Arsenic modifies the effect of folic acid in spina bifida prevention, a large hospital-based case-control study in Bangladesh

Arsenic modifies the effect of folic acid in spina bifida prevention, a large hospital-based case-control study in Bangladesh | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Arsenic modifies the effect of folic acid in spina bifida prevention, a large hospital-based case-control study in Bangladesh Chih-Fu Wei, Sudipta Kumar Mukherjee, Sheikh Muhammad Ekramullah, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3989039/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background Spina bifida, a developmental malformation of the spinal cord, is associated with high rates of mortality and disability. Although folic acid-based preventive strategies have been successful in reducing rates of spina bifida, some areas continue to be at higher risk because of chemical exposures. Bangladesh has high arsenic exposures through contaminated drinking water and high rates of spina bifida. Methods We conducted a hospital-based case-control study at the National Institute of Neurosciences & Hospital (NINS&H) in Dhaka, Bangladesh, between December 2016 and December 2022. Cases were infants under age one year with spina bifida and further classified using data from observations by neurosurgeons and available imaging. Controls were drawn from children who presented to NINS&H or Dhaka Shishu Hospital (DSH) during the same study period. Mothers reported folic acid use during pregnancy, and we assessed folate status with serum assays. Arsenic exposure was estimated in drinking water using graphite furnace atomic absorption spectrophotometry (GF-AAS) and in toenails using inductively coupled plasma mass spectrometry (ICP-MS). Results We evaluated data from 294 cases of spina bifida and 163 controls. We did not find a main effect of mother’s arsenic exposure on spina bifida risk. However, in stratified analyses, folic acid use was associated with lower odds of spina bifida (adjusted odds ratio [OR]: 0.50, 95% confidence interval [CI]: 0.25-1.00, p = 0.05) among women with toenail arsenic concentrations below the median, and no association was seen among mothers with toenail arsenic concentrations higher than median (adjusted OR: 1.09, 95% CI: 0.52–2.29, p = 0.82). Conclusions Mother’s arsenic exposure modified the protective association of folic acid with spina bifida. Increased surveillance and additional preventive strategies, such as folic acid fortification and reduction of arsenic, are needed in areas of high arsenic exposure. Arsenic Bangladesh folic acid spina bifida Figures Figure 1 BACKGROUND Spina bifida is a developmental malformation of the spinal cord that leads to increased risk of mortality and disability, including leg, bladder, and bowel dysfunction, susceptibility to infection, hydrocephalus, and cerebrospinal fluid (CSF) leakage.[ 1 ] Spina bifida is a type of neural tube defect (NTD), a group of disorders caused by failure of the neural tube to fuse in the third week of gestation.[ 1 ] A recent analysis estimated that globally, at least 213,800 − 322,000 pregnancies are affected by NTDs each year, and in low-income and middle-income countries, the prevalence of NTDs exceeds one in every 100 births.[ 2 ] Folic acid supplement use reduces the risk of spina bifida,[ 3 ] and fortification of staple foods with folic acid has been successful in decreasing spina bifida rates in multiple countries.[ 4 , 5 ] However, a substantial number of affected pregnancies occur in areas with folic acid fortification and to women known to have taken folic acid supplements,[ 6 ] and the effectiveness of folic-acid based preventive strategies varies significantly across and even within countries.[ 2 , 5 ] There is an urgent need to identify modifiable factors that may reduce the burden of this condition. Environmental exposure to arsenic is of particular concern and may be a potential contributor to spina bifida risk, especially in areas of the world with high arsenic exposures and micronutrient deficiencies. Arsenic induces neural tube defects in several animal models,[ 7 – 11 ] and arsenic toxicity is closely related to one-carbon metabolism nutrients including folate.[ 12 ] A recent systematic review showed there was inadequate evidence to determine the relationship between prenatal arsenic exposure and prevalence of NTDs,[ 13 ] but the review did not address potential interactions between arsenic and folic acid that may affect spina bifida risk. Understanding the relationship between folic acid, spina bifida, and arsenic exposure is especially important in Bangladesh, where an estimated 70 million people are chronically exposed to high concentrations of arsenic through contaminated groundwater, in what has been described as the largest mass poisoning in history.[ 14 , 15 ] In 2019, a national survey conducted by the Bangladesh Bureau of Statistics and United Nations Children’s Fund estimated that 18.6% of households in Bangladesh were exposed to source water arsenic levels > 10 µg/L and 11.8% were exposed to levels > 50 µg/L.[ 16 ] Bangladesh also has high rates of spina bifida. The country does not have systematic surveillance for birth defects, but large hospital-based studies estimate the prevalence of spina bifida to be between 10.4 and 38.2 per 10,000 births.[ 17 , 18 ] Our previous study discovered that higher water arsenic concentrations reduced the effectiveness of folic acid in spina bifida prevention.[ 19 ] Based on these findings, we used toenail arsenic as biomarker, and identified an association between father’s arsenic exposure and spina bifida.[ 20 ] In this study, we sought to ascertain whether environmental arsenic exposure is associated with higher risk of spina bifida and whether arsenic exposure modified the protective effect of folic acid supplementation in Bangladesh. METHODS Case ascertainment, and control selection We conducted a hospital-based case-control study at the National Institute of Neurosciences & Hospital (NINS&H), the primary center for spina bifida surgery in Bangladesh. Cases were infants with spina bifida who were less than one year old and had mothers who could identify their primary drinking water source during early pregnancy. Neurosurgeons at NINS&H examined all cases and reviewed medical records, operative reports, and available imaging results. Study staff recorded the subtype of spina bifida (myelomeningocele or meningocele), level of lesion (e.g., cervical, thoracic, lumbar, sacral, or lumbosacral), co-occurring anomalies, and complications present at time of enrollment, including cerebrospinal fluid leak, lesion infection, and hydrocephalus. A senior neurosurgeon (BCW) confirmed classification of cases by reviewing photographs and available imaging. We selected controls from children who presented to NINS&H or the adjacent Dhaka Shishu Hospital (DSH), a children’s hospital immediately adjacent to NINS&H with similar referral patterns. Potential controls were seen for conditions including craniosynostosis, arachnoid cyst, trauma, subdural effusion, and epilepsy. Children with brain tumors or other cancers were not eligible to be controls. When a case was enrolled, we reviewed that week’s clinic lists and identified potential controls who were within six months of age of the enrolled case. The Bangladesh Medical Research Council and the Human Research Committees at Boston Children’s Hospital (BCH), NINS&H, and DSH approved this study (BCH protocol number IRB-P00019768; BMRC registration number: 006 23 08 2016). The Harvard T.H. Chan School of Public Health ceded review to BCH (protocol number: IRB20-0780). Parents provided informed consent before enrollment. Clinical information and folate status Trained study staff interviewed patients’ families to collect demographic characteristics, patient and family medical histories, referral patterns to NINS&H, and medical histories. We measured infant’s weight and head circumference using standardized protocols, and mother’s medication and vitamin intake using a structured questionnaire that included the seven major types of folic acid-containing tablets in Bangladesh. We also collected information about the timing of initiation of vitamin use (before or after knowing about pregnancy), duration and frequency. To estimate nutritional intake during pregnancy, we administered a food frequency questionnaire previously validated in Bangladesh.[ 21 ] Folate status was additionally assessed in serum samples using Chemiluminescent Microparticle Immunoassay at NINS&H (ARCHITECT plus ci4100, Abbott Company, Abbott Park, IL, USA). Water arsenic concentration Mothers were asked to identify the primary drinking water source they used at the time they became aware of their pregnancies. Trained staff visited these sites and collected water samples in polyethylene containers. Arsenic concentrations in water were assessed at the Bangladesh University of Engineering and Technology using graphite furnace atomic absorption spectrometry (GF-AAS) with a limit of detection (LOD) of 1 µg/L.[ 22 ] For water arsenic concentrations below the limit of detection (LOD), we assigned a value of LOD/ \(\sqrt{2 }.