Prenatal polycyclic aromatic hydrocarbon (PAH) exposure may contribute to neural tube defects (NTD), but combined effects and underlying mechanisms remain unclear. We conducted a matched case-control study (128 pairs) in six high-risk counties in Shanxi Province, China (2004-2016). At delivery or pregnancy termination, placenta samples were collected. Sixteen placental PAHs and 11 hydroxylated metabolites were quantified by GC-MS/MS as biomarkers of fetal intrauterine exposure, alongside 16 DNA adducts measured by UPLC-MS/MS. Complementary ICR mouse experiments with benzo[a]pyrene (BaP, 100 mg/kg) validated mechanistic findings. Statistical analyses used conditional logistic regression, WQS and BKMR (for parent PAHs and hydroxylated metabolites, respectively), followed by mediation analysis. Results showed that increased fresh vegetable intake and kitchen-living separation significantly reduced placental PAH concentrations. A dose-dependent increase in NTD risk was observed across tertiles of measured placental PAHs. The highest versus lowest tertile was associated with increased NTD risk for total PAHs (OR=5.04, 95%CI:1.96-12.96), phenanthrene (OR=4.96, 95%CI:2.05-11.97), and low-molecular-weight PAHs (OR=4.47, 95%CI:1.87-10.67) after adjustment for confounders. In subtype analyses, higher phenanthrene, low-molecular-weight PAH and total PAH related to anencephaly, whereas benzo[b]fluoranthene, indeno[1,2,3-cd]pyrene, and 9-hydroxyfluorene were associated with spina bifida. Mixture models confirmed significant joint effects of multiple PAHs. Mediation analyses showed 5-HmdC changes explained 10.7% (phenanthrene) and 12.4% (total PAHs) of NTD risk. In mice, BaP increased NTD incidence (5.7% vs. 1.8%), stillbirths (1.8% vs. 0%), and fetal resorptions (3.6% vs. 1.8%) versus controls. BaP-exposed groups showed reduced 5-HmdC in fetal tissues, consistent with human findings. This study provides evidence that individual and mixed PAH exposures are associated with an increased risk of NTD. DNA damage, particularly 5-HmdC alterations, may partially contribute to this association. These findings further support preventive strategies, including dietary and household environmental modifications.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants with reproductive and developmental toxicity, though it remains unclear whether prenatal PCB mixture exposure can lead to neural tube defects (NTDs). We evaluate the effects of real-world PCB mixtures on NTD risk and potential mechanisms by integrating epidemiology, in vivo toxicology, and multiomics. In our case-control study (n = 482), prenatal PCB exposure was estimated from placental concentrations of 13 congeners. Multivariable logistic regression showed that higher levels of PCB-52, PCB-118, and total ΣPCBs were associated with increased NTD risk. Three mixture models associated higher placental PCB mixture levels with elevated NTD risk and identified PCB-52 and PCB-118 as major contributors. Moreover, pregnant mice were dosed with a human-relevant PCB mixture during neurulation, inducing fetal NTDs dose-dependently. Multiomics indicated activation of pyroptosis; targeted assays supported activation of the NLRP3-caspase-1-GSDMD axis with increased IL-18, along with pyroptotic ultrastructural alterations. Overall, we found prenatal PCB mixture exposure was associated with higher NTD risk, and pyroptosis may represent a teratogenic mechanism. Indoor air pollution may be a prenatal exposure source; folic acid supplementation may attenuate PCB-associated NTD risk. This study provides a generalizable framework for real-world mixture risk assessment in birth defect research.

