Vitamin D supplementation for term breastfed infants to prevent vitamin D deficiency and improve bone health

Abstract

This is a protocol for a Cochrane Review (Intervention). The objectives are as follows:

To determine the effect of vitamin D supplementation given to:

• infants compared to placebo or no intervention on vitamin D deficiency, bone density and growth in healthy term breastfed infants;

• lactating mothers compared to placebo or no intervention on vitamin D deficiency, bone density and growth in healthy term breastfed infants;

• infants compared to vitamin D supplementation given to lactating mothers on vitamin D deficiency, bone density and growth in healthy term breastfed infants;

• infants compared periods of infant sun exposure on vitamin D deficiency, bone density and growth in healthy term breastfed infants.

For each of the above comparisons:

• to determine adverse effects from vitamin D supplementation compared to placebo, no intervention or other interventions in healthy term breastfed infants.

Background

Description of the condition

Breastfeeding is the optimal source of nutrition for infants under six months of age. The World Health Organization (WHO) recommends exclusive breastfeeding for the first six months of life, followed by continued breastfeeding with complementary food until two years of age and beyond (WHO 2003). Exclusive breastfeeding means that no other fluid or food is given to the infant. It is recommended that for the duration of exclusive breastfeeding a mother's breast milk alone is sufficient to meet the energy and nutrition requirements of her infant (Butte 2001). However, there are concerns that breastfed infants may not maintain adequate vitamin D status from sunshine or their mother’s milk (Dawodu 2003; Lovell 2016). This is in part contributed by low maternal vitamin D levels (Andiran 2002), and limited exposure of infants to sunlight (NHS 2017).

It is widely accepted that vitamin D levels are low in breast milk (Hollis 1981; við Streym 2016). The reported prevalence of vitamin D insufficiency or deficiency in term breastfed infants without vitamin D supplementation ranges from 0.6% at seven months of age in Nepalese infants (Haugen 2016), to 40% at four months of age in infants in the USA (Merewood 2012), and even as high as 83% at one month of age in Qatari infants (Salameh 2016). The vast differences seen are likely to be caused by multiple factors, including geographical factors (latitude and season during measurement), skin pigmentation of the population studied, use of covered clothing and methodological differences (Kasalová 2015; Matsuoka 1992; Munns 2016).

Serum 25(OH)vitamin D (calcidiol) is the generally accepted marker of vitamin D sufficiency (IOM 2011). Though there is no universal consensus, most guidelines report that 25(OH)vitamin D of at least 50 nmol/L is adequate (EFSA 2016; IOM 2011; Munns 2016). A 25(OH)vitamin D level of 30 to 50 nmol/L is considered insufficient, while a level lower than 30 nmol/L is considered deficient (Munns 2016). (Note: 1 nmol/L = 0.4 ng/mL; IOM 2011).

Vitamin D deficiency in an infant can result in a number of bone‐related as well as 'non‐bone'‐related conditions (Wharton 2003). The bony condition resulting from vitamin D deficiency in children is nutritional rickets. Nutritional rickets is characterised by deficient mineralization of cartilage and bone, growth failure and skeletal deformity (Shore 2013a). The 'non‐bone' conditions resulting from vitamin D deficiency include seizure, myopathy and myelofibrosis (Wharton 2003). Nutritional rickets results from vitamin D deficiency, primary calcium deficiency, or both (Pettifor 2004). Two reviews on the epidemiology of nutritional rickets worldwide found that calcium deficiency may also be a major etiology of nutritional rickets in some African, Middle Eastern and Asian countries (Creo 2017; Prentice 2013). For this Cochrane Review, the term 'nutritional rickets' refers to vitamin D‐deficient nutritional rickets.

Infants with nutritional rickets often present at between three to 18 months of age, when exclusive or partial breastfeeding is predominant (Creo 2017). Prior to three months, the infant is relatively protected by placental transfer of vitamin D (Shore 2013b).

The progression of nutritional rickets can be described in three stages. Initially, low circulating calcium (hypocalcaemia) occurs as a result of reduced absorption from the gastrointestinal tract and reabsorption from bones. The hypocalcaemia is often transient, but in infants can be prolonged enough for the infant to become symptomatic, presenting with tetany or seizures. Subsequently, direct feedback to the parathyroid gland producing secondary hyperparathyroidism results in normalisation of serum calcium, but this is also accompanied by hypophosphataemia and hyperphosphaturia. If vitamin D deficiency continues, the raised parathyroid hormone (PTH) can no longer maintain calcium levels and rickets becomes more severe (Fraser 1967).