\) We tested one blank for every 50 water samples. Toenail arsenic concentration Mother’s toenail clippings were obtained from all toenails and placed in a small coin envelope. The samples were stored and shipped to the Dartmouth Trace Element Analysis Core at room temperature. Arsenic concentrations were measured using inductively coupled plasma mass spectrometry (ICP-MS) using methods that have been previously described.[ 23 ] The instrument’s LOD was 0.01 ng/g for the first 338 samples, and 0.1 ng/g for the remaining samples. For toenail arsenic concentrations below the instrument’s LOD, we assigned a value equal to the average dilution factor of toenail samples multiplied by (0.001*LOD)/ \(\sqrt{2 }.\) [20] Statistical analysis To compare the data between cases and controls, we used t-tests for continuous variables, and chi-square and Fisher's exact tests for categorical variables. We calculated Spearman correlation coefficients for the correlations of toenail and water arsenic concentrations. We employed logistic regression models to assess the associations between arsenic concentrations and folic acid use (predictors) and case status. We modeled arsenic concentrations in two ways: 1) as a continuous variable and 2) as a binary variable: above and below 10 µg/L for water (the current World Health Organization drinking water standard for arsenic), and above and below the median for toenails. We did not use conditional models because of the uneven numbers of cases and controls. Models were adjusted for potential confounders that were chosen based on prior knowledge and using directed acyclic graphs and included mother’s age, place of birth (hospital, clinic, or home) and secondhand smoke exposure. In sensitivity analyses, we restricted the cases to only those with myelomeningocele. Data were analyzed using R (version 4.0.4). RESULTS We enrolled 333 infants with initial diagnoses of spina bifida (meningocele or myelomeningocele) and 165 controls. We stopped enrolling controls and collecting water in March 2020 because of COVID-19 restrictions, and this led to an uneven number of cases and controls. Participation rates were 73% among potential families with spina bifida and 57% among potential controls. Reasons for study refusal were similar among cases and controls and primarily included concerns about giving blood. We excluded 11 cases because surgery revealed lipomeningocele and 1 case because the mother reported valproic acid use. Toenail arsenic concentrations were not available for 27 cases and 2 controls. Our final study population included 294 cases and 163 controls. Diagnoses of the controls are presented in Supplemental Table 1. Demographic information is presented in Table 1. None of the mothers reported a diagnosis of diabetes, including gestational diabetes. Prenatal folic acid use among our study population was low, but consistent with other reports from Bangladesh;[ 24 – 26 ] only 16.7% of mothers of cases in our study and 22.1% of mothers of controls reported using folic acid during pregnancy. Serum folate concentrations were higher among mothers who reported folic acid use during pregnancy compared to those who did not report folic acid use (10.20 ng/ml and 7.96 ng/ml, respectively). On average, arsenic concentrations in water were lower than seen in our previous studies in Bangladesh,[ 19 , 27 ] although some high arsenic concentrations were seen (maximum water arsenic concentration: 451 µg/L) (Table 2). We observed a moderate correlation between water and toenail arsenic concentrations (Spearman correlation coefficient: 0.45). Arsenic was detected in all toenail samples. There was no significant main effect found between water and mother’s toenail arsenic concentrations and spina bifida risk (Supplemental Table 2). However, in stratified models, we found evidence that arsenic exposure modified the effect of folic acid on spina bifida risk (Fig. 1 ). Among mothers with toenail arsenic concentrations below the median, folic acid use during pregnancy was associated with lower odds of having an infant with spina bifida (adjusted odds ratio [OR]: 0.50, 95% confidence interval [CI]: 0.25–1.00). Among women with toenail arsenic concentrations above the median, this protective association was not seen (adjusted OR: 1.09, 95% CI: 0.52–2.29). We found similar results after restricting the cases to those with myelomeningocele (Supplemental Fig. 1) (adjusted OR: 0.45, 95% CI: 0.21–0.94 vs. adjusted OR: 1.03, 95% CI: 0.48–2.23). When using water arsenic concentration as our measure of arsenic exposure, folic acid had a protective association at arsenic concentrations > 10 µg/L (adjusted OR: 0.18, 95% CI 0.04–0.93, p = 0.04), but this finding may be limited by the small numbers of reported folic acid users in this group (Supplemental Fig. 2). DISCUSSION Our study found that arsenic modifies the effect of folic acid on spina bifida prevention in a population in Bangladesh, a country with high arsenic exposures through contaminated drinking water. Among mothers with toenail arsenic concentrations below the median, folic acid was associated with a protective effect (adjusted OR:0.50, 95% CI: 0.25-1.00), but a protective effect was not observed among mothers with toenail concentrations above the median (adjusted OR:1.09, 95% CI: 0.52–2.29). We found similar evidence of effect modification when we restricted the analysis to cases with myelomeningocele, a more severe type of spina bifida. (Supplemental Fig. 1) Our results are consistent with recent studies conducted in high arsenic areas that suggest that environmental arsenic exposure may attenuate the protective benefit of folic acid supplements in preventing spina bifida. In a large case-control study of NTDs in Shanxi, China, researchers found that placental arsenic concentrations were associated with NTD-affected pregnancies among mothers who reported not taking folic acid supplements.[ 28 ] Our previous studies in Bangladesh found that as drinking water arsenic concentrations increased from 1 µg/L to 25 µg/L, the protective effect of folic acid use declined (OR: 0.22, 95% CI: 0.13, 0.37 to OR: 1.03, 95% CI: 0.55, 1.91).[ 19 ] These studies suggest that prenatal folic acid supplementation may be less effective in spina bifida prevention in populations with high arsenic exposure because of interactions between arsenic and folic acid. Animal studies have consistently shown arsenic to be a potent teratogen, inducing neural tube defects in several animal models.[ 7 – 11 ] In addition, animal studies demonstrate that arsenic-folate interactions are important in arsenic’s teratogenicity. In Folbp2 knockout mice, for example, mice with this specific defect in folate transport had higher rates of NTDs after arsenic exposure, and mice nullizygous for genes encoding proteins in cellular uptake of folate were more susceptible to arsenic-induced NTDs.[ 29 ] The interaction between arsenic and folate may be explained by arsenic’s interference with folate-related functions, such as the depletion of S-adenosylmethionine (SAM), a key methyl donor for pathways implicated in neural tube closure.[ 12 ] In experimental models, chicken embryos exposed to 100nM arsenate showed reduced SAM levels and higher rates of NTDs.[ 30 ] In humans, the strongest evidence for arsenic-folic acid interactions comes from trials in which folic acid has been shown to reduce blood arsenic concentrations.[ 31 – 33 ] Although we found evidence of effect modification by arsenic, our study did not demonstrate a primary (or main) effect of mother’s arsenic exposure on spina bifida risk, consistent with findings from a recent systematic review assessing cohort studies that had measures of prenatal arsenic exposure and spina bifida outcomes.[ 13 ] It is possible that even higher arsenic exposures are needed to demonstrate a primary effect. A recent study from an area of Turkey with high arsenic exposures reported higher levels of arsenic in plasma samples of 100 mothers of NTD cases as compared to mothers from 70 controls.[ 34 ] Survivorship bias may also account for not finding a primary effect. We enrolled cases at time of presentation for surgical care and did not capture affected pregnancies that did not continue to birth or more severely affected infants who were not brought for care. If arsenic exposure was related to these more severe outcomes, our results could represent a downward bias towards or beyond the null.[ 35 ] Another possible explanation for why we did not find a primary effect of arsenic is that arsenic exposure by itself may be insufficient, and that the addition of other risk factors, such as inadequate folate, is a necessary contribution to disrupt neural tube closure. The strengths of our study include the use of individual measures of exposure, including toenail arsenic measurements, which represent arsenic exposure from 5 to 18 months before collection,[ 36 ] and may better represent exposure during early pregnancy than blood, urine, or water samples collected after delivery. Previous work in Bangladesh in a large pregnancy cohort has shown high correlations between pregnant women’s toenail arsenic measurements during the first trimester and 1 month post-partum and also with measurements from their infant’s toenails at age 1 month.