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Funder: Rural and Environment Science and Analytical Services Division; doi: http://dx.doi.org/10.13039/100011310, Epigenetic imprinting is important for neurogenesis and brain function. Hippocampal volumes and brain hyperintensities in late life have been associated with early life circumstances. Epigenetic imprinting may underpin these associations. Methylation was measured at 982 sites in 13 imprinted locations in blood samples from a longitudinal cohort by bisulphite amplicon sequencing. Hippocampal volumes and hyperintensities were determined at age 64y and 72y using MRI. Hyperintensities were determined in white matter, grey matter and infratentorial regions. Permutation methods were used to adjust for multiple testing. At 64y, H19/IGF2 and NESPAS methylation predicted hippocampal volumes. PEG3 predicted hyperintensities in hippocampal grey matter, and white matter. GNASXL predicted grey matter hyperintensities. Changes with age were predicted for hippocampal volume (MEST1, KvDMR, L3MBTL, GNASXL), white matter (MEST1, PEG3) and hippocampal grey matter hyperintensities (MCTS2, GNASXL, NESPAS, L3MBTL, MCTS2, SNRPN, MEST1). Including childhood cognitive ability, years in education, or socioeconomic status as additional explanatory variables in regression analyses did not change the overall findings. Imprinting methylation in multiple genes predicts brain structures, and their change over time. These findings are potentially relevant to the development of novel tests of brain structure and function across the life-course, strategies to improve cognitive outcomes, and our understanding of early influences on brain development and function.

[This corrects the article DOI: 10.1371/journal.pone.0211799.].

Purpose of review Epigenetics has emerged in recent years as one of the most important biological mechanisms linking exposures across the life course to long-term health. This article reviews recent developments in our understanding of the metabolic and genetic determinants of epigenetic variation in human populations. Recent findings Epigenetic status is influenced by a range of environmental exposures, including diet and nutrition, social status, the early emotional environment, and infertility and its treatment. The period around conception is particularly sensitive to environmental exposures with evidence for effects on epigenetic imprinting within the offspring. Epigenetic status is also influenced by genotype, and genetic variation in methylene tetrahydrofolate reductase, and the DNA methytransferase and ten-eleven translocation methylcytosine dioxygenase proteins has been linked to the epigenetic status, biological function and disease. Summary Epigenetics is at the heart of a series of feedback loops linking the environment to the human genome in a way that allows crosstalk between the genome and the environment it exists within. It offers the potential for modification of adverse epigenetic states resulting from events/exposures at earlier life stages. We need to better understand the nutritional programming of epigenetic states, the persistence of these marks in time and their effect on biological function and health in current and future generations.

Cognitive abilities vary among people. About 40–50% of this variability is due to general intelligence (g), which reflects the positive correlation among individuals' scores on diverse cognitive ability tests. g is positively correlated with many life outcomes, such as education, occupational status and health, motivating the investigation of its underlying biology. In psychometric research, a distinction is made between general fluid intelligence (gF) – the ability to reason in novel situations – and general crystallized intelligence (gC) – the ability to apply acquired knowledge. This distinction is supported by developmental and cognitive neuroscience studies. Classical epidemiological studies and recent genome-wide association studies (GWASs) have established that these cognitive traits have a large genetic component. However, no robust genetic associations have been published thus far due largely to the known polygenic nature of these traits and insufficient sample sizes. Here, using two GWAS datasets, in which the polygenicity of gF and gC traits was previously confirmed, a gene- and pathway-based approach was undertaken with the aim of characterizing and differentiating their genetic architecture. Pathway analysis, using genes selected on the basis of relaxed criteria, revealed notable differences between these two traits. gF appeared to be characterized by genes affecting the quantity and quality of neurons and therefore neuronal efficiency, whereas long-term depression (LTD) seemed to underlie gC. Thus, this study supports the gF–gC distinction at the genetic level and identifies functional annotations and pathways worthy of further investigation. publishedVersion

