Current Information and Asian Perspectives on Long-Chain Polyunsaturated Fatty Acids in Pregnancy, Lactation, and Infancy: Systematic Review and Practice Recommendations from an Early Nutrition Academy Workshop

Essential polyunsaturated fatty acids (PUFA) of the omega-3 (n-3) and omega-6 (n-6) series are of critical importance during early life, and they are known to play an essential role in growth and development. Intakes in pregnancy and early life are thought to affect the quality of growth and neurological and immune function in later life.

The long-chain PUFA (LC-PUFA) eicosapentaenoic acid (EPA; n-3) and docosahexaenic acid (DHA; n-3), and arachidonic acid (AA, n-6), can be formed from the precursors α-linolenic acid (n-3) and linoleic acid (n-6), respectively. However, the rates of conversion of the precursor PUFA are low and are estimated to range from only 0.1 to 10% [1,2,3]. Moreover, the conversion rates depend on common polymorphisms in the fatty acid desaturase (FADS) gene cluster [4,5]. Pregnant and breast-feeding women with the less common genotypes have a very low ability to form EPA and DHA from ALA and AA from LA, respectively [6,7,8]. Conversion rates of precursor PUFA in infants, and particularly in premature infants, have been reported as insufficient to allow for biochemical and functional normality [9,10].

Information on the roles of LC-PUFA during pregnancy, lactation, and infancy was reviewed and intake recommendations were provided in 2007 and 2008 [11,12]. Since then, many new studies and meta-analyses have provided new information on the role of LC-PUFA in maternal and infant nutrition and on their impact on health and development. Therefore, the Early Nutrition Academy (ENA) decided to have the recent information systematically reviewed, and recommendations for practice were derived by experts in PUFA, perinatal nutrition, and health, taking into account particularly the dietary intake and conditions of Asian populations. The ENA is a nonprofit society created by and representing the partners of international research projects funded by the European Commission and the Australian National Health and Medical Research Council that perform research on food and dietetic products in pregnancy and early childhood, in part also in collaboration with commercial partners (e.g. www.project-earlynutrition.eu and www.nutrimenthe.eu). The ENA aims to promote knowledge of human nutrition in early life, to stimulate quality research in this and related areas of science, nutrition, and health, and to disseminate such knowledge.

We performed a systematic search of the literature databases of PubMed using the following search strings: (unsaturated fatty acids OR omega 3 fatty acids OR fish oils OR long-chain omega-3 fatty acids OR docosahexaenoic acids OR omega-6 fatty acids OR arachidonic acid) AND (pregnant women OR pregnancy OR lactating women OR breastfeeding OR lactation OR infants OR infancy).

The search was limited to studies in humans, published during the last 5 years (September 2008 to September 2013), in the English language and reporting functional outcomes. All systematic reviews, meta-analyses, and randomized controlled trials (RCT), were included. In addition, we also evaluated relevant observational studies. Studies that included interventions with essential fatty acids combined with micronutrients in lipid-based supplements or in fat-based flour, meant to be added to homemade meals, were not included in this review despite the fact that a number of these studies reported benefits for children's growth and development [13,14]. In addition, further relevant publications identified by the group of participating experts were considered. An expert workshop was hosted by the ENA in Singapore prior to the 8th World Congress on Developmental Origins of Health and Disease (DOHaD) in November 2013. Participants were invited based on their expertise in the areas of PUFA, perinatal nutrition, and health, while a focus was placed on experts from Asian countries. At the workshop, the evidence was reviewed and discussed in detail. All conclusions and recommendations presented in this manuscript were agreed upon by consensus.

A total of 20 systematic reviews and/or meta-analyses and 78 original reports of RCT that met the inclusion criteria were found in the systematic literature review. These 78 original reports related to 44 individual studies (13 in pregnant women, 6 in pregnant and lactating women, 2 in lactating women, 3 in preterm infants, 14 in term infants, and 6 in older infants) and are summarized in tables 1, 2, 3, 4, 5.

We evaluated a total of 21 individual studies published since 2008 identified by the systematic search. These studies investigated the impact of n-3 LC-PUFA interventions on pregnancy and longer-term outcomes. Eleven of these studies supplemented with LC-PUFA during pregnancy, 6 studies supplemented both during pregnancy and during lactation, 2 studies supplemented mothers during pregnancy and subsequently the infants after delivery, and 2 studies supplemented during lactation only. In addition, 12 systematic reviews or meta-analyses were found. Two of the studies in pregnant women published since 2008 were performed in women from low- or middle-income countries, i.e. Mexico [15] and China [16].

A systematic review of 15 RCT found that women receiving an additional n-3 LC-PUFA supply in different amounts during pregnancy delivered infants with a slightly higher birth weight (42.2 g; 95% CI 14.8-69.7), with a 26% lower risk of early preterm delivery (<34 weeks) (RR 0.74; 95% CI 0.58-0.94). In addition, supplemented women showed a trend toward a decreased risk of preterm delivery (RR 0.91; 95% CI 0.83-1.02) [17]. Three previous meta-analyses also evaluated such effects. Szajewska et al. [18] reported a significant increase in the mean pregnancy duration of 1.6 days, along with a slight increase in infant size at birth with a nonsignificant trend toward fewer preterm births before 37 weeks of gestation (RR 0.67; 95% CI 0.41-1.10, n.s.). A Cochrane review by Makrides et al. [19] included 6 randomized trials on marine oil supplementation in different amounts during pregnancy, involving 2,783 women. Women allocated to a marine oil supplement had a small but significant increase in the length of gestation of 2.6 days, along with a slightly higher infant birth weight (47 g; 95% CI 1-93). The number if early preterm births before 34 completed weeks of gestation was significantly reduced by marine oil (RR 0.69, 95% CI 0.49-0.99; 3 trials, 2,440 women) [19]. In high-risk pregnancies, n-3 LC-PUFA supplementation showed an even more marked and significant reduction in the number of early preterm births before 34 weeks of gestation (RR 0.39; 95% CI 0.18-0.84) [20].