[ 27 ] An additional strength is classification of cases by neurosurgeons using examination, imaging and observations during surgery. It is increasingly understood that NTDs are not one disorder, but instead a wide array of morphologically distinct malformations which likely each result from a different contribution of risk factors.[ 37 ] Our study is the largest study to date to investigate spina bifida only (larger studies included all NTDs), and we were able to further restrict our analyses to only myelomeningocele in sensitivity analyses. CONCLUSIONS Environmental arsenic exposure may reduce the protective effects of folic acid supplementation on spina bifida risk. Our findings raise important questions about the risk of spina bifida in arsenic-endemic areas and the effectiveness of folic acid supplements alone as the strategy to prevent spina bifida in high-arsenic areas of the world. At minimum, more surveillance for spina bifida is needed in high arsenic areas such as Bangladesh. Additional preventive measures, such as folic acid fortification of the food supply and reduction of arsenic exposure, may be needed to optimize spina bifida prevention in areas with high arsenic exposures. Abbreviations Boston Children’s Hospital, BCH; cerebrospinal fluid, CSF; confidence interval, CI; Dhaka Shishu Hospital, DSH; graphite furnace atomic absorption spectrometry, GF-AAS; inductively coupled plasma mass spectrometry, ICP-MS; limit of detection, LOD; National Institute of Neurosciences & Hospital, NINS&H; neural tube defect, NTD; odds ratio, OR; S-adenosylmethionine, SAM. Declarations Ethics approval and consent to participate The Bangladesh Medical Research Council and the Human Research Committees at Boston Children’s Hospital (BCH), NINS&H, and DSH approved this study (BCH protocol number IRB-P00019768; BMRC registration number: 006 23 08 2016). The Harvard T.H. Chan School of Public Health ceded review to BCH (protocol number: IRB20-0780). Parents provided informed consent before enrollment. Consent for publication Not applicable. Availability of data and materials Data will be made available from the corresponding author on reasonable request. Competing Interests The authors declare that they have no known competing interests. Funding This work was supported by the National Institutes of Environmental Health Sciences (grant numbers: NIEHS R01 ES026317, R21 ES030784) and the National Institute of Mental Health (NIMH T32 MH112510). In addition, this project used core facilities supported by institutional grants from NIEHS P30 ES000002. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIEHS. Authors’ contributions MM, SKM, SME, DMA, JI, MA, AR, BCW, and DCC conceived the study. MM, SKM, SME, DMA, JI, MA, AR, MNR, MZ and HSS conducted the field activities. WCF drafted the manuscript. WCF, MM, GT, LL, DG, DCC, and MW participated in the data analyses. All authors read and approved the final manuscript. Acknowledgments We would like to acknowledge our field team at NINS&H, including Shipra Rani Saha, and Shashwati Suter, Suvajit Das, Shamantha Afreen, Ansar Uddin, Abdullah Al Mahbub and Md. Ziaul Hoq, Dartmouth Trace Element Core Facility, which is supported by Dartmouth Cancer Center with NCI Cancer Center Support Grant 5P30 CA023108, as well as laboratory staff at the Harvard T.H. Chan School of Public Health, including Li Su, Danny Donato, and Erica Haley. We are particularly grateful for support from the Outstanding New Environmental Scientist advisory committee: Richard Finnell, Mary Gamble and Martha Werler. References Iskandar BJ, Finnell RH: Spina Bifida . N Engl J Med 2022, 387 (5):444-450. Blencowe H, Kancherla V, Moorthie S, Darlison MW, Modell B: Estimates of global and regional prevalence of neural tube defects for 2015: a systematic analysis . Ann N Y Acad Sci 2018, 1414 (1):31-46. 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Demir N, Başaranoğlu M, Huyut Z, Değer İ, Karaman K, Şekeroğlu MR, Tuncer O: The relationship between mother and infant plasma trace element and heavy metal levels and the risk of neural tube defect in infants . The Journal of Maternal-Fetal & Neonatal Medicine 2019, 32 (9):1433-1440. Leung M, Kioumourtzoglou MA, Raz R, Weisskopf MG: Bias due to Selection on Live Births in Studies of Environmental Exposures during Pregnancy: A Simulation Study . Environ Health Perspect 2021, 129 (4):47001. Signes-Pastor AJ, Gutierrez-Gonzalez E, Garcia-Villarino M, Rodriguez-Cabrera FD, Lopez-Moreno JJ, Varea-Jimenez E, Pastor-Barriuso R, Pollan M, Navas-Acien A, Perez-Gomez B et al : Toenails as a biomarker of exposure to arsenic: A review . Environ Res 2021, 195 :110286. Wallingford JB, Niswander LA, Shaw GM, Finnell RH: The continuing challenge of understanding, preventing, and treating neural tube defects . Science 2013, 339 (6123):1222002. Tables Table 1. Study Population Cases (n = 294) Controls (n = 163) p-value Infant characteristics Boys 157 (53.4) 104 (63.8) 0.04 Firstborn 102 (34.7) 87 (53.4) < 0.001 Gestational age (week) 37.3 (2.1) 37.0 (2.3) 0.22 Age at study visit (days) 75.5 (86.6) 185.3 (96.1) < 0.001 Myelomeningocele 249 (84.7) NA Level of spina bifida Cervical or thoracic 11 (3.7) NA Lumbar 144 (49.0) NA Sacral or lumbosacral 138 (46.9) NA Mother’s characteristics Age (years) 24.7 (4.7) 24.3 (5.0) 0.39 Education level 0.11 Illiterate 3 (1.0) 2 (1.2) Literate 55 (18.7) 22 (13.5) High school or less 164 (55.8) 83 (50.9) College 56 (19.0) 38 (23.3) University 16 (5.4) 18 (11.0) Folic acid use during early pregnancy 49 (16.7) 36 (22.1) 0.19 Living in Dhaka 153 (52.0) 89 (54.6) 0.67 Pre-pregnancy diabetes 9 (3.1) 4 (2.5) 0.78 Diagnosis of gestational diabetes 5 (1.7) 4 (2.5) 0.73 Secondhand smoke exposure 127 (43.2) 60 (36.8) 0.22 Fever episodes during pregnancy 153 (52.0) 70 (42.9) 0.06 Place of birth 0.16 Home 79 (26.9) 41 (25.2) Clinics 111 (37.8) 50 (30.7) Hospitals 104 (35.4) 72 (44.2) Mean (SD) or number (%); NA = not applicable. Table 2. Arsenic concentrations in toenails and drinking water Mean (SD) Median IQR Min Max Mother’s toenail arsenic concentration (µg/g) Cases (n = 294) 1.04 (1.58) 0.45 0.28–1.05 0.07 13.77 Controls (n = 163) 1.13 (1.94) 0.47 0.30–0.96 0.09 12.30 Water arsenic concentration (µg/L) Cases (n = 166) 15.35 (37.28) 2.00 1.00–7.00 0.71 255.00 Controls (n = 161) 20.91 (56.08) 2.00 0.71-7.00 0.71 451.00 a. Water arsenic concentrations were below the LOD (1 µg/L) for 41 cases and 52 controls. Arsenic was detected in all toenail samples, and the LOD for toenail arsenic was 0.01 ng/g for first 338 samples tested and 0.1 ng/g for remaining samples b. Water samples were not available for participants enrolled during the COVID-19 pandemic due to travel restrictions in Bangladesh. Abbreviations: IQR, interquartile range; LOD, limit of detection; SD, standard deviation. Additional Declarations No competing interests reported. Supplementary Files NTDAsfolicacidSupplMaterialv0.126January2024CFW0225cleaned.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 09 Apr, 2024 Reviews received at journal 26 Mar, 2024 Reviewers agreed at journal 12 Mar, 2024 Reviewers invited by journal 12 Mar, 2024 Editor assigned by journal 26 Feb, 2024 Submission checks completed at journal 26 Feb, 2024 First submitted to journal 25 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3989039","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":275427460,"identity":"d14d3cfa-89b0-45b0-8e36-ff6ff5ec3dac","order_by":0,"name":"Chih-Fu Wei","email":"","orcid":"","institution":"Harvard T.H. Chan School of Public Health","correspondingAuthor":false,"prefix":"","firstName":"Chih-Fu","middleName":"","lastName":"Wei","suffix":""},{"id":275427461,"identity":"5602ee6e-4441-43d2-92b6-8fd6522b1cc9","order_by":1,"name":"Sudipta Kumar Mukherjee","email":"","orcid":"","institution":"National Institute of Neurosciences \u0026 Hospital","correspondingAuthor":false,"prefix":"","firstName":"Sudipta","middleName":"Kumar","lastName":"Mukherjee","suffix":""},{"id":275427462,"identity":"b6675e2c-c9b5-4b74-a1f0-8213ff53a660","order_by":2,"name":"Sheikh Muhammad Ekramullah","email":"","orcid":"","institution":"National Institute of Neurosciences \u0026 Hospital","correspondingAuthor":false,"prefix":"","firstName":"Sheikh","middleName":"Muhammad","lastName":"Ekramullah","suffix":""},{"id":275427463,"identity":"8deb4a31-ca96-454e-a642-98fc79700e85","order_by":3,"name":"D. M. Arman","email":"","orcid":"","institution":"National Institute of Neurosciences \u0026 Hospital","correspondingAuthor":false,"prefix":"","firstName":"D.","middleName":"M.","lastName":"Arman","suffix":""},{"id":275427464,"identity":"1d75b766-3b69-484f-8947-d410fad83260","order_by":4,"name":"Md Joynul Islam","email":"","orcid":"","institution":"National Institute of Neurosciences \u0026 Hospital","correspondingAuthor":false,"prefix":"","firstName":"Md","middleName":"Joynul","lastName":"Islam","suffix":""},{"id":275427465,"identity":"332282f6-8026-425a-9556-e4fa1c88de2d","order_by":5,"name":"Mubinul Azim","email":"","orcid":"","institution":"Dhaka Shishu