Review

Maternal choline supplementation: a nutritional approach for improving offspring health? Xinyin Jiang1, Allyson A. West2, and Marie A. Caudill2 1 2

Department of Health and Nutrition Sciences, Brooklyn College, Brooklyn, NY 11210, USA Division of Nutritional Sciences, Cornell University, Ithaca 14853, NY, USA

The modulatory role of choline on the fetal epigenome and the impact of in utero choline supply on fetal programming and health are of great interest. Studies in animals and/or humans suggest that maternal choline supplementation during pregnancy benefits important physiologic systems such as offspring cognitive function, response to stress, and cerebral inhibition. Because alterations in offspring phenotype frequently coincide with epigenetic modifications and changes in gene expression, maternal choline supplementation may be a nutritional strategy to improve lifelong health of the child. Future studies are warranted to elucidate further the effect of choline on the fetal epigenome and to determine the level of maternal choline intake required for optimal offspring physiologic function. Choline and its role in fetal development The essential nutrient choline participates in several vital biological functions (see Figure I in Box 1) with key roles in fetal development [1]. During development, choline phospholipids (i.e., phosphatidylcholine and sphingomyelin) are required in large amounts for membrane biogenesis, myelination of nerve axons, cell division, tissue expansion, and lipid transport [2]. In addition, the choline-derived neurotransmitter, acetylcholine, is essential for proper organization and function of the developing brain through its effects on neurogenesis and synapse formation [3]. Notably, large amounts of acetylcholine are produced and accumulate in human placenta (see Glossary) where it functions as a signaling molecule to influence cellular differentiation and proliferation as well as parturition [4]. Finally, betaine, an oxidized metabolite of choline, is a source of methyl groups for the production of S-adenosylmethionine (SAM), which serves as a substrate for DNA and histone methyltransferases (Box 1), and is thus required for the establishment and maintenance of the fetal epigenome [5–10]. Corresponding author: Caudill, M.A. ([email protected]). Keywords: choline; development; epigenetics; fetus; programming. 1043-2760/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tem.2014.02.001

During pregnancy, maternal choline intake can affect metabolic and physiologic function of the offspring through a variety of inter-related mechanisms. With the overall goal of examining whether maternal choline supplementation Glossary Adequate intake (AI): a recommended average daily nutrient intake level that is established when a Recommended Dietary Allowance (RDA) cannot be determined due to insufficient data. The main criterion for establishing the choline AI was prevention of liver damage. Angiogenesis: the process of growing new blood vessels. Placental angiogenesis is complex with several angiogenic factors, including sFLT1 and VEGF, playing important roles. Perturbations in placental angiogenesis can impair placental and maternal vasculature function with downstream adverse effects on fetal and maternal health. Down syndrome: also known as trisomy 21, is a genetic disorder resulting from the triplication of chromosome 21. The disorder is associated with aberrant neurological symptoms, which result in intellectual disability and early onset dementia. Hippocampus: a part of the brain that plays an important role in information consolidation, short- and long-term memory formation, attention and spatial navigation. Hypothalamic–pituitary–adrenal (HPA) axis: the neuroendocrine feedback interactions between the hypothalamus, the pituitary gland, and the adrenal glands that regulate several bodily functions, including stress reactivity, digestion, immune system function, and energy expenditure. Insulin-like growth factor 2 (IGF2): a growth factor that promotes placental and fetal cellular growth and division. It is encoded by an imprinted gene which is expressed only from the paternal allele. In mice, Igf2 expression is regulated by CpG methylation of the differentially methylated region 2 (DMR2) located within the last coding exon of Igf2. DMR2 methylation generates an active chromatin conformation which increases Igf2 gene expression. One-carbon metabolism: the transfer of activated one-carbon units (e.g., methyl groups). Choline functions in one-carbon metabolism as a source of methyl groups through its oxidative products betaine, dimethylglycine, and sarcosine. Other nutrients with important roles in mediating the transfer of one-carbon units include methionine, folate, vitamin B12, vitamin B6, and riboflavin. Placenta: a fetus-derived tissue connecting the fetus to the maternal uterine wall. The placenta exchanges nutrients, metabolic substrates, and respiratory gases between maternal and fetal circulation. In addition, the placenta is an endocrine organ that secretes several hormones, including corticotropin releasing hormone (primate placentas). Preeclampsia: a serious medical condition of pregnancy characterized by high blood pressure and proteinuria. Preeclampsia and its complications may persist into the post-partum period; treatments to control preeclampsia include antiplatelet and antihypertensive medications. The pathogenesis of preeclampsia is not entirely clear; however, an imbalance between pro- and anti-angiogenic factors in the maternal vasculature may play a role. Schizophrenia: a brain disorder characterized by psychotic behaviors, hallucinations, delusions, and impaired cognitive function. The etiology of schizophrenia is complex and likely includes both genetic and environmental components. Brain chemistry, structure, and function are altered with schizophrenia. Sensory gating: describes the capacity of the brain to filter out repeat and/or unimportant environmental stimuli. Cerebral inhibition, a measure of sensory gating, is evaluated by comparing the electrophysiologic response to a pair of repeated auditory stimuli, with a weaker response to the second stimulus versus the initial stimulus, indicating cerebral inhibition. Diminished sensory gating/cerebral inhibition (i.e., inability to ignore repeat stimuli) is a trait associated with schizophrenia and inattention. Trends in Endocrinology and Metabolism, May 2014, Vol. 25, No. 5

