Folate is a source of one-carbons necessary for DNA methylation, a critical epigenetic modification necessary for genomic structure and function. The use of supplemental folic acid is widespread however; the potential influence on DNA methylation is unclear. We measured global DNA methylation using DNA extracted from samples from a population-based, double-blind randomized trial of folic acid supplementation (100, 400, 4000 µg per day) taken for 6 months; including a 3 month post-supplementation sample. We observed no changes in global DNA methylation in response to up to 4,000 µg/day for 6 months supplementation in DNA extracted from uncoagulated blood (approximates circulating blood). However, when DNA methylation was determined in coagulated samples from the same individuals at the same time, significant time, dose, and MTHFR genotype-dependent changes were observed. The baseline level of DNA methylation was the same for uncoagulated and coagulated samples; marked differences between sample types were observed only after intervention. In DNA from coagulated blood, DNA methylation decreased (-14%; P<0.001) after 1 month of supplementation and 3 months after supplement withdrawal, methylation decreased an additional 23% (P<0.001) with significant variation among individuals (max+17%; min-94%). Decreases in methylation of ≥25% (vs. <25%) after discontinuation of supplementation were strongly associated with genotype: MTHFR CC vs. TT (adjusted odds ratio [aOR] 12.9, 95%CI 6.4, 26.0). The unexpected difference in DNA methylation between DNA extracted from coagulated and uncoagulated samples in response to folic acid supplementation is an important finding for evaluating use of folic acid and investigating the potential effects of folic acid supplementation on coagulation.

DNA methylation is an epigenetic modification critical to normal genome regulation and development. The vitamin folate is a key source of the one carbon group used to methylate DNA. Because normal mammalian development is dependent on DNA methylation, there is enormous interest in assessing the potential for changes in folate intake to modulate DNA methylation both as a biomarker for folate status and as a mechanistic link to developmental disorders and chronic diseases including cancer. This review highlights the role of DNA methylation in normal genome function, how it can be altered, and the evidence of the role of folate/folic acid in these processes.

Periconceptional folic acid use can often prevent neural tube defects (NTDs). Variants of genes involved in folate metabolism in mothers and children have been associated with occurrence of NTDs. We identified Irish families with individuals affected by neural tube defects. In these families, we observed that neural tube defects and birth defects overall occurred at a higher rate in the maternal lineage compared with the paternal lineage. The goal of this study was to look for evidence for genetic effects that could explain the discrepancy in the occurrence of these birth defects in the maternal vs. paternal lineage. We genotyped blood samples from 322 individuals from NTD-affected Irish families, identified through their membership in spina bifida associations. We looked for differences in distribution in maternal vs. paternal lineages of five genetic polymorphisms: the DHFR 19 bp deletion, MTHFD1 1958G>A, MTHFR 1298A>C, MTHFR 677C>T, and SLC19A1 80A>G. In addition to looking at genotypes individually, we determined the number of genotypes associated with decreased folate metabolism in each relative ("risk genotypes") and compared the distribution of these genotypes in maternal vs. paternal relatives. Overall, maternal relatives had a higher number of genotypes associated with lower folate metabolism than paternal relatives (p = 0.017). We expected that relatives would share the same risk genotype as the individuals with NTDs and/or their mothers. However, we observed that maternal relatives had an over-abundance of any risk genotype, rather than one specific genotype. The observed genetic effects suggest an epigenetic mechanism in which decreased folate metabolism results in epigenetic alterations related to the increased rate of NTDs and other birth defects seen in the maternal lineage. Future studies on the etiology of NTDs and other birth defects could benefit from including multigenerational extended families, in order to explore potential epigenetic mechanisms.

OBJECTIVES: To examine the potential for bias in the estimate of under-5 mortality due to birth defects recently produced by the WHO and the Maternal and Child Epidemiology Estimation research group. DESIGN: Systematic analysis. METHODS: We examined the estimated number of under-5 deaths due to birth defects, the birth defect specific under-5 mortality rate, and the per cent of under-5 mortality due to birth defects, by geographic region, national income and under-5 mortality rate for three age groups from 2000 to 2019. RESULTS: The under-5 deaths per 1000 live births from birth defects fell from 3.4 (95% uncertainty interval (UI) 3.1-3.8) in 2000 to 2.9 (UI 2.6-3.3) in 2019. The per cent of all under-5 mortality attributable to birth defects increased from 4.6% (UI 4.1%-5.1%) in 2000 to 7.6% (UI 6.9%-8.6%) in 2019. There is significant variability in mortality due to birth defects by national income level. In 2019, the under-5 mortality rate due to birth defects was less in high-income countries than in low-income and middle-income countries, 1.3 (UI 1.2-1.3) and 3.0 (UI 2.8-3.4) per 1000 live births, respectively. These mortality rates correspond to 27.7% (UI 26.6%-28.8%) of all under-5 mortality in high-income countries being due to birth defects, and 7.4% (UI 6.7%-8.2%) in low-income and middle-income countries. CONCLUSIONS: While the under-5 mortality due to birth defects is declining, the per cent of under-5 mortality attributable to birth defects has increased, with significant variability across regions globally. The estimates in low-income and middle-income countries are likely underestimated due to the nature of the WHO estimates, which are based in part on verbal autopsy studies and should be taken as a minimum estimate. Given these limitations, comprehensive and systematic estimates of the mortality burden due to birth defects are needed to estimate the actual burden.

BACKGROUND: Folate, including the folic acid form, is a key component of the one-carbon metabolic pathway used for DNA methylation. Changes in DNA methylation patterns during critical development periods are associated with disease outcomes and are associated with changes in nutritional status in pregnancy. The long-term impact of periconceptional folic acid supplementation on DNA methylation patterns is unknown. OBJECTIVES: To determine the long-term impact of periconceptional folic acid supplementation on DNA methylation patterns, we examined the association of the recommended dosage (400 μg/d) and time period (periconceptional before pregnancy through first trimester) of folic acid supplementation with the DNA methylation patterns in the offspring at age 14-17 y compared with offspring with no supplementation. METHODS: Two geographic sites in China from the 1993-1995 Community Intervention Program of folic acid supplementation were selected for the follow-up study. DNA methylation at 402,730 CpG sites was assessed using saliva samples from 89 mothers and 179 adolescents (89 male). The mean age at saliva collection was 40 y among mothers (range: 35-54 y) and 15 y among adolescents (range: 14-17 y). Epigenome-wide analyses were conducted to assess the interactions of periconceptional folic acid exposure, the 5,10-methylenetetrahydrofolate reductase (MTHFR)-C677T genotype, and epigenome-wide DNA methylation controlling for offspring sex, geographic region, and background cell composition in the saliva. RESULTS: In the primary outcome, no significant differences were observed in epigenome-wide methylation patterns between adolescents exposed and those non-exposed to maternal periconceptional folic acid supplementation after adjustment for potential confounders [false discovery rate (FDR) P values < 0.05]. The MTHFR-C677T genotype did not modify this lack of association (FDR P values < 0.05). CONCLUSIONS: Overall, there were no differences in DNA methylation between adolescents who were exposed during the critical developmental window and those not exposed to the recommended periconceptional/first-trimester dosage of folic acid.

