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Background: Folate and vitamin B12 (B12) are cofactors in folate-mediated 1-carbon metabolism (FOCM), a metabolic network that supports synthesis of nucleotides (including thymidylate [dTMP]) and methionine. FOCM impairments such as a deficiency or imbalance of cofactors can perturb dTMP synthesis, causing uracil misincorporation into DNA., Objective: The purpose of this study was to determine how reduced expression of the B12-dependent enzyme methionine synthase (MTR) and excess dietary folic acid interact to affect folate distribution and markers of genome stability in mouse tissues., Methods: Heterozygous Mtr knockout mice (Mtr +/- ) model the FOCM-specific effects of B12 deficiency. Folate accumulation and vitamer distribution, genomic uracil concentrations, and phosphorylated histone H2AX (γH2AX) immunostaining were measured in male Mtr +/+ and Mtr +/- mice weaned to either a folate-sufficient control (C) diet (2 mg/kg folic acid) or a high folic acid (HFA) diet (20 mg/kg folic acid) for 7 wk., Results: Exposure to the HFA diet led to tissue-specific patterns of folate accumulation, with plasma, colon, kidney, and skeletal muscle exhibiting increased folate concentrations compared with control. Liver total folate did not differ. Although unmetabolized folic acid (UMFA) increased 10-fold in mouse plasma with HFA diet, UMFA accounted for <0.2% of total folate in liver and colon tissue. Exposure to HFA diet resulted in a shift in folate distribution in colon tissue with higher 5-methyl-THF and lower formyl-THF than in control mice. Mtr heterozygosity did not impact folate accumulation or distribution in any tissue. Mice on HFA diet exhibited higher uracil in genomic DNA and γH2AX foci in colon. Similar differences were not seen in liver., Conclusions: This study demonstrates that folic acid, even when consumed at high doses, does not meaningfully accumulate in mouse tissues, although high-dose folic acid shifts folate distribution and increases uracil accumulation in genomic DNA in colon tissue., Competing Interests: Conflict of interest The authors report no conflict of interest., (Copyright © 2024 American Society for Nutrition. All rights reserved.)

Background: Folic acid (FA) supplementation may attenuate the associations between gestational exposure to certain chemicals and autism or autistic-like behaviors, but to our knowledge, this has not been assessed for lead., Objectives: We examined whether the relationship between gestational blood-lead levels (BLLs) and autistic-like behaviors was modified by gestational plasma total folate concentrations, FA supplementation, and maternal methylenetetrahydrofolate reductase ( MTHFR ) 677C>T genotype., Methods: We used data from the Maternal-Infant Research on Environmental Chemicals study (2008-2011), a Canadian pregnancy and birth cohort study. Childhood autistic-like behaviors were documented in 601 children 3-4 y of age with the Social Responsiveness Scale-2 (SRS-2), where higher scores denote more autistic-like behaviors. We measured BLLs and plasma total folate concentrations during the first and third trimesters of pregnancy. We also estimated gestational FA supplementation via surveys and genotyped the maternal MTHFR 677C>T single nucleotide polymorphism (SNP). We estimated the confounder-adjusted associations between log 2 -transformed BLLs and SRS-2 scores by two indicators of folate exposure and maternal MTHFR 677C>T genotype using linear regression., Results: Third-trimester BLLs were associated with increased SRS-2 scores [ β a d j = 3.3 ; 95% confidence interval (CI): 1.1, 5.5] among participants with low ( < 10 th percentile), third-trimester, plasma total folate concentrations, but BLL-SRS-2 associations were null ( β a d j = - 0.3 ; 95% CI: - 1.2 , 0.5) among those in the middle category ( ≥ 10 th and < 80 th percentiles) ( p -interaction < 0.001 ). FA supplementation also attenuated these associations. Both folate indicators modified first-trimester BLL-SRS-2 associations, but to a lesser extent. Third-trimester BLL-SRS-2 associations were slightly stronger among participants who were homozygous for the T (minor) allele of the MTHFR 677C>T SNP ( β a d j = 0.9 ; 95% CI: - 1.2 , 3.1) than those without the T allele ( β a d j = - 0.3 ; 95% CI: - 1.3 , 0.7), but the difference was not statistically significant ( p -interaction = 0.28 )., Discussion: Folate may modify the associations between gestational lead exposure and childhood autistic-like behaviors, suggesting that it mitigates the neurotoxic effects of prenatal lead exposure. https://doi.org/10.1289/EHP14479.

Background: Folate and vitamin B12 status during pregnancy are important for maternal and neonatal health. Maternal intake and prepregnancy body mass index (ppBMI) can influence biomarker status., Objectives: This study aimed to, throughout pregnancy; 1) assess folate and B12 status including serum total folate, plasma total vitamin B12, and homocysteine (tHcy); 2) examine how these biomarkers are associated with intakes of folate and B12 and with ppBMI; and 3) determine predictors of serum total folate and plasma total vitamin B12., Methods: In each trimester (T1, T2, and T3), food and supplement intakes of 79 French-Canadian pregnant individuals were assessed by 3 dietary recalls (R24W) and a supplement use questionnaire. Fasting blood samples were collected. Serum total folate and plasma total vitamin B12 and tHcy were assessed by immunoassay (Siemens ADVIA Centaur XP)., Results: Participants were 32.1 ± 3.7 y and had a mean ppBMI of 25.7 ± 5.8 kg/m 2 . Serum total folate concentrations were high (>45.3 nmol/L, T1: 75.4 ± 55.1, T2: 69.1 ± 44.8, T3: 72.1 ± 52.1, P = 0.48). Mean plasma total vitamin B12 concentrations were >220 pmol/L (T1: 428 ± 175, T2: 321 ± 116, T3: 336 ± 128, P < 0.0001). Mean tHcy concentrations were <11 μmol/L across trimesters. Most participants (79.6%-86.1%) had a total folic acid intake above the Tolerable Upper Intake Level (UL, >1000 μg/d). Supplement use accounted for 71.9%-76.1% and 35.3%-41.8% of total folic acid and vitamin B12 intakes, respectively. The ppBMI was not correlated with serum total folate (P > 0.1) but was weakly correlated with and predicted lower plasma total vitamin B12 in T3 (r = -0.23, P = 0.04; r 2 = 0.08, standardized beta [sβ] = -0.24, P = 0.01). Higher folic acid intakes from supplements predicted higher serum total folate (T1: r 2 = 0.05, sβ = 0.15, P = 0.04, T2: r 2 = 0.28, sβ = 0.56, P = 0.01, T3: r 2 = 0.19, sβ = 0.44, P < 0.0001)., Conclusions: Most pregnant individuals had elevated serum total folate concentrations, reflecting total folic acid intakes above the UL driven by supplement use. Vitamin B12 concentrations were generally adequate and differed by ppBMI and pregnancy stage., (Copyright © 2023 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

Transparent reporting of nutrition research promotes rigor, reproducibility, and relevance to human nutrition. We performed a scoping review of recent articles reporting dietary folate interventions in mice as a case study to determine the reporting frequency of generic study design items (i.e., sex, strain, and age) and nutrition-specific items (i.e., base diet composition, intervention doses, duration, and exposure verification) in basic nutrition research. We identified 798 original research articles in the EMBASE, Medline, Food Science and Technology Abstracts (FSTA), Global Health, and International Pharmaceutical Abstracts (IPA) databases published between January 2009 and July 2021 in which a dietary folic acid (FA) intervention was used in mice. We identified 312 original peer-reviewed articles including 191 studies in nonpregnant and 126 in pregnant mice. Most studies reported sex (99%), strain (99%), and age (83%). The majority of studies used C57BL/6 (53%) or BALB/c (11%) mice aged 3-9 wk. Nonpregnancy studies were more likely to use only male mice (57%). Dietary FA interventions varied considerably and overlapped: deficiency (0-3 mg/kg), control (0-16 mg/kg), and supplemented (0-50 mg/kg). Only 63% of studies used an open-formula base diet with a declared FA content and 60% of studies verified FA exposure using folate status biomarkers. The duration of intervention ranged from 1 to 104 wk for nonpregnancy studies. The duration of intervention for pregnancy studies was 1-19 wk, occurring variably before pregnancy and/or during pregnancy and/or lactation. Overall, 17% of studies did not report ≥1 generic study design item(s) and 40% did not report ≥1 nutrition-specific study design item(s). The variability and frequent lack of reporting of important generic and nutrition-specific study design details in nutrition studies limit their generalizability, reproducibility, and interpretation. The use of reporting checklists for animal research would enhance reporting quality of key study design and conduct factors in animal-based nutrition research., (© Her Majesty the Queen in Right of Canada, as represented by the Minister of Health, 2022.)