Diagnosis of rickets is made from a combination of clinical features, radiological findings and biochemical abnormalities. The radiological (x‐ray) findings that are most diagnostic of rickets are those that demonstrate disordered mineralization and ossification of the physes, described as metaphyseal splaying. These are best seen in the metaphysis of fast‐growing bones, such as the distal ulnar and radius, distal femur, proximal and distal tibia, proximal humerus and anterior ends of middle ribs. Other findings include osteopenia and deformities (Shore 2013b). Due to increased bone activity, raised alkaline phosphatase (ALP) and PTH are commonly found. Hypocalcaemia may not be present as this is dependant on the stage of rickets development (Fraser 1967). Specifically for vitamin D‐deficient rickets, the 25(OH)vitamin D levels are less than 30 nmol/L (Munns 2016).

Nutritional rickets can be treated by replacement of vitamin D and calcium (Misra 2008). However in the case of nutritional rickets, much of the damage caused by the deficiency, such as the skeletal deformity, is not correctable. Therefore, it is important to prevent nutritional rickets in vulnerable groups, such as breastfed infants.

Other than bone health, vitamin D has also been implicated in other conditions, such as improving immunity, prevention of cardiovascular disease, prevention of certain types of malignancies and mental health protection (Pludowski 2013). However, it is beyond the scope of this Cochrane Review to consider these outcomes.

Description of the intervention

Vitamin D, also known as ‘the sunshine vitamin’ is a pro‐hormone rather than a ‘vitamin’. It has two physiologically active forms, vitamin D₂ (ergocalciferol) and vitamin D₃ (cholecalciferol). Vitamin D₂ (VD2) is formed from ultraviolet (UV) radiation in plants and yeast (thus the source is from food), while vitamin D₃ (VD3) is synthesised in the skin (epidermis to dermis) from 7‐dehydrocholesterol. The synthesis of VD3 is a two‐step process, with the formation of pre‐VD3 using UVB (spectral range 290 to 320 nm) and subsequent thermal isomerization into VD3. Once formed it is bound to vitamin D‐binding protein for transport into the circulation (Holick 1980). Both VD2 and VD3 subsequently undergo similar metabolic pathways and are physiologically equivalent in function (Shore 2013a).

Vitamin D is considered a pro‐hormone because it requires further metabolism in order to function. VD2 and VD3 undergo hydroxylation in the liver to form 25(OH)vitamin D (calcidiol) and is further hydroxylated in the renal tubules to form of 1,25(OH)₂vitamin D (calcitriol), which is the active form of vitamin D (Shore 2013a). However, 25(OH)vitamin D (calcidiol) is the most plentiful and stable vitamin D metabolite in the human body and thus used for measurement of vitamin D level in the body (Adams 2010).

Vitamin D supplements come in two forms, either plant‐based VD2 or animal‐based VD3 (Wagner 2008). VD3 is frequently preferred over VD2 as it has greater efficacy in raising circulating levels of 25(OH)vitamin D and is more sustained (Armas 2004; Oliveri 2015). VD3 is extensively used as part of milk formula or food fortification (Holick 1992).

For infants, supplements are available either in combination with other vitamins or alone (Wagner 2008). Sole vitamin D supplements are preferable over combination vitamin preparations to allow adequate vitamin D dosing without overdose of other vitamins (Wagner 2008). The recommended dose for vitamin D supplementation of infants is between 340 IU and 400 IU per day, starting from birth up till one‐year age (Health Canada 2012; NICE 2014; Wagner 2008). At these amounts the risk of vitamin D toxicity is low (IOM 2011). As vitamin D is found in breast milk, it is possible to supplement the breastfeeding mother with vitamin D, thus indirectly supplementing the infant (Haggerty 2010). However, doses of about 6400 IU/day are needed in the lactating mother to have adequate excretion into human milk (Haggerty 2010).

Vitamin D toxicity has been defined as hypercalcaemia, a 25(OH)vitamin D level exceeding 250 nmol/L associated with hypercalcuria and suppressed PTH (Munns 2016). Clinically, it may result in growth retardation and symptoms of hypercalcaemia (IOM 2011). Toxicity only occurs with dietary intake, not sun exposure (Holick 1981).

