Omega-3 Long-Chain Polyunsaturated Fatty Acids for Extremely Preterm Infants: A Systematic Review

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1 Omega-3 Long-Chain Polyunsaturated Fatty Acids for Extremely Preterm Infants: A Systematic Review abstract BACKGROUND AND OBJECTIVE: Omega-3 long chain polyunsaturated fatty acid (LCPUFA) exposure can be associated with reduced neonatal morbidities. We systematically review the evidence for the benefits of omega-3 LCPUFAs for reducing neonatal morbidities in extremely preterm infants. METHODS: Data sources were PubMed, Embase, Center for Reviews and Dissemination, and the Cochrane Register of Controlled Trials. Original studies were selected that included infants born at,29 weeks gestation, those published until May 2013, and those that evaluated the relationship between omega-3 LCPUFA supplementation and major adverse neonatal outcomes. Data were extracted on study design and outcome. Effect estimates were pooled. RESULTS: Of the 1876 studies identified, 18 randomized controlled trials (RCTs) and 6 observational studies met the defined criteria. No RCT specifically targeted a population of extremely preterm infants. Based on RCTs, omega-3 LCPUFA was not associated with a decreased risk of bronchopulmonary dysplasia in infants overall (pooled risk ratio [RR] 0.97, 95% confidence interval [CI] ], 12 studies, n = 2809 infants); however, when considering RCTs that include only infants born at #32 weeks gestation, a trend toward a reduction in the risk of bronchopulmonary dysplasia (pooled RR 0.88, 95% CI , 7 studies, n = 1156 infants) and a reduction in the risk of necrotizing enterocolitis (pooled RR 0.50, 95% CI , 5 studies, n = 900 infants) was observed with LCPUFA. CONCLUSIONS: Large-scale interventional studies are required to determine the clinical benefits of omega-3 LCPUFA, specifically in extremely preterm infants, during the neonatal period. Pediatrics 2014;134: AUTHORS: Peiyin Zhang, MS-2, a Pascal M. Lavoie, MD, PhD, b Thierry Lacaze-Masmonteil, MD, PhD, c Marc Rhainds, MD, PhD, d and Isabelle Marc, MD, PhD a a Department of Pediatrics, Centre Mère-Enfant, and d Health Technology Assessment Unit, CHU de Quebec, Laval University, Quebec, Canada; b Department of Pediatrics, Children s and Women s Health Centre, University of British Columbia, Vancouver, Canada; and c Department of Pediatrics, Children s Hospital of Eastern Ontario, Ottawa University, Ottawa, Canada KEY WORDS omega-3, fatty acids, systematic review, preterm, infants, neonatal ABBREVIATIONS ALA a-linolenic acid BPD bronchopulmonary dysplasia CI confidence interval DHA docosahexaenoic acid DINO Docosahexaenoic acid for the Improvement in Neurodevelopmental Outcome GA gestational age IVH intraventricular hemorrhage LCPUFA long-chain polyunsaturated fatty acid NEC necrotizing enterocolitis PMA postmenstrual age RCT randomized controlled trial ROP retinopathy of prematurity RR risk ratio Dr Marc conceptualized and designed the protocol, screened the titles and abstracts, and retrieved articles for inclusion and quality, performed the analyses, drafted the initial manuscript, and approved the final manuscript as submitted; Ms Zhang critically reviewed the protocol, screened the titles and abstracts and retrieved articles for inclusion and quality, drafted the tables and figures, and reviewed the final manuscript as submitted; Dr Lavoie screened the articles for quality, revised the manuscript, participated in the redaction of the final manuscript, and reviewed the final manuscript as submitted; Dr Rhainds designed and conducted the systematic database search, reviewed the manuscript, and approved the final manuscript as submitted; and Dr Lacaze-Masmonteil reviewed the manuscript and approved the final manuscript as submitted. doi: /peds Accepted for publication Apr 22, 2014 Address correspondence to Isabelle Marc, MD, PhD, Département de Pédiatrie, Université Laval, Centre Mère Enfant du CHUQ, 2705 bd Laurier, Québec G1V 4G2, Canada. isabelle. marc@crchul.ulaval.ca (Continued on last page) 120 ZHANG et al