BACKGROUND: Neural tube defects (NTDs) encompass a variety of distinct types. We assessed if the preventive effect of folic acid (FA) varied by NTD type and infant sex. METHODS: We examined all pregnancies with NTD status confirmation from a pregnancy-monitoring system in selected locations in northern and southern regions of China between 1993 and 1996. Women who took 400 μg of FA daily during 42 days after last menstrual period were considered FA users. We analyzed NTD prevalence by FA use status, NTD type, geographic region, and infant sex. RESULTS: Among 626,042 pregnancies, 700 were affected by an NTD. Among FA nonusers, 65 pregnancies (8.8 per 10,000) in the north and 51 pregnancies (1.2 per 10,000) in the south were affected by one of the two rare NTDs, that is, craniorachischisis, iniencephaly. FA use prevented occurrence of these two rare NTDs and reduced the prevalence of spina bifida (SB) by 78% (from 17.9 to 3.9 per 10,000) in the north and 51% (from 2.4 to 1.2 per 10,000) in the south. Among FA users, SB prevalence, including SB with high lesion level, was significantly reduced in both geographic regions. FA use reduced prevalence of anencephaly and encephalocele by 85% and 50%, respectively in the north, while it did not reduce the prevalence of these two NTDs in the south. There was a greater reduction in NTD prevalence in female than in male infants and fetuses. CONCLUSIONS: This is the first study to show that FA prevents the entire spectrum of NTD types.

BACKGROUND: Arsenic is a common environmental toxin. Exposure to arsenic (particularly its inorganic form) through contaminated food and drinking water is an important public health burden worldwide, and is associated with increased risk of neurotoxicity, congenital anomalies, cancer, and adverse neurodevelopment in children. Arsenic is excreted following methylation reactions, which are mediated by folate. Provision of folate through folic acid supplements could facilitate arsenic methylation and excretion, thereby reducing arsenic toxicity. OBJECTIVES: To assess the effects of provision of folic acid (through fortified foods or supplements), alone or in combination with other nutrients, in lessening the burden of arsenic-related health outcomes and reducing arsenic toxicity in arsenic-exposed populations. SEARCH METHODS: In September 2020, we searched CENTRAL, MEDLINE, Embase, 10 other international databases, nine regional databases, and two trials registers. SELECTION CRITERIA: Randomised controlled trials (RCTs) and quasi-RCTs comparing the provision of folic acid (at any dose or duration), alone or in combination with other nutrients or nutrient supplements, with no intervention, placebo, unfortified food, or the same nutrient or supplements without folic acid, in arsenic-exposed populations of all ages and genders. DATA COLLECTION AND ANALYSIS: We used standard methodological procedures expected by Cochrane. MAIN RESULTS: We included two RCTs with 822 adults exposed to arsenic-contaminated drinking water in Bangladesh. The RCTs compared 400 µg/d (FA400) or 800 µg/d (FA800) folic acid supplements, given for 12 or 24 weeks, with placebo. One RCT, a multi-armed trial, compared FA400 plus creatine (3 g/d) to creatine alone. We judged both RCTs at low risk of bias in all domains. Due to differences in co-intervention, arsenic exposure, and participants' nutritional status, we could not conduct meta-analyses, and therefore, provide a narrative description of the data. Neither RCT reported on cancer, all-cause mortality, neurocognitive function, or congenital anomalies. Folic acid supplements alone versus placebo Blood arsenic. In arsenic-exposed individuals, FA likely reduces blood arsenic concentrations compared to placebo (2 studies, 536 participants; moderate-certainty evidence). For folate-deficient and folate-replete participants who received arsenic-removal water filters as a co-intervention, FA800 reduced blood arsenic levels more than placebo (percentage change (%change) in geometric mean (GM) FA800 -17.8%, 95% confidence intervals (CI) -25.0 to -9.8; placebo GM -9.5%, 95% CI -16.5 to -1.8; 1 study, 406 participants). In one study with 130 participants with low baseline plasma folate, FA400 reduced total blood arsenic (%change FA400 mean (M) -13.62%, standard error (SE) ± 2.87; placebo M -2.49%, SE ± 3.25), and monomethylarsonic acid (MMA) concentrations (%change FA400 M -22.24%, SE ± 2.86; placebo M -1.24%, SE ± 3.59) more than placebo. Inorganic arsenic (InAs) concentrations reduced in both groups (%change FA400 M -18.54%, SE ± 3.60; placebo M -10.61%, SE ± 3.38). There was little to no change in dimethylarsinic acid (DMA) in either group. Urinary arsenic. In arsenic-exposed individuals, FA likely reduces the proportion of total urinary arsenic excreted as InAs (%InAs) and MMA (%MMA) and increases the proportion excreted as DMA (%DMA) to a greater extent than placebo (2 studies, 546 participants; moderate-certainty evidence), suggesting that FA enhances arsenic methylation. In a mixed folate-deficient and folate-replete population (1 study, 352 participants) receiving arsenic-removal water filters as a co-intervention, groups receiving FA had a greater decrease in %InAs (within-person change FA400 M -0.09%, 95% CI -0.17 to -0.01; FA800 M -0.14%, 95% CI -0.21 to -0.06; placebo M 0.05%, 95% CI 0.00 to 0.10), a greater decrease in %MMA (within-person change FA400 M -1.80%, 95% CI -2.53 to -1.07; FA800 M -2.60%, 95% CI -3.35 to -1.85; placebo M 0.15%, 95% CI -0.37 to 0.68), and a greater increase in %DMA (within-person change FA400 M 3.25%, 95% CI 1.81 to 4.68; FA800 M 4.57%, 95% CI 3.20 to 5.95; placebo M -1.17%, 95% CI -2.18 to -0.17), compared to placebo. In 194 participants with low baseline plasma folate, FA reduced %InAs (%change FA400 M -0.31%, SE ± 0.04; placebo M -0.13%, SE ± 0.04) and %MMA (%change FA400 M -2.6%, SE ± 0.37; placebo M -0.71%, SE ± 0.43), and increased %DMA (%change FA400 M 5.9%, SE ± 0.82; placebo M 2.14%, SE ± 0.71), more than placebo. Plasma homocysteine: In arsenic-exposed individuals, FA400 likely reduces homocysteine concentrations to a greater extent than placebo (2 studies, 448 participants; moderate-certainty evidence), in the mixed folate-deficient and folate-replete population receiving arsenic-removal water filters as a co-intervention (%change in GM FA400 -23.4%, 95% CI -27.1 to -19.5; placebo -1.3%, 95% CI -5.3 to 3.1; 1 study, 254 participants), and participants with low baseline plasma folate (within-person change FA400 M -3.06 µmol/L, SE ± 3.51; placebo M -0.05 µmol/L, SE ± 4.31; 1 study, 194 participants). FA supplements plus other nutrient supplements versus nutrient supplements alone In arsenic-exposed individuals who received arsenic-removal water filters as a co-intervention, FA400 plus creatine may reduce blood arsenic concentrations more than creatine alone (%change in GM FA400 + creatine -14%, 95% CI -22.2 to -5.0; creatine -7.0%, 95% CI -14.8 to 1.5; 1 study, 204 participants; low-certainty evidence); may not change urinary arsenic methylation indices (FA400 + creatine: %InAs M 13.2%, SE ± 7.0; %MMA M 10.8, SE ± 4.1; %DMA M 76, SE ± 7.8; creatine: %InAs M 14.8, SE ± 5.5; %MMA M 12.8, SE ± 4.0; %DMA M 72.4, SE ±7.6; 1 study, 190 participants; low-certainty evidence); and may reduce homocysteine concentrations to a greater extent (%change in GM FA400 + creatinine -21%, 95% CI -25.2 to -16.4; creatine -4.3%, 95% CI -9.0 to 0.7; 1 study, 204 participants; low-certainty evidence) than creatine alone. AUTHORS' CONCLUSIONS: There is moderate-certainty evidence that FA supplements may benefit blood arsenic concentration, urinary arsenic methylation profiles, and plasma homocysteine concentration versus placebo. There is low-certainty evidence that FA supplements plus other nutrients may benefit blood arsenic and plasma homocysteine concentrations versus nutrients alone. No studies reported on cancer, all-cause mortality, neurocognitive function, or congenital anomalies. Given the limited number of RCTs, more studies conducted in diverse settings are needed to assess the effects of FA on arsenic-related health outcomes and arsenic toxicity in arsenic-exposed adults and children.

