Maternal Folic Acid Supplementation and Neural Tube Defects in Northeast Iran: Insights for Developing Countries

Abstract

Background

Neural tube defects (NTDs) are among the most common severe congenital malformations, caused by incomplete closure of the neural tube in the third and fourth weeks after conception. This study aimed to investigate potential maternal and environmental risk factors associated with NTDs in newborns under the coverage of Mashhad University of Medical Sciences.

Methods

A descriptive case-control study was conducted using the Integrated Health Information System. All NTDs newborns (n=126) were case identified while three times of the number of mothers of healthy newborns (n=378) were picked as controls. Maternal and neonatal factors like folic acid supplementation, maternal age, BMI, parity, smoking, passive smoking, consanguinity, and neonatal sex were collected and processed with SPSS 27 software. Chi-square, Fisher’s exact test, and logistic regression were employed.

Results

Among mothers of NTD-affected newborns, 20.6% reported folic acid supplementation versus 31.5% in controls (P=0.02). The gender of the newborn, mother’s age, BMI, number of children, education level, kinship, history of having affected child, and history of stillbirth showed no significant difference between cases and controls. Logistic regression results showed that not taking folic acid increased the likelihood of NTDs by more than two times (OR=2.11, P=0.006).

Conclusion

Maternal folic acid supplementation was significantly associated with reduced risk of NTDs. No significant relationship was found with the other factors studied.

Introduction

Neural tube defects (NTDs) are counted among the most serious congenital anomalies - their occurrence is due to the incomplete closure of the neural tube during the third and fourth weeks of embryo development.¹ The NTDs – mainly spina bifida, anencephaly, and encephalocele- not only cause heavy morbidity but also, in many cases, lead to long-term disability and mortality worldwide.^1–3 The prevalence of NTDs varies geographically, with estimates in Iran ranging from 1.01 to 8.29 per 1000 live births.^1,4

NTDs have a multifactorial etiology involving genetic predisposition, maternal nutritional status, environmental exposures, and lifestyle factors. Among the factors, supplementation of mothers with folic acid is the most accepted prevention measure. If mothers have adequate folic acid intake before conception and during early pregnancy, the risk of NTDs can be reduced by as much as 70%.^5–7 However, this benefit is very well established but still, the usage of folic acid is not optimal in many regions including Iran. Data from national studies indicate that preconception folic acid use is between 15% and 35%, which is much lower compared to reported rates in high-income countries where such programs as supplementation and food fortification are prevalent.^7,8 As highlighted in comprehensive reviews by van Gool et al (2018),⁹ and Osterhues et al 2013,¹⁰ the efficacy of periconceptional folic acid in reducing NTD risk is one of the most robust findings in preventive medicine.

The low rate is caused by several factors, for example, women of reproductive age not knowing about the need for folic acid, being unable to get prenatal care, economic hardship, and cultural customs.¹¹ In Iran, the economic impact of international sanctions has further strained access to nutritional supplements, potentially exacerbating deficiencies during critical periods of early pregnancy. Social taboos and misconceptions about supplement use may also hinder consistent consumption.^12,13

Although several research papers have discussed maternal risk factors for NTDs all over the world, there is no new valuable information coming from Mashhad and its vicinity. On top of that, the socio-economic, systemic factors like access to healthcare and availability of supplements have not been implicated in the use of folic acid in a greater number of studies.^14,15 To reduce the number of NTDs, the public health interventions need to be well designed and this will not happen unless the mentioned gaps are properly filled. Jin (2023) further emphasizes in the Journal of the American Medical Association that despite clear evidence, implementation gaps persist globally.¹⁶

Globally, the debate on mandatory fortification continues; for example, while Australia and New Zealand have successfully implemented fortification programs,¹⁷ Europe still lacks a unified policy,^18,19 and South Africa’s programme has provided critical insights for middle‑income countries.²⁰ Although several research papers have discussed maternal risk factors for NTDs all over the world, there is no new valuable information coming from Mashhad and its vicinity. On top of that, the socio‑economic and systemic factors like access to healthcare and availability of supplements have not been implicated in the use of folic acid in a greater number of studies.^14,15 To reduce the number of NTDs, public health interventions need to be well designed, and this will not happen unless the mentioned gaps are properly filled. In Iran, the economic impact of international sanctions has further strained access to nutritional supplements, potentially exacerbating deficiencies during critical periods of early pregnancy

This study aimed to investigate potential maternal risk factors associated with NTDs among newborns under the coverage of Mashhad University of Medical Sciences, with a particular focus on the prevalence and protective association of folic acid supplementation, contextualised within socio‑economic, cultural, and healthcare system factors.