Three studies published after 2008 confirmed the positive effects of n-3 LC-PUFA interventions during pregnancy on birth-size, with larger birth weights and head circumferences in infants of supplemented mothers [15,21,22]. In one of these studies in Mexico, these effects were only seen in firstborn infants [15]. In a small study of 48 pregnant women who had consumed cereal-based food fortified with DHA, a lower ponderal index but no effect on birth weight or length were reported [23]. Four studies have been published since 2008 reporting on gestational age at birth and the risk of early preterm delivery. Makrides et al. [24] performed a double-blind RCT in 2,399 women with singleton pregnancies supplemented from mid-gestation to birth with DHA-rich fish oil capsules providing 800 mg DHA/day or matched vegetable oil capsules without DHA. The adjusted RR for early preterm birth before 34 weeks of gestation was significantly reduced to 0.49 (95% CI 0.25-0.94; p = 0.03), while the mean gestational age was prolonged by 1 day (p = 0.05) [24]. Carlson et al. [22] performed a double-blind RCT supplementing women during the second half of pregnancy with 600 mg DHA daily or placebo. Supplementation led to a longer mean duration of gestation by 2.9 days (p = 0.041), along with a higher birth weight (plus 172 g; p = 0.004), length (plus 0.7 cm; p = 0.022), and head circumference (plus 0.5 cm; p = 0.012). Early preterm births before 34 weeks of gestation were markedly reduced from 4.8% in controls to 0.6% in the intervention groups (p = 0.025), and the length of hospital stay in infants born preterm was shorter (8.9 vs. 40.8 days; p = 0.026) [22]. No safety issues occurred. In contrast, 2 other studies that provided lesser amounts of n-3 LC-PUFA did not observe a beneficial effect of supplementation on premature birth [15,25]. These studies that did not observe a benefit of supplementation were performed in Mexican women and in women with major depressive disorders. Only one study reported on pregnancy complications as an outcome. In Mexico, no impact was found on gestational diabetes and preeclampsia after maternal supplementation with DHA in pregnancy [26].

A cross-sectional study in rural, poor Southern Indian women found a significant positive correlation between EPA/DHA intake during pregnancy and birth weight [27]. Women whose fish intake was in the lowest tertile in the third trimester of pregnancy had a significantly increased adjusted odds ratio for the risk of low birth weight compared to those in the highest tertile, but there was no difference in gestational duration.

In conclusion, the results of 4 meta-analyses and 2 recent large RCT consistently showed a protective effect of n-3 LC-PUFA supplementation during pregnancy with respect to a reduction in the incidence of early preterm births (<34 weeks gestation). The impressive effect sizes of the reductions were 26% in the most recent meta-analysis and 51 and 87.5%, respectively, in 2 more recent large RCT using higher n-3 LC-PUFA dosages (600 or 800 mg DHA/day). Two other studies found no significant effects. No dose-response studies with direct comparisons of the effects of different dosages on early preterm birth have been performed. However, the greater effect sizes observed in the recent studies using 600-800 mg DHA/day compared to the earlier studies providing lower dosages are suggestive of a possible greater protective effect at higher intake levels, whereas no adverse effects were reported with the higher dosages. n-3 LC-PUFA supplementation in pregnancy also led to a small increase in infant size at birth, whereas there was no evidence of relevant untoward effects. Given that early preterm birth before 34 weeks of gestation markedly increases infant short- and long-term morbidity as well as mortality, it is advisable that pregnant women aim to achieve a regular supply of preformed n-3 LC-PUFA from foods such as oily fish and seafood or from supplements. A regular supply prior to pregnancy may also be beneficial since it contributes to the accumulation of body stores of n-3 LC-PUFA that can be utilized and thus contribute to securing protective blood and tissue levels of n-3 LC-PUFA during pregnancy (and during lactation; see below).

In several observational cohort studies, beneficial effects of n-3 or fish intake during pregnancy and/or lactation on the developmental outcomes and cognition of the offspring up to 14 years of age have been reported, even after adjusting for potential confounding factors [28,29,30].

However, the findings or outcomes from RCT are conflicting. A meta-analysis of 11 RCT involving 5,277 participants found no significant differences in standardized psychometric test scores for cognitive, language, or motor development in the offspring of mothers who had been supplemented with LC-PUFA during pregnancy [31]. There was a suggestion of higher cognitive development scores at later ages (2-5 years) in children of supplemented mothers derived from 2 trials. No results on visual outcomes could be calculated because of the variety of visual assessments used in the different studies.

Five studies have been published since 2008 evaluating cognitive outcomes and visual acuity after n-3 LC-PUFA supplementation during pregnancy; one of these studies extended the supplementation to the newborns until they reached 6 months of age [32]. Three of these studies did not observe beneficial effects of supplementation on the overall cognitive test scores at 18 months [24] or 6.5 years of age [33], or on visual development at 3-6 or 4 months of age [34,35]. A study in 67 pregnant women given a daily supplement of 600 mg DHA/day observed higher fetal heart rate variability measured at 24, 32, and 36 weeks of gestation and higher scores on a newborn neonatal behavioral assessment scale [36]. A study in 48 pregnant women in the USA who consumed cereal-based DHA-fortified food during pregnancy observed fewer sleep arousals in the infants of supplemented women, which the authors identified as an early marker of improved neurodevelopment [37].

Observational studies found a link between LC-PUFA status markers during pregnancy and at birth, reflecting a maternal supply before and during pregnancy, and later neurodevelopmental outcomes. Steer et al. [8] explored the link between maternal red blood cell contents of LC-PUFA in pregnancy and children's IQ at about 8 years in 2,839 mother-child pairs from the Avon Longitudinal Study of Parents and Children in the UK. Low maternal AA levels in pregnancy were linked to lower performance IQ, i.e. -2.0 points (95% CI -3.5 to -0.6; p = 0.007; increase in R² = 0.27%), and low levels of DHA (22:6n-3) with a reduction of full scale IQ of -1.5 points (95% CI -2.9 to -0.1; p = 0.031; R² = 0.15%). Kohlboeck et al. [38] reported a longitudinal cohort study with analysis of venous cord blood glycerophospholipid fatty acids at birth and behavior at age 10 years in 416 children in Munich, Germany. A 1% increase in DHA in cord blood serum decreased total behavioral difficulties by (exp)β_adj = 0.93 (SE 0.02; p = 0.0001) and hyperactivity or inattentiveness by (exp)β_adj = 0.94 (SE 0.03; p = 0.04). Higher LC-PUFA concentrations in cord blood serum were associated with fewer emotional symptoms [(exp)β_adj = 0.95, SE 0.03; p = 0.01], and similarly higher AA concentrations were associated with fewer emotional symptoms [(exp)β_adj = 0.94, SE 0.03; p = 0.03]. The results of these observational studies together suggest that the maternal AA and DHA status is relevant for fetal neural development and the long-term development of cognitive and emotional outcomes.

