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

Abstract Background Dietary supplementation with prebiotic oligosaccharides to modulate the intestinal microbiome has been proposed as a strategy to reduce the risk of necrotising enterocolitis (NEC) and associated mortality and morbidity in very preterm or very low birth weight (VLBW) infants. Objectives To assess the benefits and harms of enteral supplementation with prebiotics (versus placebo or no treatment) for preventing NEC and associated morbidity and mortality in very preterm or VLBW infants. Search methods We searched the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, Embase, the Maternity and Infant Care database and the Cumulative Index to Nursing and Allied Health Literature (CINAHL), from the earliest records 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 prebiotics with placebo or no prebiotics in very preterm (< 32 weeks' gestation) or VLBW (< 1500 g) infants. The primary outcomes were NEC and all‐cause mortality, and the secondary outcomes were late‐onset invasive infection, duration of hospitalisation since birth, and neurodevelopmental impairment. Data collection and analysis Two review authors separately evaluated risk of bias of the trials, extracted data, and synthesised effect estimates using risk ratio (RR), risk difference (RD), and mean difference (MD), with associated 95% confidence intervals (CIs). The primary outcomes of interest were NEC and all‐cause mortality; our secondary outcome measures were late‐onset (> 48 hours after birth) invasive infection, duration of hospitalisation, and neurodevelopmental impairment. We used the GRADE approach to assess the level of certainty of the evidence. Main results We included seven trials in which a total of 705 infants participated. All the trials were small (mean sample size 100). Lack of clarity on methods to conceal allocation and mask caregivers or investigators were potential sources of bias in three of the trials. The studied prebiotics were fructo‐ and galacto‐oligosaccharides, inulin, and lactulose, typically administered daily with enteral feeds during birth hospitalisation. Meta‐analyses of data from seven trials (686 infants) suggest that prebiotics may result in little or no difference in NEC (RR 0.97, 95% CI 0.60 to 1.56; RD none fewer per 1000, 95% CI 50 fewer to 40 more; low‐certainty evidence), all‐cause mortality (RR 0.43, 95% CI 0.20 to 0.92; 40 per 1000 fewer, 95% CI 70 fewer to none fewer; low‐certainty evidence), or late‐onset invasive infection (RR 0.79, 95% CI 0.60 to 1.06; 50 per 1000 fewer, 95% CI 100 fewer to 10 more; low‐certainty evidence) prior to hospital discharge. The certainty of this evidence is low because of concerns about the risk of bias in some trials and the imprecision of the effect size estimates. The data available from one trial provided only very low‐certainty evidence about the effect of prebiotics on measures of neurodevelopmental impairment (Bayley Scales of Infant Development (BSID) Mental Development Index score < 85: RR 0.84, 95% CI 0.25 to 2.90; very low‐certainty evidence; BSID Psychomotor Development Index score < 85: RR 0.24, 95% 0.03 to 2.00; very low‐certainty evidence; cerebral palsy: RR 0.35, 95% CI 0.01 to 8.35; very low‐certainty evidence). Authors' conclusions The available trial data provide low‐certainty evidence about the effects of prebiotics on the risk of NEC, all‐cause mortality before discharge, and invasive infection, and very low‐certainty evidence about the effect on neurodevelopmental impairment for very preterm or VLBW infants. Our confidence in the effect estimates is limited; the true effects may be substantially different. Large, high‐quality trials are needed to provide evidence of sufficient validity to inform policy and practice decisions.


A B S T R A C T Background
Dietary supplementation with prebiotic oligosaccharides to modulate the intestinal microbiome has been proposed as a strategy to reduce the risk of necrotising enterocolitis (NEC) and associated mortality and morbidity in very preterm or very low birth weight (VLBW) infants.

Objectives
To assess the benefits and harms of enteral supplementation with prebiotics (versus placebo or no treatment) for preventing NEC and associated morbidity and mortality in very preterm or VLBW infants.

Search methods
We searched the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, Embase, the Maternity and Infant Care database and the Cumulative Index to Nursing and Allied Health Literature (CINAHL), from the earliest records 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 prebiotics with placebo or no prebiotics in very preterm (< 32 weeks' gestation) or VLBW (< 1500 g) infants.The primary outcomes were NEC and all-cause mortality, and the secondary outcomes were late-onset invasive infection, duration of hospitalisation since birth, and neurodevelopmental impairment.

Data collection and analysis
Two review authors separately evaluated risk of bias of the trials, extracted data, and synthesised e ect estimates using risk ratio (RR), risk di erence (RD), and mean di erence (MD), with associated 95% confidence intervals (CIs).The primary outcomes of interest were NEC and all-cause mortality; our secondary outcome measures were late-onset (> 48 hours a er birth) invasive infection, duration of hospitalisation, and neurodevelopmental impairment.We used the GRADE approach to assess the level of certainty of the evidence.