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Box 1. The role of choline in methylation To serve as a methyl donor, choline is first oxidized to betaine via a two-step reaction mediated by choline dehydrogenase and betaine aldehyde dehydrogenase (Figure I). One of the three methyl groups associated with betaine is subsequently transferred to homocysteine, forming methionine and dimethylglycine in a reaction catalyzed by betaine homocysteine N-methyltransferase (BHMT). The methionine produced by homocysteine remethylation can then be converted to Sadenosylmethionine (SAM), which is the universal methyl donor that

transfers its methyl group to various molecules, including DNA, proteins, and lipids, using over 50 methyltransferases. Because BHMT is only expressed in liver and to a lesser extent kidney, the use of betaine/choline as a methyl donor mainly occurs in these two organs. However, choline-derived methyl groups are made available to other tissues following their uptake of plasma methionine and SAM – which were initially generated via the hepatic or renal BHMT reaction [32,36,37].

CH3 CH3

– N+ – CH2 – CH2 – OH CH3

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Folate-mediated one-carbon metabolism

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• Synapse formaon

• Membrane biogenesis

• Placental development

• Myelinaon

Hcy

Betaine • Methyl group donaon

SAM

• Cell division • Lipid transport

Key: Methyl group

Histone methylaon

DNA methylaon

TRENDS in Endocrinology & Metabolism

Figure I. Biological functions of choline. Choline is the substrate for the synthesis of acetylcholine, phospholipids (e.g., phosphatidylcholine and sphingomyelin), and betaine. The functions of these choline derivatives are listed in their respective boxes. Betaine donates a methyl group to homocysteine (Hcy) to form methionine, which is converted to the universal methyl donor S-adenosylmethionine (SAM) and dimethylglycine (DMG). SAM-derived methyl groups are used for the synthesis of several metabolites (e.g., creatine, neurotransmitters, hormones, phosphatidylcholine) and for the methylation of DNA and histones. DMG and its demethylated derivative, sarcosine, may then be used as a source of one-carbon units for folate-mediated one-carbon metabolism.

may be a nutritional strategy for improving offspring health, this article will assess: choline-mediated changes on the fetal epigenome and on gene expression and thus fetal development; the supply of, and demand for, choline during pregnancy; and the impact of maternal choline supplementation on offspring physiologic systems. Maternal choline affects the fetal epigenome and developmental programming The prenatal period is associated with the establishment and maintenance of the epigenome (Box 2). Following conception, DNA methylation patterns of gametes are mostly abolished, and de novo methylation is required to establish an appropriate gene-silencing pattern. DNA methylation is tightly linked to histone modifications through the activities of methyl-binding proteins and histone-modifying proteins which, together with DNA methylation, orchestrate the spatial- and timesensitive epigenomic alterations that occur during fetal development [11]. 264

The fetal epigenome exhibits substantial plasticity: it can be altered by various maternal environmental factors including nutrition (e.g., starvation, methyl donor supply, protein availability) [12–14], maternal stress [15], seasonality [16], pollutants and chemicals (e.g., bisphenol A, lead, arsenic, or pesticides) [17–20], and substance abuse (e.g., alcohol and tobacco) [21,22]. The environmental conditions of the fetus can profoundly influence its biology and longterm health, a process known as developmental programming of adult disease. The pathways most sensitive to programming by in utero exposures include those related to cardiovascular diseases [23], type 2 diabetes [24,25], obesity [26], immunological diseases [27], and neural function [28]. Interestingly, developmental programming of adult disease differs between male and female offspring with males often exhibiting greater sensitivity to the maternal in utero environment [29–31]. Maternal choline supply during pregnancy has been shown to modify the epigenome of fetal liver [8,10] and brain [5,6,10] in animals, as well as the placenta and fetal

