CASP9 on 1p36 located about 3 Mb from PAX7, a recognized risk gene. Several regions gave suggestive evidence of G × E interaction in the 1 df test. For example, for G × Smoking interaction, the 1 df test suggested markers in MUSK on 9q31.3 from meta-analysis. Markers near SLCO3A1 also showed suggestive evidence in the 1 df test for G × Alcohol interaction, and rs41117 near RETREG1 (a.k.a. FAM134B) also gave suggestive significance in the meta-analysis of the 1 df test for G × Vitamin interaction. While it remains quite difficult to obtain definitive evidence for G × E interaction in genome-wide studies, perhaps due to small effect sizes of individual genes combined with low exposure rates, this analysis of two large case–parent trio studies argues for considering possible G × E interaction in any comprehensive study of complex and heterogeneous disorders such as OFC.Two large studies of case–parent trios ascertained through a proband with a non-syndromic orofacial cleft  were used to test for possible gene–environment (G × E) interaction between genome-wide markers (both observed and imputed) and self-reported maternal exposure to smoking, alcohol consumption, and multivitamin supplementation during pregnancy. The parent studies were as follows: GENEVA, which included 1,939 case–parent trios recruited largely through treatment centers in Europe, the United States, and Asia, and 1,443 case–parent trios from the Pittsburgh Orofacial Cleft Study (POFC) also ascertained through a proband with an OFC including three major racial/ethnic groups . Exposure rates to these environmental risk factors  varied across studies and among racial/ethnic groups, creating substantial differences in power to detect G × E interaction, but the trio design should minimize spurious results due to population stratification. The GENEVA and POFC studies were analyzed separately, and a meta-analysis was conducted across both studies to test for G × E interaction using the 2 df test of gene and G × E interaction and the 1 df test for G × E interaction alone. The 2 df test confirmed effects for several recognized risk genes, suggesting modest G × E effects. This analysis did reveal suggestive evidence for G × Vitamin interaction for  Orofacial clefts (OFCs) are the most common craniofacial malformations in humans, affecting approximately one per 1,000 live births . OFCs arGRHL3) has been clearly identified as associated with risk to CP [largely limited to populations of European ancestry  can explain about a quarter of the estimated heritability in risk to CL/P based on twin and family studies (Genome-wide association studies (GWAS) using both case–control  and caseancestry ]. A receancestry . Of thes studies , which h studies . Thus, aGRID2 and ELAVL2 genes showed evidence of G × E interaction with maternal smoking in influencing the risk of CL/P among mothers of European ancestry  consortium and case–parent trios drawn from the Pittsburgh Orofacial Cleft (POFC) study. Both studies have genome-wide marker data available and additional markers were imputed against the same reference panel (1000G phase 3 v5). The case–parent trio design provides a unique advantage when analyzing samples from distinct populations for a relatively rare disorder. Unlike a cohort study with randomly ascertained individuals or the more conventional case–control study design, the case–parent trio design is robust to spurious signals arising from population stratification , which can occur whenever samples from multiple populations are combined. We used the genotypic transmission disequilibrium test (gTDT) to test for possible G × E interactions considering three common maternal exposures .The samples in the GENEVA consortium include case–parent trios from multiple populations combined in a GWAS of non-syndromic OFC. Case–parent trios were recruited largely through surgical treatment centers by multiple investigators from Europe (Norway), the United States  and Asia  . PhenotyThe POFC study included case–parent trios ascertained through a proband with an isolated CL/P or CP from multiple populations and a large number of OFC cases and ethnically matched controls from some of these same populations , 2017. HSimilar to the GENEVA study, the three environmental risk factors  were obtained through direct maternal interview. Exposure to ETS, however, was not available in the POFC data. The research protocol was approved by the IRBs at the University of Pittsburgh and all participating institutions, and informed consent was obtained from all participants.1 on the Illumina Human610 Quadv1_B array, which includes 589,945 SNPs through the NHGRI GENEVA program and submitted to dbGaP (2 accession number phs000094.v2.p1). To take advantage of more efficient imputation tools and larger reference panels, we re-imputed genotypes on the GENEVA dataset using the Michigan Imputation Server  for 1,939 complete case–parent trios .In the GENEVA study, DNA was genotyped at the Center for Inherited Disease Researchn Server  after drn Server  while con Server . This imith POFC . All impphs000774.v2.p1), and similar quality control filtering was used to remove rare and poor-quality SNPs. Genomic coordinates were given in human genome build 37 (hg19). Genotype data were pre-phased with SHAPEIT taking family structure into account . However, reduced power due to genetic heterogeneity is counter-balanced by improved power due to increased sample size when genetic sharing between CL/P and CP exists  was used on common SNPs in the combined set of all OFC trios from GENEVA and POFC separately. For a common bi-allelic marker, a conditional logistic model can be used to test the null hypothesis of independence between each common SNP and disease . In this article, we assume an additive genetic model and consider the conditional logistic model that models the association between each common SNP and its interaction with a maternal exposure and OFC:For our G × E interaction analyses, we considered three self-reported maternal exposures: maternal smoking, alcohol consumption, and multivitamin supplementation. Note that ETS was not available in both GENEVA and POFC, and hence not studied in this analysis. We used the gTDT in the R  package  for this package . The triY_0 is the disease status of the child ; Y_l is the disease status of the lth pseudo-control ; G_0 is the genotype of the child (case) at the marker coded additively as 0, 1, or 2; G_l is the genotype of the lth pseudo-control at this same marker; and E is a binary environmental variable denoting presence/absence of a maternal exposure during pregnancy. Basically, G_1, G2, and G_3 represent possible SNP genotypes the observed case did not inherit from the parents. We first performed a 2 df χ2 test of the null hypothesis H0:βG=0,βGE=0 to identify markers with either a main effect, or a G × E interaction effect, or both. To focus exclusively on G × E interaction between a marker and a maternal exposure, we conducted a 1 df χ2 test of the null hypothesis where trio package), then the overall meta-analyzed estimates are i=1,2. We calculated these meta-analyzed estimates meta (v4.13.0) . For the 2 df G × E interaction test, we meta-analyzed using the approach described in 2 test of H0:βG=0,βGE=0 by jointly meta-analyzing estimates https://github.com/RayDebashree/GxE.Finally, we conducted a combined analysis of the GENEVA and the POFC studies using inverse-variance weighted fixed effect meta-analysis. The closed form solutions of the coefficients and their standard errors from the gTDT model discussed above enable computationally efficient genome-wide meta-analysis across both studies. In particular, for the 1 df G × E interaction test, if v4.13.0)  and appl5 . This meta-analysis does show recognized risk genes for OFCs are not obscured in this 2 df joint test of G and G × E interaction.It has been suggested the 2 df joint test for gene (G) and G × E interaction could provide more power to detect genes influencing risk to complex and heterogeneous diseases when there is any possibility of G × E interaction . Figure GE). For example, the signal for SNPs near PAX7 on 1p36 almost achieved genome-wide significance for the joint test of G and G × Smoking  and when G and G × Vitamin (p = 8.6 × 10–6) were analyzed in this joint test . Specifically, SNP rs4646022 yielded suggestive significance for G and G × Vitamin interaction in this 2 df test (p = 3.1 × 10–7). CASP9 and PAX7 for the 2 df joint test of G and G × E interaction for each of the three maternal exposures. PAX7 and virtually no signal in the region of CASP9 . CASP9.There are some differences among these results from meta-analysis across the three exposures considered  in a similar meta-analysis over both the GENEVA and the POFC studies (p < 10–6) of possible G × Smoking interaction and may warrant further exploration. p value, and frequency in each racial/ethnic group. To focus explicitly on tests of G × E interaction, we used the 1 df test for H studies . While nFGF12 and an intronic SNP