Main results
We included seven trials in which a total of 705 infants participated.All the trials were small (mean sample size 100).Lack of clarity on methods to conceal allocation and mask caregivers or investigators were potential sources of bias in three of the trials.The studied prebiotics were fructo-and galacto-oligosaccharides, inulin, and lactulose, typically administered daily with enteral feeds during birth hospitalisation.

Background
Very preterm (born more than eight weeks early) and very low birth weight (less than 1.5 kg) infants are at risk of developing necrotising enterocolitis, a severe condition where some lining of the infant's bowel becomes inflamed and dies.This condition is associated with death, serious infection, and long-term disability and developmental problems.One way to help prevent necrotising enterocolitis may be to add prebiotics (non-digestible sugar chains to support intestinal colonisation with healthy 'probiotic' bacteria) to milk feeds.

What did we do?
We searched for trials that looked examined the e ect of prebiotics on the risk of necrotising enterocolitis in very preterm or very low birth weight infants.We compared and summarised the results of the trials and rated our confidence in the evidence, based on factors such as study methods and sizes.

B A C K G R O U N D
This review assesses the trial evidence for the e ectiveness of enteral supplementation with prebiotic oligosaccharides for preventing necrotising enterocolitis (NEC) in very preterm or very low birth weight (VLBW) infants.Other Cochrane Reviews intend to assess the evidence for prebiotics in combination with probiotics ('synbiotics') or probiotics alone (Sharif 2020;Sharif 2021).

Description of the condition
Necrotising enterocolitis (NEC) is a syndrome of acute intestinal necrosis, which a ects about one in 20 very preterm (born before 32 weeks' gestation) or VLBW (birth weight less than 1500 g) infants (Horbar 2012).The risk factors for NEC include being extremely preterm (born before 28 weeks' gestation) or extremely low birth weight (ELBW; birth weight less than 1000 g), and intrauterine growth restriction or compromise indicated by absent or reversed end-diastolic flow velocities (AREDFV) in antenatal Doppler studies of the umbilical artery (Samuels 2017).Infants who develop NEC experience more episodes of severe infection, have lower levels of nutrient intake, grow more slowly, and have longer durations of hospital stay than gestation-comparable infants who do not (Battersby 2018;Berrington 2012).The associated mortality rate is about 20%, and, in infants who survive NEC -especially if it is associated with bloodstream infections -there is a high risk of neurodevelopmental problems and disabilities (Hickey 2018).
The pathogenesis of NEC is incompletely understood, but intestinal dysbiosis, infection and inflammation are thought to contribute (Eaton 2017;Mara 2018;Stewart 2016).Evidence exists that the pattern, diversity and stability of the intestinal microbiome (microbial life and genes) is associated with the risk of developing NEC (Masi 2019;Olm 2019;Stewart 2012;Warner 2016).Feeding with human milk compared with cow-milk formula reduces the risk of NEC in very preterm or VLBW infants (Cleminson 2015; Quigley 2019).One putative mechanism for this protective e ect is that 'prebiotic' oligosaccharides, which are abundant in human milk (but not in standard formula), promote the growth of non-pathogenic probiotic microorganisms, such as lactobacilli and bifidobacteria.These modulate the intestinal microbiome and enhance mucosal barrier functions (Embleton 2017;Granger 2020;Walsh 2019).Compared with human milkfed term infants, however, very preterm or VLBW infants tend to harbour fewer intestinal probiotic microorganisms, and more potential pathogens, which might be due to the dysbiotic e ects of antibiotic exposure and enteral fasting during the early neonatal period (Stewart 2017).

Description of the intervention
Prebiotics are a diverse family of complex glycans (chains of polymerised carbohydrates) that promote intestinal colonisation by probiotic microorganisms (Davani-Davari 2019; Gibson 2017).Human milk contains numerous prebiotic substances, predominantly galacto-oligosaccharides and fructooligosaccharides (based on the sugars galactose and fructose, respectively), that influence the intestinal microbiome in preterm infants (Boehm 2008;Nolan 2020).More than 150 di erent prebiotic oligosaccharides have been detected in human milk, with about 20 of these accounting for almost all human milk oligosaccharide content in most women.The pattern of human milk oligosaccharides produced varies markedly between individual women, and can vary temporally (depending on the stage of lactation) within an individual woman (Austin 2019;Durham 2021;Smilowitz 2013).
Newborn infants do not digest human milk oligosaccharides.Rather, these are primarily nutrient sources for intestinal probiotic microorganisms, particularly bifidobacteria (Alcon-Giner 2020; Jost 2015).Emerging evidence suggests that specific humanmilk oligosaccharides can promote probiotic predominance and reduce intestinal dysbiosis in very preterm infants (Masi 2021;Underwood 2015).Manufactured or plant-based (for example, inulin) prebiotic oligosaccharides are less heterogeneous than natural human-milk oligosaccharides, typically consisting of chains of galactose or fructose, usually with a terminal glucose monomer (Johnson-Henry 2016).These include lactulose, a nonabsorbable disaccharide synthesised from galactose and fructose (MacGillivray 1959).Evidence exists that giving supplemental, synthetic, prebiotic oligosaccharides to formula-fed very preterm infants stimulates the growth of an intestinal microflora that is similar to that found in infants fed with maternal milk (Autran 2018;Boehm 2008;Kapiki 2007;Veereman-Wauters 2011).Prebiotic oligosaccharides are added as ingredients to some cow-milk formulas for feeding newborn infants for whom su icient human milk is not available (Salminen 2020).Studies using animal models, however, have not provided consistent evidence of e icacy in preventing NEC-like syndromes (Nolan 2020).