How the intervention might work

Human bone is first formed as cartilage and, later, bone tissue is laid down to replace the cartilage. This process is called bone mineralization or ossification. As the infant grows, bones undergo longitudinal and radial growth and a process of modelling‐remodeling takes place (Clarke 2008). Vitamin D plays an important role in these processes. The primary action of vitamin D is to increase the absorption of calcium from the gastrointestinal tract (Elder 2014). It also mobilises calcium from bone with the help of PTH by way of increasing osteoclastic bone resorption (Shore 2013a). In addition, vitamin D also increases the kidney's distal tubules reabsorption of calcium together with the action of PTH (IOM 2011). The net action of vitamin D is to increase serum calcium levels.

Good bone mineralization during early childhood and adolescent is the foundation of stronger bones later in life preventing fractures and osteoporosis (Winzenberg 2013a). Aquisition of bone mineral content is greatest in the first year of life (Koo 2013). Therefore, it is hypothesised that prevention of vitamin D deficiency by supplementation of breastfed infants should lead to better bone health in future.

Why it is important to do this review

Vitamin D deficiency and nutritional rickets among breastfed infants are not uncommon. A review of the global incidence of nutritional rickets in the last 10 years found it is an important global health problem (Creo 2017). With increasing efforts to promote exclusive breastfeeding of infants from birth to six months old (WHO 2003), it is important the risk of vitamin D deficiency in these infants is addressed.

Vitamin D supplementation of term breast‐fed infants has been recommended by the American Academy of Pediatrics (AAP), Institute of Medicine, Canada Health and NICE Guidelines (Health Canada 2012; IOM 2011; NICE 2014; Wagner 2008). These guidelines state that breast‐fed infants should start supplements by one month of life. Adherence to these guidelines is influenced by the recommendations of individual physicians or other healthcare professionals (Crocker 2011; Taylor 2010; Umaretiya 2017). However, when surveyed, the most common reasons given for low adherence to guidelines by physicians or mothers included "breast milk has all the nutrients a baby needs" and "nutritional rickets is not an important disease" (Perrine 2010; Taylor 2010; Umaretiya 2017). Breastfeeding advocates have also expressed concerns that the suggestion that breast milk may be vitamin D‐deficient and thus require additional supplementation may imply that artificial feeding is superior to breastfeeding (Heinig 2003).

There are two Cochrane Reviews and a Cochrane protocol on vitamin D supplementation for children and pregnant women (De‐Regil 2016; Winzenberg 2010; Winzenberg 2013b). A review on interventions to prevent nutritional rickets in term‐born children reported few data specific to term breastfed infants (Lerch 2007). This review aims to focus on evidence from randomised controlled trials (RCTs), specifically for term breastfed infants for the role of vitamin D supplementation to prevent vitamin D deficiency and improve bone health.

Objectives

To determine the effect of vitamin D supplementation given to:

• infants compared to placebo or no intervention on vitamin D deficiency, bone density and growth in healthy term breastfed infants;

• lactating mothers compared to placebo or no intervention on vitamin D deficiency, bone density and growth in healthy term breastfed infants;

• infants compared to vitamin D supplementation given to lactating mothers on vitamin D deficiency, bone density and growth in healthy term breastfed infants;

• infants compared periods of infant sun exposure on vitamin D deficiency, bone density and growth in healthy term breastfed infants.

For each of the above comparisons:

• to determine adverse effects from vitamin D supplementation compared to placebo, no intervention or other interventions in healthy term breastfed infants.

Methods

Criteria for considering studies for this review

Types of studies

RCTs or quasi‐RCTs. We will exclude cross over studies. We will consider unpublished studies or studies reported only as abstracts as eligible for inclusion, if the methods and data can be confirmed by the review author team.

Types of participants

Term healthy infants who are breastfeeding (exclusive or partial), from birth to six months of age.

Types of interventions

Vitamin D supplement, either as a single preparation or combined with other vitamins, given directly to the infant or lactating mother. We will not apply a minimum duration of supplementation. We plan to perform the following separate comparisons:

• vitamin D given to infants versus placebo or no treatment;

• vitamin D given to lactating mothers versus placebo or no treatment;

• vitamin D given to infants versus vitamin D given to lactating mothers;

• vitamin D given to infants versus periods of infant sun exposure.

Types of outcome measures

Primary outcomes

• Low bone mineral density measured by dual x‐ray absorptiometry (DXA) or other validated technique (Pezzuti 2017);

• vitamin D deficiency based on serum 25‐hydroxy vitamin D levels (sufficiency > 50 nmol/L; insufficiency 30 to 50 nmol/L; deficiency < 30 nmol/L) (Munns 2016);

• nutritional rickets defined as clinical symptoms or signs; and/or radiological signs (including reduced mineralization and ossification of the physes and metaphyseal splaying); and/or biochemical changes (raised PTH and alkaline phosphatase, hypophosphataemia and hyperphosphaturia with or without hypocalcaemia) (Munns 2016);

• adverse effects including vitamin D toxicity (defined as hypercalcaemia and serum 25 OH D > 250 nmol/L, with hypercalciuria and suppressed PTH) (Munns 2016).