Congenital heart defects (CHDs) have complex causes, with prenatal exposure to environmental metals emerging as an increasing public health concern due to its potential impact on fetal heart development. Pregnant women are regularly exposed to mixtures of metals. However, few comprehensive studies have examined the joint effects of such exposure on the fetus during the critical window of heart development. To evaluate the association between maternal exposure to both toxic metals (mercury, lead, and arsenic) and essential trace elements (copper, iron, selenium, manganese, and nickel) at everyday or low levels during the periconceptional period and risk for fetal CHDs using individual and mixed approaches. Metal concentrations in maternal hair samples were measured in 170 mothers whose fetuses exhibited CHDs and 170 healthy controls. Logistic regression was used to assess the association between individual metals and CHD risk. The effects of metal mixtures were explored using quantile g-computation and Bayesian kernel machine regression (BKMR). Interactions among mercury, lead, and selenium were also evaluated using both methods. The logistic regression showed that mercury was positively associated with the risk of CHDs, while nickel was inversely associated it. Quantile g-computation identified mercury and nickel as the factors with the strongest positive and negative associations with CHD risk, respectively. BKMR suggested no joint effects of exposure to the eight-metal mixture. No significant interactions were observed. Maternal periconceptional hair concentrations of mercury and nickel are associated with CHD risk, with high mercury and low nickel having detrimental effects. These findings provide a reference for developing intervention strategies to reduce metal exposure and help prevent CHDs.

No epidemiological studies have systematically evaluated the associations between prenatal exposure to organophosphate esters and polychlorinated biphenyls and the risk of congenital heart disease (CHD) in offspring. Moreover, the potential modifying role of maternal B-vitamin status in persistent organic pollutants (POPs)-CHD associations has not been examined. We therefore investigated the cardiotoxic effects of prenatal POPs exposure and evaluated effect modification by maternal B-vitamin levels. A multicenter case-control study was conducted in China from 2016 to 2021, including 425 participants. Thirty POPs and seven plasma B vitamins were quantified using high-resolution mass spectrometry. Single-exposure associations were examined by logistic regression, while multipollutant effects were assessed through Bayesian kernel machine regression (BKMR) and Weighted Quantile Sum (WQS) models. Potential effect modification by B vitamins was systematically evaluated. Prenatal exposure to p-cresyl diphenyl phosphate was associated with an increased risk of CHD (odds ratio [OR] = 1.36, 95

Neural tube defects (NTDs) represent severe congenital malformations whose environmental determinants remain incompletely understood. Per- and polyfluoroalkyl substances (PFAS), persistent environmental contaminants capable of crossing the placental barrier during critical developmental windows, have been linked to various adverse birth outcomes, yet their association with NTDs remains unclear. We combined population-based, animal, and cellular approaches to establish associations and explore relevant mechanisms. In a case-control study of 271 NTD and 391 controls, placental PFAS concentrations were significantly elevated (41.29 vs. 22.36 ng/g, P < 0.001). High exposure was associated with markedly increased NTD risk (OR=14.13, 95 % CI: 4.39-45.49). Three mixture modeling approaches (BKMR, WQS, Qgcomp) consistently identified PFOS, PFDA, and PFHxS as dominant contributors, with weights of 35 %, 14 %, and 12 %, respectively. To validate causality, we exposed pregnant mice to this human-relevant PFAS mixture, which induced dose-dependent NTD phenotypes in the high-dose group (16.18 % vs. 2.67 %) accompanied by embryonic iron accumulation, oxidative stress, and dysregulation of ferroptosis and autophagy pathways, suggesting these pathways mediate PFAS developmental toxicity. Mechanistic validation in hPSCs confirmed that PFAS exposure reduced viability and suppressed GPX4 while increasing lipid peroxidation. Critically, pharmacological interventions revealed distinct rescue mechanisms. Ferrostatin-1 restored GPX4/SLC7A11 expression and reduced lipid peroxidation, while chloroquine blocked LC3B activation, prevented NCOA4-mediated ferritin degradation, and attenuated iron release, demonstrating that PFAS-induced ferroptosis operates through an autophagy-mediated mechanism wherein excessive autophagy promotes ferritinophagy and subsequent iron-driven cell death. This study identifies PFOS, PFDA, and PFHxS as potential developmental toxicants and highlight ferroptosis and autophagy as potential therapeutic targets, suggesting the need for further evaluation of PFAS regulation to protect prenatal health.

