality control reassurances with regard to product integrity and safety. Furthermore, investigators could consider whether trials using synbiotics (combinations of probiotics with prebiotics such as human milk oligosaccharides and other milk glycans) are merited alongside trials of probiotics, or as part of an adaptive design (Underwood 2019).

Although individual infant randomisation is preferred for statistical and analytical reasons, it is possible that cross‐contamination of the trial organisms to infants in the control group will limit the power of the trial to detect an effect (as may have happened in Costeloe 2015). Randomising at the neonatal care centre level (cluster‐randomised controlled trial) obviates this problem but inflates the sample size requirement considerably because of intracluster correlation of outcomes.

What's new

Date	Event	Description
18 July 2023	New citation required but conclusions have not changed	Probiotics may reduce the risk of necrotising enterocolitis, but the certainty of the evidence is low.
18 July 2023	New search has been performed	We searched the literature in July 2022. We included four new trials (Alshaikh 2022; Matin 2022; Moreno‐Sanz 2022; Sowden 2022).

History

Protocol first published: Issue 4, 2005
Review first published: Issue 1, 2008

Date	Event	Description
4 October 2020	New citation required and conclusions have changed	Probiotics may reduce the risk of necrotising enterocolitis, but the certainty of the evidence is "low".
4 October 2020	New search has been performed	Inclusion criteria modified to include only very preterm (< 32 weeks' gestation) or very low birth weight infants (< 1500 g) with pre‐specified analyses for extremely preterm (< 28 weeks' gestation) or extremely low birth weight (< 1000 g) infants. The literature was searched in February 2020. Thirty‐two new published trials were identified.
1 October 2013	New search has been performed	This updates Al Faleh 2011
1 October 2013	New citation required but conclusions have not changed	Updated search identified eight new trials for inclusion in this review update.
3 November 2010	New search has been performed	This updates the review "Probiotics for prevention of necrotizing enterocolitis in preterm infants" published in the Cochrane Database of Systematic Reviews (Al Faleh 2008). New authorship: Khalid AlFaleh, Jasim Anabrees, Dirk Bassler, Turki Al‐Kharfi. Updated search identified seven new trials for inclusion in this review update.
3 November 2010	New citation required and conclusions have changed	With the addition of seven new trials to this update, it brings the total to sixteen eligible trials randomizing 2842 infants. The previous review included nine eligible trials, randomizing 1425 infants.
12 November 2008	Feedback has been incorporated	Feedback incorporated
22 July 2008	Amended	Converted to new review format.

Appendices

Appendix 1. Electronic search methodology

Search date: 5 July 2022

Cochrane Register of Controlled Trials (CENTRAL)

#1 [mh Probiotics]

#2 (probiotic*):ti,ab,kw

#3 [mh Bifidobacterium]

#4 (bifidobacterium*):ti,ab,kw

#5 [mh Lactobacillus]

#6 (lactobacill*):ti,ab,kw

#7 ([mh ^Saccharomyces] or [mh ^"Saccharomyces boulardii"] or [mh ^"Saccharomyces cerevisiae"])

#8 [mh ^"Saccharomyces boulardii"]

#9 (Saccharomyces):ti,ab,kw

#10 #1 OR #2 OR #3 OR #4 OR #5 OR #6 OR #7 OR #8 OR #9

#11 [mh Prebiotics]

#12 (prebiotic*):ti,ab,kw

#13 [mh Oligosaccharides]

#14 (oligosaccharide*):ti,ab,kw

#15 [mh Inulin]

#16 (inulin*):ti,ab,kw

#17 ((fructooligosaccharide* or fructo NEXT oligosaccharide* or FOS or FOSs or galacto NEXT oligosaccharide* or galactooligosaccharide*)):ti,ab,kw

#18 [mh Lactoferrin]

#19 (lactoferrin*):ti,ab,kw

#20 [mh Lactulose] 439

#21 (lactulose*):ti,ab,kw

#22 #11 OR #12 OR #13 OR #14 OR #15 OR #16 OR #17 OR #18 OR #19 or #20 or #21

#23 [mh Synbiotics]

#24 (synbiotic*):ti,ab,kw

#25 (((probiotic* and prebiotic*) NEAR/4 combin*)):ti,ab,kw

#26 #23 OR #24 OR #25

#27 #10 OR #22 OR #26

#28 [mh "Infant, Newborn"]

#29 [mh "Premature Birth"]

#30 neonat*:ti,ab,kw

#31 neo NEXT nat*:ti,ab,kw

#32 newborn or new NEXT born* or newly NEXT born*:ti,ab,kw

#33 preterm or preterms or pre NEXT term or pre NEXT terms:ti,ab,kw

#34 preemie* or premie or premies:ti,ab,kw

#35 prematur* NEAR/3 (birth* or born or deliver*):ti,ab,kw

#36 low NEAR/3 (birthweight* or birth NEXT weight*):ti,ab,kw

#37 lbw or vlbw or elbw:ti,ab,kw

#38 infan* or baby or babies:ti,ab,kw

#39 #28 or #29 or #30 or #31 or #32 or #33 or #34 or #35 or #36 or #37 or #38

#40 #27 AND #39 in Trials

CINAHL via EBSCO

S35 S31 AND S34

S34 S32 OR S33

S33 TX ( (neonat* or neo nat*) ) OR TX ( (newborn* or new born* or newly born*) ) OR TX ( (preterm or preterms or pre term or pre terms) ) OR TX ( (preemie$ or premie or premies) ) OR TX ( (prematur* N3 (birth* or born or deliver*)) ) OR TX ( (low N3 (birthweight* or birth weight*)) ) OR TX ( (lbw or vlbw or elbw) ) OR TX infan* OR TX ( (baby or babies) )

S32 (MH "Infant, Newborn+")

S31 S22 AND S30

S30 S28 not S29

S29 ( MH animals+ OR MH (animal studies) OR TI (animal model*) ) NOT MH (human) 194,413

S28 S23 OR S24 OR S25 OR S26 OR S27

S27 AB (cluster W3 RCT)

S26 MH placebos OR PT randomized controlled trial OR AB control W5 group OR MH crossover design OR MH comparative studies

S25 MH sample size AND AB ( (assigned OR allocated OR control) )

S24 TI ( (randomised OR randomized) ) OR AB random* OR TI trial

S23 MH Randomized Controlled Trials OR MH double‐blind studies OR MH single‐blind studies OR MH random assignment OR MH pretest‐posttest design OR MH cluster sample

S22 S9 OR S18 OR S21

S21 S19 OR S20

S20 TI ( (probiotic* and prebiotic*) N4 combin* ) OR AB ( (probiotic* and prebiotic*) N4 combin* )

S19 TI Synbiotic* OR AB Synbiotic*

S18 S10 OR S11 OR S12 OR S13 OR S14 OR S15 OR S16 OR S17

S17 TI Lactoferrin OR AB Lactoferrin

S16 TI fructooligosaccharide* OR AB fructooligosaccharide* OR TI fructo‐oligosaccharide* OR AB fructo‐oligosaccharide* OR TI galactooligosaccharide* OR AB galactooligosaccharide* OR TI galacto‐oligosaccharide* OR AB galacto‐oligosaccharide*

S15 TI Inulin OR AB Inulin

S14 TI lactulose* OR AB lactulose*

S13 TI Oligosaccharides OR AB Oligosaccharides

S12 MH "Oligosaccharides"

S11 TI Prebiotic* OR AB Prebiotic*

S10 MH "Prebiotics"

S9 S1 OR S2 OR S3 OR S4 OR S5 OR S6 OR S7 OR S8

S8 TI Saccharomyces OR AB Saccharomyces

S7 MH "Saccharomyces"

S6 TI lactobacillus OR AB lactobacillus

S5 (MH "Lactobacillus") OR (MH "Lactobacillus Acidophilus")

S4 TI bifidobacterium* OR AB bifidobacterium*

S3 MH "Bifidobacterium"

S2 TI probiotic* OR AB probiotic*

S1 MH "Probiotics"

Embase via Ovid <1974 to 2022 July 1>

1 Probiotic Agent/

2 probiotic$.ti,ab,kw.

3 exp bifidobacterium/

4 bifidobacterium$.ti,ab,kw.

5 exp lactobacillus/

6 lactobacill$.ti,ab,kw.

7 Saccharomyces/ or Saccharomyces boulardii/ or Saccharomyces cerevisiae/

8 Saccharomyces$.ti,ab,kw.

9 or/1‐8

10 Prebiotic Agent/

11 prebiotic$.ti,ab,kw.

12 exp Oligosaccharide/

13 oligosaccharide$.ti,ab,kw.

14 Galactose oligosaccharide/

15 (galacto‐oligosaccharide$ or galactooligosaccharide$).ti,ab,kw.

16 Fructose Oligosaccharide/

17 (fructooligosaccharide$ or fructo‐oligosaccharide$ or FOS or FOSs).ti,ab,kw.

18 Lactulose/

19 lactulose$.ti,ab,kw.

20 Inulin/

21 inulin$.ti,ab,kw.

22 Lactoferrin/

23 lactoferrin$.ti,ab,kw.

24 or/10‐23

25 Synbiotic Agent/

26 synbiotic$.ti,ab,kw.

27 ((probiotic$ and prebiotic$) adj4 combin$).ti,ab,kw.

28 25 or 26 or 27

29 9 or 24 or 28

30 Newborn/

31 Prematurity/

32 (neonat$ or neo nat$).ti,ab.

33 (newborn$ or new born$ or newly born$).ti,ab.

34 (preterm or preterms or pre term or pre terms).ti,ab.

35 (preemie$ or premie or premies).ti,ab.

36 (prematur$ adj3 (birth$ or born or deliver$)).ti,ab.

37 (low adj3 (birthweight$ or birth weight$)).ti,ab.

38 (lbw or vlbw or elbw).ti,ab.

39 infan$.ti,ab.

40 (baby or babies).ti,ab.

41 or/30‐40

42 Randomized controlled trial/

43 Controlled clinical study/

44 Random$.ti,ab.

45 randomization/

46 intermethod comparison/

47 placebo.ti,ab.

48 (compare or compared or comparison).ti.

49 ((evaluated or evaluate or evaluating or assessed or assess) and (compare or compared or comparing or comparison)).ab.

50 (open adj label).ti,ab.

51 ((double or single or doubly or singly) adj (blind or blinded or blindly)).ti,ab.

52 double blind procedure/

53 parallel group$1.ti,ab.

54 (crossover or cross over).ti,ab.

55 ((assign$ or match or matched or allocation) adj5 (alternate or group$1 or intervention$1 or patient$1 or subject$1 or participant$1)).ti,ab.

56 (assigned or allocated).ti,ab.

57 (controlled adj7 (study or design or trial)).ti,ab.

58 (volunteer or volunteers).ti,ab.

59 human experiment/

60 trial.ti.

61 or/42‐60

62 (random$ adj sampl$ adj7 ("cross section$" or questionnaire$1 or survey$ or database$1)).ti,ab. not (comparative study/ or controlled study/ or randomi?ed controlled.ti,ab. or randomly assigned.ti,ab.)