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Box 2. Mechanics of epigenetic modification The epigenome refers to epigenetic marks (e.g., DNA methylation) that modify the activity of the genome without altering its DNA sequence [26]. Epigenetic modifications can exert lasting effects on gene expression and physiologic systems [12,27,28,89] and may be inherited by future generations [90–92]. DNA methylation is the most common form of epigenetic modification and involves the addition of a methyl group to the 50 position of the pyrimidine ring of the cytosine base within cytosine-phosphate-guanine (CpG) dinucleotides. Although most of the CpG dinucleotides in the genome are methylated, promoter regions of genes frequently contain consecutive CpGs (i.e., CpG islands) that are hypomethylated. CpG islands play an important role in regulating gene expression [11] because their methylation is usually related to gene silencing (e.g., methylation inhibits the access of proteins, such as transcription factors, to the gene). CpG methylation is mediated by a family of

cord leukocytes in humans (see below) [32]. For example, fetuses of rodent mothers on a choline-deficient (vs control) diet during pregnancy exhibited hypermethylation of hepatic insulin-like growth factor 2 (Igf2), a gene involved in placental and fetal growth [8]. In the same study, fetal livers from choline-deficient (vs control) mothers exhibited hypomethylation and higher expression of Dnmt1 [DNA methyltransferase (cytosine-5) 1], and this correlated with the epigenetic and expression changes of hepatic Igf2, suggesting that maternal choline supply altered Igf2 methylation and expression through its effects on Dnmt1 [8]. In summary, maternal choline intake during pregnancy can influence offspring DNA methylation status and gene expression patterns in a complex manner and may initiate long-term developmental changes with lasting effects on offspring health. Choline supply during fetal development Choline derived from the maternal diet as well as phosphatidylcholine produced endogenously by the maternal liver is used to meet the needs of the developing fetus (Figure 1). Dietary choline can be obtained from plant and animal source foods; however, animal source foods contain significantly more choline per gram of food than plant source foods [2]. Choline can also be produced de novo in the maternal liver by phosphatidylethanolamine Nmethyltransferase (PEMT), an enzyme that sequentially methylates phosphatidylethanolamine (a non-choline-containing molecule) to phosphatidylcholine using SAM as the methyl donor. PEMT synthesis of phosphatidylcholine is upregulated during pregnancy through the binding of estrogen (which rises during pregnancy) to its response elements within the regulatory region of the PEMT gene [33]. Nevertheless, despite enhanced synthesis of endogenous choline during pregnancy, pregnant animals consuming a normal chow diet deplete their choline stores [34], underscoring the high demand for choline in this reproductive state and the importance of an adequate maternal choline intake. Choline is made available to the fetus via placental uptake of phosphatidylcholine-containing lipoproteins and free choline from maternal circulation, the levels of which rise in maternal blood throughout pregnancy [2,35]. Choline-derived methyl groups can also enter the placenta via the uptake of methionine and/or SAM originally

DNA methyltransferases, including DNMT1 which maintains DNA methylation patterns during cellular division, and DNMT3a and DNMT3b which catalyze de novo methylation during embryonic development [11]. Histone modifications (e.g., acetylation and methylation) are also common epigenetic marks. These modifications usually occur on lysine residues and change histone structure, thereby altering access of transcription factors to chromosomes [93]. Histone acetylation usually coincides with an open chromatin structure and transcriptional activation, whereas histone methylation associates with transcriptional repression, or activation, depending on the amino acid being methylated. Because epigenetic mechanisms regulate many important biological processes (through effects on gene expression and genome stability), these mechanisms are also involved in many diseases including cancer and diabetes.

produced in maternal liver [32,36,37]. Within the placenta, choline is used to synthesize acetylcholine, whereas choline-derived methyl groups can be used to methylate the placental genome [32,38]. Large amounts of choline are transferred to the fetus – as indicated by fetal cord blood choline concentrations that are 3–5 times higher than those in the maternal circulation [35,39]. High demand for choline during pregnancy The increased need for choline across gestation is best illustrated by the pronounced depletion of choline-derived methyl donors in pregnant (vs nonpregnant) women. Plasma betaine decreases by 60% during the second trimester [39,40] and becomes a stronger predictor of homocysteine, a risk factor for obstetric complications when elevated, than folate [39]. In a feeding study by Yan et al. [35], pregnant (vs nonpregnant) women consuming 480 mg choline/day [a level slightly above the current adequate intake (AI) of 450 mg/day] exhibited substantially lower circulating concentrations of several cholinederived methyl donors including 55% lower betaine, 38% lower dimethylglycine, and 49% lower sarcosine, across the third trimester. This pregnancy-induced depletion of choline-derived methyl donors arises in part from increased use of betaine as a methyl donor [35,41] and decreased production of betaine from choline due to the preferential partitioning of choline towards the cytidine diphosphate-choline (CDP-choline) pathway for phosphatidylcholine synthesis [41]. A reduced supply of cholinederived methyl donors may impair homocysteine remethylation (leading to elevations in homocysteine and depletions in SAM) and perturb folate mediated one-carbon metabolism (e.g., nucleotide biosynthesis and cellular methylation reactions) because dimethylglycine and sarcosine supply this metabolic network with one-carbon moieties [42] (see Figure I in Box 1). Pregnant women harboring genetic variants that increase choline requirements may be particularly susceptible to choline inadequacy during this reproductive state (Box 3). Notably, increased consumption of dietary choline during pregnancy can improve biomarkers of choline metabolism. For example, consumption of 930 versus 480 mg choline/day by third-trimester pregnant women led to higher circulating concentrations of several cholinederived methyl donors [35] and restored choline partitioning 265