In 1998 a Tolerable Upper Intake Level (UL) for folic acid was established based on case reports from the 1940s suggesting that high-dosage folic acid intake, used to treat patients with pernicious anemia, had the potential to precipitate or speed-up the development of neurological problems. This UL has been employed in the decision-making process used by more than 80 countries to establish programs to fortify staple foods with folic acid to prevent neural tube birth defects. Some have claimed that this UL is flawed and has become an obstacle to the wider adoption of neural tube defect prevention programs and have called for re-evaluation of the scientific validity of this UL. Case reports cannot establish causality, but they can reveal patterns in the timing of the onset and treatment of patients with pernicious anemia. These patterns can be compared with secular trends of usual medical practice for the treatment of pernicious anemia and with the changes in usage of folic acid preparations, including recommended therapeutic dosage and precautions for its usage surrounding the synthesis of folic acid in 1945 and vitamin B12 in 1948. Folic acid package inserts, early editions of hematology textbooks, and international expert reports provide valuable historical information. The recommended therapeutic daily dosage for folic acid of 5-20 mg was unchanged from 1946 through to 1971. The likely cause of the neurological problems encountered is the development of vitamin B12 neuropathy when pernicious anemia was treated with high-dosage folic acid before vitamin B12 was widely available in the early 1950s. Thus, the historical record does not provide compelling evidence that folic acid can potentially cause neurologic complications among those with low vitamin B12 status and lends support for reconsidering the basis for the UL of folic acid.

BACKGROUND: For women of reproductive age, a population-level red blood cell (RBC) folate concentration below the threshold 906 nmol/L or 400 ng/mL indicates folate insufficiency and suboptimal neural tube defect (NTD) prevention. A corresponding population plasma/serum folate concentration threshold for optimal NTD prevention has not been established. OBJECTIVE: The aim of this study was to examine the association between plasma and RBC folate concentrations and estimated a population plasma folate insufficiency threshold (pf-IT) corresponding to the RBC folate insufficiency threshold (RBCf-IT) of 906 nmol/L. METHODS: We analyzed data on women of reproductive age (n = 1673) who participated in a population-based, randomized folic acid supplementation trial in northern China. Of these women, 565 women with anemia and/or vitamin B-12 deficiency were ineligible for folic acid intervention (nonintervention group); the other 1108 received folic acid supplementation for 6 mo (intervention group). We developed a Bayesian linear model to estimate the pf-IT corresponding to RBCf-IT by time from supplementation initiation, folic acid dosage, methyltetrahydrofolate reductase (MTHFR) genotype, body mass index (BMI), vitamin B-12 status, or anemia status. RESULTS: Using plasma and RBC folate concentrations of the intervention group, the estimated median pf-IT was 25.5 nmol/L (95% credible interval: 24.6, 26.4). The median pf-ITs were similar between the baseline and postsupplementation samples (25.7 compared with 25.2 nmol/L) but differed moderately (+/-3-4 nmol/L) by MTHFR genotype and BMI. Using the full population-based baseline sample (intervention and nonintervention), the median pf-IT was higher for women with vitamin B-12 deficiency (34.6 nmol/L) and marginal deficiency (29.8 nmol/L) compared with the sufficient group (25.6 nmol/L). CONCLUSIONS: The relation between RBC and plasma folate concentrations was modified by BMI and genotype and substantially by low plasma vitamin B-12. This suggests that the threshold of 25.5 nmol/L for optimal NTD prevention may be appropriate in populations with similar characteristics, but it should not be used in vitamin B-12 insufficient populations. This trial was registered at clinicaltrials.gov as NCT00207558.