Two studies evaluated the impact of n-3 LC-PUFA supplementation during pregnancy and extended during the first 3 months postpartum on children's later cognitive development at 18 months [39] or at 7 years of age [40]. No beneficial effects of supplementation were found on mental development index (MDI) scores at 18 months or on cognitive scores on the Kaufman Assessment Battery for Children (KABC) test at 7 years.

LC-PUFA supplementation in breast-feeding women was reviewed in a Cochrane analysis [41]. A pooled analysis of outcomes in 5 clustered areas of neurodevelopment, i.e. language development, intelligence/problem-solving ability, psychomotor development, motor development, and child attention, reported no significant overall effects in the rather heterogeneous studies, except for improved sustained attention at 5 years observed in a study supplementing breast-feeding women for 4 months with 200 mg DHA/day [42].

Since 2008, two studies have been published evaluating the impact of supplementation during breast-feeding. Jensen et al. [42] followed children of mothers who had been supplemented with 200 mg DHA/day during the first 4 months of lactation and at the children's age of 5 years observed higher scores on the sustained-attention subscale in the children of supplemented mothers. Previously reported findings from the same study [43] demonstrated a better performance of children of DHA-supplemented mothers on tests of psychomotor development at 30 months of age, which might be explained by improved sustained attention as documented at the older age. Another trial in breast-feeding Danish women [44] evaluated the impact of supplementation during the first 4 months of lactation on cognitive test scores at 7 years of age. That study found a faster speed of information processing in children of previously supplemented mothers, and lower scores for inhibitory control/working memory in children with a higher DHA status at 4 months.

Further evidence has emerged from analysis of the interaction of breast-feeding and genotype effects. Steer et al. [45] explored the interaction of postnatal breast-feeding and variation in the genotypes for FADS enzymes with regard to IQ scores assessed at about 8 years of age in 5,934 children born in the early 1990s in the UK. Breast-feeding was associated with higher IQ scores than bottle-feeding, which was not LC-PUFA supplemented or enriched at the time of the study. In children with a FADS genotype linked to a low endogenous LC-PUFA synthesis, breast-feeding supplying LC-PUFA provided an added benefit of more than 4 IQ points at school age compared to infants with a genotype supporting a more active LC-PUFA formation. Similarly, Morales et al. [46] found that Spanish children who had been previously bottle-fed formula without added LC-PUFA had an 8- to 9-point disadvantage in cognitive scores assessed at 14 months or at 4 years if there were homozygous for FADS genotypes linked to a low endogenous LC-PUFA synthesis compared to a genotype leading to more active LC-PUFA formation. Assuming that FADS genotypes are distributed at random in the population and are not related to the decision to breast-feed (the concept of ‘mendelian randomization'), these data support a causal relationship between LC-PUFA supply during lactation and status in infancy and later cognitive achievements.

We conclude that the totality of recent RCT which are heterogeneous in outcomes and methods of assessment does not provide conclusive evidence of the benefits of added LC-PUFA supplementation during pregnancy and/or lactation for early cognitive or visual outcomes in offspring. However, the results of some high-quality randomized trials and observational as well as gene-nutrient interaction studies point to biologically important beneficial effects of an enhanced maternal pre- and postnatal LC-PUFA status on later attention, information processing, and cognitive performance.

The LC-PUFA content of human breast milk is not only derived from the current dietary intake [47], but a large proportion also comes from maternal body stores of LC-PUFA that have been previously deposited [48]. Therefore, it appears desirable to aim at securing an adequate maternal pre- and postnatal LC-PUFA status also to ensure an adequate LC-PUFA supply to the breast-fed infant to support child development.

A systematic review that included 5 RCT on the effect of perinatal n-3 fatty acid supplementation on inflammatory markers and allergic diseases concluded that n-3 PUFA supplementation during pregnancy reduced the 12-month prevalence of positive egg skin prick tests and childhood asthma [49]. Since 2008, six studies have been published evaluating markers of immune response and/or allergic diseases in children of mothers supplemented with n-3 LC-PUFA or oily fish during pregnancy. All of these studies reported some positive findings on selected immune markers and/or the incidence of (allergic) diseases in the children of supplemented mothers. Supplementation with LC-PUFA or consumption of 2 portions of oily fish/week during pregnancy resulted in improvements in neonatal [50,51,52,53] and maternal [53,54] immune responses, with attenuation of allergic inflammation, perhaps influenced by the infant allergic status.

Infants of supplemented mothers also showed lower rates of atopic eczema at 1 year of age [55,56] and allergic asthma at 19 years of age [57]. A decreased occurrence of common colds and a shorter duration of common respiratory illnesses in the first 6 months of life were observed in infants of Mexican mothers supplemented with 400 mg algal DHA/day during pregnancy [58]. Four studies supplemented mothers during pregnancy and extended the supplementation during the first 3 or 4 months postpartum and reported positive effects on immune responses or allergic diseases. Furuhjelm et al. [59] reported a lower prevalence of food allergy and a cumulative incidence of immunoglobulin E (IgE)-associated diseases in the first 2 years of life after supplementing 145 at-risk pregnant women in Sweden with a provision of DHA and EPA during pregnancy and the first 3.5 months of breast-feeding [59]. Granot et al. [60] found changes in CD4 and CD8 cells compatible with attenuation of the proinflammatory response in the infants of 60 pregnant women supplemented with 400 mg DHA/day from early gestation until 4 months postpartum. A lower prevalence of atopic dermatitis (AD) was also observed in Finnish infants whose mothers had been supplemented with black currant seed oil during pregnancy until the cessation of breast-feeding, suggesting a possible beneficial role also for n-6 PUFA [61]. Follow-up of adolescents whose mothers had been randomized to supplementation with fish oil or olive oil capsules during pregnancy showed that fish oil provision in pregnancy markedly reduced the risk of asthma and particularly of allergic asthma at the age of 16 years [57].

Observational studies also reported a protective effect of fish intake in pregnancy and infancy on the allergic disease risk. Sausenthaler et al. [62] assessed 2,641 children and their mothers in a prospective birth cohort study in Germany (LISA). Higher maternal fish consumption (which provides n-3 LC-PUFA) during the last 4 weeks of pregnancy significantly reduced the risk of child eczema up to the age of 2 years (adjusted OR 0.75; 95% CI 0.57-0.98). Similarly, a higher fish consumption by pregnant women was linked to a lower allergic sensitization of their children in Italy [63], less doctor-diagnosed eczema in children in The Netherlands [64], and less eczema, allergic sensitization, and atopic wheezing in Mexican children [65].