rs2186801 in the 9q31.3 region containing the MUSK (muscle associated receptor tyrosine kinase) gene both gave such suggestive evidence when testing for G × Smoking interaction. The top signal near FGF12 is questionable, however, because nearby SNPs did not show any supporting evidence of linkage and association (see R2 = 0.84). As indels are intrinsically more difficult to call, extreme caution should be used when interpreting suggestive evidence of possible G × Smoking interaction. Also, the frequency of the allele associated with any effect on risk showed considerable variability across racial/ethnic groups  at position 191,830,067 on 3q28-q29 near tion see , and thic groups . The pea on risk . This imSLCO3A1 on 15q26 achieved suggestive evidence of linkage and association . CASP9 gene is of interest because it has been previously associated with risk of OFC  the threshold for “suggestive” significance (p = 1.02 × 10–6) in this 1 df test for G × Vitamin interaction  in tion see . CautionRETREG1 (a.k.a. FAM134B) on 5p15.1 also achieved suggestive evidence for G × Vitamin interaction in this 1 df test. Another imputed SNP rs41117 located near While it is widely accepted that both genes and environmental risk factors influence risk to OFC, it is quite difficult to formally test for statistical interaction between the two . StatistIRF6, GRHL3, and ARHGAP29 regions , but also just due to different exposure rates. If sub-populations differ in both their allele frequencies and their exposure rates , an estimation approach can be used to estimate mean exposure rates within distinct sub-populations, which may protect from spurious results in tests for G × E interaction . HoweverPAX7 (1p36.13), ABCA4 (1q22), IRF6 (1q32.2), DCAF4L2 (8q21.3), 8q.24 (gene desert), VAX1 (10q25.2), NTN1 (17p13.1), and MAFB (20q12). This is reassuring and argues that testing for possible G × E interaction will not conceal genetic risk factors when they do exist. Also, G × E interaction may not be an overwhelming risk factor for OFC controlled by these well-recognized risk genes.The 2 df test from the meta-analysis of all OFC case–parent trios revealed many recognized risk genes for CL/P, the predominant form of OFC in this study. Confirmed risk genes include the following: PAX7 but interestingly when G × Vitamin interaction was included in the conditional logistic model for the gTDT, a rather distinct suggestive peak becomes apparent a short physical distance from PAX7 (see CASP9 (caspase 9) is of particular interest because it was previously identified as a potential risk gene for OFC based on a sequencing study of members of multiplex cleft families from Syria (CASP9 (predicted to be pathogenic) occurred in three homozygous family members with an OFC as well as other affected relatives who were heterozygous. CASP9 is directly involved in an apoptotic signaling pathway shown to result in a facial cleft phenotype in mouse models  on 9p31.3 showed well-defined evidence against the null hypothesis , providing support for its involvement in fetal development.When we focused on the 1 df test for evidence of G × E interaction alone, no markers achieved genome-wide significance, but several gave “suggestive” evidence and some of these are worthy of further consideration. Several SNPs in and near pothesis . The hig on risk . MutatioSLCO3A1 (solute carrier organic anion transporter family member 3A1) on 15q26 yielded suggestive evidence G × Alcohol interaction (SLCO3A1 may be associated with nicotine dependence (Two imputed SNPs (rs8031462 and rs4777824) near eraction . The soleraction . These gpendence  and bloopendence .RETREG1  on 5p15.1 is a cis-Golgi transmembrane protein, and mutations in this gene lead to the production of an impaired gene product, which is unable to act as an autophagy receptor and leads to hereditary sensory and autonomic neuropathy in humans . This gene can also act as a tumor suppressor in colorectal adenocarcinoma and an oncogene in esophageal squamous cell carcinoma, and loss-of-function mutations can control viral replication  together is unusual, it is reassuring that the estimated effect sizes in www.ncbi.nlm.nih.gov/gap) through dbGaP accession number phs000094.vl.pl (GENEVA) and accession number phs000774.v2.p1 (POFC), respectively.The datasets analyzed for this study can be found in dbGaP at  of each participating site, both domestic and foreign. For the GENEVA study, IRB at the Johns Hopkins Bloomberg School of Public Health and at each participating recruitment site aooroved. The research protocol for the POFC was approved by the IRB at the University of Pittsburgh and all participating institutions. Informed consent was obtained from all participants. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin for minor children.WZ did the re-imputation of the GENEVA genome-wide markers and conducted the statistical analyses of this dataset. SV ran the analyses of POFC data and the meta-analysis, summarized the findings, and generated the visualizations. JH managed the GENEVA dataset and directed quality control after imputation. MM, EF, SMW, and EL conducted data acquisition and analysis for the POFC. TB, JH, AS, IR, and MT conducted data acquisition and analysis for GENEVA. DR, MT, and IR designed and executed the statistical analysis. TB, DR, and WZ wrote the manuscript with input from JH, SV, MT, MM, IR, and AS. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The role of allium vegetables or garlic supplements on reducing cancer risk was inconsistent between laboratory study findings and related epidemiologic studies.Studies assessing the effect of allium vegetables and garlic supplement consumption on cancer risk were included in our meta-analysis. We used fixed- or random-effects models to pool effect measures to evaluate the highest and lowest consumption. A dose-response regression analysis was used to assess the association between allium vegetables, garlic supplements, and cancer risk.P for nonlinearity = 0.958, P for linearity = 0.907).In a pooled analysis of 22 studies with 25 reports on allium vegetables, a high consumption of allium vegetables showed no significant association with cancer risk  in a fixed-effects model. Similarly, garlic supplements were not found to be significantly associated with an increased risk of cancer  in a random-effects model involving a pooled analysis of 10 studies with 11 reports. Consumption of allium vegetables did not significantly correspond with cancer risk (In this meta-analysis, we found no evidence that higher consumption of allium vegetables or garlic supplements reduced the risk of cancer; however, this finding requires further validation.https://www.crd.york.ac.uk/prospero/#recordDetails, identifier: CRD42021246947. Over the past decade, the search for dietary factors with effects on cancer prevention have led to the rapid expansion of the field of dietary patterns and cancer. Studies of individual nutrients and phytochemicals have revealed associations between certain dietary factors and cancer risk , there were 19.3 million new cancer cases and almost 10.0 million cancer deaths in 2020  . The proAfter preliminary searches, we decided to include prospective studies, and took the pooled analysis of retrospective studies as our next work. Thus, we only considered trials to be eligible if they involved prospective study designs, including cohort, case-cohort, or intervention studies; if they investigated the relationship between allium vegetables or garlic supplements and malignancy risk; and if they provided or allowed for the calculation of relative risks (RR) with 95% confidence intervals (CIs).We excluded studies if they were designed as nonhuman studies, case reports, or case series or had a retrospective study design, such as a case-control study; if they were editorials, or letters without the analyses of primary data; or, if they did not provide sufficient data; if they did not use an analytic epidemiologic design.ClinicalTrials.gov and the World Health Organization International Clinical Trials Registry Platform (WHO ICTRP). To maximize the identification of relevant studies, the reference lists of eligible studies were also reviewed. If multiple studies remained with the same or partially overlapping populations, we extracted the data with the largest sample size or the longest follow-up duration. If duplicate studies offered results related to different outcomes, we included them all in the pooled analysis of specific outcomes. An English-language restriction was also included in our search strategy.One of the authors (Q.F. Zhang) performed the search of several databases: Medline, Embase, and Web of Science from inception to May 1, 2021. The following combined search terms were used: MeSH headings and keywords relating to “neoplasms,” the MeSH heading “allium,” and keywords related to the allium vegetables and garlic supplements. Ongoing or unpublished eligible studies were also searched through Two researchers (Q. F. Zhang and Q. Zhao) independently