Assessing omega-3 fatty acid supplementation during pregnancy and lactation to optimize maternal mental health and childhood cognitive development

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1 Clinical Lipidology ISSN: (Print) (Online) Journal homepage: Assessing omega-3 fatty acid supplementation during pregnancy and lactation to optimize maternal mental health and childhood cognitive development Chelsea M Klemens, Kataneh Salari & Ellen L Mozurkewich To cite this article: Chelsea M Klemens, Kataneh Salari & Ellen L Mozurkewich (2012) Assessing omega-3 fatty acid supplementation during pregnancy and lactation to optimize maternal mental health and childhood cognitive development, Clinical Lipidology, 7:1, To link to this article: Copyright 2012 Future Medicine Ltd Published online: 18 Jan Submit your article to this journal Article views: 92 View related articles Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 24 December 2017, At: 13:48

2 Review Assessing omega-3 fatty acid supplementation during pregnancy and lactation to optimize maternal mental health and childhood cognitive development The omega-3 fatty acid, docosahexaenoic acid, is an important building block of the CNS, and its availability during pregnancy and lactation may influence maternal mental health and, later, childhood developmental outcomes. Epidemiological and observational studies have supported a role for omega-3 fatty acids in optimizing child development and maternal mental health. However, results of randomized intervention studies have not consistently shown beneficial effects for omega-3 fatty acid supplementation during pregnancy and lactation. This review summarizes the biological plausibility, as well as the available evidence from observational and randomized controlled trials, of omega-3 supplementation in pregnancy and lactation. KEYWORDS: child development mental health omega-3 fatty acid placenta Chelsea M Klemens 1, Kataneh Salari 2 & Ellen L Mozurkewich* Industrialization has led to major changes in the western diet that have increased the percentage of dietary calories obtained from saturated fats and omega-6 fatty acids, and decreased the percentage of calories obtained through omega-3 fatty acids [1]. In contrast with a previous ratio of omega-6:omega-3 fats of approximately 2:1, the modern diet has an omega-6:omega-3 ratio of 20 30:1 [1]. This imbalance has led to speculation that these dietary changes may be responsible for the observed increase in mood disorders and depression over the past half a century [2]. The omega-3 fatty acid docosahexaenoic acid (DHA), is an essential substrate for the development of the brain and CNS during fetal development, and DHA is preferentially transferred across the placenta to meet this need [3]. This selective transfer may leave mothers relatively depleted of DHA stores; low DHA levels have been identified as a risk factor for perinatal depression [3,4]. Because of a potential preventive or therapeutic role for omega-3 fatty acids in perinatal depression at a time when there is a concern to avoid the potential harms of pharmacotherapy, there is a growing interest to explore any role for maternal supplementation in the prevention or treatment of perinatal depression [5]. Maternal omega-3 fatty acid supplementation has also been proposed as a means to optimize the cognitive development of the fetus, although the data surrounding this potential benefit are also very mixed [6]. We conducted this review to summarize the recent literature on omega-3 fatty acid supplementation during pregnancy and lactation on maternal mental health and child development. Nomenclature Essential polyunsaturated fatty acids (PUFAs) are termed essential because they cannot be synthesized by the human body [7]. The omega-3 and omega-6 fatty acids comprise the two main families of essential fatty acids [8]. The omega-3 and omega-6 fatty acids are so named because of the position of the double bond from the methyl end of each molecule [9]. The first double bond of the omega-6 series is located at the sixth carbon atom. This configuration applies to linoleic acid (LA), the 18 carbon parent compound and its 20-carbon derivative, arachidonic acid (ARA) [8]. Similarly, the 18 carbon parent compound of the omega-3 series is a-linolenic acid (ALA); its metabolic derivatives are eicosapentaenoic acid (EPA) (20 carbon), which in turn may be elongated to form DHA (22 carbon). Thus the PUFAs LA and ALA are elongated and desaturated to the long-chain PUFAs ARA, EPA and DHA [8] Both long-chain PUFAs in the omega-3 series share the first double bond at the third carbon atom [8,9]. The essential fatty acid LA, of the omega-6 series, is found in grains and vegetable oils and is the precursor of ARA [1]. ARA is synthesized in the human body by a process of enzymatic 1 Michigan State University, College of Human Medicine, East Lansing, MI 48824, USA 2 Division of Maternal Fetal Medicine, Department of Obstetrics & Gynecology, University of Michigan, F4835, Box 0264 Mott Hospital, 1500 E Medical Center Drive, Ann Arbor, MI , USA *Author for correspondence: Division of Maternal Fetal Medicine, School of Medicine, Department of Obstetrics & Gynecology, MSC , 1 University of New Mexico, Albuquerque, NM , USA Tel.: Fax: mozurk@med.umich.edu part of /CLP Future Medicine Ltd Clin. Lipidol. (2012) 7(1), ISSN

3 Review Klemens, Salari & Mozurkewich elongation and desaturation, and is also supplied in the diet in red meat and meat products [10,11]. Similarly, the essential fatty acid parent of the omega-3 series, ALA, is present in flax seed, rapeseed oil and green leafy vegetables [12]. ALA undergoes elongation and desaturation to the long-chain PUFAs of the omega-3 series, EPA and DHA (Figure 1) [10]. Although humans are able to metabolically convert the essential fatty acids to ARA, EPA and DHA via fatty acid desaturase enzymes, this process is relatively inefficient, and is unable to meet the maternal and fetal needs for DHA [3,10,13]. Thus DHA and EPA in the human diet are largely supplied through eating oily fish [11,14]. However, industrial contamination with mercury has led the US FDA to issue dietary advice to pregnant and lactating women, suggesting restriction of intake to two or fewer fish meals (~340 g) per week [14,101]. Thus, pregnant women are at risk for lower levels of DHA [3]. This relative fatty acid deficiency is thought to play a role in maternal mental health during pregnancy and the postpartum period. 