Background: Achieving optimal folate status during early gestation reduces the risk of neural tube defects (NTDs). While inadequate folate intake remains a concern, it is becoming increasingly common for individuals to consume higher than recommended doses of folic acid (FA) with minimal additional benefit., Objective: Here, we sought to investigate the determinants, including FA supplement dose and use, of plasma total and individual folate vitamer concentrations in the first and third trimesters of pregnancy., Methods: Using data from the Maternal-Infant Research on Environmental Chemicals (MIREC) Study, a cohort exposed to mandatory FA fortification, we measured plasma total folate and individual folate vitamer [5-methyltetrahydrofolate (5-methylTHF), unmetabolized FA (UMFA), and non-methyl folates (sum of THF, 5-formylTHF, 5,10-methenyl-THF)] concentrations in the first and third trimesters (n = 1,893). Using linear mixed models, we estimated associations between plasma folate concentrations, total daily supplemental FA intake, plasma vitamin B-12 concentrations, and multiple demographic, maternal, and reproductive factors., Results: Almost 95% of MIREC study participants met or exceeded the recommended daily supplemental FA intake from supplements (≥400 μg/d), with approximately 25% consuming more than the Tolerable Upper Intake Level (>1000 μg/d). Over 99% of MIREC participants had a plasma total folate status indicative of maximal NTD risk reduction (25.5 nmol/L) regardless of FA supplement dose. UMFA was detected in almost all participants, with higher concentrations associated with higher FA doses. Determinants of adequate FA supplement intake and folate status associated with reduced NTD risk included indicators of higher socioeconomic position, higher maternal age, nulliparity, and lower prepregnancy BMI., Conclusions: In the context of mandatory FA fortification, our data indicate that higher-than-recommended FA doses are unwarranted, with the exception of individuals at higher risk for NTDs. Ideally, prenatal supplements would contain 400 rather than 1000 µg FA, thereby enabling the consumption of optimal and safe FA doses., (© Crown copyright 2022.)

Gestational arsenic exposure adversely impacts child health. Folate-mediated 1-carbon metabolism facilitates urinary excretion of arsenic and may prevent arsenic-related adverse health outcomes. We investigated the potential for maternal folate status to modify associations between gestational arsenic exposure and child health. We used data from 364 mother-child pairs in the MIREC study, a prospective pan-Canadian cohort. During pregnancy, we measured first trimester urinary arsenic concentrations, plasma folate biomarkers, and folic acid supplementation intake. At age 3 years, we evaluated twelve neurodevelopmental and anthropometric features. Using latent profile analysis and multinomial regression, we developed phenotypic profiles of child health, estimated covariate-adjusted associations between arsenic and these phenotypic profiles, and evaluated whether folate status modified these associations. We identified three phenotypic profiles of neurodevelopment and three of anthropometry, ranging from less to more optimal child health. Gestational arsenic was associated with decreased odds of optimal neurodevelopment. Maternal folate status did not modify associations of arsenic with neurodevelopmental phenotypic profiles, but gestational arsenic was associated with increased odds of excess adiposity among those who exceed recommendations for folic acid (>1000 μg/day). However, arsenic exposure was low and folate status was high. Gestational arsenic exposure may adversely impact child neurodevelopment and anthropometry, and maternal folate status may not modify these associations; however, future work should examine these associations in more arsenic-exposed or lower folate-status populations.

Competing Interests: Competing interests: We have read and understood BMJ policy on declaration of interests and declare the following: RWP has received personal fees for consultancies unrelated to this topic from Amgen, Biogen, Merck, Nant Pharma, Pfizer, and Reckitt Benckiser. All other authors have no conflicts to disclose.

Background: Periconceptional folic acid (FA) supplementation is recommended to prevent neural tube defects; however, the extent to which recommendations are met through dietary sources and supplements is not clear., Objectives: Our objective was to evaluate the dietary and supplemental intakes of FA in a Canadian pregnancy cohort and to determine the proportions of pregnant women exceeding the Estimated Average Requirement (EAR) and Tolerable Upper Intake Level (UL)., Methods: FACT (the Folic Acid Clinical Trial) was an international multicenter, randomized, double-blinded, placebo-controlled, phase III trial investigating FA for the prevention of pre-eclampsia in high-risk pregnancies. Participants were enrolled from Canadian sites at 8-16 weeks of gestation. Dietary and supplemental FA intake data were collected through participant interviews and FFQs at the time of FACT enrollment. Categorical data were summarized as n (%) and continuous data as median (IQR)., Results: This study included 1198 participants. Participants consumed 485 μg dietary folate equivalents (DFE)/d (IQR: 370-630 μg DFE/d) from dietary sources of folate and FA. Through diet alone, 43.4% of participants consumed ≥520 μg DFE/d, the EAR for pregnant individuals. Of the 91.9% of participants who consumed daily FA supplements, 0.4% consumed <400 μg FA/d and 96.0% consumed ≥1000 μg/d, the UL for FA. Median (IQR) total folate intake was 2167 μg DFE/d (2032-2325 μg DFE/d); 95.3% of participants met or exceeded the EAR from all sources, but 1069 (89.2%) participants exceeded the UL., Conclusions: The majority of participants in this Canadian pregnancy cohort did not consume the recommended amount of folate from dietary sources. However, most prenatal supplements contained 1000 μg FA, resulting in the majority of women exceeding the UL. With no additional benefit associated with FA intakes beyond the UL for most women, modification of prenatal supplement formulations may be warranted to ensure women meet but do not exceed recommended FA intakes.FACT was registered at clinicaltrials.gov as NCT01355159 and at isrctn.com as ISRCTN23781770., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Background: Periconceptional folic acid (FA) supplementation is recommended to prevent the occurrence of neural tube defects. Currently, most over-the-counter FA supplements in Canada and the United States contain 1 mg FA and some women are prescribed 5 mg FA/d. High-dose FA is hypothesized to impair 1-carbon metabolism. We aimed to determine folate and 1-carbon metabolism biomarkers in pregnant women exposed to 1 mg or 5 mg FA., Objectives: This was an ancillary study within the Folic Acid Clinical Trial (FACT), a randomized, double-blinded, placebo-controlled, phase III trial designed to assess the efficacy of high-dose FA to prevent preeclampsia., Methods: For FACT, women were randomized at 8-16 gestational weeks to receive daily 4.0 mg FA (high dose) or placebo (low dose) plus their usual supplementation (≤1.1 mg). Women were recruited from 3 Canadian FACT centers and provided nonfasting blood samples at 24-26 gestational weeks for measurement of RBC and serum total folate, serum unmetabolized FA (UMFA), tetrahydrofolate (THF), 5-methylTHF, 5-formylTHF, 5,10-methenylTHF, and MeFox (pyrazino-s-triazine derivative of 4α-hydroxy-5-methylTHF, a 5-methylTHF oxidation product); total vitamins B-12 and B-6; and plasma total homocysteine. Group differences were determined using χ2, Fisher exact, and Wilcoxon rank-sum tests., Results: Nineteen (38%) women received high-dose FA and 31 (62%) received low-dose FA. The median RBC folate concentration was 2701 (IQR: 2243-3032) nmol/L and did not differ between groups. The high-dose group had higher serum total folate (median: 148.4 nmol/L, IQR: 110.4-181.2; P = 0.007), UMFA (median: 4.6 nmol/L, IQR: 2.5-33.8; P = 0.008), and 5-methylTHF (median: 126.6 nmol/L, IQR: 98.8-158.6; P = 0.03) compared with the low-dose group (median: 122.8 nmol/L, IQR: 99.5-136.0; median: 1.9 nmol/L, IQR: 0.9-4.1; median: 108.6 nmol/L, IQR: 96.4-123.2, respectively). Other biomarkers of 1-carbon metabolism did not differ., Conclusions: High-dose FA supplementation in early pregnancy increases maternal serum folate but not RBC folate concentrations, suggesting tissue saturation. Higher UMFA concentrations in women receiving high-dose FA supplements suggest that these doses are supraphysiologic but with no evidence of altered 1-carbon metabolism., (© Crown copyright 2021.)