Neural tube defects (NTDs) exhibit a multifaceted etiology. Limited research has assessed the association between exposure to metallic and non-metallic elements and the incidence of NTDs at the elementomic level. Our study included 40 women with NTD-affected pregnancies and 119 controls in northern China's Shanxi province. Inductively coupled plasma mass spectrometry was employed to quantify 45 elements in umbilical cord serum samples collected from all participants. Thirteen elements were excluded from subsequent analysis due to a detection rate below 60%. We employed three machine learning selection models, Boruta, Least Absolute Shrinkage and Selection Operator, and Extreme Gradient Boosting, to jointly identify the key exposure elements as zinc (Zn), sodium (Na), molybdenum (Mo), cerium (Ce), barium (Ba) and titanium (Ti). Bayesian kernel machine regression assessed both single and combined effects of these elements on NTD risk, with individual element effects confirmed via logistic regression. Our results showed a non-significant trend toward higher NTD risk with rising concentrations of the mixture of these six elements. When Ce concentration increased from the 25th to the 75th percentile, a statistically significant elevated risk of NTDs was observed, after adjusting for the concentrations of the other five metals at the 25th, 50th and 75th percentiles. Logistic regression analysis further identified a significant association between Ce exposure and NTD risk, with an odds ratio (95% CI) of 1.96 (1.10-3.49). No interactions among the elements were identified. In conclusion, our results indicate that higher Ce concentrations are significantly associated with NTD risk.

Neural tube defects (NTDs) are one of the most common birth defects. Phthalates and bisphenols are ubiquitous endocrine-disrupting chemicals (EDCs), which can disrupt the endocrine system by altering hormone levels. However, there is no study on the associations between co-exposure to phthalate metabolites and bisphenols and risk of fetal NTDs. A case–control study was conducted among 286 participants in China from 2005 to 2016. Liquid chromatography–tandem mass spectrometry was used to detect the levels of 25 EDCs in placenta. Logistic regression and multipollutant models were used to evaluate the single and co-exposure effects of EDCs on NTD risk, with maternal age, gestational weeks at placenta collection, pre-pregnancy body mass index, history of birth defects, folate supplementation during pregnancy, and smoking status as covariates. This study also assessed whether gestational exposure to mono-(2-ethylhexyl) phthalate (MEHP) in pregnant mice induces NTDs in offspring. Individual exposure to high levels of MEHP, mono-octyl phthalate, and mono-cyclohexyl phthalate was associated with increased NTD risk [odds ratio (OR) = 2.89, 95

Organochlorine pesticides (OCPs) are persistent environmental contaminants linked to adverse health outcomes, including neural tube defects, though their mechanisms remain unclear. This study investigated the effects of OCP exposure on homocysteine metabolism by depleting folate through a cohort study (103 pre-folic acid (FA) supplementation and 83 post-FA supplementation women) and animal experiments. In pre-FA supplementation women, concentrations of most OCPs were significantly associated with decreased plasma folate and elevated homocysteine. Bayesian Kernel Machine Regression (BKMR) confirmed an inverse combined effect of OCP mixtures on folate and a positive effect on homocysteine. Rat models confirmed the findings in humans, demonstrating that DDT exposure decreased folate levels and increased homocysteine levels. Conversely, in post-FA supplementation women, these associations disappeared. Further analysis revealed that FA supplementation exhibited significant inverse associations with several OCPs, including α-Hexachlorocyclohexane (α-HCH), β-Hexachlorocyclohexane (β-HCH), Endosulfan-β (β-ES), Heptachlor epoxide (HEPCE), isodrin, dieldrin, o,p'-Dichlorodiphenyltrichloroethane (o,p-DDT), o,p'-Dichlorodiphenyldichloroethylene (o,p-DDE), p,p'-Dichlorodiphenyldichloroethylene (p,p-DDE), and o,p'-Dichlorodiphenyldichloroethane (o,p-DDD). Mediation analysis indicated that folate partially mediated the effect of OCPs on homocysteine (mediation proportion: 13-27 %). While FA supplementation effectively accelerated the metabolism of OCPs in rat. In conclusion, OCP exposure elevates homocysteine levels by depleting folate. FA supplementation counteracts this effect by reducing OCP accumulation in vivo. These findings underscore the importance of nutritional status-particularly folate levels-in assessing OCP-related health risks. Future research should explore the molecular mechanisms by which OCPs disrupt folate metabolism and how nutritional interventions can mitigate associated health risks.