2 REVIEW ARTICLE Despitemajorimprovementsinperinatal and neonatal care over the past few decades, extremely preterm infants continue to face high morbidity and mortality.innorthamerica,1of8ofthese infants do not survive and 15% of survivors suffer major long-term neurodevelopmental disabilities. In these vulnerable infants, the neonatal period is a critical time that can set the stage for lifelong morbidities, such as respiratory diseases and cognitive, motor, and behavioral impairments. During this time, preventive measures can have great and lasting impact. 1 4 Bronchopulmonary dysplasia (BPD) is one of the most common and most serious complications occurring in extremely preterm infants. 5 BPD occurs in 45% of infants born at,29 weeks of gestation and clinically presents with persistent need for supplemental oxygen or respiratory support beyond the neonatal period. 1 The disease is characterized by impaired pulmonary vascular and alveolar development. A dysregulation of inflammation has been proposed to play a central role in the etiology of lung injury characteristic of this disease. Inflammatory responses are well known to be modulated by longchain polyunsaturated fatty acids (LCPUFAs), including omega-3 fatty acids, such as docosahexaenoic acid (DHA).Italsohasbeenproventhat extremely preterm infants are deficient in LCPUFAs, particularly in DHA. 6,7 Therefore, it is possible that supplementing the diet of extremely preterm infants with DHA could reduce inflammation that predisposes to BPD and other major neonatal diseases Establishing a causal link between exposure to omega-3 lipids and neonatal outcomes in high-risk preterm infants has important clinical implications in neonatal care. Recent studies have investigated associations between reduced nutritional omega-3 during the perinatal or neonatal period and adverse neonatal outcomes, with the hope that supplementation may improve those outcomes However, whether supplementing in omega-3 improves neonatal health in extremely preterm infants is unknown. The objective of this study was to conduct a systematic review of observational studies and randomized controlled trials and, where possible, a meta-analysis, to examine whether omega-3 LCPUFA exposure or its supplementation (compared with no exposure or a placebo group) reduces the incidence of BPD and other major adverse neonatal outcomes in extremely preterm infants. METHODS The protocol of this review has been registered in PROSPERO (#CRD with the participation of a multidisciplinary team, including neonatologists, epidemiologists, and research methodologists. We used the Preferred Reporting Items for Systematic Reviews and Meta-Analysis statement for reporting the review. 14 Search Strategy and Study Selection: We searched PubMed, Embase up to May 2013, and specialized sites (eg, Center for Reviews and Dissemination, Cochrane Register of Controlled Trials) by using MeSH terms and text words, adapting the search strategy if needed for each database. The search strategy on PubMed was as follows: #1 Infant, Premature [Mesh] OR Infant, Very Low Birth Weight [Mesh] OR ((preterm OR premature) AND (infant* OR newborn* OR neonat*)) #2: Fish Oils [Mesh] OR fish oil OR n-3 OR omega-3 OR omega-3 OR PUFA OR polyunsaturated fatty acid* OR marine oil OR algal oil OR Docosahexaenoic Acids [Mesh] OR docosahexaenoic acid* OR DHA #3: inclusion of strategy #1 AND #2. In Embase, the strategy search used the terms #1: prematurity /exp OR very low birth weight /exp OR (preterm OR premature AND (infant* OR newborn* OR neonat*)) #2: fish oil /exp OR polyunsaturated fatty acid /exp OR omega-3 fatty acid / exp OR docosahexaenoic acid /exp OR fish oil OR pufa OR polyunsaturated fatty acid OR n-39 OR omega-39 OR marine oil OR algal oil #3: #1 AND #2. We developed search strategies with terms related to the exposure/ intervention (omega-3) and the target population (preterm) and did not use any filter for randomized controlled trials (RCTs) or cohort studies to maximize the sensitivity of the search strategies. There was no language restriction. Finally, we looked at reference lists and citations of relevant articles (previous reviews and included studies) to identify any additional eligible studies. All citations were then combined and duplicates were excluded. Studies were excluded if they were conference abstracts, reviews, or case reports. The bibliographies of review articles were manually searched for potentially relevant citations that were not detected by the electronic search. Eligibility Criteria We considered all observational studies (case-control, cohort, cross-sectional) or randomized controlled trials assessing the effects of omega-3 LCPUFA on mortality or adverse neonatal outcomes in infant populations including exclusively or in part extremely preterm infants. Extremely preterm infants were defined as infants with a gestational age (GA) of,29 weeks of gestation. For the purpose of this study, studies were included that reported any omega-3 LCPUFA exposure in the infant (eg, PEDIATRICS Volume 134, Number 1, July