Background: While cancer is common, its incidence varies widely by tissue. These differences are attributable to variable risk factors, such as environmental exposure, genetic inheritance, and lifetime number of stem cell divisions in a tissue. Folate deficiency is generally associated with increased risk for colorectal cancer (CRC) and acute lymphocytic leukemia (ALL). Conversely, high folic acid (FA) intake has also been associated with higher CRC risk., Objective: Our objective was to compare the effect of folate intake on mutant frequency (MF) and types of mutations in the colon and bone marrow of mice., Methods: Five-week-old MutaMouse male mice were fed a deficient (0 mg FA/kg), control (2 mg FA/kg), or supplemented (8 mg FA/kg) diet for 20 wk. Tissue MF was assessed using the lacZ mutant assay and comparisons made by 2-factor ANOVA. LacZ mutant plaques were sequenced using next-generation sequencing, and diet-specific mutation profiles within each tissue were compared by Fisher's exact test., Results: In the colon, the MF was 1.5-fold and 1.3-fold higher in mice fed the supplemented diet compared with mice fed the control (P = 0.001) and deficient (P = 0.008) diets, respectively. This contrasted with the bone marrow MF in the same mice where the MF was 1.7-fold and 1.6-fold higher in mice fed the deficient diet compared with mice fed the control (P = 0.02) and supplemented (P = 0.03) diets, respectively. Mutation profiles and signatures (mutation context) were tissue-specific., Conclusions: Our data indicate that dietary folate intake affects mutagenesis in a tissue- and dose-specific manner in mice. Mutation profiles were generally tissue- but not dose-specific, suggesting that altered cellular folate status appears to interact with endogenous mutagenic mechanisms in each tissue to create a permissive context in which specific mutation types accumulate. These data illuminate potential mechanisms underpinning differences in observed associations between folate intake/status and cancer., (© Crown copyright 2021.)

Folate, an essential nutrient found naturally in foods in a reduced form, is present in dietary supplements and fortified foods in an oxidized synthetic form (folic acid). There is widespread agreement that maintaining adequate folate status is critical to prevent diseases due to folate inadequacy (e.g., anemia, birth defects, and cancer). However, there are concerns of potential adverse effects of excess folic acid intake and/or elevated folate status, with the original concern focused on exacerbation of clinical effects of vitamin B-12 deficiency and its role in neurocognitive health. More recently, animal and observational studies have suggested potential adverse effects on cancer risk, birth outcomes, and other diseases. Observations indicating adverse effects from excess folic acid intake, elevated folate status, and unmetabolized folic acid (UMFA) remain inconclusive; the data do not provide the evidence needed to affect public health recommendations. Moreover, strong biological and mechanistic premises connecting elevated folic acid intake, UMFA, and/or high folate status to adverse health outcomes are lacking. However, the body of evidence on potential adverse health outcomes indicates the need for comprehensive research to clarify these issues and bridge knowledge gaps. Three key research questions encompass the additional research needed to establish whether high folic acid or total folate intake contributes to disease risk. 1) Does UMFA affect biological pathways leading to adverse health effects? 2) Does elevated folate status resulting from any form of folate intake affect vitamin B-12 function and its roles in sustaining health? 3) Does elevated folate intake, regardless of form, affect biological pathways leading to adverse health effects other than those linked to vitamin B-12 function? This article summarizes the proceedings of an August 2019 NIH expert workshop focused on addressing these research areas., (Published by Oxford University Press on behalf of the American Society for Nutrition 2020.)

Prenatal exposure to persistent organic pollutants (POPs) has been associated with the development of metabolic syndrome-related diseases in offspring. According to epidemiological studies, father's transmission of environmental effects in addition to mother's can influence offspring health. Moreover, maternal prenatal dietary folic acid (FA) may beneficially impact offspring health. The objective is to investigate whether prenatal FA supplementation can overcome the deleterious effects of prenatal exposure to POPs on lipid homeostasis and inflammation in three generations of male rat descendants through the paternal lineage. Female Sprague-Dawley rats (F0) were exposed to a POPs mixture (or corn oil) +/- FA supplementation for 9 weeks before and during gestation. F1 and F2 males were mated with untreated females. Plasma and hepatic lipids were measured in F1, F2, and F3 males after 12-h fast. Gene expression of inflammatory cytokines was determined by qPCR in epididymal adipose tissue. In F1 males, prenatal POPs exposure increased plasma lipids at 14 weeks old and hepatic lipids at 28 weeks old and prenatal FA supplementation decreased plasma total cholesterol at 14 weeks old. Prenatal POPs exposure decreased plasma triglycerides at 14 weeks old in F2 males. No change was observed in inflammatory markers. Our results show an impact of the paternal lineage on lipid homeostasis in rats up to the F2 male generation. FA supplementation of the F0 diet, regardless of POPs exposure, lowered plasma cholesterol in F1 males but failed to attenuate the deleterious effects of prenatal POPs exposure on plasma and hepatic lipids in F1 males.

The paternal environment is thought to influence sperm quality and future progeny may also be impacted. We hypothesized that prenatal exposure to environmentally-relevant contaminants impairs male reproduction, altering embryo gene expression over multiple generations. Folic acid (FA) can improve sperm quality and pregnancy outcomes, thus we further hypothesized that FA mitigates the contaminants. Sprague-Dawley F0 female rats treated with persistent organic pollutants (POPs) or corn oil and fed basal or supplemented FA diets, then used to yield four generations of litters. Only F0 females received POPs and/or FA treatments. In utero POPs exposure altered sperm parameters in F1, which were partly rescued by FA supplementation. Paternal exposure to POPs reduced sperm quality in F2 males, and the fertility of F3 males was modified by both POPs and FA. Ancestral FA supplementation improved sperm parameters of F4 males, while the POPs effect diminished. Intriguingly, F3 males had the poorest pregnancy outcomes and generated the embryos with the most significantly differentially expressed genes. Early-life exposure to POPs harms male reproduction across multiple generations. FA supplementation partly mitigated the impact of POPs. The two-cell embryo transcriptome is susceptible to paternal environment and could be the foundation for later pregnancy outcomes.

Background: Maternal nutrition and genetics are determinants of breast-milk nutrient composition and, as such, are determinants of the nutritional exposure of breastfed infants., Objectives: The aim of this study was to determine whether common maternal single nucleotide polymorphisms (SNPs) in folate-dependent enzymes are associated with breast-milk folate content in a cohort of mothers enrolled in the Maternal-Infant Research on Environmental Chemicals (MIREC) study., Methods: The MIREC study is a Canadian prospective pregnancy cohort study that recruited 2001 participants between 2008 and 2011. Five folate-related SNPs-MTHFR 677C>T (rs1801133), MTHFR 1298A>C (rs1801131), MTHFR 1793G>A (rs2274976), MTR 2756A>G (rs1805087), and MTRR 66A>G (rs1801394)-were genotyped. Breast milk was sampled ∼1 mo postpartum, and tetrahydrofolate (THF), 5-methyl-THF, 5-formyl-THF, 5,10-methenyl-THF, and unmetabolized folic acid (UMFA) were measured using liquid chromatography-tandem mass spectrometry in a subset of participants (n = 551). Associations were assessed using Wald's test. Associations were considered significant if P ≤ 0.01 (Bonferroni correction for multiple testing)., Results: None of the SNPs were associated with total breast-milk folate. However, the MTHFR 677C>T SNP was associated with breast-milk UMFA (R2 = 0.01; unadjusted P = 0.004), explaining a small portion of total variance; this association remained significant when adjusted for other covariates, including supplemental folic acid consumption. The MTHFR 1793G>A and MTRR 66A>G SNPs tended to be associated with 5-methyl-THF (R2 = 0.008, P = 0.04) and reduced folates (THF + 5-methyl-THF + 5-formyl-THF + 5,10-methenyl-THF; R2 = 0.01, P = 0.02), respectively., Conclusions: We found that total breast-milk folate content was not associated with any of the folate-related SNPs examined. The association between the MTHFR 677C>T SNP and breast-milk UMFA, albeit modest, highlights the need to better understand the determinants of breast-milk folate and the impact they might have on milk folate bioavailability., (© Crown copyright 2019.)