STUDY QUESTION Is DNA methylation in buccal cell DNA from children born following IVF (in vitro fertilization) and ICSI (intra-cytoplasmic sperm injection) different from that of spontaneously conceived children? SUMMARY ANSWER DNA methylation in the imprinted gene, small nuclear ribonucleoprotein polypeptide N (SNRPN), was higher in children conceived by ICSI and in those born to women with the longest duration of infertility regardless of the method of conception. WHAT IS KNOWN ALREADY Fertility treatment is associated with a small but significant increase in the risk of a range of adverse obstetric outcomes, birth defects and longer term sequelae, but the biological basis for this is unknown. A growing evidence base suggests that epigenetics may play a role in subfertility and the link between fertility and health. STUDY DESIGN, SIZE, DURATION In this retrospective cohort study of children born between 2002 and 2008, we measured DNA methylation in paternally expressed gene 3 (PEG3), insulin-like growth factor II (IGF2), SNRPN, long interspersed nuclear element 1 (LINE1) and the insulin gene (INS) in buccal cell DNA from children born following IVF (n = 49) and ICSI (n = 20) and compared them with a matched spontaneous conception group (n = 86). PARTICIPANTS/MATERIALS, SETTING, METHODS Participants were identified from the Aberdeen Maternity and Neonatal Databank and IVF and ICSI pregnancies were matched to spontaneous conception pregnancies on year of birth and maternal age at delivery. Only singleton pregnancies following fresh embryo transfer were included. DNA methylation was determined by pyrosequencing. Regression with adjustment for covariates was used to determine the effect of infertility on offspring DNA methylation. MAIN RESULTS AND THE ROLE OF CHANCE SNRPN methylation in the offspring was linked to fertility treatment in the parents. This effect was specific to children conceived using ICSI and was apparent in the comparison of ICSI versus spontaneous conception (1.03%; 95% CI 0.10, 1.97; P = 0.031), ICSI versus standard IVF (1.13%; 95% CI 0.04, 2.23; P = 0.043) and ICSI versus standard IVF and spontaneous conception (1.05; 95% CI 0.15, 1.94; P = 0.023). In all comparisons, the use of ICSI was associated with a higher level of SNRPN methylation in the offspring. A higher level of SNRPN methylation in the offspring was also associated with a longer duration of infertility in the parents. This was observed in all cases of infertility (0.18% per year of infertility; 95% CI 0.02, 0.33; P = 0.026) and after excluding ICSI cases (0.21% per year of infertility; 95% CI 0.04, 0.37; P = 0.017). There was a significant increase in the level of LINE1 methylation with age between birth and 7 years (0.77% per year; 95% CI 0.49, 1.05; P < 0.001). Methylation in the INS gene decreased significantly over the same period (−0.46% per year; 95% CI −0.89, −0.03; P = 0.035). There was no evidence from this cross-sectional data that methylation within the imprinted genes changed over the first 7 years of life. LIMITATIONS, REASONS FOR CAUTION The ICSI sample size was limited but the groups were carefully selected and well matched and the SNRPN findings were consistent across different outcomes. WIDER IMPLICATIONS OF THE FINDINGS The results of this study provide support for a role for epigenetics, and imprinting in particular, in fertility. The specific changes point to possible long-term consequences of fertility treatment for the health and fertility of future generations. STUDY FUNDING/COMPETING INTEREST(S) The authors report no conflict of interest in relation to this work. Funding was provided by the University of Aberdeen and the Scottish Government.

Background: Epigenetic regulation of imprinted genes and transposable elements has been implicated in human disease and may be affected by maternal diet. Objective: The objective was to determine the effect on offspring epigenetic status of nutritional and genetic factors that influence folate exposure in pregnancy. Design: We measured folate intake from diet, the use of folic acid supplements and the period of consumption, maternal and cord red blood cell (RBC) folate, and genotypes for 5 methylation cycle enzymes in a prospective cohort study of pregnancies in the United Kingdom between 2000 and 2006. We related these to offspring methylation status within 3 maternally methylated imprinted genes: paternally expressed gene 3 (PEG3), insulin-like growth factor 2 (IGF2), and small nuclear ribonucleoprotein polypeptide N, and the long interspersed nuclear element 1 (LINE-1) in genomic DNA extracted from whole blood in 913 pregnancies. Results: Supplement use after 12 wk of gestation was associated with a higher level of methylation in IGF2 (+0.7%; 95% CI: 0.02, 1.4; P = 0.044) and reduced methylation in both PEG3 (20.5%; 95% CI: 20.9, 20.1; P = 0.018) and LINE-1 (20.3%; 95% CI: 20.6, 20.04; P = 0.029). The same pattern was observed in relation to RBC folate in the cord blood at birth: IGF2 (P = 0.038), PEG3 (P , 0.001), and LINE-1 (P , 0.001). LINE-1 methylation was related to maternal RBC folate (P = 0.001) at 19 wk. No effect of supplement use up to 12 wk (current recommendation) was found. Conclusions: Folic acid use after 12 wk of gestation influences offspring repeat element and imprinted gene methylation. We need to understand the consequences of these epigenetic effects. Am J Clin Nutr 2013;97:94‐9.

Cognitive decline is a feared aspect of growing old. It is a major contributor to lower quality of life and loss of independence in old age. We investigated the genetic contribution to individual differences in nonpathological cognitive ageing in five cohorts of older adults. We undertook a genome-wide association analysis using 549 692 single-nucleotide polymorphisms (SNPs) in 3511 unrelated adults in the Cognitive Ageing Genetics in England and Scotland (CAGES) project. These individuals have detailed longitudinal cognitive data from which phenotypes measuring each individual's cognitive changes were constructed. One SNP - rs2075650, located in TOMM40 (translocase of the outer mitochondrial membrane 40 homolog) - had a genome-wide significant association with cognitive ageing (P=2.5 × 10 -8). This result was replicated in a meta-analysis of three independent Swedish cohorts (P=2.41 × 10 -6). An Apolipoprotein E (APOE) haplotype (adjacent to TOMM40), previously associated with cognitive ageing, had a significant effect on cognitive ageing in the CAGES sample (P=2.18 × 10 -8; females, P=1.66 × 10 -11; males, P=0.01). Fine SNP mapping of the TOMM40/APOE region identified both APOE (rs429358; P=3.66 × 10 -11) and TOMM40 (rs11556505; P=2.45 × 10 -8) as loci that were associated with cognitive ageing. Imputation and conditional analyses in the discovery and replication cohorts strongly suggest that this effect is due to APOE (rs429358). Functional genomic analysis indicated that SNPs in the TOMM40/APOE region have a functional, regulatory non-protein-coding effect. The APOE region is significantly associated with nonpathological cognitive ageing. The identity and mechanism of one or multiple causal variants remain unclear. © 2014 Macmillan Publishers Limited.