3 measured on blood concentration) or any omega-3 LCPUFA supplementation directly to the infant in the neonatal period or through the mother during pregnancy or lactation. The intervention or exposure was compared with other standard interventions, placebo, or any other control levels of omega-3 LCPUFA exposure. The primary outcome of interest in this study was BPD-free survival at 36 weeks postmenstrual age (PMA), with BPD defined as the need for oxygen and/or ventilation at 36 weeks PMA. Nevertheless, studies reporting BPD with any definition were considered in the review. Studies also were included even if they did not include data on our primary outcome as long as they included other relevant secondary neonatal outcomes listed in the next sentence. Secondary outcomes (as defined in the original publications) comprised death; duration of ventilation or oxygen support; length of hospitalization; or occurrence of intraventricular hemorrhage (IVH), periventricular leukomalacia, necrotizing enterocolitis (NEC), infections, retinopathy of prematurity (ROP), or hemodynamically significant patent ductus arteriosus. Mortality was defined as death from any cause. Because these specific outcomes of interest might not have been reported in the title or the abstract, we did not narrow our initial search to include them. Instead, we did filter out databases to include any of these outcomes when reviewing articles at the stage of thorough reading. Data Extraction Process Two reviewers (P.Z., I.M.) independently screened the titles for potential relevance. Only titles that were mutually agreed as being not relevant were excluded. The 2 reviewers then screened abstractsand,finally, full-text articles for inclusion/exclusion criteria. To reach consensus, reviewers resolved disagreement between themselves by further discussion. Once the articles were selected for the purpose of full-text review, the 2 investigators independently reviewed the articles and extracted the following information by using a specific form: year and country of the study; study population; study design; type of the interventions or exposure (source of omega-3, dose, mode of administration, timing, association with another LCPUFA); comparison groups, such as a placebo or another nutrient or another dosage or mode of administration; number of exposed/unexposed or cases/controls; definitions and criteria used for BPD and each of the neonatal outcomes; and dichotomous variables or continuous data. The risk of bias was assessed by examining the sample selected, recruitment method, completeness of follow-up, andblinding according to the guidelines for assessing non-rcts proposed by the Ohio Department of Mental Health and adapted in French by us (available at evaluation/uetmis). RCTs were assessed by the checklist proposed by COMPUS Adapted SIGN 50 ( methodology/checklists.html), To indicate the summary judgment of quality of each study, we used the summary ratings of unsatisfactory, satisfactory, or very satisfactory. Definitions of Neonatal Outcomes Neonatal outcomes were defined by the presence or absence of the diagnosis basedonstandardcriteriaorthecriteria as reported in the studies. When.1 category was available for a given neonatal outcome (eg, none, mild, moderate, severe) and when these categories were mutually exclusive, we combined numbers to provide an effect estimate for any definition of the neonatal outcome. Otherwise, we extracted data matchingwiththemoreseverecategory of the outcome. If there were.2 comparison groups (eg, different sources of LCPUFA versus a placebo), we combined the data of the experimental groups if they involved similar intervention; furthermore, these data were compared with the data of the reference group (eg, placebo). Finally, in cases in which outcome data were available for subgroups of preterm infants as part of the larger studied group, only the data from the subgroup that was the closest to our population of interest (ie, preterm infants of,29 weeks gestation) were included in the combined analyses for this outcome. For example, results for the outcome BPD in the subgroup of infants with a birth weight,1250 g available from Manley et al were included in the meta-analysis instead of the results for the preterm infants of,33 weeks as reported in Makrides et al Synthesis of Results We classified studies according to their design and also by neonatal outcomes. For dichotomous data, we presented results as summary risk ratio (RR) with 95% confidence intervals (CIs). Continuous data were not pooled, because they were measured only in a minority of studies. We used fixed-effect metaanalysis for combining data where it was reasonable to assume that studies were estimating the same underlying treatment effect (ie, where the trials population interventions and methods were judged sufficiently similar). If there was a clinical heterogeneity or if we detected substantial statistical heterogeneity, we used random-effects meta-analysis to produce an overall summary. Heterogeneity of the effect estimates was assessed by calculating the I 2 statistic, 12 and significant heterogeneity was defined per protocol as I 2 $50%. Prespecified subgroup analyses were performed where possible for the infant GA (studies including preterm infants of,37 weeks versus studies targeting preterm infants of #32 weeks of gestation), the DHA dose, 122 ZHANG et al

4 REVIEW ARTICLE timing and duration of exposure, the definition or severity of the outcomes, the mode of administration, and for the comparison between omega-3 provided alone or in association with another LCPUFA (ie, omega-6 LCPUFA). RESULTS Overview of Included Studies Using our search strategy, we identified and screened 1876 citations. Fig 1 describes the process of selection for the final 24 studies included in the qualitative analysis. Characteristics of the 24 studies are summarized in Table 1. Eighteen of these studies were RCTs ,15 29 Among nonrandomized studies, 4 were interventional studies with a control group and the remaining 2 studies 8,34 did not test an intervention because they examined the association of BPD with LCPUFA concentrations in blood or in tracheal aspirates. Fifteen RCTs presented dichotomous outcomes. Finally, 13 studies (7 RCTs) assessed outcomes in a population of preterm infants of #32 weeks of gestation. No RCT or study subanalyses were performed exclusively in extremely preterm infants (,29 weeks of gestation). FIGURE 1 Flowchart of studies through the review process. Characteristics of Exposure and Outcomes In randomized and nonrandomized interventional studies, there was heterogeneity between studies regarding the sources of omega-3 (fish, alga, egg), their mode of administration (intravenous or enteral), the vehicle used for supplementation (formula or breast milk), the timing of exposure (started within 10 days after birth or later, but usually provided at least up to 36 weeks of PMA), the DHA percentage in formula or breast milk (usually,0.40% of total fatty acids [with high dose defined as DHA concentration of 1% of total fatty acids in the experimental group product]), or the cointerventions (exposure to omega-3 LCPUFA alone, generally DHA, or in association with other nutrients, usually in combination with arachidonic acid) (Table 1). Except for 1 study, 30 the DHA intakes (dosage in mg/kg/d) based on the daily amount of feeds in the first weeks of life and the percentage of DHA in formula or breast milk were not reported. No RCTs reported survival free of BPD as an outcome. None of the studies reported BPD as a primary outcome. Most of the studies (n = 15, including 12 RCTs) defined BPD according to an oxygen requirement up to 28 days of postnatal age, or at 36 weeks PMA, but none of these studies used a physiologic definition 35 (Table 2). Of all the observational studies or RCTs reporting neonatal outcomes as dichotomous variables, the association between omega-3 LCPUFA and any definition of BPD was examined in 18 studies (combined in 12 RCTs), with 1 RCT 23 reporting a composite outcome including BPD. Similarly, NEC was reported in 14 studies (combined in 11 RCTs), IVH in 12 studies (combined in 7 RCTs), infections in 15 studies (combined in 10 RCTs), ROP in 10 studies (combined in 6 RCTs), and death in 8 studies (combined in 6 RCTs). Several studies examined.1 neonatal outcome at a time (Table 1). Only a few studies reported other relevant neonatal outcomes, such as number of days of ventilation, or the use or duration of supplementation oxygen, 13,20,22,27,28 patent ductus arteriosus, 12,20,23,30 32 cholestasis, 31,32 or number of days of hospital admission. 8,11,13,18,31,32 Effect Estimates for Dichotomous Neonatal Outcomes For each neonatal outcome of interest, combined effect estimates were calculated where possible and are outlined in Figs 2, 3, 4, and 5. Heterogeneity of effect estimates was reported. PEDIATRICS Volume 134, Number 1, July