Secondary outcomes

• Lowest serum 25(OH)vitamin D level (nmol/L) up to six months of age;

• serum 25(OH)vitamin D level (nmol/L) at latest time reported during treatment to six months of age;

• fracture (radiologically confirmed);

• osteomalacia ‐ low bone mineral density reported on x‐ray;

• infant growth at latest time measured:

  • weight gain (g/kg per day);

  • linear/height growth (cm/week);

  • head circumference (cm/week);

• change of standardised growth at latest time measured:

  • change in weight z‐score;

  • change in length z‐score;

  • change in head circumference z‐score;

• size at latest time measured:

  • weight;

  • length/height;

  • head circumference.

Search methods for identification of studies

Electronic searches

We will use the standard search strategy for the Cochrane Neonatal Review Group. We will search the Cochrane Neonatal Review Group Specialised Register, the Cochrane Central Register of Controlled Trials (CENTRAL 2017, current issue) in the Cochrane Library; MEDLINE via PubMed (1946 to current); Embase (1947 to current); and CINAHL (1982 to current) using the following search strategy in Appendix 1. We will also search trial registries, including the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/en/) and ClinicalTrials.gov (clinicaltrials.gov/) for ongoing trials. We will not set date or language restrictions for searches.

Searching other resources

We will search the reference lists of any identified reviews and included trials for references to other trials. We will also search abstracts and conference proceedings of the Pediatric Academic Societies (PAS), the Perinatal Society of Australia and New Zealand (PSANZ), the European Society for Pediatric Endocrinology, the Pediatric Endocrine Society (PES), the Asia Pacific Pediatric Endocrine Society (APPES), the Japanese Society for Pediatric Endocrinology (JSPE), the Sociedad Latino‐Americana de Endo‐crinología Pediátrica (SLEP), the Australasian Pediatric Endocrine Group (APEG), the Indian Society for Pediatric and Adolescent Endocrinology (ISPAE), the African Society for Pediatric and Adolescent Endocrinology (ASPAE), the Chinese Society of Pediatric Endocrinology and Metabolism (CSPEM), the British Nutrition Society, and the European Society for Pediatric Gastroenterology Hepatology and Nutrition (ESPGHAN). We will contact experts in the field for any unpublished studies.

Data collection and analysis

Selection of studies

Two review authors will assess titles and abstracts of all citations retrieved from the literature search to determine eligibility. We will resolve any differences in opinion through consensus or by consulting a third review author as arbiter. We will retrieve the full‐text articles versions of potentially eligible articles or when inadequate information is provided in the abstract. We will list any full‐text articles excluded after full‐text assessment in the ‘Characteristics of excluded studies' tables. Included studies will be listed in the ‘Characteristics of included studies' tables. We will record the study selection process in a PRISMA flow diagram.

Data extraction and management

We will independently extract data from the included trials using specially designed data extraction forms. We will request additional unpublished information from the authors of original reports. We will enter and crosscheck data using Review Manager 5 (RevMan 5) software (RevMan 2014), and will compare extracted data for any differences. If noted, we will resolve differences through discussion and consensus.

Assessment of risk of bias in included studies

Two review authors will independently assess risk of bias (low, high or unclear) of all included trials using the Cochrane ‘Risk of bias' tool (Higgins 2011) for the following domains:

• selection bias;

• performance bias;

• attrition bias;

• reporting bias;

• any other bias.

We will resolve any disagreements through discussion or by consulting a third review author. See Appendix 2 for a detailed description of ‘Risk of bias' assessment for each domain.

Measures of treatment effect

We will analyses study results using RevMan 5 (RevMan 2014). We will report continuous outcomes using mean difference (MD) and dichotomous outcomes as risk ratios (RR) and risk difference (RD) with 95% confidence intervals (CI). For results that are statistically significant, we will use 1/RD to calculate the number needed to treat for an additional benefit outcome (NNTB) or the number needed to treat for an additional harmful outcome (NNTH).

Unit of analysis issues

The unit of analysis will be the participating infant in individually RCTs. We do not anticipate any cluster‐RCTs will meet the inclusion criteria. However, if included, we will use the following method.