Deposition of fat in the fetus increases exponentially with gestational age, reaching its maximal rate—around 7 g/day or 90% of energy deposition—at term. In late pregnancy, many women consuming contemporary Western diets may not be able to meet the fetal demand for n-3 long chain polyunsaturated fatty acids (LCPUFAs) from the diet alone. Numerous mechanisms have evolved to protect human offspring from extreme variation or deficiency in the maternal diet during pregnancy. Maternal adipose tissue is an important source of LCPUFA. Temporal changes in placental function are synchronized with maternal metabolic and physiological changes to ensure a continuous supply of n-3 and n-6 LCPUFA-enriched fat to the fetus. LCPUFA storage in fetal adipose tissue provides an important source of LCPUFA during the critical first months of postnatal life. An appreciation of these adaptations is important in any nutritional strategy designed to improve the availability of fatty acids to the fetus.

Summary Background There is a need to understand what affects the success of in-vitro fertilisation (IVF) and the rate of resulting twin births so that pregnancy rates can be improved and multiple gestations avoided. Our aim was to assess the role of B vitamins and genetics. Methods We did a prospective cohort study of 602 women undergoing fertility treatment. We assessed intake of folate and vitamin B12 with a questionnaire and measured their plasma and red-blood-cell concentrations by radioimmunoassay. We measured five B-vitamin-related gene variants in women who received treatment and in 932 women who conceived naturally. Findings The likelihood of a twin birth after IVF rose with increased concentrations of plasma folate (1·52, 1·01–2·28; p=0·032) and red-cell folate (1·28, 1·00–1·65; p=0·039). There was no association between folate and vitamin B12 levels and likelihood of a successful pregnancy. Women homozygous for the 1298 CC variant of methylenetetrahydro-folate reductase ( MTHFR ), rather than the AA variant, were less likely to produce a livebirth after IVF (0·24, 0·08–0·71; p=0·003) or to have had a previous pregnancy (0·42, 0·21–0·81; p=0·008). Interpretation Our findings suggest that MTHFR genotype is linked to a woman's potential to produce healthy embryos (possibly through interaction with genes related to DNA methylation). In women likely to have a successful IVF pregnancy, high folate status increases the likelihood of twin birth after multiple embryo transfer. Proposals to fortify the UK diet with folic acid could lead to an increase in the number of twins born after IVF.

Twins can be used to investigate the biological basis for observed associations between birth weight and later disease risk, as they experiencein uterogrowth restriction compared with singletons, which can differ in magnitude within twin pairs despite partial or total genetic identity. In the present study, sixty monozygotic and seventy-one dizygotic same-sex twin pairs aged 19–50 years and eighty-nine singleton controls matched for age, gestational age, sex, maternal age and parity were recruited from an obstetric database. Associations between fasting lipid levels and birth weight were assessed by linear regression with adjustment for possible confounding factors. Twins were significantly lighter at birth but were not significantly different in adult height, weight or lipid levels from the singleton controls. There was a significant inverse association between birth weight and both total and LDL-cholesterol levels among singleton controls (−0·53mmol/l per kg (95% CI −0·97, −0·09),P=0·02 and −0·39mmol/l per kg (95% CI −0·76, −0·02),P=0·04, respectively), but there was no significant association between birth weight and lipid levels in either unpaired or within-pair analysis of twins. The results suggest that thein uterogrowth restriction and early catch-up growth experienced by twins does not increase the risk of an atherogenic lipid profile in adult life.

More than 90 per cent of the fat deposition in the fetus occurs in the last 10 weeks of pregnancy during which it increases exponentially to reach a rate of accretion of around 7g/day close to term. All of the n -3 and n -6 fatty acid structure acquired by the fetus has to cross the placenta and fetal blood is enriched in long chain polyunsaturated fatty acids (LCPUFA) relative to the maternal supply. The placenta may regulate its own fatty acid substrate supply via the action of placental leptin on maternal adipose tissue. Fatty acids cross the microvillous and basal membranes by simple diffusion and via the action of membrane bound and cytosolic fatty acid binding proteins (FABPs). The direction and magnitude of fatty acid flux is mainly dictated by the relative abundance of available binding sites. The fatty acid mix delivered to the fetus is largely determined by the fatty acid composition of the maternal blood although the placenta is able to preferentially transfer the important PUFA to the fetus as a result of selective uptake by the syncytiotrophoblast, intracellular metabolic channelling of individual fatty acids, and selective export to the fetal circulation. Placental FABP polymorphisms may affect these processes. There is little evidence to suggest that placental delivery of fatty acids limits normal fetal growth although the importance of the in utero supply may be to support post-natal development as most of the LCPUFA accumulated by the fetus is stored in the adipose tissue for use in early post-natal life.