18:2 omega-6 Linoleic acid 18:3 omega-6 γ-linoleic acid 20:3 omega-6 20:4 omega-6 Arachidonic acid 6-desaturase FADS2 Elongase ELOVL5 5-desaturase FADS1 Biological activities of omega-3 fatty acids in the adult brain & their relationship to maternal depression in pregnancy & postpartum Lipids are the most predominant substances in the mammalian brain [15]. These lipids are in the form of saturated, monounsaturated and PUFAs. DHA is the most abundant of all omega-3 fatty acids in the brain, comprising 10 20% of the entire fatty acid composition [15]. In the brain and retina, DHA is found primarily in the form of membrane phospholipids and regulates membrane fluidity and membrane-bound enzymes [16]. These DHA-induced property changes in the membrane impact cell signaling by altering the binding or release of neurotransmitters [17]. More specifically, in cultured rat astrocytes, DHA has been implicated in the inhibition of glutamate uptake by cortical astrocytes [18]. There have been a number of published animal studies modeling maternal dietary shortages of omega-3 fatty acids during gestation, investigating the effect of this deprivation on 18:3 omega-3 α-linolenic acid 18:4 omega-3 20:4 omega-3 20:5 omega-3 Eicosapentaenoic acid Elongase ELOVL5, ELOVL2 22:5 omega-3 ELOVL2 24:5 omega-3 FADS2 22:6 omega-3 Docosahexaenoic acid CS 24:6 omega-3 Figure 1. Conversion of linoleic acid and a-linolenic acid to long-chain polyunsaturated fatty acids. 94 Clin. Lipidol. (2012) 7(1)

4 Assessing omega-3 fatty acid supplementation to optimize maternal & childhood cognition Review the maternal brain. In one such study, Chalon et al. demonstrated that dietary deficiency of ALA across two generations of breeding female rats resulted in dysregulation of dopamine and serotonin transmission in maternal rat brains [19]. In another series of dietary deprivation studies, Levant et al. showed that when dietary deficiency of omega-3 fatty acids is combined with the depletion of DHA that occurs during pregnancy and lactation in the rat model, specific brain regions lose more DHA than others [20]. In an experimental model involving female rats fed a low ALA diet across two reproductive cycles, Levant et al. showed that the frontal cortex and temporal lobes (the areas involved in cognition and affect) as well as the caudate putamen appear to suffer the greatest reduction in DHA levels, suggesting that certain neuronal systems are more sensitive to DHA depletion [20]. In a subsequent set of experiments, the same team of investigators constructed a rodent model of DHA depletion and perinatal depression [21]. In this rat model, female rats in the intervention group were fed ALA deficient diets across two reproductive cycles; control animals were fed a diet with adequate ALA [21]. This dietary manipulation resulted in decreased brain DHA content in the intervention group compared to controls, which was associated with decreased hippocampal BDNF gene expression, altered serotonin content in some brain regions, and increased hypothalamic pituitary axis response to stress [21]. In a later set of experiments using the same model, these investigators found that animals fed the ALA-deficient diet had a 20 22% reduction in brain DHA compared with controls. DHA depletion in the brains of the parous rats was shown to be associated with a decrease in the density of D2-like dopamine receptors in the ventral striatum [22]. These findings are thought to replicate observed biochemical changes in the brains of depressed humans [21,22]. The actions of DHA as a neuroprotectant have also been described through its effect on phosphatidylserine synthesis [23]. Phosphatidyl serine is made from DHA-containing substrates and is important in preventing inappropriate cell death and supporting neuronal differentiation [23]. In retinal pigment epithelial cells, DHA also acts indirectly to protect against oxidative stress through the enzymatic production of neuroprotectin D1 (NPD1) [24]. NPD1 increases production of antiapoptotic proteins and decreases proapoptotic proteins of the Bcl-2 family [24]. NPD1 also works through neuroprotective gene expression mechanisms that suppress the ability of Ab42 to activate proinflammatory genes [16]. A large body of evidence has shown that DHA actually modulates several genes with diverse functions that include cell proliferation, DNA binding, transcriptional regulation, transport, cell adhesion and a number of others. This demonstrates that DHA is capable of significantly impacting cell development, function and maturation [9,25,26]. Another important role for DHA is the modulation of inflammation in the brain [25]. Animal models have demonstrated that during states of neuroinflammation, microglial cells are firstresponders, acting to initiate a series of events that eventually leads to the breakdown of membrane glycerophospholipids and the release of ARA and DHA [25]. ARA is then oxidized into proinflammatory prostaglandins, leukotrienes and thromboxanes [25]. DHA, on the other hand, is enzymatically converted into D-series resolvins and neuroprotectins, which inhibit the formation of these proinflammatory prostaglandins, leukotrienes and thromboxanes [25]. DHA and its metabolites also act to decrease inflammation by inhibiting NF kb, a transcription factor whose activation upregulates proinflammatory cytokine production. Its inhibition prevents the release of cytokines and curbs leuko cyte activity [25]. While neuroinflammation does serve a protective purpose against CNS insults, DHA acts to modulate the inflammatory effect of ARA-derived inflammatory mediators and therefore protects the brain from unopposed inflammation [25]. The ability of omega-3 fatty acids to modulate the inflammatory response, combined with the established relationship between inflammation and depression, has provided a link between omega-3 fatty acids and mental health. Recent evidence suggests that inflammation plays a major role in the pathogenesis of depression; elevated levels of proinflammatory cytokines have been observed in many depressed individuals [27]. An association between elevation of inflammatory cytokines and depression in nonpregnant and nonpostpartum individuals has been well established [28,29]; however, relatively few studies have investigated whether the inflammatory response might play a role in perinatal depression [30,31]. In a longitudinal observational study of 91 healthy pregnant women, Maes and colleagues assessed maternal serum cytokines IL-6, IL