Despite widespread human exposure to tin, its health effects remain poorly understood. This study examined the role of tin in neural tube defects (NTDs) through a case-control study and animal experiments. Tin levels in maternal serum and placentas were analyzed in 200 NTD cases and 400 controls to explore potential associations. Elevated tin concentrations in maternal serum were associated with an increased risk of NTDs, with an odds ratio of 2.31 (95% CI, 1.13-4.75), with similar associations found for placental tin and tributyltin (TBT) exposure. In animal experiments, pregnant mice exposed to 10-40 mg/kg TBT exhibited a 25.5%-27.6% incidence of fetal NTDs. Maternal TBT exposure increased oxidative stress and apoptosis in embryonic neural tissues. Antibody microarray analysis prioritized MAPK signaling as the dominant perturbed pathway. Subsequent western blot and RT-qPCR analysis convergently validated TBT-induced MAPK hyperactivation. Vitamin E supplementation had antagonistic effects, reducing these harmful outcomes. These findings suggest that prenatal tin exposure is a significant risk factor for NTDs. The teratogenic effect of TBT appears to be mediated by enhanced oxidative stress, activation of MAPK signaling, and apoptosis in the developing neural tube, processes that can be mitigated by Vitamin E supplementation. Thus, tin exposure during pregnancy is associated with an increased risk of fetal NTDs, and animal models demonstrate that TBT can induce these defects through specific biological pathways. This research highlights the need for further investigation into tin exposure and its potential health impacts on fetal development.

Objective Exposure to polycyclic aromatic hydrocarbons（PAHs） or metal（loid）s individually has been associated with neural tube defects（NTDs）.However,the impacts of PAH and metal（loid） co-exposure and potential interaction effects on NTD risk remain unclear.We conducted a case-control study in China among population with a high prevalence of NTDs to investigate the combined effects of PAH and metal（loid） exposures on the risk of NTD.Methods Cases included 80 women who gave birth to offspring with NTDs,whereas controls were 50women who delivered infants with no congenital malformations.We analyzed the levels of placental PAHs using gas chromatography and mass spectrometry,PAH-DNA adducts with 32 P-post-labeling method,and metal（loid）s with an inductively coupled plasma mass spectrometer.Unconditional logistic regression was employed to estimate the associations between individual exposures and NTDs.Least absolute shrinkage and selection operator（LASSO） penalized regression models were used to select a subset of exposures,while additive interaction models were used to identify interaction effects.Results In the single-exposure models,we found that eight PAHs,PAH-DNA adducts,and 28 metal（loid）s were associated with NTDs.Pyrene,selenium,molybdenum,cadmium,uranium,and rubidium were selected through LASSO regression and were statistically associated with NTDs in the multiple-exposure models.Women with high levels of pyrene and molybdenum or pyrene and selenium exhibited significantly increased risk of having offspring with NTDs,indicating that these combinations may have synergistic effects on the risk of NTDs.Conclusion Our findings suggest that individual PAHs and metal（loid）s,as well as their interactions,may be associated with the risk of NTDs,which warrants further investigation.

Cleft lip and/or palate (CL/P) is a congenital facial defect. Polychlorinated biphenyls (PCBs) are recognized as both persistent organic pollutants and endocrine-disrupting chemicals, which can cross the placental barrier, leading to an increased risk of adverse pregnancy outcomes. However, there is no study to evaluate the association of prenatal exposure to PCBs with CL/P risk in offspring. Therefore, we investigated a multicenter case-control study, comprising 149 cases and 292 controls, to explore the association of prenatal exposure to single PCB and a PCB mixture with CL/P risk in offspring. Thirteen PCBs were measured using a gas chromatograph coupled with a triple-quad mass spectrometer. The single exposure analysis indicated a significant statistical association of placental 2,2',5,5'-tetrachlorobiphenyl and 3,4,4',5-tetrachlorobiphenyl levels with CL/P risk (OR = 1.23, 95 % CI: 1.03, 1.48; OR = 1.20, 95 % CI: 1.03, 1.41, respectively). Three multipollutant models demonstrated a positive association between prenatal exposure to the PCB mixture and CL/P risk, with 2,2',5,5'-tetrachlorobiphenyl and 3,4,4',5-tetrachlorobiphenyl being the primary contributors to this risk. This study pioneers the investigation of the association between prenatal exposure to PCBs and the risk for CL/P, which not only deepens our understanding of the toxic effects of PCBs but also serves as a stark reminder of the urgent need to intensify efforts to remediate contaminated environments.