screened all titles and abstracts of studies meeting the inclusion criteria. They then obtained full-text versions and performed further screening if studies were deemed eligible. We resolved any disagreements by consensus.Two independent researchers (Q.F. Zhang and Y. Shen) used a standard form to extract data from the included studies, including first author, publication year, study design, geographic region, sample size , duration of follow-up, demographics of participants, tumor site, exposure, and effect sizes with 95% CIs of highest vs. lowest consumption category. We extracted effect sizes that represented the greatest degree of control for potential confounders. If person-years were not presented, they were using the number of subjects multiplied by the mean duration of follow-up. When a study reported an outcome of interest but without estimates, we tried to contact the author for related data. We resolved any disagreements by consensus.http://www.ohri.ca/programs/clinical_epidemiology/oxford.asp). Publication bias was assessed qualitatively by visual estimation of funnel plots and quantitatively by calculation of Egger's test  independently assessed the quality of included studies by the Newcastle-Ottawa Scale , we used a fixed-effects model to pool outcomes; otherwise, a random-effects model was used (I2 ≥ 50%).The primary outcome for this meta-analysis was the incidence of cancer. We considered the RRs and 95% CI as the effect sizes for all studies. The association between allium vegetables/garlic supplements and cancer risk was quantified by pooling the RRs for the highest vs. lowest consumption category using random-effects or fixed-effects models according to the extent of between-study heterogeneity. The extent of heterogeneity was quantified by the Q test  and I2 s. Garlic supplements were also not found to be significantly associated with an increased risk of cancer  in a random-effects model involving a pooled analysis of 10 studies   .In an analysis of individual allium vegetables, garlic intake was investigated in seven studies , 41, 42 The association between allium vegetable consumption and cancer risk were similarly nonsignificant in other subgroup analyses based on the number of cases, sex, and location. However, a stratified analysis based on the duration of follow-up period indicated that a high amount of allium vegetable consumption was marginally associated with a decreased risk of cancer in patients with a follow-up period of less than 7 years  .Moreover, in the subgroup analysis of garlic supplements, the consumption of garlic supplements was not associated with cancer risk according to the number of cases, sex, follow-up period, or tumor site. Stratified by study location, however, garlic supplement consumption was marginally associated with a decreased cancer risk in Chinese patients ; however, only two reports were pooled in this subgroup.2 = 17.6%; Ph = 0.219) after omitting the report by Meng, 2013 (1.1)  after omitting the report by Schulz, 2005  after omitting the report by Satia, 2009  after omitting the report by Walter, 2010 (As shown in 13 (1.1)  to a higia, 2009  to a higer, 2010 , which wP for interaction = 0.017; Our univariate meta-regression analysis found no statistically significant differences, revealing that the analyzed stratification factors were not a source of heterogeneity. However, we did find that the cancer risk was significantly higher in studies with allium vegetable consumption and a longer duration of follow-up  detected no significant small-study effects  varied across the included studies, making it impossible to accurately compare equivalent consumption. Also, the included studies only reported use or nonuse, leading to the impossibility of assessing the dose-response relationship between garlic supplements and cancer risk. Second, although we pooled effect sizes adjusting for most established risk factors, the causes of cancer incidence are complicated and sometimes unclear, which may have introduced confounders that could have influenced our findings. Third, many laboratory studies have demonstrated the role of garlic components in inhibiting tumor growth and progression, suggesting that they might also play a role in prolonging the survival of cancer patients. However, whether allium vegetables or supplements could reduce cancer mobility was not analyzed in the current study, limiting the implications of our findings. Last, though prospective studies are less likely to be influenced by recall or selection bias, well-conducted retrospective studies provide less-biased evidence. The addition of retrospective studies in the pooled analysis was believed to provide more detailed information and strength the reliability of the findings, which was also our next work.Cancer incidence and mortality is a major burden in most countries worldwide. Currently, not only treatment but also prevention of cancer has been of great concern to prolong lifespan of population. Considering the anticancer effects of certain active compounds present in allium vegetables, such as organosulfur, researchers have shown an increasingly interest in the ability of these substances to prevent cancer. Our findings suggest that neither allium vegetables or supplements have clinically relevant effects on cancer prevention; therefore, there is little evidence to support the idea that allium vegetables or supplements can reduce cancer in incidence. Additional large, randomized controlled trials are needed to confirm these results.Overall, the current meta-analysis found that higher consumption of allium vegetables or garlic supplements were not associated with a decrease in cancer risk. Randomized controlled trials or well-designed cohort studies are warranted to confirm these findings.The original contributions presented in the study are included in the article/Material preparation, data collection, and analysis were performed by QifZ, QinZ, YS, FZ, and YZ. The first draft of the manuscript was written by QifZ and QinZ. All authors commented on previous versions of the manuscript, read and approved the final manuscript, and contributed to the study conception and design.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Preeclampsia has genetic correlation. Many studies have shown that microRNA (miRNA) polymorphism is highly associated with preeclampsia, but the results are inconsistent. The purpose of this study is to systematically evaluate the relationship between miRNA polymorphism and preeclampsia.In this study, the search time is set from the establishment of the database on January 2021. The search database include China National Knowledge Infrastructure (CNKI), Wanfang, VIP and China Biology Medicine disc (CBM), PubMed, EMBASE, and Web of Science, and the Cochrane Library. The subjects are case-control studies on the relationship between miRNA polymorphism and preeclampsia. The language is limited to English and Chinese. The data of the included study are extracted and the literature quality is evaluated by 2 researchers independently. The data are statistically analyzed through Stata 16.0 software. We also predicted the miRNA secondary structure and the binding sites of miRNA interaction with its target genesThis review will be disseminated in print by peer-review.This study will provide evidence-based medicine to elucidate the genetic tendency of preeclampsia.Private information from individuals will not be published. This systematic review also does not involve endangering participant rights. Ethical approval will not be required. The results may be published in a peer-reviewed journal or disseminated at relevant conferences.DOI 10.17605/OSF.IO/MJY2X. Meanwhile, placental ischemia and changes in maternal immune response may play important roles in the development of preeclampsia. Although extensive studies have been carried out, accurate diagnostic markers for preeclampsia,9 is still scarce.Preeclampsia is a multisystem disease that occurs in 2% to 10% of pregnant women and is the leading cause of maternal and perinatal morbidity and mortality. By regulating many target genes, miRNA participates in the process of immune response and plays a very important role in the occurrence and development of autoimmune diseases.,12 miRNA not only plays a significant role in the normal growth and differentiation of body cells, but also participates in pathological processes such as inflammatory reaction and carcinogenesis. At the same time, miRNA plays an important role in the occurrence and development of malignant tumors such as liver cancer, breast cancer, and rectal cancer.,14 Trophoblast invasion is very similar to tumor cell infiltration during pregnancy. Recent studies have proved that miRNA is also important in the occurrence and development of preeclampsia. miRNA stably exists in blood and can be detected in patients’ serum to observe its expression.MicroRNA (miRNA) is a non-coding single-stranded RNA and participates in the regulation of apoptosis, proliferation, differentiation, ontogeny, and other life activities. Therefore, changing the level of miRNA may lead to the abnormal expression of target genes, abnormal placental function, and subsequent complications such as preeclampsia.,18 By targeting genes in inflammatory response, miRNA-499 has potential inflammatory inhibitory effects and may play a regulatory role under the hypoxic-ischemic condition.,20 The relationship between miRNA gene variation and many diseases has been confirmed, including the effects on pregnancy complications such as preeclampsia.