Recently, Standl et al. [66] reported a marked breast-feeding-gene interaction effect on doctor-diagnosed asthma up to the age of 10 years. In children with an FADS genotype resulting in low LC-PUFA synthesis, longer breast-feeding, which would support an improved LC-PUFA status in infancy, reduced the asthma risk by 57-61%, whereas there was no significant effect of breast-feeding in children homozygous for the major genetic allele [66]. These studies lend further support to the conclusion that the LC-PUFA status in early life is very important for long-term protection against childhood allergies.

With respect to the infants' anti-infectious response, increased IgG titers to common childhood vaccines were found in infants after LC-PUFA supplementation during pregnancy [59]. Moreover, daily supplementation of 400 mg DHA from algal oil compared to placebo from 18-22 weeks of gestation to birth in a double-blind RCT in Mexican pregnant women showed in infants (n = 834) beneficial effects of maternal DHA supplementation on a reduced rate of respiratory tract infections at the age of 1 month (RR 0.76; 95% CI 0.58-1.00) and on the infant disease burden at the ages of 1, 3, and 6 months [58]. For example, at the age of 6 months, the duration of fever was reduced by 20%, nasal secretions by 13%, dyspnea by 54%, skin rashes by 23%, and other symptoms by 25%. These effects might be mediated by modulation of DNA methylation and Th1/Th2 balance [67].

In summary, current evidence suggests a beneficial effect of oily fish intake or supplementation with fish oils or with DHA-rich algal oil during pregnancy on immune responses involved in allergic inflammation, on the incidence of allergic conditions, and on anti-infectious protection and the infection disease risk in children.

One systematic review included 10 articles (cohort studies, RCT, and pilot trials) that evaluated the impact of maternal n-3 fatty acid supplementation on perinatal maternal depression. Inconsistent results were observed, with 6 studies finding no association, 2 showing mixed results, and 2 reporting a positive association between n-3 PUFA and a reduced incidence of maternal perinatal depression [68]. The authors concluded that the heterogeneity of the results could be explained by the heterogeneity of the studies, with different study durations and time periods and other differences in PUFA interventions.

Four studies evaluated maternal depression outcomes after supplementation with n-3 LC-PUFA during pregnancy and lactation. Three of those studies [24,69,70] did not observe differences in maternal depressive symptoms after supplementation. A small study in 36 pregnant women in China with diagnosed major depressive disorders found significantly lower depression scores after an 8-week daily supplementation during pregnancy with 3.4 g n-3 PUFA [16].

In summary, evidence for the role of maternal n-3 LC-PUFA supplementation in perinatal depression is inconclusive. More research is needed to confirm or refute the observation that mothers with more severe risks of depression may benefit from supplementation.

Maternal supplementation with 400 mg/day DHA from algal oil during pregnancy in Mexico increased infants' length at 18 months of age, but only in firstborn children [34]. No impact of cod liver oil supplementation during pregnancy and the first 3 months after delivery was found on children's height, weight, or BMI at 7 years of age [40]. Maternal AA and n-6 LC-PUFA concentrations during gestation and lactation were negatively related to the BMI and the ponderal index at 1 year of age [21], but no differences in abdominal fat mass or fat distribution were observed in infants of German mothers supplemented with 1,200 mg n-3 LC-PUFA from the 15th week gestation until 4 months postpartum [71].

Three systematic reviews evaluated the impact of maternal n-3 fatty acid supplementation on children's later body composition or body weight. The reviews included studies that supplemented mothers during pregnancy, lactation, or both. Inverse associations between maternal n-3 LC-PUFA intake or supplementation and the children's later body composition (adiposity, BMI, or body weight) were observed in some but not all studies [72,73,74]. A recent review including 6 controlled trials of pre- or postnatal n-3 LC-PUFA supplementation concluded that currently there is little evidence to support the hypothesis that LC-PUFA supplementation during pregnancy and lactation prevents childhood obesity [75].

Two controlled studies evaluated markers of early cardiovascular disease (CVD) at 7 and 19 years of age, respectively, in children whose mothers had been supplemented with fish oil or placebo during pregnancy [76,77,78,79]. No differences in adiposity, plasma lipids, plasma lipoproteins, blood pressure, heart rate, or heart rate variability were reported in Danish children aged 19 years whose mothers had been randomized to receiving daily fish oil, olive oil, or no oil during pregnancy. Adverse effects on blood pressure, energy intake, and physical activity at 7 years of age were observed in boys, but not in girls, after fish oil supplementation during the first 4 months of lactation [76]. In contrast, lower umbilical cord blood insulin concentrations were observed in infants of mothers who had been supplemented with cereal-based DHA-fortified food during pregnancy, but that study was small and suffered from methodological flaws [23].

Two studies evaluated markers of oxidative stress and did not report any differences between pregnant mothers supplemented with LC-PUFA or oily fish and nonsupplemented mothers [80,81].

In summary, the current evidence for a possible programming effect of maternal n-3 fatty acid supplementation on child growth, obesity risk, and early markers of CVD is limited and does not allow firm conclusions.

A total of 14 original studies that evaluated the impact of n-3 LC-PUFA supplementation during infancy in term infants were included. In addition, we evaluated 6 systematic reviews and meta-analyses that reported findings in term infants.

All meta-analyses in this area are limited by a large degree of heterogeneity between the included studies, in particular regarding interventions, dosages, selected outcomes, and methods of outcome assessment. Moreover, the available intervention trials did not adjust for genetic variation in PUFA metabolism (FADS genotypes). A meta-analysis of 15 randomized studies on the effects of LC-PUFA supplementation in term infants did not show significant beneficial effects of supplementation on either mental or psychomotor development [82]. This finding was repeated in another meta-analysis including 12 trials involving 1,802 infants and demonstrating no significant effect of LC-PUFA supplementation of formula on early infant visual development and cognition [83,84]. Outcomes regarding visual acuity were inconsistent: no overall benefits were reported in a Cochrane review, with 5 out of 9 studies not showing a beneficial effect [82]. In contrast, a meta-analysis involving 1,949 infants from 19 studies found a significant benefit for infants' visual acuity after LC-PUFA supplementation up to 12 months of age [83]. Studies that provided higher doses of DHA (at least 0.32% in formula) and AA (at least 0.65% in formula) for a longer duration (>1 year) and measured visual acuity with electrophysiological tests were more likely to show beneficial effects of supplementation in term infants [82].