63 Cross‐sectional study/ not (randomized controlled trial/ or controlled clinical study/ or controlled study/ or randomi?ed controlled.ti,ab. or control group$1.ti,ab.)

64 (((case adj control$) and random$) not randomi?ed controlled).ti,ab.

65 (Systematic review not (trial or study)).ti.

66 (nonrandom$ not random$).ti,ab.

67 "Random field$".ti,ab.

68 (random cluster adj3 sampl$).ti,ab.

69 (review.ab. and review.pt.) not trial.ti.

70 "we searched".ab. and (review.ti. or review.pt.)

71 "update review".ab.

72 (databases adj4 searched).ab.

73 (rat or rats or mouse or mice or swine or porcine or murine or sheep or lambs or pigs or piglets or rabbit or rabbits or cat or cats or dog or dogs or cattle or bovine or monkey or monkeys or trout or marmoset$1).ti. and animal experiment/

74 Animal experiment/ not (human experiment/ or human/)

75 or/62‐74

76 61 not 75

77 29 and 41 and 76

Maternity & Infant Care Database (MIDIRS) via OVID <1971 to June 14, 2022>

1 probiotic$.ti,ab,de.

2 bifidobacterium$.ti,ab,de.

3 lactobacill$.ti,ab,de.

4 Saccharomyces$.ti,ab,de.

5 or/1‐4

6 prebiotic$.ti,ab,de.

7 oligosaccharide$.ti,ab,de.

8 inulin$.ti,ab,de.

9 (fructooligosaccharide$ or fructo‐oligosaccharide$ or FOS or FOSs).ti,ab,de.

10 (galactooligosaccharide$ or galacto‐oligosaccharide$).ti,ab,de.

11 lactoferrin$.ti,ab,de.

12 lactulose$.ti,ab,de.

13 or/6‐12

14 synbiotic$.ti,ab,de.

15 ((probiotic$ and prebiotic$) adj4 combin$).ti,ab,de.

16 14 or 15

17 5 or 13 or 16

18 (neonat$ or neo nat$).ti,ab.

19 (newborn$ or new born$ or newly born$).ti,ab.

20 (preterm or preterms or pre term or pre terms).ti,ab.

21 (preemie$ or premie or premies).ti,ab.

22 (prematur$ adj3 (birth$ or born or deliver$)).ti,ab.

23 (low adj3 (birthweight$ or birth weight$)).ti,ab.

24 (lbw or vlbw or elbw).ti,ab.

25 infan$.ti,ab.

26 (baby or babies).ti,ab.

27 or/18‐26

28 17 and 27

29 limit 28 to randomised controlled trial

Ovid MEDLINE(R) ALL <1946 to July 1, 2022>

1 Probiotics/

2 probiotic$.ti,ab,kw.

3 exp bifidobacterium/

4 bifidobacterium$.ti,ab,kw.

5 exp lactobacillus/

6 lactobacill$.ti,ab,kw.

7 Saccharomyces/ or Saccharomyces boulardii/ or Saccharomyces cerevisiae/

8 Saccharomyces$.ti,ab,kw.

9 or/1‐8

10 Prebiotics/

11 prebiotic$.ti,ab,kw.

12 Oligosaccharides/

13 oligosaccharide$.ti,ab,kw.

14 (galactooligosaccharides or galacto‐oligosaccharides).ti,ab,kw.

15 (fructooligosaccharide$ or fructo‐oligosaccharide$ or FOS or FOSs).ti,ab,kw.

16 Lactulose/

17 lactulose$.ti,ab,kw.

18 Inulin/

19 inulin$.ti,ab,kw.

20 Lactoferrin/

21 lactoferrin$.ti,ab,kw.

22 or/10‐21

23 Synbiotics/

24 synbiotic$.ti,ab,kw.

25 ((probiotic$ and prebiotic$) adj4 combin$).ti,ab,kw. (374)

26 or/23‐25

27 9 or 22 or 26

28 exp Infant, Newborn/

29 Premature Birth/

30 (neonat$ or neo nat$).ti,ab.

31 (newborn$ or new born$ or newly born$).ti,ab.

32 (preterm or preterms or pre term or pre terms).ti,ab.

33 (preemie$ or premie or premies).ti,ab.

34 (prematur$ adj3 (birth$ or born or deliver$)).ti,ab.

35 (low adj3 (birthweight$ or birth weight$)).ti,ab.

36 (lbw or vlbw or elbw).ti,ab.

37 infan$.ti,ab.

38 (baby or babies).ti,ab.

39 or/28‐38

40 randomized controlled trial.pt.

41 controlled clinical trial.pt.

42 randomized.ab.

43 placebo.ab.

44 drug therapy.fs.

45 randomly.ab.

46 trial.ab.

47 groups.ab.

48 or/40‐47

49 exp animals/ not humans.sh.

50 48 not 49

51 27 and 39 and 50

‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐

Trials registers

Search date: 5th July 2022

WHO ICTRP via https://trialsearch.who.int/

Search 1 of 2: 
Condition: (infant* OR baby OR babies OR premature or neonate* OR new born OR preterm OR
low birth weight OR low birthweight OR LBW OR VLBW or ELBW) AND Intervention: (probiotic*
OR bifidobacterium OR lactobacillus OR saccharomyces OR prebiotic* OR oligosaccharide* OR
galactooligosaccharide* OR galacto‐oligosaccharide*)
Recruitment Status: ALL

Search 2 of 2: 
Condition: (infant* OR baby OR babies OR premature or neonate* OR new born OR preterm OR
low birth weight OR low birthweight OR LBW OR VLBW or ELBW) AND Intervention:
(fructooligosaccharide* OR fructo‐oligosaccharide* OR FOS OR lactulose OR inulin OR lactoferrin
OR synbiotics)
Recruitment Status: ALL

Clinical Trials.gov via https://clinicaltrials.gov/

Search 1 of 2: 
Other terms: (infant OR baby OR premature OR neonate OR "new born" OR preterm OR "low
birth weight" OR LBW OR VLBW OR ELBW) AND (probiotics OR bifidobacterium OR lactobacillus
OR saccharomyces OR prebiotics OR oligosaccharides OR galactooligosaccharides)

Search 2 of 2: 
Other terms: (infant OR baby OR premature OR neonate OR "new born" OR preterm OR "low
birth weight" OR LBW OR VLBW OR ELBW) AND (fructooligosaccharide OR fos OR lactulose OR
inulin OR lactoferrin OR synbiotics)

Appendix 2. Risk of bias tool (RoB 1)

Sequence generation (checking for possible selection bias). Was the allocation sequence adequately generated?

For each included study, we categorised the method used to generate the allocation sequence as:

• low risk: any truly random process (e.g. random number table, computer random number generator);

• high risk: any non‐random process (e.g. odd or even date of birth, hospital, or clinic record number); or

• unclear risk.

Allocation concealment (checking for possible selection bias). Was allocation adequately concealed?

For each included study, we categorised the method used to conceal the allocation sequence as:

• low risk (e.g. telephone or central randomisation, consecutively numbered sealed envelopes);

• high risk (open random allocation, unsealed or non‐opaque envelopes, alternation, date of birth); or

• unclear risk.

Blinding of personnel (checking for possible performance bias). Was knowledge of the allocated intervention adequately prevented during the study?

For each included study, we categorised the methods used to blind study participants and personnel from knowledge of which intervention a participant received. Blinding was assessed separately for different outcomes or class of outcomes. We categorised the methods as:

• low risk, high risk, or unclear risk for personnel.

Blinding of outcome assessment (checking for possible detection bias). Was knowledge of the allocated intervention adequately prevented at the time of outcome assessment?

For each included study, we categorised the methods used to blind outcome assessment. Blinding was assessed separately for different outcomes or class of outcomes. We categorised the methods as:

• low risk for outcome assessors;

• high risk for outcome assessors; or

• unclear risk for outcome assessors.

Incomplete outcome data (checking for possible attrition bias through withdrawals, dropouts, protocol deviations). Were incomplete outcome data adequately addressed?

For each included study and for each outcome, we described the completeness of data including attrition and exclusions from the analysis. We noted whether attrition and exclusions were reported, the numbers included in the analysis at each stage (compared with the total randomised participants), reasons for attrition or exclusion where reported, and whether missing data were balanced across groups or were related to outcomes. Where sufficient information was reported or supplied by the trial authors, we re‐included missing data in the analyses. We categorised the methods as:

• low risk (< 20% missing data);

• high risk (≥ 20% missing data); or

• unclear risk.

Selective reporting bias. Are reports of the study free of suggestion of selective outcome reporting?

For each included study, we described how we investigated the possibility of selective outcome reporting bias and what we found. For studies in which study protocols were published in advance, we compared prespecified outcomes versus outcomes eventually reported in the published results. If the study protocol was not published in advance, we contacted study authors to gain access to the study protocol. We assessed the methods as:

• low risk (where it is clear that all the study's prespecified outcomes and all expected outcomes of interest to the review have been reported);

• high risk (where not all the study's prespecified outcomes have been reported; one or more reported primary outcomes were not prespecified outcomes of interest and are reported incompletely and so cannot be used; study fails to include results of a key outcome that would have been expected to have been reported); or

• unclear risk.

Data and analyses

Comparison 1. Probiotics versus placebo or no probiotics (very preterm or very low birth weight infants).