Hospital","correspondingAuthor":false,"prefix":"","firstName":"Mubinul","middleName":"","lastName":"Azim","suffix":""},{"id":275427466,"identity":"e4d06426-933e-438f-9de3-37adfbd2ac1a","order_by":6,"name":"Asifur Rahman","email":"","orcid":"","institution":"Bangabandhu Sheikh Mujib Medical University (BSMMU)","correspondingAuthor":false,"prefix":"","firstName":"Asifur","middleName":"","lastName":"Rahman","suffix":""},{"id":275427467,"identity":"e77863cb-64fd-4795-b428-d2348d0222c0","order_by":7,"name":"Md Nafaur Rahman","email":"","orcid":"","institution":"National Institute of Neurosciences \u0026 Hospital","correspondingAuthor":false,"prefix":"","firstName":"Md","middleName":"Nafaur","lastName":"Rahman","suffix":""},{"id":275427468,"identity":"843a957e-7067-4c28-92c1-cf7de3ed8f6d","order_by":8,"name":"Md. Ziauddin","email":"","orcid":"","institution":"National Institute of Neurosciences \u0026 Hospital","correspondingAuthor":false,"prefix":"","firstName":"Md.","middleName":"","lastName":"Ziauddin","suffix":""},{"id":275427469,"identity":"6455d99f-9214-48c6-8d74-fdc747fe160f","order_by":9,"name":"Gwen Tindula","email":"","orcid":"","institution":"Stanford University","correspondingAuthor":false,"prefix":"","firstName":"Gwen","middleName":"","lastName":"Tindula","suffix":""},{"id":275427470,"identity":"9aaed14b-fa31-40ff-acf2-9f6e09d8247f","order_by":10,"name":"Hafiza Sultana Suchanda","email":"","orcid":"","institution":"National Institute of Neurosciences \u0026 Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hafiza","middleName":"Sultana","lastName":"Suchanda","suffix":""},{"id":275427471,"identity":"1381bf29-c8b6-4a47-886a-4daab91a47ce","order_by":11,"name":"Diana F. Gomberg","email":"","orcid":"","institution":"Boston Children’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Diana","middleName":"F.","lastName":"Gomberg","suffix":""},{"id":275427472,"identity":"724c5422-1d28-4af2-b2af-c33d5d0111be","order_by":12,"name":"Marc G. Weisskopf","email":"","orcid":"","institution":"Harvard T.H. Chan School of Public Health","correspondingAuthor":false,"prefix":"","firstName":"Marc","middleName":"G.","lastName":"Weisskopf","suffix":""},{"id":275427473,"identity":"075bf716-2d40-495c-b366-70aa2fd88aef","order_by":13,"name":"Liming Liang","email":"","orcid":"","institution":"Harvard T.H. Chan School of Public Health","correspondingAuthor":false,"prefix":"","firstName":"Liming","middleName":"","lastName":"Liang","suffix":""},{"id":275427474,"identity":"578cb817-7227-4acb-ae5c-6368e951b3bb","order_by":14,"name":"Benjamin C. Warf","email":"","orcid":"","institution":"Boston Children’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Benjamin","middleName":"C.","lastName":"Warf","suffix":""},{"id":275427475,"identity":"14ea2b0c-2859-4ae3-9422-60a990063bdc","order_by":15,"name":"David C. Christiani","email":"","orcid":"","institution":"Harvard T.H. Chan School of Public Health","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"C.","lastName":"Christiani","suffix":""},{"id":275427476,"identity":"2d277aa4-73f6-4023-801a-5443f727ca12","order_by":16,"name":"Maitreyi Mazumdar","email":"data:image/png;base64,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","orcid":"","institution":"Harvard T.H. Chan School of Public Health","correspondingAuthor":true,"prefix":"","firstName":"Maitreyi","middleName":"","lastName":"Mazumdar","suffix":""}],"badges":[],"createdAt":"2024-02-25 21:30:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3989039/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3989039/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51819993,"identity":"f711aa18-7313-4dfd-9082-18a10af8894f","added_by":"auto","created_at":"2024-02-29 15:58:39","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1486408,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eAssociation between prenatal folic acid use and spina bifida risk, by mother’s toenail arsenic concentration.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAdjusted for mother’s age (years), place of birth (hospital, clinic, or homes), and secondhand smoke exposure. The cutoff for mother’s toenail arsenic concentrations was defined by the median of study population (0.46 µg/g toenail). Abbreviations: CI, confidence interval; OR, odds ratio.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3989039/v1/1feadc195382bc5316e6ddfb.jpeg"},{"id":51820885,"identity":"1204f8f4-c80b-42b1-9e6f-0c7b1c709801","added_by":"auto","created_at":"2024-02-29 16:06:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1104063,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3989039/v1/2faae495-f387-4b0f-90a6-b6e3b167e467.pdf"},{"id":51819994,"identity":"08acaa4c-3454-4d68-ac91-614832c27b3d","added_by":"auto","created_at":"2024-02-29 15:58:40","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":197749,"visible":true,"origin":"","legend":"","description":"","filename":"NTDAsfolicacidSupplMaterialv0.126January2024CFW0225cleaned.docx","url":"https://assets-eu.researchsquare.com/files/rs-3989039/v1/ccb5a4ea26c26430cf33cdc0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Arsenic modifies the effect of folic acid in spina bifida prevention, a large hospital-based case-control study in Bangladesh","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eSpina bifida is a developmental malformation of the spinal cord that leads to increased risk of mortality and disability, including leg, bladder, and bowel dysfunction, susceptibility to infection, hydrocephalus, and cerebrospinal fluid (CSF) leakage.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] Spina bifida is a type of neural tube defect (NTD), a group of disorders caused by failure of the neural tube to fuse in the third week of gestation.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] A recent analysis estimated that globally, at least 213,800\u0026thinsp;\u0026minus;\u0026thinsp;322,000 pregnancies are affected by NTDs each year, and in low-income and middle-income countries, the prevalence of NTDs exceeds one in every 100 births.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eFolic acid supplement use reduces the risk of spina bifida,[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and fortification of staple foods with folic acid has been successful in decreasing spina bifida rates in multiple countries.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] However, a substantial number of affected pregnancies occur in areas with folic acid fortification and to women known to have taken folic acid supplements,[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and the effectiveness of folic-acid based preventive strategies varies significantly across and even within countries.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] There is an urgent need to identify modifiable factors that may reduce the burden of this condition.\u003c/p\u003e \u003cp\u003eEnvironmental exposure to arsenic is of particular concern and may be a potential contributor to spina bifida risk, especially in areas of the world with high arsenic exposures and micronutrient deficiencies. Arsenic induces neural tube defects in several animal models,[\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and arsenic toxicity is closely related to one-carbon metabolism nutrients including folate.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] A recent systematic review showed there was inadequate evidence to determine the relationship between prenatal arsenic exposure and prevalence of NTDs,[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] but the review did not address potential interactions between arsenic and folic acid that may affect spina bifida risk.\u003c/p\u003e \u003cp\u003eUnderstanding the relationship between folic acid, spina bifida, and arsenic exposure is especially important in Bangladesh, where an estimated 70\u0026nbsp;million people are chronically exposed to high concentrations of arsenic through contaminated groundwater, in what has been described as the largest mass poisoning in history.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] In 2019, a national survey conducted by the Bangladesh Bureau of Statistics and United Nations Children\u0026rsquo;s Fund estimated that 18.6% of households in Bangladesh were exposed to source water arsenic levels\u0026thinsp;\u0026gt;\u0026thinsp;10 \u0026micro;g/L and 11.8% were exposed to levels\u0026thinsp;\u0026gt;\u0026thinsp;50 \u0026micro;g/L.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] Bangladesh also has high rates of spina bifida. The country does not have systematic surveillance for birth defects, but large hospital-based studies estimate the prevalence of spina bifida to be between 10.4 and 38.2 per 10,000 births.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eOur previous study discovered that higher water arsenic concentrations reduced the effectiveness of folic acid in spina bifida prevention.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] Based on these findings, we used toenail arsenic as biomarker, and identified an association between father\u0026rsquo;s arsenic exposure and spina bifida.