5 TABLE 1 Characteristics of the Studies Including Extremely Premature Infants and Reporting the Effects of Omega 3 LCPUFA (Alone or in Association With ARA) on Adverse Neonatal Outcomes Studies Participants Control Group Treatment Group Timing Dichotomous Outcomes (Any Definition) or Equivalent Continuous Outcomes Death BPD NEC IVH ROP Infections RCTs Carlson 1992, United States preterms Standard formula Formula enriched with marine oil GA,32 wk BW,1400 g DHA 0.20% Carlson 1996, United States preterms Standard formula EPA 0.30% ARA Not reported Formula enriched with marine oil GA,30 wk DHA 0.20% EPA 0.06% ARA Not reported Carlson 1998, United States preterms Standard formula GA #32 wk Formula enriched with egg phospholipids BW g DHA 0.13% EPA 0.00% ARA 0.41% Clandinin 2005, Canada preterms Standard formula Formula enriched with either fish and fungal oil DHA 0.33% EPA 0.1% ARA 0.67% GA, 35 wk or Algal oil DHA 0.33% EPA 0.00% ARA 0.67% Fewtrell 2002, United Kingdom preterms Standard formula GA, 37 wk Formula enriched with egg phospholipids BW,1750 g DHA 0.17% EPA 0.04% ARA 0.31% Fewtrell 2004, United Kingdom preterms Standard formula GA,35 wk Formula enriched with borage and fish oil: BW #2000 g DHA 0.50% EPA 0.10% ARA 0.04% Foreman-van Drongelen 1996, 43 preterms Standard formula Netherlands 22 GA,37 wk Formula enriched with algal and fungal oils BW,1800 g DHA 0.30% EPA 0.00% ARA 0.61% Groh-Wargo 2005, United States preterms Standard formula Formula enriched with either fish and fungal oil GA,33 wk BW g DHA 0.27% From enteral feeding.110 kcal/k g/j (day 9 to day 40) to discharge #day 5 to discharge day 5 to discharge $day 14 of enteral intake (mean day30) to hospital discharge Day 10 to discharge neonatal unit 1st days of life (mean day 14) to 9 mo after term,day 10 to 3 mo Within 72 h of first enteral feeding, by day28to1yca 124 ZHANG et al

6 REVIEW ARTICLE TABLE 1 Continued Studies Participants Control Group Treatment Group Timing Dichotomous Outcomes (Any Definition) or Equivalent Continuous Outcomes Death BPD NEC IVH ROP Infections Harper 2010, United States pregnant women with a history of previous preterm birth Placebo mineral oil Henriksen 2008, Norway preterms Daily infant supplementation GA,31 wk with 0.5 ml control oil BW,1500 g (no DHA, no ARA) /100 ml human milk (mother or donor) Innis 2002, Canada preterms Standard formula,32 wk EPA 0.10% ARA 0.43% or fish oil and egg triglycerides DHA 0.24% EPA 0.00% ARA 0.41% Mother daily omega-3 fish oil supplementation (1200 mg EPA and 800 mg DHA) Daily infant supplementation with 0.5 ml study oil (DHA 32 mg, ARA 31 mg) /100 ml of human milk (mother or donor) Formula enriched with either alga oil (DHA alone) BW g DHA 0.34% EPA not reported ARA 0.00% or alga oil (DHA+ARA) DHA 0.34% EPA not reported ARA 0.60% Koletzko 1995, Germany preterms Formula without LCPUFA Formula enriched with GA,37 wk egg-tg and evening primrose oil Makrides 2010, Australia pregnant women,21 wk of gestation During pregnancy within wk to 36 wk of gestation or delivery From enteral feeding.100 ml /kg/d to day 63 post delivery From enteral feeding 50 kcal/kg/d for at least 28 d BW #1850 g DHA 0.30% From full enteral feeding Daily supplementation with vegetable oil EPA 0.03% ARA 0.50% Daily supplementation with omega-3 fish oil (100 mg EPA and 800 mg DHA) ($130 ml milk/kg/d) for at least 21 d During pregnancy from 21 wk of gestation until birth Manley 2011 substudy 657 preterms Breast milk Breast milk Within 5 d of any enteral of Makrides 2009, Australia 11 GA,33 wk Subgroup of 294 preterms,1200 g DHA 0.26% DHA 1.07% % EPA 0.08% EPA 0.16% % ARA 0.45% ARA 0.43% % following mother daily supplementation with six 500-mg soy oil capsules following mother daily supplementation with six 500-mg DHA-rich tuna oil (43%) capsules feeding to expected date of delivery PEDIATRICS Volume 134, Number 1, July