Study Question: Could clinically-relevant moderate and/or high dose maternal folic acid supplementation prevent aberrant developmental and epigenetic outcomes associated with assisted reproductive technologies (ART)?, Summary Answer: Our results demonstrate dose-dependent and sex-specific effects of folic acid supplementation in ART and provide evidence that moderate dose supplements may be optimal for both sexes., What Is Known Already: Children conceived using ART are at an increased risk for growth and genomic imprinting disorders, often associated with DNA methylation defects. Folic acid supplementation is recommended during pregnancy to prevent adverse offspring outcomes; however, the effects of folic acid supplementation in ART remain unclear., Study Design, Size, Duration: Outbred female mice were fed three folic acid-supplemented diets, control (rodent daily recommended intake or DRI; CD), moderate (4-fold DRI; 4FASD) or high (10-fold DRI; 10FASD) dose, for six weeks prior to ART and throughout gestation. Mouse ART involved a combination of superovulation, in vitro fertilisation, embryo culture and embryo transfer., Participants/materials, Setting, Methods: Midgestation embryos and placentas (n = 74-99/group) were collected; embryos were assessed for developmental delay and gross morphological abnormalities and embryos and placentas were examined for epigenetic defects. We assessed methylation at four imprinted genes (Snrpn, Kcnq1ot1, Peg1 and H19) in matched midgestation embryos and placentas (n = 31-32/group) using bisulfite pyrosequencing. In addition, we examined genome-wide DNA methylation patterns in placentas (n = 6 normal placentas per sex/group) and embryos (n = 6 normal female embryos/group; n = 3 delayed female embryos/group) using reduced representation bisulfite sequencing (RRBS)., Main Results and the Role of Chance: Moderate, but not high dose supplementation, was associated with a decrease in the proportion of developmentally delayed embryos. Although moderate dose folic acid supplementation reduced DNA methylation variance at certain imprinted genes in embryonic and placental tissues, high dose supplementation exacerbated the negative effects of ART at imprinted loci. Furthermore, folic acid supplements resolved female-biased aberrant imprinted gene methylation. Supplementation was more effective at correcting ART-induced genome-wide methylation defects in male versus female placentas; however, folic acid supplementation also led to additional methylation perturbations which were more pronounced in males., Large-Scale Data: The RRBS data from this study have been submitted to the NCBI Gene Expression Omnibus under the accession number GSE123143., Limitations Reasons for Caution: Although the combination of mouse ART utilised in this study consisted of techniques commonly used in human fertility clinics, there may be species differences. Therefore, human studies, designed to determine the optimal levels of folic acid supplementation for ART pregnancies, and taking into account foetal sex, are warranted., Wider Implications of the Findings: Taken together, our findings support moderation in the dose of folic acid supplements taken during ART., Study Funding/competing Interest(s): This work was funded by the Canadian Institutes of Health Research (FDN-148425). The authors declare no conflict of interest., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: We sought to assess the recent trend in NTD prevalence at birth in the post-folic acid food fortification era and to identify the maternal risk factors associated with that trend., Methods: We carried out a population-based study of all livebirths and stillbirths (including late pregnancy terminations) delivered in hospitals in Canada (excluding Quebec) from 2004 to 2015 (n = 3 439 330). We examined NTD birth prevalence by year, multiple pregnancy, maternal age, parity, pregestational diabetes, chronic illness, and problematic substance use. Poisson regression was used to quantify the association between spina bifida and cranial defects and maternal characteristics and other risk factors., Results: We identified 1517 non-chromosomal NTDs, yielding a birth prevalence of 4.4 per 10 000 total births. NTD prevalence rose from 3.6 in 2004 to 4.6 per 10 000 in 2015 (P trend  = 0.03). Among NTD subtypes, only spina bifida showed a temporal increase (P trend  = 0.03). Birth prevalence of spina bifida was higher among younger mothers, those with type 2 diabetes (rate ratio (RR) 3.74, 95% confidence interval (CI) 2.21, 6.35), chronic illness (RR 3.16, 95% CI 1.97, 5.07), and problematic substance use (RR 1.88, 95% CI 1.31, 2.71). Adjusting for risk factors attenuated the significant temporal trend in spina bifida (unadjusted average annual prevalence ratio (aAAPR) 1.016, 95% CI 1.001, 1.032; adjusted AAPR 1.014, 95% CI 0.998, 1.029)., Conclusions: Increases in the frequency of maternal risk factors such as pregestational diabetes mellitus, substance use, and chronic illness may be partly responsible for the recent rise in NTDs, particularly spina bifida., (© 2019 John Wiley & Sons Ltd.)

The Government of Canada and the Society of Obstetricians and Gynaecologists of Canada both recommend a daily multivitamin supplement containing 400 µg folic acid (FA) for the primary prevention of neural tube defects among low-risk women from before conception and throughout lactation. Prenatal supplements marketed and prescribed in Canada typically exceed the recommended dose, usually providing ≥1000 µg FA/d. This high daily dose, coupled with staple-food FA fortification, has resulted in the observation of very high blood folate concentrations among reproductive-aged women consuming FA-containing supplements. The long-term consequences of high folate status on fetal development are unknown; however, evidence from animal studies and some human epidemiologic data suggest potential adverse consequences. To address this issue, a workshop was convened with the overall goal to identify challenges and solutions to aligning supplemental FA intakes with current evidence-based recommendations. Thirty-eight stakeholders from academia, industry, government, and health professional groups participated. Group discussions facilitated the identification and prioritization of 5 key challenges for which solutions and implementation strategies were proposed. The 5 themes encompassed clarity and harmonization of evidence-based guidelines, reformulation or relabeling of FA-containing supplements, access to FA for all women, knowledge dissemination strategies and education of the public and health care professionals, and attitude change to overcome the perception of "more is better." A combination of the proposed implementation strategies involving all key stakeholders and directed to health care professionals and the public may enable a sustainable change to align FA intake during the periconceptional period with evidence-based recommendations.