Methods

Study Design and Population

This descriptive case-control study was conducted using data from the Integrated Health Information System, which covers all pregnant women under the care of Mashhad University of Medical Sciences. The study population included mothers who delivered newborns between January 2016 and December 2024. NTDs were diagnosed by neonatologists and confirmed through clinical examination and ultrasonographic findings. For each case, three mothers of healthy newborns were randomly selected as controls using systematic random sampling from the same health information system, matched by health centre catchment area and delivery date within ±3 months. Sample size was calculated based on an expected exposure prevalence of 30% in controls, a 20% difference between groups, 80% power, and α=0.05, yielding a minimum of 124 cases and 372 controls.

Inclusion and Exclusion Criteria

Only mothers with complete demographic and clinical data available in the system were selected as participants for the study. Data quality was a concern, so mothers who lacked information regarding key variables like folic acid supplementation, parity, age, or the outcome of the newborn were omitted from the analysis.

Data Collection

Data were extracted from the Integrated Health Information System and included maternal and neonatal variables: maternal age, body mass index (BMI), parity, education level, consanguinity, smoking habits, passive smoking exposure, folic acid supplementation, and neonatal sex. Data were collected using standardized checklists completed by trained healthcare providers at health centers and verified in the system prior to analysis. Folic acid supplementation was defined as reported intake of 400–800 µg of folic acid daily, initiated at least one month before conception and continued through the first trimester.

Statistical Analysis

Data were analyzed using SPSS version 27 (IBM Corp., Armonk, NY, USA). Descriptive statistics, including frequencies and percentages, were calculated for categorical variables, while means and standard deviations were used for continuous variables. Associations between categorical variables and NTD occurrence were assessed using the chi-square test or Fisher’s exact test, as appropriate. Independent t-tests were used to compare continuous variables between cases and controls. The logistic regression model was adjusted for all variables listed in Table 1, including maternal age, BMI, parity, education, consanguinity, neonatal sex, history of prior affected child, history of stillbirth, passive smoking, and maternal smoking. Model fit was assessed using the Hosmer‑Lemeshow goodness‑of‑fit test (χ²=8.24, df=8, P=0.41). Statistical power may be limited for rare exposures such as maternal smoking, passive smoking, history of stillbirth, and prior affected child. A P-value of less than 0.05 was considered statistically significant.

Table 1.

Maternal and Neonatal Characteristics of NTDs and Controls (n=504)

Characteristic	NTDs (n=126)	Controls (n=378)	P-value
Neonatal Sex			0.837
Male	66 (52.4%)	194 (51.3%)
Female	60 (47.6%)	184 (48.7%)
History of Prior Affected Child			0.603*
Yes	2 (1.6%)	3 (0.8%)
No	124 (98.4%)	375 (99.2%)
History of Stillbirth			0.374*
Yes	3 (2.4%)	4 (1.1%)
No	123 (97.6%)	374 (98.9%)
Folic Acid Supplementation			0.02
Yes	26 (20.6%)	119 (31.5%)
No	100 (79.4%)	259 (68.5%)
Maternal Smoking			1.00*
Yes	1 (0.8%)	4 (1.1%)
No	125 (99.2%)	374 (98.9%)
Passive Smoking			0.261*
Yes	2 (1.6%)	2 (0.5%)
No	124 (98.4%)	376 (99.5%)
Maternal Education			0.216
<High School	96 (80.7%)	272 (74.7%)
≥High School	23 (19.3%)	92 (25.3%)
Parental Consanguinity			0.259
Yes	42 (34.1%)	107 (28.8%)
No	81 (65.9%)	265 (71.2%)
Maternal Age (years, mean ± SD)	32.72 ± 6.88	32.36 ± 6.76	0.619
BMI (kg/m2, mean ± SD)	24.96 ± 5.36	24.85 ± 4.95	0.854
Parity (mean ± SD)	2.41 ± 1.92	2.08 ± 1.63	0.091

Notes: *=Fisher’s exact test was used where expected cell counts were less than 5. Missing data resulted in denominators of 119 for maternal education (126 cases, 378 controls) and 123 for consanguinity (126 cases, 378 controls).