7 TABLE 1 Continued Studies Participants Control Group Treatment Group Timing Dichotomous Outcomes (Any Definition) or Equivalent Continuous Outcomes Death BPD NEC IVH ROP Infections O Connor 2001, 470 preterms Standard formula United States 26 GA,30 wk Formula enriched with either fish and fungal oil BW,1800 g DHA 0.26% EPA 0.08% ARA 0.42% or egg-tg and fish oil DHA 0.24% EPA 0.00% ARA 0.41% Skouroliakou 2010, Greece preterms Intravenous lipid emulsion GA,32 wk BW,1500 g containing conventional soybean lipid (Intralipid) Tomsits 2010, Hungary preterms Intravenous lipid emulsion GA #34 wk containing conventional BW g soybean lipid (Intralipid 20%) Vanderhoof 1999, 288 preterms Standard formula United States 29 Intravenous lipid emulsion SMOFlipid, containing lipids from soya bean oil (30%), olive oil (25%) and fish oil (15%) and medium-chain TG (30%) Intravenous lipid emulsion SMOFlipid, containing lipids from soya bean oil (30%), olive oil (25%) and fish oil (15%) and medium-chain TG (30%) Formula enriched with fish oil BW g DHA 0.35% EPA not reported ARA 0.50% Observational studies and interventional clinical trials Within 72 h of the first enteral feeding to term From day 1 2 postbirth to at least day 14 From day 3 7 postbirth to day 14 Approximately up to 3 4 wk before full enteral feeding until 48 wk postconceptional age Marc 2011, Canada preterms Breast milk Breast milk From day 3 7 post GA #29 wk DHA 0.10% % DHA 1.20% % birth to 36 wk PMA EPA 0.00% % EPA 0.00% % ARA 0.30% % AA 0.40% % Following supplementation of mother with DHA-rich algal oil (DHA 1.2 g/d) Martin 2011, United States 8 88 preterms None Fatty acids profiles First day of birth to 1 mo postnatal GA,30 wk Pawlik 2011, Poland preterms Intravenous lipid emulsion GA,32 wk containing soybean and olive oil emulsion (20% Clinoleic) Intravenous lipid emulsion containing 50% of soybean and olive oil emulsion (20% Clinoleic) Day 1 after birth to relay with enteral nutrition 126 ZHANG et al

8 REVIEW ARTICLE TABLE 1 Continued Studies Participants Control Group Treatment Group Timing Dichotomous Outcomes (Any Definition) or Equivalent Continuous Outcomes Death BPD NEC IVH ROP Infections and 50% of fish-oil emulsion (10% Omegaven) Day 1 after birth to relay with enteral nutrition Intravenous lipid with emulsion 50% fish oil emulsion (Omegaven 10%) 50% soybean and olive oil emulsion (Clinoleic 20%) Pawlik 2011, Poland preterms Intravenous lipid emulsion GA,32 wk soybean and olive oil BW,1250 g emulsion (Clinoleic 20%) Day 1 after birth and in the following 4 d Rudiger 2000, Germany preterms None Tracheal and pharyngeal GA,32 wk amount of PUFA First or second day after birth for at least 7 d Parenteral lipid emulsions of medium-chain TG and omega-3 PUFA (SMOFlipid,) Skouroliakou 2012, Greece preterms Parenteral lipid emulsions GA wk of soybean- lipid BW,2500 g (Intralipid) Subgroups: 129 very low BW BW,1500 g 153 low BW BW g reported outcomes; ARA, arachidonic acid; BW, birth weight; CA, corrected age; EPA, eicosapentaenoic acid; PUFA, polyunsaturated fatty acid; TG, Triglycerids. Although most of the nonrandomized interventional studies were in favor of the intervention, they were not pooled because of the clinical and statistical heterogeneity between the studies with regard to design, DHA exposure, and methodology (Table 1). Among RCTs reporting the association between omega-3 LCPUFA and BPD (n = 12), the between-study variance accounted for 1% of the total variance (Fig 2A). The overall effect of omega-3 total fatty acids was not significant (pooled RR 0.97, 95% CI ). None of the 12 RCTs reported an increased risk of BPD in the experimental group. Four-point estimates yielded RRs,1, 3 of which had CIs that extended above 1. As part of a large study including preterm infants of,33 weeks gestation, 15 a subgroup analysis in the low birth weight preterm infants (birth weight,1200 g, n = 294) found that a high dose of DHA was protective for the development of BPD (adjusted RR 0.75, 95% CI ). 11 In this study, DHA was delivered early after birth to the preterm infants through breast milk from their mothers, who had been supplemented with DHA during their lactation period. According to these data, a nearly significant lower risk for BPD was found in subgroup analyses of RCTs targeting only the preterm infants born at #32 weeks of gestation (n =7 RCTs; Fig 2A) or reporting an intervention delivering omega-3 LCPUFA alone (n = 5 RCTs; Fig 2B). In RCTs assessing the effect of LCPUFA on death during the neonatal period(rr 0.76, 95% CI ), the betweenstudy variance for effect estimates was 44% of the total variance (Fig 3). With the exception of 2 studies with RRs.1, all other studies had RRs of,1 but with several studies reporting large 95% CI. The RR of NEC (Fig 4A), after combining the 5 RCTs reporting the effects of omega-3 LCPUFA in preterm infants PEDIATRICS Volume 134, Number 1, July