Stroke is a leading cause of disability and death world-wide and nutrition is a modifiable risk factor for stroke. Metheylenetetrahydrofolate reductase (MTHFR) is an enzyme involved in the metabolism of folic acid, a B-vitamin. In humans, a polymorphism in MTHFR (677C→T) is linked to increased risk of stroke, but the mechanisms remain unknown. The Mthfr +/- mice mimic a phenotype described in humans at bp677. Using this mouse model, the aim of this study was to investigate the impact of MTHFR deficiency on stroke outcome. Male Mthfr +/- and wildtype littermate control mice were aged (~1.5-year-old) and trained on the single pellet reaching task. After which the sensorimotor cortex was then damaged using photothrombosis (PT), a model for ischemic stroke. Post-operatively, animals were tested for skilled motor function, and brain tissue was processed to assess cell death. Mthfr +/- mice were impaired in skilled reaching 2-weeks after stroke but showed some recovery at 5-weeks compared to wild types after PT damage. Within the ischemic brain, there was increased expression of active caspase-3 and reduced levels of phospho-AKT in neurons of Mthfr +/- mice. Recent data suggests that astrocytes may play a significant role after damage, the impact of MTHFR and ischemic investigated the impact of MTHFR-deficiency on astrocyte function. MTHFR-deficient primary astrocytes showed reduced cell viability after exposure to hypoxia compared to controls. Increased immunofluorescence staining of active caspase-3 and hypoxia-inducible factor 1-alpha were also observed. The data suggest that MTHFR deficiency decreases recovery after stroke by reducing neuronal and astrocyte viability., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Folate deficiency causes megaloblastic anemia and neural tube defects, and is also associated with some cancers. In vitro, folate deficiency increases mutation frequency and genome instability, as well as exacerbates the mutagenic potential of known environmental mutagens. Conversely, it remains unclear whether or not elevated folic acid (FA) intakes are beneficial or detrimental to the induction of DNA mutations and by proxy human health. We used the MutaMouse transgenic model to examine the in vivo effects of FA deficient, control, and supplemented diets on somatic DNA mutant frequency (MF) and genome instability in hematopoietic cells. We also examined the interaction between FA intake and exposure to the known mutagen N-ethyl-N-nitrosourea (ENU) on MF. Male mice were fed the experimental diets for 20 weeks from weaning. Half of the mice from each diet group were gavaged with 50 mg/kg body weight ENU after 10 weeks on diet and remained on their respective diet for an additional 10 weeks. Mice fed a FA-deficient diet had a 1.3-fold increase in normochromatic erythrocyte micronucleus (MN) frequency (P = 0.034), and a doubling of bone marrow lacZ MF (P = 0.035), compared to control-fed mice. Mice exposed to ENU showed significantly higher bone marrow lacZ and Pig-a MF, but there was no effect of FA intake on ENU-induced MF. These data indicate that FA deficiency increases mutations and MN formation in highly proliferative somatic cells, but that FA intake does not mitigate ENU-induced mutations. Also, FA intake above adequacy had no beneficial or detrimental effect on mutations or MN formation. Environ. Mol. Mutagen. 59:366-374, 2018. © 2018 Her Majesty the Queen in Right of Canada 2018., (© 2018 Her Majesty the Queen in Right of Canada 2018.)

Study Question: Do paternal exposures to folic acid deficient (FD), and/or folic acid supplemented (FS) diets, throughout germ cell development adversely affect male germ cells and consequently offspring health outcomes?, Summary Answer: Male mice exposed over their lifetimes to both FD and FS diets showed decreased sperm counts and altered imprinted gene methylation with evidence of transmission of adverse effects to the offspring, including increased postnatal-preweaning mortality and variability in imprinted gene methylation., What Is known Already: There is increasing evidence that disruptions in male germ cell epigenetic reprogramming are associated with offspring abnormalities and intergenerational disease. The fetal period is the critical time of DNA methylation pattern acquisition for developing male germ cells and an adequate supply of methyl donors is required. In addition, DNA methylation patterns continue to be remodeled during postnatal spermatogenesis. Previous studies have shown that lifetime (prenatal and postnatal) folic acid deficiency can alter the sperm epigenome and increase the incidence of fetal morphological abnormalities., Study Design, Size, Duration: Female BALB/c mice (F0) were placed on one of four amino-acid defined diets for 4 weeks before pregnancy and throughout pregnancy and lactation: folic acid control (Ctrl; 2 mg/kg), 7-fold folic acid deficient (7FD; 0.3 mg/kg), 10-fold high FS (10FS, 20 mg/kg) or 20-fold high FS (20FS, 40 mg/kg) diets. F1 males were weaned to their respective prenatal diets to allow for diet exposure during all windows of germline epigenetic reprogramming: the erasure, re-establishment and maintenance phases., Participants/materials, Settings, Methods: F0 females were mated with chow-fed males to produce F1 litters whose germ cells were exposed to the diets throughout embryonic development. F1 males were subsequently mated with chow-fed female mice. Two F2 litters, unexposed to the experimental diets, were generated from each F1 male; one litter was collected at embryonic day (E)18.5 and one delivered and followed postnatally. DNA methylation at a global level and at the differentially methylated regions of imprinted genes (H19, Imprinted Maternally Expressed Transcript (Non-Protein Coding)-H19, Small Nuclear Ribonucleoprotein Polypeptide N-Snrpn, KCNQ1 Opposite Strand/Antisense Transcript 1 (Non-Protein Coding)-Kcnq1ot1, Paternally Expressed Gene 1-Peg1 and Paternally Expressed Gene 3-Peg3) was assessed by luminometric methylation analysis and bisulfite pyrosequencing, respectively, in F1 sperm, F2 E18.5 placenta and F2 E18.5 brain cortex., Main Results and the Role of Chance: F1 males exhibited lower sperm counts following lifetime exposure to both folic acid deficiency and the highest dose of folic acid supplementation (20FS), (both P < 0.05). Post-implantation losses were increased amongst F2 E18.5 day litters from 20FS exposed F1 males (P < 0.05). F2 litters derived from both 7FD and 20FS exposed F1 males had significantly higher postnatal-preweaning pup death (both P < 0.05). Sperm from 10FS exposed males had increased variance in methylation across imprinted gene H19, P < 0.05; increased variance at a few sites within H19 was also found for the 7FD and 20FS groups (P < 0.05). While the 20FS diet resulted in inter-individual alterations in methylation across the imprinted genes Snrpn and Peg3 in F2 E18.5 placenta, ≥50% of individual sites tested in Peg1 and/or Peg3 were affected in the 7FD and 10FS groups. Inter-individual alterations in Peg1 methylation were found in F2 E18.5 day 10FS group brain cortex (P < 0.05)., Large Scale Data: Not applicable., Limitations Reasons for Caution: The cause of the increase in postnatal-preweaning mortality was not investigated post-mortem. Further studies are required to understand the mechanisms underlying the adverse effects of folic acid deficiency and supplementation on developing male germ cells. Genome-wide DNA and histone methylome studies as well as gene expression studies are required to better understand the links between folic acid exposures, an altered germ cell epigenome and offspring outcomes., Wider Implications of the Findings: The findings of this study provide further support for paternally transmitted environmental effects. The results indicate that both folic acid deficiency and high dose supplementation can be detrimental to germ cell development and reproductive fitness, in part by altering DNA methylation in sperm., Study Funding and Competing Interests: This study was supported by a grant to J.M.T. from the Canadian Institutes of Health Research (CIHR #89944). The authors declare they have no conflicts of interest., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Background: Folate requirements increase during pregnancy and lactation. It is recommended that women who could become pregnant, are pregnant, or are lactating consume a folic acid (FA)-containing supplement. Objectives: We sought to determine breast-milk total folate and unmetabolized folic acid (UMFA) contents and their relation with FA-supplement use and doses in a cohort of Canadian mothers who were enrolled in the MIREC (Maternal-Infant Research on Environmental Chemicals) study. Design: Breast-milk tetrahydrofolate (THF), 5-methyl-THF, 5-formyl-THF, 5,10-methenyl-THF, and UMFA were measured with the use of liquid chromatography-tandem mass spectrometry ( n = 561). Total daily supplemental FA intake was based on self-reported FA-supplement use. Results: UMFA was detectable in the milk of 96.1% of the women. Total daily FA intake from supplements was associated with breast folate concentration and species. Breast-milk total folate was 18% higher ( P < 0.001) in supplement users ( n = 401) than in nonusers ( n = 160), a difference driven by women consuming >400 μg FA/d ( P ≤ 0.004). 5-Methyl-THF was 19% lower ( P < 0.001) and UMFA was 126% higher ( P < 0.001) in supplement users than in nonusers. Women who consumed >400 μg FA/d had proportionally lower 5-methyl-THF and higher UMFA than did women who consumed ≤400 μg FA/d. Conclusions: FA-supplement use was associated with modestly higher breast-milk total folate. Detectable breast-milk UMFA was nearly ubiquitous, including in women who did not consume an FA supplement. Breast-milk UMFA was proportionally higher than 5-methyl-THF in women who consumed >400 μg FA/d, thereby suggesting that higher doses exceed the physiologic capacity to metabolize FA and result in the preferential uptake of FA in breast milk. Therefore, FA-supplement doses >400 μg may not be warranted, especially in populations for whom FA fortification is mandatory., (© 2017 American Society for Nutrition.)