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] In this study, we sought to ascertain whether environmental arsenic exposure is associated with higher risk of spina bifida and whether arsenic exposure modified the protective effect of folic acid supplementation in Bangladesh.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCase ascertainment, and control selection\u003c/h2\u003e \u003cp\u003eWe conducted a hospital-based case-control study at the National Institute of Neurosciences \u0026amp; Hospital (NINS\u0026amp;H), the primary center for spina bifida surgery in Bangladesh. Cases were infants with spina bifida who were less than one year old and had mothers who could identify their primary drinking water source during early pregnancy. Neurosurgeons at NINS\u0026amp;H examined all cases and reviewed medical records, operative reports, and available imaging results. Study staff recorded the subtype of spina bifida (myelomeningocele or meningocele), level of lesion (e.g., cervical, thoracic, lumbar, sacral, or lumbosacral), co-occurring anomalies, and complications present at time of enrollment, including cerebrospinal fluid leak, lesion infection, and hydrocephalus. A senior neurosurgeon (BCW) confirmed classification of cases by reviewing photographs and available imaging.\u003c/p\u003e \u003cp\u003eWe selected controls from children who presented to NINS\u0026amp;H or the adjacent Dhaka Shishu Hospital (DSH), a children\u0026rsquo;s hospital immediately adjacent to NINS\u0026amp;H with similar referral patterns. Potential controls were seen for conditions including craniosynostosis, arachnoid cyst, trauma, subdural effusion, and epilepsy. Children with brain tumors or other cancers were not eligible to be controls. When a case was enrolled, we reviewed that week\u0026rsquo;s clinic lists and identified potential controls who were within six months of age of the enrolled case. The Bangladesh Medical Research Council and the Human Research Committees at Boston Children\u0026rsquo;s Hospital (BCH), NINS\u0026amp;H, and DSH approved this study (BCH protocol number IRB-P00019768; BMRC registration number: 006 23 08 2016). The Harvard T.H. Chan School of Public Health ceded review to BCH (protocol number: IRB20-0780). Parents provided informed consent before enrollment.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eClinical information and folate status\u003c/h2\u003e \u003cp\u003eTrained study staff interviewed patients\u0026rsquo; families to collect demographic characteristics, patient and family medical histories, referral patterns to NINS\u0026amp;H, and medical histories. We measured infant\u0026rsquo;s weight and head circumference using standardized protocols, and mother\u0026rsquo;s medication and vitamin intake using a structured questionnaire that included the seven major types of folic acid-containing tablets in Bangladesh. We also collected information about the timing of initiation of vitamin use (before or after knowing about pregnancy), duration and frequency. To estimate nutritional intake during pregnancy, we administered a food frequency questionnaire previously validated in Bangladesh.[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] Folate status was additionally assessed in serum samples using Chemiluminescent Microparticle Immunoassay at NINS\u0026amp;H (ARCHITECT plus ci4100, Abbott Company, Abbott Park, IL, USA).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eWater arsenic concentration\u003c/h2\u003e \u003cp\u003eMothers were asked to identify the primary drinking water source they used at the time they became aware of their pregnancies. Trained staff visited these sites and collected water samples in polyethylene containers. Arsenic concentrations in water were assessed at the Bangladesh University of Engineering and Technology using graphite furnace atomic absorption spectrometry (GF-AAS) with a limit of detection (LOD) of 1 \u0026micro;g/L.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] For water arsenic concentrations below the limit of detection (LOD), we assigned a value of LOD/\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\sqrt{2 }.\\)\u003c/span\u003e\u003c/span\u003eWe tested one blank for every 50 water samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eToenail arsenic concentration\u003c/h2\u003e \u003cp\u003eMother\u0026rsquo;s toenail clippings were obtained from all toenails and placed in a small coin envelope. The samples were stored and shipped to the Dartmouth Trace Element Analysis Core at room temperature. Arsenic concentrations were measured using inductively coupled plasma mass spectrometry (ICP-MS) using methods that have been previously described.[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] The instrument\u0026rsquo;s LOD was 0.01 ng/g for the first 338 samples, and 0.1 ng/g for the remaining samples. For toenail arsenic concentrations below the instrument\u0026rsquo;s LOD, we assigned a value equal to the average dilution factor of toenail samples multiplied by (0.001*LOD)/\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\sqrt{2 }.\\)\u003c/span\u003e\u003c/span\u003e[20]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eTo compare the data between cases and controls, we used t-tests for continuous variables, and chi-square and Fisher's exact tests for categorical variables. We calculated Spearman correlation coefficients for the correlations of toenail and water arsenic concentrations.\u003c/p\u003e \u003cp\u003eWe employed logistic regression models to assess the associations between arsenic concentrations and folic acid use (predictors) and case status. We modeled arsenic concentrations in two ways: 1) as a continuous variable and 2) as a binary variable: above and below 10 \u0026micro;g/L for water (the current World Health Organization drinking water standard for arsenic), and above and below the median for toenails. We did not use conditional models because of the uneven numbers of cases and controls. Models were adjusted for potential confounders that were chosen based on prior knowledge and using directed acyclic graphs and included mother\u0026rsquo;s age, place of birth (hospital, clinic, or home) and secondhand smoke exposure. In sensitivity analyses, we restricted the cases to only those with myelomeningocele. Data were analyzed using R (version 4.0.4).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eWe enrolled 333 infants with initial diagnoses of spina bifida (meningocele or myelomeningocele) and 165 controls. We stopped enrolling controls and collecting water in March 2020 because of COVID-19 restrictions, and this led to an uneven number of cases and controls. Participation rates were 73% among potential families with spina bifida and 57% among potential controls. Reasons for study refusal were similar among cases and controls and primarily included concerns about giving blood. We excluded 11 cases because surgery revealed lipomeningocele and 1 case because the mother reported valproic acid use. Toenail arsenic concentrations were not available for 27 cases and 2 controls. Our final study population included 294 cases and 163 controls. Diagnoses of the controls are presented in Supplemental Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003eDemographic information is presented in Table\u0026nbsp;1. None of the mothers reported a diagnosis of diabetes, including gestational diabetes. Prenatal folic acid use among our study population was low, but consistent with other reports from Bangladesh;[\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] only 16.7% of mothers of cases in our study and 22.1% of mothers of controls reported using folic acid during pregnancy. Serum folate concentrations were higher among mothers who reported folic acid use during pregnancy compared to those who did not report folic acid use (10.20 ng/ml and 7.96 ng/ml, respectively). On average, arsenic concentrations in water were lower than seen in our previous studies in Bangladesh,[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] although some high arsenic concentrations were seen (maximum water arsenic concentration: 451 \u0026micro;g/L) (Table\u0026nbsp;2). We observed a moderate correlation between water and toenail arsenic concentrations (Spearman correlation coefficient: 0.45). Arsenic was detected in all toenail samples.\u003c/p\u003e \u003cp\u003eThere was no significant main effect found between water and mother\u0026rsquo;s toenail arsenic concentrations and spina bifida risk (Supplemental Table\u0026nbsp;2). However, in stratified models, we found evidence that arsenic exposure modified the effect of folic acid on spina bifida risk (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among mothers with toenail arsenic concentrations below the median, folic acid use during pregnancy was associated with lower odds of having an infant with spina bifida (adjusted odds ratio [OR]: 0.50, 95% confidence interval [CI]: 0.25\u0026ndash;1.00). Among women with toenail arsenic concentrations above the median, this protective association was not seen (adjusted OR: 1.09, 95% CI: 0.52\u0026ndash;2.29). We found similar results after restricting the cases to those with myelomeningocele (Supplemental Fig.