The threshold for population-level optimal red blood cell (RBC) folate concentration among women of reproductive age for the prevention of neural tube defects has been estimated at 906 nmol/L; however, the dose-response relationship between folic acid intake and blood folate concentrations is uncharacterized. To estimate the magnitude of blood folate concentration increase in response to specific dosages of folic acid under steady-state conditions (as could be achieved with food fortification), a systematic review of the literature and meta-analysis was conducted. Of the 14,002 records we identified, 533 were selected for full-text review, and data were extracted from 108 articles. The steady-state concentrations (homeostasis) of both serum/plasma and RBC folate concentrations were estimated using a Bayesian meta-analytic approach and one-compartment physiologically-based pharmacokinetic models. RBC folate concentrations increased 1.78 fold (95% credible interval (CI): 1.66, 1.93) from baseline to steady-state at 375(-)570 microg folic acid/day, and it took a median of 36 weeks of folic acid intake (95% CI: 27, 52) to achieve steady-state RBC folate concentrations. Based on regression analysis, we estimate that serum/plasma folate concentrations increased 11.6% (95% CI: 8.4, 14.9) for every 100 microg/day folic acid intake. These results will help programs plan and monitor folic acid fortification programs.

OBJECTIVE: To determine the feasibility of long-term prospective follow-up and ascertainment of cancer in offspring and mothers from the 1993-1995 Chinese Community Intervention Program that provided folic acid supplements before and during early pregnancy to reduce neural tube defects. DESIGN: Feasibility pilot study for a prospective cohort study. SETTING: Families residing during 2012-2013 in one rural and one urban county from 21 counties in 3 provinces in China included in the Community Intervention Program campaign. PARTICIPANTS: The feasibility study targeted 560 families, including 280 from the rural and 280 from the urban county included in the large original study; about half of mothers in each group had taken and half had not taken folic acid supplements. INTERVENTION: The planned new study is observational. PRIMARY AND SECONDARY OUTCOME MEASURES: Primary: incidence of paediatric cancers in offspring; secondary: other chronic diseases in offspring and chronic diseases in mothers RESULTS: Only 3.4% of pilot study families could not be found, 3.9% had moved out of the study area and 8.8% refused to participate. Interviews were completed by 82% of mothers, 79% of fathers and 83% of offspring in the 560 families. Almost all mothers and offspring who were interviewed also participated in anthropometric measurements. We found notable urban-rural differences in sociodemographic and lifestyle characteristics of the parents, but fewer differences among the offspring. In eight catchment area hospitals, we identified a broad range of paediatric cancers diagnosed during 1994-2013, although paediatric brain tumours, lymphomas and rarer cancers were likely under-represented. CONCLUSIONS: Overall, 20 years after the original Community Intervention Program, the pilot study achieved high levels of follow-up and family member interview participation, and identified substantial numbers of paediatric malignancies during 1994-2013 in catchment area hospitals. Next steps and strategies for overcoming limitations are described.

This article reports the study design, methodological issues and early results of a pilot study testing methods for collecting nutrition, physical activity, and ultraviolet (UV) radiation exposure data in a groundbreaking study in China. Epidemiological studies suggest that exposures across the entire life course, including in utero, early childhood, and adolescence, may be important in the etiology of adult cancers and other chronic diseases. The Chinese Children and Families Cohort Study intends to follow-up subjects from the 1993 to 1995 Community Intervention Program of folic acid supplementation for the prevention of neural tube defects. This cohort is unique in that only folic acid exposure during pregnancy varies between groups as other supplements were not available, and there were nutrient deficiencies in the populations. Prior to launching a large-scale follow-up effort, a pilot study was conducted to assess the feasibility of recontacting original study participants to collect extensive diet, physical activity, and UV radiation exposure data in this population. The pilot study included 92 mothers and 184 adolescent children aged 14 to 17 years from 1 urban and 1 rural Community Intervention Program site. Subjects completed a Food Frequency Questionnaire, a 3-day food record, a physical activity questionnaire, a 3-day sun exposure diary together with 3 days of personal UV dosimetry, and 7 days of pedometry measurements and provided blood, saliva, and toenail samples. Grip strength and body composition measurements were taken, and ambient solar UV radiation was monitored in both study sites. While most of the assessments were successful, future studies would likely require different dietary intake instruments. The purpose of this report is to describe the study design and methodological issues emerging from this pilot work relevant for the follow-up of this large birth cohort.

Background: The US CDC and the Institute of Medicine recommend that women capable of becoming pregnant consume >/=400 microg synthetic folic acid/d to prevent neural tube defects (NTDs). The United States has 3 sources of folic acid: fortified enriched cereal grain products (ECGPs), fortified ready-to-eat (RTE) cereals, and dietary supplements. Objective: Our objectives were as follows: 1) to estimate the usual daily folic acid intake and distributions of red blood cell (RBC) folate concentrations among women consuming folic acid from different sources; 2) to assess the usual daily total folic acid intake associated with optimal RBC folate concentrations for NTD prevention; 3) to predict NTD prevalence; and 4) to estimate the number of preventable folate-sensitive NTDs. Design: NHANES data (2007-2012) for nonpregnant women of reproductive age (12-49 y) were used to estimate usual daily intakes of synthetic folic acid and natural food folate. We applied existing models of the relation between RBC folate concentrations and NTD risk to predict NTD prevalence. Results: Based on the distribution of overall RBC folate concentrations (4783 women), the predicted NTD prevalence was 7.3/10,000 live births [95% uncertainty interval (UI): 5.5-9.4/10,000 live births]. Women consuming folic acid from ECGPs as their only source had lower usual daily total folic acid intakes (median: 115 microg/d; IQR: 79-156 microg/d), lower RBC folate concentrations (median: 881 nmol/L; IQR: 704-1108 nmol/L), and higher predicted NTD prevalence (8.5/10,000 live births; 95% UI: 6.4-10.8/10,000 live births) compared with women consuming additional folic acid from diet or supplements. If women who currently consume folic acid from ECGPs only (48% of women) consumed additional folic acid sources, 345 (95% UI: 0-821) to 701 (95% UI: 242-1189) additional NTDs/y could be prevented. Conclusions: This analysis supports current recommendations and does not indicate any need for higher intakes of folic acid to achieve optimal NTD prevention. Ensuring 400 microg/d intake of folic acid prior to pregnancy has the potential to increase the number of babies born without an NTD.