Objective: To examine the association between BMI and folate concentrations in serum and red blood cells (RBC) in pregnant women., Design: A cross-sectional comparison of folate concentrations in serum and RBC sampled simultaneously from the same individual., Setting: The Ottawa Hospital and Kingston General Hospital, Ontario, Canada., Subjects: Pregnant women recruited between 12 and 20 weeks of gestation., Results: A total of 869 pregnant women recruited from April 2008 to April 2009 were included in the final analysis. Serum folate was inversely associated and RBC folate positively associated with BMI, after adjusting for folic acid supplementation, age, gestational age at blood sample collection, race, maternal education, annual income, smoking and MTHFR 677C→T genotype. In stratified analyses, this differential association was significant in women with the MTHFR CC variant. In women with the CT and TT variants, the differential associations were in the same direction but not significant. Folic acid supplementation during pregnancy did not alter the differential association of BMI with serum and RBC folate concentration. This indicates that the current RBC folate cut-off approach for assessing risk of neural tube defects in obese women may be limited., Conclusions: BMI is inversely associated with serum folate and positively associated with RBC folate in pregnant women, especially for those with the MTHFR CC variant.

Introduction: The combined intake of folic acid (FA) from prenatal multivitamin supplements and fortified foods can result in FA intake values that exceed the tolerable upper intake level (UL). It is unclear what impact FA intake above the UL may have on the feto-placental unit. Our objective was to determine the effects of increasing concentrations of FA on trophoblast health and function in vitro., Methods: Two human placental cell lines [HTR-8/SVneo (n = 5 experiments) and BeWo (n = 5 experiments)] and human placenta tissue explants (n = 6 experiments) were exposed to increasing concentrations of FA (2-2000 ng/mL) for 48-h. Intracellular total folate concentration, trophoblast proliferation, viability, apoptosis, placenta cell invasion and β-hCG hormone release were assessed., Results: Exposure to increasing FA concentrations resulted in higher intracellular total folate in placental cell lines and tissue explants (p < 0.05); yet, only minimal effects of excess folic acid were observed on the primary indicators of placental health and function studied. Specifically, treatment with excess folic acid (2000 ng/mL) resulted in reduced cellular viability in the villous trophoblast BeWo cell line and increased rates of proliferation in the HT8-8/SVneo extravillous trophoblast cell line (p < 0.05). Further, deficient concentrations of folic acid (2 ng/mL) resulted in decreased cell viability and invasive capabilities of the HTR-8/SVneo extravillous trophoblast cell line (p < 0.05)., Discussion: Our results demonstrate that placental health and function may be compromised in conditions of folate deficiency, and not necessarily in conditions of excess FA. This finding supports the recommendation of prenatal folic acid supplementation in the North American population. Further work aimed at clarifying the therapeutic window of FA intake in the obstetrical population is warranted., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: Prenatal folic acid supplementation or maternal folate sufficiency may protect the offspring from obesity and insulin resistance. This study aims to summarize the findings of association between prenatal folic acid supplementation/maternal folate sufficiency and obesity/insulin resistance in the offspring., Methods: Twelve databases were searched for both published and unpublished work of prenatal folic acid supplementation/maternal folate status up to 1 July 2014. Experimental and observational studies on animals and human beings were included based on the eligibility criteria. There were no limits to the time period and language of publication. The study quality was assessed with a 10-Point Scale for Scientific Methodology., Results: The search identified 2548 records. Nine animal studies and five human studies satisfied search criteria were included. Five of these nine animal studies showed a protective effect of folic acid. Of the five human studies, one showed a protective effect of folic acid, two showed a harmful effect and two showed uncertain results., Conclusions: Data from both animal studies and human studies are inconsistent. Future researches with sophisticated designs are needed to demonstrate the potential protective effect of maternal folate on obesity/insulin resistance in the offspring in animal models and human pregnancies.

Dietary folate is a major source of methyl groups required for DNA methylation, an epigenetic modification that is actively maintained and remodeled during spermatogenesis. While high-dose folic acid supplementation (up to 10 times the daily recommended dose) has been shown to improve sperm parameters in infertile men, the effects of supplementation on the sperm epigenome are unknown. To assess the impact of 6 months of high-dose folic acid supplementation on the sperm epigenome, we studied 30 men with idiopathic infertility. Blood folate concentrations increased significantly after supplementation with no significant improvements in sperm parameters. Methylation levels of the differentially methylated regions of several imprinted loci (H19, DLK1/GTL2, MEST, SNRPN, PLAGL1, KCNQ1OT1) were normal both before and after supplementation. Reduced representation bisulfite sequencing (RRBS) revealed a significant global loss of methylation across different regions of the sperm genome. The most marked loss of DNA methylation was found in sperm from patients homozygous for the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, a common polymorphism in a key enzyme required for folate metabolism. RRBS analysis also showed that most of the differentially methylated tiles were located in DNA repeats, low CpG-density and intergenic regions. Ingenuity Pathway Analysis revealed that methylation of promoter regions was altered in several genes involved in cancer and neurobehavioral disorders including CBFA2T3, PTPN6, COL18A1, ALDH2, UBE4B, ERBB2, GABRB3, CNTNAP4 and NIPA1. Our data reveal alterations of the human sperm epigenome associated with high-dose folic acid supplementation, effects that were exacerbated by a common polymorphism in MTHFR., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Mandatory folic acid fortification of white-wheat flour and selected other grain products has reduced the prevalence of neural tube defects in Canada; however, the fortification of whole-wheat flour is not permitted., Objective: The objective of this study was to model the impact of adding folic acid to whole-wheat flour on the folate intake distribution of Canadians., Methods: Twenty-four-hour dietary recall and supplement intake data (n = 35,107) collected in the 2004 Canadian Community Health Survey 2.2 were used to calculate the prevalence of folate inadequacy (POFI) and the proportion of folic acid intakes above the Tolerable Upper Intake Level (UL). In model 1, folic acid was added to whole-wheat flour-containing foods in amounts comparable to those that are mandatory for white-wheat flour-containing foods. In model 2, a 50% overage of folic acid fortification was considered. Models 3 and 4 included assessment of folate intake distributions in adult whole-wheat consumers with or without a fortification overage. SIDE (Software for Intake Distribution Estimation; Department of Statistics and Center for Agricultural and Rural Development, Iowa State University) was used to estimate usual folate intakes., Results: Mean folate intakes increased by ∼ 5% in all sex and age groups when whole-wheat foods were fortified (models 1 and 2; P < 0.0001). Folic acid fortification of whole-wheat flour-containing foods did not change the POFI or percentage of intakes above the UL in the general population, whether in supplement users or nonusers. Among whole-wheat consumers, the POFI was reduced by 10 percentage points after fortification of whole-wheat flour-containing foods (95% CIs did not overlap). The percentage of whole-wheat consumers with intakes above the UL did not change., Conclusion: Although folic acid fortification of whole-wheat flour-containing foods is unlikely to change the POFI or proportion of folic acid intakes above the UL in the general Canadian population, this fortification strategy may reduce the POFI in adult whole-wheat consumers., (© 2015 American Society for Nutrition.)