9 TABLE 2 The Reporting of BPD Studies Definition Carlson No definition for BPD Carlson Supplemented oxygen for.28 d and/or pulmonary changes on radiologic examination Carlson Supplemental oxygen on day 28 of age with radiographic changes Clandinin Supplemental oxygen at 36 wk PMA with severe or chronic radiologic changes on chest x-ray Fewtrell Oxygen requirement.30% for.28 d Fewtrell Respiratory assistance (ventilation, CPAP, or supplemental oxygen) at 36 wk Foreman-Van Drongelen No definition for BPD Groh-Wargo Composite outcomes (Need for supplemental oxygen beyond 1 mo postnatal or 36 wk corrected age (CA) OR patent ductus arteriosus OR IVH) Harper Requirement for supplemental oxygen at 36 wk CA for neonates born before 34 wk Henriksen Need for respiratory support (no mention of timing); days with oxygen Manley Oxygen required at 36 wk PMA O Connor Supplemental oxygen beyond 1-mo postnatal age or at 36 wk PMA Skouroliakou Days of ventilation (no dichotomous variable was reported) Tomsits Days of supplemental oxygen (no dichotomous variable was reported) Vanderhoof No definition for BPD Marc I Supplemental oxygen required at 36 wk PMA Martin Supplemental oxygen required at 36 wk PMA Pawlik Oxygen requirement and/ or respiratory support at 36 wk CA Pawlik Oxygen requirement and/or respiratory support at 36 wk CA Rudiger Need for mechanical ventilation or oxygen therapy at day 28 of age with radiologic signs of BPD Skouroliakou Need for supplementary oxygen at day 28 of age and classified as mild, moderate, and severe according to the need for supplemental oxygen at 36 wk PMA or at 56 d postnatally CA, corrected age; CPAP, continuous positive airway pressure. Quality Analysis Heterogeneity in study quality, design, and reporting made quality evaluation difficult, especially for somercts. The inclusion/exclusion criteria often excluded more at-risk populations. Randomization process was poorly reported in 5 RCTs. Depending on the report of randomization, percentage of follow-up loss for the main outcome, and blinding, the study quality was judged as unsatisfactory, satisfactory, or very satisfactory for 5, 7, and 6, respectively, of the 18 RCTs. Definition of the various neonatal outcomes and timing of measures according to the beginning of the intervention varied substantiallyacross studies. Analyses were often not reported to beperformed in intentionto-treat. The number of dropouts and the reasons for subjects lost to followup were not clearly reported. The 6 observational studies were classified as satisfactory. born at #32 weeks of gestation, was in favor of the intervention, although this relative risk was not statistically significant (RR 0.50, 95% CI ; I 2 = 0%). Nevertheless, the effect of administering omega-3 to these preterm infants was significantly different and more beneficial where the intervention focused on this population rather than on the older neonate population. There was no clear trend in risk of ROP (n = 6 RCTs, RR 1.10, 95% CI ; I 2 = 0%), infections (n = 10 RCTs, RR 1.10, 95% CI ; I 2 = 0%), and IVH of any grade (n = 7 RCTs, RR 0.96, 95% CI ; I 2 = 0%) between the preterm infants receiving a placebo or omega-3 LCPUFA-supplemented nutrition overall, or among subgroups according to GA analyses (Fig 4A and Fig 5). When taking into consideration the severity of IVH (grade 3 4; n =5 studies; RR 1.37, 95% CI ; I 2 = 0%) or the severity of the ROP (grade 3 4 or need for treatment; n = 3 studies; RR 0.86, 95% CI ; I 2 = 0%), results did not change significantly. Reporting of Continuous and Other Dichotomous Outcomes Five studies individually reported no differences in the numbers of days of hospital admission (Table 1). A few studies reported either continuous or dichotomous outcomes relating to the neonatal outcomes of interest. The data available were for (1) BPD severity (1 observational study), (2) days of ventilation or with oxygen, (3) occurrence of periventricular leukomalacia (3 studies), and (4) type of infections (systemic, confirmed). The study results did not reach statistical significance. Pooling was not performed because of the small numbers of studies for these outcomes. DISCUSSION The main finding of this study is the potential protective effect of omega-3 LCPUFA supplementation on BPD and NEC when examining RCTs exclusively targeting preterm infants born at #32 weeks of gestation. This finding is of major clinical relevance mainly because infants born at the extreme lower end of prematurity are at particularly high risk for a nutritional deficit in omega-3 lipids, potentially predisposing to adverse neonatal outcomes. These results also are in accordance with observational studies, indicating an association between DHA levels and adverse neonatal outcomes in very preterm infants. 8,30,33 Preterm infants are nutritionally deficient in DHA. Daily nutritional DHA requirements of extremely preterm infants are estimated at the range of 40 to 60 mg/kg. 36 An essential nutrient, the a-linolenic acid (ALA) is the major n-3 fatty acid. In the body, ALA is metabolized 128 ZHANG et al