Outcome or subgroup title	No. of studies	No. of participants	Statistical method	Effect size
1.1 Necrotising enterocolitis	57	10918	Risk Ratio (M‐H, Fixed, 95% CI)	0.54 [0.46, 0.65]
1.1.1 Bifidobacterium spp.	14	2988	Risk Ratio (M‐H, Fixed, 95% CI)	0.72 [0.54, 0.96]
1.1.2 Lactobacillus spp.	13	2052	Risk Ratio (M‐H, Fixed, 95% CI)	0.45 [0.28, 0.71]
1.1.3 Sacchromyces spp.	4	621	Risk Ratio (M‐H, Fixed, 95% CI)	0.82 [0.44, 1.50]
1.1.4 Bacillus spp.	2	465	Risk Ratio (M‐H, Fixed, 95% CI)	0.61 [0.23, 1.61]
1.1.5 Bifidobacterium spp. plus Lactobacillus spp.	13	2303	Risk Ratio (M‐H, Fixed, 95% CI)	0.39 [0.25, 0.62]
1.1.6 Bifidobacterium spp. plus Streptococcus spp.	2	1244	Risk Ratio (M‐H, Fixed, 95% CI)	0.36 [0.19, 0.68]
1.1.7 Bifidobacterium spp. plus Lactobacillus spp. plus Sacchromyces spp.	4	583	Risk Ratio (M‐H, Fixed, 95% CI)	0.67 [0.28, 1.58]
1.1.8 Bifidobacterium spp. plus Lactobacillus spp. plus Streptococcus spp.	5	662	Risk Ratio (M‐H, Fixed, 95% CI)	0.42 [0.22, 0.77]
1.2 All‐cause mortality	54	10484	Risk Ratio (M‐H, Fixed, 95% CI)	0.77 [0.66, 0.90]
1.2.1 Bifidobacterium spp.	12	2761	Risk Ratio (M‐H, Fixed, 95% CI)	0.79 [0.58, 1.09]
1.2.2 Lactobacillus spp.	13	2052	Risk Ratio (M‐H, Fixed, 95% CI)	0.91 [0.60, 1.37]
1.2.3 Sacchromyces spp.	3	534	Risk Ratio (M‐H, Fixed, 95% CI)	1.12 [0.46, 2.70]
1.2.4 Bacillus spp.	2	465	Risk Ratio (M‐H, Fixed, 95% CI)	0.87 [0.45, 1.69]
1.2.5 Bifidobacterium spp. plus Lactobacillus spp.	14	2333	Risk Ratio (M‐H, Fixed, 95% CI)	0.64 [0.48, 0.85]
1.2.6 Bifidobacterium spp. plus Streptococcus spp.	2	1244	Risk Ratio (M‐H, Fixed, 95% CI)	0.84 [0.52, 1.35]
1.2.7 Bifidobacterium spp. plus Lactobacillus spp. plus Sacchromyces spp.	4	583	Risk Ratio (M‐H, Fixed, 95% CI)	0.67 [0.30, 1.49]
1.2.8 Bifidobacterium spp. plus Lactobacillus spp. plus Streptococcus spp.	4	512	Risk Ratio (M‐H, Fixed, 95% CI)	0.74 [0.39, 1.42]
1.3 Late‐onset invasive infection	49	9876	Risk Ratio (M‐H, Fixed, 95% CI)	0.89 [0.82, 0.97]
1.3.1 Bifidobacterium spp.	12	2736	Risk Ratio (M‐H, Fixed, 95% CI)	0.84 [0.70, 1.02]
1.3.2 Lactobacillus spp.	12	2022	Risk Ratio (M‐H, Fixed, 95% CI)	0.93 [0.74, 1.17]
1.3.3 Sacchromyces spp.	4	621	Risk Ratio (M‐H, Fixed, 95% CI)	0.84 [0.58, 1.22]
1.3.4 Bacillus spp.	2	465	Risk Ratio (M‐H, Fixed, 95% CI)	1.00 [0.67, 1.51]
1.3.5 Bifidobacterium spp. plus Lactobacillus spp.	11	1975	Risk Ratio (M‐H, Fixed, 95% CI)	0.94 [0.81, 1.11]
1.3.6 Bifidobacterium spp. plus Streptococcus spp.	2	1244	Risk Ratio (M‐H, Fixed, 95% CI)	0.92 [0.72, 1.17]
1.3.7 Bifidobacterium spp. plus Lactobacillus spp. plus Sacchromyces spp.	4	583	Risk Ratio (M‐H, Fixed, 95% CI)	0.79 [0.53, 1.18]
1.3.8 Bifidobacterium spp. plus Lactobacillus spp. plus Streptococcus spp.	2	230	Risk Ratio (M‐H, Fixed, 95% CI)	0.79 [0.63, 1.00]
1.4 Duration of hospitalisation from birth (days)	24	5572	Mean Difference (IV, Fixed, 95% CI)	‐1.68 [‐3.08, ‐0.28]
1.4.1 Bifidobacterium spp.	4	1945	Mean Difference (IV, Fixed, 95% CI)	0.51 [‐2.98, 4.01]
1.4.2 Lactobacillus spp.	5	269	Mean Difference (IV, Fixed, 95% CI)	‐3.65 [‐7.76, 0.45]
1.4.3 Sacchromyces spp.	2	470	Mean Difference (IV, Fixed, 95% CI)	‐2.88 [‐8.06, 2.29]
1.4.4 Bifidobacterium spp. plus Lactobacillus spp.	8	1327	Mean Difference (IV, Fixed, 95% CI)	‐0.90 [‐3.28, 1.47]
1.4.5 Bifidobacterium spp. plus Streptococcus spp.	1	1044	Mean Difference (IV, Fixed, 95% CI)	‐3.00 [‐6.28, 0.28]
1.4.6 Bifidobacterium spp. plus Lactobacillus spp. plus Sacchromyces spp.	2	231	Mean Difference (IV, Fixed, 95% CI)	‐5.65 [‐11.68, 0.38]
1.4.7 Bifidobacterium spp. plus Lactobacillus spp. plus Streptococcus spp.	2	286	Mean Difference (IV, Fixed, 95% CI)	0.65 [‐4.86, 6.16]
1.5 Neurodevelopmental impairment	5	1518	Risk Ratio (M‐H, Fixed, 95% CI)	1.03 [0.84, 1.26]
1.5.1 Bifidobacterium spp.	1	162	Risk Ratio (M‐H, Fixed, 95% CI)	0.77 [0.34, 1.72]
1.5.2 Lactobacillus spp.	1	249	Risk Ratio (M‐H, Fixed, 95% CI)	1.01 [0.69, 1.48]
1.5.3 Bacillus spp.	1	174	Risk Ratio (M‐H, Fixed, 95% CI)	1.09 [0.58, 2.07]
1.5.4 Bifidobacterium spp. plus Streptococcus spp.	1	664	Risk Ratio (M‐H, Fixed, 95% CI)	0.97 [0.69, 1.36]
1.5.5 Bifidobacterium spp. plus Lactobacillus spp.	1	269	Risk Ratio (M‐H, Fixed, 95% CI)	1.27 [0.81, 1.98]
1.6 Cerebral palsy	5	1512	Risk Ratio (M‐H, Fixed, 95% CI)	1.13 [0.74, 1.72]
1.6.1 Bifidobacterium spp.	1	156	Risk Ratio (M‐H, Fixed, 95% CI)	0.38 [0.10, 1.36]
1.6.2 Lactobacillus spp.	1	249	Risk Ratio (M‐H, Fixed, 95% CI)	0.92 [0.40, 2.08]
1.6.3 Bacillus spp.	1	174	Risk Ratio (M‐H, Fixed, 95% CI)	2.05 [0.38, 10.88]
1.6.4 Bifidobacterium spp. plus Streptococcus spp.	1	664	Risk Ratio (M‐H, Fixed, 95% CI)	1.32 [0.67, 2.58]
1.6.5 Bifidobacterium spp. plus Lactobacillus spp.	1	269	Risk Ratio (M‐H, Fixed, 95% CI)	2.28 [0.62, 8.41]
1.7 Visual impairment	4	1356	Risk Ratio (M‐H, Fixed, 95% CI)	0.50 [0.14, 1.80]
1.7.1 Bifidobacterium spp.	1	174	Risk Ratio (M‐H, Fixed, 95% CI)	0.51 [0.05, 5.54]
1.7.2 Lactobacillus spp.	1	0	Risk Ratio (M‐H, Fixed, 95% CI)	Not estimable
1.7.3 Bifidobacterium spp. plus Streptococcus spp.	1	664	Risk Ratio (M‐H, Fixed, 95% CI)	2.91 [0.12, 71.21]
1.7.4 Bifidobacterium spp. plus Lactobacillus spp.	1	269	Risk Ratio (M‐H, Fixed, 95% CI)	0.21 [0.02, 1.89]
1.8 Hearing impairment	4	1356	Risk Ratio (M‐H, Fixed, 95% CI)	0.46 [0.18, 1.17]
1.8.1 Bifidobacterium spp.	1	174	Risk Ratio (M‐H, Fixed, 95% CI)	1.02 [0.07, 16.10]
1.8.2 Lactobacillus spp.	1	249	Risk Ratio (M‐H, Fixed, 95% CI)	3.02 [0.12, 73.52]
1.8.3 Bifidobacterium spp. plus Streptococcus spp.	1	664	Risk Ratio (M‐H, Fixed, 95% CI)	0.18 [0.04, 0.79]
1.8.4 Bifidobacterium spp. plus Lactobacillus spp.	1	269	Risk Ratio (M‐H, Fixed, 95% CI)	1.71 [0.16, 18.64]
1.9 Cognitive performance	1	52	Mean Difference (IV, Fixed, 95% CI)	‐1.00 [‐6.38, 4.38]

Comparison 2. Probiotics versus placebo or no probiotics (extremely preterm or extremely low birth weight infants).

Outcome or subgroup title	No. of studies	No. of participants	Statistical method	Effect size
2.1 Necrotising enterocolitis	10	1836	Risk Ratio (M‐H, Fixed, 95% CI)	0.92 [0.69, 1.22]
2.1.1 Bifidobacterium spp.	2	665	Risk Ratio (M‐H, Fixed, 95% CI)	1.00 [0.70, 1.43]
2.1.2 Lactobacillus spp.	2	330	Risk Ratio (M‐H, Fixed, 95% CI)	0.73 [0.36, 1.48]
2.1.3 Bacillus spp.	1	0	Risk Ratio (M‐H, Fixed, 95% CI)	Not estimable
2.1.4 Bifidobacterium spp. plus Lactobacillus spp.	4	247	Risk Ratio (M‐H, Fixed, 95% CI)	1.22 [0.38, 3.92]
2.1.5 Bifidobacterium spp. plus Streptococcus spp.	1	474	Risk Ratio (M‐H, Fixed, 95% CI)	0.73 [0.33, 1.60]
2.2 All‐cause mortality	7	1723	Risk Ratio (M‐H, Fixed, 95% CI)	0.92 [0.72, 1.18]
2.2.1 Bifidobacterium spp.	1	474	Risk Ratio (M‐H, Fixed, 95% CI)	0.98 [0.60, 1.61]
2.2.2 Lactobacillus spp.	2	330	Risk Ratio (M‐H, Fixed, 95% CI)	0.78 [0.42, 1.42]
2.2.3 Bacillus clausii	1	120	Risk Ratio (M‐H, Fixed, 95% CI)	0.86 [0.36, 2.08]
2.2.4 Bifidobacterium spp. plus Lactobacillus spp.	2	162	Risk Ratio (M‐H, Fixed, 95% CI)	1.22 [0.36, 4.10]
2.2.5 Bifidobacterium spp. plus Streptococcus spp.	1	637	Risk Ratio (M‐H, Fixed, 95% CI)	0.94 [0.65, 1.35]
2.3 Late‐onset invasive infection	7	1533	Risk Ratio (M‐H, Fixed, 95% CI)	0.93 [0.78, 1.09]
2.3.1 Bifidobacterium spp.	2	642	Risk Ratio (M‐H, Fixed, 95% CI)	1.00 [0.73, 1.37]
2.3.2 Lactobacillus spp.	1	134	Risk Ratio (M‐H, Fixed, 95% CI)	1.05 [0.67, 1.66]
2.3.3 Bacillus clausii	1	120	Risk Ratio (M‐H, Fixed, 95% CI)	0.80 [0.43, 1.47]
2.3.4 Bifidobacterium spp. plus Lactobacillus spp.	2	163	Risk Ratio (M‐H, Fixed, 95% CI)	1.12 [0.65, 1.92]
2.3.5 Bifidobacterium spp. plus Streptococcus spp.	1	474	Risk Ratio (M‐H, Fixed, 95% CI)	0.82 [0.64, 1.06]
2.4 Duration of hospitalisation from birth (days)	2	84	Mean Difference (IV, Random, 95% CI)	2.18 [‐13.84, 18.21]
2.4.1 Bifidobacterium spp. plus Lactobacillus spp.	2	84	Mean Difference (IV, Random, 95% CI)	2.18 [‐13.84, 18.21]

Comparison 3. Subgroup analysis by type of feeding.