Folate is an essential B vitamin required for de novo purine and thymidylate synthesis, and for the remethylation of homocysteine to form methionine. Folate deficiency has been associated with placenta-related pregnancy complications, as have SNP in genes of the folate-dependent enzymes, methionine synthase (MTR) and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1). We aimed to determine the effect of altered folate metabolism on placental cell proliferation, viability and invasive capacity and on progesterone and human chorionic gonadotropin (hCG) secretion. Human placental choriocarcinoma (JEG-3) cells cultured in low folic acid (FA) (2 nM) demonstrated 13% (P<0.001) and 26% (P<0.001) lower proliferation, 5.5% (P=0.025) and 7.5% (P=0.004) lower invasion capacity, and 5 to 7.5% (P=0.004-0.025) lower viability compared with control (20 nM) or supplemented (100 nM) cells, respectively. FA concentration had no effect on progesterone or hCG secretion. Small interfering RNA (siRNA) knockdown of MTR gene and protein expression resulted in 17.7% (P<0.0001) lower proliferation and 61% (P=0.014) higher progesterone secretion, but had no effect on cell invasion and hCG secretion. siRNA knockdown of MTHFD1 gene expression in the absence of detectable changes in protein expression resulted in 10.3% (P=0.001) lower cell proliferation, but had no effect on cell invasion and progesterone or hCG secretion. Our data indicate that impaired folate metabolism can result in lower trophoblast proliferation, and could alter viability, invasion capacity and progesterone secretion, which may explain in part the observed associations between folate and placenta-related complications.

Folate is an essential B vitamin required for the de novo synthesis of purines, thymidylate and methionine. Folate deficiency can lead to mutations and genome instability, and has been shown to exacerbate the genotoxic potential of environmental toxins. We hypothesized that a folic acid (FA) deficient diet would induce genotoxicity in mice as measured by the Pig-a mutant phenotype (CD24-) and micronuclei (MN) in reticulocytes (RET) and red blood cells/normochromatic erythrocytes (RBC/NCE). Male Balb/c mice were fed a FA deficient (0 mg/kg), control (2 mg/kg) or supplemented (6 mg/kg) diet from weaning for 18 wk. Mice fed the deficient diet had 70% lower liver folate (p < 0.001), 1.8 fold higher MN-RET (p < 0.001), and 1.5 fold higher MN-NCE (p < 0.001) than mice fed the control diet. RET(CD24-) and RBC(CD24-) frequencies were not different between mice fed the deficient and control diets. Compared to mice fed the FA supplemented diet, mice fed the deficient diet had 73% lower liver folate (p < 0.001), a 2.0 fold increase in MN-RET (p < 0.001), a 1.6 fold increase in MN-NCE (p < 0.001) and 3.8 fold increase in RBC(CD24-) frequency (p = 0.011). RET(CD24-) frequency did not differ between mice fed the deficient and supplemented diets. Our data suggest that FA adequacy protects against mutagenesis at the Pig-a locus and MN induction in the red blood cells of mice., Competing Interests: Author disclosure The authors declare no conflicts of interest., (Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.)

Background: Genetic variation can cause variable responses to environmental stimuli. A number of single-nucleotide polymorphisms (SNPs) have been associated with B vitamin status or chronic diseases related to vitamin B-12 and folate metabolism., Objective: Our objective was to identify associations between common SNPs in genes related to folate and vitamin B-12 metabolism or associated with B vitamin-related chronic diseases and biomarkers of nutrient status in a population exposed to folic acid fortification., Design: A panel of 116 SNPs was sequenced by using the Sequenom iPLEX Gold platform in a sample of 3114 adults aged 20-79 y from the Canadian Health Measures Survey, cycle 1. Associations between these SNPs and red blood cell (RBC) folate, serum vitamin B-12, and plasma total homocysteine were determined., Results: Twenty-one SNPs and 6 haplotype blocks were associated with RBC folate, serum vitamin B-12, and/or plasma homocysteine concentrations. Vitamin status was associated mainly with SNPs in genes directly involved in vitamin absorption/uptake (CUBN, CD320), transport (TCN1, TCN2), or metabolism (BHMT2, CBS, MTHFR, MUT, SHMT1). Other SNPs included those in the DNMT2, DPEP1, FUT2, NOX4, and PON1 genes., Conclusions: We identified novel associations between SNPs in CD320 and DNMT2, which had been previously associated with neural tube defects, and vitamin B-12 status, as well as between SNPs in SHMT1, which had been previously associated with colorectal cancer and cardiovascular disease risk, and RBC folate status. These novel associations provide a plausible metabolic rationale for the association of these SNPs with B vitamin-related diseases. We also observed a novel association between an SNP in CUBN with RBC folate and confirmed the association of a number of SNPs with B vitamin status in this large cross-sectional study., (© 2015 American Society for Nutrition.)

It is now established that the mitochondrial production of formate is a major process in the endogenous generation of folate-linked one-carbon groups. We have developed an in vivo approach involving the constant infusion of [(13)C]formate until isotopic steady state is attained to measure the rate of endogenous formate production in rats fed on either a folate-replete or folate-deficient diet. Formate was produced at a rate of 76 μmol·h(-1)·100 g of body weight(-1) in the folate-replete rats, and this was decreased by 44% in folate-deficient rats. This decreased formate production was confirmed in isolated rat liver mitochondria where formate production from serine, the principal precursor of one-carbon groups, was decreased by 85%, although formate production from sarcosine and dimethylglycine (choline metabolites) was significantly increased. We attribute this unexpected result to the demonstrated production of formaldehyde by sarcosine dehydrogenase and dimethylglycine dehydrogenase from their respective substrates in the absence of tetrahydrofolate and subsequent formation of formate by formaldehyde dehydrogenase. Comparison of formate production with the ingestion of dietary formate precursors (serine, glycine, tryptophan, histidine, methionine, and choline) showed that ∼75% of these precursors were converted to formate, indicating that formate is a significant, although underappreciated end product of choline and amino acid oxidation. Ingestion of a high protein diet did not result in increased production of formate, suggesting a regulation of the conversion of these precursors at the mitochondrial level to formate., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objective: Mandatory folic acid fortification was implemented in Canada in 1998 to reduce the risk of neural tube defects (NTD). Our objective was to assess the relationship between socio-demographic factors and folate status in non-supplement-consuming Canadian women of childbearing age., Methods: Data on demographic factors, lifestyle factors, physical measures and red blood cell (RBC) folate concentration were collected from 1,008 non-supplement-consuming women aged 15-49 years in the Canadian Health Measures Survey (2007-2009). RBC folate ³906 nmol/L was used as a cut-off for optimal folate status for protection from NTD., Results: Approximately 75% of non-supplement consuming women had an RBC folate concentration ³906 nmol/L. Young age (15-19 years), White ethnicity, less than secondary education, lowest income adequacy, smoking and high body mass index were associated with a higher prevalence of lower folate status. After adjustment, only young age (adjusted odds ratio [OR] 1.99-95% confidence interval [CI]: 1.25-3.18) was associated with lower folate status. Less than secondary education (adjusted OR 5.66, 95% CI: 1.10-29.04) and lowest income adequacy (adjusted OR 4.77, 95% CI: 1.06-21.49) were associated with lower folate status in women aged 15-24 and 25-49 years, respectively., Conclusions: Many risk factors for lower folate status identified before food fortification was implemented were not associated with folate status in our representative sample of non-supplement-consuming Canadian women. However, younger women, women aged 15-24 with less than secondary education and women aged 25-49 with low income adequacy remain at risk of lower folate status, supporting the continued promotion of folic acid supplement use to women of childbearing age.

Folate metabolism affects DNA synthesis, methylation, mutation rates, genomic stability, and gene expression, which are altered in colon cancer. Serine hydroxymethyltransferase 1 (SHMT1) regulates thymidylate (dTMP) biosynthesis and uracil accumulation in DNA, and as such affects genome stability. Previously, we showed that decreased SHMT1 expression in Shmt1 knockout mice (Shmt1(-/+)) or its impaired nuclear localization, as occurs in mice over-expressing an Shmt1 transgene (Shmt1(tg+)), results in elevated uracil incorporation into DNA, which could affect colon cancer risk. We used these 2 models to determine the effect of altered SHMT1 expression and localization, and its interaction with folate insufficiency, on azoxymethane (AOM)-induced colon cancer in mice. Shmt1(-/+) and Shmt1(tg+) mice were weaned to a control or folate-and-choline-deficient (FCD) diet and fed the diet for 28 or 32 wk, respectively. At 6 wk of age, mice were injected weekly for 6 wk with 10 mg/kg AOM (w/v in saline). Colon uracil concentrations in nuclear DNA were elevated 2-7 fold in Shmt1(-/+) and Shmt1(tg+) mice. However, colon tumor incidence and numbers were not dependent on SHMT1 expression in Shmt1(-/+) or Shmt1(-/-) mice. The FCD diet reduced tumor load independent of Shmt1 genotype. In contrast, Shmt1(tg+) mice exhibited a 30% reduction in tumor incidence, a 50% reduction in tumor number, and a 60% reduction in tumor load compared with wild-type mice independent of dietary folate intake. Our data indicate that uracil accumulation in DNA does not predict tumor number in AOM-mediated carcinogenesis. Furthermore, enrichment of SHMT1 in the cytoplasm, as observed in Shmt1(tg+) mice, protects against AOM-mediated carcinogenesis independent of its role in nuclear de novo dTMP biosynthesis.