10 REVIEW ARTICLE FIGURE 2 Meta-analysis comparing the effects of LCPUFA supplementation on BPD. A, According to all RCTsand a subgroupof RCTs including preterm infants of #32 weeks of gestation. B, According to a subgroup of RCTs evaluating an intervention that exclusively supplement in omega-3 LCPUFA. to either eicosapentaenoic acid or DHA. As an important constituent of cell membranes, DHA is highly concentrated in several tissues, including the brain (23% of total body stores in a term infant), muscles, and stored adipose tissue. 37 In extremely preterm infants, DHA stores represent,10% of the total body stores in lipids when compared with a full-term neonate. 38,39 Preterm infants can synthesize small amounts of DHA from the omega-3 dietary precursor PEDIATRICS Volume 134, Number 1, July

11 FIGURE 3 Meta-analysis comparing the effects of LCPUFA supplementation on death. ALA, via the desaturation-elongation pathway. 40 However, because of limited fat stores, considerable oxidation, and increased requirements to support rapidly growing tissues, availability of DHA is insufficient during the neonatal period. 38,41 44 In extreme preterm infants, high systemic inflammatory biomarkers are measured in the earliest hours after birth and for extended periods of time during the neonatal period, particularly in infants exposed to mechanical ventilation and supplemental O Preterm infants also have a limited ability to counteract inflammatory and oxidative stress during the neonatal period. 50 Sustained oxidative stress and inflammation are key factors contributing to the development of BPD as well as other neonatal complications. Dietary LCPUFA, particularly DHA and eicosapentaenoic acid are important modulators of inflammation. Considering the anti-inflammatory effect of DHA, and of its downstream metabolite eicosanoids, 51,52 authors have argued that DHA deficits in preterm infants may have considerable, underestimated adverse effects during the neonatal period. 9,10,53,54 In light of the strong association between elevated inflammation and both shortand long-term morbidities in extreme preterm infants, 48,55 we postulate that a DHA supplementation or a decrease in the omega-6/omega-3 reduces the occurrence of these major adverse neonatal outcomes, including BPD. Of note, when specifically examining studies in which omega-6 lipids were coadministered with DHA, we detected no additional benefit on BPD compared with studies using DHA alone (Fig 2B), indicating a selective role of DHA or the ratio omega-6/omega-3. Biological Plausibility Interventions that modulate DHA levels have been shown to reduce pulmonary inflammation in animal models, although data are lacking in humans. Indeed, intravenous omega-3 lipids improve the redox potential of glutathione and reduce lung inflammation in the neonatal guinea pig model. 52 Maternal DHA supplementation reduces inflammation in the neonatal mouse hyperoxia model of BPD. 56 In extremely preterm infants, reduced serum DHA during the first postnatal month was associated with increased need for supplemental oxygen at 36 weeks PMA (odds ratio 2.5, 95% CI ). 8 The ratio of n6-linoleic acid to n3-dha in these infants was highly predictive of lung disease, confirming the importance of balanced ratios of LCPUFA and potential selective role of DHA in protecting against BPD. 8 A balanced LCPUFA ratio was also protective against late-onset infections, providing an additional protective mechanism against adverse neonatal outcomes. 8 Altogether, our data strongly suggest that low DHA contributes to a dysregulation of inflammation, predisposing to major neonatal morbidities, and that restoring an adequate ratio in LCPUFA may prevent the adverse effects of an excessive inflammatory and oxidative stress exposure repeatedly associated with lung injury in extreme preterm infants. This nutritional approach has gained interest recently, since publication of the Docosahexaenoic acid for the Improvement in Neurodevelopmental Outcome (DINO) trial 15 and of a secondary analysis in low birth weight infants suggesting an effect of omega-3 LCPUFA to reduce the need for oxygen at 36 weeks PMA in this population. 11 The DINO trial substantially influences but does not dominate the meta-analytic findings. Therefore, since the last review in 2008, 57 this review is valuable for placing the DINO trial results in a broader context, and to highlight the need for a definitive trial testing the impact of LCPUFA on neonatal morbidities. Limitations Although it is the aim of this review to estimate treatment effects with more precision, the major limitation with our systematic review and meta-analysis of RCTs is the low occurrence of reported events on the outcomes of interest. This is mainly due to the large GA range of preterm infants included in the studies, and especially to the inclusion of a large 130 ZHANG et al