–23 Mohseni et al displayed that the expression of miRNA is higher in patients with preeclampsia and cardiac remodeling, suggesting that miRNA may play a role in cardiomyocyte biology. Alipour et al reported that miRNA is associated with recurrent pregnancy loss, some of which may be closely related to preeclampsia. Jeon et al,27 found that miRNA polymorphism is associated with idiopathic, recurrent, and spontaneous abortion and fetus.In different stages of pregnancy, a large number of miRNAs were found, and expression profiles were different between preeclampsia and normal placenta. Therefore, we conducted a meta-analysis to examine the accurate correlation between miRNA polymorphism and susceptibility to preeclampsia.The abnormal expression of miRNA plays an important role in the occurrence and development of preeclampsia, which clearly indicates that miRNA polymorphism can be applied as a biomarker to evaluate the risk of preeclampsia. Many studies have explored the relationship between miRNA polymorphism and the risk of preeclampsia. However, the results of these studies are not consistent.22.1 Moreover, it has been registered on open science framework (OSF) on January 2021 (Registration number: DOI 10.17605/OSF.IO/MJY2X).This protocol of systematic review and meta-analysis have been drafted under the guidance of the preferred reporting items for systematic reviews and meta-analyses protocols (PRISMA-P).2.2Since the program does not include the recruitment of patients and the collection of personal information, it does not require the approval of the Ethics Committee.2.3(1)The subjects were clinically diagnosed as preeclampsia with systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg, and proteinuria ≥0.3 g for 24 hours or random urine protein ≥(+) after 20 weeks of pregnancy;(2)The exposure factor was miRNA mutation;(3)Outcome measures were the risk of preeclampsia;(4)The control group involved pregnant women without hypertension;(5)The study was a case-control study;(6)Languages were limited to Chinese and English;(7)Allele or genotype distribution frequency data were available;(8)The distribution frequency of genotype conformed to Hardy-Weinberg's law.2.4(1)Repeated published researches;(2)In articles, the published literatures were abstracts or reviews, the data of the article were incomplete or incorrect, and the complete data cannot be obtained after contacting the author(s);(3)The study failed to provide detailed data on the frequency of genotypes;(4)Literatures had no relevant outcome indicators;(5)The objects of studying were not from human beings.2.5The search database include China National Knowledge Infrastructure (CNKI), Wanfang, VIP and China Biology Medicine disc, PubMed, EMBASE, and Web of Science and the Cochrane Library. All the literatures about the relationship between miRNA polymorphism and preeclampsia will be collected from the establishment of the database on January 2021. Taking PubMed as an example, the retrieval strategy is exhibited in Table 2.6Two researchers independently complete the literature screening, exclude the studies that obviously do not meet the inclusion criteria, and further read the abstracts and the full texts to determine whether they meet the inclusion criteria. The data included in the literature will be extracted and cross-checked. Disagreement should be solved by consulting a third researcher, thus reaching a consensus. The extracted data include: the first author, the number of years of publication, the country of publication, the race of the study population, the basic characteristics of the study population , the distribution of each phenotype of genes (whether obeying the law of Hardy-Weinberg equilibrium), and so on. The literature screening process is illustrated in Fig. 2.7The case-control study will be evaluated by Newcastle-Ottawa Scale (NOS), including 3 columns and 8 items with a total of 9 points, and the evaluation criteria ≥6 is regarded as high quality.2.82.8.1P ≥ .05). Meta-analysis is carried out by using STATA.16 software . The binary classification variables will be expressed as odds ratio (OR) and 95% confidence interval (CI). Heterogeneity is analyzed by conducting Q test and I2, and the heterogeneity is evaluated based on the I2 value. If P > .1 and I2 < 50%, the heterogeneity among the included studies is small, so the fixed effect model is applied for analysis. If P < .1 and I2 ≥ 50%, the heterogeneity among the included studies is obvious, and the sources of heterogeneity are analyzed. Random effect model will be utilized for analysis. RNAfold web server (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi/) was used to perform in silico analyses for the prediction of each miRNA secondary structure, harboring selected single nucleotide polymorphisms. miRNA binding sites were predicted by miRanda (http://www.mirbase.org/).Before evaluating the correlation between miRNA polymorphism and preeclampsia, chi-squared will be adopted to test whether the genotype distribution in the control group is in accordance with Hardy-Weinberg genetic equilibrium (2.8.2If the data of the required study are incomplete or not reported in the study, the researcher will contact the first author or other authors of the study by phone or email. If the required data are not available, we will perform descriptive analysis instead of meta-analysis and exclude these studies if necessary.2.8.3In order to deal with the heterogeneity among different studies, subgroup analyses will be conducted on the basis of ethnicity.2.8.4In order to test the stability of the meta-analysis results, we will adopt the one-by-one exclusion method to analyze the sensitivity of the results.2.8.5P < .05, it is considered to have obvious publication bias.A funnel chart will be used to qualitatively identify publication bias, and Egger and Begg tests will be applied to quantitatively evaluate publication bias. If the funnel diagram is asymmetrical, with 3 The incidence of preeclampsia is 2.5%, accounting for 6% of perinatal maternal death.,32 The exact pathogenesis of preeclampsia is still unclear. Many scholars believe that preeclampsia has a familial genetic tendency and is a polygenic genetic disease. miRNA is a single-stranded RNA that does not encode a protein. At present, many studies have revealed that a close relationship between miRNA and the occurrence and development of preeclampsia can be found, while the relationship between miRNA polymorphism and the occurrence of preeclampsia is controversial. This study comprehensively searched the case-control study on the relationship between miRNA polymorphism and the risk of preeclampsia, and made a quantitative comprehensive evaluation by carrying out meta-analysis to provide evidence-based reference for the study on the etiology of preeclampsia.Preeclampsia is the occurrence of hypertension and proteinuria after 20 weeks of pregnancy with normal blood pressure before pregnancy. Preeclampsia is one of the hypertensive disorders of pregnancy and an idiopathic disease of pregnancy. Meanwhile, it is closely related to the maternal and fetal outcome.This study also has some limitations. First of all, ethnic groups of meta-analysis are wide, while researches on single race are few. Population confounding factors may affect the credibility of the results in this study. Second, the method adopted in this study is meta-analysis that belongs to the secondary analysis. Therefore, the quality of this study mainly depends on the overall quality of the included studies, which is an inevitable problem of the secondary analysis. Third, due to the limitation of the number of studies included, there may be some publication bias in this meta-analysis.To sum up, this meat-analysis will provide the correlation between miRNA polymorphism and the risk of preeclampsia. In view of the limitation in terms of the number of included studies, this conclusion needs to be verified through further large sample and high-quality studies, so as to clarify the relationship between gene polymorphism and preeclampsia.Conceptualization: Tao Li, Guolin He.Data curation: Tao Li, Yihong Chen.Funding acquisition: Guolin He.Investigation: Guolin He.Methodology: Yihong Chen.Project administration: Guolin He.Resources: Yihong Chen.Software: Yihong Chen, Yi Lai.Supervision: Yi Lai, Guoqian He.Validation: Yi Lai, Guoqian He.Visualization: Yi Lai.Writing – original draft: Tao Li, Guolin He.Writing – review & editing: Tao Li, Guolin He.


Our findings support the idea that EBV is related to laryngeal carcinoma. However, further studies are needed before recognizing a definitive etiological role of EBV in the development and/or progression of laryngeal carcinomas.Over the past few decades, several publications have investigated the role of Epstein-Barr virus (EBV) in head and neck squamous cell carcinomas, and an increasing number of them have shown its presence in laryngeal tumors. The purpose of this meta-analysis was to evaluate the association of EBV with laryngeal carcinoma. The search was carried out in two databases, Scopus and PubMed, using the following terms: “Epstein-Barr virus” and “laryngeal carcinoma”. A total of 187 records were found, of which 31 were selected for meeting the inclusion and exclusion criteria. The meta-analysis yielded an overall pooled prevalence of 43.72% : 34.35–53.08). Studies carried out in Europe and Eurasia had slightly higher pooled prevalence than other subgroups, while the prevalence of studies performed in developed countries was higher than in developing countries (46.37% vs. 34.02%). Furthermore, laryngeal carcinoma occurred almost three times as often among EBV-infected individuals compared to those without EBV infection (odds ratio = 2.86 (95% CI: 1.18–6.90); Begg’s test,  According to the global estimates from the International Agency for Research on Cancer (IARC), for 2018, there were 177,422 new cases and 94,771 deaths due to cancers of the larynx. Both values  represented about 1.0% of all 36 cancers analyzed [Although it is well known that tobacco smoking and alcohol consumption are the major risk factors for the development of laryngeal SCC, infectious agents may also be implicated in the pathophysiology of some cases ,4. In thAdditionally, various genotypes of human papillomavirus (HPV) have been found in samples of laryngeal SCC . Thus, tBřicháček et al.  were theIn the present study, we performed a systematic review with meta-analysis of articles available in the international literature on the association of EBV with carcinomas of the larynx, in order to answer the following research question: Are EBV-infected individuals at increased risk for the development and/or progression of laryngeal carcinoma? Characteristics of the studies, such as reported prevalence, techniques used, detection targets, and other relevant aspects for understanding of the relationship between EBV and laryngeal carcinomas, were evaluated.n = 8) [n = 5) [n = 3) [n = 1) [n = 1) [n = 1) [The 31 systematically reviewed studies were carried out in 19 different countries in Europe n = 12) [ 12,13,1,18,19,20n = 8) ,26,27,28 [n = 5) ,30,31,32 [n = 3) ,5,33, Oc [n = 1) , South A [n = 1)  Table 1n = 8 [n n = 16) [n = 14) [n = 10) [The studies have detected EBV by PCR-based techniques n = 16) ,32,33,34 ,32,33,3[n = 14) ,31,32,35[n = 10) ,29,30,35[n = 10) ,28.Some articles also explored the presence of other viruses. In order to investigate the possible association between viral (co)infection and a laryngeal carcinogenesis, these data were also considered in the present systematic review and summarized in p < 0.001). p < 0.001). Subgroups that used the in situ hybridization and PCR-based technique yielded the following pooled prevalence: 49.58%  and 39.31% , respectively.Collectively, the studies included in the present review gathered 1292 distinct specimens from patients with laryngeal carcinoma. Twenty-nine studies could be submitted to meta-analysis, of which eight studies presented a prevalence of 0 or 100% and were automatically excluded from the analysis. The overall pooled prevalence was 43.72% .p < 0.001) was higher than in developing countries.Studies carried out in Europe and Eurasia had slightly higher pooled prevalence than other subgroups, whereas the prevalence of studies performed in developed countries ; in comparison, for the control group composed of non-malignant lesions, the chance of developing LC in EBV-positive individuals was nearly twice as common as in the non-infected group (OR = 1.94 (1.18–3.21); p = 0.299). The results of the p-value and I-squared statistics in the latter analysis revealed low heterogeneity (I2 < 50% and p > 0.05), therefore indicating the fixed-effects model as the most suitable, and both models had similar odds ratio results . The overall result showed values closely related to those of the second subgroup (OR = 2.18 (95% CI: 1.33–3.59); p = 0.007). p = 0.843) and Egger’s test (p = 0.866). No evidence of publication bias was observed.In our sample, 15 case-control studies were identified and used in the odds ratio analysis ,30,33,35The hypothesis that viral infections are associated with the development of human cancers has been studied since the early 1960s, when the first human oncovirus was discovered in Burkitt’s lymphoma: the Epstein-Barr virus . In 1973One year later, the first study evaluating the involvement of EBV in laryngeal carcinoma was published; however, the authors failed to show any significant antibody titer differences between carcinoma and control groups, leading them to suggest that EBV is not etiologically related to LC . PerhapsIn the same year, Callaghan et al.  found thThereafter, other studies have sought to assess the relationship between EBV and laryngeal carcinoma, and the number of publications on this subject gradually increased in the following decades, but in a smaller proportion compared to other EBV-related tumors . As shown in Among the studies included, some were not able to detect EBV in their samples, either by ISH ,22,26,31It is worth mentioning that studies such as Yang et al.  and de ORegarding the studies employing PCR-based methods, most demonstrated positivity for EBV with an average prevalence around 50%. However, these results face the same dilemma observed in carcinomas from other anatomical sites, that is, the real meaning of the expressive PCR positivities given the impossibility of determining the precise source of viral DNA, whether from tumor or non-malignant cells . In this regard, when PCR-based methods are used for EBV detection, the results must be considered with caution because it has been estimated that EBV latently infected B-cells may be found in circulating cells in healthy individuals, and thus false-positive results must be considered ,3. In a In the present review, even without applying a time limitation filter in the searches, the number of articles retrieved evaluating the involvement of EBV in LC is still small. Moreover, many studies enrolled small samples, some applied indirect EBV detection methods , while only a few were able to demonstrate the viral genome or its products (transcripts or proteins) in tumor cells, and there is a lack of information on the distribution patterns of staining. In addition, there are some relevant confounding variables, such as alcohol consumption and tobacco smoking that are traditional risk factors for HNSCC, as well as other oncogenic viruses that are also found in this site, sometimes in coinfections, which compromise the assessment of the role of EBV in the initiation and/or progression of laryngeal carcinomas. In this context, Mulder et al.  were unaTherefore, some authors recognize that the role of EBV in the pathogenesis of laryngeal carcinoma is still unclear ,25,32,33In a Turkish study, the identification of EBV DNA in 50% of patients with laryngeal carcinoma and in 41.2% of the vocal cord nodules indicates, according to the authors, that this agent does not seem to be directly associated with the pathogenesis of LC, but suggests a causal relationship involving EBV and proliferative diseases in the larynx . In the Three studies could not find any significant difference between EBV DNA positivity and smoking