Cluster‐randomised trials

We will make adjustments to the standard errors using the methods described in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011, Section 16.3.6) using an estimate of the intracluster correlation co‐efficient (ICC) derived from the trial (if possible), from a similar trial or from a study with a similar population. If we use ICC values from other sources, we will report this and conduct sensitivity analyses to investigate the effect of variation in the ICC. We will consider it reasonable to combine the results from both cluster‐RCTs and individually RCTs if there is little heterogeneity between the study designs and the interaction between the effect of the intervention and the choice of randomisation unit is considered to be unlikely.

Trials with more than two treatment groups

If we identify trials with more than two intervention groups, we will only include eligible groups. We will combine intervention groups if we consider doses comparable where appropriate. If the control group is shared by two or more study arms, we will divide the control group over the number of relevant subgroup categories to avoid double counting participants.

Dealing with missing data

We will obtain missing data from the trial authors when possible. Where we are unable to obtain missing data, we will examine the effect of excluding trials with substantial missing data (e.g. greater than 10% losses) in sensitivity analyses.

We will attempt to overcome potential bias from missing data (greater than 10% losses) using one or more of the following approaches:

• whenever possible, we will contact the original trial investigators to request missing data;

• we will perform sensitivity analyses to assess how sensitive the results are to reasonable changes in the assumptions that are made (e.g. the effect of excluding trials with substantial missing data (greater than 10% losses);

• we will address the potential impact of missing data (greater than 10% losses) upon the findings of the review in the ‘Discussion' section.

Assessment of heterogeneity

We will use RevMan 5 to assess the heterogeneity of treatment effects between trials (RevMan 2014). We will undertake this assessment using the following two formal statistical models:

• Chi² test, to assess whether observed variability in effect sizes between studies is greater than would be expected by chance. As this test has low power when few studies are included in the meta‐analysis, we will set the probability at the 10% level of significance;

• I² statistic, to ensure that pooling of data is valid. We will grade the degree of heterogeneity as follows: none (< 25%); low (25% to 49%); moderate (50% to 74%); or high (≥ 75%). When we find evidence of apparent or statistical heterogeneity, we will assess the source of heterogeneity by performing sensitivity and subgroup analyses, while looking for evidence of bias or methodological differences between trials.

Assessment of reporting biases

If we identify 10 or more studies that include a specific intervention (comparison) and report on the same outcome, we will assess reporting and publication biases by examining the degree of asymmetry of a funnel plot in RevMan 5 (RevMan 2014).

Data synthesis

If we identify two or more studies that are homogenous, we will perform a meta‐analysis using RevMan 5 (RevMan 2014). We will use a fixed‐effect model for analysis as recommended by the Cochrane Neonatal Group (neonatal.cochrane.org/resources‐review‐authors). For studies that are clinically distinct we will not combine the studies for meta‐analysis and instead will present a narrative description of the study results. The narrative description will include the general direction, size, consistency and strength of evidence of effect of each individual study but we will not attempt to compare the effects of each study or draw an overall conclusion.

Subgroup analysis and investigation of heterogeneity

If sufficient data are available, we will explore potential sources of clinical heterogeneity by analysing whether results differed for infants at:

• high risk of vitamin D deficiency due to: pigmentation, covering or avoidance of sun exposure, and/or latitude, versus lower risk;

• seasonality of supplementation (winter versus non‐winter);

• supplementation with plant‐based VD2 versus animal‐based VD3;

• dose of vitamin D (200 to 400 iU; 400 to 800 iU; > 800 iU per day);

• duration of vitamin D supplementation (< 1 month; 1 to 2 months; 2 to 4 months; 4 to 6 months); and

• timing of commencement of vitamin D supplementation (from birth; 1 to 2 months; 3 to 4 months; 5 to 6 months).

Sensitivity analysis

We will explore methodological heterogeneity if sufficient data are available by performing sensitivity analyses. Where possible, we will conduct sensitivity analyses to assess any change in the direction of effect caused by inclusion of studies of lower quality, based on assessment of: allocation concealment, adequate randomisation, blinding of treatment, greater than 10% loss to follow‐up, and intention‐to‐treat analyses.

‘Summary of findings' table

We will use the GRADE approach, as outlined in the GRADE Handbook (Schünemann 2013), to assess the quality of evidence for the following (clinically relevant) outcomes:

• serum 25(OH)vitamin D level;

• number of infants diagnosed with nutritional rickets;

• bone mineral density;

• adverse effect.