The role of the placenta in controlling the supply of fatty acids to the fetus was investigated in term placentas from non-smokers (n5), smokers (>ten cigarettes/d;n5) and after addition of ethanol at 2 mg/ml (n4). The maternal side was of the placenta was perfusedex vivofor 90 min with a physiological mixture of fatty acids and fatty acid:human albumin ratio. There was no effect of smoking on the transfer of linoleic (LA, 18 : 2n-6), α-linolenic (αLN, 18 : 3n-3), arachidonic (AA, 20 : 4n-6) or docosahexaenoic acid (DHA, 22 : 6n-3), expressed per perfused area (calculated from H218O exchange). However, the presence of ethanol in the perfusate at a concentration of 2 mg/ml significantly reduced (Pn-3 polyunsaturated fatty acids, αLN and DHA. This specific effect of ethanol on αLN and DHA also resulted in an altered selectivity for transfer of individual fatty acids. In the non-smoking control group the placenta selectively transferred polyunsaturated fatty acids to the fetus in the order DHA>AA>αLN>LA. The order of selectivity was unaltered in placentas from smokers, but the addition of ethanol to the perfusion medium altered the order of selectivity to AA>αLN>LA>DHA. The presence of ethanol in the perfusate was also associated with a significant reduction (P218O. These results suggest that the presence of ethanol at a concentration of 2 mg/ml may reduce the availability of polyunsaturated fatty acids to the developing fetus.

The objective of this study was to determine whether the ability of the human placenta to transfer glucose and fatty acids is related to normal fetal growth. The intrinsic nutrient transport capacity of the placenta was measured under standardized conditions during in vitro perfusion of 30 human term placentas and related to birth weight (range 2640–4640g), birth weight centile (8th–99th), ponderal index (2.43–3.69), placental weight (418–1030g) and placental:fetal weight (0.14–0.31). There was no statistically significant change in the rate of nutrient transfer per placenta or per kg fetal weight, with birth weight, birth weight centile, ponderal index, placental weight and placental:fetal weight. There was a weak but significant relationship ( P =0.020, r 2 =9 per cent) between the ratio of glucose to fatty acid transport and birth weight centile, largely due to the high ratio found in the lowest birth weight quartile where the babies are thinnest. This study provides no evidence that placental nutrient transport capacity limits fetal growth across a wide range of birth weights in normal pregnancies. It is proposed that the fetus itself may regulate placental nutrient transport in vivo via the fetal cardiac output and the rate of fetal nutrient utilization.

The aim of this study was to determine the effect of fatty acid intake and insulin dependent diabetes on the fatty acid composition of maternal erythrocytes, the placenta and cord. Fatty acid intake (from food frequency questionnaire) and the fatty acid composition of maternal erythrocytes, the placenta and cord from pregnant vegetarians (n = 4) and insulin dependent diabetics (n = 5) was compared with pregnant omnivores (n = 10). There was a significantly lower intake of n-6 long chain polyunsaturated fatty acid (LCPUFA) (-75% P < 0.01) and n-3 LCPUFA (-92% P < 0.01) and increased ratio of n-6/n-3 LCPUFA in the vegetarians (103%; P < 0.001). The concentrations of 22:4 n-6 (+28%; P < 0.05) and 22:5 n-3 (+40%; P < 0.05) were higher in vegetarian erythrocytes. Placental 18:2 n-6 (+26.9%; P < 0.05) 18:3 n-3 (+139%; P < 0.05) and 22:5 n-3 (+24%; P < 0.05) were increased while 20:5 n-3 (-36%; P < 0.05), 22:6 n-3 (-16%; P = 0.059), and the ratios of 20:4 n-6/18:2 n-6 (P < 0.01) and 22:6 n-3/18:3 n-3 were reduced. 22:6 n-6 (-49%; P < 0.05) and total n-3 LCPUFA (-11%; P < 0.01) were reduced in vegetarian cord. For the diabetic mothers, all of the n-6 LCPUFA and n-3 LCPUFA were reduced in the maternal erythrocytes; 22:4 n-6 (-42%; P < 0.05), 22:5 n-6 (-46%; P < 0.05) and 22:6 n-3 (-41%; P < 0.05). For the diabetic placenta and cord the general pattern of n-3 LCPUFA was the same as that in the vegetarians. In the vegetarian mothers, the PUFA profiles in the maternal erythrocytes, placenta and cord are consistent with an elevation in the rate of LCPUFA synthesis in order to make up the relative deficit in LCPUFA intake. However, it may be that the higher level of desaturase activity is not able to overcome the dietary deficit of 22-6 n-3 and 22:6 n-6. Despite the fact that the dietary LCPUFA intake in the pregnant diabetic was comparable with that in the pregnant 'normal' omnivore mothers, the pattern of PUFA in the tissues resembled that of the vegetarian mothers.

The role of the placenta in controlling the supply of fatty acids to the fetus was investigated in term placentae (n = 9) from normal pregnancies. The maternal side was perfused ex vivo for 90 min with a modified Krebs Ringer solution containing a physiological mixture of fatty acids and ratio of fatty acid to human albumin. There was no evidence of chain elongation and desaturation of the essential fatty acids. Relative to the value for oleic acid, the rate of transfer to the fetal circulation was: 1.30 +/- 0.02 (P0.001) for linoleic acid, 1.61 +/- 0.09 (P = 0.002) for alpha-linolenic acid, 0.67 +/- 0.10 (P = 0.033) for arachidonic acid and 2.10 +/- 0.16 (P = 0.003) for docosahexaenoic acid. For tissue accumulation the values were 1.47 +/- 0.39 (P0.001) for linoleic acid, 2.24 +/- 0.37 (P = 0.027) for alpha-linolenic acid, 9.84 +/- 1.03 (P = 0.001) for arachidonic acid, and 3.01 +/- 0.79 (P = 0.064) for docosahexaenoic acid. The order of selectivity for transfer from the maternal to the fetal circulation was docosahexaenoicalpha-linoleniclinoleicoleicarachidonic acid. Such a mechanism would allow the preferential transfer of docosahexaenoic acid and the essential fatty acids to the fetal circulation, thereby protecting the polyunsaturated fatty acid supply to the fetus during a critical period of development.