Nine studies have been published since 2008 evaluating the impact of LC-PUFA supplementation in term infants on several aspects of cognitive development. Two of these studies [85,86] have already been included in the 3 meta-analyses [82,83,84].

Studies have evaluated mental development, visual acuity, or motor development during infancy or in young childhood and the results have been mixed: one study providing high-dose fish oil with n-3 LC-PUFA but without AA did not observe beneficial effects on Bayley Scale of Infant Development (BSID) scores [87], one observed a beneficial effect on the mental development index [85], and another study reported beneficial effects on visual acuity [86] when 3 different DHA dosage groups were combined. These data may indicate that effects at younger ages may be too subtle to detect with smaller sample sizes. Supplementation with LC-PUFA in term infants shortened the time to achieving a milestone of motor development (sitting without support) [88].

Four studies assessed cognitive development at school age when the children were 6 or 9 years of age. Two of these studies [89,90] reported beneficial effects on various aspects of cognition (attention, fine-grained tasks, speed of processing, and problem solving), suggesting that the effects of LC-PUFA supplementation in early life may be better detectable at a later age in more specific or fine-grained tasks. The other 2 studies, however, reported no beneficial effects on selected outcomes in LC-PUFA-supplemented children [91,92].

In summary, among the recently published studies, some studies supplementing formula for term infants with LC-PUFA found benefits with regard to visual, motor, and cognitive outcomes in early childhood, and even more pronounced benefits in later childhood. However, other studies found no significant beneficial effects. There was no evidence of untoward effects. There is a trend toward a greater likelihood of benefit with higher dosages (DHA ≥0.32% and AA ≥0.66%) and a longer duration of a higher postnatal LC-PUFA supplementation (up to 1 year of age). As discussed before, further evidence for the benefits of a postnatal LC-PUFA supply is derived from gene-nutrition interaction studies that report greater benefits of breast-feeding, which provides preformed LC-PUFA, in infants genetically determined to have a lower endogenous LC-PUFA synthesis [45,46].

A systematic review of 13 studies in term infants found no effects of LC-PUFA supplementation on growth [82]. Rosenfeld et al. [93] performed a meta-analysis based on individual patient data of 901 children from 4 RCT of formula milk with and without LC-PUFA and found no evidence that supplementation affects children's growth at 18 months of age. A previous meta-analysis including 14 trials with 1,846 infants showed no significant effect of LC-PUFA supplementation with infant formula on infant weight, length, or head circumference at any assessment age [94]. Of interest, that meta-analysis reported that supplementation of infant formulae with n-3 LC-PUFA but no AA showed a mean reduction in plasma AA of 25% relative to the control groups [94]. The potential biological relevance of these reduced AA levels is uncertain.

Since 2008, five studies with LC-PUFA supplementation in term infants have reported effects on growth and/or the evolution of the BMI during infancy [95,96,97,98] or at 9 years of age [99]. None of these studies observed differences in anthropometric measurements between supplemented and nonsupplemented infants, and there was no effect of fish-oil supplementation on growth hormone IGF-1 production in a study in Denmark [98].

In summary, the available studies published to date do not provide evidence that LC-PUFA supplied to term infants would change infant or later childhood growth or obesity risk.

Studies evaluating the effect of LC-PUFA as well as other nutrient interventions on AD in children were recently reviewed [100]. The authors concluded that γ-linolenic acid supplementation in healthy infants seems to reduce the severity of AD. Supplementation with prebiotics and black currant seed oil (γ-linolenic acid and n-3 fatty acids) was effective in reducing the development of AD.

We found 4 studies published since 2008 that evaluated the impact of LC-PUFA supplementation in term infants on immune markers and/or allergies. Three of these studies found beneficial effects of supplementation on immune markers [101,102] or allergic diseases [103]. D'Vaz et al. [102] randomized 420 term infants with a high risk of allergy to a daily supplement of fish oil (280 mg DHA, 100 mg EPA) or placebo from birth until 6 months of age. They found lower allergen-specific Th2 responses and elevated Th1 responses in the fish oil group, suggesting a potentially allergy-protective effect on immune function [102]. Improved immune parameter responses after LC-PUFA supplementation were also observed in formula-fed term infants at 16 weeks of age [101]. In contrast, no differences in responses to childhood vaccines were observed after n-3 LC-PUFA and AA supplementation in 12-month-old formula-fed term infants in Australia [97].

Term infants receiving different dosages of DHA (0.32-0.36% fatty acids) and AA (0.64-0.72%) from 1-9 days of life until 12 months of age had lower odds of developing lower respiratory tract infections, wheezing/asthma, or other allergic diseases than controls, regardless of the dosage of DHA received [103]. No differences in childhood allergic diseases were observed in infants with a high atopy risk at 6 months of age, despite the fact that immune parameters had been altered [104], suggesting that longer follow-up times may be required before effects on disease become apparent.

Protective effects of fish intake with complementary feeding in infancy on allergic conditions were found in 2 large prospective birth cohort studies in Scandinavia. In a study in 4,921 infants, introduction of fish prior to the infant age of 9 months reduced the eczema risk at 1 year (RR 0.76; 95% CI 0.62-0.94; p = 0.009) [105]. In 4,089 children followed from birth, provision of more than 2-3 portions of fish per month in infancy compared to less the 2 portions per month at the age of 4 years led to a significantly reduced risk of asthma (RR 0.68; 95% CI 0.51-0.92), eczema (RR 0.69; 95% CI 0.57-0.84), allergic rhinitis (RR 0.57; 95% CI 0.45-0.73), any allergic disease (RR 0.65; 95% CI 0.54-0.77), and allergic sensitization (RR 0.66; 95% CI 0.53-0.82) [106].

Further support for the conclusion that the LC-PUFA status in infancy impacts the allergic disease risk stems from the observation of marked protective effects of breast-feeding for at least 3 months, which reduces the risk of physician-diagnosed asthma at up to 10 years of age in subgroups defined by LC-PUFA metabolism. Breast-feeding with the provision of LC-PUFA reduced the asthma risk up to 10 years of age by about one half in children who had an FADS genotype determining a low LC-PUFA synthesis.