12 REVIEW ARTICLE FIGURE 4 Meta-analysis comparingtheeffects oflcpufasupplementationon(a) NEC and(b) ROPaccordingtoallRCTsandasubgroupof RCTsincludingpreterminfantsof #32 weeks of gestation. proportion of older preterm infants who are relatively lower risk of neonatal complications. Furthermore, in none of the studies were neonatal outcomes stated as the primary outcomes, with older studies mainly reporting neonatal complications as safety outcomes only. Finally, the studies varied widely in terms of methodology quality, sample size, DHA doses, source and administration protocol, and in the populations studied. Because the daily amount of feeds administered to infants was not reported in most studies, it is impossible to extrapolate a dose-effect PEDIATRICS Volume 134, Number 1, July

13 FIGURE 5 Meta-analysiscomparingtheeffectsofLCPUFAsupplementationon(A) neonatalinfectionaccordingtoallrctsandasubgroupofrctsincludingpreterminfants of #32 weeks of gestation, and (B) IVH. of DHA (eg, in mg/kg body weight) required to achieve a therapeutic benefit. IMPLICATIONS AND CONCLUSIONS According to results of preclinical experimental animal studies, as well as observational studies in humans, DHA supplementation is safe and likely to be beneficial in extremely preterm infants. This systematic review highlights potential benefits of DHA in the prevention of BPD or NEC when specifically targeting the younger, most immature preterm infants immature preterm infants. No detrimental effect was reported with omega-3 LCPUFA in this age group. In current clinical practice, enteral supplementation remains the best strategy for optimizing fat absorption and bioavailability in extremely preterm infants. Future studies focused on an early supplementation of DHA are required to establish more definitively the clinical benefits relative to therapeutic effects of different methods of supplementation of DHA for extremely premature infants, with the prospect of reducing neonatal mortality and morbidities in this age group. Overall, our results support theneed fordefinitiverctstoexamine the effectiveness of omega-3 LCPUFA versus placebo in reducing the occurrence of BPD and of other adverse neonatal complications in extremely preterm infants. Of note, a large RCT is ongoing testing the effect of a direct oral supplementation of extremely preterminfantswithfishoil(n3ro:n-3 LCPUFA for improvement in Respiratory Outcomes; trial # ). 132 ZHANG et al

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Ann Neurol. 2009;66(2): Rogers LK, Valentine CJ, Pennell M, et al. Maternal docosahexaenoic acid supplementation decreases lung inflammation in hyperoxia-exposed newborn mice. J Nutr. 2011;141(2): Smithers LG, Gibson RA, McPhee A, Makrides M. Effect of long-chain polyunsaturated fatty acid supplementation of preterm infants on disease risk and neurodevelopment: a systematic review of randomized controlled trials. Am J Clin Nutr. 2008;87(4): (Continued from first page) PEDIATRICS (ISSN Numbers: Print, ; Online, ). Copyright 2014 by the American Academy of Pediatrics FINANCIAL DISCLOSURE: The authors have indicated they have no financial relationships relevant to this article to disclose. FUNDING: Ms Zhang was supported by a summer bursary from the Fondation des Étoiles. Dr Marc was supported by a clinician research award from the Fonds de Recherche Québec Santé, Canada. Dr Lavoie is funded by a Michael Smith Foundation Career Investigator Award and a Child and Family Research Institute Clinician-Scientist Award. POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose. 134 ZHANG et al

16 Omega-3 Long-Chain Polyunsaturated Fatty Acids for Extremely Preterm Infants: A Systematic Review Peiyin Zhang, Pascal M. Lavoie, Thierry Lacaze-Masmonteil, Marc Rhainds and Isabelle Marc Pediatrics 2014;134;120 DOI: /peds originally published online June 9, 2014; Updated Information & Services References Subspecialty Collections Permissions & Licensing Reprints including high resolution figures, can be found at: This article cites 55 articles, 16 of which you can access for free at: This article, along with others on similar topics, appears in the following collection(s): Fetus/Newborn Infant sub Neonatology Nutrition Information about reproducing this article in parts (figures, tables) or in its entirety can be found online at: Information about ordering reprints can be found online:

17 Omega-3 Long-Chain Polyunsaturated Fatty Acids for Extremely Preterm Infants: A Systematic Review Peiyin Zhang, Pascal M. Lavoie, Thierry Lacaze-Masmonteil, Marc Rhainds and Isabelle Marc Pediatrics 2014;134;120 DOI: /peds originally published online June 9, 2014; The online version of this article, along with updated information and services, is located on the World Wide Web at: Pediatrics is the official journal of the American Academy of Pediatrics. A monthly publication, it has been published continuously since Pediatrics is owned, published, and trademarked by the American Academy of Pediatrics, 141 Northwest Point Boulevard, Elk Grove Village, Illinois, Copyright 2014 by the American Academy of Pediatrics. All rights reserved. Print ISSN: