Maternal periconceptional folic acid supplementation reduced risks of non-syndromic oral clefts in offspring

Maternal periconceptional folic acid supplementation (FAS) has been documented to be associated with decreased risk of nonsyndromic oral clefts (NsOC). However, the results remain inconclusive. In this population-based case–control study of 807 singletons affected by NsOC and 8070 healthy neonates who were born between October 2010 and September 2015 in Chengdu, China, we examined the association of maternal FAS with the risk of nonsyndromic cleft lip with or without cleft palate (NsCL/P), and cleft palate (NsCP). Unconditional logistic regression analysis was used to estimate the crude and adjusted odds ratios (ORs) and 95% confidential intervals (CI). Significant associations were found between maternal periconceptional FAS and decreased risk of NsCL/P (aOR = 0.41, 95% CI 0.33–0.51). This protective effect was also detected for NsCL (aOR = 0.42, 95% CI 0.30–0.58) and NsCLP (aOR = 0.41, 95% CI 0.31–0.54). Both maternal FAS started before and after the last menstrual period (LMP) were inversely associated with NsCL/P (before LMP, aOR = 0.43, 95% CI 0.33–0.56; after LMP, aOR = 0.41, 95% CI 0.33–0.51). The association between NsCP and maternal FAS initiating before LMP was also found (aOR = 0.52, 95% CI 0.30–0.90). The findings suggest that maternal periconceptional FAS can reduce the risk of each subtype of NsCL/P in offspring, while the potential effect on NsCP needs further investigations.

. General characteristics of the study subjects. NsCL/P, nonsyndromic cleft lip with or without cleft palate cases; NsCL, nonsyndromic cleft lip cases; NsCLP, nonsyndromic cleft lip with cleft palate cases; NsCP, nonsyndromic cleft palate cases. # The infant sex records with one missing value on NsCLPs. a The environmental exposure in the first trimester was whether the mothers had smoking, alcohol drinking, drug abuse, or exposure to radiation and hazardous substances in the first trimester. b For those cases of termination of pregnancy, their gestational age at the time of termination of pregnancy were regarded as birth week. www.nature.com/scientificreports/ 35 years of age at the time of delivery. NsCL/P mothers were less educated (≤ 9 years), or more overweighted (BMI ≥ 24.0) than control mothers. Much more NsCL/P mothers exposed to environmental risks. Male predominance in NsCL/P and female excess in NsCP were identified. No significant difference was found between cases and controls regarding the distribution of parity, and maternal medical conditions in the first trimester.  Table 3, the crude odds ratio of maternal periconceptional FAS for NsCL/P was 0.39 (95% CI 0.32-0.48), and the adjusted OR was 0.41 (95% CI 0.33-0.51).

Association of maternal FAS and NsOC. As shown in
Further stratification analysis showed that the associations remained significant for NsCL (aOR = 0.42, 95% CI 0.30-0.58) and NsCLP (aOR = 0.41, 95% CI 0.31-0.54). When analyzing according to the initiating time of maternal FAS, both maternal preconception and post-conception use could reduce the risks of NsCL/P and the subtypes (Table 3). Notably, maternal preconception FAS appeared to lower the risk of NsCP (aOR = 0.52, 95% CI 0.30-0.90). But the association between maternal periconceptional FAS and NsCP was nonsignificant. Several known risk factors such as advanced maternal age, higher maternal BMI, living in rural areas, exposure to environmental risks, nulliparity and male infants were positively associated with NsCL/P. Females were identified as risk factors of NsCP (Supplementary Table 1).