habits ,32,33. TInterestingly, two studies failed to detect EBV by EBER-ISH in laryngeal lymphoepithelial carcinomas, denoting the peculiarity of this rare type of neoplasm ,31. AcuñDespite the controversy, some authors consider that EBV plays a role in the development of LC ,23,25,27Although the possibility of false-negative cases is admitted in those studies investigating the presence of EBV in samples taken from FFPET, since formalin is a known inhibitor for PCR ,27, in oInterestingly, the developed countries demonstrated higher pooled prevalence than developing ones. Although there is little information in this regard, one could argue that the difference observed between developing and developed countries might be related to the period of primary EBV infection. According to Ali et al. , due to In a recent case-control study, the EBV DNA positivity detected by qPCR was significantly higher in LC group (52% vs. 20%), showing that individuals with laryngeal EBV DNA positivity had a greater risk of developing LC (unadjusted OR = 4.3). Also, EBV DNA presence was positively correlated with BCL-2 expression (an anti-apoptotic protein) . Althougp = 0.004).Despite the debate on the high sensitivity of PCR-based methods which may incur false-positive results, in a multivariate analysis performed by Lee et al. , EBV DNARegarding histological approaches, although evidence on the location of EBV is scarce, some studies have shown distinct nuclear staining by ISH in tumor cells of laryngeal SCC ,28,35. IThe cases in which EBV DNA was found but viral protein was not detected ,32 may iSome experiments have indicated that EBV infection may affect the immune response in the context of laryngeal carcinogenesis. For instance, the findings of Klatka et al.  point toThe importance of studies focusing on the impact of coinfections on the malignant transformation of the laryngeal epithelium have also been highlight, which may contribute to clarifying their association with the carcinogenic process and to guide prophylactic strategies ,17,18. IIn our analysis, five studies reported EBV and HPV coinfections ,15,17,18Although the role of EBV/HPV coinfection has been hypothesized, the coexistence of both viruses has not been proven to have an unfavorable implication in the prognosis of LC patients . In addiOther viruses detected in LC include HSV-1, CMV, HHV-8, HIV, BKV, and MCPV ,17,18,35In general, the limitations of the present study were related to the use of only two databases and the inclusion of articles exclusively published in English that may have restricted the number of records found. Moreover, as studies on EBV in laryngeal carcinoma are less common, a time period was not established in the searches, resulting in the selection of a greater number of articles using indirect methods for EBV detection, which along with the small sample sizes observed in some works and diversity of techniques employed are likely the main causes of the heterogeneity verified in our meta-analysis. Finally, the review being centered on observational studies, due the nature of the investigated subject, may have downgraded the strength of the current evidence.To investigate the association of EBV with laryngeal carcinoma, we conducted a systematic review with a meta-analysis of publications that were retrieved from the electronic scientific databases PubMed and Scopus, using the following search terms: “Epstein-Barr virus” and “laryngeal carcinoma”. In the PubMed database, the search was set to include all fields, while in Scopus the search was conducted based on the title, abstract, and keywords. In view of the limited number of articles available, no filter was applied for the time period; for this reason, the search covered a relatively long period (November 1973 to 3 September 2021).The manuscripts were selected primarily through the analysis of titles and abstracts. The publications were reviewed independently by three authors and submitted to the following inclusion criteria: (i) articles written in English; (ii) articles addressing the association between carcinoma of the larynx and EBV; and (iii) original studies. Publications were excluded which (i) employed no EBV detection method (direct or indirect); (ii) were not classified as original research ; (iii) involved only squamous cell carcinomas from other locations in the upper aerodigestive tract and did not include any laryngeal cases, or combined the LC cases with tumors from other anatomical sites without presenting the values separately; (iv) were based on animal samples or models; or (v) were repeated documents. Any disagreement during the selection of records was resolved by the senior author.Initially, we found 109 records in PubMed and 78 in Scopus. Fifty-nine duplicate records were excluded. After filtering for language and type of study and evaluating the connection of the content with the proposed aim, 31 articles were included in the systematic review . Full-te2) and the Q statistics, with the level of statistical significance set at 5% (p < 0.05). The EBV pooled prevalence was initially estimated with a fixed-effects model. However, due to the significant level of heterogeneity observed, we conducted a second analysis using the random-effects model of Der Simonian and Laird [The statistical analysis was performed with the software Stata, version 12.0 , using the following commands: “metan”, “metafunnel”, and “metabias” . First, nd Laird . StudiesTo our knowledge, the present study represents the first systematic review with meta-analysis of the literature on the association of EBV with carcinoma of the larynx, with relevant evidence such as the expressive pooled EBV prevalence of 43.7% in samples from LC patients and the fact that EBV-infected individuals are 2.86 times more likely to develop laryngeal carcinoma. Despite the controversies, our findings indicate a possible role for EBV as a risk (co)factor in the development and/or progression of LC. Moreover, these tumors seem to share similarities with other HNSCCs, as well as with anogenital and breast carcinomas in which EBV can be found alone or in coinfection with high-risk genotypes of HPV, strengthening the thesis that both oncoviruses can cooperate in the epithelial transformation. There may also be an influence of EBV in the immunological response in the context of laryngeal carcinoma, providing an appropriate environment for the establishment of the LC, by promoting tumor growth and by suppressing anti-tumor responses. On the other hand, unlike other EBV-related carcinomas, there are still few studies in the literature dealing with this topic in the larynx; consequently, the location and distribution of EBV in the tissue remain controversial and there is a lack of information about its transcriptional activity, and, in turn, about the expression pattern of viral genes, whose available data only allow us to assume that EBERs are expressed in these tumors. Hence, further studies are needed in order to elucidate the inconsistencies. In this sense, large and standardized case-control and cohort prospective studies, focusing on cases unrelated to confounding factors for the upper aerodigestive tract, combining different techniques and targets for EBV detection, may contribute to the understanding of the role played in this context. Also, in vitro studies are particularly important to investigate the effects of EBV on laryngeal carcinoma cell lines and possible underlying tumor mechanisms. Taken together, the present findings suggest that EBV may be associated with laryngeal carcinomas and contribute to expanding the knowledge on this oncovirus, in addition to encouraging new studies which, ultimately, may improve the clinical management and prevention of LC cases.