How the intervention might work
The principal mechanism of action of supplemental prebiotics is likely to be the enhancement of probiotic microorganism growth and intestinal colonisation (Nolan 2020 While there is some trial-based evidence that enteral administration of exogenous probiotics reduces the risk of NEC and associated mortality and morbidity in very preterm or VLBW infants, concerns exist that e ect size estimates are inflated by publication bias (Sharif 2020).Another major barrier to use of probiotic supplementation is uncertainty about the optimal constitution of products, as well as availability, and regulatory and licensing issues (Berrington 2019;Du ield 2019;Fleming 2019;Pell 2019;Vermeulen 2020).Furthermore, although existing data are reassuring with regard to safety, probiotic bacteraemia or fungaemia (the potentially problematic presence of live bacteria/fungi in the bloodstream) and other adverse e ects have been reported in preterm infants (Bertelli 2015;Esaiassen 2016;Zbinden 2015).

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Why it is important to do this review
Necrotising enterocolitis and its associated complicationsparticularly invasive infection -are the commonest causes of mortality and serious morbidity beyond the early neonatal period in very preterm or VLBW infants (Berrington 2012).
It is plausible that prebiotic supplementation might promote endogenous probiotic growth and colonisation, and reduce the risk of NEC and its associated morbidity and mortality (with fewer risks than exogenous probiotic supplementation).Appraising and synthesising the trial evidence about the e ectiveness and safety of prebiotic supplementation could inform practice, policy and research.

O B J E C T I V E S
To assess the benefits and harms of enteral supplementation with prebiotics (versus placebo or no treatment) for preventing NEC and associated morbidity and mortality in very preterm or VLBW infants.

M E T H O D S
Criteria for considering studies for this review

Types of studies
We included randomised or quasi-randomised (predictable allocation) controlled trials, including cluster-randomised controlled trials.Cross-over studies were not eligible for inclusion.

Types of interventions
The interventions of interest were prophylactic enteral prebiotics: any combination or dose of prebiotic oligosaccharides (galactooligosaccharides (GOS); fructo-oligosaccharides (FOS); inulin; or lactulose), commenced within 14 days of birth and continued (at least) daily for (at least) one week was eligible, versus placebo or no prebiotic.
We did not include trials of synbiotics (combinations of probiotics and prebiotics), or trials of other substances that may have some prebiotic properties, for example lactoferrin.The e ectiveness of these interventions is addressed in other Cochrane Reviews (Pammi 2020;Sharif 2021).

Types of outcome measures
We focused on assessing e ects on infant-and family-important outcomes, principally neonatal morbidities that plausibly a ect rates of mortality or neurodisability.We did not include surrogate outcomes such as stool colonisation patterns.

Primary outcomes
• NEC before discharge from hospital, confirmed at surgery or autopsy or using standardised clinical and radiological criteria (VON 2020): • at least one of: bilious gastric aspirate or emesis; or abdominal distention; or blood in stool; and

Search methods for identification of studies
We used the criteria and standard methods of Cochrane Neonatal, as set out in our protocol (Sharif 2021).

Electronic searches
We searched the following electronic databases using a combination of text words and MeSH terms described in Appendix 1: • We limited the search outputs with filters for clinical trials as recommended in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2020).We did not apply any language restrictions.
We searched clinical trials registries for ongoing or recently completed trials (clinicaltrials.gov; the World Health Organization's International Trial Registry Platform (www.who.int/clinical-trialsregistry-platform), and the ISRCTN Registry (www.isrctn.com)).

Searching other resources
We examined the reference lists of any articles selected for inclusion in this review.

Data collection and analysis
We used the standard methods of Cochrane Neonatal as set out in our protocol (Sharif 2021).

Selection of studies
Two review authors (SS and WM) independently screened the titles and abstracts of all studies and assessed the full articles for all potentially relevant trials.We excluded those reports that did not meet all the inclusion criteria, and we stated the reasons for exclusion.We discussed disagreements until consensus was achieved, with referral to a third author (SO or PTH) for final decision as necessary.