The human diet has undergone profound changes over recent generations and this trend is likely to accelerate in the 21st century. Innovations in food technology, new ways of producing and processing foods and the increasing use of artificial vitamins and novel ingredients are changing the human diet in ways that our dietary monitoring systems struggle to keep pace with. There is a growing awareness of the importance of diet, but little understanding of how these changes may affect the health of current and future generations. Epigenetic programming, and specifically the persistence of functional epigenetic states following nutritional exposure, is particularly relevant to the issue of dietary change. Epigenetics is emerging as perhaps the most important mechanism through which diet and nutrition can directly influence the genome and there is now considerable evidence for nutritional epigenetic programming of health and the response to diet itself. A number of nutrients and food components that are changing in the human diet have been shown to produce epigenetic states that are stable across different timescales. We need to better understand the nutritional programming of epigenetic states, the persistence of these marks in time and their effect on biological function and the response to diet.

The main nutritional role of the placenta is to provide the correct mix of nutrients in sufficient quantities to support fetal growth and development throughout pregnancy while coping with wide variations in maternal nutrient intake between pregnancies and temporal variations within a pregnancy. The placenta represents a nutritional ‘bottleneck’ where competition for nutrient transporters and metabolic selectivity, acting in concert with maternal adaptations, allows the placenta to regulate the nutrient mix within the fetal circulation. The fetus itself also plays an active role in regulating key aspects of placental transport in order to meet its own nutrient requirements.

Epigenetic regulation is central to genome structure and function. Epigenetic status varies between individuals, and there is increasing awareness of the importance of this variation in health and disease. Epigenetic mechanisms include DNA methylation, histone modification, and regulation by noncoding RNAs. Epigenetic control is central to the way in which the genome interacts with, and responds to, the environment and even potentially the way in which the genome can influence its own environment via effects on behavior. The substrates for epigenetic reactions (acetyl and methyl groups) are central to nutritional metabolism, and there is ample evidence for nutritional effects on the epigenome. Challenges in human nutritional epigenetics research include the problem of tissue-specific epigenomes and heterogeneity of response by epigenetic loci. The promise of nutritional epigenetics is that it will help elucidate the way in which nutrition can influence health through direct effects on the genome.

Contributors. 1. Introduction (Mihai D. Niculescu and Paul Haggarty). PART I: Fundamental Principles in Epigenetics. SECTION A: Epigenetic Mechanisms. 2. DNA Methylation (Natalia V. Cucu). 3. Chromatin Modifications (Sandra B. Hake). 4. Roles of RNAi and Other MicroRNAs in the Regulation of Epigenetic Processes (Muller Fabbri). 5. Epigenetic Inheritance: Both Mitotic and Meiotic (Nina J. Kaminen-Ahola, Arttu I. Ahola, and Emma Whitelaw). SECTION B: Development Epigenetics. 6. Developmental Epigenetics: Roles in Embryonic Development (Liliana Burliba a and Lucian Gavril ). SECTION C: Epigenetic Mechanisms in Disease. 7. Epigenetics, Nutrition, and Cancer (Amy R. Johnson). 8. Metabolic Syndrome, Obesity, and Diabetes (Karen D. Corbin). 9. Autoimmunity (Donna Ray and Raymond Yung). 10. Cardiovascular Diseases (Aurelian Bidulescu and Methode Bacanamwo). PART II: Nutritional Status and Specific Nutrients. SECTION A: Maternal Nutrition and Fetal Development. 11. Maternal Nutrition and Developmental Outcomes (Shannon L. Haley, Laurie J. Moyer-Mileur, Robert H. Lane, and Lisa A. Joss-Moore). SECTION B: Role of Specific Nutrients. 12. Folate, Vitamin B12, and Vitamin B6 (Patrick J. Stover). 13. Dietary Choline, Betaine, Methionine, and Epigenetic Mechanisms Influencing Brain Development (Steven H. Zeisel). 14. Epigenetic Regulation by Retinoids (Amandio Vieira). 15. We are WhatWe Eat: How Nutritional Compounds Such As Isoflavones Shape Our Epigenome (Carlos M. Guerrero-Bosagna and Susan J. Clark). 16. Isothiocyanates and Polyphenols (Nigel J. Belshaw and Ian T. Johnson). SECTION C: Macronutrient Intakes. 17. The Effect of Maternal Macronutrient Intake on Phenotype Induction and Epigenetic Gene Regulation (Karen A. Lillycrop, Mark A. Hanson, and Graham C. Burdge). SECTION D: Environmental Exposures. 18. Epigenetic Manifestation of Environmental Exposures (Dana C. Dolinoy, Olivia S. Anderson, and Laura S. Rozek). SECTION E: Epidemiology of Nutritional Epigenetics. 19. Epigenetics, Nutrition, and Reproduction: Short- and Long-Term Consequences (Paul Haggarty). 20. Nutrition, Epigenetics, and Cancer: An Epidemiological Perspective (Audrey Jung and Ellen Kampman). Index.