BACKGROUND: Previous studies noted associations between birth defects and some antibiotics (e.g., nitrofurantoin, sulfonamides) but not others (e.g., penicillins). It is unclear if previous findings were due to antibiotic use, infections, or chance. To control for potential confounding by indication, we examined associations between antibiotic use and birth defects, among women reporting urinary tract infections (UTIs). METHODS: The National Birth Defects Prevention Study is a multi-site, population-based case-control study. Case infants/fetuses have any of over 30 major birth defects and controls are live-born infants without major birth defects. We analyzed pregnancies from 1997 to 2011 to estimate the association between maternally reported periconceptional (month before conception through the third month of pregnancy) use of nitrofurantoin, trimethoprim-sulfamethoxazole, or cephalosporins and specific birth defects, among women with periconceptional UTIs. Women with periconceptional UTIs who reported penicillin use served as the comparator. RESULTS: Periconceptional UTIs were reported by 7.8% (2029/26,068) of case and 6.7% (686/10,198) of control mothers. Most (68.2% of case, 66.6% of control mothers) also reported antibiotic use. Among 608 case and 231 control mothers reporting at least one periconceptional UTI and certain antibiotic use, compared with penicillin, nitrofurantoin use was associated with oral clefts in the offspring (adjusted odds ratio, 1.97 [95% confidence interval, 1.10-3.53]), trimethoprim-sulfamethoxazole use with esophageal atresia (5.31 [1.39-20.24]) and diaphragmatic hernia (5.09 [1.20-21.69]), and cephalosporin use with anorectal atresia/stenosis (5.01 [1.34-18.76]). CONCLUSION: Periconceptional exposure to some antibiotics might increase the risk for certain birth defects. However, because individual birth defects are rare, absolute risks should drive treatment decisions.,

There is renewed interest in health benefits of folic acid supplementation since the China Stroke Prevention Primary Prevention Trial (CSPPT) showed the potential benefits of folic acid in preventing stroke in Chinese adults with hypertension.1 It has long been known that folic acid prevents neural tube defects, which are among the most severe and debilitating congenital birth defects worldwide. The CSPPT was terminated early when 800 μg of folic acid in combination with 10 mg of enalapril significantly reduced primary stroke incidence compared with the enalapril alone treatment arm. This finding was unexpected as other studies had failed to find a preventive effect of folic acid supplementation on cardiovascular events.2 In this issue of JAMA Internal Medicine, Xu et al3 report findings from a prespecified CSPPT substudy that demonstrated that the folic acid–enalapril combination was more effective than enalapril alone in the secondary prevention of renal function decline among Chinese adults with hypertension across a spectrum of mild to moderate chronic kidney disease (CKD). | Folic acid is commonly administered to treat hyperhomocysteinemia, but the benefits of homocysteine lowering on the prevention or management of most chronic diseases associated with hyperhomocysteinemia remains unproven.2 In the renal substudy of the CSPPT,3 Xu and colleagues report that 42% of participants with CKD exhibited hyperhomocysteinemia (serum homocysteine ≥15 μM) and 24% had diabetes, compared with only 26% with hyperhomocysteinemia and 12% with diabetes in those without CKD. Individuals who were homozygous for the MTHFR C677T polymorphism, a genetic variant that expresses an enzyme with reduced 5-methyltetra-hydrofolate synthesis activity, exhibited the greatest reduction in serum homocysteine following folic acid–enalapril treatment because their baseline levels of circulating homocysteine were higher. This finding is consistent with other studies suggesting that the current recommended dietary intake for folate may not be adequate for individuals homozygous for the MTHFR 677T variant when homocysteine lowering is used as the functional biomarker to assess folate status.4

BACKGROUND: Twinning has been associated with many types of birth defects, although previous studies have had inconsistent findings. Many studies lack information about potential confounders, particularly use of fertility treatment. Our objective was to assess the association between twinning and birth defects in the National Birth Defects Prevention Study (NBDPS). METHODS: We used data from the NBDPS, a population-based, case-control study of major birth defects in the USA, to evaluate associations between twinning and birth defects. The study population included mothers of twin and singleton controls (live-born infants without major birth defects), and cases (fetuses or infants with a major birth defect) born October 1997-December 2007. Adjusted ORs and 95% CIs were estimated using multivariable logistic regression stratified by use of fertility treatment. Twin sex-pairing data and a simulation approach were used to estimate the zygosity of twins. RESULTS: In the unassisted conception stratum, we observed significant positive associations between twinning and 29 of 45 defect groups. The largest effect estimates were observed for multiple ventricular septal defects and cloacal exstrophy. Among mothers reporting any use of fertility treatments, we observed a significant association with twinning for 5 of 25 defect groups, with the largest effect estimates for hypoplastic left heart syndrome and omphalocele. OR estimates in the estimated monozygotic stratum were generally further from the null than in the dizygotic stratum. CONCLUSIONS: Compared with singletons, a wide range of birth defects are significantly more common among twins. Birth defect risk in twins may be differential by use of fertility treatment.

In this issue of the American Journal of Preventive Medicine, Cui et al.1 examined the congenital anomaly (CA)–specific under-5 mortality rate in China from 1996 to 2013 and found a substantial reduction in this rate from 407.7 per 100,000 live births in 1996 to 217.4 per 100,000 live births in 2013. In addition, they also found that the proportion of total under-5 mortality due to CAs (also referred to as birth defects) was increasing but were unable to include CA-specific data on stillbirths and terminations of pregnancy. | The Millennium Development Goal (MDG)-4, established in 2000, aimed to reduce under-5 mortality rate by two thirds by 2015.2 Globally, the under-5 mortality rate has decreased by 53%, from an estimated rate of 90 deaths per 1,000 live births in 1990 to 43 deaths per 1,000 live births in 2015.2 However, the MDG-4 goal of a two-thirds reduction in under-5 mortality rate was not achieved despite great progress made during the past few decades.2 In January 2016, the MDGs were replaced by a new framework, the Sustainable Development Goals (SDGs), within which a new target to end preventable deaths of newborns and children under age 5 years was established with all countries aiming to reduce the neonatal mortality rate to 12 per 1,000 live births or fewer and the under-5 mortality rate to 25 per 1,000 live births or fewer by 2030.3 In order to achieve this SDG, a major cause of neonatal and under-5 mortality, birth defects, must be addressed. More global efforts need to be undertaken in the prevention of birth defects to realize a reduction in neonatal and under-5 mortality.