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Data extraction and management
Two authors (SS, SO or WM) extracted data independently, using a form to aid extraction of information on design, methodology, participants, interventions, outcomes and treatment e ects from each included study.We discussed disagreements until we reached a consensus.If data from the study reports were insu icient, we contacted the report 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) (Higgins 2011) for the following domains: • sequence generation (selection bias); • allocation concealment (selection bias); • blinding of participants and personnel (performance bias); • blinding of outcome assessment (detection bias); • incomplete outcome data (attrition bias); • selective reporting (reporting bias); • any other bias (principally baseline imbalance).
Had any disagreements occurred, we planned to resolve these 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, where groups of individuals rather than individuals were randomised to the di erent 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 e ect
We analysed the treatment e ects in the individual trials and reported the risk ratio (RR) and risk di erence (RD) for dichotomous data and the mean di erence (MD) for continuous data, with respective 95% confidence intervals (CI).We planned to determine the number needed to treat for an additional beneficial outcome (NNTB) or an additional harmful outcome (NNTH) for analyses with a statistically significant di erence in the RD.

Unit of analysis issues
The unit of analysis was the participating infant in individually randomized trials and the neonatal unit (or subunit) for clusterrandomised trials.For cluster-randomised trials, we planned to undertake analyses at the level of the individual while accounting for the clustering in the data using the methods recommended in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2020).

Dealing with missing data
We planned to request additional data from trial investigators when data on important outcomes were missing or reported unclearly.If unavailable, we planned to undertake sensitivity analyses to assess the potential impact on outcomes by excluding those trials with > 20% missing data.

Assessment of heterogeneity
We examined the treatment e ects of individual trials and heterogeneity between trial results by inspecting the forest plots.We calculated the I statistic for each analysis to quantify inconsistency across studies and describe the percentage of variability in e ect estimates that may be due to heterogeneity rather than to sampling error.If we detected high levels of heterogeneity (I > 75%), we planned to explore the possible sources in subgroup analyses.

Assessment of reporting biases
If at least 10 trials were included in a meta-analysis, we planned to examine a funnel plot for asymmetry visually and with Harbord's modification of Egger's test (Harbord 2006).

Data synthesis
We used a fixed-e ect inverse variance meta-analysis for combining data where trials examined the same intervention and the populations and methods of the trials were judged to be similar.

Subgroup analysis and investigation of heterogeneity
When high heterogeneity was detected (I 2 > 75%), we planned to examine the potential causes in subgroup analyses for the primary outcomes, specifically: • type of prebiotic: GOS/FOS; inulin; or lactulose; • type of enteral feeding permitted for participating infants: human milk, formula, or both; • trials in which most (> 50%) participants were extremely low birth weight (ELBW; < 1000 g) or extremely preterm (< 28 weeks' gestation at birth) versus trials in which most infants were ≥ 28 weeks' gestation at birth or birth weight ≥ 1000 g; • trials which restricted participation to infants with intrauterine growth restriction or absent or reversed end-diastolic flow velocities in the foetal aorta or umbilical artery versus trials which did not do so.

Sensitivity analysis
We planned to undertake sensitivity analyses to determine how estimates are a ected by including only studies at low risk of bias: (i) selection bias (adequate randomisation and allocation concealment), (ii) detection or performance bias (adequate masking of intervention and measurement), (iii) attrition bias (< 20% loss to follow-up for primary outcome assessment), and (iv) reporting bias (selective reporting).

Summary of findings and assessment of the certainty of the evidence
Two authors (PTH, SO or WM) used the GRADE approach to assess the certainty of the evidence for e ects on NEC, all-cause mortality before hospital discharge, late-onset invasive infection, and measures of neurodevelopmental impairment a er 12 months' post-term neurological evaluations, developmental scores, and classifications of disability, including cerebral palsy and auditory and visual impairment (Schünemann 2013;Walsh 2021).
We considered evidence from randomised controlled trials as high certainty but downgraded the evidence certainty by one level for serious (or two levels for very serious) limitations based upon the Cochrane Database of Systematic Reviews following domains: design (study limitations), inconsistency across studies, indirectness of the evidence, imprecision of estimates, and presence of publication bias.This approach results in an assessment of the certainty of a body of evidence as one of four grades: • High certainty: further research is very unlikely to change our confidence in the estimate of e ect.• Moderate certainty: further research is likely to have an important impact on our confidence in the estimate of e ect and may change the estimate.• Low certainty: further research is very likely to have an important impact on our confidence in the estimate of e ect and is likely to change the estimate.
• Very low certainty: we are very uncertain about the estimate.
We used GRADEpro GDT to create summary of findings table and to report the certainty of the evidence.

Results of the search
A er the removal of duplicates from the search results, we screened 3282 titles and abstracts.We evaluated 14 articles sourced as fulltext reports (Figure 1) and of these, we included seven studies.No ongoing studies were identified.