5 Review Klemens, Salari & Mozurkewich receptor (IL-6R), gp130 (the IL-6 signaling protein), IL-1R antagonist (IL-1RA) and leukemia inhibitory factor receptor before delivery and 1 and 3 days after delivery [32]. The women also completed the Spielberger State Trait Anxiety Inventory (STAI) and the Zung Depression Rating Scale (ZDS) at each time point [32]. Although there was no overall relationship between the inflammatory markers studied and the absolute values of depression or anxiety scores, the investigators found that mothers with greater postnatal increases in the ZDS had significantly higher levels of IL-6 and IL-1RA. Similarly, mothers with greater increases in the STAI had significantly higher IL-6 and IL-6R [32]. Subsequently, Corwin and colleagues investigated the relationship between depressive symptoms and inflammatory markers in 25 healthy postpartum women [30]. Participants completed the Centers for Epidemiologic Studies Depression Scale on day 28 following delivery; a score of >11 was used as a measure of significant depressive symptoms [30]. Participants also gave urine samples for measurement of IL-1b and IL-6 on days 7, 14 and 28. The authors found that women with significant depressive symptoms on day 28 had significantly elevated IL-1b levels on day 14, compared with women without significant depressive symptoms [30]. In observational studies among nonpregnant individuals, higher plasma omega-3 fatty acid levels have been found to be associated with lower levels of inflammatory cytokines and higher levels of anti-inflammatory cytokines. For example, in a community-based sample of 1123 individuals plasma PUFA levels and circulating inflammatory markers, it was found that PUFAs, especially omega-3 fatty acids, were independently associated with lower levels of proinflammatory markers (IL-6, IL-1RA, TNF-a and C-reactive protein) and higher levels of anti-inflammatory markers (soluble IL-6R, IL-10 and TGF-b) [33]. As DHA is a major building block of the brain, studies of the effects of omega-3 fatty acids on brain function have, in the main, focused on DHA and omitted discussion of EPA. EPA has been detected in trace amounts in the brain where it may be enzymatically converted to DHA or participate in a host of biochemical activities [34,35]. In an animal model in which 3 week-old male rats were fed highly purified ethyl EPA, EPA was shown to modulate synaptic plasticity and activates the PI3-kinase/Akt pathway activities that are thought to protect against neurodegeneration [34]. Like DHA, EPA may suppress the inflammatory response. One study comparing 8 weeks of dietary supplementation with fish oils with varying ratios of EPA:DHA among female mice (from 6 weeks of age onward) has suggested that suppression of proinflammatory cytokine production may be greatest with those oils with a high EPA:DHA ratio [36]. However, this relationship has not been confirmed in all studies. For example, an in vitro study comparing effects of EPA with DHA on liposaccharide-stimulated THP-1 macrophages, found that pretreatment of cell cultures with DHA suppressed proinflammatory cytokine production (IL-1b, IL-6 and TNF-a) more efficiently than pretreatment with EPA [37]. Therefore, it is uncertain what composition of omega-3 fatty acids would be most beneficial in decreasing inflammation and improving mental health. EPA is also the metabolic precursor of the E-series resolvins; resolvin E1 (RvE1) and RvE2 [38]. RvE1 possesses potent anti-inflammatory activities at nanomolar levels, reducing PMN infiltration and synthesis of proinflammatory cytokines in a dorsal pouch model [38]. In a mouse model of inflammatory pain, RvE1 modulated synaptic plasticity in the dorsal horn of the spinal cord [39]; however, the activities of RvE1 in the brain have not been well characterized. Epidemiology of depression in pregnancy & postpartum & dietary contributors Postpartum depression occurs in approximately 10 20% of childbearing women and can be associated with significant morbidity [40]. A significant proportion of women (5 10%) will also experience a major depressive episode during pregnancy [40]. While the etiology of perinatal depression is quite complex and most likely multifactorial, an area of recent interest is the role of nutrition in depression [5]. Plausible links between nutrition and mood have been previously reported for several vitamins, nutrients and more recently PUFAs [17]. Without an adequate nutritional supply of omega-3 fatty acid rich foods, mothers can experience lower levels of these fatty acids during pregnancy [17]. Some observational studies have reported slower recovery rates of maternal DHA status in women with postpartum depression compared to nondepressed control subjects [4]. 96 Clin. Lipidol. (2012) 7(1)

6 Assessing omega-3 fatty acid supplementation to optimize maternal & childhood cognition Review In a crossnational ecological study including 23 countries, both lower rates of seafood intake and lower concentrations of DHA in breast milk predicted higher prevalence rates of postpartum depression [41]. Similarly, Golding et al. reported high levels of depressive symptoms in mothers with lower intake of omega-3, with as much as a 50% increased risk of depression in mothers who ate no seafood compared to those who consumed a substantial amount of seafood [42]. Intervention studies of long-chain PUFA supplementation & maternal depression Despite observational studies linking relative DHA deficiency in pregnancy and lactation with perinatal depression, results of intervention studies of omega-3 fatty acid supplementation have been mixed, with a few positive studies, but many not showing clear evidence of benefits [43]. Recently, Jans and colleagues conducted a meta-analysis of seven double blind, randomized controlled trials with a total of 612 participants that evaluated the effect of omega-3 fatty acids for prevention or treatment of depression in pregnancy and postpartum. The included studies involved comparison of oils containing differing combinations of DHA and EPA with placebo during pregnancy [43]. In their aggregate analysis of these trials, the authors did not demonstrate any benefits of omega-3 fatty acid supplementation during pregnancy for prevention or treatment of perinatal depression [43]. The trials included in this analysis included four in which participants were healthy, and three trials in which participants were suffering from major depressive disorder at baseline [43]. In contrast to the studies that aimed to prevent depression, the authors