In summary, fish intake and an enhanced LC-PUFA status in infancy appear to have a protective effect on the development of allergic diseases in childhood.

de Jong et al. [91] measured the CVD risk at 9 years of age in term infants who had been supplemented with LC-PUFA formula (n = 146; 0.45% AA and 0.30% DHA) or standard formula (n = 169) during the first 2 months of life. No differences in blood pressure or heart rate were observed between the 2 intervention groups.

In a study in Thailand, 144 healthy term infants were supplemented with whey-based formula containing LC-PUFA and oligosaccharides or placebo formula from 30 days to 4 months of life [95]. Beneficial effects on gastrointestinal comfort (gastric transit time, intestinal transit time, hard stools, and stool microbiota) were reported.

Colombo et al. [107] randomized 133 infants born at term to receive for the first year of life formula either with no LC-PUFA or with DHA at levels of 0.32, 0.64, or 0.96% fatty acids combined with 0.64% AA. All LC-PUFA-enriched formula groups showed a reduced heart rate, without a recognizable dose-response relationship to levels of DHA intake [107]. These results confirm previous observations by Pivik et al. [108] who reported higher heart rates and lower values for heart rate variability measures in infants fed diets without DHA compared to DHA-containing formula. The effects appeared from 4 months onwards and were interpreted as increased parasympathetic tone in infants receiving DHA. In adults, a decreased heart rate is considered a positive health outcome.

Five studies published since 2008 have assessed the effect of LC-PUFA supplementation in older infants (aged >3 months) and young children. One of these studies was performed in a disadvantaged population of infants from a low-income country [109]. Overall, very few positive effects of LC-PUFA supplementation in older infants and young children have been reported since 2008. In Gambia, LC-PUFA supplementation in healthy infants from 3 to 9 months of age increased the mid-upper-arm circumference and skinfold thickness at 12 months of age [109], whereas no growth effect was observed in Danish infants supplemented with fish oil from 9 to 18 months of age [110]. No effects on morbidity or gut integrity were found in the Gambia study [109]. Fish oil supplementation modified the fecal microflora in a subgroup of infants from the Danish study who had been weaned prior to the onset of supplementation [111].

A study in Australian children randomized to active diet interventions to increase n-3 and decrease n-6 intakes from weaning until 5 years of age or controls and did not find changes in blood pressure or arterial structure at 8 years of age [112].

Observations in infants who had been supplemented with fish oil from 9 to 12 months of age showed a 6% longer mean RR interval in fish-oil-supplemented boys (p = 0.007). Irrespectively of gender, there was an association between RR interval changes and erythrocyte n-3 PUFA (p < 0.001), suggesting a beneficial effect of fish oil supplementation in older infants on early markers of CVD risk [113]. Fish oil supplementation also induced beneficial effects on free-play scores and looking behavior and decreased the systolic blood pressure [114].

We evaluated data from 3 trials in preterm infants that published results since 2008. Most studies in preterm infants evaluated the effects of DHA supplies with human milk or formula near a 0.2-0.3% fat content. This amount is considered insufficient to support the average daily whole-body DHA accretion in excess of 50 mg/kg of body weight that occurs in utero, which requires a DHA supply of about 1% human milk or formula fat [115]. In a large RCT, Makrides et al. [115] randomized 657 preterm infants to a conventional (0.3%) or higher (1%) DHA supply, along with about 0.6% AA, from day 2 to day 4 of life until a term-corrected age. The findings of that trial were reported in a number of different publications [116,117,118,119]. In addition, one meta-analysis specifically reported on the effects of LC-PUFA supplementation in preterm infants [120].

Four out of 7 studies included in the meta-analysis [120] did not show a beneficial effect of supplementation on cognitive outcomes evaluated at 12 or 18 months of age. The 3 trials that showed beneficial effects on cognitive development all used the newer version of the BSID, suggesting that other methods might not have been sensitive or precise enough to detect effects. No beneficial effects of LC-PUFA supplementation on visual acuity were seen in the 16 studies included in the meta-analysis that evaluated visual acuity [120].

Two of the 3 studies that reported outcomes on child neurodevelopment showed no beneficial effects of LC-PUFA supplementation in preterm infants on cognitive outcomes at an early age [18 months; school age (language)] or behavior at 3 or 5 years of age (general cognitive tests at 10 years of age) [115,121,122]. However, beneficial effects of supplementation were observed in certain subgroups. For instance, girls provided a high DHA intake (1%) along with AA had higher MDI scores at 18 months of age [115]. In the total population, the occurrence of significant mental delay (mental developmental index <70) at 18 months was markedly reduced by one half (p = 0.03), which is considered a major benefit for the developmental outcome.

Another study in children aged 10 years found higher language scores in children who previously as preterm infants had been randomized to a DHA-containing formula compared to controls who did not get DHA in early life [122]. In addition, children who received DHA formula and no breast milk had better IQ and memory scores at 10 years of age, whereas this effect was not apparent in those who received formula with breast milk (which provides DHA) [122]. Beneficial effects of supplementation on recognition and problem solving at 6 months of age were also observed in a group of very preterm infants (birth weight <1,500 g) who had been supplemented with DHA and AA in the first 9 weeks of life [123]. Better visual acuity at 4 months' corrected age was observed after DHA-supplemented formula in Australian preterm infants [124].

In summary, while there is considerable heterogeneity among the available studies, there are consistent indications that an LC-PUFA supply to preterm infants can have benefits for visual and cognitive outcomes, with evidence from one large high-quality trial indicating DHA dose dependency. Certain subgroups of preterm infants, e.g. with a lower birth weight, may achieve a greater benefit from a preformed LC-PUFA supply.

Preterm infants fed with high-DHA formula during early infancy in the Australia DINO trial were 0.7 cm longer at 18 months of age, and those born with a higher birth weight (≥1,250 g) also had an increased later weight [118]. Higher body weights at 10 years of age in girls, but not in boys, were also observed in children who had been born preterm and had received LC-PUFA-supplemented formula until 9 months of age [122]. However, these results were not confirmed by a meta-analysis, which did not find effects of providing LC-PUFA to preterm infants on weight, length, or head circumference at 12 months (4 studies) or 18 months (2 studies) in spite of an increased weight and length at 2 months postterm in supplemented infants [120].

In summary, there are conflicting data on the effects of LC-PUFA supply to preterm infants on postnatal growth, with some data indicating potential benefits for length growth in early childhood.