Population
In total, 705 infants participated in the included trials (mean 100).Three trials enrolled only very preterm or VLBW infants.Four trials enrolled infants of gestational age up to 32 weeks', and because the average gestation at birth was < 32 weeks', or the average birth weight < 1500 g, we included these trials (Boehm 2002;Hascoët 2022;Modi 2010;Riskin 2010).One trial excluded infants who were born with birth weight below the 10th percentile for the reference population ("small-for-gestation") (Modi 2010).None of the trials specified exclusion of infants who had evidence of absent or reversed end-diastolic flow velocities detected on antenatal Doppler studies of the foetal aorta or umbilical artery.
In most trials, participating infants were permitted human milk or formula feeding.One trial enrolled infants who received human milk only (Armanian 2014), and one trial enrolled only formula-fed participants (Boehm 2002).
Most trials started prebiotic (and placebo if used) supplements during the first week a er birth when enteral feeding with human milk or formula was tolerated.In five of the trials, prebiotics or placebo were administered daily until discharge from hospital (Armanian 2014;Dilli 2015;Hascoët 2022;Modi 2010;Riskin 2010).
In two trials the intervention was continued for four weeks (Boehm 2002; van den Berg 2010).

Outcomes
All the trials reported the number of infants who developed NEC, all-cause mortality, and late-onset invasive infection.In one trial, none of the participants experienced any of these outcomes (Boehm 2002).Other in-hospital outcomes reported included time to establish full enteral feeding, rate of weight gain, and duration of hospital stay.Only one of the trials reported neurodevelopmental outcomes (van den Berg 2010).
One trial was excluded because participating infants did not commence supplements until beyond the neonatal period, when the risk of the outcomes for this review occurring was already much reduced (Mihatsch 2006).

Risk of bias in included studies
Risk of bias assessments and judgements are described in Characteristics of included studies and are summarised in Figure 2.

Allocation
Most trial reports described methods to generate random sequences (typically computer-generated) and to ensure adequate allocation concealment (typically sealed opaque envelopes).One report did not describe the methods used to randomise infants (Boehm 2002).One trial was quasi-randomised (high risk) with treatment allocation based on the infant's case file number (Armanian 2014).

Prebiotics to prevent necrotising enterocolitis in very preterm or very low birth weight infants (Review)
Copyright © 2023 The Authors.2014).

Incomplete outcome data
Six trials reported complete or near-complete assessments of primary outcomes (Boehm 2002;Dilli 2015;Hascoët 2022;Modi 2010;Riskin 2010;van den Berg 2010).In one trial, primary outcome data were not available for more than one-quarter of participants in the intervention group (Armanian 2014).

Selective reporting
Although trial protocols were not available for most trials, selective reporting bias was not considered a major threat given that all relevant clinical outcomes were reported.

Other potential sources of bias
We did not find evidence of between-group baseline di erences in participant characteristics or demographics in six trials (Armanian 2014;Boehm 2002;Dilli 2015;Hascoët 2022;Modi 2010;van den Berg 2010).In one trial, the mean birth weight and gestational age di ered substantially between the groups (Riskin 2010).These di erences were not explained in the report.

E ects of interventions
See: Summary of findings 1 Prebiotics compared to control in very preterm or very low birth weight infants

NEC
Meta-analysis of data from seven trials (686 infants) suggests that prebiotics may result in little or no di erence in NEC prior to hospital discharge (Analysis 1.1; Figure 3):

Subgroup analysis for heterogeneity
In the absence of high levels of heterogeneity (I = 0%), we did not undertake subgroup analyses (Subgroup analysis and investigation of heterogeneity).
Using the GRADE approach, we assessed the certainty of the evidence to be 'low'.We downgraded evidence certainty by one level for study limitations and one level for imprecision of the e ect estimate (Summary of findings 1).

All-cause mortality before hospital discharge
Meta-analysis of data from seven trials (686 infants) suggests that prebiotics may result in little or no di erence in all-cause mortality prior to hospital discharge (Analysis 1.2; Figure 4

Subgroup analysis for heterogeneity
In the absence of high levels of heterogeneity (I = 22%), we did not undertake subgroup analyses (Subgroup analysis and investigation of heterogeneity).
We assessed the certainty of evidence to be 'low'.We downgraded evidence certainty by one level for study limitations and by one level for imprecision (Summary of findings 1).

Late-onset invasive infection
Meta-analysis of data from seven trials (686 infants) suggests that prebiotics may result in little or no di erence in late-onset invasive infection prior to hospital discharge (Analysis 1.3; Figure 5):

Subgroup analysis for heterogeneity
In the absence of high levels of heterogeneity (I = 0%), we did not undertake subgroup analyses (Subgroup analysis and investigation of heterogeneity).
We assessed the certainty of evidence to be 'low'.We downgraded evidence certainty by one level for study limitations and by one level for imprecision (Summary of findings 1).

Duration of hospitalisation
Two trials reported a shorter median duration of hospitalisation with prebiotics versus control: *Meta-analysis was not possible as standard errors were not reported.