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Maternal liver Choline

Betaine CDP-choline pathway

CDP-choline pathway

Met

SAM PEMT pathway

PC

Placenta PC

Choline

Met

SAM

G

C G C G C C G C G

DNA

Acetylcholine

Fetus

Key:

Methyl group TRENDS in Endocrinology & Metabolism

Figure 1. Choline metabolism and transfer among maternal, placental, and fetal compartments. In maternal liver, choline provides methyl groups for methionine (Met) and S-adenosylmethionine (SAM) synthesis. Phosphatidylcholine (PC) is synthesized via the cytidine diphosphate (CDP)-choline pathway and by the phosphatidylethanolamine N-methyltransferase (PEMT) de novo pathway. Choline metabolites are transported to the placenta, where SAM is used for methylation reactions including DNA methylation, and choline is used to synthesize PC and acetylcholine. Large amounts of free choline are transferred to the developing fetus. Choline can also enter the fetal compartment as PC (via lipoproteins made within the placenta [94]) and as acetylcholine.

Box 3. Genetic variation impacts choline utilization and requirement Single nucleotide polymorphisms (SNPs) in several genes encoding choline- and folate-metabolizing enzymes affect choline production, metabolism, and utilization, thus influencing dietary choline requirement. For example, SNPs in the PEMT (rs12325817) [95], CHDH (rs12676) [95], and 5,10-methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) (rs2236225) [96] genes increase susceptibility to liver and muscle dysfunction, indicators of choline insufficiency, among study participants consuming a low-choline diet [95]. In addition, homozygosity for the methylenetetrahydrofolate reductase (MTHFR) 677 C>T (rs1801133) variant alters choline biomarkers in circulation [97,98] and increases the use of choline as a methyl donor [99] among folate-deficient men. Finally, mouse models of BHMT and CHDH deletion suggest functional SNPs in these genes could have deleterious effects on one-carbon metabolism [100,101] and male fertility [101] in humans. In summary, individuals with SNPs that reduce efficient utilization and/or production of choline (e.g., CHDH rs12676, PEMT SNP rs12325817), or increase reliance on choline-derived methyl groups (e.g., MTHFD1 rs2236225, MTHFR rs1801133), need more dietary choline than individuals without such SNPs to satisfy somatic choline requirements and optimize health outcomes. 266

between the CDP-choline and betaine pathways (which compete for choline as a substrate) to a nonpregnant state [41]. In summary, the demand for choline during pregnancy appears to be very high and likely exceeds current choline intake recommendations. At present, an estimated 90% of pregnant women in the US are not meeting recommended choline intake levels [43] and most prenatal vitamins do not contain this essential micronutrient. Given its crucial role in fetal development, insufficient consumption of choline during pregnancy would be expected to have adverse health consequences for mother and child. Functional consequences of maternal choline supply during pregnancy Supplementing the maternal diet with extra choline during pregnancy has been shown to improve a variety of developmental and health outcomes in the offspring. The majority of this research has been conducted in rodent models; however, effects of maternal choline intake on health-related metabolic and physiologic processes in humans have emerged, indicating proof of concept for the benefits of maternal choline supplementation during pregnancy in humans.