Nonsyndromic cleft lip and/or palate (NSCL/P) is a common congenital malformation with a complex etiology involving many environmental factors. Organophosphate flame retardants (OPFRs) are a class of emerging contaminants that are associated with adverse reproductive and developmental outcomes, but their potential role in NSCL/P risk remains unexplored. A total of 134 NSCL/P cases and 292 controls were recruited in China between 2005 and 2021. The concentrations of six target OPFRs in placentas were quantified using gas chromatography–tandem mass spectrometry. Logistic regression, Bayesian kernel machine regression (BKMR), and weighted quantile sum (WQS) regression were employed to examine the relationships between OPFR exposure and NSCL/P risk. In the logistic regression model, high TBP level was associated with an increased risk for NSCL/P, while low TCPP level was linked to elevated risk for NSCL/P. The BKMR model revealed a significant joint effect of mixed exposure to OPFRs on the increased risk for NSCL/P, with TBP associated with a significant increase in NSCL/P risk. In the WQS model, the WQS index was positively correlated with the risk for NSCL/P [odds ratio (OR) = 1.15, 95

Background: As emerging environmental contaminants, antibiotics pose potential threats to human health, in particular to pregnant women and infants. However, the potential harm of inadvertent antibiotic exposure (IAE) is often disregarded in light of the focus on intentional antibiotic use during pregnancy. Currently, little is known about the effects of IAE during pregnancy on fetal neural tube development. Methods: In this case-control study, we used questionnaire data from 855 subjects to investigate the effects of intentional antibiotic use in early pregnancy on neural tube defects (NTDs). Then we tested for placental antibiotics in mothers who had not intentionally used antibiotics, and the compounds were detected in 379 subjects; these were considered IAE cases. We assessed the association between IAE during pregnancy and fetal NTDs using both multivariable logistic and multi-pollutant exposure models. We also analyzed the correlation between maternal dietary habits and placental antibiotics to explore possible sources of IAE. Results: Only 50 of 855 participants (5.8%) intentionally used antibiotics and such use showed no significant association with NTD risk (odds ratio [OR] = 1.92, confidence interval [95%CI] = [0.66, 5.59]). However, 14 of 15 placental antibiotics were detected in 378 of 379 subjects (99.7%) and multivariable logistic analysis indicated that high levels of placental macrolides were significantly associated with increased NTD risk (4.42 [2.01 -10.45]). Multi-pollutant exposure analysis suggested an increase in NTD risk with an increase in exposure to a mixture of placental antibiotics, among which macrolides were the most important contributor. In addition, the level of placental macrolides was positively correlated with the intake frequency of milk. Finally, mothers who drank river, well, or pond water had higher levels of placental macrolides than those who drank only tap water. Conclusions: Intentional antibiotic use during early pregnancy may not be associated with NTDs, while IAE during pregnancy is associated with higher NTD risk in offspring. Macrolides are crucial risk factors. Milk, and river, well, or pond water may be important sources of IAE.

Background: Congenital heart defects (CHDs) have a complex etiology, and environmental factors play an important role in their occurrence. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous chemicals, and some have teratogenic potential. However, few studies have examined PAHs exposure and CHD risk. We investigated the association between PAHs in maternal scalp hair and CHD risk. Methods: A case-control study involving 170 severe CHD cases and 170 healthy controls was conducted, and the concentrations of 11 PAHs in maternal hair grown during the periconceptional period were quantified. A generalized linear mixed model (GLMM) was used to determine the effects of each PAHs on the risk for CHDs. Weighted quantile sum (WQS) regression and Bayesian kernel machine regression (BKMR) were used to assess the overall effects of the 11-PAHs mixture on the risk for CHDs. Results: The median concentration of chrysene (CHR) was higher in CHD cases (9.75 ng/g) than in controls (6.50 ng/g). In GLMM, higher levels of CHR were associated with a 4.88-fold greater risk for CHDs (95 % confidence interval [CI]: 2.69-8.89). In WQS regression, higher levels of PAHs mixture were associated with a 2.03-fold greater CHD risk (95 % CI: 1.75-2.31), and CHR had the highest weighting (weighted 0.9346). In BKMR, CHD risks increased steadily with the levels of the PAHs mixture. CHR showed a toxic effect when the other PAHs were fixed at their 25(th), 50(th), or 75(th) percentile. No interactions among PAHs were found. Conclusions: When examined individually, a high concentration of CHR in periconceptional maternal hair was associated with an increased risk for CHDs. When considering the 11 PAHs together, higher levels of the PAHs mixture were associated with increased odds of CHD occurrence.