Two review authors will independently assess the quality of the evidence for each of the outcomes listed above. We will consider evidence from RCTs as high quality, but will downgrade the quality of the evidence by one level for serious (or two levels for very serious) limitations based upon the following: design (risk of bias), consistency across studies, directness of the evidence, precision of estimates and presence of publication bias. We will use the GRADEpro Guideline Development Tool to create a ‘Summary of findings’ table to report the quality of the evidence (GRADEpro GDT 2014).

The GRADE approach results in an assessment of the quality of a body of evidence to one of four grades:

• high: we are very confident that the true effect lies close to that of the estimate of the effect;

• moderate: 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: our confidence in the effect estimate is limited: the true effect may be substantially different from the estimate of the effect;

• very low: we have very little confidence in the effect estimate: the true effect is likely to be substantially different from the estimate of effect.

Appendices

Appendix 1. Search strategies

(Vitamin D OR ergocalciferols OR cholecalciferol) AND (breastfeed* OR breast feed* OR breastfed OR lactation) adapted for database combined with database‐specific terms:

Cochrane Central Register of Controlled Trials (CENTRAL) in the Cochrane Library

(infant OR newborn OR neonate OR neonatal OR preterm OR very low birth weight OR low birth weight OR VLBW OR LBW)

PubMed

((infant, newborn[MeSH] OR newborn OR neonate OR neonatal OR preterm OR low birth weight OR VLBW OR LBW or infan* or neonat*) AND (randomized controlled trial [pt] OR controlled clinical trial [pt] OR randomized [tiab] OR placebo [tiab] OR drug therapy [sh] OR randomly [tiab] OR trial [tiab] OR groups [tiab]) NOT (animals [mh] NOT humans [mh]))

Embase

(infant, newborn OR newborn OR neonate OR neonatal OR preterm OR very low birth weight OR low birth weight OR VLBW OR LBW OR Newborn OR infan* OR neonat*) AND (human NOT animal) AND (randomized controlled trial OR controlled clinical trial OR randomized OR placebo OR clinical trials as topic OR randomly OR trial or clinical trial)

CINAHL

(infant, newborn OR newborn OR neonate OR neonatal OR preterm OR low birth weight OR VLBW OR LBW or Newborn or infan* or neonat*) AND (randomized controlled trial OR controlled clinical trial OR randomized OR placebo OR clinical trials as topic OR randomly OR trial OR PT clinical trial)

Appendix 2. ‘Risk of bias' tool

We will evaluate the following issues and enter these into the ‘Risk of bias' table.

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

For each included study, we will categorise 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);

• unclear risk.

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

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

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

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

• unclear risk.

3. Blinding (checking for possible performance bias). Was knowledge of the allocated intervention adequately prevented during the study? At study entry? At the time of outcome assessment?

For each included study, we will categorise the methods used to blind study participants and personnel from knowledge of which intervention a participant received. We will assess blinding separately for different outcomes or classes of outcomes. We will categorise the methods as:

• low risk, high risk or unclear risk for participants;

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

• low risk, high risk or unclear risk for outcome assessors.

4. 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 will describe the completeness of data including attrition and exclusions from the analysis. We will note 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 will re‐include missing data in the analyses. We will categorise the methods as:

• low risk (< 20% missing data);

• high risk (≥ 20% missing data);

• unclear risk.

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

For each included study, we will describe how we investigated the possibility of selective outcome reporting bias and what we found. We will assess the methods as:

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

• high risk (where not all the study’s pre‐specified outcomes have been reported; one or more reported primary outcomes were not pre‐specified outcomes of interest 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);

• unclear risk.

6. Other sources of bias. Was the study apparently free of other problems that could put it at a high risk of bias?

For each included study, we will describe any important concerns we had about other possible sources of bias (for example, whether there was a potential source of bias related to the specific study design or whether the trial was stopped early due to some data‐dependent process). We will assess whether each study was free of other problems that could put it at risk of bias as:

• low risk;

• high risk;

• unclear risk.

If needed, we will explore the impact of the level of bias through undertaking sensitivity analyses.

Contributions of authors

MLT wrote the protocol with assistance from DO and SA.

Sources of support

Internal sources

• No sources of support supplied

External sources

• Australian Satellite of the Cochrane Neonatal Review Group, Australia.

  Supported by a bridging funding grant from Cochrane 2017‐8.

Declarations of interest

May Loong Tan has no known conflicts of interest. Steven A Abrams has no known conflicts of interest. David A Osborn has no known conflicts of interest.

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