Review Cognitive function Maternal nutrition can affect offspring cognitive function throughout the life course [44,45]. In rodents, a large body of research has consistently demonstrated that maternal choline supplementation during pregnancy enhances hippocampal function and improves performance on cognitive/ behavioral tests, particularly those evaluating spatial learning and memory or attention (reviewed by McCann et al. [46]). Longitudinal assessment of cognitive function in adult rats born to mothers supplemented with choline during pregnancy demonstrated better performance on spatial maze tasks at all ages compared to rats born to control or choline-deficient mothers. Moreover, an agerelated decrease in maze performance was observed only among rats born to control or choline-deficient mothers, indicating that maternal choline supplementation prevents age-related cognitive decline in these animals (reviewed by Meck and Williams [3]). Extra maternal choline during pregnancy has also been shown to exert lasting neuroprotective effects [47], and attenuate cognitive impairment associated with seizures [48–50] and prenatal alcohol exposure [51,52] in animals. Although not fully elucidated, the mechanism through which maternal choline intake affects offspring cognitive function appears to be multifaceted. First, choline-derived phospholipids (e.g., phosphatidylcholine) are crucial constituents of cellular membranes, and are in high demand during periods of rapid cell division and neuron myelination. Maternal choline intake can modulate phosphatidylcholine content in fetal brain [53] and thus affect structural integrity, function, and cell signaling. Second, the neurotransmitter acetylcholine is present in neural progenitor cells and functions in cholinergic neurotransmission [54]. Maternal choline supplementation of rodent mothers during pregnancy alters acetylcholine metabolism in offspring brain, with long-lasting effects on brain organization and function [55]. Maternal choline availability in rodents also alters cellular proliferation, differentiation, and apoptosis in the fetal hippocampus [56], as well as hippocampal morphology [57], neurogenesis [58], and long-term potentiation [59] in postnatal offspring hippocampus. Third, the persistent effects of maternal choline supplementation on offspring cognition strongly suggest an epigenetic mechanism that is mediated by the role of choline as a methyl donor [60]. Indeed, compared with control, a choline-deficient diet during pregnancy was shown to decrease fetal hippocampal DNA methylation of the Cdkn3 (cyclin-dependent kinase inhibitor 3) gene and increase expression of its protein product, kinase-associated phosphatase (Kap), a cell cycle regulator that inhibits cell proliferation [7]. Finally, choline supplementation in premenopausal women (and likely pregnant women in early gestation) increases the production of PEMT-derived phosphatidylcholine [41,61] which is enriched with docosahexaenoic acid (DHA) [62,63], a long-chain polyunsaturated fatty acid with crucial roles in brain development [64]. In human pregnancy, PEMT-phosphatidylcholine (vs phosphatidylcholine produced by the CDP-choline pathway, see Figure 1) is preferentially partitioned from the maternal to the fetal compartment [41]. Thus, supplementing the maternal diet with extra choline during early pregnancy

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may increase supply of choline and DHA to the developing fetus thereby improving brain development and ultimately cognitive function. Based on the beneficial effects of maternal choline supplementation on offspring cognitive function in rodents, the relationship between maternal choline intake/status during pregnancy and cognitive endpoints in human offspring are being explored. Wu et al. [65] found that maternal plasma concentrations of choline and betaine at 12 weeks of gestation were positively associated with measures of early cognitive development at age 18 months, indicating a possible benefit of choline supply on cognitive function in humans. Similarly, Boeke et al. [66] reported children born to mothers in the highest quartile of choline intake performed significantly better on a measure of visual memory at age 7 years. However, results of three other studies, including a randomized control trial supplementing mothers with choline from the second trimester through 3 months post-partum [67] failed to detect a benefit of higher maternal choline intake [67– 69]. The mixed findings of the human studies may be attributed to various factors including the age of the child at the time of assessment, the type of cognitive tests with which the children were assessed (e.g., IQ tests versus tests of memory or attention), the index of maternal choline intake or status, and/or additional confounders that may obscure the effect of choline nutrition during gestation on cognitive performance (e.g., socioeconomic status). Perhaps most importantly, it is very possible that the studies failing to detect a benefit of increased maternal choline intake on child cognitive function did not administer tests which sufficiently challenge memory or attentional function; animal studies have clearly shown that the benefits of increased maternal choline intake are only seen for tests which place great demands on these specific functions [46]. Overall, given the compelling animal data and encouraging results from some human studies, further examination of the relationship between maternal choline intake and cognitive function in child- and adulthood is warranted. Congenital defects and developmental disorders The effect of maternal choline intake and supplementation on the occurrence and severity of offspring congenital defects and developmental disorders has also been investigated. For example, Shaw et al. [70,71] have shown that maternal choline intake and choline concentrations in maternal circulation are inversely associated with offspring neural tube defect risk in humans, independently of folate. This protective effect of higher maternal choline on neural tube closure may be linked to the roles of choline in one-carbon metabolism and nervous system development. In a randomized placebo-controlled trial, Ross et al. [72] evaluated the effect of supplementing pregnant women with 900 mg choline/day (vs placebo) from the second trimester through parturition, and infants with 100 mg phosphatidylcholine/day (vs placebo) through 3 months post-partum, on infant cerebral inhibition – a measure of sensory gating that is diminished in the pathophysiology of schizophrenia. Perinatal choline supplementation (vs 267