Neurodevelopmental impairment
One trial assessed neurodevelopmental impairment in surviving children at the corrected age of two years (van den Berg 2010).Outcomes were assessed in 76 infants (75% of eligible participants).
The evidence if very uncertain about the e ect of prebiotics on the median Bayley Scales of Infant Development (second or third edition) Index scores: • RR 0.84, 95% CI 0.25 to 2.90; • RD 20 per 1000 fewer, 95% CI 170 fewer to 120 more.
The evidence is very uncertain about the e ect of prebiotics on the proportion of infants with Bayley Scales of Infant Development PDI scores < 85 (Analysis 1.5): • RR 0.24, 95% 0.03 to 2.00; • RD 90 per 1000 fewer, 95% CI 220 fewer to 30 more.
The evidence is very uncertain about the e ect of prebiotics on the proportion of infants diagnosed with cerebral palsy (Analysis 1.6): • RR 0.35, 95% CI 0.01 to 8.35; • RD 30 per 1000 fewer, 95% CI 90 fewer to 40 more.
None of the infants had auditory and visual impairment.
We assessed the certainty of evidence to be 'very low' because of study limitations and serious imprecision of e ect estimate (Summary of findings 1).

Sensitivity analyses of trials at low risk of bias
We undertook sensitivity meta-analyses of data from the three trials (467 infants) at low risk of bias across all domains (Dilli 2015; Modi 2010; van den Berg 2010).These showed similar results compared with the primary analyses:

Summary of main results
Meta-analyses of data from seven trials suggests that enteral supplementation with prebiotics may result in little or no di erence in NEC, all-cause mortality, or late-onset invasive infection prior to hospital discharge, but the evidence is of low certainty.Four trials reported a shorter median duration of hospitalisation with prebiotics versus control, and two trials did not show a di erence.These trials did not provide data to permit meta-analysis.Only one trial assessed neurodevelopmental impairment and the evidence of e ect is of very low certainty.

Overall completeness and applicability of evidence
These data are likely to be relevant to current practice since all the included trials were conducted during the past 25 years in neonatal care facilities across a variety of settings (Iran, Germany, Turkey, England, Israel, Netherlands, France).The risk of developing NEC amongst infants in both the control and intervention groups was about 5% to 10%, similar to incidence estimates from recent observational studies (Battersby 2018;Horbar 2012).While most participants were very preterm or VLBW infants, few were extremely preterm or ELBW.However, only one of the trials specifically excluded infants born 'small for gestational age' (Modi 2010).None excluded infants who had evidence of absent or reversed end-diastolic flow velocities in antenatal Doppler studies of the umbilical artery or foetal aorta, increasing the applicability of the review findings to these populations of very preterm of VLBW infants at high risk of NEC and associated mortality and morbidity.
The trials used a variety of prebiotics.The most commonly assessed formulations were plant-derived and synthetic galactooligosaccharides and fructo-oligosaccharides constituted to mimic oligosaccharides found in human milk (Armanian 2014;Boehm 2002;Dilli 2015;Modi 2010;van den Berg 2010).One trial assessed lactulose, a synthetic fructose-galactose disaccharide

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Cochrane Database of Systematic Reviews (Riskin 2010).Only one trial assessed human milk oligosaccharides (2′-fucosyllactose and lacto-N-neotetraose) (Hascoët 2022).These were mostly commercially-available products supplied by the manufacturer for use in the trial.A better understanding of the mechanisms and events occurring at the intestinal epithelial and mucosal level may help to determine which prebiotics optimally supports a putatively beneficial microbiome in very preterm or VLBW infants (Abbas 2021; Autran 2018).
The type of enteral feeds that infants receive might influence the e ects of prebiotic supplementation (Quigley 2019).One trial permitted only human milk feeding, two trials recruited formulafed infants, while in the other three trials infants could be fed with human milk or formula, or both.In the absence of high levels of heterogeneity, we did not undertake any subgroup analyses by type of milk feeding.Any such analysis, furthermore, would need to be interpreted cautiously as the data available were insu icient to define subgroups at an infant (rather than trial) level.The possibility remains that infants who receive human milk as their predominant source of nutrition might not gain added benefit from prebiotics supplementation since their milk is already rich in human milk oligosaccharides that enhance probiotic growth and colonisation (Nolan 2020).