did find some evidence for effectiveness in the three studies that employed omega-3 fatty acid supplementation among subjects who were depressed at trial entry rather than among subjects who were healthy at baseline [43]. Subsequent to these findings was the publication of a recent large multicenter study by Makrides et al. [44]. This study randomized 2399 women in five Australian maternity hospitals to receive either 800 mg daily of DHA or placebo from study entry in the second trimester of pregnancy until delivery [44]. Participants were healthy women who were not selected based on depressive symptoms or predisposition to depression [44]. The primary outcome of the study was Edinburgh Postnatal Depression Scale Score (EPDS) at 6 weeks and 6 months postpartum [44]. The authors did not find any difference in the proportion of mothers reporting significant depressive symptoms (EPDS >12) or new diagnosis of depression [44]. Among women with depressive symptoms at trial entry, significant depressive symptoms at 6 months postpartum did not differ between groups [44]. The individual studies reviewed by Jans, as well as the more recent Makrides trial are summarized in Table 1 [44 51]. There are several potential reasons that this study might have not shown effectiveness for the omega-3 fatty acid supplementation intervention. First, the supplementation was carried only through birth of the infant, and not until 6 weeks and 6 months postpartum when the outcome was assessed [44]. Second, it is possible that the selection of DHA rather than EPA as the study intervention led to the negative result. Alternatively, it is possible that the ratio of EPA:DHA may be of the greatest importance in the regulation of mood. This may explain why most of the extant trials of fish oil supplementation during pregnancy have shown no effect on mood disorders, in that most of these trials used DHA-predominant oils, or fish oils with a low EPA:DHA ratio (Table 1). Two systematic reviews of studies concerning EPA and DHA for treating depression and other mood disorders in mixed pregnant and nonpregnant subjects have suggested that EPA, but not DHA, is effective in treating this disorder [52,53]. Indeed, Martins noted that trials in which fish oil contained at least 50% EPA were more likely to show benefits than trials in which the studied fish oils were DHApredominant [53]. Martins noted that trials utilizing nearly pure EPA were most likely to show benefits [53]. However, no studies directly comparing these two fatty acids in pregnant patients are currently available. A double-blind, randomized pilot trial comparing EPA-rich fish oil with DHA-rich fish oil and with placebo in pregnant women selected based on risk for depression is currently underway [54]. Biological activities of omega-3 fatty acids in the developing brain In parallel to the interest in omega-3 fatty acids for maternal mental health, there has been growing work exploring the role of these lipids in the optimization of fetal and postnatal mental development. Significant DHA 97

7 Review Klemens, Salari & Mozurkewich Table 1. Summary of randomized controlled trials of omega-3 fatty acids for the prevention or treatment of perinatal depression. Study Year Inclusion criteria Participants Duration of supplementation Llorente et al Healthy lactating women Krauss- Etschmann et al Healthy pregnant women Rees et al Major depressive disorder diagnosed at baseline Freeman et al Major depressive disorder diagnosed at baseline Su et al Major depressive disorder diagnosed at baseline 138 (37 drop-outs) 4 months during the postpartum period 311 From week 22 until delivery 26 Pregnancy and/or postpartum Mattes et al Healthy 98 Pregnancy (from 20 weeks gestation until delivery) Doornbos et al Healthy 182 Pregnancy and postpartum Makrides et al Healthy 2399 From <21 weeks until delivery Composition and dose Outcome measures Results (significance) Ref. 200 mg DHA; placebo not described 500 mg DHA and 150 mg EPA/400 µg 5-MTHF/both/placebo; placebo not described 6 g fish oil (27.3% DHA, 6.8% EPA), 80 mg vitamin E or placebo (sunola oil) 59 Pregnancy or Supportive psychotherapy postpartum ± 1.1 g EPA plus 0.8 g DHA or placebo; maize oil with 1% fish oil placebo 36 Pregnancy 2.2 g EPA plus 1.2 g DHA or placebo (olive oil ethyl esters) 4 g fish oil (56% DHA and 27.7% EPA) vs placebo (olive oil) 220 mg DHA vs 220 mg DHA mg ARA vs placebo (soy oil) 800 mg DHA plus 100 mg EPA or vegetable oil BDI, EPDS and SCID No significant differences [45] EPDS No significant differences among the groups EPDS, HAM-D and MADRS No difference between groups EPDS and HAM-D No difference between groups HAM-D, EPDS and BDI Significantly higher response rate and lower HAM-D, EPDS and BDI scores with intervention BDI No difference between groups [46] [47] [48] EPDS No difference in outcomes [51] EPDS >12 No difference in outcomes [44] 5-MTHF: 5-methyltetrahydrofolate; ARA: Arachidonic acid; BDI: Beck Depression Inventory; DHA: Docosahexaenoic acid; EPA: Eicosapentaenoic acid; EPDS: Edinburgh Postnatal Depression Scale; HAM-D: Hamilton Rating Scale for Depression; MADRS: Montgomery Åsberg Depression Rating Scale; SCID: Structured Clinical Interview for DSM-IV. Adapted with permission from [43]. [49] [50] 98 Clin. Lipidol. (2012) 7(1)

8 Assessing omega-3 fatty acid supplementation to optimize maternal & childhood cognition Review accumulation occurs in the developing brain during the third intrauterine trimester and the first year of life [55,56]. DHA is an important ingredient in the synthesis of membrane phospholipids that occurs with neurogenesis. During pregnancy, most DHA supplied to the fetus originates from maternal sources, either from diet or mobilization of fat stores, and is transferred across the placenta. ALA transferred across the placenta may also be converted, to a limited degree, to DHA in the fetal compartment [57]. Postpartum, the infant receives DHA through breast milk as well as from PUFA-fortified formula. In a study of human mother infant dyads, maternal red blood cell omega-6:omega-3 ratios increased during the course of lactation, while DHA content of milk over a similar time scale did not show a decrease, suggesting that DHA continues to be transferred through breast milk to the infant to support the needs of ongoing brain development [58]. Although an optimal level of dietary DHA for fetal development has not been established, there is some evidence that the developing fetus may be vulnerable to the effects of low maternal