Discussion
In this population-based case-control study, we demonstrated that maternal periconceptional FAS was associated with a reduced risk of overall NsCL/P, and the reduced risk varied by cleft subtype and supplementation initiation timing. We did not identify the preventive effect of periconceptional FAS for NsCP, whereas we observed a significant association between maternal FAS started before LMP and NsCP, suggesting that earlier or longer supplementation may be protective. Table 2. Maternal periconceptional folic acid supplementation in cases and controls. NsCL/P, nonsyndromic cleft lip with or without cleft palate cases; NsCL, nonsyndromic cleft lip cases; NsCLP, nonsyndromic cleft lip with cleft palate cases; NsCP, nonsyndromic cleft palate cases. a Significant differences were found between controls, the overall and each subtype of nonsyndromic oral clefts. b Non-users were defined as not having taken folic acid supplements or taken it continuously less than 1 month during the periconceptional period. c Periconception users were defined as having taken folic acid supplements regularly at least 1 month during the periconceptional period. d Preconception users were defined as having taken folic acid supplements regularly at least 1 month during the periconceptional period and started before their last menstrual period. e Post-Conception users were defined as having taken folic acid supplements regularly at least 1 month during the periconceptional period and started on or after their last menstrual period.   www.nature.com/scientificreports/ Consistent with most previous studies in some western [9][10][11][12][13][14][15][16][17]36 and Asian non-Chinese populations 19,20 , our results showed that maternal periconceptional FAS could reduce the risk of overall NsCL/P by approximately 60% regardless of the initiation timing. This preventive effect was observed for both NsCL and NsCLP. Similar associations have been noted in several epidemiological studies in China 18,30-34. These investigations were hospital-based and mainly focused on the environmental risk factors rather than the effect of supplementation. So far, only one cohort study specifically evaluated the preventive effect of maternal FAS on oral clefts in China, and found that in the north of China maternal supplementation started before the LMP reduced the risk of NsCL/P (NsCL/P, aRR = 0.69, 95% CI 0.55-0.87; NsCL, aRR = 0.26, 95% CI 0.07-0.98; NsCLP, aRR = 0.19, 95% CI 0.07-0.50), while such effect was neither observed in the south of China nor for supplementation started on or after the LMP 18,31-35 . Another population-based case-control study in Shaanxi province reported a preventive effect of optimal FAS for overall birth defects (OR = 0.71, 95% CI 0.57-0.89), but not for oral clefts 26 . The authors thought that differences in study design, supplementation initiation timing and small sample size could explain differences studies 18,26 . A recent meta-analysis revealed the preventive effect of maternal FAS in early pregnancy against NsCL/P and NsCP, and identified publication bias in previously published researches 7 . However, another systematic review seemed not to support the preventive effect of daily FAS on oral clefts 30 . It can be seen that inconsistences among various studies are obvious. In fact, there is still a lack of reliable evidence on the preventive effect of folic acid on oral clefts, especially on the dose and initiation timing of supplementation.
Nonsyndromic cleft palate has been regarded as a distinct condition from NsCL/P on embryologic origin and etiology. As noted in several previous investigation 6,11,18,21,31 , this study found a negative but nonsignificant association of maternal periconceptional FAS with NsCP. Interestingly, maternal FAS starting before LMP was significantly associated with decreased risk of NsCP, suggesting that earlier use may work or supplementation initiation timing may play a part. A few studies reported the preventive effect of FAS on NsCP 7,32 . Compared with NsCL/P, researches on FAS and NsCP produced more conflicting results. A cohort study in Norway reported an adjusted RR of 0.84 (95% CI 0.66-1.06) for maternal use of Vitamins/folic acid and NsCP, and a stronger association (RR = 0.63, 95% CI 0.45-0.88) for cleft palate in combination with other malformations 24 . On the contrary, a case-control study conducted in Northern Netherlands 27 and another cohort study 28 in Japan identified maternal FAS/multivitamin supplement use during the first trimester as a risk factor of NsCP. Whether the preventive effect of maternal FAS against NsCP can be observed in a certain population depends on various factors, including genetic background, maternal dietary folic acid intake, serum folate level and the compliance with FAS, etc. Considering the small number of NsCP cases in our analysis, the findings about maternal FAS and NsCP need to be further studied. These controversial results may be due to the heterogeneities in research design, population, sample size, exposure assessment, and other potential confounders 7,18,21,31-35 . Overall, more prospective studies are needed to elucidate the relationship between maternal FAS and NsCP.
Several strengths and limitations should be considered when interpreting our study results. The populationbased nature and large sample size of Chinese cleft cases of the study ensure robust OR estimates for NsCL/P subtypes. In addition, the prospectively collected exposure data could minimize recall bias. When calculating the OR estimates for NsCP, there were potential bias due to low detection rate of syndromic CP but it could be minimal. Though information of maternal dietary and multivitamin intake was not available for analysis, the associations are less likely to be distorted because the OR estimates were based on randomly selected control data with adjustment for known risk factors such as maternal illness, social factors, environmental exposures, and maternal illness.
In conclusion, our study provides additional evidence that maternal FAS during periconceptional period can reduce the risk of NsCL/P. Larger-sample-size studies are warranted to elucidate the association with NsCP and to determine whether women can benefit more from supplementation starting before LMP. It is of paramount importance for women of childbearing age to become aware of that maternal FAS can not only reduce the risk of NTD, but also reduce the risk of NsCL/P.

Methods
Data source and study subjects. Data for this study were abstracted from the CMIHS. According to the "Maternal and Infant Health Care Protocol of Sichuan Province" 37 , every pregnant woman was required to have at least five prenatal medical examinations during her pregnancy and three postnatal clinical visits (on the 7th, 28th and 42nd day after delivery). The results of examinations or visits, risk factors (maternal diseases, family history, other medical conditions), and pregnancy outcomes (spontaneous abortion, stillbirth, live birth, birth defects, etc.), were recorded in the CMIHS system. The medical examination, data collecting, checking and auditing were performed by well-trained obstetricians and nurses. CMIHS adopted the diagnosis criterions, case ascertainment, quality assurance, data collection and encoding of birth defects proposed by Chinese Birth Defects Monitoring Network (CBDMN) 38 . In detail, nonsyndromic clefting referred a cleft case without any non-cleft malformations, while the syndromic cleft was defined as a case occurred in association with other congenital anomalies. The diagnosis was made by at least one obstetrician or pediatrician, and confirmed by a senior doctor at the hospital level. Following the CBDMN guide, the diagnosis was finally coded by CMIHS stuff according to International Classification of Diseases, Tenth Revision. Prenatally diagnosed cases must be rechecked and confirmed after birth. Between October 2010 and September 2015, 807 singletons with NsOCs were included in as the cases, for each case ten controls were randomly selected from the healthy singletons born in the same period.
Folic acid intake and covariates. Briefly, women who joined in the prevention program were followed once a month by local community healthcare workers, and their registration dates, last menstrual period (LMP), dates of starting and ending use, and the information of folic acid supplementation (folic acid alone) were www.nature.com/scientificreports/ recorded. In the CMIHS system, a woman's FAS information was linked to her medical records once she had her first prenatal exam in any of the local hospitals. In this study, pregnant women who regularly took folic acid (400 μg/day) during the periconceptional period for at least 1 month were defined as "periconception users". Of them, those who started intake before their LMP were termed as "preconception users", while those who started on or after their LMP were named as "postconception users". On the contrary, pregnant women who did not have folic acid intake, or took it continuously less than 1 month were considered as "non-users". Other variables included maternal age, nationality, education, residence, parity, medical condition and environmental exposures in the first trimester. Specifically, the maternal medical condition referred to any of such conditions as positive result of syphilis, human immunodeficiency virus and hepatitis B virus testing, anemia, chronic kidney, liver and heart diseases, diabetes (type I or II), primary hypertension disorders. The environmental exposure was an indicator (yes or no) to show whether the mothers had smoking, alcohol drinking, drug abuse, or exposure to radiation and hazardous substances. The variables of infants included gestational age, date of birth, birth weight, sex, birth defects and infant outcomes. Data used in this study were extracted anonymously, with a few variables as identifiers (e.g. date of birth). This research was approved by the Ethics Committee of West China Second University Hospital, Sichuan University. All methods were performed in accordance with relevant regulations and the individual informed consent was waived.
Statistical analyses. Differences in maternal and infant characteristics between cases and controls were examined with Pearson chi-square tests for categorical variables. Non-conditional logistic regression analysis was used to calculate the crude and adjusted odds ratios (ORs) and 95% confidence intervals (CI). Statistical analyses were performed with R 3.5.3 (R Development Core Team 2019) and packages "rms".