Abbreviations: NTDs, Neural Tube Defects; SD, Standard Deviation; BMI, Body Mass Index.

Ethical Considerations

The study protocol was approved by the Ethics Committee of Mashhad University of Medical Sciences (Approval number: IR.MUMS.MEDICAL.REC.1402.473; Date: 14/11/1402). All data were anonymized to maintain participant confidentiality, and the study was conducted in accordance with the ethical principles of the Declaration of Helsinki.

Results

A total of 504 mothers were included in the study, comprising 126 cases (mothers of newborns with neural tube defects, NTDs) and 378 controls (mothers of healthy newborns). The distribution of maternal and neonatal characteristics is summarized below.

Neonatal Sex

Among the NTD cases, 66 (52.4%) were male and 60 (47.6%) were female, while in the control group, 194 (51.3%) were male and 184 (48.7%) were female. Statistical analysis using the chi-square test indicated no significant difference in neonatal sex distribution between cases and controls (P=0.837).

Maternal History of Affected Child

Two mothers (1.6%) in the case group reported a history of a previously affected child compared to three mothers (0.8%) in the control group. Given the low expected frequencies, Fisher’s exact test was used, showing no statistically significant association between prior affected child and NTD risk (P=0.603).

Maternal History of Stillbirth

Three mothers (2.4%) in the case group and four mothers (1.1%) in the control group reported a history of stillbirth. Fisher’s exact test indicated no significant difference between the groups regarding this variable (P=0.374).

Folic Acid Supplementation

Among mothers of NTD-affected newborns, only 26 (20.6%) reported folic acid supplementation, compared to 119 (31.5%) in the control group. Chi-square analysis showed this difference was statistically significant (P=0.02), indicating a lower prevalence of folic acid supplementation among mothers of affected newborns. Logistic regression analysis further demonstrated that lack of folic acid supplementation (reference category: No supplementation) was associated with an increased risk of NTDs (OR=2.11; 95% CI: 1.24‑3.58; P=0.006) (Table 2).

Table 2.

Logistic Regression Analysis of Maternal Factors Associated with Neural Tube Defects

Variable	B	Wald	P-value	OR (Exp(B))	95% CI for OR
Folic Acid Supplementation (Yes vs No)	0.748	7.55	0.006	2.11	1.24–3.59
Maternal Age (years)	0.043	0.24	0.624	1.04	0.94–1.15
BMI (kg/m2)	0.023	0.14	0.905	1.02	0.91–1.14
Parity	0.188	0.55	0.458	1.21	0.78–1.87
Maternal Education (≥High School vs <High School)	−0.010	0.005	0.945	0.99	0.58–1.69
Parental Consanguinity (Yes vs No)	0.169	0.25	0.617	1.18	0.74–1.87
Neonatal Sex (Male vs Female)	−0.010	0.001	0.970	0.99	0.63–1.55
History of Prior Affected Child	0.062	0.005	0.943	1.06	0.16–7.02
History of Stillbirth	0.629	0.768	0.381	1.88	0.51–6.88
Passive Smoking (Yes vs No)	0.042	0.015	0.902	1.04	0.17–6.47
Maternal Smoking (Yes vs No)	−0.016	0.001	0.970	0.98	0.11–8.82

Notes: Reference category for Folic Acid Supplementation is “No”. Reference category for Maternal Education is “< High School”. Reference category for all categorical variables is the baseline group as indicated in parentheses.

Abbreviations: OR, Odds Ratio; CI, Confidence Interval; BMI, Body Mass Index.

Maternal Smoking and Passive Smoking

Only one mother (0.8%) in the case group and four mothers (1.1%) in the control group reported smoking, while two mothers (1.6%) in the case group and two mothers (0.5%) in the control group reported passive smoking exposure. Fisher’s exact test showed no significant differences for smoking (P=1.00) or passive smoking (P=0.261) between groups.