It has been proposed that altered epigenetic status at imprinted loci may play a role in the early programming of disease susceptibility (1) . The ultimate methyl donor for methylation reactions is the folate-methylation cycle and feeding pregnant dams diets deficient in methyl donors results in altered regulation of specific genes in the offspring which are under imprinting control (2) . The aim of this study was to determine whether a similar phenomenon may occur in human pregnancy. The study was approved by Grampian Research Ethics Committee and all participants gave informed written consent. We collected data on dietary and supplement intake and cord blood DNA from pregnancies sequentially recruited at Aberdeen Maternity Hospital. Energyadjusted intake of the B-vitamins (thiamine, niacin, biotin, riboflavin, folate, B6 and B12) from the diet was assessed at 19 weeks gestation using a self-administered food frequency questionnaire (3) . The use of folic acid supplements (type/brand, amount per day, timing and duration of consumption in relation to stage of pregnancy) were also recorded. Multiple methylation sites were measured in three imprinted genes (IGF2, n 374; SNRPN, n 363; PEG3, n 377) and LINE1 (n 372) which was thought to contribute to the differential expression of paternally and maternally imprinted genes. Methylation was determined by pyrosequencing using a PyroMark MD system (Qiagen, Crawley, UK) after bisulphite conversion of DNA using Epitect Bisulfite kits (Qiagen, Crawley, UK). Statistical analysis was carried out using STATA 11MP (Stata Corp, College Station, TX, USA). All the B vitamins from diet were included in the regression model. There was no evidence that dietary intake of B vitamins was related to LINE1 or SNRPN methylation. Dietary intake of riboflavin, vitamin B6 and folate were associated with the average methylation status of both IGF2 (riboflavin, P = 0.012; vitamin B6, P = 0.047; folate, P = 0.024; model r 2 = 3.9%) and PEG3 (riboflavin, P = 0.016; vitamin B6, P = 0.014; folate, P = 0.001; model r 2 = 2.6%). Although responsive to the same nutrients, there were differences between PEG3 and IGF2 in the sign of the response. Separate analysis of supplement use provided no evidence for an effect on the methylation of folic acid intake in line with current advice. The way in which parental diet influences the methylation of individual genes and the health consequences for the offspring merit further study. Further work on this phenomenon in larger numbers of pregnancies is proposed.

BACKGROUND: Little is known of fatty acid metabolism in human embryos. This information would be useful in developing metabolic tests of embryo quality and improving embryo culture media. METHODS: The fatty acid composition of human embryos and their ability to accumulate 13 C labelled fatty acids was assessed in relation to the stage of development using gas-chromatography and combustion-isotope-ratio-mass spectrometry. RESULTS: Compared with embryos which did not develop beyond the 4-cell stage, those that did had significantly higher concentrations of the unsaturates, linoleic (12% versus 3%; P = 0.02) and oleic (14% versus 7%; P = 0.02), and a lower concentration of total saturates (62% versus 77%; P = 0.04). There was uptake of both 13 C linoleic and palmitic, but the developmental pattern was different for each fatty acid. The net accumulation in pmol/embryo/24h for palmitic was 1 at the 2-cell to

The fetus has an absolute requirement for the n-3/n-6 fatty acids and docosahexaenoic acid (22:6 n-3; DHA) in particular is essential for the development of the brain and retina. Most of the fat deposition in the fetus occurs in the last 10 weeks of pregnancy. The likely rate of DHA utilisation during late pregnancy cannot be met from dietary sources alone in a significant proportion of mothers. De novo synthesis makes up some of the shortfall but the available evidence suggests that the maternal adipose tissue makes a significant contribution to placental transport to the fetus. The placenta plays a crucial role in mobilising the maternal adipose tissue and actively concentrating and channelling the important n-3/n-6 fatty acids to the fetus via multiple mechanisms including selective uptake by the syncytiotrophoblast, intracellular metabolic channelling, and selective export to the fetal circulation. These mechanisms protect the fetus against low long-chain polyunsaturated fatty acid (LCPUFA) intakes in the last trimester of pregnancy and have the effect of reducing the maternal dietary requirement for preformed DHA at this time. As a result of these adaptations, small changes in the composition of the habitual maternal diet before pregnancy are likely to be more effective in improving LCPUFA delivery to the fetus than large dietary changes in late pregnancy. There is little evidence that DHA intake/status in the second half of pregnancy affects visual and cognitive function in the offspring, but more studies are needed, particularly in children born to vegetarian and vegan and mothers who may have very low intakes of DHA.

Objective: To study the effects of ω-3 and ω-6 polyunsaturated fatty acid (PUFA) on in vitro proliferation of endometrial cells and their production of the cytokine interleukin-8 (IL-8). Design: In vitro study. Setting: Obstetrics and gynecology department, University of Aberdeen. Patient(s): Women attending an infertility clinic. Intervention(s): In vitro cell cultures using culture mediums supplemented with normal and high ratios of ω-3 PUFA and ω-6 PUFA. Main Outcome Measure(s): In vitro survival and production of IL-8 by dispersed endometrial cells. Result(s): In vitro survival of endometrial cells from women with and without endometriosis was significantly reduced in the presence of high ω-3:ω-6 PUFA ratios compared with cells incubated in the absence of fatty acids, in balanced ω-3:ω-6 PUFA ratios, and in high ω-6:ω-3 PUFA ratios. Endometrial cells from women with endometriosis secreted higher concentrations of IL-8, especially in the presence of high ω-3:ω-6 PUFA ratios. Conclusion(s): ω-3 PUFA may have a suppressive effect on the in vitro survival of endometrial cells and ω-3 PUFA be useful in the management of endometriosis by reducing the inflammatory response and modulating cytokine function.

