Associations between maternal stressful life events experiences and risk of neural tube defects in offspring: a systematic review and meta-analysis

Abstract Objectives Multiple factors associated with neural tube defects (NTDs) risk have been identified, yet there is little evidence on the possible effects of maternal stressful life events. In this study, we aimed to investigate the association between stressful life events during the periconceptional period and risk of NTDs in offspring. Methods Relevant literature was searched in PubMed, Springer Link, ScienceDirect, and Cochrane Library up to July 2023. The pooled odds ratio (OR) and 95% confidence interval (CI) of NTDs risk with maternal stressful life events were estimated using a random effects model. Publication bias was assessed using Egger’s regression asymmetry test and Begg’s rank correlation test with Begg’s funnel plot. Results Analysis results showed that mothers who experienced stressful life events during the periconceptional period were at greater risk of having NTDs offspring (OR: 1.37, 95% CI: 1.08–1.73) than those who did not. In subgroup analysis, the pooled OR was 1.37 (1.13–1.67) and 1.73 (0.36–8.32) for with and without adjusting for folic acid supplementation in each included study, while was 1.37 (1.13–1.67) and 1.64 (0.39–6.88) for exposure time of three months preconception until three months post conception and one year preconception until three months post conception, respectively. Conclusions This study suggests that maternal stressful life events during the periconceptional period are significantly associated with higher NTDs risk in offspring. Tailored approaches for evaluating the risk and policy of NTDs among women of childbearing age should emphasize individual stressful experiences before and during early pregnancy.


Introduction
Neural tube defects (NTDs) are major structural birth defects caused by defective closure of the neural tube, leading to infant death and lifelong disabilities in children [1].Worldwide, the average prevalence of NTDs was estimated to be 2.0 per 1000 births, accounting for about 0.21-0.32 million affected pregnancies annually [2].NTDs are highly prevalent globally, but their exact causes remain unclear.In recent decades, maternal folate deficiency before conception and during early pregnancy has been suggested as the most important preventable risk factor for NTDs [3].However, previous findings have shown that appropriate consumption of folic acid could only prevent 50-70% of NTDs [4].Regarding the effects of folic acid fortification of staple foods, a recent study estimated that only approximately 22% of all folic acid-preventable NTDs cases globally were prevented [5].Similarly, the Shanxi Province of northern China ranks among the highest prevalence of NTDs worldwide.Although there has been a substantial decrease since the national folic acid supplementation program in 2009, the overall prevalence of NTDs in this area has remained high over the recent decade [6,7].In light of these findings, to better improve the current situation of NTDs prevention, it is important to understand more related factors contributing to the occurrence of NTDs.
NTDs have been suggested to be affected by multiple factors, including both genetic and environmental factors [8].Although the exact etiology of NTDs remains elusive, epidemiological studies have proposed that various maternal lifestyles and environmental exposures may be involved, including smoking, obesity, poor sleep, zinc deficiency, passive smoking, and air pollution [9][10][11][12][13][14].In addition to these traditional risk factors, several stress-related psychological factors, such as emotional stress and stressful life events, have also been implicated in the risk of adverse birth outcomes, including NTDs [15][16][17][18].Experimental studies have also indicated that psychological stress can induce congenital malformations through elevated corticosteroid [19,20].However, overall, available epidemiological evidence on the effect of psychological stress linked to NTDs risk is not yet well-established, and specifically, stressful life events are no exception [21].Accordingly, more highquality evidence is required to confirm this association.In this systematic review and meta-analysis, we calculated the pooled effects to investigate the association between maternal stressful life events during the periconceptional period and the risk of NTDs in offspring, aiming at providing support for more initiation of NTDs prevention measures.

Search strategy
This study followed the Meta-analysis of Observational Studies in Epidemiology (MOOSE) guidelines [22].Relevant literature has focused on the association between maternal stressful life events and NTDs risk in offspring using four databases (PubMed, Springer Link, ScienceDirect, and Cochrane Library) up to July 2023.No language limit was set in the literature search, and only articles published in English were included in this study.The main search terms of exposure factor included "stress", "life events", "stressful life events, "major life events", "psychological stress", "risk factor", "determinant", and "predictor" were screened with OR.The study outcome was searched by keywords for "neural tube defects," "NTDs," "anencephaly," "spina bifida," "encephalocele," "congenital malformations," "birth defects," and combining these with OR.Then, the exposure and outcome search items were linked using AND.The reference lists of the retrieved studies were also screened.

Eligibility criteria
The selected studies included in this analysis met the following eligibility criteria: (1) examining the association between maternal stressful life events and NTDs (any of anencephaly, spina bifida, and encephalocele) risk in offspring; (2) with a case-control or cohort study design; (3) reporting the odds ratio (OR) or relative risk (RR) with 95% confidence interval (CI) for with vs. without experiencing stressful life events.