Review placebo) yielded more infants within the normal cerebral inhibition range at the 5th postnatal week, indicating that choline supplementation during pregnancy and early postnatal life facilitates appropriate sensory gating development [72]. A possible mechanism involves CHRNA7 [cholinergic receptor, nicotinic, a7 (neuronal)] a candidate gene for schizophrenia [73] that encodes the a7-nicotinic acetylcholine receptor, which is crucial for hippocampal acetylcholine signaling and the development of cerebral inhibition. Infant CHRNA7 rs3087454 genotype was correlated with cerebral inhibition in the placebo group, but not in the treatment group, indicating that supplemental choline during the perinatal period may mitigate impaired functionality of the acetylcholine signaling system associated with CHRNA7 genetic variants, and reduce the risk of schizophrenia [72]. Using the Ts65Dn mouse model of Down syndrome, Strupp and colleagues [74,75] report that maternal choline supplementation during pregnancy and lactation substantially lessens the neurocognitive dysfunction seen in this genetic disorder. Specifically, trisomic offspring born to mothers that consumed extra choline performed better on tasks assessing attention, spatial cognition, or emotional regulation compared to unsupplemented Down syndrome mice, and on some tests performed similarly to their normal (disomic) littermate controls [74,75]. Maternal choline supplementation significantly increased adult offspring hippocampal neurogenesis [75] and increased the number and size of cholinergic neurons in the medial septum (which degenerate in Down syndrome with the onset of Alzheimer-like neuropathology) [76]. Importantly, both of these neural indices correlated with the performance of the mice in a test of spatial learning and memory [75,76], indicating functional relationships. Although much remains to be learned about the mechanisms by which maternal choline supplementation produces these beneficial effects in this Down syndrome mouse model, it is very likely that the basic mechanisms are the same as those that provide cognitive benefit in normal animals and other disease models (see previous text). The findings collectively encourage investigations of choline supplementation during human pregnancy as a strategy to alleviate cognitive impairment in Down syndrome offspring, and possibly reduce the incidence of Alzheimer’s disease in light of the early-onset Alzheimer-like neuropathology and dementia seen in Down syndrome [74]. Cancer Maternal choline supplementation during pregnancy may also affect cancer risk and severity in offspring. With a rat model of chemically induced breast cancer, Kovacheva et al. [9] investigated the effect of maternal choline intake (i.e., choline-deficient, control, and choline-supplemented) on mammary tumor growth among daughter offspring. Although 70% of offspring developed tumors regardless of maternal choline intake, the tumors of choline-supplemented offspring grew at a significantly slower rate compared with the choline-deficient group and exhibited a modified gene expression profile characteristic of improved survival humans [9]; genes associated with a favorable 268

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prognosis in human cancers were overexpressed whereas those associated with aggressive disease were underexpressed in the offspring of choline-supplemented dams. In addition, DNA methylation of the tumor-suppressor gene stratifin (Sfn) was proportional to maternal choline supply, suggesting that modulation of tumor growth and gene expression by maternal choline intake involved an epigenetic mechanism [9]. A similar study by Cho et al. [77] found that supplementing the maternal diet with several methyl nutrients (i.e., choline, betaine, methionine, and folate) also suppressed mammary carcinogenesis among daughter offspring. Maternal methyl donor intake did not affect global DNA methylation in offspring tumors; however, the expression of genes associated with tumor initiation and development (histone deacetylase 1, Hdac1; and methyl CpG binding protein 2, Mecp2) was reduced in offspring tumors of methyl-supplemented mothers. Taken together, these studies suggest that maternal methyl nutrient (e.g., choline) supplementation may reduce the risk of breast cancer development and severity among daughter offspring by altering the fetal epigenome. Hypothalamic–pituitary–adrenal (HPA) axis programming A recent human study by Jiang et al. [32] reported that a higher maternal choline intake may attenuate offspring HPA axis stress reactivity. In this controlled feeding study, pregnant women were randomized to consume 480 or 930 mg choline/day for 12 weeks during the third trimester of pregnancy [35]. Infants born to mothers who consumed 930 (vs 480) mg choline/day exhibited 33% lower cord venous plasma cortisol, a product of the HPA axis that is produced in response to stress [32]. Corticotropin releasing hormone (CRH) is a main stimulator of the HPA axis, and thus of cortisol production. During pregnancy, the placenta produces large amounts of CRH which can subsequently enter the fetal compartment and stimulate the fetal HPA axis [78]. CRH expression is regulated by DNA methylation, and expression patterns are established early in life [28,79,80]. Notably, Jiang et al. [32] detected higher CRH promoter methylation and lower CRH mRNA abundance in placentas from mothers consuming 930 versus 480 mg choline/day [32]. In addition, methylation status of cord leukocyte CRH and glucocorticoid receptor (GR) were lower in infants of mothers consuming more choline; effects which appear to be secondary responses to changes in placental CRH expression and reduced circulating cortisol in the fetus [32]. These data collectively imply that maternal choline supplementation may lower offspring circulating cortisol by altering the methylation state of cortisol-regulating genes in the placental and fetal compartments (Figure 2). Animal and human data show that a heightened HPA axis response increases vulnerability to stress-induced illnesses, such as hypertension and type 2 diabetes, throughout the life course [28,81]. Thus, the epigenetically mediated decrease in HPA axis reactivity in babies of mothers consuming roughly double the choline AI suggests maternal choline supplementation as a possible strategy for reducing the lifetime risk of stress-related diseases in