Outcome or subgroup title	No. of studies	No. of participants	Statistical method	Effect size
3.1 Necrotising enterocolitis	57	10918	Risk Ratio (M‐H, Fixed, 95% CI)	0.54 [0.46, 0.65]
3.1.1 Human milk only	9	1038	Risk Ratio (M‐H, Fixed, 95% CI)	0.30 [0.16, 0.57]
3.1.2 Mixed: human milk, formula, or both	44	9626	Risk Ratio (M‐H, Fixed, 95% CI)	0.58 [0.48, 0.70]
3.1.3 Formula only	4	254	Risk Ratio (M‐H, Fixed, 95% CI)	0.43 [0.16, 1.18]
3.2 All‐cause mortality	54	10484	Risk Ratio (M‐H, Fixed, 95% CI)	0.77 [0.66, 0.90]
3.2.1 Human milk only	9	1038	Risk Ratio (M‐H, Fixed, 95% CI)	0.64 [0.41, 1.00]
3.2.2 Mixed: human milk, formula, or both	42	9279	Risk Ratio (M‐H, Fixed, 95% CI)	0.80 [0.68, 0.95]
3.2.3 Formula only	3	167	Risk Ratio (M‐H, Fixed, 95% CI)	0.22 [0.04, 1.21]
3.3 Late‐onset invasive infection	49	9876	Risk Ratio (M‐H, Fixed, 95% CI)	0.89 [0.82, 0.97]
3.3.1 Human milk only	9	1038	Risk Ratio (M‐H, Fixed, 95% CI)	0.73 [0.58, 0.93]
3.3.2 Mixed: human milk, formula, or both	37	8614	Risk Ratio (M‐H, Fixed, 95% CI)	0.92 [0.84, 1.01]
3.3.3 Formula only	3	224	Risk Ratio (M‐H, Fixed, 95% CI)	0.41 [0.11, 1.49]
3.4 Duration of hospitalisation from birth (days)	24	5572	Mean Difference (IV, Fixed, 95% CI)	‐1.68 [‐3.08, ‐0.28]
3.4.1 Human milk only	5	418	Mean Difference (IV, Fixed, 95% CI)	‐4.09 [‐7.09, ‐1.09]
3.4.2 Mixed: human milk, formula, or both	17	5064	Mean Difference (IV, Fixed, 95% CI)	‐0.72 [‐2.34, 0.90]
3.4.3 Formula only	2	90	Mean Difference (IV, Fixed, 95% CI)	‐7.38 [‐14.96, 0.21]

Comparison 4. Sensitivity analyses: trials at low risk of bias.

Outcome or subgroup title	No. of studies	No. of participants	Statistical method	Effect size
4.1 Necrotising enterocolitis	17	4649	Risk Ratio (M‐H, Fixed, 95% CI)	0.70 [0.55, 0.89]
4.2 All‐cause mortality	17	4649	Risk Ratio (M‐H, Fixed, 95% CI)	0.86 [0.69, 1.07]
4.3 Late‐onset invasive infection	17	4649	Risk Ratio (M‐H, Fixed, 95% CI)	0.89 [0.78, 1.01]
4.4 Duration of hospitalisation from birth (days)	7	2838	Mean Difference (IV, Random, 95% CI)	‐2.45 [‐5.45, 0.56]
4.5 Neurodevelopmental impairment	2	913	Risk Ratio (M‐H, Fixed, 95% CI)	0.99 [0.76, 1.27]
4.6 Cerebral palsy	2	913	Risk Ratio (M‐H, Fixed, 95% CI)	1.14 [0.68, 1.92]
4.7 Visual impairment	2	913	Risk Ratio (M‐H, Fixed, 95% CI)	2.91 [0.12, 71.21]
4.8 Hearing impairment	2	913	Risk Ratio (M‐H, Fixed, 95% CI)	0.30 [0.09, 0.98]

Characteristics of studies

Characteristics of included studies [ordered by study ID]

Agarwal 2003.

Study characteristics
Methods	RCT
Participants	39 VLBW infants
Interventions	Probiotics (n = 24): Lactobacillus rhamnosus GG once daily with human milk or formula for 21 days or discharge from hospital Control (n = 15): unsupplemented milk feeds
Outcomes	Stool colonisation patterns NEC, death, and infection not reported
Notes	Setting: India (1999–2000) Funding: UK National Institute for Health (Fogarty Grant TW‐00601) and Conagra Foods Inc., USA (supplied intervention)
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	Not described
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	High risk	Unmasked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Unlikely
Selective reporting (reporting bias)	Unclear risk	No clinical outcomes reported
Other bias	Low risk	No evidence of baseline imbalance

Al‐Hosni 2012.

Study characteristics
Methods	RCT
Participants	101 ELBW infants (appropriate for gestational age)
Interventions	Probiotic (n = 50): Lactobacillus rhamnosus GG and Bifidobacterium infantis added to the first milk feed and continued once daily until discharge or until 34 weeks' postmenstrual age Control (n = 51): unsupplemented milk feeds
Outcomes	Weight gain NEC Death Infection
Notes	Setting: USA (2009–2011) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	Not described
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	Unclear risk	Unsupplemented milk feeds; not placebo‐controlled
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence baseline imbalance

Alshaikh 2022.

Study characteristics
Methods	RCT
Participants	62 ELBW infants
Interventions	Probiotics (n = 31): Bifidobacterium breve (1.2 billion CFU), B. bifidum (800 million CFU), B. longum subsp. infantis (600 million CFU), B. longum subsp. longum (400 million CFU), and Lactobacillus rhamnosus (1.0 billion CFU) with expressed breast milk or formula feeds once daily until 37 weeks' postmenstrual age Control (n = 31): unsupplemented milk feeds
Outcomes	NEC Infection Death Time to full enteral feeds Length of hospital stay
Notes	Setting: Canada (2017–2019) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Sealed opaque envelope with group allocation
Blinding (performance bias and detection bias) All outcomes	High risk	Control infants received unsupplemented milk feeds; not placebo‐controlled
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Bin‐Nun 2005.

Study characteristics
Methods	RCT
Participants	145 VLBW infants
Interventions	Probiotics (n = 72): "Lactobacillus bifidus" (likely Bifidobacterium bifidum), Streptococcus thermophilus, and B. infantis added to expressed breast milk or formula enteral feeds daily until 36 weeks' postmenstrual age Control (n = 73): unsupplemented milk feeds
Outcomes	NEC Death Infection Time to full enteral feeds
Notes	Setting: Israel (2001–2004) Funding: Solgar, Wyeth (manufacturer of intervention)
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	Not described
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	Unclear risk	Unsupplemented milk feeds; not placebo‐controlled
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Unclear risk	Data published in an abstract form on 2 previous occasions at the Society of Pediatrics Research (SPR 2003, 2005) with different inclusion criteria and clinical outcomes
Other bias	Low risk	No evidence of baseline imbalance

Braga 2011.

Study characteristics
Methods	RCT
Participants	231 VLBW infants (birth weight 750 g–1500 g)
Interventions	Probiotics (n = 119): Lactobacillus casei and Bifidobacterium breve (Yakult ‐ LB) in human milk once daily until day 30 or hospital discharge Control (n = 112): unsupplemented milk feeds
Outcomes	NEC Death Infection Days to full enteral feeds Duration hospital stay
Notes	Setting: Brazil (2007–2008) Funding: public/state External Study Committee terminated trial early "for a clear benefit" after enrolment of 231 infants.
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Sealed envelope with group allocation
Blinding (performance bias and detection bias) All outcomes	Unclear risk	Unsupplemented milk feeds; not placebo‐controlled
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Chandrashekar 2018.

Study characteristics
Methods	Quasi‐RCT
Participants	145 preterm infants of gestation < 34 weeks' (most participants were very preterm or VLBW)
Interventions	Probiotics (n = 72): Lactobacillus acidophilus, L. rhamnosus, Bifidobacterium longum, and Saccharomyces boulardii with human milk or formula feeds until discharge from hospital Control (n = 73): unsupplemented milk feeds
Outcomes	NEC Death Infection Duration of hospitalisation
Notes	Setting: India (2014–2015) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	High risk	Quote: "Simple random sampling method"
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	High risk	Unmasked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Near‐complete (5 participants withdrawn pre‐analysis)
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Chowdhury 2016.

Study characteristics
Methods	RCT
Participants	119 VLBW Infants (28–33 weeks' gestation)
Interventions	Probiotics (n = 60): "Cap TS6" containing Lactobacillus rhamnosus GG, L. paracasei, L. casei, L. acidophilus, Lactococcus latis, Bifidobacterium bifidum, B. longum, B. infantis in human milk once daily until discharge Control (n = 59): unsupplemented milk feeds
Outcomes	NEC Death Infection days to achieve full enteral feeding Length of hospital stay
Notes	Setting: Bangladesh (2012–2015) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	High risk	First allocation by lottery, then alternate allocation
Allocation concealment (selection bias)	High risk	Unconcealed
Blinding (performance bias and detection bias) All outcomes	High risk	Unmasked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Chrzanowska‐Liszewska 2012.

Study characteristics
Methods	RCT
Participants	47 very preterm infants (birth weight > 1000 g)
Interventions	Probiotics (n = 21): Lactobacillus rhamnosus GG added to formula, once daily until day 42 Control (n = 26): maltodextrin placebo added to formula
Outcomes	Microflora of stool measured on day 7, 21, and 42 NEC Death Infection (data provided by trial authors on request)
Notes	Setting: Poland (2008–2009) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Coded capsules containing probiotics or placebo
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Costalos 2003.

Study characteristics
Methods	RCT
Participants	87 formula‐fed infants (gestational age at birth 28 to 32 weeks)
Interventions	Probiotics (n = 51): Saccharomyces boulardii added to formula every 12 hours during the first week of life when enteral feeds are tolerated for 30 days Control (n = 36): maltodextrin placebo
Outcomes	NEC Death Infection Weight gain
Notes	Setting Greece (period of study not specified) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	Not described
Allocation concealment (selection bias)	Low risk	Cards with allocation in sealed envelopes
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Near‐complete (5 infants with incomplete data not included in analyses)
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Costeloe 2015.

Study characteristics
Methods	RCT
Participants	1310 infants born before 31 weeks' gestation
Interventions	Probiotics (n = 650): Bifidobacterium breve BBG‐001 once daily until 36 weeks’ postmenstrual age or discharge from hospital Control (n = 660): corn starch placebo
Outcomes	NEC Death Infection
Notes	Setting: UK (24 neonatal units; 2010–2013) Funding: by UK National Institute for Health Research Health Technology Assessment programme (ISRCTN 05511098)
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Web‐based
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	No
Other bias	Low risk	No evidence of baseline imbalance

Dani 2002.