The Food and Drug Administration mandated that by 1998, all enriched cereal grain products (ECGP) be fortified with folic acid in order to prevent the occurrence of neural tube defects. The Institute of Medicine established the tolerable upper intake level (UL) for folic acid (1000 microg/day for adults) in 1998. We characterized U.S. adults with usual daily folic acid intake exceeding the UL. Using NHANES 2003-2010 data, we estimated the percentage of 18,321 non-pregnant adults with usual daily folic acid intake exceeding the UL, and among them, we calculated the weighted percentage by sex, age, race/ethnicity, sources of folic acid intake, supplement use and median usual daily folic acid intakes. Overall, 2.7% (standard error 0.6%) of participants had usual daily intake exceeding the UL for folic acid; 62.2% were women; 86.3% were non-Hispanic whites; and 98.5% took supplements containing folic acid. When stratified by sex and age groups among those with usual daily folic acid intake exceeding the UL, 20.8% were women aged 19-39 years. Those with usual daily intake exceeding the folic acid UL were more likely to be female, non-Hispanic white, supplement users or to have at least one chronic medical condition compared to those not exceeding the folic acid UL. Among those with usual daily folic acid intake exceeding the UL who also took supplements, 86.6% took on average >400 microg of folic acid/day from supplements. Everyone with usual daily folic acid intake exceeding the UL consumed folic acid from multiple sources. No one in our study population had usual daily folic acid intake exceeding the UL through consumption of mandatorily-fortified enriched cereal grain products alone. Voluntary consumption of supplements containing folic acid is the main factor associated with usual daily intake exceeding the folic acid UL.

INTRODUCTION: Although fortification of food with folic acid has been calculated to be cost saving in the U.S., updated estimates are needed. This analysis calculates new estimates from the societal perspective of net cost savings per year associated with mandatory folic acid fortification of enriched cereal grain products in the U.S. that was implemented during 1997-1998. METHODS: Estimates of annual numbers of live-born spina bifida cases in 1995-1996 relative to 1999-2011 based on birth defects surveillance data were combined during 2015 with published estimates of the present value of lifetime direct costs updated in 2014 U.S. dollars for a live-born infant with spina bifida to estimate avoided direct costs and net cost savings. RESULTS: The fortification mandate is estimated to have reduced the annual number of U.S. live-born spina bifida cases by 767, with a lower-bound estimate of 614. The present value of mean direct lifetime cost per infant with spina bifida is estimated to be $791,900, or $577,000 excluding caregiving costs. Using a best estimate of numbers of avoided live-born spina bifida cases, fortification is estimated to reduce the present value of total direct costs for each year's birth cohort by $603 million more than the cost of fortification. A lower-bound estimate of cost savings using conservative assumptions, including the upper-bound estimate of fortification cost, is $299 million. CONCLUSIONS: The estimates of cost savings are larger than previously reported, even using conservative assumptions. The analysis can also inform assessments of folic acid fortification in other countries.

The Biomarkers of Nutrition for Development (BOND) project is designed to provide evidence-based advice to anyone with an interest in the role of nutrition in health. Specifically, the BOND program provides state-of-the-art information and service with regard to selection, use, and interpretation of biomarkers of nutrient exposure, status, function, and effect. To accomplish this objective, expert panels are recruited to evaluate the literature and to draft comprehensive reports on the current state of the art with regard to specific nutrient biology and available biomarkers for assessing nutrients in body tissues at the individual and population level. Phase I of the BOND project includes the evaluation of biomarkers for 6 nutrients: iodine, iron, zinc, folate, vitamin A, and vitamin B-12. This review represents the second in the series of reviews and covers all relevant aspects of folate biology and biomarkers. The article is organized to provide the reader with a full appreciation of folate's history as a public health issue, its biology, and an overview of available biomarkers (serum folate, RBC folate, and plasma homocysteine concentrations) and their interpretation across a range of clinical and population-based uses. The article also includes a list of priority research needs for advancing the area of folate biomarkers related to nutritional health status and development.

Neural tube defects (NTDs) such as spina bifida, anencephaly, and encephalocele are serious birth defects of the brain and spine that occur during the first month of pregnancy when the neural tube fails to close completely. Randomized controlled trials and observational studies have shown that adequate daily consumption of folic acid before and during early pregnancy considerably reduces the risk for NTDs. The U.S. Public Health Service recommends that women capable of becoming pregnant consume 400 microg of folic acid daily for NTD prevention. Furthermore, fortification of staple foods (e.g., wheat flour) with folic acid has decreased folate-sensitive NTD prevalence in multiple settings and is a highly cost-effective intervention.

Serum and erythrocyte (RBC) total folate are indicators of folate status. No nationally representative population data exist for folate forms. We measured the serum folate forms (5-methyltetrahydrofolate (5-methylTHF), unmetabolised folic acid (UMFA), non-methyl folate (sum of tetrahydrofolate (THF), 5-formyltetrahydrofolate (5-formylTHF), 5,10-methenyltetrahydrofolate (5,10-methenylTHF)) and MeFox (5-methylTHF oxidation product)) by HPLC-MS/MS and RBC total folate by microbiologic assay in US population ≥ 1 year (n approximately 7500) participating in the National Health and Nutrition Examination Survey 2011-2. Data analysis for serum total folate was conducted including and excluding MeFox. Concentrations (geometric mean; detection rate) of 5-methylTHF (37.5 nmol/l; 100 %), UMFA (1.21 nmol/l; 99.9 %), MeFox (1.53 nmol/l; 98.8 %), and THF (1.01 nmol/l; 85.2 %) were mostly detectable. 5-FormylTHF (3.6 %) and 5,10-methenylTHF (4.4 %) were rarely detected. The biggest contributor to serum total folate was 5-methylTHF (86.7 %); UMFA (4.0 %), non-methyl folate (4.7 %) and MeFox (4.5 %) contributed smaller amounts. Age was positively related to MeFox, but showed a U-shaped pattern for other folates. We generally noted sex and race/ethnic biomarker differences and weak (Spearman's r< 0.4) but significant (P< 0.05) correlations with physiological and lifestyle variables. Fasting, kidney function, smoking and alcohol intake showed negative associations. BMI and body surface area showed positive associations with MeFox but negative associations with other folates. All biomarkers showed significantly higher concentrations with recent folic acid-containing dietary supplement use. These first-time population data for serum folate forms generally show similar associations with demographic, physiological and lifestyle variables as serum total folate. Patterns observed for MeFox may suggest altered folate metabolism dependent on biological characteristics.