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Maternal liver Choline

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CRH

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Corsol Producon and in circulaon

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Methyl group TRENDS in Endocrinology & Metabolism

Figure 2. Proposed mechanism through which maternal choline intake affects circulating cortisol in the fetus. Maternal choline supplementation increases choline and its downstream metabolites betaine, methionine (Met), and S-adenosylmethionine (SAM) in the maternal liver. Methionine and SAM are transported to the placenta, where the methyl groups are used to increase corticotropin-releasing hormone (CRH) promoter region cytosine-phosphate-guanine (CpG) dinucleotide methylation, and decrease CRH expression. Less placental CRH is transferred to fetal circulation, and this decreases stimulation of the fetal HPA axis, resulting in reduced pituitary production of adrenocorticotropic hormone (ACTH) and decreased adrenal production of cortisol.

offspring. Nonetheless, future studies are warranted to determine whether the effects of extra maternal choline intake on HPA axis reactivity extend beyond the prenatal period. Placental angiogenesis Further exploring outcomes in the study where third-trimester pregnant women consumed controlled doses of 930 or 480 mg choline/day [35], Jiang et al. [38] reported that placentas from mothers consuming 930 mg choline/day (vs 480 mg/day) had 30% reduced transcript abundance of sFLT1 (soluble fms-like tyrosine kinase-1), an anti-angiogenic factor that is elevated in preeclampsia. Notably, protein concentrations of sFLT1 in maternal circulation were also reduced by 30% in the higher choline intake group, indicating that supplementing the maternal diet with extra choline ultimately lowered the amount of placenta-produced sFLT1 released into maternal circulation [38]. In the maternal circulation, sFLT1 sequesters vascular endothelial growth factor (VEGF), a pro-angiogenic protein that is required for endothelial health [82]. Sequestering of VEGF by sFLT1 inhibits VEGF signaling in the

maternal vasculature, thereby contributing to maternal endothelial cell dysfunction and the clinical abnormalities of preeclampsia, including hypertension, proteinuria, and glomerular endotheliosis [83]. Thus, supplementing the maternal diet with extra choline may be a nutritional strategy for mitigating some of the maternal endothelial cell perturbations that occur in diseases of placental dysfunction (Figure 3). The mechanism through which a higher maternal choline intake lowered placental production of sFLT1 has not been elucidated. Although the expression of sFLT1 is regulated (at least in part) by promoter methylation of the FLT1 gene [84], maternal choline intake did not modify the methylation state of this region [38], suggesting that mechanisms other than methylation could be influencing sFLT1 expression. In this regard, sFLT1 production was positively associated with placental acetylcholine concentrations and the expression levels of the placental acetylcholine receptor [i.e., cholinergic receptor, muscarinic 4 (CHRM4)], indicating that the changes in sFLT1 gene expression may be mediated by acetylcholine signaling [38]. Another possible route by which choline supply can 269

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(A) Preeclampsia

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Figure 3. Proposed mechanism through which maternal choline supplementation may restore maternal endothelial function. Maternal choline supplementation decreases the production of placental sFLT1 (soluble fms-like tyrosine kinase-1), a possible mediator of preeclampsia and maternal endothelial cell dysfunction. (A) Under the condition of preeclampsia, placental production of sFLT1 (from the FLT1 gene) increases. More sFLT1 is released into maternal circulation where it sequesters vascular endothelial growth factor (VEGF), prevents VEGF binding to its receptor (i.e., membrane-bound FLT1, mFLT1) on maternal endothelial cells, and contributes to endothelial cell dysfunction and the clinical manifestations of preeclampsia. (B) Maternal choline supplementation may restore maternal endothelial function by reducing placental sFLT1 production and increasing the availability of VEGF for binding to mFLT1 on maternal endothelial cells.