Maternal Education

Among mothers of affected newborns, 96 (80.7%) had education below a high school diploma, and 23 (19.3%) had a high school diploma or higher. In the control group, 272 (74.7%) had education below a diploma, and 92 (25.3%) had a diploma or higher. No statistically significant difference was observed between groups regarding maternal education level (P=0.216).

Parental Consanguinity

Parental consanguinity was reported in 42 (34.1%) of cases and 107 (28.8%) of controls. Chi-square analysis showed no significant difference between cases and controls (P=0.259).

Maternal Age, BMI, and Parity

The mean maternal age in the case group was 32.72 ± 6.88 years and 32.36 ± 6.76 years in the control group (P=0.619). Mean BMI was 24.96 ± 5.36 in cases and 24.85 ± 4.95 in controls (P=0.854). The mean number of pregnancies was 2.41 ± 1.92 in cases and 2.08 ± 1.63 in controls (P=0.091). None of these continuous variables demonstrated statistically significant differences between groups.

Summary of Risk Factors

Overall, logistic regression analysis identified lack of maternal folic acid supplementation as a significant risk factor for NTDs, while other maternal and neonatal variables including maternal age, BMI, parity, education, smoking, passive smoking, neonatal sex, parental consanguinity, history of stillbirth, and prior affected child were not significantly associated with the risk of NTDs.

Discussion

This study demonstrated a significant protective association of maternal folic acid supplementation against neural tube defects (NTDs). Among mothers of NTD-affected newborns, only 20.6% reported using folic acid compared to 31.5% in the control group, underscoring a concerningly low rate of supplementation in this population. Logistic regression analysis further confirmed that lack of folic acid supplementation was associated with more than a twofold increase in the risk of NTDs (OR = 2.11; 95% CI: 1.24‑3.58; P = 0.006). On the other hand, variables regarding the mother and baby such as maternal age, body mass index (BMI), education level, number of previous children, being related to the father or mother, sex of the baby, and a history of previous children with the same disability or miscarriages were not significantly related to the risk of neural tube defects (NTD) in our study. These results indicate that folic acid supplementation is the most reliable and modifiable protective factor that has been detected. However, non‑significant findings for demographic and obstetric factors do not mean that these factors have no role in NTD etiology; they may be masked by sample size or population‑specific effects. This finding aligns with the extensive evidence base reviewed by Czeizel et al (2013),²¹ who detailed the role of folate in preventing both NTDs and congenital heart defects. The optimal dosing strategy has been debated; Dolin et al (2018) concluded that 4 mg daily for recurrent NTDs is often unnecessary, as standard 0.4–0.8 mg doses are sufficient for primary prevention.²²

In comparison with previous studies, Louden et al (2020) conducted a large case‑control study investigating the impact of alcohol consumption on neural tube defects, including 1922 cases and 11,251 controls, and found no significant association between pre‑pregnancy alcohol use and risk. In contrast, Farzaneh Zaheri in 2014 in western Iran, with 46 cases and 138 controls, reported alcohol consumption as a significant risk factor.²³ In our study, both the case and control groups did not have any alcohol drinking, which locked the analysis. This possibly indicates underreporting influenced by social norms regarding alcohol consumption in Iran. Population differences, sample size variations, and cultural reporting biases may explain these differing findings. What is more, recent data point out that BMIs are of moderate effectiveness in increasing the rate of abstinence during pregnancy and in lessening the negative effects such as preterm birth, but Popova et al still stress that there is a need for more studies in developing and middle-income countries, for younger mothers, and in multi-ethnic groups to ascertain their greater effectiveness.²⁴ Our sample’s low smoking rate might be the reason for the inconsistency, and statistical power may be limited for such rare exposures.