DHA supply. Studies carried out in the rat model have shown that maternal diets deficient in omega-3 fatty acid during gestation may alter neurogenesis and the morphology of telencephalic structures in the embryo [57]. In a study comparing brain development of rat embryos, pregnant rats were fed a diet deficient in ALA, with a high omega-6:omega-3 ratio, or an ALA-adequate control diet [57]. The respective diets were initiated 2 weeks prior to mating and continued through embryonic day 19. The investigators noted that brains of the embryonic pups of mothers eating the deficient diet had a 55 65% reduction in DHA content [57]. There was a compensatory increase in ARA noted. The cerebral hemispheres of the omega-3 fatty aciddeficient rat embryos were noticeably smaller than that of controls due to a decrease in size of their cortical plate, primordial hippocampus and dentate gyrus [57]. Another study compared neurons of embryonic rat hippocampi under conditions of maternal dietary deficiency of omega-3 fatty acids (fed with 0.09 wt% of LA) versus an omega-3 fatty acid adequate diet (2.5 wt% LA plus 0.9 wt% DHA) [59]. Hippocampal neurons from the omega-3 fatty acid-deficient embryos showed inhibited neurite growth and synaptogenesis, a decrease in synapsins and glutamate receptor subunits and impairment of long-term potentiation. By contrast, the brains of 18 day-old embryos from dams that had received a DHAand LA-adequate diet manifested increased neurite growth and synapsin formation as well as increased levels of pre- and postsynaptic proteins, including glutamate receptors [59]. This potentiation of neural growth by DHA has also been demonstrated in vitro with neural stem cells of 15.5 day-old rat embryos cultured with or without DHA [60]. Those cultured with DHA showed more morphologically mature neurons an increased number of Tuj1-positive neurons and an increased incorporation ratio of 5-bromo-2 -deoxyuridine a mitotic division marker. DHA also appeared to accelerate the transition of neural stem cells from undifferentiated to differentiated by promoting cellcycle exit. The number of pyknotic cells was also decreased on day 7 in DHA-supplemented cultures, suggesting that DHA may suppress apoptotic cell death and increase the viability of young neurons [60]. In a later study, in a rat model of hypothyroidism-induced neuronal apoptosis, omega-3 fatty acid (EPA-rich marine oil) supplementation of pregnant and lactating hypothyroid maternal rats significantly decreased DNA fragmentation and caspase-3 activation in the cerebellums of the hypothyroid pups whose mothers had been supplemented, compared with cerebellums of pups born to hypothyroid dams that had not been supplemented [61]. Additionally, supplementation decreased the levels of proapoptotic proteins Bcl-2 and Bax, and increased the levels of antiapoptotic proteins Bcl-2 and Bcl-xL in the developing cerebellum [61]. The rat model has also been used to demonstrate the detrimental effects of maternal omega-3 fatty acid deficiency on dopaminergic regulatory proteins in the developing brain [62]. Kuperstein et al. fed dams either a LA adequate or deficient diet from the second postconception day through lactation. Neonatal rats were weaned on the 21st postnatal day, then sacrificed on the 28th postnatal day. Decreased levels of tyrosine hydroxylase (the rate-limiting enzyme in dopamine synthesis), the vesicular monoamine transporter (VMAT-2) and VMAT-associated vesicles in the hippocampus were seen in omega-3 fatty acid-deficient offspring [62]. Dopamine receptors DAR1 and DAR2 were markedly increased in the cortex and striatum. This study also found that omega-3 fatty acid deficiency in the developing brain increased microglia activation and 99

9 Review Klemens, Salari & Mozurkewich NF kb levels, suggesting that there may be a correlation between omega-3 fatty acid deficiency, oxidative stress and dysregulation of dopaminergic components [62]. Although DHA is critical for optimal development of the fetal nervous system and its deficiency may result in adverse effects on neurodevelopment, excess levels of DHA during fetal life may also have undesired consequences [7,63]. Davis-Bruno et al. recently conducted a comprehensive review of the literature surrounding DHA, EPA and ARA in pregnancy and lactation in animal models. Based on their review of animal studies, they note that deficiencies of DHA and/or EPA may result in neuronal arborization deficits [7]. They hypothesized that preterm infants are particularly vulnerable to the effects of DHA deficiency and that this deficiency may underlie the vascular fragility leading to intraventricular hemorrhage [7]. Their review of the animal literature also suggested that although adequacy of DHA is necessary for optimal neurodevelopment, DHA excess may be detrimental, by increasing oxidative stress and apoptosis [7]. The authors concluded that although balancing the ratio of DHA:ARA is necessary for optimal neurodevelopment, it is unclear whether maternal omega-3 fatty acid supplementation during pregnancy and lactation or fortification of infant formula is beneficial [7]. Recent research in animal models has also explored a possible role for DHA supplementation for neuroprotection, with a view toward the potential use of DHA to prevent childhood disability resulting from intrapartum hypoxia ischemia [64 66]. Indeed, in a study in which intra-amniotic DHA was administered to maternal rats whose fetuses were experiencing global ischemic stress during intrauterine life, DHA was shown to confer neuroprotection through its free radical scavenging abilities [64]. Fetal rats that experienced ischemic stress during gestations in which their mothers had been fed a diet deficient in omega-3 fatty acids (LA) were observed to have an overexpression of a multitude of genes coding for receptors for neurotransmitters, especially DAR1 and DAR2 [64]. Likewise, in a series of experiments in neonatal rats (postnatal day 7), DHA albumin administered before or after experimental hypoxia ischemia was shown to improve forepaw placement scores compared with control animals who received albumin alone [65,66]. Similarly, in a study in which 7 day-old rat pups underwent hypoxia ischemia potentiated by lipopolysaccharide-induced brain inflammation, pretreatment with DHA conferred neuroprotection and resulted in improved function after brain insult [67]. To date, any preventive or therapeutic role for DHA awaits further translational investigation and clinical trials. Mode