Study characteristics
Methods	RCT
Participants	585 VLBW infants (or < 33 weeks' gestation at birth)
Interventions	Probiotics (n = 295): Lactobacillus rhamnosus GG added to milk (human or formula) feeds once daily until hospital discharge Control (n = 290): maltodextrin placebo
Outcomes	NEC Death Infection Duration of hospitalisation
Notes	Setting: Italy (12 centres; study period not specified) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	Not described
Allocation concealment (selection bias)	Low risk	Sealed envelope containing allocation
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked (placebo‐controlled)
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Dashti 2014.

Study characteristics
Methods	RCT
Participants	136 preterm infants of birth weight 700–1800 g (most participants very preterm or VLBW)
Interventions	Probiotics (n = 69): Lactobacillus acidophilus, L. rhamnosus, L. bulgaricus, L. casei, Streptococcus thermophilus, Bifidobacterium longum, B. breve added to milk feeds once daily until hospital discharge Control (n = 67): placebo powder (not described)
Outcomes	NEC Death Infection data sought from trial authors in July 2020
Notes	Setting: Iran (2010–2011) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	Not described
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked (placebo‐controlled)
Incomplete outcome data (attrition bias) All outcomes	Unclear risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Demirel 2013.

Study characteristics
Methods	RCT
Participants	271 VLBW infants (gestational age ≤ 32 weeks at birth)
Interventions	Probiotics (n = 135): Saccharomyces boulardii added to human milk or formula once a day, starting with the 1st feed, until hospital discharge Control (n = 136): unsupplemented milk
Outcomes	NEC Death Infection
Notes	Setting: Turkey (2011) Funding: not stated ClinicalTrials.gov identifier NCT01315821
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Allocations sealed in opaque, sequentially‐numbered envelopes
Blinding (performance bias and detection bias) All outcomes	High risk	Unmasked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Dilli 2015.

Study characteristics
Methods	RCT
Participants	200 very preterm or VLBW infants
Interventions	Probiotics (n = 100): Bifidobacterium lactis added to human milk or formula once daily for 8 weeks (or hospital discharge) Control (n= 100): maltodextrin powder placebo
Outcomes	NEC Death Infection Length of hospital stay
Notes	Setting: Turkey (5 centres; 2011–2014) Funding: not stated This was a 4‐arm RCT; 2 other groups received prebiotics (n = 100) and synbiotics (n > 100)
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Sealed opaque envelopes
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked (placebo‐controlled)
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	High risk	Mean birth weight higher in intervention group (1236 g) than in control group (1147 g)

Dutta 2015.

Study characteristics
Methods	RCT
Participants	149 infants (27 to 33 weeks' gestation at birth)
Interventions	Probiotics (n = 114): Lactobacillus acidophilus, L. rhamnosus, Bifidobacterium longum, and Saccharomyces boulardii (3 groups: "low‐dose" (109) for 21 days or "high‐dose" (1010) 2 times daily with human milk or formula feeds for 14 or 21 days) Control (n = 35): maltodextrin placebo for 21 days
Outcomes	Probiotic stool colonisation NEC Mortality Infection
Notes	Setting: India (study period not stated) Funding: Aristo Pharmaceuticals Pvt Ltd, Madhya Pradesh, India provided the sachets of probiotics and placebo free of cost.
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Fernández‐Carrocera 2013.

Study characteristics
Methods	RCT
Participants	150 VLBW infants
Interventions	Probiotics (n = 75): Lactobacillus rhamnosus, L. casei, L. plantarum, L acidophilus, Bifidobacterium infantis, and Streptococcus thermophilus added to human milk or formula (duration of intervention not stated) Control (n = 75): unsupplemented milk feeds
Outcomes	NEC Death Infection
Notes	Setting: Mexico (2007–2010) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Random number table
Allocation concealment (selection bias)	Low risk	Staff unable to predict allocation by number
Blinding (performance bias and detection bias) All outcomes	Unclear risk	Unsupplemented milk feeds; not placebo‐controlled
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Unclear risk	Birth weight and gestation (or other neonatal baseline characteristics) not reported

Fujii 2006.

Study characteristics
Methods	Quasi‐RCT
Participants	19 preterm infants (most very preterm or VLBW)
Interventions	Probiotics (n = 11): Bifidobacterium breve 2 times daily with human milk or formula feeds until hospital discharge Control (n = 8): unsupplemented milk feeds
Outcomes	Cytokine levels in plasma NEC Death Infection
Notes	Setting: Japan (2000–2002) Funding: Morinaja Milk industry and Meiji Dairies (manufactured intervention)
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	Not described
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	High risk	Unmasked
Incomplete outcome data (attrition bias) All outcomes	Unclear risk	Complete
Selective reporting (reporting bias)	Unclear risk	Unclear
Other bias	Low risk	No evidence of baseline imbalance

Hariharan 2016.

Study characteristics
Methods	RCT
Participants	196 very preterm infants with birth weight < 1250 g
Interventions	Probiotics (n = 93): Lactobacillus acidophilus, Bifidobacterium bifidum, and Saccharomyces boulardii 2 times daily in milk feeds for 6 weeks Control (n = 103): unsupplemented feeds
Outcomes	NEC Death Infection
Notes	Setting: India (study period not stated) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	Not described
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	High risk	Unmasked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Unclear risk	Unclear
Other bias	Low risk	No evidence of baseline imbalance

Hays 2015.

Study characteristics
Methods	RCT
Participants	199 very preterm infants (gestation at birth 25–1 weeks), and birth weight 700–1600 g that was appropriate for gestational age
Interventions	Probiotics (3 groups; n = 145): Bifidobacterium lactis, B. longum, or both once daily in sterile water for 4 to 6 weeks (depending on gestation at birth) Control (n = 52): maltodextrin placebo
Outcomes	NEC Death Infection
Notes	France (3 centres; 2007–2010) Funding: Nestle France (Marne‐la‐Vallee, France) and Nestec (Vevey, Switzerland)
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Consecutively numbered, sealed, opaque envelopes
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked (placebo‐controlled)
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Hernandez‐Enriquez 2016.

Study characteristics
Methods	RCT
Participants	44 preterm infants < 34 weeks' gestation or ≤ 1550 g birth weight (most infants very preterm or VLBW)
Interventions	Intervention (n = 24): Lactobacillus reuteri once daily for first 10 days after birth Control (n = 20): placebo (sterile water)
Outcomes	NEC Death Infection (data provided by trial authors on request)
Notes	Setting: Mexico (2012–2013) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	Quote: "Simple randomisation sequence"
Allocation concealment (selection bias)	Low risk	Sealed opaque envelopes
Blinding (performance bias and detection bias) All outcomes	High risk	Unmasked
Incomplete outcome data (attrition bias) All outcomes	Unclear risk	Complete
Selective reporting (reporting bias)	Unclear risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Hikaru 2010.

Study characteristics
Methods	RCT
Participants	208 VLBW infants
Interventions	Probiotics (n = 108): Bifidobacterium breve in human milk or formula once daily until discharge from the intensive care unit Control (n = 100): unsupplemented milk feeds
Outcomes	Infection NEC not reported
Notes	Setting: Japan (2001–2013) Funding: Morinaga Milk Industry Co. Ltd. (supplied Bifidobacterium breve preparation)
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	Not described
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	High risk	Unmasked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Unclear risk	Unclear
Other bias	Low risk	No evidence of baseline imbalance

Huang 2009.

Study characteristics
Methods	RCT
Participants	183 VLBW infants who survived 7 days after birth and began enteral feeding
Interventions	Probiotics (n = 95): Bifidobacterium adolescentis twice daily with milk feeds daily for 7 days Control (n = 88): unsupplemented milk feeds
Outcomes	NEC (unclear whether death or infection assessed)
Notes	Setting: China (single‐centre, study dates not stated) Translation from Chinese courtesy of Yuan Chi
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	Not described
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	High risk	Unmasked
Incomplete outcome data (attrition bias) All outcomes	Unclear risk	Unable to assess
Selective reporting (reporting bias)	Unclear risk	Mortality and infection not reported
Other bias	Low risk	No evidence of baseline imbalance

Indrio 2017.

Study characteristics
Methods	RCT
Participants	60 preterm infants of gestational age 28 to 32 weeks at birth
Interventions	Probiotics (n = 30): Lactobacillus reuteri suspended in sunflower and medium‐chain triglyceride oils, given once daily until day 30 Control (n = 30): identical oils without probiotics
Outcomes	NEC Death Infection Duration of hospital stay (data provided by trial authors on request)
Notes	Setting: Italy (2011–2012) Funding: University of Bari, Italy ClinicalTrials.gov identifier NCT00985816
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked (placebo‐controlled)
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Unclear risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Jacobs 2013.

Study characteristics
Methods	RCT
Participants	1099 very preterm VLBW infants
Interventions	Probiotics (n = 548): Bifidobacterium infantis, Streptococcus thermophilus, and B. lactis once daily in human milk or formula until discharge from hospital or term‐corrected age. Control (n = 551): maltodextrin powder placebo
Outcomes	NEC Death Infection Infection with a probiotic species Duration of hospitalisation from birth Major neurodevelopmental impairment: moderate or severe cerebral palsy, Bayley‐III Motor Composite Scale < –2 SD (or Movement Assessment Battery for Children < 15th centile if > 42 months post‐term), Bayley‐III Composite Cognitive or Language Scales < –2 SD (or Wechsler Preschool and Primary Scale of Intelligence Full Scale Intelligence Quotient < –2 SD if > 42 months post‐term), blindness, or deafness
Notes	Setting: Australasia (10 centres; 2007–2011) Funding: National Health and Research Medical Council, Australia
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Central allocation
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked (placebo‐controlled)
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete for in‐hospital outcomes (48% for neurodevelopmental assessment)
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Kanic 2015.

Study characteristics
Methods	RCT
Participants	80 VLBW infants
Interventions	Probiotics (n = 40): Lactobacillus acidophilus, Enterococcus faecium, and Bifidobacterium infantis 2 times daily with milk feeds until discharge from hospital Control (n = 40): unsupplemented milk feeds
Outcomes	NEC Death Infection Duration of hospitalisation from birth
Notes	Setting: Slovenia (2008–2011) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	High risk	Alternate allocation (quote: "quasi‐randomised")
Allocation concealment (selection bias)	High risk	Unconcealed
Blinding (performance bias and detection bias) All outcomes	High risk	Unmasked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Kitajima 1997.

Study characteristics
Methods	RCT
Participants	91 VLBW infants
Interventions	Probiotics (n = 45): Bifidobacterium breve in distilled water once daily for 28 days Control (n = 46): distilled water
Outcomes	Probiotic colonisation of stool Date on NEC, death, and infection provided by trial authors on request
Notes	Setting: Japan (1990–1991) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	Not described
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	High risk	Unmasked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Near‐complete (4 participants not included in analyses)
Selective reporting (reporting bias)	Low risk	Data
Other bias	Low risk	No evidence of baseline imbalance

Li 2019.

Study characteristics
Methods	RCT
Participants	30 VLBW infants
Interventions	Probiotics (n = 16): Lactobacillus plantarum, Bifidobacterium longum, and B. bifidum once daily with milk feeds until 36 weeks' postmenstrual age Control (n = 14): 5% glucose solution
Outcomes	Change of gut microbiota Correlation of gut microbial composition Levels of cytokines NEC, death, infection not reported; trial author contacted in May 2020
Notes	Setting: China (2014–2015) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Unclear risk	Quote: "Concealed by the principal investigator according to sequential numbers"
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked (intervention and control solutions identical)
Incomplete outcome data (attrition bias) All outcomes	High risk	> 50% outcome data unreported
Selective reporting (reporting bias)	Unclear risk	Unable to determine
Other bias	Low risk	No evidence of baseline imbalance

Lin 2005.

Study characteristics
Methods	RCT
Participants	367 VLBW infants
Interventions	Probiotics (n = 180): Lactobacillus acidophilus and Bifidobacterium infantis (Infloran) 2 times daily with human milk until discharge from hospital Control (n = 187): unsupplemented milk feeds
Outcomes	NEC Death Infection Duration of hospitalisation Neurodevelopmental impairment at aged 3 years, defined as 1 or more of: BSID‐II MDI < 70, PDI < 70, bilateral blindness, bilateral hearing impairment requiring amplification, or moderate or severe cerebral palsy (requiring ambulatory assistance)
Notes	Setting: Taiwan (1999–2003) Funding: Research Department of China Medical University Hospital
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Random‐number table
Allocation concealment (selection bias)	Low risk	Opaque, sequentially numbered, sealed envelopes
Blinding (performance bias and detection bias) All outcomes	Unclear risk	Unsupplemented milk feeds; not placebo‐controlled (investigators aware of allocation)
Incomplete outcome data (attrition bias) All outcomes	Low risk	Near‐complete (90% for neurodevelopmental assessments)
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Lin 2008.

Study characteristics
Methods	RCT
Participants	434 VLBW infants
Interventions	Probiotics (n = 217): Bifidobacterium bifidum and Lactobacillus acidophilus, added to human milk or formula 2 times daily for 6 weeks Control (n = 217): unsupplemented milk feeds
Outcomes	NEC Death Infection
Notes	Setting: Taiwan (7 centres: 2005–2007) Funding: National Science Council of Taiwan ClinicalTrials.gov identifier NCT00540033
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Allocated centrally
Blinding (performance bias and detection bias) All outcomes	Unclear risk	Unsupplemented milk feeds; not placebo‐controlled
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Manzoni 2006.

Study characteristics
Methods	RCT
Participants	80 VLBW infants
Interventions	Probiotics (n = 39): Lactobacillus casei subspecies rhamnosus with human milk until 6 weeks or hospital discharge Control (n = 41): unsupplemented milk feeds
Outcomes	NEC Death Infection
Notes	Setting: Italy (2004–2005) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	High risk	Unmasked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Manzoni 2009.

Study characteristics
Methods	RCT
Participants	485 VLBW infants
Interventions	Probiotics (n = 238): Lactobacillus casei subspecies rhamnosus with human milk or formula until 4 weeks (VLBW) or 6 weeks (ELBW) plus bovine lactoferrin (100 mg/day) Control (n = 247): bovine lactoferrin alone All doses including placebo were diluted in prepared milk to maintain masking
Outcomes	NEC Death Infection
Notes	Setting: Italy (11 centres; 2007–2008) Funding: Dicofarm SpA (manufacturer of intervention)
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Pharmacy allocation (remote)
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Unclear risk	Data for invasive infection in complete cohort not reported in primary publication (available to derive from later publications)
Other bias	Low risk	No evidence of baseline imbalance

Matin 2022.

Study characteristics
Methods	RCT
Participants	52 VLBW infants (1000–1500 g)
Interventions	Probiotics (n = 26): Lactobacillus paracasei once daily with maternal milk for 28 days Control (n = 26): potato starch placebo
Outcomes	Serum bilirubin levels Duration of hospitalisation NEC Death Infection
Notes	Setting: Iran (2021) Funding: Tabriz University of Medical Sciences (Grant numbers 66400) Iranian Registry of Clinical Trials registration number IRCT20100414003706N38
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Pharmacy‐allocated
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked (placebo‐controlled)
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Mihatsch 2010.

Study characteristics
Methods	RCT
Participants	180 VLBW infants (< 30 weeks' gestation)
Interventions	Probiotics (n = 91): Bifidobacterium lactis BB12 mixed with powdered fortifier in human milk or formula once daily for 6 weeks Control (n = 89): powdered fortifier placebo
Outcomes	NEC Death Infection
Notes	Setting: Germany (2000–2003) Funding: Nestlé AG, Frankfurt, Germany
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Sealed envelopes
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked (placebo‐controlled)
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Millar 1993.

Study characteristics
Methods	RCT
Participants	20 infants < 33 weeks' gestation (most participants very preterm or VLBW)
Interventions	Probiotics (n = 10): Lactobacillus rhamnosus GG mixed with human milk or formula 2 times daily for 14 days, starting with first feed Control (n = 10): unsupplemented milk feeds
Outcomes	Stool colonisation Invasive infection Data for NEC and death provided by trial authors on request
Notes	Setting: UK (1991–1992) Funding: Wessex Medical Trust
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	Not described
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	Unclear risk	Unsupplemented milk feeds; not placebo‐controlled
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Mohan 2006.

Study characteristics
Methods	RCT
Participants	69 preterm infants (most participants were very preterm or VLBW)
Interventions	Probiotics (n = 37): Bifidobacterium lactis in milk feeds from first day after birth for 21 days Control (n = 32): unsupplemented milk feeds
Outcomes	No clinical outcomes presented in the published data Data for NEC, death, and infection provided by trial authors on request
Notes	Setting: Germany (2003–2005) Funding: Nestlé, Konolfingen, Switzerland
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Unclear risk	Central allocation (web‐based)
Blinding (performance bias and detection bias) All outcomes	Unclear risk	Unsupplemented milk feeds; not placebo‐controlled
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Moreno‐Sanz 2022.

Study characteristics
Methods	RCT
Participants	30 very preterm infants
Interventions	Probiotics (n = 15) Ligilactobacillus salivarius subsp infantis and Bifidobacterium longum Placebo (n = 15): placebo not specified, but described as having "appearance, colour, and taste [...] identical to the probiotic"
Outcomes	Stool colonisation patterns
Notes	Setting: not stated Funding: Probisearch SLU Outcome data not reported; further information sought from investigators but not available (August 2022)
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked (placebo‐controlled)
Incomplete outcome data (attrition bias) All outcomes	Low risk	Near‐complete assessment (3 withdrawals)
Selective reporting (reporting bias)	Unclear risk	Primary outcome as stool colonisation patterns; clinical outcomes not reported
Other bias	Low risk	No evidence of baseline imbalance

Oncel 2014.

Study characteristics
Methods	RCT
Participants	424 VLBW infants (and gestational age ≤ 32 weeks at birth)
Interventions	Probiotics (n = 213) Lactobacillus reuteri DSM 17938 once daily with milk feeds until discharge from hospital Placebo (n = 211): placebo containing only oil base
Outcomes	NEC Death Infection Culture‐proven infection with L. reuteri Duration of hospitalisation (presented as median/range) Neurodevelopmental impairment at 18–24 months, defined as 1 or more of: BSID‐II MDI < 70, PDI < 70, moderate‐to‐severe cerebral palsy, bilateral hearing impairment, or bilateral blindness
Notes	Setting: Turkey (2012–2013) Funding: not stated ClinicalTrials.gov identifier NCT01531179
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Opaque, sequentially numbered sealed envelopes
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Near‐complete (8 participants withdrawn by family) for in‐hospital outcomes (68% for neurodevelopmental assessment)
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Oshiro 2019.

Study characteristics
Methods	RCT
Participants	35 VLBW infants
Interventions	Probiotics (n = 17): Bifidobacterium breve BBG‐01 in human milk feeds once daily during the hospital stay Control (n = 18): "placebo" (not specified)
Outcomes	NEC Death Infection Weight gain
Notes	Setting: Japan (2015–2017) Funding: Yakult Honsha Company, Japan (manufacturer of intervention) Additional data via personal communication with Dr Yuichiro Yamashiro UMIN Registration No. UMIN000005412
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Sealed, opaque envelopes
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked (probiotic added to milk by dieticians who were not involved in the care of the infant)
Incomplete outcome data (attrition bias) All outcomes	Low risk	Masked
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Patole 2014.

Study characteristics
Methods	RCT
Participants	159 VLBW infants (< 33 weeks' gestation at birth)
Interventions	Probiotics (n = 79): Bifidobacterium breve M‐16V in milk feeds once daily until term equivalent Control (n = 80): maltodextrin placebo
Outcomes	Probiotic colonisation of stool NEC Death Infection Blood culture‐positive sepsis by B. breve M‐16V Neurodevelopmental outcomes provided by trial authors on request
Notes	Setting: Australia (2009–2012) Funding: Morinaga Milk Industry Company, Japan supplied the product free for the trial
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Opaque, sealed, coded envelopes
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked (placebo‐controlled)
Incomplete outcome data (attrition bias) All outcomes	Low risk	Near‐complete (6 infants withdrawn)
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Rehman 2018.

Study characteristics
Methods	RCT
Participants	146 VLBW preterm infants (gestational age at birth > 26 weeks)
Interventions	Probiotics (n = 70): Bifidobacterium spp. (not specified), Lactobacilli acidophilhis, Streptococcus thermophilus, and L. delbrueckii with human milk or formula 2 times daily until hospital discharge Control (n = 70): unsupplemented milk feeds
Outcomes	NEC Death (data provided by trial authors on request)
Notes	Setting: Pakistan (2014) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	High risk	Unmasked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Unclear risk	Infection not reported
Other bias	Low risk	No evidence of baseline imbalance

Ren 2010.

Study characteristics
Methods	RCT
Participants	150 preterm infants (most participants were very preterm)
Interventions	Probiotics (n = 79): Bifidobacterium infantis, Lactobacillus acidophilus, Bacillus cereus, and Enterococcus faecalis in milk feeds twice daily from day 7 after birth for 7 days (route translated as "oral or nasal", presumed to mean orogastric or nasogastric tube) Control (n = 80): unsupplemented milk feeds
Outcomes	NEC
Notes	Setting: China (single‐centre, 2006–2008) Funding: not stated Translation from Chinese courtesy of Yuan Chi
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Quote: "Drawing lots"
Allocation concealment (selection bias)	Unclear risk	Safeguards unclear
Blinding (performance bias and detection bias) All outcomes	High risk	Unmasked
Incomplete outcome data (attrition bias) All outcomes	Unclear risk	Unable to assess
Selective reporting (reporting bias)	Unclear risk	Mortality and infection not reported
Other bias	Unclear risk	Baseline characteristics not reported

Reuman 1986.

Study characteristics
Methods	Quasi‐RCT
Participants	30 very preterm infants (birth weight < 2000 g)
Interventions	Probiotics (n = 15): Lactobacillus acidophilus in formula daily for 28 days Control (n = 15): unsupplemented formula feeds
Outcomes	Stool colonisation NEC Death Duration of hospitalisation Rate of weight gain
Notes	Setting: USA (early 1980s) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	High risk	Random number charts and the last digit of the infant's chart number, then alternate allocation of next infant
Allocation concealment (selection bias)	High risk	Unconcealed
Blinding (performance bias and detection bias) All outcomes	High risk	Unmasked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Unclear risk	Infection not reported
Other bias	Low risk	No evidence of baseline imbalance

Rougé 2009.

Study characteristics
Methods	RCT
Participants	94 very preterm or VLBW infants
Interventions	Probiotics (n = 45): Lactobacillus rhamnosus GG and Bifidobacterium longum with human milk or formula once daily until discharge from hospital Control (n = 49): maltodextrin placebo
Outcomes	NEC Death Infection Duration of hospital stay
Notes	Setting: France (2005–2007) Funding: Programme Hospitalier de Recherche Clinique of the French Ministry of Health
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Centrally allocated
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked (placebo‐controlled)
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Roy 2014.

Study characteristics
Methods	RCT
Participants	112 preterm VLBW infants
Interventions	Probiotics (n = 56): Lactobacillus acidophilus, Bifidobacterium longum, B. bifidum, and B. lactis 2 times daily with human milk for 6 weeks or until discharged from hospital Control (n = 56): sterile water as "placebo"
Outcomes	NEC Death Infection
Notes	Setting: India (2012–2013) Funding: none
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Centrally allocated
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Sadowska‐Krawczenko 2012.

Study characteristics
Methods	RCT
Participants	55 very preterm or VLBW infants
Interventions	Probiotics (n = 30): Lactobacillus rhamnosus 2 times daily in 2 mL of 5% dextrose until discharge from hospital Control (n = 25): maltodextrin placebo
Outcomes	NEC Death Infection
Notes	Setting: Poland (2008–2009) Funding: Biomed Lublin, Poland supplied the intervention
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Central allocation
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked (placebo‐controlled)
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	High risk	Median birthweight higher in the experimental group (1034 g) than in controls (900 g)

Saengtawesin 2014.

Study characteristics
Methods	RCT
Participants	60 VLBW infants with gestational age ≤ 34 weeks at birth
Interventions	Probiotics (n = 31): Lactobacillus acidophilus and Bifidobacterium bifidum (Infloran) once daily with human milk or formula until 6 weeks or hospital discharge Control (n = 29): unsupplemented milk feeds
Outcomes	NEC Death Infection Probiotic "sepsis" Duration of hospitalisation
Notes	Setting: Thailand (2012–2013) Funding: Queen Sirikit National Institute of Child Health, Perinatal Society of Thailand and DKSH (Thailand) Limited
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	Not described
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	High risk	Unmasked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Samanta 2009.

Study characteristics
Methods	RCT
Participants	186 very preterm or VLBW infants
Interventions	Probiotics (n = 91): Bifidobacteria infantis, B. bifidum, B. longum, and Lactobacillus acidophilus with human milk 2 times daily until hospital discharge Control (n = 95): unsupplemented human milk feeds
Outcomes	NEC Death Infection Duration of hospital stay
Notes	Setting: India (2007–2008) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	Not described
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	High risk	Unmasked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Sari 2011.

Study characteristics
Methods	RCT
Participants	221 VLBW infants (gestational age < 33 weeks at birth)
Interventions	Probiotics (n = 110): Lactobacillus sporogenes in human milk or formula once daily until discharge from hospital Control (n = 111): unsupplemented milk feeds
Outcomes	NEC Death Infection Rate of weight gain Neurodevelopmental impairment at 18–24 months post‐term, defined as 1 or more of: BSID‐II MDI < 70, PDI < 70, cerebral palsy, bilateral blindness, or hearing impairment requiring amplification in both ears
Notes	Setting: Turkey (2008–2009) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Opaque, sequentially numbered, sealed envelopes
Blinding (performance bias and detection bias) All outcomes	Unclear risk	Caregivers masked, investigators not masked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete for in‐hospital outcomes (84% for neurodevelopmental assessment)
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Serce 2013.

Study characteristics
Methods	RCT
Participants	208 very preterm or VLBW infants
Interventions	Probiotics (n = 104): Saccharomyces boulardii in human milk or formula once daily until discharge from hospital Control (n = 104): unsupplemented milk feeds
Outcomes	NEC Death Infection Rate of weight gain Duration of hospitalisation Culture proven Saccharomyces boulardii "sepsis"
Notes	Setting: Turkey (2010–2011) Funding: Biocodex supplied the intervention
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Opaque, sequentially‐numbered, sealed envelopes
Blinding (performance bias and detection bias) All outcomes	Unclear risk	Unsupplemented milk feeds; not placebo‐controlled
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Shadkam 2015.

Study characteristics
Methods	RCT
Participants	60 preterm infants born at 28–34 weeks' gestation with birth weight 1000–1800 g (most participants were very preterm or VLBW)
Interventions	Probiotics (n = 30): Lactobacillus reuteri DSM 17938 2 times daily with human milk until full enteral feeding was reached (about 2 weeks) Control (n =30): unsupplemented milk feeds
Outcomes	NEC Death Infection
Notes	Setting: Iran (2012–2013) Funding: Shahid Sadughi University, Iran
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Random allocation software
Allocation concealment (selection bias)	Unclear risk	No information on concealment
Blinding (performance bias and detection bias) All outcomes	Unclear risk	Unsupplemented milk feeds; not placebo‐controlled
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Shashidhar 2017.

Study characteristics
Methods	RCT
Participants	104 VLBW infants
Interventions	Probiotics (n = 52): Lactobacillus acidophilus, L. rhamnosus, Bifidobacterium longum, and Saccharomyces boulardii (Darolac) once daily in human milk until discharge from hospital Control (n = 52): unsupplemented milk feeds
Outcomes	NEC Death Duration of hospital stay
Notes	Setting India (2012–2013) Funding: not stated
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Sequentially‐numbered, opaque, sealed envelopes
Blinding (performance bias and detection bias) All outcomes	Unclear risk	Unsupplemented milk feeds; not placebo‐controlled
Incomplete outcome data (attrition bias) All outcomes	Low risk	Near‐complete (3 infants in each group withdrawn)
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Sowden 2022.

Study characteristics
Methods	RCT
Participants	200 VLBW infants
Interventions	Probiotics (n = 100) Lactobacillus acidophilus, Bifidobacterium bifidum (0.67 CFUs), and B. infantis (Labinic) once daily in human milk or formula for 28 days Control (n = 100): medium chain triglyceride oil and Aerosil 2000 placebo (Aerosil 2000 is a stabiliser used in probiotic and placebo formulations)
Outcomes	NEC Mortality Feed intolerance
Notes	Setting: South Africa (2021) Funding: South African Medical Research Council, Harry Crossley Foundation, and VU University of Amsterdam
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	Quote: "Random number allocation"
Allocation concealment (selection bias)	Unclear risk	Quote: "Consecutive sampling"
Blinding (performance bias and detection bias) All outcomes	Low risk	Placebo‐controlled
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Unclear risk	Mortality not reported
Other bias	Low risk	No evidence of baseline imbalance

Stratiki 2007.

Study characteristics
Methods	RCT
Participants	77 preterm infants with gestational age > 26 weeks at birth (most infants were very preterm or VLBW)
Interventions	Probiotics (n = 41): Bifidobacterium lactis supplemented formula for 30 days Control (n = 36): unsupplemented formula feeds
Outcomes	Stool colonisation Intestinal permeability NEC Death Infection Rate of weight gain
Notes	Setting: Greece (2004–2005) Funding: Nestlé, Vevey provide the B. lactis supplemented formula
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Random numbers generator
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	Unclear risk	Unsupplemented milk feeds; not placebo‐controlled
Incomplete outcome data (attrition bias) All outcomes	Low risk	Near‐complete (3 infants not included in analyses)
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Strus 2018.

Study characteristics
Methods	RCT
Participants	181 preterm infants ≤ 34 weeks' gestation and birth weight 750–1800 g (most infants were very preterm or VLBW)
Interventions	Probiotics (n = 90): Lactobacillus rhamnosus KL53A and Bifidobacterium breve PB04 in milk feeds for 6 weeks or until hospital discharge Control (n = 91): maltodextrin placebo
Outcomes	Stool colonisation NEC Death Infection
Notes	Setting: Poland (2012–2013) Funding: IBSS BIOMED S.A., Krakow, Poland ClinicalTrials.gov identifier NCT02073214
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated sequence
Allocation concealment (selection bias)	Low risk	Centrally allocated
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked (placebo‐controlled)
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Tewari 2015.

Study characteristics
Methods	RCT
Participants	244 preterm infants < 34 weeks' gestation at birth (most infants were very preterm or VLBW)
Interventions	Probiotics (n = 121): Bacillus clausii 3 times daily with human milk for 6 weeks, or until discharge or death or occurrence of late‐onset invasive infection Control (n = 123): sterile water placebo (probiotic and the placebo were identical in appearance)
Outcomes	NEC Death Infection Duration of hospital stay
Notes	Setting: India (2012–2014) Funding: Enterogermina, Sanofi‐Aventis, Italy supplied the intervention
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Web‐based
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Totsu 2014.

Study characteristics
Methods	Cluster‐RCT
Participants	283 VLBW infants in 19 neonatal centres
Interventions	Probiotics (n = 10 centres; 153 infants*): Bifidobacterium bifidum with human milk or formula feeds 2 times daily until infant reached 2000 g bodyweight Control (n = 9 centres; 130 infants*): maltodextrin placebo *Inter‐cluster correlation correction of data for inclusion in meta‐analyses achieved by dividing numerators and denominator by the design effect (1.2779): Probiotics: adjusted n = 120 for in‐hospital outcomes; n = 80 for neurodevelopmental assessment outcomes Control: adjusted n = 102 for in‐hospital outcomes; n = 82 for neurodevelopmental assessment outcomes
Outcomes	NEC Death Infection Duration of hospital stay Rate of weight gain Neurodevelopmental impairment at 18 months, defined as Kyoto Scale of Psychological Development 2001 developmental quotient < 70, hearing (bilateral aids) or visual impairment, cerebral palsy (Gross Motor Function Classification System level II or greater)
Notes	Setting: Japan (19 centres; 2010–2011) Funding: Meiji, Tokyo, Japan
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated (stratified by "patient volume" of centre)
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked (placebo‐controlled)
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete for in‐hospital outcomes (73% for neurodevelopmental assessment)
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	High risk	Mean birthweight and gestation were similar, but antenatal corticosteroid exposure was higher in the intervention group (66%) than controls (51%).

Van Niekerk 2014.

Study characteristics
Methods	RCT
Participants	184 VLBW infants (< 1250 g)
Interventions	Probiotics (n = 91): Lactobacillus rhamnosusGG and Bifidobacterium infantis daily with human milk feeds for 4 weeks Control (n = 93): MCT oil placebo in milk feeds
Outcomes	NEC Death Infection
Notes	Setting: South Africa (2011–2012) Funding: National Research Foundation, Nestle Nutrition Institute Africa, Medical Research Council, and the Faculty of Medicine and Health Sciences, Stellenbosch University ClinicalTrials.gov identifier NCT01868737
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Independent statistician‐generated
Allocation concealment (selection bias)	Low risk	Pharmacy allocation (stratified by maternal HIV status)
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked (placebo‐controlled)
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Wang 2007.

Study characteristics
Methods	Quasi‐RCT
Participants	44 VLBW infants
Interventions	Probiotics (n = 22): Bifidobacterium breve in milk feeds 2 times daily until hospital discharge Control (n = 33): unsupplemented milk feeds
Outcomes	Short‐chain fatty acid and faecal lactic acid concentration Infection NEC data provided by trial authors on request
Notes	Setting: Japan (2001–2004) Funding: intervention provided by Morinaga Milk Industry, Kanagawa, Japan
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	High risk	Alternate allocation
Allocation concealment (selection bias)	High risk	Unconcealed
Blinding (performance bias and detection bias) All outcomes	High risk	Unmasked
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely (did not aim to assess clinical outcomes)
Other bias	Low risk	No evidence of baseline imbalance

Wejryd 2019.

Study characteristics
Methods	RCT
Participants	141 ELBW infants (gestational age at birth < 28 weeks)
Interventions	Probiotics (n = 72): Lactobacillus reuteri DSM 17938 once daily with human milk until 36 weeks' postmenstrual age Control (n = 69): maltodextrin placebo
Outcomes	Time to full enteral feeds NEC Death Infection
Notes	Setting: Sweden (10 centres; 2012–2015) Funding: Swedish Research Council, the Swedish Society for Medical Research, the Swedish Society of Medicine, the Research Council for the South‐East Sweden, ALF Grants, Region Ostergotland, the Ekhaga Foundation, and BioGaia AB ClinicalTrials.gov identifier NCT01603368
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Computer‐generated
Allocation concealment (selection bias)	Low risk	Centrally coded by sequential study number
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked (placebo‐controlled)
Incomplete outcome data (attrition bias) All outcomes	Low risk	Complete
Selective reporting (reporting bias)	Low risk	Unlikely
Other bias	Low risk	No evidence of baseline imbalance

Zeber‐Lubecka 2016.

Study characteristics
Methods	RCT
Participants	55 preterm infant < 33 weeks' gestation (most infants were very preterm or VLBW)
Interventions	Probiotics (n = 28): Saccharomyces boulardii once daily with human milk or formula feeds for six weeks Control (n = 27): maltodextrin placebo
Outcomes	Stool microbiomic structure Data for NEC, death, and infection provided by trial authors on request (no events)
Notes	Setting: Poland (study period not stated) Funding: The National Science Centre, Poland
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	Not described (quote: "randomly divided")
Allocation concealment (selection bias)	Unclear risk	Not described
Blinding (performance bias and detection bias) All outcomes	Low risk	Masked (placebo‐controlled)
Incomplete outcome data (attrition bias) All outcomes	High risk	Missing data from each group (10 from probiotics and 6 from placebo) not accounted for
Selective reporting (reporting bias)	Low risk	Unlikely (primary aim to study intestinal microbiome)
Other bias	Low risk	No evidence of baseline imbalance

BG‐01: Bifidobacterium breve; BSID: the Bayley Scales of Infant Development; CFU: colony forming units; ELBW: extremely low birth weight; g: gram(s); MCT: medium chain triglycerides; MDI: Mental Developmental Index; NEC: necrotising enterocolitis; PDI: Psychomotor Development Index; RCT: randomised controlled trial; SD: standard deviation; VLBW: very low birth weight.

Characteristics of excluded studies [ordered by study ID]

Study	Reason for exclusion
Ahmed 2022	Most participants not very preterm or VLBW.
Arora 2017	Most participants not very preterm or VLBW.
Awad 2010	Most participants not very preterm or VLBW.
Chi 2019	Not an RCT.
Dasopoulou 2015	RCT of prebiotics.
Deng 2010	Most participants not very preterm or VLBW.
Denkel 2016	Not an RCT.
Di 2010	Most participants not very preterm or VLBW.
Dongol‐Singh 2017	Most participants not very preterm or VLBW.
Hua 2014	Most participants not very preterm or VLBW.
Hussain 2016	Most participants not very preterm or VLBW.
Ke 2008	Most participants not very preterm or VLBW.
Koksal 2015	RCT of synbiotics.
Moles 2015	A pilot study in 5 infants.
Partty 2013	Most participants not very preterm or VLBW.
Qiao 2017	Most participants not very preterm or VLBW.
Rojas 2012	Most participants not very preterm or VLBW.
Romeo 2011	Most participants not very preterm or VLBW.
Shujie 2011	Most participants not very preterm or VLBW.
Sinha 2015	Most participants not very preterm or VLBW.
Thanhaeuser 2014	Not an RCT.
Uhlemann 1999	Most participants not very preterm or VLBW.
Underwood 2014	RCT of prebiotics
Xu 2016	Most participants not very preterm or VLBW.
Zhou 2012	Most participants not very preterm or VLBW.
Zhuang 2007	Most participants not very preterm or VLBW.

RCT: randomised controlled trial; VLBW: very low birth weight.

Characteristics of studies awaiting classification [ordered by study ID]

Coleta 2013.

Methods	RCT
Participants	60 preterm infants
Interventions	Lactobacillus reuteri
Outcomes	Feed tolerance
Notes	Setting: Romania (study period not stated) Funding: not stated Unlikely to have been reported fully (unable to contact investigators)

Kaban 2019.

Methods	RCT
Participants	94 infants of gestation 28 – 34 weeks and birthweight 1000 – 1800 g
Interventions	Lactobacillus reuteri
Outcomes	NEC Infection Death
Notes	Setting: not stated Funding: not stated Data for very preterm or VLBW infants sought from investigators (August 2022)

Niazi 2021.

Methods	RCT
Participants	260 VLBW infants
Interventions	Probiotics (not specified)
Outcomes	NEC Death
Notes	Setting: not stated Funding: not stated Outcome data not reported; further information sought from investigators (August 2022)

Punnahitananda 2006.

Methods	RCT (unclear)
Participants	VLBW infants
Interventions	Lactobacillus acidophilus and Bifidobacterium infantis
Outcomes	NEC Infection Time to full enteral feeding
Notes	Setting: not stated Funding: not stated Data presented at 14th Congress of the Federation of Asia Oceania Perinatal Societies, 2006, Bangkok, Thailand. Possible RCT (unclear); report not available.

Reznikov 2022.

Methods	RCT (unclear)
Participants	19 very preterm or VLBW infants
Interventions	Bifidobacterium lactis, B. infantis, and Streptococcus thermophilus
Outcomes	Plasma levels of immune system/inflammatory biomarkers
Notes	Setting: not stated Funding: Abbott Nutrition Presented as conference abstract (report not available). Possible RCT (unclear); investigators contacted for further information (August 2022)

NEC: necrotising enterocolitis; RCT: randomised controlled trial; VLBW: very low birth weight.

Characteristics of ongoing studies [ordered by study ID]

Marißen 2019.

Study name	Efficacy of Bifidobacterium longum, B. infantis and Lactobacillus acidophilus probiotics to prevent gut dysbiosis in preterm infants of 28‐ 32 weeks' gestation: a randomised, placebo‐controlled, double‐blind, multicentre trial: the PRIMAL Clinical Study protocol
Methods	RCT
Participants	Preterm infants (28 to 32 weeks')
Interventions	Bifidobacterium longum, B. infantis, and Lactobacillus acidophilus
Outcomes	Stool colonisation
Starting date	2020
Contact information	Christoph Hartel, Department of Paediatrics, University of Lübeck, Germany
Notes	Setting: not stated Trial registration number DRKS00013197

NCT00977912.

Study name	Necrotizing enterocolitis (Nec) and B. Lactis in premature babies
Methods	RCT
Participants	VLBW infants
Interventions	B. lactis for 6 weeks
Outcomes	NEC Antibiotic administration Stool microbiology
Starting date	November 2009
Contact information	Dr Peter Cooper, University of Witwatetersrand & Charlotte Maxek Johannestburg Academic Hospital, Zambia
Notes	Setting: not stated "Terminated" in 2013; unlikely to have been completed (not reported)

NCT01181791.

Study name	Effects of Lactobacillus reuteri in premature infants (reuteri)
Methods	RCT
Participants	VLBW infant
Interventions	Lactobacillus reuteri during hospitalisation
Outcomes	Time to reach full enteral feeds Stool colonisation Intestinal immunological response
Starting date	2010
Contact information	Teresa del Moral, University of Miami
Notes	Setting: Chile "Terminated" because of slow recruitment; unlikely to have been reported

NCT01375309.

Study name	Bifidobacterium supplementation for very low birth weight infants (Bifido(RCT))
Methods	RCT
Participants	VLBW infants
Interventions	Bifidobacterium bifidum (duration not clear)
Outcomes	Time to full enteral feeding Weight gain NEC
Starting date	2011
Contact information	Satoshi Kusuda, Professor of Neonatology, Tokyo Women's Medical University
Notes	Setting: not stated "Completed" 2012; unlikely to have been reported

NCT04541771.

Study name	The role of Lactobacillus reuteri in preventing necrotizing enterocolitis (NEC) in pre‐term infants (NEC)
Methods	RCT
Participants	Preterm infants (28 to 34 weeks')
Interventions	Lactobacillus reuteri until 35 weeks of gestation or discharged from hospital
Outcomes	NEC Infection
Starting date	2020
Contact information	Dr Summera Tabasum, The Children Complex & The Institute of Child Health, Multan
Notes	Setting: not stated

NEC: necrotising enterocolitis; RCT: randomised controlled trial; VLBW: very low birth weight.

Differences between protocol and review

See AlFaleh 2005 (review protocol).

In the 2023 update:

• we updated the search strategy;

• we included four new trials (Alshaikh 2022; Matin 2022; Moreno‐Sanz 2022; Sowden 2022); and

• we updated the risk of bias assessments.

Contributions of authors

SS, NM, SJO, and WM contributed to the development of the protocol.
SS, SJO, and WM screened the search results.
SS, SJO, MXRR, and WM performed the risk of bias and GRADE assessments.
SS, NM, SJO, and WM undertook data extraction and analysis.
SJO and WM arbitrated inclusion and data extraction disagreements.
NM undertook analyses for small‐study bias.
SS, NM, SJO, MXRR, and WM contributed to the development of the final review and approved the final version to be published.

Sources of support

Internal sources

• Centre for Reviews and Dissemination, University of York, UK

  Host department

• Department of Clinical Epidemiology and Biostatistics. Faculty of Medicine. Pontificia Universidad Javeriana, Colombia

  Host department

External sources

• Vermont Oxford Network, USA

  Cochrane Neonatal Reviews are produced with support from Vermont Oxford Network, a worldwide collaboration of health professionals dedicated to providing evidence‐based care of the highest quality for newborn infants and their families.

Declarations of interest

SS has no conflicts of interest to declare.
NM is Deputy Co‐ordinating Editor for Cochrane Common Mental Disorders but was not involved in the editorial process or decision‐making for this review.
SJO has no conflicts of interest to declare.
MXRR is an Associate Editor of Cochrane Neonatal but was not involved in the editorial process or decision‐making for this review.
WM is a Co‐ordinating Editor of Cochrane Neonatal but was not involved in the editorial process or decision‐making for this review.

New search for studies and content updated (no change to conclusions)