Extending beyond lifestyle exposures, our analysis also examined several demographic and maternal characteristics. Our findings showed no significant difference in neonatal sex between groups, consistent with Galalipour et al,² and Talebian et al.²⁵ However, Nili et al²⁶ in Tehran and Behrooz et al²⁷ in Khuzestan reported higher incidence among female infants. Similarly, maternal age and BMI showed no significant association in our study, aligning with Masoudi et al.²⁸ Conversely, McMahon et al found that high maternal BMI doubled the risk of neural tube defects.²⁹ The differences in the data could be attributed to various factors such as geographical areas, the number of samples taken, or differences in the way the data were reported. Additional to our research, systematic reviews that were carried out recently have pointed out several maternal risk factors with different strength of evidence: hyperthermia, influenza, obesity, and passive smoking have been classified as suggestive or highly suggestive contributors to neural tube defects, while low maternal vitamin B12 has been reported as a potential risk factor supported by weaker evidence. These findings together emphasize the complex nature of NTD etiology, where demographic factors and modifiable exposures like nutrition, infection, and lifestyle can be critically important in varying populations.³⁰

Alongside demographic and exposure-related factors, sociocultural variables also warrant consideration. Maternal education level showed no significant association in our study, consistent with Galalipour et al⁵ and Gedefaw et al, though Canfield et al reported a protective association of higher education.^8,31 Regarding parental consanguinity, 34.1% of cases and 28.8% of controls in our study had consanguineous parents, reflecting a higher prevalence compared to developed countries. No significant difference was observed between the two groups, consistent with Zaheri et al.²³ In contrast, Behrooz et al²⁷ and Nasri et al in Tunisia identified consanguinity as a significant risk factor.³² Such discrepancies not only point out the need to take into account cultural and genetic contexts in neural tube defect research but also give reasons for it. For example, El Goundali et al revealed through their systematic review of consanguinity in the Arab population that this practice is strongly connected with congenital anomalies and therefore bringing up the cultural and genetic significance of this factor in Middle Eastern areas.³³ From a mechanistic perspective, Harris et al,³⁴ has provided crucial insights using mouse mutants to explain failure of neural tube closure. Furthermore, Obeid et al (2013),³⁵ explored 5‑methyltetrahydrofolate as a viable alternative to folic acid, particularly for individuals with MTHFR polymorphisms.

Finally, we evaluated obstetric history and maternal health conditions. In our study, there was no significant association between a past history of abnormal newborns or stillbirths and the present case, which is in agreement with Gedefaw et al.⁸ Zaheri et al recognised previous NTDs as a risk factor,²³ while Behrooz et al indicated that most mothers had no such history.²⁷ Talebian et al cited miscarriage and stillbirth as important risk factors,²⁵ and Gashaw et al also identified these as risk factors,³⁶ while Tirsit et al recorded higher rates among affected mothers.^36,37 Chronic hypertension could not be analyzed in our study due to low prevalence, though Weber et al identified it as a risk factor for spina bifida.³⁸ Gravidity showed no significant association, consistent with Nasri et al, though multiparity was reported as a risk factor.³² These findings suggest that obstetric history and maternal comorbidities may vary in importance across populations, a pattern of inconsistency also observed for other exposures. Koçak et al³⁹ found a significant prevalence of congenital heart disease in NTD patients. Long‑term survival remains a concern; Deniz et al (2024) identified key mortality predictors in myelomeningocele,⁴⁰ while Turgut et al (2022) correlated defect size with morbidity.⁴¹ Surgical complications, such as cerebrospinal fluid leakage leading to severe hyponatremia,⁴² and advanced reconstructive approaches remain critical.⁴³ Additionally, Cayli et al (2002) highlighted unusual presentations of occult spinal dysraphism.⁴⁴

Smoking and passive smoking showed no significant association in our study. Zaheri et al similarly found no link with active smoking but reported passive smoking as a risk factor. Meng et al concluded passive smoking posed greater risk than active smoking,⁴⁵ while Elahi et al reported strong associations with both.⁴⁶ Our sample’s low smoking rate might be the reason for the inconsistency. Consistent with previous studies, our findings reaffirm the significant protective association of maternal folic acid supplementation against neural tube defects, with risk reductions ranging from 60% to over 70% in well-conducted trials and population studies.^47,48 The 2023 US Preventive Services Task Force recommendation endorses daily supplementation of 0.4 to 0.8 mg folic acid for individuals planning or capable of pregnancy,⁴⁹ supported by strong evidence of NTD risk reduction across diverse populations without associated harms.⁵⁰ Despite this, compliance remains suboptimal in many regions, due to limited awareness, cultural barriers, and economic challenges a pattern consistent with broader social contagion and syndemic frameworks that highlight how social networks, trust, and structural determinants shape preventive health behaviors.⁵¹ Public health strategies integrating supplementation with food fortification and continuous education are essential to boost adherence and effectively prevent NTDs globally and in Middle Eastern countries like Iran. It is necessary to improve coverage and prevent NTDs by addressing these gaps through education, subsidization, fortification, and monitoring systems. Dean et al (2020) documented a significant reduction in NTD prevalence post‑fortification in the US.⁵² Similarly, Australian, New Zealand¹⁷ and South African²⁰ experiences provide robust evidence that mandatory fortification is achievable and effective. However, the European context remains fragmented; Turner (2018),¹⁹ and EBCOG have repeatedly called for mandatory fortification to address persisting inequities.¹⁸