Inflammatory bowel disease (IBD) is a risk factor for the development of colon cancer. Environmental factors including diet and the microflora influence disease outcome. Folate and homocysteine have been associated with IBD-mediated colon cancer but their roles remain unclear. We used a model of chemically induced ulcerative colitis (dextran sodium sulphate (DSS)) with or without the colon carcinogen azoxymethane (AOM) to determine the impact of dietary folic acid (FA) on colonic microflora and the development of colon tumours. Male mice (n 15 per group) were fed a FA-deficient (0 mg/kg), control (2 mg/kg) or FA-supplemented (8 mg/kg) diet for 12 weeks. Folate status was dependent on the diet (P< 0·001) and colitis-induced treatment (P= 0·04) such that mice with colitis had lower circulating folate. FA had a minimal effect on tumour initiation, growth and progression, although FA-containing diets tended to be associated with a higher tumour prevalence in DSS-treated mice (7-20 v. 0%, P= 0·08) and the development of more tumours in the distal colon of AOM-treated mice (13-83% increase, P= 0·09). Folate deficiency was associated with hyperhomocysteinaemia (P< 0·001) but homocysteine negatively correlated with tumour number (r - 0·58, P= 0·02) and load (r - 0·57, P= 0·02). FA had no effect on the intestinal microflora. The present data indicate that FA intake has no or little effect on IBD or IBD-mediated colon cancer in this model and that hyperhomocysteinaemia is a biomarker of dietary status and malabsorption rather than a cause of IBD-mediated colon cancer.

To date, fewer than 50 mutagens have been studied for their ability to cause heritable mutations. The majority of those studied are classical mutagens like radiation and anti-cancer drugs. Very little is known about the dietary variables influencing germline mutation rates. Folate is essential for DNA synthesis and methylation and can impact chromatin structure. We therefore determined the effects of folic acid-deficient (0mg/kg), control (2mg/kg) and supplemented (6mg/kg) diets in early development and during lactation or post-weaning on mutation rates and chromatin quality in sperm of adult male Balb/c mice. The sperm chromatin structure assay and mutation frequencies at expanded simple tandem repeats (ESTRs) were used to evaluate germline DNA integrity. Treatment of a subset of mice fed the control diet with the mutagen ethylnitrosourea (ENU) at 8 weeks of age was included as a positive control. ENU treated mice exhibited decreased cauda sperm counts, increased DNA fragmentation and increased ESTR mutation frequencies relative to non-ENU treated mice fed the control diet. Male mice weaned to the folic acid deficient diet had decreased cauda sperm numbers, increased DNA fragmentation index, and increased ESTR mutation frequency. Folic acid deficiency in early development did not lead to changes in sperm counts or chromatin integrity in adult mice. Folic acid supplementation in early development or post-weaning did not affect germ cell measures. Therefore, adequate folic acid intake in adulthood is important for preventing chromatin damage and mutation in the male germline. Folic acid supplementation at the level achieved in this study does not improve nor is it detrimental to male germline chromatin integrity., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Folate deficiency can cause chromosome damage, which could result from reduced de novo thymidylate synthesis or DNA hypomethylation. High folic acid intake has been hypothesized to inhibit folate-dependent one-carbon metabolism, which could also lead to DNA damage. A large proportion of the general population may have high folic acid intakes. In this study, 2 experiments were conducted to examine the effects of folate on chromosome damage. First, male mice were fed folic acid-deficient (D) (0 mg folic acid/kg diet), control (C) (2 mg/kg), or folic acid-supplemented (S) (6 mg folic acid/kg diet) diets from weaning to maturity. Second, female mice were fed the D, C, or S diet throughout pregnancy, lactation, and breeding for 3 generations; male mice from the F3 generation were fed the same diet as their mothers from weaning, producing D, C, and S F3 male mice. RBC micronucleus frequencies, a measure of chromosome damage or aneuploidy, were determined for both experimental groups. In mice fed diets from weaning to maturity, erythrocyte micronucleus frequency was 24% greater in D compared with C mice. F3 mice fed diet D had 260% and 174% greater reticulocyte and erythrocyte micronucleus frequencies compared with F3 C mice, respectively. The S diets did not affect micronucleus frequency, suggesting that excess folic acid at this level does not promote or protect against chromosome damage. The results suggest that chronic exposure to folic acid at the levels similar to those achieved through fortification is unlikely to be clastogenic or aneugenic.

Background: Vitamin B-12 is an important cofactor required for nucleotide and amino acid metabolism. Vitamin B-12 deficiency causes anemia and neurologic abnormalities-a cause for concern for the elderly, who are at increased risk of vitamin B-12 malabsorption. Vitamin B-12 deficiency is also associated with an increased risk of neural tube defects and hyperhomocysteinemia. The metabolism of vitamin B-12 and folate is interdependent, which makes it of public health interest to monitor biomarkers of vitamin B-12, folate, and homocysteine in a folic acid-fortified population., Objective: The objective was to determine the vitamin B-12, folate, and homocysteine status of the Canadian population in the period after folic acid fortification was initiated., Design: Blood was collected from a nationally representative sample of ∼5600 participants aged 6-79 y in the Canadian Health Measures Survey during 2007-2009 and was analyzed for serum vitamin B-12, red blood cell folate, and plasma total homocysteine (tHcy)., Results: A total of 4.6% of Canadians were vitamin B-12 deficient (<148 pmol/L). Folate deficiency (<320 nmol/L) was essentially nonexistent. Obese individuals were less likely to be vitamin B-12 adequate than were individuals with a normal BMI. A total of 94.9% of Canadians had a normal tHcy status (≤13 μmol/L), and individuals with normal tHcy were more likely to be vitamin B-12 adequate and to have high folate status (>1090 nmol/L)., Conclusions: Approximately 5% of Canadians are vitamin B-12 deficient. One percent of adult Canadians have metabolic vitamin B-12 deficiency, as evidenced by combined vitamin B-12 deficiency and high tHcy status. In a folate-replete population, vitamin B-12 is a major determinant of tHcy.

Cytoplasmic folate-mediated one carbon (1C) metabolism functions to carry and activate single carbons for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine. C1 tetrahydrofolate (THF) synthase, encoded by Mthfd1, is an entry point of 1Cs into folate metabolism through its formyl-THF synthetase (FTHFS) activity that catalyzes the ATP-dependent conversion of formate and THF to 10-formyl-THF. Disruption of FTHFS activity by the insertion of a gene trap vector into the Mthfd1 gene results in embryonic lethality in mice. Mthfd1gt/+ mice demonstrated lower hepatic adenosylmethionine levels, which is consistent with formate serving as a source of 1Cs for cellular methylation reactions. Surprisingly, Mthfd1gt/+ mice exhibited decreased levels of uracil in nuclear DNA, indicating enhanced de novo thymidylate synthesis, and suggesting that serine hydroxymethyltransferase and FTHFS compete for a limiting pool of unsubstituted THF. This study demonstrates the essentiality of the Mthfd1 gene and indicates that formate-derived 1Cs are utilized for de novo purine synthesis and the remethylation of homocysteine in liver. Further, the depletion of cytoplasmic FTHFS activity enhances thymidylate synthesis, affirming the competition between thymidylate synthesis and homocysteine remethylation for THF cofactors.