\u0026nbsp;1) (adjusted OR: 0.45, 95% CI: 0.21\u0026ndash;0.94 vs. adjusted OR: 1.03, 95% CI: 0.48\u0026ndash;2.23). When using water arsenic concentration as our measure of arsenic exposure, folic acid had a protective association at arsenic concentrations\u0026thinsp;\u0026gt;\u0026thinsp;10 \u0026micro;g/L (adjusted OR: 0.18, 95% CI 0.04\u0026ndash;0.93, p\u0026thinsp;=\u0026thinsp;0.04), but this finding may be limited by the small numbers of reported folic acid users in this group (Supplemental Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur study found that arsenic modifies the effect of folic acid on spina bifida prevention in a population in Bangladesh, a country with high arsenic exposures through contaminated drinking water. Among mothers with toenail arsenic concentrations below the median, folic acid was associated with a protective effect (adjusted OR:0.50, 95% CI: 0.25-1.00), but a protective effect was not observed among mothers with toenail concentrations above the median (adjusted OR:1.09, 95% CI: 0.52\u0026ndash;2.29). We found similar evidence of effect modification when we restricted the analysis to cases with myelomeningocele, a more severe type of spina bifida. (Supplemental Fig.\u0026nbsp;1)\u003c/p\u003e \u003cp\u003eOur results are consistent with recent studies conducted in high arsenic areas that suggest that environmental arsenic exposure may attenuate the protective benefit of folic acid supplements in preventing spina bifida. In a large case-control study of NTDs in Shanxi, China, researchers found that placental arsenic concentrations were associated with NTD-affected pregnancies among mothers who reported not taking folic acid supplements.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] Our previous studies in Bangladesh found that as drinking water arsenic concentrations increased from 1 \u0026micro;g/L to 25 \u0026micro;g/L, the protective effect of folic acid use declined (OR: 0.22, 95% CI: 0.13, 0.37 to OR: 1.03, 95% CI: 0.55, 1.91).[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] These studies suggest that prenatal folic acid supplementation may be less effective in spina bifida prevention in populations with high arsenic exposure because of interactions between arsenic and folic acid.\u003c/p\u003e \u003cp\u003eAnimal studies have consistently shown arsenic to be a potent teratogen, inducing neural tube defects in several animal models.[\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] In addition, animal studies demonstrate that arsenic-folate interactions are important in arsenic\u0026rsquo;s teratogenicity. In \u003cem\u003eFolbp2\u003c/em\u003e knockout mice, for example, mice with this specific defect in folate transport had higher rates of NTDs after arsenic exposure, and mice nullizygous for genes encoding proteins in cellular uptake of folate were more susceptible to arsenic-induced NTDs.[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] The interaction between arsenic and folate may be explained by arsenic\u0026rsquo;s interference with folate-related functions, such as the depletion of S-adenosylmethionine (SAM), a key methyl donor for pathways implicated in neural tube closure.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] In experimental models, chicken embryos exposed to 100nM arsenate showed reduced SAM levels and higher rates of NTDs.[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] In humans, the strongest evidence for arsenic-folic acid interactions comes from trials in which folic acid has been shown to reduce blood arsenic concentrations.[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eAlthough we found evidence of effect modification by arsenic, our study did not demonstrate a primary (or main) effect of mother\u0026rsquo;s arsenic exposure on spina bifida risk, consistent with findings from a recent systematic review assessing cohort studies that had measures of prenatal arsenic exposure and spina bifida outcomes.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] It is possible that even higher arsenic exposures are needed to demonstrate a primary effect. A recent study from an area of Turkey with high arsenic exposures reported higher levels of arsenic in plasma samples of 100 mothers of NTD cases as compared to mothers from 70 controls.[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] Survivorship bias may also account for not finding a primary effect. We enrolled cases at time of presentation for surgical care and did not capture affected pregnancies that did not continue to birth or more severely affected infants who were not brought for care. If arsenic exposure was related to these more severe outcomes, our results could represent a downward bias towards or beyond the null.[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] Another possible explanation for why we did not find a primary effect of arsenic is that arsenic exposure by itself may be insufficient, and that the addition of other risk factors, such as inadequate folate, is a necessary contribution to disrupt neural tube closure.\u003c/p\u003e \u003cp\u003eThe strengths of our study include the use of individual measures of exposure, including toenail arsenic measurements, which represent arsenic exposure from 5 to 18 months before collection,[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and may better represent exposure during early pregnancy than blood, urine, or water samples collected after delivery. Previous work in Bangladesh in a large pregnancy cohort has shown high correlations between pregnant women\u0026rsquo;s toenail arsenic measurements during the first trimester and 1 month post-partum and also with measurements from their infant\u0026rsquo;s toenails at age 1 month.[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] An additional strength is classification of cases by neurosurgeons using examination, imaging and observations during surgery. It is increasingly understood that NTDs are not one disorder, but instead a wide array of morphologically distinct malformations which likely each result from a different contribution of risk factors.[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] Our study is the largest study to date to investigate spina bifida only (larger studies included all NTDs), and we were able to further restrict our analyses to only myelomeningocele in sensitivity analyses.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eEnvironmental arsenic exposure may reduce the protective effects of folic acid supplementation on spina bifida risk. Our findings raise important questions about the risk of spina bifida in arsenic-endemic areas and the effectiveness of folic acid supplements alone as the strategy to prevent spina bifida in high-arsenic areas of the world. At minimum, more surveillance for spina bifida is needed in high arsenic areas such as Bangladesh. Additional preventive measures, such as folic acid fortification of the food supply and reduction of arsenic exposure, may be needed to optimize spina bifida prevention in areas with high arsenic exposures.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBoston Children\u0026rsquo;s Hospital, BCH; cerebrospinal fluid, CSF; confidence interval, CI; Dhaka Shishu Hospital, DSH; graphite furnace atomic absorption spectrometry, GF-AAS; inductively coupled plasma mass spectrometry, ICP-MS; limit of detection, LOD; National Institute of Neurosciences \u0026amp; Hospital, NINS\u0026amp;H; neural tube defect, NTD; odds ratio, OR; S-adenosylmethionine, SAM.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Bangladesh Medical Research Council and the Human Research Committees at Boston Children\u0026rsquo;s Hospital (BCH), NINS\u0026amp;H, and DSH approved this study (BCH protocol number IRB-P00019768; BMRC registration number: 006 23 08 2016). The Harvard T.H. Chan School of Public Health ceded review to BCH (protocol number: IRB20-0780). Parents provided informed consent before enrollment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Institutes of Environmental Health Sciences (grant numbers: NIEHS R01 ES026317, R21 ES030784) and the National Institute of Mental Health (NIMH T32 MH112510). In addition, this project used core facilities supported by institutional grants from NIEHS P30 ES000002. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIEHS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMM, SKM, SME, DMA, JI, MA, AR, BCW, and DCC conceived the study. MM, SKM, SME, DMA, JI, MA, AR, MNR, MZ and HSS conducted the field activities. WCF drafted the manuscript. WCF, MM, GT, LL, DG, DCC, and MW participated in the data analyses. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge our field team at NINS\u0026amp;H, including Shipra Rani Saha, and Shashwati Suter, Suvajit Das, Shamantha Afreen, Ansar Uddin, Abdullah Al Mahbub and Md. Ziaul Hoq, Dartmouth Trace Element Core Facility, which is supported by Dartmouth Cancer Center with NCI Cancer Center Support Grant 5P30 CA023108, as well as laboratory staff at the Harvard T.H. Chan School of Public Health, including Li Su, Danny Donato, and Erica Haley. We are particularly grateful for support from the Outstanding New Environmental Scientist advisory committee: Richard Finnell, Mary Gamble and Martha Werler.