Public Health and Economic Implications

Neural tube defects (NTDs) are one of the main reasons for the economic strain that is put on families and health insurance companies. In the case of developing countries like Iran, for example, the children who have NTDs will be needing medical care, surgeries, and rehabilitation throughout their lives. Furthermore, they will also need special education. All these requirements will mean that the families will be spending a great deal of money on direct medical expenses and will also be losing money indirectly due to staying at home with the child and losing productivity.⁵³

The low rate of folic acid supplementation in the present study – 20.6% for mothers of newborns with defects and 31.5% for controls implies that many NTD cases continue to be unreported which are actually preventable, adding unnecessary burden to healthcare delivery. The cost of folic acid fortification is very affordable (under $10 per woman annually), whereas the lifetime cost of caring for a child with spina bifida in a middle‑income country may exceed $100,000.⁵⁴ Thus, closing the supplementation gap we observed would have substantial economic benefits.

The low rate of folic acid supplementation in the present study 20.6% for mothers of newborns with defects and 31.5% for controls hints that a lot of NTD cases continue to be unreported which are actually preventable and thus add additional burden to the delivery of healthcare. In countries, like Iran, where economic sanctions are imposed, the escalation in the cost of supplements might also be a hindrance to how accessible they are, thus widening the gap between the rich and poor in terms of health and finances.⁵⁵

Research done in various places around the globe and in the region has affirmed that the fortification and supplementation programs, if implemented nationwide, would be among the most cost-effective ways to tackle the problem of NTDs.⁵⁶ Conducting modeling studies in Iran predicts that the complete allocation of folic acid to the entire population could avert as much as 60% of NTD cases, and thus the healthcare sector would save a lot and also the disability costs of the patients in the long run would be reduced.⁵⁷

Economic evidence therefore supports strengthening maternal supplementation programs, subsidizing folic acid access, and integrating educational campaigns into prenatal care services, particularly in regions affected by economic challenges or limited healthcare infrastructure.

The findings draw attention to the fact that prenatal education must be reinforced, consistent folic acid supplementation must be promoted, and early maternal screening and intervention programs must be improved. Strengthening comprehensive prenatal care will have a significant impact on the prevention of NTDs and the enhancement of the overall health of mothers and children.

Strengths and Limitations

The research gets a significant advantage due to its large sample size, and its three-to-one control-to-case ratio provides even more statistical power. On the other hand, the major limitations of the research include possible errors in reporting, underreporting of smoking and alcohol consumption, and the inability to evaluate ethnicity or other environmental factors.

Recommendations

Future research should aim to improve the accuracy and completeness of maternal health data within national reporting systems. Inclusion of paternal and environmental variables is recommended to provide a more comprehensive understanding of NTD risk factors. Furthermore, prospective cohort studies are needed to establish causal relationships between maternal factors and NTD occurrence.

Conclusion

Maternal folic acid supplementation was significantly associated with reduced risk of neural tube defects in newborns, emphasising its critical role in prenatal health. While other maternal and neonatal factors did not show significant associations in this study, these findings reinforce the importance of adequate folic acid intake during pregnancy as a key preventive measure. Given the observational case‑control design, these findings are limited to the variables examined and the population studied in Northeast Iran. Further prospective studies are needed to establish causal relationships.

Data Sharing Statement

The datasets used and/or analyzed during the current study are not publicly available due to ethical and confidentiality restrictions but are available from the corresponding author on reasonable request.

Consent for Publication

The patients filled the informed consent form for participation and publication.

Disclosure

The authors declare no conflicts of interest relevant to this manuscript.