An underlying assumption when estimating total energy expenditure (TEE) using doubly labelled water (DLW) is that the injected isotopes (lsO and 2H) leave the body only in the form of CO, and H20. However, both isotopes have additional routes of loss. We quantified the loss of 2H (i) attached to faecal solids and (ii) by sequestration into newly synthesised fat in reindeer (Rangifer tarandus tarandus). Estimates of the errors caused by these processes were applied to data from DLW studies with reindeer in summer and in winter. Given the net rate of faecal dry matter output and lipid synthesis in the present study, ignoring both sources of error caused the TEE of reindeer to be underestimated by approximately 5% in winter and approximately 9% in summer. The separate effect of each source of error was evaluated in summer. If ignored, loss of 2H through sequestration alone caused TEE to be underestimated by approximately 3.7%. Similarly, if ignored, loss of 2H attached to faecal solids alone caused TEE to be underestimated by approximately 5.9%.

The role of the placenta in controlling the supply of fatty acids to the fetus was investigated in term placentas (n = 5) from normal pregnancies. The maternal side was perfused ex vivo for 90 min with a modified Krebs Ringer solution containing a physiological mixture of fatty acids – designed to mimic the composition of non-esterified fatty acids (NEFA) measured in the last trimester of pregnancy (n = 10) – and ratio of fatty acid to human albumin. The selectivity for α-linolenic acid (αLN) transfer to the fetal circulation was not significantly different from that observed when using the triglyceride (TG) composition (1.21 ± 0.04), but significantly different for AA (1.43 ± 0.12; p < 0.001) and docosahexaenoic acid (DHA; 2.02 ± 0.09; p = 0.048). The absolute rate of transfer (nmol · ml–1) compared to that using the TG maternal perfusate composition was significantly different for LA (0.562 ± 0.038; p = 0.50), αLN (0.130 ± 0.009; p < 0.001), arachidonic acid (AA; 0.218 ± 0.022; p = 0.001) and DHA (0.383 ± 0.04; p < 0.001). Thus, placental selectivity for αLN and DHA appears to be relatively unresponsive to changes in the mixture of fatty acids in the maternal circulation but the selectivity for AA increased with the increase in the maternal AA concentration. For an 8-fold increase in the concentration of DHA in the maternal circulation there was a 13-fold increase in the transfer of DHA to the fetal circulation. For a 2-fold increase in the concentration of AA, transfer was increased 8-fold. For a 1.3-fold increase in the concentration of αLN, transfer was increased 2.1-fold. These results suggest that the maternal concentration of individual fatty acids, and hence the composition of the maternal diet, can have large effects on polyunsaturated/long-chain polyunsaturated fatty acids delivery to the fetus.

The investigators conducted a prospective cohort study of 602 women treated for infertility who underwent in vitro fertilization (IVF). The intake of folate and vitamin B 12 was estimated from responses to a questionnaire, and plasma and red blood cell concentrations were determined by radioimmunoassay. In addition, buccal cells were sampled to measure 4 B vitamin-related gene variants in the study women and also in 932 women who conceived naturally. Fertility was ascribed to a male factor in 44% of cases and to a female factor in 33%; in the remaining 23% of cases, no cause was apparent. The mean duration of infertility was 5 years. The median energy-adjusted intakes of folate and vitamin B 12 from food were 311 μg and 6.5 μg, respectively, in women having IVF, figures that were similar to those reported for nonpregnant women of the same age and geographic area. Nearly three fourths of women reported taking the recommended daily dose of folic acid, 400 μg daily. Overall folate intake correlated significantly with age-adjusted plasma and red cell folate concentrations. Neither the intake nor blood level of folate and vitamin B 12 was significantly related to the rate of live births or pregnancy loss after IVF. Total folate intake was significantly associated with twin births as were high plasma B 12 levels. Twin births were most frequent in connection with relatively low age plus a high plasma folate level. Women found to be homozygous for the 1298 CC variant of methylenetetrahydrofolate reductase (MTHFR) rather than the AA variant were less likely to produce a living infant after IVF and also less likely to have had a previous pregnancy. There was no indication that MTHFR A1298C interacted with folate or vitamin B 12 intake to determine the outcome of fertility treatment. These findings suggest that in women who are likely to have a successful pregnancy after IVF, high folate status makes a twin birth more likely when multiple embryos are transferred. The practice of fortifying the diet with folic acid might lead to more twin births after IVF.