Data extraction
Two authors independently assessed the eligibility of studies and extracted information from each eligible study.The information included (1) name of the first author, (2) year of publication, (3) participants and sample size, (4) data source and location, (5) study design, (6) definition of measures, and (7) adjusted confounders.The quality of each study was assessed using the 9-star Newcastle-Ottawa Scale [23], which is a validated scale for observational and nonrandomized studies in meta-analyses.This scale assessed the selection of the study sample (for case-control or cohort studies, maximum of four points), comparability of the sample groups (maximum of two points), and ascertainment of outcome (for cohort studies, maximum of three points).

Statistical analysis
Heterogeneity between studies was assessed using the Q-test and I 2 statistic [24].For the Q-test, a p value <.05 was considered statistically significant.Low, moderate, and high degrees of heterogeneity corresponded to I 2 values of 25, 50, and 75%, respectively.If there was significant heterogeneity, a randomeffects model was used to assign the weight of each study according to the DerSimonian-Laird method [25].If there was no evidence of heterogeneity, we used a fixed-effects model with effect estimates given equal weight to the inverse variance of the study.To test the robustness of the present meta-analysis results, a sensitivity analysis was performed by excluding outliers.Publication bias was assessed using both Egger's regression asymmetry test and Begg's rank correlation test (p value <.05, considered statistically significant) with Begg's funnel plot.To explore the possible sources of heterogeneity, we also carried out a subgroup analysis based on (1) whether the effect of maternal stressful life events on NTDs was adjusted for the factor of folic acid supplementation in each included study; and (2) the time window of assessing the maternal stressful life events (three months preconception until three months post conception, one year preconception until three months post conception).STATA Version 11 software (Stata Corp LP, College Station, TX) was used for all the statistical analyses.

Literature screening and characteristics
A flowchart of the literature selection process is presented in Figure 1.After retrieving relevant databases, 2243 potentially human articles were identified.Based on the titles and abstracts, 1656 articles were excluded.Among the remaining 587 articles retrieved for eligibility, 566 articles were further excluded because they were duplicates, did not include the exposure factor of stressful life events, were review and RCT studies, and were not written in English.Of the remaining 21 articles, 11 articles that did not include the outcome of NTDs (any of anencephaly, spina bifida, and encephalocele), three articles that did not provide primary or adjusted data, and one article with an inappropriate reference group were also excluded.Finally, six articles including one cohort and five case-control studies [26][27][28][29][30][31] were included in this meta-analysis.Detailed information on the included studies is shown in Table 1.All studies reported the final estimates with adjustments for specific confounders.The quality scores of the studies ranged from 5 to 7 stars, according to the Newcastle-Ottawa Scale (Supplementary Tables 1 and 2).
Meta-analysis of the association between maternal stressful life events experiences and the risk of NTDs in offspring.
A total of 3056 NTDs cases were included in this meta-analysis.A formal test for heterogeneity yielded a significant result (p ¼ .011,I 2 ¼ 66.1%); therefore, a random-effects model was used to calculate the OR of NTDs risk with maternal stressful life events.The pooled analysis showed that mothers with stressful life event experiences during the periconceptional period were at greater risk of having offspring with NTDs (OR: 1.37, 95% CI: 1.08-1.73)than those without (Figure 2).After excluding the outliers, the sensitivity analysis results showed that the OR was 1.40 (1.23-1.59)without any between-study heterogeneity (p ¼ .643,I 2 ¼ 0.0%).To explore the source of heterogeneity between studies, we performed a subgroup analysis.Specifically, based on whether the effect of maternal stressful life events on NTDs risk was adjusted for folic acid supplementation, the pooled OR was 1.37 (1.13-1.67)and 1.73 (0.36-8.32) with and without adjusting for folic acid supplementation in each included study, respectively (Figure 3); based on the time window of assessing maternal stressful life events, the pooled OR was 1.37 (1.13-1.67)and 1.64 (0.39-6.88) for the periconceptional period of three months preconception until three months post conception and one year preconception unit three months post conception, respectively (Figure 4).

Publication bias
Regarding the assessment of publication bias, neither Egger's regression asymmetry test (p ¼ .399)nor Begg's rank correlation test (p ¼ .452)yielded a statistically significant result.In addition, Begg's funnel plot showed no evidence of publication bias (Figure 5).