of placental transport of fatty acids The fetus is able to convert ALA to DHA, but this ability decreases with advancing gestational age [68]. Therefore the fetus is largely dependent on the mother to transfer preformed DHA across the placenta [68]. There are several factors influencing fatty acid transport across the placenta; these include maternal fatty acid status, placental function, as well as fatty acid transport proteins (FATP) and fatty acid binding proteins (FABP) [68]. Fatty acids from the maternal circulation pass across placenta cell membranes via passive diffusion as well as by active transport through a combination of binding proteins. These are termed plasma membrane fatty acid binding protein (FABPpm/GOT2), FABPs and FATPs, and fatty acid translocase (FAT/CD36) [69]. In a secondary analysis of data from a double-blind randomized DHA supplementation trial, Larque et al. demonstrated that FATP-1 and FATP-4 are most important for the transplacental transport of DHA to the fetal compartment [70]. Long-chain PUFA transport across the placenta is important because the fetus is able to elongate and desaturate the essential fatty acids ALA and LA to DHA and ARA, respectively, only to a limited degree [69]. Interestingly, infants born preterm are able to convert EFA to longchain PUFAs to a greater degree than infants born at term, although fetal capacity to synthesize ARA exceeds fetal ability to synthesize DHA [2]. Thus, transport of the long-chain PUFAs is necessary to meet the needs of the developing brain and CNS [69]. Maternal & fetal concentrations of essential fatty acids & long-chain PUFAs Several investigations have demonstrated that fetal and maternal plasma and red blood cell levels of essential fatty acids and long-chain PUFAs are highly correlated [68]. Notably, fetal longchain PUFA levels typically exceed maternal levels [42,68]. These findings have demonstrated that the long-chain PUFAs are preferentially transported across the placenta [68]. Similarly, DHA is preferentially transported relative to ARA [44,68]. The placenta is unable to elongate and desaturate 100 Clin. Lipidol. (2012) 7(1)

10 Assessing omega-3 fatty acid supplementation to optimize maternal & childhood cognition Review essential fatty acids to long-chain PUFAs [2,71]. Thus the fetus must rely upon transport of preformed ALA and DHA from the maternal compartment [71]. Fetal accretion of the longchain PUFAs DHA and ARA is dependent upon adequate provision of DHA and ARA preformed in the maternal diet, as the fetus only converts ALA to DHA to a limited degree [69]. However, not all placental transport reflects recent maternal diet. Long-chain PUFA stores laid down in maternal adipose tissue during early fetal life are mobilized via lipolysis in the third trimester for transport to the fetus [71]. Current knowledge of the activities of FABPs suggests that that DHA is transported preferentially across the placenta compared with other long-chain PUFAs and EFA in the following hierarchical fashion: DHA > ARA > ALA > LA [2,71]. The relative efficiency of placental transport of fatty acids is most likely influenced by individual genetic variations [2]. Additionally, fatty acid transport to the fetal compartment is responsive to variations in maternal blood fatty acid status [72]. In an observational study of Tanzanian mothers with low, intermediate and high fish intake, Kuipers et al. compared maternal and fetal red blood cell fatty acid composition, as well as the fatty acid composition of fetal cord blood at delivery and infant DHA status at 3 months of age [72]. The investigators observed that in conditions of low-to-intermediate maternal fish intake, biomagnification of DHA via placental transport occurs [72]. By contrast, in conditions of high maternal fish intake, bioattenuation of transport across the placenta occurs, suggesting that there is a DHA equilibrium that is optimal for fetal growth and development [72]. The authors speculate that rather than an optimal DHA level, an appropriate DHA:ARA ratio is necessary for fetal health [72]. However, they concede that the optimal maternal and fetal DHA levels are not known [72]. In the absence of definitive data, they recommend adhering to the consensus guidelines of Koletzko et al., recommending maternal DHA intake of at least 200 mg/day [10]. Other factors that may influence fatty acid transfer to the fetal compartment Fatty acid transfer across the placenta may also be influenced by a number of pregnancy complications, including gestational diabetes mellitus and intrauterine growth restriction [71]. For example, available studies demonstrate that placental long-chain PUFA uptake is increased but placental transport of long-chain PUFA to the fetal compartment is impaired in pregnancies complicated by gestational diabetes mellitus [71]. Similarly, in pregnancies complicated by intrauterine growth restriction, fetal cord blood ratios of the long-chain PUFAs DHA and ARA and the EFA precursors LA and ALA have been demonstrated to be significantly altered [45,46,71]. Findings of nonrandomized & randomized studies of omega-3 fatty acid supplementation of maternal diet or supplemented infant formula & child development Observational studies of prenatal dietary omega-3 fatty acid intake & child development In a longitudinal cohort study of over 11,000 pregnant women in the UK, Hibbeln et al. assessed maternal seafood intake by use of a food frequency questionnaire administered at 32 weeks gestation [14]. Cognitive development of children born to mothers enrolled in the study was assessed by means of parental questionnaires administered by post 6, 18, 30 and 42 months of age and by IQ testing at 8 years of age [14]. The authors found that children born to mothers who ate no seafood or whose seafood consumption was less than 340 g per week during pregnancy were at increased risk to have a verbal IQ score in the lowest quartile (no seafood consumption odds ratio: 1.48, 95% CI: ; some: 1.09, 95% CI: ; overall trend, p = 0.004) [14]. That is greater maternal intake of seafood during pregnancy was associated with lower risk of suboptimum verbal IQ [14]. In this study, seafood consumption above the 340 g dietary limit currently advocated by the FDA was actually associated with improved neurocognitive outcomes compared with consumption of lesser amounts or no seafood [14]. More recently, a prospective longitudinal observational study of 154 Inuit children reported that higher umbilical cord blood DHA levels at birth were associated with improved measures of memory function at school age (mean 11.3 years) [73]. Interventional studies of maternal supplementation during pregnancy & lactation Several randomized trials have been undertaken to assess the potential benefits of either maternal or infant omega-3 fatty acid supplementation in child development. A randomized placebo- controlled trial of 98 pregnant women 101