Folate is found naturally in foods or as synthetic folic acid in dietary supplements and fortified foods. Adequate periconceptional folic acid intake can prevent neural tube defects. Folate intake impacts blood folate concentration; however, the dose-response between natural food folate and blood folate concentrations has not been well described. We estimated this association among healthy females. A systematic literature review identified studies (1 1992-3 2014) with both natural food folate intake alone and blood folate concentration among females aged 12-49 years. Bayesian methods were used to estimate regression model parameters describing the association between natural food folate intake and subsequent blood folate concentration. Seven controlled trials and 29 observational studies met the inclusion criteria. For the six studies using microbiologic assay (MA) included in the meta-analysis, we estimate that a 6% (95% Credible Interval (CrI): 4%, 9%) increase in red blood cell (RBC) folate concentration and a 7% (95% CrI: 1%, 12%) increase in serum/plasma folate concentration can occur for every 10% increase in natural food folate intake. Using modeled results, we estimate that a natural food folate intake of ≥450 mug dietary folate equivalents (DFE)/day could achieve the lower bound of an RBC folate concentration (~1050 nmol/L) associated with the lowest risk of a neural tube defect. Natural food folate intake affects blood folate concentration and adequate intakes could help women achieve a RBC folate concentration associated with a risk of 6 neural tube defects/10,000 live births.

BACKGROUND: The methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphism is a risk factor for neural tube defects. The T allele produces an enzyme with reduced folate-processing capacity, which has been associated with lower blood folate concentrations. OBJECTIVE: We assessed the association between MTHFR C677T genotypes and blood folate concentrations among healthy women aged 12-49 y. DESIGN: We conducted a systematic review of the literature published from January 1992 to March 2014 to identify trials and observational studies that reported serum, plasma, or red blood cell (RBC) folate concentrations and MTHFR C677T genotype. We conducted a meta-analysis for estimates of percentage differences in blood folate concentrations between genotypes. RESULTS: Forty studies met the inclusion criteria. Of the 6 studies that used the microbiologic assay (MA) to measure serum or plasma (S/P) and RBC folate concentrations, the percentage difference between genotypes showed a clear pattern of CC > CT > TT. The percentage difference was greatest for CC > TT [S/P: 13%; 95% credible interval (CrI): 7%, 18%; RBC: 16%; 95% CrI: 12%, 20%] followed by CC > CT (S/P: 7%; 95% CrI: 1%, 12%; RBC: 8%; 95% CrI: 4%, 12%) and CT > TT (S/P: 6%; 95% CrI: 1%, 11%; RBC: 9%; 95% CrI: 5%, 13%). S/P folate concentrations measured by using protein-binding assays (PBAs) also showed this pattern but to a greater extent (e.g., CC > TT: 20%; 95% CrI: 17%, 22%). In contrast, RBC folate concentrations measured by using PBAs did not show the same pattern and are presented in the Supplemental Material only. CONCLUSIONS: Meta-analysis results (limited to the MA, the recommended population assessment method) indicated a consistent percentage difference in S/P and RBC folate concentrations across MTHFR C677T genotypes. Lower blood folate concentrations associated with this polymorphism could have implications for a population-level risk of neural tube defects.

Prevention of maternal-to-child transmission (MTCT) of HIV remains a priority, as globally, approximately 700 infants are newly infected with HIV each day [1]. Remarkable progress has been made in the past decade in reducing MTCT, with one million infants prevented from acquiring HIV between 2003 and 2013 because of maternal and infant antiretroviral prophylaxis [2]. However, limited safety data currently exist on potential adverse outcomes among HIV-infected pregnant women and their infants after exposure to combination antiretroviral therapy (cART) before and throughout pregnancy [3]. | In the United States and other high-resource countries, pregnant HIV-infected women receive cART starting usually early in pregnancy and, as a result, MTCT of HIV has been nearly eliminated in the past decade, with rates decreased from approximately 25% to currently 1% [4,5]. The advent of more potent and better-tolerated antiretroviral drugs has led to a strong push toward earlier initiation of cART, including amongst women of reproductive age [6–8]. Thus, an increasing number of HIV-infected women are already taking cART when conception occurs. In resource-limited settings, initiating cART has previously been recommended only for pregnant women with more advanced HIV disease (i.e. CD4+ cell count below 200 cells/μl or symptomatic HIV with CD4+ cell count <350 cells/μl) [9]. However, the 2013 WHO consolidated guidelines recommend that all HIV-infected pregnant women initiate cART regardless of CD4+ cell count, and if breastfeeding, continue cART throughout breastfeeding [10,11]. Women may either continue lifelong treatment regardless of clinical status or stop if they do not yet meet country-specific treatment eligibility criteria (‘option B’). An increasing number of countries are in the process of adopting lifelong treatment (the so-called ‘option B+’) for all pregnant women found to be HIV-infected [12–14].

OBJECTIVE: To determine an optimal population red blood cell (RBC) folate concentration for the prevention of neural tube birth defects. DESIGN: Bayesian model. SETTING: Data from two population based studies in China. PARTICIPANTS: 247 831 participants in a prospective community intervention project in China (1993-95) to prevent neural tube defects with 400 mug/day folic acid supplementation and 1194 participants in a population based randomized trial (2003-05) to evaluate the effect of folic acid supplementation on blood folate concentration among Chinese women of reproductive age. INTERVENTION: Folic acid supplementation (400 mug/day). MAIN OUTCOME MEASURES: Estimated RBC folate concentration at time of neural tube closure (day 28 of gestation) and risk of neural tube defects. RESULTS: Risk of neural tube defects was high at the lowest estimated RBC folate concentrations (for example, 25.4 (95% uncertainty interval 20.8 to 31.2) neural tube defects per 10 000 births at 500 nmol/L) and decreased as estimated RBC folate concentration increased. Risk of neural tube defects was substantially attenuated at estimated RBC folate concentrations above about 1000 nmol/L (for example, 6 neural tube defects per 10 000 births at 1180 (1050 to 1340) nmol/L). The modeled dose-response relation was consistent with the existing literature. In addition, neural tube defect risk estimates developed using the proposed model and population level RBC information were consistent with the prevalence of neural tube defects in the US population before and after food fortification with folic acid. CONCLUSIONS: A threshold for "optimal" population RBC folate concentration for the prevention of neural tube defects could be defined (for example, approximately 1000 nmol/L). Population based RBC folate concentrations, as a biomarker for risk of neural tube defects, can be used to facilitate evaluation of prevention programs as well as to identify subpopulations at elevated risk for a neural tube defect affected pregnancy due to folate insufficiency.