Discussion
The association between maternal exposure to stressful life events and the risk of adverse birth outcomes has long been debated [15,18].Given that NTDs are common severe birth defects, it is essential to extend our knowledge to relationships with the occurrence of NTDs.Using a systematic review and meta-analysis method, this study attempted to determine the pooled effect of maternal stressful life events on the risk of NTDs in the offspring.Overall, findings from the present analysis suggested that mothers who experienced    stressful life events periconceptionally were at greater risk of having NTDs offspring (OR: 1.37, 95% CI: 1.08-1.73)compared to those who never experienced them after adjusting for potential confounders.Considering the differences in various terms of NTDs types, data collection periods, stressful life events items assessed, and population selections across studies, we conducted a sensitivity analysis; however, the analysis yielded relatively consistent results.Since research on maternal stressful life events and NTDs risk in offspring has been sparse, findings from the current study are still comparable to the existing literature.In a previous meta-analysis exploring the maternal, paternal, and neonatal risk factors for NTDs,  the authors reported that stressful life events were significantly associated with NTDs risk, and the effect estimate (OR: 1.61, 95% CI: 1.24-2.08)[21] was similar to our result, while the literature search strategies and eligibility criteria differed.In addition, the authors only reported the overall effect of maternal stressful life events and did not conduct any further subgroup analyses with significant heterogeneity.
The mechanisms by which maternal stressful life events may result in NTDs remain inconclusive.Some findings have proposed the hypothesis that psychological stress could induce NTDs by elevating cortisone hormone and homocysteine level [19,20,32].Other mechanisms, such as cigarette smoking and reduced nutrient intake, are involved in stress-related lifestyle changes [28].Further work is needed to elucidate other possible mechanisms in the future.
Meanwhile, to explore the possible sources of heterogeneity between studies and the mediating effects on the association between maternal stressful life events and NTDs risk imposed by potential factors, we also carried out a subgroup analysis based on whether the major NTD-related factor of folic acid supplementation was adjusted for in each study and the different time windows of exposure assessment.The overall significant association was confirmed with lower heterogeneity when adjusting for folic acid supplementation and was limited to a relatively narrower time window of exposure assessment (three months preconception until three months post conception).Based on these findings, we speculated that the effect of maternal stressful life events on NTDs risk might be mediated by folic acid supplementation and the timing of exposure, which should be considered and evaluated in further studies.In addition, although the current human epidemiological studies regarding the time window of psychological stress are inadequate, some research has found interesting associations between different pregnancy periods of stress exposure and adverse birth outcomes [33,34].Given that neural tube formation is completed very early in pregnancy, when evaluating the effect of maternal stressful life events on NTDs risk, more detailed and narrower time windows of the exposure assessment are warranted.
Despite these findings, this meta-analysis study had some limitations.First, our analysis only included six studies published in English.Although there was no evidence of publication bias, according to prior knowledge, statistical tests had limited power for detecting bias, especially for small numbers of studies [35].Second, there was still a moderate level of heterogeneity between studies in the subgroup analysis after adjusting for the major NTD-related factor of folic acid, which suggested that unknown confounders might exist.Third, due to the limited number of studies and NTDs cases included in the analysis, the observed association between maternal stressful life events during the periconceptional period and the risk of NTDs should be interpreted with caution.Fourth, recall bias was inevitable in the measurement of maternal stressful life events, as most of the included studies had a case-control design.Additionally, some studies evaluated only one or three items of stressful life events, such as close relative death, separated or divorced, and losing a job, which might limit the assessment of the association.
In conclusion, the findings from this meta-analysis suggested that maternal stressful life events during the periconceptional period were significantly associated with a higher NTDs risk in offspring.In particular, our analysis results showed that folic acid supplementation and the time window of exposure to stressful life events played an important role in this association.Based on these findings, we recommend that the development of relevant approaches to evaluate the risk of NTDs and strategies preventing the occurrence of NTDs among women of childbearing age should seriously consider the individual experiences of stressful life events, especially at the critical time window.

Figure 1 .
Figure 1.Selection of studies for inclusion in meta-analysis through searching PubMed, Springer Link, ScienceDirect, and Cochrane Library up to July 2022.

Figure 2 .
Figure 2. Effect of maternal stressful life events during periconceptional period on risk of neural tube defects in offspring.OR: odds ratio; CI: confidence interval.Diamond represents the pooled effects of the overall group of included studies.

Figure 3 .
Figure3.Effect of maternal stressful life events during periconceptional period on risk of neural tube defects in offspring stratified by whether folic acid supplementation was adjusted for in each included study (1: yes, 0: no).OR: odds ratio, CI: confidence interval.Diamonds represent the pooled effects of the overall/each group of included studies.

Figure 4 .
Figure 4. Effect of maternal stressful life events during periconceptional period on risk of neural tube defects in offspring stratified by time window of exposure (1: three months preconception until three months post conception, 0: one year preconception until three months post conception).OR: odds ratio, CI: confidence interval.Diamonds represent the pooled effects of the overall/ each group of included studies.

Table 1 .
Information of the studies included in the meta-analysis.