11 Review Klemens, Salari & Mozurkewich who received either fish oil or olive oil from 20 weeks gestation until delivery revealed significantly higher scores for hand eye coordination in children whose mothers received fish oil versus those who received placebo [74]. Similarly, a randomized intervention trial of cod liver oil supplementation of 82 women during pregnancy and lactation showed improvements in verbal IQ at 4 years of age compared with children born to 62 women who received corn oil [75]. However, in a later follow-up evaluation of children born in the trial, Kaufman Assessment Battery for Children scores at 7 years of age did not differ between the groups [76]. The available trials of omega-3 fatty acid supplementation during pregnancy have been recently summarized in two systematic reviews [6,77]. In a systematic review of 13 trials of omega-3 fatty acid supplementation during pregnancy and/or lactation that aimed to demonstrate improvement in fetal/infant cognitive development, Dziechciarz et al. found that evidence from the available randomized controlled trials has not demonstrated a clear and consistent benefit for omega-3 fatty acid supplementation in pregnancy on infant cognitive development [6]. They noted that although individual trials reported improvements in limited domains on intellectual tests, these improvements were not consistent across trials, nor did they tend to persist over time [6]. Similarly, a Cochrane systematic review of omega-3 fatty acid supplementation during lactation (six trials, 1280 women) found that supplementation during lactation had no significant effect on childrens neurodevelopment [77]. The trials included in the Dziechciarz systematic review, as well as the subsequent trials described below, are summarized in Table 2 [44,74 76,78 86]. Subsequent to the search dates of these systematic reviews, several additional trials evaluating the effects of maternal omega-3 fatty acid supplementation on offspring s cognitive development have been published. A large multicenter trial of DHA supplementation (800 mg/day) did not show any benefit for the intervention on the infants cognitive function at 18 months of life [44]. A total of 2399 women with singleton pregnancies who were enrolled before 21 weeks gestation were randomly assigned to receive either DHA or vegetable oil, which was continued until birth. A subset of 726 infants born to women participating in the trial was assessed for developmental outcome using the Bayley Scales of Infant and Toddler Development at 18 months of age. There were no differences found in mean cognitive composite score or mean language composite score between the groups. Children born to mothers who received DHA supplementation were significantly less likely to have a cognitive standard score <85. This group difference was most significant among boys. However, an unexpected and unexplained finding of this trial was that girls born to mothers receiving DHA had a lower language standardized score than girls born to mothers who received placebo; although there was no difference in language standardized score for the total group. These findings require further study in long-term follow-up of children born in this trial [44]. Another recent large, randomized controlled trial involved 315 healthy pregnant women, of whom 161 completed follow-up. Participants were supplemented with fish oil (500 mg DHA plus 150 mg EPA), 400 µg/day of 5-methyltetrahydrofolate, both or placebo from 20 weeks until delivery. This study did not show any beneficial or harmful effects of the omega-3 fatty acid supplementation on children s developmental outcomes at 4, 5.5 or 6.5 years of age [84,85]. Likewise, a smaller randomized, blinded study (n = 114) comparing low dose prenatal supplementation with DHA (220 mg), DHA plus ARA (220 mg DHA mg ARA), or placebo from 15.6 to 17.4 weeks gestation through pregnancy and the first 3 months of lactation failed to show any difference in neurodevelopmental outcome at 18 months of age [86]. Secondary analyses of data from randomized controlled trials Despite negative results from the primary analyses from these trials, several authors have noted associations between omega-3 fatty acid levels and cognitive development in secondary analyses that were not carried out on an intent-to-treat basis; for example, in a secondary analysis of data from the study by Helland et al., maternal plasma phospholipid ALA, DHA and omega 3:omega-6 ratios at 35 weeks gestation were positively correlated with sequential processing scores at 7 years of age [75,76]. EPA from umbilical cord blood was also positively correlated with the sequential processing score [76]. Similarly, in a secondary analysis of data from the trial conducted by Escolano-Margarit et al., higher levels of DHA in the cord blood of infants born to study participants were shown by regression analysis to be associated with greater likelihood of optimal cognitive development at 102 Clin. Lipidol. (2012) 7(1)

12 Assessing omega-3 fatty acid supplementation to optimize maternal & childhood cognition Review Table 2. Summary of randomized controlled trials of maternal omega-3 fatty acid supplementation during pregnancy and/or lactation for optimization of infant cognitive development. Author Year Participants Duration of supplementation Dunstan et al. Judge et al From 20 weeks gestation until delivery From 24 weeks gestation until delivery Tofail et al (249 assessed) Jensen et al. Lauritzen et al. and Cheatham et al. Helland et al. From 25 weeks gestation until delivery For 4 months after 2005, , 2003, 2008 delivery in breastfeeding mothers 122 For 4 months after delivery in breastfeeding mothers 341 From 18 weeks until 3 months after delivery Intervention Control Outcome Length of follow-up Fish oil (DHA 2.2 g/day plus EPA 1.1 g/day) DHA-containing cereal bars (~214 mg/day DHA) Fish oil containing 1.2 g DHA and 1.8 g EPA daily Algal oil capsules ~200 mg DHA/day Microencapsulated fish oil added to muesli bars or to cookies or as capsules containing 900 mg/day of DHA, low content of EPA Cod liver oil containing