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eIskandar BJ, Finnell RH: \u003cstrong\u003eSpina Bifida\u003c/strong\u003e. \u003cem\u003eN Engl J Med \u003c/em\u003e2022, \u003cstrong\u003e387\u003c/strong\u003e(5):444-450.\u003c/li\u003e\n\u003cli\u003eBlencowe H, Kancherla V, Moorthie S, Darlison MW, Modell B: \u003cstrong\u003eEstimates of global and regional prevalence of neural tube defects for 2015: a systematic analysis\u003c/strong\u003e. \u003cem\u003eAnn N Y Acad Sci \u003c/em\u003e2018, \u003cstrong\u003e1414\u003c/strong\u003e(1):31-46.\u003c/li\u003e\n\u003cli\u003eDe-Regil LM, Pena-Rosas JP, Fernandez-Gaxiola AC, Rayco-Solon P: \u003cstrong\u003eEffects and safety of periconceptional oral folate supplementation for preventing birth defects\u003c/strong\u003e. \u003cem\u003eCochrane 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biomarkers of prenatal exposure to arsenic and manganese\u003c/strong\u003e. \u003cem\u003eJ Expo Sci Environ Epidemiol \u003c/em\u003e2015, \u003cstrong\u003e25\u003c/strong\u003e(6):639-648.\u003c/li\u003e\n\u003cli\u003ePi X, Wang C, Yin S, Jin L, Li Z, Wang L, Liu J, Zhang Y, Ren A: \u003cstrong\u003eArsenic Exposure, Periconceptional Folic Acid Supplementation, and the Risk for Neural Tube Defects: A Case\u0026ndash;Control Study\u003c/strong\u003e. \u003cem\u003eExposure and Health \u003c/em\u003e2022, \u003cstrong\u003e15\u003c/strong\u003e(1):245-254.\u003c/li\u003e\n\u003cli\u003eWlodarczyk BJ, Cabrera RM, Hill DS, Bozinov D, Zhu H, Finnell RH: \u003cstrong\u003eArsenic-induced gene expression changes in the neural tube of folate transport defective mouse embryos\u003c/strong\u003e. \u003cem\u003eNeurotoxicology \u003c/em\u003e2006, \u003cstrong\u003e27\u003c/strong\u003e(4):547-557.\u003c/li\u003e\n\u003cli\u003eHan ZJ, Song G, Cui Y, Xia HF, Ma X: \u003cstrong\u003eOxidative stress is implicated in arsenic-induced neural tube defects in chick embryos\u003c/strong\u003e. \u003cem\u003eInt J Dev Neurosci \u003c/em\u003e2011, \u003cstrong\u003e29\u003c/strong\u003e(7):673-680.\u003c/li\u003e\n\u003cli\u003eGamble MV, Liu X, Ahsan H, Pilsner JR, Ilievski V, Slavkovich V, Parvez F, Chen Y, Levy D, Factor-Litvak P\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eFolate and arsenic metabolism: a double-blind, placebo-controlled folic acid-supplementation trial in Bangladesh\u003c/strong\u003e. \u003cem\u003eAm J Clin Nutr \u003c/em\u003e2006, \u003cstrong\u003e84\u003c/strong\u003e(5):1093-1101.\u003c/li\u003e\n\u003cli\u003eGamble MV, Liu X, Slavkovich V, Pilsner JR, Ilievski V, Factor-Litvak P, Levy D, Alam S, Islam M, Parvez F\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eFolic acid supplementation lowers blood arsenic\u003c/strong\u003e. \u003cem\u003eAm J Clin Nutr \u003c/em\u003e2007, \u003cstrong\u003e86\u003c/strong\u003e(4):1202-1209.\u003c/li\u003e\n\u003cli\u003ePeters BA, Hall MN, Liu X, Parvez F, Sanchez TR, van Geen A, Mey JL, Siddique AB, Shahriar H, Uddin MN\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eFolic Acid and Creatine as Therapeutic Approaches to Lower Blood Arsenic: A Randomized Controlled Trial\u003c/strong\u003e. \u003cem\u003eEnviron Health Perspect \u003c/em\u003e2015, \u003cstrong\u003e123\u003c/strong\u003e(12):1294-1301.\u003c/li\u003e\n\u003cli\u003eDemir N, Başaranoğlu M, Huyut Z, Değer İ, Karaman K, Şekeroğlu MR, Tuncer O: \u003cstrong\u003eThe relationship between mother and infant plasma trace element and heavy metal levels and the risk of neural tube defect in infants\u003c/strong\u003e. \u003cem\u003eThe Journal of Maternal-Fetal \u0026amp; Neonatal Medicine \u003c/em\u003e2019, \u003cstrong\u003e32\u003c/strong\u003e(9):1433-1440.\u003c/li\u003e\n\u003cli\u003eLeung M, Kioumourtzoglou MA, Raz R, Weisskopf MG: \u003cstrong\u003eBias due to Selection on Live Births in Studies of Environmental Exposures during Pregnancy: A Simulation Study\u003c/strong\u003e. \u003cem\u003eEnviron Health Perspect \u003c/em\u003e2021, \u003cstrong\u003e129\u003c/strong\u003e(4):47001.\u003c/li\u003e\n\u003cli\u003eSignes-Pastor AJ, Gutierrez-Gonzalez E, Garcia-Villarino M, Rodriguez-Cabrera FD, Lopez-Moreno JJ, Varea-Jimenez E, Pastor-Barriuso R, Pollan M, Navas-Acien A, Perez-Gomez B\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eToenails as a biomarker of exposure to arsenic: A review\u003c/strong\u003e. \u003cem\u003eEnviron Res \u003c/em\u003e2021, \u003cstrong\u003e195\u003c/strong\u003e:110286.\u003c/li\u003e\n\u003cli\u003eWallingford JB, Niswander LA, Shaw GM, Finnell RH: \u003cstrong\u003eThe continuing challenge of understanding, preventing, and treating neural tube defects\u003c/strong\u003e. \u003cem\u003eScience \u003c/em\u003e2013, \u003cstrong\u003e339\u003c/strong\u003e(6123):1222002.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e \u003cem\u003eStudy Population\u003c/em\u003e\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Taba\" border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eCases (n\u0026thinsp;=\u0026thinsp;294)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eControls (n\u0026thinsp;=\u0026thinsp;163)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003ep-value\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"BoldItalic\"\u003eInfant characteristics\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eBoys\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e157 (53.4)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e104 (63.8)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.04\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eFirstborn\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e102 (34.7)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e87 (53.4)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.001\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eGestational age (week)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e37.3 (2.1)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e37.0 (2.3)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.22\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eAge at study visit (days)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e75.5 (86.6)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e185.3 (96.1)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.001\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eMyelomeningocele\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e249 (84.7)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eNA\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eLevel of spina bifida\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eCervical or thoracic\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e11 (3.7)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eNA\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eLumbar\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e144 (49.0)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eNA\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eSacral or lumbosacral\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e138 (46.9)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eNA\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"BoldItalic\"\u003eMother\u0026rsquo;s characteristics\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eAge (years)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e24.7 (4.7)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e24.3 (5.0)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.39\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eEducation level\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.11\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eIlliterate\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e3 (1.0)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e2 (1.2)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eLiterate\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e55 (18.7)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e22 (13.5)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eHigh school or less\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e164 (55.8)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e83 (50.9)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eCollege\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e56 (19.0)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e38 (23.3)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eUniversity\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e16 (5.4)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e18 (11.0)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eFolic acid use during early pregnancy\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e49 (16.7)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e36 (22.1)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.19\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eLiving in Dhaka\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e153 (52.0)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e89 (54.6)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.67\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003ePre-pregnancy