A reduced incidence of reported hay fever at 12 and 18 months of age and a reduction in chronic lung disease in boys and in infants born with a birth weight <1,250 g were observed in preterm infants randomized to a higher DHA supply (1%) along with AA as compared to a lower DHA supply (0.3%) in the previously cited study by Makrides and coworkers [117]. An earlier study in preterm infants demonstrated comparable lymphocyte populations, cytokine production, and antigen maturity between infants receiving LC-PUFA-supplemented formula and those receiving human milk, whereas infants receiving an unsupplemented formula differed in these immune parameters [101,125].

Follow-up of a subgroup of previously preterm born children at the age of 10 years did not show differences in growth or blood pressure, whereas a subgroup analysis among girls who had received formula with added DHA but no AA postnatally had a slightly higher blood pressure, but there were no significant differences after adjustment for current body weight [126].

In summary, the reported positive effects of early LC-PUFA supplementation in preterm infants on chronic lung disease and the immune response are promising and should prompt further investigation.

Previously, scientific experts and learned societies recommended that pregnant and lactating women should achieve an average daily intake of at least 200 mg DHA, which can be reached by eating 2 portions of fish per week if this includes oily fish [11,12,127,128]. Alternatively, the use of supplements has been recommended for women that do not achieve this level of fish consumption. There is a broad consensus that breast-feeding, which provides preformed LC-PUFA, is the optimal choice for infant feeding [129. ]It was also concluded that formulae for term infants should contain DHA at levels between 0.2 and 0.5 weight percent of total fat, with the minimum amount of AA at least equivalent to the content of DHA. The dietary LC-PUFA supply should continue after the first 6 months of life, but no quantitative recommendations were provided since there was not sufficient information at that time.

The European Food Safety Authority (EFSA) determined adequate intakes per day of 250 mg for EPA plus DHA for adults based on cardiovascular considerations and an added 100-200 mg preformed DHA during pregnancy and lactation to compensate for oxidative losses of maternal dietary DHA and accumulation of DHA in the body fat of the fetus/infant [130]. In the year 2010, the EFSA further recommended a DHA intake of approximately 20-50 mg/day for young infants aged 0-6 months and an intake of 100 mg DHA at 6-24 months to support optimal growth and development. More recently, in 2013, the EFSA considered the following nutrient intakes adequate for the majority of infants and young children: 100 mg DHA/day and 140 mg AA/day from birth to age <6 months, 100 mg DHA/day from 6 to <24 months, and 250 mg EPA + DHA/day after the age of 24 months [131]. Recently, the EFSA shared a draft opinion on the compositional requirements of infant and follow-on formulae and advised that both infant and follow-on formulae should contain 20-50 mg DHA/100 kcal (at an assumed mean fat content of 5.2 g/100 kcal, ∼0.38-0.96% of fat), while it was not considered necessary to set a minimum requirement for AA content [132]. However, this panel notes that no adequate clinical studies have evaluated the suitability and safety of feeding infant formula from birth with DHA contents of up to about 1% fat and no content of AA. Therefore, the EFSA proposal is not supported by this panel.

In 2010, the Food and Agriculture Organization of the United Nations (FAO) concluded that for pregnant and lactating females the minimum intake for optimal adult health and fetal and infant development is 0.3 g/day EPA + DHA, of which at least 0.2 g/day should be DHA [133]. For infants aged 0-6 months, the FAO recommended a DHA intake of 0.1-0.18% of energy intake and an AA intake of 0.2-0.3% of energy intake based on human-milk composition. For infants above the age of 6 months, a DHA supply of 10-12 mg/kg/day was recommended without specifying a requirement for AA [133].

Recent recommendations for the compositional requirements of follow-up formula suitable for feeding from the age of about 6 months onwards stipulate an optional DHA content of up to 1% fatty acids, while the evidence available was considered insufficient to set a minimal quantitative requirement for added AA in follow-up formula that provides DHA [134]. This was based on the consideration that the ability to maintain AA stores from endogenous synthesis increases during the second half of the first year of life [135], and that dietary preformed AA is usually provided by a variety of complementary foods during the second half of the first year of life.

For preterm infants, daily intakes of 12-30 mg DHA and 18-42 mg AA per kilogram of bodyweight or 11-27 mg DHA/100 kcal of energy intake and 16-39 mg AA/100 kcal were recommended by the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) in 2009 [136], though these recommendations were developed before the full information on the trial in preterm infants evaluating the suitability and safety of higher dosages [117,118,137] had become available. It was also recommended that the ratio of AA to DHA should be in the range of 1.0-2.0 to 1 (wt/wt), and the EPA (20:5n-3) supply should not exceed 30% of the DHA supply [136]. A recent international expert group with contributors from all 5 continents reviewed the nutritional needs of very-low-birth weight infants (birth weight <1,500 g). They concluded that very-low-birth weight infants should receive per kilogram and per day between 385 and 1,540 mg linoleic acid and >55 mg α-linolenic acid. Intakes of DHA ranging from 18 to 60 mg and intakes of AA ranging from 18 to 45 mg were considered reasonable, while EPA intakes should not exceed 20 mg; however, DHA intakes of 55-60 mg, along with AA intakes of 35-45 mg, were considered preferable (all per kg and per day) [138].

Many populations in Asia have traditionally regularly consumed fish and seafood, and hence they have had a relatively high intake of preformed EPA and DHA. However, in recent years the fish intake has tended to markedly decrease while the meat consumption has increased, with a resulting decreased intake of DHA and EPA and an increase in AA intake (fig. 1), particularly among younger women (fig. 2), as documented in the Japanese National Health and Nutrition Survey [139] and in the Korean National Health and Nutrition Examination Survey [140,141].Vegetarian and vegan diets are commonly followed not only in India but also in other Asian countries and have recently gained popularity particularly among young women of childbearing age [142,143,144,145]. Vegetarian diets provide very little, and vegan diets provide basically no, preformed LC-PUFA. DHA supplementation is advisable, and vegetarian sources of DHA from algal oils are available.

A recent review evaluated the essential fatty acid intakes and status of pregnant and lactating women, infants, and children in developing countries and reported on findings from Asian countries [146].

In pregnant women, the lowest DHA intakes were reported in India in the third trimester of pregnancy at only 11 mg [27], and in Bangladesh the DHA intake was only 30 mg/day [147].

The mean concentration of DHA in breast milk is quite variable in Asian countries where there is data available. Some countries have reported lower levels of DHA in breast milk, e.g. 0.23% in Nepal [148], and 0.30% in Bangladesh [147], whereas higher DHA levels than the approximately 0.3% typically found in breast milk in Western countries [149] were reported in the Philippines (0.74 ± 0.05%) and a coastal area of Southeastern China (0.61 ± 0.46%) [150] and about 1% was reported in Japan [151]. These data reflect that the DHA content in human milk directly responds to the maternal dietary DHA intake [47].