Quality of the evidence
We used GRADE methods to assess the certainty of the evidence for e ects on NEC, all-cause mortality, late-onset invasive infection, and neurodevelopmental impairment (Summary of findings 1).We downgraded the certainty of the evidence because of methodological weaknesses (risk of bias) in three of the trials (Armanian 2014;Boehm 2002;Riskin 2010).These included uncertainty about measures to conceal allocation and to mask parents, caregivers, and clinical assessors that may have introduced selection, performance and detection biases.In one trial, there was unexplained baseline imbalance with the mean gestational age and birth weight higher in intervention than control groups.A priori, therefore, infants in the intervention group were at lower average risk than control infants of NEC, all-cause mortality, late-onset invasive infection, and neurodevelopmental impairment, potentially leading to over-estimates of e ect sizes.However, prespecified sensitivity analyses of the three trials (467 infants) at low risk of bias across all domains showed e ects consistent with those in the primary meta-analyses that included all the trials (Dilli 2015; Modi 2010; van den Berg 2010).
The other reason for downgrading the certainty of the evidence was the existence of substantial imprecision in estimates of e ect, with meta-analyses generating 95% CI that included large benefit as well as small or no benefit or harm.Estimates of e ect were imprecise, especially for less common outcomes, including all-cause mortality prior to hospital discharge, where the 95% CI ranged from an NNTB from 80 fewer to none fewer per 1000 infants given prebiotic supplements.Such imprecise estimates of e ect are unlikely to meaningfully inform decision-making in this context.

Potential biases in the review process
We used the standard methods of Cochrane Neonatal to minimise potential biases in the review process.Two authors performed the literature search independently and combined results.We contacted study investigators to clarify inclusion criteria where necessary, and to provide unpublished data and missing information.Following full-text screening, we excluded six studies because of the characteristics of their participant populations (term or near-term infants rather than very preterm infants).We made one marginal decision to exclude another study on the grounds that participating very preterm infants commenced prebiotic supplements only when fully-fed; mean day 36 for prebiotics, day 53 for maltodextrin placebo (Mihatsch 2006).Although this was not a prespecified exclusion criterion, we agreed that the study di ered substantially from the review's intent, that is, focused primarily on preventing necrotising enterocolitis.
An important concern with the review process is the possibility that the findings are subject to publication and other reporting biases.Data from trials which show statistically significant or potentially important e ects tend to be more readily available for inclusion in meta-analyses (Gale 2020).Publication bias, as well as other sources of small-study bias, is an important contributor to inflation of e ect size estimates in meta-analyses of interventions to improve outcomes in very preterm or VLBW infants (Young 2021).For example, the Cochrane Review of probiotics to prevent NEC in very preterm or VLBW infants showed a large reduction in the risk of NEC, but the funnel plot and regression analysis indicated that publication bias was likely to have inflated the pooled e ect size estimate (Sharif 2020).In this review, we could not assess whether publication bias (or related small study biases) exaggerated the e ect size since the meta-analyses contained insu icient data points (fewer than 10) to make funnel plot inspection and regression analysis valid and reliable; that is, able to distinguish real asymmetry from chance asymmetry (Higgins 2020).Although we attempted to minimise the threat of publication bias by screening the reference lists of included trials and related reviews and searching the proceedings of the major international perinatal conferences to identify trial reports that are not published in full form in academic journals, we cannot be sure that other trials have been undertaken but not reported.

Agreements and disagreements with other studies or reviews
We are aware of one other systematic review that assessed the trial evidence for prebiotics supplementation in preterm infants (Srinivasjois 2013).Although this review employed less stringent inclusion criteria than our review has (for example, including trials in which term infants participated), the findings were similar, that is, suggesting that prebiotic supplementation has little or no e ect on the risk of NEC or associated morbidity.
Other Cochrane Reviews have addressed whether probiotics alone or synbiotics (probiotics combined with prebiotics) a ect the risk of NEC (Sharif 2020;Sharif 2021).Meta-analyses of data from trials of probiotic or synbiotics supplementation suggested a reduction in the risk of NEC and associated morbidity and all-cause mortality for very preterm or VLBW infants.Similar to the findings in this review, however, concerns about trial quality, heterogeneity of interventions, imprecision, and publication bias, as well as the paucity of data for extremely preterm or ELBW infants, means that these findings are of low certainty, and should be interpreted and applied cautiously.

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Implications for practice
The available trial data provide low-certainty evidence about the e ects of prebiotics on the risk of necrotising enterocolitis (NEC), all-cause mortality before discharge, and invasive infection, and very low-certainty evidence about the e ect on neurodevelopmental impairment, for very preterm or very low birth weight (VLBW) infants.Our confidence in the e ect estimates is limited; further research is very likely to have an important impact on the estimates of e ect.In addition to concern about biases in the existing trials, a major barrier to implementing the findings is that existing analyses are not able to determine reliably the optimal constitution of prebiotic supplements (as well as doses, timing of introduction, duration of use) for routine prophylactic use.A variety of commercially available prebiotic preparations are in use in a minority of neonatal units internationally, but widespread use is limited by availability and regulatory and licensing issues.

Implications for research
Given the low level of certainty about whether (and which) prebiotics a ect important outcomes in very preterm or VLBW infants, further high-quality randomised, placebo-controlled trials are needed to provide evidence of su icient validity to inform policy and practice.Such trials are likely to need to recruit several thousands of infant participants to reliably detect plausible e ects on uncommon outcomes such as NEC and mortality prior to hospital discharge (Gale 2020).Ideally, trials should attempt to ensure that caregivers and assessors are masked to the intervention, as investigation and diagnosis of NEC, late-onset invasive infection and neurodevelopmental impairment can be subjective and can be associated with the inter-rater variation.While it may be appropriate to be broadly inclusive of very preterm and VLBW infant participants, trials should ensure su icient power to assess e ects in extremely preterm or extremely low birth weight (ELBW) infants, infants born 'small for gestational age', or with evidence of absent or reversed end-diastolic flow velocities in antenatal Doppler studies of the umbilical artery or foetal aorta.Trials, furthermore, should be powered to explore interactions with the type of enteral feed (human milk versus cow-milk formula) received (Quigley 2019).Investigators need to consider which types of prebiotic to evaluate in trials, including perhaps those specific human milk oligosaccharides that have been associated with a lower risk of NEC in preterm infants (Masi 2021), and whether trials using prebiotics are merited alongside trials of probiotics and synbiotics as part of a factorial or an adaptive design (Underwood 2019).

A C K N O W L E D G E M E N T S
Cochrane Neonatal supported the authors in the development of this review.We thank Colleen Ovelman and Jane Cracknell (former Managing Editors), Michelle Fiander (Information Specialist and current Managing Editor), and Roger Soll (Co-ordinating editor).William McGuire is a member of Cochrane Neonatal but was not involved in the editorial process or decision-making for this review.
We thank Melissa Harden (Information Specialist, Centre for Reviews and Dissemination, University of York, UK) for the search strategies and database management.
The following people conducted the editorial process for this review.
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Figure 2 .
Figure 2. Risk of bias graph: review authors' judgements about each risk of bias item presented as percentages across all included studies

Figure 3 .
Figure 3. Forest plot: e ects of prebiotics versus control on necrotising enterocolitis sequence generation (selection bias) (B) Allocation concealment (selection bias) (C) Blinding of participants and personnel (performance bias) (D) Blinding of outcome assessment (detection bias) (E) Incomplete outcome data (attrition bias) (F) Selective reporting (reporting bias) (G) Other bias Prebiotics to prevent necrotising enterocolitis in very preterm or very low birth weight infants (Review) Copyright © 2023 The Authors.Cochrane Database of Systematic Reviews published by John Wiley & Sons, Ltd. on behalf of The Cochrane Collaboration.

•
Mental Development Index (MDI): 95 (range 80 to 115) versus 100 (range 65 to 115); • Psychomotor Development Index (PDI): 100 (range 71 to 130) versus 97 (range 69 to 145).The evidence is very uncertain about the e ect of prebiotics on the proportion of infants with Bayley Scales of Infant Development MDI score < 85 (indicative of developmental delay) (Analysis 1.4): Prebiotics to prevent necrotising enterocolitis in very preterm or very low birth weight infants (Review)Copyright © 2023 The Authors.Cochrane Database of Systematic Reviews published by John Wiley & Sons, Ltd. on behalf of The Cochrane Collaboration.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$1).ti.and animal experiment/ Animal experiment/ not (human experiment/ or human/Care Database (MIDIRS) via OVID <1971 to 14 June 2022> probiotic$.ti,ab,de.bifidobacterium$.ti,ab,de.lactobacill$.ti,ab,de.Saccharomyces$.ti,ab,de.
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95% CI) Outcomes Risk with control Risk with prebiotics
* ( Cochrane Database of Systematic Reviews published by John Wiley & Sons, Ltd. on behalf of The Cochrane Collaboration.Riskin 2010; van den Berg 2010).The other trial did not mask parents, caregivers, or clinical investigators (Armanian

Prebiotics to prevent necrotising enterocolitis in very preterm or very low birth weight infants (Review)
Copyright © 2023 The Authors.Cochrane Database of Systematic Reviews published by John Wiley & Sons, Ltd. on behalf of The Cochrane Collaboration.

to prevent necrotising enterocolitis in very preterm or very low birth weight infants (Review)
Copyright © 2023 The Authors.Cochrane Database of Systematic Reviews published by John Wiley & Sons, Ltd. on behalf of The Cochrane Collaboration.

to prevent necrotising enterocolitis in very preterm or very low birth weight infants (Review)
Copyright © 2023 The Authors.Cochrane Database of Systematic Reviews published by John Wiley & Sons, Ltd. on behalf of The Cochrane Collaboration.

to prevent necrotising enterocolitis in very preterm or very low birth weight infants (Review)
Copyright © 2023 The Authors.Cochrane Database of Systematic Reviews published by John Wiley & Sons, Ltd. on behalf of The Cochrane Collaboration.

to prevent necrotising enterocolitis in very preterm or very low birth weight infants (Review)
Copyright © 2023 The Authors.Cochrane Database of Systematic Reviews published by John Wiley & Sons, Ltd. on behalf of The Cochrane Collaboration.

Prebiotics to prevent necrotising enterocolitis in very preterm or very low birth weight infants (Review)
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