Human exposure to chromium (Cr) is common but little is known about its adverse effects on pregnancy outcomes. This study aimed to explore the association between Cr exposure and the risk of neural tube defects (NTDs) and the underlying mechanisms of Cr-induced NTDs. 593 controls and 408 NTD cases with placentas were included in this study. Chromium trichloride (Cr(III)) and potassium dichromate(Cr(VI)) were intragastrically administered to pregnant mice and the number of NTDs was recorded. The odds ratio for total NTDs in the highest exposure group in placenta was 4.18(95% confidence interval (CI), 1.97-8.84). The incidence of fetal NTDs in mice administered with Cr(III) showed a dose-response relationship. Cr(VI) didn’t show teratogenicity of NTDs whereas increased the stillbirth rate. Prenatal exposure to Cr(III) increased levels of oxidative stress and apoptosis in fetal mice. RNA-sequencing results indicated significant enrichment of the MAPK pathway. RT-qPCR and western blot analysis revealed that Cr(III) induced increased expression of p-JNK, p-P38, and Casp3. Toxicological effects can be partly antagonized by antioxidant supplementation. High chromium exposure was associated with increased human NTD risks. Excessive Cr(III) exposure can induce NTDs in fetal mice by increasing apoptosis through upgrading oxidative stress and then activating JNK/P38 MAPK signaling pathway.

Neural tube defects (NTDs) are a group of severe congenital malformations of the central nervous system, for which environmental causes are not fully known. In a human case–control study, we compared lead (Pb) levels in umbilical cord blood serum from 40 NTD cases and 119 controls. In a toxicology study, we treated Institute of Cancer Research (ICR) mice with different dosage of Pb acetate orally from gestational day (GD) 6.5 through 9.5. At GD10.5, mouse embryos were harvested and examined. RNA-sequencing was conducted on the embryos to detect mRNA profiles. siRNAs were used to pull down target genes in human embryonic stem cells to examine the effects of the aberrant gene expression on neural cells. A higher Pb concentration in cord serum was associated with the odds of NTDs [odds ratio (OR): 4.66 (1.69–15.24)]. Lead acetate induced NTDs in 10.6

Congenital heart disease (CHD) is the most prevalent congenital malformation worldwide, and the association between per- and polyfluoroalkyl substances (PFASs) exposure and CHD in population has only received limited study. Therefore, we conducted a multicenter case -control study to explore the associations between prenatal exposure to individual PFASs, and also a PFAS mixture, and CHD risk, including 185 CHDs and 247 controls in China from 2016 to 2021. Thirteen PFASs in maternal plasma were quantified using liquid chromatographytandem mass spectrometry. Logistic regression and two multipollutant models (Bayesian kernel machine regression [BKMR] and quantile g -computation [qgcomp]) were used to assess the potential associations between any individual PFAS, and also a PFAS mixture, and CHD risk. After adjusting for potential confounders, logistic regression indicated significant associations between elevated levels of perfluorononanoic acid (odds ratio [OR] = 1.30, 95% confidence intervals [CI]: 1.07 -1.58), perfluorodecanoic acid (OR =2.07, 95%CI: 1.32 -3.26), and perfluoroundecanoic acid (OR =2.86, 95%CI:1.45 -5.65) and CHD risk. The BKMR model and qgcomp approach identified that a significant positive association between the PFAS mixture and risk for CHD. These findings provide essential evidence that there is indeed a health crisis associated with PFASs and that it is linked to CHD.