Very few studies have reported intake or status data of infants from Asian countries, and intakes vary significantly across the region and within countries. In Bangladesh, the total fat intakes were extremely low at 19.5% of energy in breast-fed children and only 12.7% of energy in non-breast-fed children at 24-35 months of age [147], whereas in children aged 1-3 years in rural areas from the Yunnan Province in China the mean fat intake was 24 ± 7% of energy [152].

Only 2 studies reported status data of infants and children from Asian countries. Low DHA levels in RBC were reported in Pakistani infants, corresponding to a low DHA content in maternal milk [153]. In an intervention study, the baseline fatty acids statuses of 6-month-old Cambodian and Italian infants were compared. Cambodian infants had lower baseline levels of LA, comparable ALA levels, and higher levels of AA + EPA + DHA in blood compared to their Italian counterparts [154]. Subsequent multiple micronutrient supplementation in those Cambodian infants resulted in significantly higher levels of DHA at 18 months of age compared to infants in the other intervention groups who received only iron-folic acid or placebo, respectively.

This review revealed a general scarcity of data on n-3 LC-PUFA supply and benefits in Asian populations. Future research should include this region, acknowledging its large variety of people and dietary habits. With many countries experiencing a rapid nutritional transition, future research should include indicators of chronic diseases such as gestational diabetes and programming effects on future CVD risk factors.

Since the responses to supplementation are likely to depend on current intakes and on nutritional status, future research evaluating the benefits of LC-PUFA supplementation should control for baseline nutritional status and intakes, as well as genetic variation, e.g. in the FADS genotype. Studies evaluating child development should use task-specific, sensitive tests focusing on areas likely to benefit from DHA and AA supplementation, including, for example, sustained attention and memory recognition.

Recent evidence indicates that n-3 LC-PUFA supplementation during pregnancy reduces the risk of early preterm birth prior to 34 weeks of gestation, which would predict a major benefit for infant morbidity and mortality. It is recommended that pregnant women should aim to achieve an added minimum average daily supply of 200 mg DHA over and above the intake level recommended for adult general health, resulting in a total DHA intake of at least 300 mg/day. Based on the comparison of studies with different dosages, it seems possible that higher intakes (600-800 mg DHA/day) may provide greater protection than lower dosages, but direct comparative dose-effect evaluations are not available.

Some but not all studies evaluating the effects of DHA or EPA + DHA supplementation in pregnant and lactating women and in term infants indicate possible beneficial effects on later child visual and cognitive and emotional development. The LC-PUFA supply and fish intake in pregnancy and infancy seem to positively affect the development of immune responses involved in allergic reactions, and reduce the risk of allergic diseases (asthma and eczema). Further support for the beneficial effects of early LC-PUFA status on later cognition and asthma risk is provided by recent diet-gene interaction studies.

Breast-feeding is recommended as the preferred choice for infant feeding. Breast-feeding women should aim to achieve a minimum average daily supply of 200 mg DHA, which is expected to result in a milk DHA content of 0.3% of fatty acids [47]. During the first months of life, term infants should receive 100 mg DHA/day and 140 mg AA/day, and hence infant formula should provide at least 0.3% of fatty acids as DHA along with AA. We recommend a continued supply of 100 mg DHA/day during the second 6 months of life. We do not provide quantitative advice on AA intake with follow-on formula fed once complimentary foods have been introduced due to a lack of sufficient data and due to considerable variation in the amounts of AA provided by complimentary foods within and among countries.

There are indications of benefits of supplying DHA in combination with AA to preterm infants with respect to cognitive and visual development and in infants with a birth weight <1,250 g with respect to the prevention of neurodevelopmental handicaps and chronic lung disease, with apparent greater benefits provided by a higher compared to a lower DHA supply (1 vs. 0.35% of the total fatty acid intake). For very-low-birth weight infants (birth weight <1,500 g), we support the recent recommendations [138] that intakes per kilogram per day of DHA ranging from 18 to 60 mg and of AA ranging from 18 to 45 mg are reasonable, and EPA intakes should not exceed 20 mg EPA; while intakes per kg per day of 55 to 60 mg DHA (∼1% of the total fatty acid intake), along with 35-45 mg AA (∼0.6-0.75% of the total fatty acid intake) are considered preferable.

The authors thank Prof. Sir Peter Gluckman (University of Auckland, New Zealand) for facilitating the organization of the workshop prior to the DOHaD Conference of 2013 in Singapore, and for opening the workshop with a review presentation on the role of nutrition in the developmental origins of adult health. Thanks also go to Felicitas Maier (Ludwig Maximilians University of Munich, Germany) for her dedicated help and support in organizing the consensus meeting and for her contribution to the electronic literature search.

The development of this publication was made possible by partial financial support from the Commission of the European Community, the specific RTD Program Quality of Life and Management of Living Resources, within the 7th Framework Program, research grant No. FP7/2007-13 (EarlyNutrition Project; project-earlynutrition.eu), and European Research Council Advanced Grant ERC-2012-AdG - No. 322605 META-GROWTH. This paper does not necessarily reflect the views of the Commission and in no way anticipates the future policy in this area. An additional unconditional grant was provided to the charitable ENA (www.early-nutrition.org) by DSM Nutritional Products AG, Kaiseraugst, Switzerland.

Christopher Boey, Cristina Campoy, Namsoo Chang, Sadhana Joshi, Christine Prell, Maria Antonia Guillermo-Tuazon, Quak, Damayanti Rusli Sjarif, and Sarayut Supapannachart report no conflicts of interest. Susan E. Carlson has received financial support from DSM, Mead Johnson Nutrition, Wyeth Nutrition, and Sequoia. Berthold Koletzko is a member of the National Breastfeeding Committee and tends to be biased towards breast-feeding. The Ludwig Maximilians University of Munich and its employee Berthold Koletzko have received support for scientific and educational activities from Abbott, Danone, DSM, Fonterra, Hipp, Mead Johnson, and Nestlé, predominantly as part of publically funded research projects with support of the European Commission or German governmental research granting agencies. Saskia Osendarp worked for Unilever until January 2012. Yuichiro Yamashiro has received research support from Yakult Honsha Co., Ltd., Tokyo, Japan.