DHA g/day plus EPA g/day, vitamins A, D and E Olive oil 2.7 g/day GMDS; PPVT; CBC (age 2.5 years) Corn oil-containing cereal bars Soy oil placebo; LA 2.25 g/day plus ALA 0.27 g/day Vegetable oil capsules: LA 56.3%; ALA 3.9% Microencapsulated olive oil added to muesli bars or cookie or as capsules Corn oil containing LA g/day, ALA g/day, vitamins A, D and E Two-step problem-solving test from Willatts; FTII (9 months) BSID, WMBA (10 months) PSI and MDI (30 months); GDI (12 and 30 months); CLAMS, visual motor problem-solving (12 and 30 months); CAT (12 and 30 months) IPT at 9 months; CDI at 1 and 2 years; ST and SDQ at 7 years FTII at 6 and 9 months, K-ABC at 4 years; K-ABC at 7 years Results (significance) 2.5 years Children in supplemented group had improved hand eye coordination 9 months Significant improvement in problem-solving tasks; no difference in FTII 10 months No significant differences 30 months PSI at 30 months of age significantly increased compared with controls 7 years Passive vocabulary reduced in fish oil group compared with controls at 1 year of age; no difference at 2 years of age; no difference at 7 years of age 7 years No difference in outcomes at 6 and 9 months; improvements in verbal IQ scores of children whose mothers received DHA; no difference in outcomes at 7 years of age 5-MTHF: 5-methyltetrahydrofolate; ALA: a-linolenic acid; Bayley III: Bayley Scales of Infant and Toddler Development; BSID: Bayley Scale of Infant Development II; CAT: Clinical Adaptive Test; CBC: Child Behavior Checklist; CDI: MacArthur Communicative Development Inventory; CLAMS: Clinical Linguistic and Auditory Milestone Scale; DHA: Docosahexaenoic acid; EPA: Eicosapentaenoic acid; FTII: Fagan Test of Infant Intelligence; GDI: Gesell Developmental Inventory; GMDS: Griffiths Mental Development Scales; HE: Hempel Examination; IPT: Infant Planning Test; K-ABC: Kaufman Assessment Battery for Children; LA: Linoleic acid; MDI: Bayley Mental Development Index; PPVT: Peabody Picture Vocabulary Test; PSI: Bayley Psychomotor Development Index; SDQ: Strengths and Difficulties Questionnaire; ST: Stroop Task; TE: Touwen Examination; WMBA: Wolke Modified Behavioral Assessment. Adapted with permission from [6]. Ref. [74] [78] [79] [80] [81,82] [75,76,83] 103

13 Review Klemens, Salari & Mozurkewich Table 2. Summary of randomized controlled trials of maternal omega-3 fatty acid supplementation during pregnancy and/or lactation for optimization of infant cognitive development (cont.). Results (significance) Ref. Intervention Control Outcome Length of follow-up Author Year Participants Duration of supplementation [44] Vegetable oil Bayley III (18 months) 18 months No difference in outcomes at 18 months 800 mg DHA plus 100 mg EPA 500 mg DHA and 150 mg EPA/400 µg 5-MTHF/both From <21 weeks until delivery 6.5 years No significant differences [84,85] HE (4 years); TE (5.5 years); K-ABC (5.5 and 6.5 years) Placebo not described From 20th week of pregnancy until delivery 18 months No difference in outcomes [86] Soy oil placebo HE, BSID (both at 18 months) Makrides et al. Escolano- Margarit et al. and Campoy et al. van Goor et al. DHA (220 mg), DHA plus ARA (220 mg DHA plus 220 mg ARA) From weeks gestation through pregnancy and the first 3 months of lactation 5-MTHF: 5-methyltetrahydrofolate; ALA: a-linolenic acid; Bayley III: Bayley Scales of Infant and Toddler Development; BSID: Bayley Scale of Infant Development II; CAT: Clinical Adaptive Tests; CBC: Child Behavior Checklist; CDI: MacArthur Communicative Development Inventory; CLAMS: Clinical Linguistic and Auditory Milestone Scale; DHA: docosahexaenoic acid; EPA: Eicosapentaenoic acid; FTII: Fagan Test of infant intelligence; GDI: Gesell Developmental Inventory; GMDS: Griffiths Mental Development Scales; HE: Hempel Examination; IPT: Infant Planning Test; K-ABC: Kaufman Assessment Battery for Children; LA: Linoleic acid; MDI: Bayley Mental Development Index; PPVT: Peabody Picture Vocabulary Test; PSI: Bayley Psychomotor Development Index; SDQ: Strengths and Difficulties Questionnaire; ST: Stroop Task; TE: Touwen Examination; WMBA: Wolke Modified Behavioral Assessment; Adapted from [6]. 5.5 years of age [84]. The discrepancies between the results of the primary and secondary analyses suggest that differences in compliance and in fatty acid metabolism among subjects may have influenced the outcomes reported. Interventional studies of postnatal supplementation of children born preterm In a study involving 657 infants born preterm, Makrides et al, found that supplementing infant formula with DHA or supplementing lactating mother s breast milk via dietary tuna oil did not alter the infants mental development index (MDI) at 18 months of age overall [87]. However, girls born to mothers randomized to the high DHA group were at reduced risk for significantly delayed cognitive development; similarly, the mean MDI among girls assigned to the high DHA group was higher than the MDI of girls assigned to the standard DHA group. [87]. By contrast, a Cochrane review that included 17 trials of supplementation of infant formula with long-chain PUFAs did not show any beneficial effects for supplementation on visual acuity or neurocognitive development [88]. The longchain PUFA supplements under study in the included trials were, in most cases, a mixture of omega-3 and omega-6 fatty acids [88]. Interventional studies of postnatal supplementation of children born at term In a postnatal dietary supplementation study, Agostoni and coworkers randomized 1160 healthy neonates born at term to receive either 20 mg liquid DHA or placebo daily, starting at 24 h after hospital discharge and continuing daily through 1 year of life [89]. All infants also received 400 units of vitamin D3 per day. The primary outcome for this study was achievement on four gross motor milestones: sitting without support, hands and knees crawling, standing without support and walking [89]. Although infants assigned to receive DHA were able to sit without support approximately 1 week earlier on average than infants receiving placebo, there were no differences in any other outcome of interest [89]. A recent Cochrane review on long-chain PUFA supplementation of formula for term infants analyzed the results of 15 randomized controlled trials [90]. Supplemented infant formula contained DHA alone or DHA plus ARA. The authors concluded that results of the most well-conducted randomized controlled trials have not shown beneficial effects of long-chain 104 Clin. Lipidol. (2012) 7(1)