Peripheral blood S100B levels may have potential as a useful biomarker for ASD.The S100 calcium-binding protein beta subunit (S100B) protein, which mostly exists in the central nervous system, is commonly noted as a marker of neuronal damage. We conducted the first systematic review with meta-analysis to compare peripheral blood S100B levels in individuals with ASD with those in healthy controls. A systematic search was carried out for studies published before May 5, 2020. In total, this meta-analysis involved ten studies with 822 participants and 451 cases. The meta-analysis revealed that individuals with ASD had higher peripheral blood S100B levels than healthy controls [standardized mean difference (SMD) = 0.97, 95% confidence interval (95% CI) = 0.41–1.53;  Autism spectrum disorder (ASD) comprises neurodevelopmental disorders specified by stereotypic behaviour, limited social interaction, and impaired communication. The worldwide prevalence of ASD is approximately 1%  protein is commonly noted as a marker of neuronal damage and blood–brain barrier dysfunction  an evaluation of peripheral blood S100B levels in individuals with ASD and (2) a description of S100B levels in the median and range, the mean and standard deviation (SD), or the mean and standard error of the mean (SEM).Exclusion criteria were (1) animal studies, reviews, abstracts, and letters; (2) study populations duplicated in another study; (3) studies that did not measure peripheral blood S100B levels, including pharmacology, genetics, brain imaging, and postmortem studies; and (4) studies that showed peripheral blood S100B levels in dot plot or histogram format, with no numerical results provided.Data on the author, number of samples, number of males, participant age and publication year were extracted. Data on country, ASD diagnostic criteria, adjusted confounders, study design, analytical technology, biomaterial, S100B and measurement unit were also extracted. Different measurement units  were used in the studies. All S100B levels were reported in pg/ml in this meta-analysis. If data were expressed in terms of the median and range, the sample mean and SD were calculated according to the method of Wan et al.  Stang . The stu2 and Q statistics. The I2 values of 0.25, 0.5, and 0.75 revealed small, medium and high heterogeneity, respectively.The standard mean difference (SMD) was summarized to calculate peripheral blood S100B levels in ASD. Heterogeneity was determined by the IPublication bias was confirmed by funnel plot analysis as well as by Begg’s and Egger’s tests. Subgroup analyses were carried out by using study design, biomaterial, analytical technology, and geographic location. Meta-regressions were conducted to evaluate whether sample size, mean age, publication year, and gender affected the outcome. Sensitivity analysis was carried out by the omission of studies one by one to evaluate whether any study changed the overall outcome. Stata 12.0 and Review Manager 5.1.2 software were used to perform the statistical tests.We first conducted a systematic search and identified 16 studies from PubMed, 29 studies from Embase, two studies from the Cochrane Library, 38 studies from Scopus, 26 studies from the Web of Science, 16 studies from China National Knowledge Infrastructure, nine studies from the Wanfang database, and two additional studies from the scanning of references. After reviewing titles and abstracts, 13 papers on peripheral blood S100B levels in ASD were selected for full-text review. Subsequently, one study was excluded due to its irrelevant topic. One report without sufficient data was excluded. One report was excluded for not being published in English. Eventually, 10 studies with 822 participants and 451 cases met the criteria for inclusion in the present meta-analysis    = 0.41–1.53; The quality evaluation results showed that the ten studies were high quality. The mean score was 7.7 Table .Table 2Qp = 0.37; Egger’s test, p = 0.07).Asymmetry was found by funnel plot analysis Fig. . Begg’s p < 0.001) for cross-sectional studies and 0.12  for case–control study. Further subgroup analysis of biomaterials showed that individuals with ASD had higher peripheral blood S100B levels in serum [0.62 ] but not in plasma [2.63 ]. In addition, we conducted subgroup analysis on the basis of which analysis techniques were applied. The effect values of S100B levels in individuals with ASD were 0.94  for WB and 0.97  for ELISA. We also carried out subgroup analysis in terms of geographic locations. The results demonstrated that individuals with ASD had higher peripheral blood S100B levels in other countries [0.5 ] but not in China [0.6 ] or Turkey [2.09 ]  significantly influenced the results Table .Table 4MSensitivity analysis revealed that the overall results were not affected by a particular study.To our knowledge, this is the first meta-analysis to explore peripheral blood S100B levels in ASD. Pooling the ten studies, 822 participants and 451 cases were encompassed. Our study showed that individuals with ASD had higher peripheral blood S100B levels than healthy controls. Sensitivity analysis indicated that the outcome was not unduly influenced by a particular study. In addition, no obvious publication bias was observed. This study demonstrated that individuals with ASD had higher peripheral blood S100B levels.High levels of heterogeneity were detected in this study. Subgroup analyses revealed that different study designs contributed to the heterogeneity. Individuals with ASD who had higher peripheral blood S100B levels were detected in cross-sectional studies but not in case–control study, indicating that the association may be affected by the study design. But this analysis may have been underpowered owing to only one case–control study was included in the subgroup. This potential confounding factor needs to be considered in the future analysis of S100B in individuals with ASD.Individuals with ASD had higher S100B levels when measured in serum but not when measured in plasma. In the meta-analysis, most studies provided serum S100B levels; therefore, enough data were available to conclude that ASD is correlated with elevated serum S100B levels. Evidence that changes in serum S100B levels may reflect changes occurring in the brain has been reported (Vos et al. Interestingly, the peripheral blood S100B levels were higher in individuals with ASD than in healthy controls when measured by ELISA but not when measured by WB, indicating that different assay methods may affect the correlation. Assay properties such as sensitivity and inter-assay and intra-assay coefficients of variability should be considered. In addition, differences in the way samples are collected and handled might contribute to the discrepancies in reported S100B levels in individuals with ASD. But only one study utilized WB in the subgroup, the result was not robust. Further investigations are needed to explore the correlation between S100B levels measured by WB and ASD.In addition, individuals with ASD were discovered to have higher peripheral blood S100B levels than healthy controls in other countries, but there was no difference between the groups in China or Turkey. Genetic factors, environmental factors, lifestyle and economic conditions may contribute to this difference.Furthermore, some clinical confounders were not analyzed in our meta-analysis, such as BMI, leptin and other adipose-related factors, may contribute to the observed heterogeneity. Scientists have reported that S100B levels were closely correlated with BMI, leptin and other adipose-related factors (Steiner et al. The mechanisms of increased S100B in ASD are not completely clear, but several mechanisms have been proposed. First, autoimmunity may be involved in the development of ASD (Edmiston et al. This meta-analysis had the following strengths. First, this is the first meta-analysis carried out to evaluate peripheral blood S100B levels in ASD. Second, no significant publication bias existed, indicating the high reliability of the results. Third, the sensitivity analysis found that no study could change the outcome, demonstrating that the conclusion was robust.However, there were some limitations. First, because of the small sample size, studies with larger samples are required to illustrate the role of peripheral blood S100B levels in ASD. Second, since only a few studies analyzed the severity of ASD and S100B levels, the correlation between them was not evaluated. Future studies should pay attention to this subject. Third, the meta-analysis provides us with comprehensive results mainly from cross-sectional studies. Therefore, whether S100B levels are the cause or the result of ASD development remains unclear. Fourth, significant heterogeneity was found in this meta-analysis. Random-effects, subgroup and regression analyses were performed, and different study designs explained the heterogeneity. But residual confounding factors such as a wide range of mean S100B values reported across studies, which may be related with the different measurement units and different analytical technologies used in the included studies, are still a problem worthy of attention. Therefore, heterogeneity may influence the accuracy of the final results.Despite these limitations, this study may have clinical application value for using S100B levels in the early identification and evaluation of ASD progression. This meta-analysis provides clinical evidence that individuals with ASD have increased peripheral blood S100B levels. Scientists have reported that changes in peripheral blood S100B levels may reflect changes occurring in the brain (Vos et al. This analysis provides evidence that individuals with ASD have increased peripheral blood S100B levels. Peripheral blood S100B levels may have potential as a useful biomarker for ASD. Studies with large samples are warranted to investigate the severity of the clinical symptoms and the causal relationship between ASD and S100B levels.