Whether folic acid fortification and supplementation at the population level have led to a higher prevalence of vitamin B-12 deficiency in the absence of anemia remains to be examined among a nationally representative sample of older U.S. adults. We assessed the prevalence of low vitamin B-12 status in the absence of anemia or macrocytosis before and after fortification among adults aged >50 y using cross-sectional data from the NHANES 1991-1994 (prefortification) and 2001-2006 (postfortification). We compared the prefortification and postfortification prevalence of multiple outcomes, including serum vitamin B-12 deficiency (<148 pmol/L) and marginal deficiency (148-258 pmol/L) with and without anemia (hemoglobin <130 g/L for men, <120 g/L for women) and with and without macrocytosis (mean cell volume >100 fL) using multinomial logistic regression, adjusting for age, sex, ethnicity, body mass index, C-reactive protein, and vitamin B-12 supplement use. Prefortification and postfortification serum vitamin B-12 deficiency without anemia [4.0 vs. 3.9%; adjusted prevalence ratio (aPR) (95% CI): 0.98 (0.67, 1.44)] or without macrocytosis [4.2 vs. 4.1%; aPR (95% CI): 0.96 (0.65, 1.43)] remained unchanged. Marginal deficiency without anemia [25.1 vs. 20.7%; aPR (95% CI): 0.82 (0.72, 0.95)] or without macrocytosis [25.9 vs. 21.3%; aPR (95% CI): 0.82 (0.72, 0.94)] were both significantly lower after fortification. After fortification, higher folic acid intake was associated with a lower prevalence of low serum B-12 status in the absence of anemia or macrocytosis. Results suggest that the prevalence of low serum B-12 status in the absence of anemia or macrocytosis among older U.S. adults did not increase after fortification. Thus, at the population level, we found no evidence to support concerns that folic acid adversely affected the clinical presentation of vitamin B-12 deficiency among older adults.

BACKGROUND: Childhood asthma has become a critical public health problem because of its high morbidity and increasing prevalence. The impact of nutrition and other exposures during pregnancy on long-term health and development of children has been of increasing interest. OBJECTIVE: We performed a systematic review and meta-analysis of the association of folate and folic acid intake during pregnancy and risk of asthma and other allergic outcomes in children. DESIGN: We performed a systematic search of 8 electronic databases for articles that examined the association between prenatal folate or folic acid exposure and risk of asthma and other allergic outcomes (eg, allergy, eczema, and atopic dermatitis) in childhood. We performed a meta-analysis by using a random-effects model to derive a summary risk estimate of studies with similar exposure timing, exposure assessment, and outcomes. RESULTS: Our meta-analysis provided no evidence of an association between maternal folic acid supplement use (compared with no use) in the prepregnancy period through the first trimester and asthma in childhood (summary risk estimate: 1.01; 95% CI: 0.78, 1.30). Because of substantial heterogeneity in exposures and outcomes, it was not possible to generate summary measures for other folate indicators (eg, blood folate concentrations) and asthma or allergy-related outcomes; however, the preponderance of primary risk estimates was not elevated. CONCLUSIONS: Our findings do not support an association between periconceptional folic acid supplementation and increased risk of asthma in children. However, because of the limited number and types of studies in the literature, additional research is needed.

BACKGROUND: The United States implemented mandatory folic acid fortification of enriched cereal grains in 1998. Although several studies have documented the resulting decrease in anemia and folate deficiency, to our knowledge, no one has determined the prevalence of folate-deficiency anemia after fortification. OBJECTIVE: We determined the prevalence of folate deficiency and folate-deficiency anemia within a sample of the Reasons for Geographic and Racial Differences in Stroke (REGARDS) cohort. DESIGN: The REGARDS cohort is a prospective cohort of 30,239 black and white participants living in the contiguous United States. We measured serum folate concentrations in a random sample of 1546 REGARDS participants aged ≥50 y with baseline hemoglobin and red blood cell mean corpuscular volume measurements. Folate deficiency was defined as a serum folate concentration <6.6 nmol/L (<3.0 ng/mL), and anemia was defined as a hemoglobin concentration <13 g/dL in men and <12 g/dL in nonpregnant women (WHO criteria). Folate-deficiency anemia was defined as the presence of both folate deficiency and anemia. RESULTS: The mean hemoglobin concentration was 13.6 g/dL, and 15.9% of subjects had anemia. The median serum folate concentration was 34.2 nmol/L (15.1 ng/mL), and only 2 of 1546 participants 0.1%) were folate deficient. Both subjects were African American women with markedly elevated C-reactive protein concentrations, macrocytosis, and normal serum cobalamin concentrations; only one subject was anemic. Overall, the prevalence of folate-deficiency anemia was <0.1% (1 of 1546 subjects). CONCLUSION: Our data suggest that, after mandatory folic acid fortification, the prevalence of folate-deficiency anemia is nearly nonexistent in a community-dwelling population in the United States.

Autism spectrum disorders (ASDs) are a group of neurodevelopmental disorders characterized by significant impairments in social interaction and communication and by repetitive, restrictive, and stereotyped patterns of behavior.1 The most serious of the conditions comprising ASDs is autistic disorder, because it is likely to co-occur with intellectual disability and a range of medical, behavioral, and psychiatric complications.1 The prevalence of ASDs is estimated to be approximately 1% of children.2 Because of increasing temporal trends in autism prevalence and the clinical and behavioral challenges of the condition, understanding risk factors, determining potential causes and prevention, and evaluating treatment options are high priorities for researchers, parents, advocates, clinicians, and educators. | Although ASDs are heritable,3 the clinical presentation can vary widely between family members and among affected individuals. Genetic factors (eg, fragile X syndrome, 15q11-13 duplications, and other de novo and inherited genetic variants) are the best studied of the risk factors for ASD that have thus far been identified.3,4 Additionally some prenatal, obstetric, and environmental exposures (eg, parental age, prenatal drug exposure, and infections) have been associated with increased risk for ASDs.5 The evidence to date suggests that ASDs are associated with a combination of multiple genetic and environmental risk factors leading to variable clinical presentations.1,3-5 This heterogeneity is a major obstacle to researchers. Identified risk factors are implicated in only a minority of ASD cases, and the etiology of ASDs remains largely unknown.