influence angiogenic processes is via protein kinase C (PKC). Specifically, in a human trophoblast cell culture model, moderate choline deficiency (vs choline sufficiency) elevated sFLT1 transcript abundance and impaired angiogenesis [85], whereas addition of a PKC inhibitor to the cells cultured in the choline-deficient medium lowered sFLT1 transcript abundance and restored angiogenesis [85]. Possible adverse effects of excess maternal choline intake during pregnancy on offspring health Studies supplementing choline to pregnant women have not found negative outcomes [35,72,86] and, to the best of our knowledge, no adverse effects of consuming extra choline alone during pregnancy in human or animals have been reported. However, a few animal studies suggest that consumption of a diet enriched with several methyl nutrients (i.e., choline, betaine, folic acid, and vitamin B12) during pregnancy may increase offspring risk of particular diseases. Schaible et al. [87] reported increased susceptibility to colitis in offspring of rodent mothers who had consumed a methyl nutrient-supplemented diet during pregnancy. Mechanistic studies revealed modified DNA methylation at loci associated with irritable bowel disease as well as gene expression patterns consistent with altered immune response in colonic mucosa of offspring of methylsupplemented mothers [87]. Similarly, maternal methyl nutrient supplementation during pregnancy increased severity of asthma-like allergic airway disease in mouse 270

offspring; hypermethylation and decreased expression of Runx3 (runt related transcription factor 3), a regulator of lymphocyte development, supported an epigenetic mechanism for the increased severity of allergic airway disease observed in offspring of methyl nutrient-supplemented mothers [88]. Overall, these findings indicate that there may be a U-shaped curve for specific health outcomes in response to methyl donor availability during early development, such that suboptimal outcomes may be seen with levels that are too low or too high. More studies are needed Box 4. Outstanding questions  Is supplementing the maternal diet with extra choline at the population level a nutritional approach for: reducing risk of pregnancy-related health disorders such as intrauterine growth restriction and preeclampsia, attenuating offspring HPA-reactivity to stress, and/or lowering the prevalence of schizophrenia?  Can prenatal, or early postnatal, choline supplementation improve cognitive function in normal infants and in those affected with Down syndrome or other disorders known to impair cognition (e.g., autism)?  What metabolic and physiologic systems in the offspring are influenced by maternal choline intake during pregnancy?  What are the genomic and epigenomic mechanisms through which maternal choline availability influences physiologic processes in the offspring?  Do the choline-mediated changes in the fetal epigenome and/or readouts of genomic activity observed at birth influence health outcomes later in life?  How much choline do pregnant women need to consume to optimize fetal and maternal health outcomes?

Review to discern a safe upper limit for methyl nutrient intake during pregnancy that improves target health outcomes without compromising others. Outstanding questions are listed in Box 4. Concluding remarks and future perspectives Supplementing the maternal diet with extra choline during pregnancy modulates the genomic expression of fetusderived tissues, and may beneficially influence several physiologic processes in the offspring (e.g., brain development, HPA-stress reactivity) and improve offspring health (e.g., cognitive function, lower risk of developmental and chronic diseases). The mechanism through which extra choline during pregnancy exerts its effects on offspring development and health is complex, and likely involves many factors including acetylcholine signaling, phospholipid availability, delivery of DHA, and epigenetic modifications. The epigenome of the placenta is especially responsive to maternal choline supplementation and appears to be an important mediator of the effect of choline on physiologic systems in human offspring. Because epigenetic modifications and changes in gene expression in early development can persist through adulthood, maternal choline supplementation may be a nutritional strategy to improve lifelong health of the child. Nonetheless, findings from the human research detailed herein are limited in their application to the population-atlarge by several factors, including: (i) small sample sizes; (ii) cessation of data acquisition at delivery or within the first postpartum year; (iii) sparse and mixed findings on the relationship between maternal choline supplementation and cognitive function in humans; and (iv) an incomplete understanding of genetic variants that may increase choline requirements during the perinatal period. Largescale choline intervention studies across human gestation with multiple years of follow-up and acquisition of genomic, epigenetic, and clinical readouts are warranted to determine: (i) the chronological and underlying causal relationships (e.g., in most cases, it is unclear whether the epigenetic alteration precedes, and contributes to, the functional outcome); (ii) the stability of the epigenetic modifications and/or physiologic alterations (e.g., at present, it is difficult to predict the effects of prenatal choline availability on gene expression during postnatal life); and (iii) the level of maternal choline intake, alone and in combination with other methyl-nutrients, that is necessary to promote optimal maternal and offspring health. Acknowledgments The authors would like to thank Barbara Strupp PhD and Jian Yan PhD for their critical review of the manuscript as well as the three anonymous reviewers for their astute comments and suggestions.

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