Background Birth weight and prematurity are important obstetric outcomes linked to lifelong health. We studied a large birth cohort to look for evidence of epigenetic involvement in birth outcomes. Methods We investigated the association between birth weight, length, placental weight and duration of gestation and four candidate variants in 1,236 mothers and 1,073 newborns; DNMT1 (rs2162560), DNMT3A (rs734693), DNMT3B (rs2424913) and DNMT3L (rs7354779). We measured methylation of LINE1 and the imprinted genes, PEG3, SNRPN, and IGF2, in cord blood. Results The minor DNMT3L allele in the baby was associated with higher birth weight (+54 95% CI 10,99 g; p = 0.016), birth length (+0.23 95% CI 0.04,0.42 cm; p = 0.017), placental weight, (+18 95% CI 3,33 g; p = 0.017), and reduced risk of being in the lowest birth weight decile (p = 0.018) or requiring neonatal care (p = 0.039). The DNMT3B minor allele in the mother was associated with an increased risk of prematurity (p = 0.001). Placental size was related to PEG3 (p

Energy expenditure was measured over 10 d using the doubly-labelled water (DLW) and activity diary methods in summer and winter in subjects with ‘light’ occupations but leisure activities which ranged from ‘non-active’ to ‘very active’. The basal metabolic rate (BMR) and the energy cost of activities were determined by indirect calorimetry. The Department of Health (1991) predicted BMR for the group (6·89 (SD 0·30) MJ/d; n 18) was not significantly different from the measured value (7·17 (SD 0·70) MJ/d; n 18). The range of DLW-derived expenditure values within the group was BMR X 1·41 to 2·41. The largest seasonal change within individuals was BMR X 0·5. The energy expenditure of the group as a whole was lower in winter (BMR X 1·88; SD 0·33; n 9) than summer (BMR X 2·01; SD 0·30; n 9) though the difference was not statistically significant. The average summer and winter DLW-derived expenditure was BMR X 1·96 (SD 0·31; n 17). The activity diary estimate of expenditure was BMR X 1·79 (SD 0·32; n 17). In a subset of the group who were representative of the most active 26% of all adult males in the UK, the DLW-derived expenditure was BMR X 2·08 (SD 0·24; n 11). This is higher than the highest Department of Health (1991) estimate of BMR X 1·6 for individuals in light occupations. The measured energy costs of low-intensity activities were similar to those presented in the Department of Health (1991) report but the value determined for running (BMR X 13·08; SD 2·4; n 6) was higher than the highest value in the report (BMR X 6 to 8). The results indicate that the recent Department of Health (1991) reference values for energy may underestimate the expenditure of a significant proportion of the UK population largely because the energy costs of activity used in the report to calculate expenditure do not accurately reflect those achieved during active leisure in individuals who take regular exercise.

The doubly labeled water (DLW) technique allows the CO2 production (rCO2) of free living animals to be estimated from the difference between the turnover of 2H2O and H218O in the body water. A fundamental assumption of this technique is that neither of the isotopes used are lost in products other than CO2 and H2O. We found, however, that 2H was lost in both exchangeable and nonexchangeable positions in the feces of sheep. Negligible amounts of 18O were lost in exchangeable positions. 2H losses led to a 0.75% (SE 0.06, n = 4) overestimation of the measured 2H2O flux, leading to an average error in rCO2 estimates of 20.3 l/day. For a typical rCO2 rate of 370 l/day, this would amount to an error of approximately 5% (range -7.0 to -4.3%, n = 4). Correction factors to account for this loss were presented. The error in rCO2 due to 2H sequestration into fat was calculated to be at most 2.1 l/day or about -0.66% in lambs with a rCO2 of 320 l/day. In a triply labeled water (TLW) study the maximum error in the estimation of fractionated evaporative water loss (X) would lead to a 0.81% underestimation of rCO2. We recommend that during a DLW study involving ruminant animals the correction factors presented here be used to compensate for 2H loss in feces. This may be particularly important where the diet has a high roughage content leading to a significant fecal dry matter production.

The doubly labelled water (DLW) method was used to measure total energy expenditure (TEE) in three male reindeer (Rangifer tarandus tarandus) aged 22 months in winter (February) while the animals were living unrestricted at natural mountain pasture in northern Norway (69°20'N). The concentrations of 2H and l8O were measured in water extracted from samples of faeces collecred from the animals 0.4 and 11.2 days after injection of the isotopes. Calculated rates of water flux and CO2-production were adjusted to compensate for estimated losses of 2H in faecal solids and in methane produced by microbial fermentation of forage in the rumen. The mean specific TEE in the three animals was 3.057 W.kg-1 (range 2.436 - 3.728 W.kg1). This value is 64% higher than TEE measured by the DLW method in four captive, non-pregnant adult female reindeer in winter and probably mainly reflects higher levels of locomotor activity in the free-living animals. Previous estimates of TEE in free-living Rangifer in winter based on factorial models range from 3.038 W.kg-1 in female woodland caribou (R. t. caribou) to 1.813 W.kg-1 in female Svalbard reindeer (R. t. platyrhynchus). Thus, it seems that existing factorial models are unlikely to overestimate TEE in reindeer/caribou: they may, instead, be unduly conservative. While the present study serves as a general validation of the factorial approach, we suggest that the route to progress in the understanding of field energetics in wild ungulates is via application of the DLW method.

This study explored the effects of mild infestation with Giardia on energy intake and expenditure at rest and in activity in an urban Mexican population. Ten boys aged 6-10 y living in low-income sectors in northwest Mexico who had Giardia infestation were recruited. Energy intake, basal metabolic rate (BMR), and total free-living expenditure (TEE) measured by the doubly labeled water method were determined for 7 d during both infestation and after treatment. There was no significant difference in recorded energy intake between the two periods (7.76 and 7.70 MJ/d; P = 0.847). BMR showed no significant change in response to treatment; values were 4.79 and 4.86 MJ/d (P = 0.03). The mean TEE increased by almost 1 MJ/d in the Giardia-free period. This increase was observed in 8 of the 10 subjects; however, the overall change was not statistically significant (P = 0.08).