Our previous study found a positive relationship between fetal nickel exposure and the risk of OFCs. The teratogenic mechanism of nickel is not clear. In this study, we aim to examine the mediating effect of DNA methylation on the association of nickel(Ni) exposure with NSOFC in fetuses. 10 cases and 10 controls was used for screening target gene by Illumina Infinium Methylation EPIC(850k) BeadChip. 36 cases and 78 controls was conducted to determine DNA methylation level of selected gene in umbilical cord blood by Mass spectrometry assay. Mediation analysis was used to evaluate the potential mediating effect of selected gene methylation on the relation between concentrations of Ni and the risk for NSOFC. In the discovery stage, ZEB1 gene was identified to be hypermethylated in both nickel exposure and NSOFC group for validation. In the verification stage, the overall average methylation level of ZEB1 was significant higher in NSOFC cases(median=8.70, interquartile range(IQR): 5.75-11.53) as compared to controls (median=5.35, IQR: 4.30-7.78). The risk for NSOFC was increased by 1.43-fold with hypermethylation of ZEB1. Significant correlation was observed between concentrations of Ni in umbilical cord and methylation level of ZEB1. The hypermethylation of ZEB1 had a mediating effect by 20.47% of total effect of Ni on NSOFC risk. Hypermethylation of ZEB1 is associated with the risk for NSOFC and may partially explain the association between Ni exposure and NSOFC risk. Our findings provide new insights into the epigenetic mechanisms underlying NSOFC and suggesting potential targets for future therapeutic interventions.

Maternal exposure to various metallic and non-metallic elements has been linked to the occurrence of orofacial clefts (OFCs), yet there remains a dearth of comprehensive research on the potential ramifications of simultaneous exposure to multiple elements. In this study, we investigated the individual and combined effects of element exposure on OFCs in a cohort of 168 pregnant women (49 cases and 119 controls) in the Shanxi province of northern China from 2010 to 2015. Cord serum samples were obtained from all participants to analyze the levels of 32 elements using inductively coupled plasma-mass spectrometry. The study examined the independent correlation between element concentrations and OFCs using two machine screening models, Boruta and Least Absolute Shrinkage and Selection Operator. Bayesian kernel machine regression (BKMR) was utilized to determine the combined effects of key exposure elements on OFCs and to clarify the interaction between exposed elements through the generalized additive model (GAM). The screening models identified lead (Pb), tin (Sn), iron (Fe), and cesium (Cs) as the most significant risk factors for OFC development in offspring. In the BKMR model, the probability of OFCs increased with higher overall levels of these risk elements, with Pb emerging as the primary contributor to the combined effect of the mixture. The findings of the GAM indicated that the combined exposure to Pb and Sn had a synergistic effect on the risk of developing OFCs. Analysis of elemental exposure in umbilical cord serum suggested that Pb exposure may have detrimental effects on OFC development in offspring, which may be further intensified by a synergistic interaction between Sn and Pb in the occurrence of OFCs.

Phthalate metabolites and bisphenols can cause adverse pregnancy outcomes. However, there is no study to evaluate the associations of prenatal exposure to phthalate metabolites and bisphenols with non-syndromic cleft lip and/or palate (NSCL/P) risk in offspring. A population-based case-control study was conducted in a multicenter setting from 2005 to 2021, enrolling 448 pregnant women. Seven phthalate metabolites and six bisphenols were quantified in placenta using liquid chromatography-tandem mass spectrometry. In the logistic regression analysis, high levels of mono-ethyl phthalate, mono-cyclohexyl phthalate, mono-octyl phthalate, bisphenol A, bisphenol AF, bisphenol AP, and fluorene-9-bisphenol were associated with increased NSCL/P risk with odds ratios (95% confidence intervals) of 1.86(1.07,3.25), 6.56(3.47,12.39), 8.49(4.44,16.24), 8.34(4.32,16.08), 3.19(1.81,5.62), 2.78(1.59,4.86), and 5.16(2.82,9.44). The Bayesian kernel machine regression model revealed that co-exposure to phthalate metabolites and bisphenols was associated with increased NSCL/P risk. Similarly, quantile-based g-computation analysis indicated that each quantile increase in mixture concentration was positively related to higher risk for NSCL/P [odds ratio (95% confidence interval) = 2.98(1.97,4.51)]. This study provides novel evidence that prenatal single and co-exposure to phthalate metabolites and bisphenols were associated with increased NSCL/P risk, suggesting that exposure to phthalate metabolites and bisphenols during pregnancy should be minimized to reduce the incidence of NSCL/P in offspring.