diabetes\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e9 (3.1)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e4 (2.5)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.78\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eDiagnosis of gestational diabetes\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e5 (1.7)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e4 (2.5)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.73\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eSecondhand smoke exposure\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e127 (43.2)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e60 (36.8)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.22\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eFever episodes during pregnancy\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e153 (52.0)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e70 (42.9)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.06\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003ePlace of birth\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.16\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eHome\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e79 (26.9)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e41 (25.2)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eClinics\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e111 (37.8)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e50 (30.7)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eHospitals\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e104 (35.4)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e72 (44.2)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eMean (SD) or number (%); NA\u0026thinsp;=\u0026thinsp;not applicable.\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. \u003c/strong\u003e\u003cem\u003eArsenic concentrations in toenails and drinking water\u003c/em\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tabb\" border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eMean (SD)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eMedian\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eIQR\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eMin\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eMax\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"BoldItalic\"\u003eMother\u0026rsquo;s toenail arsenic concentration (\u0026micro;g/g)\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eCases (n\u0026thinsp;=\u0026thinsp;294)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e1.04 (1.58)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.45\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.28\u0026ndash;1.05\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.07\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e13.77\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eControls (n\u0026thinsp;=\u0026thinsp;163)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e1.13 (1.94)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.47\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.30\u0026ndash;0.96\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.09\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e12.30\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"3\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"BoldItalic\"\u003eWater arsenic concentration (\u0026micro;g/L)\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eCases (n\u0026thinsp;=\u0026thinsp;166)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e15.35 (37.28)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e2.00\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e1.00\u0026ndash;7.00\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.71\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e255.00\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eControls (n\u0026thinsp;=\u0026thinsp;161)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e20.91 (56.08)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e2.00\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.71-7.00\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.71\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e451.00\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"6\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003ea. Water arsenic concentrations were below the LOD (1 \u0026micro;g/L) for 41 cases and 52 controls. Arsenic was detected in all toenail samples, and the LOD for toenail arsenic was 0.01 ng/g for first 338 samples tested and 0.1 ng/g for remaining samples\u003c/div\u003e\n\u003cdiv class=\"SimplePara\"\u003eb. Water samples were not available for participants enrolled during the COVID-19 pandemic due to travel restrictions in Bangladesh.\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"6\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eAbbreviations: IQR, interquartile range; LOD, limit of detection; SD, standard deviation.\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"enhe","sideBox":"Learn more about [Environmental Health](http://ehjournal.biomedcentral.com)","snPcode":"12940","submissionUrl":"https://submission.nature.com/new-submission/12940/3","title":"Environmental Health","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Arsenic, Bangladesh, folic acid, spina bifida","lastPublishedDoi":"10.21203/rs.3.rs-3989039/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3989039/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSpina bifida, a developmental malformation of the spinal cord, is associated with high rates of mortality and disability. Although folic acid-based preventive strategies have been successful in reducing rates of spina bifida, some areas continue to be at higher risk because of chemical exposures. Bangladesh has high arsenic exposures through contaminated drinking water and high rates of spina bifida.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe conducted a hospital-based case-control study at the National Institute of Neurosciences \u0026amp; Hospital (NINS\u0026amp;H) in Dhaka, Bangladesh, between December 2016 and December 2022. Cases were infants under age one year with spina bifida and further classified using data from observations by neurosurgeons and available imaging. Controls were drawn from children who presented to NINS\u0026amp;H or Dhaka Shishu Hospital (DSH) during the same study period. Mothers reported folic acid use during pregnancy, and we assessed folate status with serum assays. Arsenic exposure was estimated in drinking water using graphite furnace atomic absorption spectrophotometry (GF-AAS) and in toenails using inductively coupled plasma mass spectrometry (ICP-MS).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe evaluated data from 294 cases of spina bifida and 163 controls. We did not find a main effect of mother\u0026rsquo;s arsenic exposure on spina bifida risk. However, in stratified analyses, folic acid use was associated with lower odds of spina bifida (adjusted odds ratio [OR]: 0.50, 95% confidence interval [CI]: 0.25-1.00, p\u0026thinsp;=\u0026thinsp;0.05) among women with toenail arsenic concentrations below the median, and no association was seen among mothers with toenail arsenic concentrations higher than median (adjusted OR: 1.09, 95% CI: 0.52\u0026ndash;2.29, p\u0026thinsp;=\u0026thinsp;0.82).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eMother\u0026rsquo;s arsenic exposure modified the protective association of folic acid with spina bifida. Increased surveillance and additional preventive strategies, such as folic acid fortification and reduction of arsenic, are needed in areas of high arsenic exposure.\u003c/p\u003e","manuscriptTitle":"Arsenic modifies the effect of folic acid in spina bifida prevention, a large hospital-based case-control study in Bangladesh","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-29 15:58:35","doi":"10.21203/rs.3.rs-3989039/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-09T22:39:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-26T07:30:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1d4ffe4c-f1bf-49f3-9f07-0e7f75a071a4","date":"2024-03-12T17:30:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-12T17:14:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-26T22:26:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-26T22:26:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Health","date":"2024-02-25T21:11:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"enhe","sideBox":"Learn more about [Environmental Health](http://ehjournal.biomedcentral.com)","snPcode":"12940","submissionUrl":"https://submission.nature.com/new-submission/12940/3","title":"Environmental Health","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b4bfebb9-c64a-4fdc-bcd1-68be03df1a4d","owner":[],"postedDate":"February 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-19T13:08:22+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-29 15:58:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3989039","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3989039","identity":"rs-3989039","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}