Fever during pregnancy is a relatively common and most often trivial event. However, under specific conditions, it could affect significantly fetal brain development. Few studies, with inconsistent results, investigated whether fever, regardless the pathogen, could represent a risk factor for neurodevelopmental disorders (NDD) in the offspring. We aimed to explore further this question by performing a systematic review and meta-analysis.Peer-reviewed studies exploring the occurrence of NDD in offspring after a fetal exposure to maternal fever were included. We specifically considered the impact of fever severity and duration, taking into consideration some confounding variables such as the use of antipyretic during pregnancy, the trimester in which the fever arose, the maternal age or smoking at time of gestation. MEDLINE, EMBASE, PsycINFO, Cochrane and Web of Science were searched without language restriction. PRISMA recommendations were followed. Odds ratio (OR) were pooled using random-effects meta-analysis. Heterogeneity in effect size across studies was studied using random-effects meta-regression analysis. (PROSPERO CRD42020182801).We finally considered ten studies gathering a total of 10,304 children with NDD. Among them, 1394 were exposed to fever during pregnancy. The selected studies were divided into 5 case–control studies and 5 cohort studies. Maternal exposure to fever during pregnancy increased the risk of NDD in offspring with an OR of 1.24 [95% CI: 1.12–1.38]. Secondary analysis revealed an increased risk for NDD when fever occurred during the first trimester of gestation [OR 1.13–95% CI: 1.02–1.26].We excluded studies that considered infections with no evidence of fever. Another potential limitation may be the possible heterogeneity between study designs (cohorts and case–control).Additional evidence supported the association between fever during pregnancy and increased risk for NDD in offspring. Careful monitoring should be considered for children born from mothers with a febrile episode during pregnancy .The online version contains supplementary material available at 10.1186/s13229-021-00464-4. Neurodevelopmental disorders (NDD) are a heterogeneous group of illnesses that begin during a child’s early brain development  and rangFever during pregnancy is common, with approximately 20% of women reporting at least one febrile episode , 9. DespHowever, the literature on fever during pregnancy remains vague on factors modulating the risk of developing a NDD in offspring. For example, the period of pregnancy during which fetal vulnerability is greatest is poorly known. Based on animal models of MIA and taking into account the development of the fetal brain in utero, we assumed that the end of the first trimester and the beginning of the second trimester of pregnancy could represent the period of greatest risk , 20–22. Herein, we present the results of a systematic review and meta-analysis of the literature which explored the association between prenatal exposure to fever, no matter the cause, and NDD outcomes in the offspring.We performed a systematic review and meta-analysis following the PRISMA recommendations  . StudiesWe searched MEDLINE , EMBASE , PsycINFO , Cochrane Central Register of Controlled Trials , and Web of Science Core Collection  without any restrictions on language, ethnic origins, date or article type. Both cohort and case–control studies were included. The research was based on a combination of medical subject heading (Table The eligibility process was conducted in two separate stages: (1) two researchers (SA and PE) independently screened all non-duplicated references initially retrieved as potentially pertinent and excluded those clearly not pertinent based on title or abstract. A final list was agreed with discrepancies resolved by consensus between the two authors. When consensus was not reached, a third, senior researcher (RD) acted as arbitrator; (2) the full-text version of the articles passing stage 1 screening was downloaded and assessed for eligibility by the two researchers, independently. Discrepancies were resolved by consensus between the two researchers and, if needed, the third senior researcher also acted as arbitrator. When required, corresponding authors were contacted to clarify study eligibility.PROSPERO (protocol number: PROSPERO CRD42020182801).The protocol for the present systematic review/meta-analysis was pre-registered on the international Prospective Register of Systematic Reviews The main outcome measure was the OR expressing the association between fever in mothers during pregnancy and NDD in the offspring. To further estimate the risk of NDD in the offspring, we performed secondary outcome analysis by taking into account: (1) the types of NDD and the criteria used for their definition; (2) the fever related information regarding the trimester in which it occurred, the prescription of antipyretic medication, and its duration (in days) and the highest fever reached during pregnancy; (3) the offspring’s related main confounding variables including age, sex, week gestational age (WGA) at delivery; and (4) the mothers’ related information: age at conception, smoking during pregnancy and personal history of psychiatric disorders. In addition to these variables, we extracted some information regarding authors, year of publication, country in which the study was conducted.To assess the quality of studies included in the meta-analysis, SA and PE used a modified version of the Newcastle Ottawa Scale (NOS) , 31. Bri2 statistic and Tau2. The I2 score represents the percentage of variation across studies due to heterogeneity, and ranges from zero to 100%; higher values indicate greater heterogeneity across studies   = 11, p < 0.001, I2 = 61.02] and we observed a funnel plot asymmetry , with an OR of 1.15 [95% CI 1.01–1.31], and a low evidence of heterogeneity . We also observed a positive association for DD (p = 0.03) with an OR of 1.23 [95% CI 1.07–1.41] and low heterogeneity . There was no possibility to calculate the OR for ADHD, with only two studies, nor for DCD, with only one study.Additional statistical analyses were carried out to stratify by NDD type. There was a positive association for ASD (p = 0.01) for case control studies taken alone with an OR of 1.19 [95% CI 1.03–1.38], with no evidence of heterogeneity . There was also a positive association for cohort studies (p = 0.004) with an OR of 1.20 [95% CI 1.06–1.36], with high evidence of heterogeneity .Stratification by type of study design were also carried out. We found a positive association (p = 0.02) with an OR of 1.24 [95% CI 1.02–1.49], without heterogeneity .We carried out cross stratification between study design and specific NDD type. Only ASD in case control studies were taken into account due to the lack of data. We found a positive association (p = 0.024) was noted during the first trimester of pregnancy , without evidence of heterogeneity   when fever exceeded 3 days in the mother [OR 1.24 (95% CI 0.98–1.57)] compared to a shorter duration . We observed heterogeneity values for a duration longer than 3 days , but not when the studies reported a fever duration shorter than 3 days . Funnel plots showed no publication bias . There was however evidence for heterogeneity in the studies , even in the absence of publication bias  on the risk of NDD in offspring, we performed a meta-regression. The analysis showed a non-significant effect of prolonged duration (p = 0.313), with evidence of heterogeneity . Similarly, the meta-regression model (k = 7) for the use of antipyretic medications confirmed the previous findings with no significant association (p = 0.264) and evidence of heterogeneity   was a significant covariate (p = 0.014), with no heterogeneity   with evidence of heterogeneity . Similarly, no association was observed with maternal smoking during pregnancy  with evidence of heterogeneity .To further examine the impact of fever duration  of 1.24 [95% CI: 1.12–1.38], specifically during the first trimester of pregnancy . This risk was particularly relevant for ASD  and DD .The increased risk of NDD may directly rise from the increased levels of cytokines and imbalance in T cell populations associated with fever, occurring in a critical window of the fetal brain development , 50. ThiInterestingly, we found a significant association between fever during the first trimester of pregnancy and the risk of NDD, in contrast with the last trimester of gestation. Even though the underlying neurobiological hypothesis was not clearly defined, the first two trimesters, specifically between 13 and 27 WGA, entailed an immune profile for appropriate fetal brain growth, characterized by a secretion of Transforming Growth Factor β (TGF-β). The association of fever with increased levels of both Il-6 and TGF-β may directly affect maternal immune cell homeostasis leading to defects in the fetal brain architecture development , 54.Surprisingly, we did not observe that fever duration or anti-pyretic medications during pregnancy influenced the neurodevelopmental risk , 27, 44.The meta-regression we ran on maternal confounding factors reported a significant cross-effect for maternal age over 35 years old. This was consistent with previous findings that provided evidence for advanced maternal age as a substantial risk factor for NDD, specifically ASD . We wereEmerging infections drew attention to the impact of fever on the offspring of infected pregnant women . The recAlthough our results were congruent with previous findings in the literature, our meta-analysis should be considered in light of its limitations. First, the inclusion process excluded studies that considered exposure to infections without information on fever. As suggested previously, distinguishing fever outcomes from those of infections might be difficult, giving the fact that both conditions could enhance MIA , 26. FevIn conclusion, our results provided further evidence for an association between maternal fever during pregnancy and NDD risk in the offspring. In line with our results, and in the absence of consensus, it is wise to treat maternal fever accordingly, in order to prevent the previously described subsequent neuroinflammation . In the Additional file 1. Full data extracted and funnel plots.


PPARG) contains thousands of polymorphic loci, among them two polymorphisms (rs10865710 and rs7649970) in the promoter region and two polymorphisms (rs1801282 and rs3856806) in the exonic region were widely reported to be significantly associated with coronary artery disease (CAD). Mechanistically, PPARG polymorphisms lead to abnormal expression of PPARG gene and/or dysfunction of PPARγ protein, causing metabolic disorders such as hypercholesterolemia and hypertriglyceridemia, and thereby increasing susceptibility to CAD.Being activated by endogenous and exogenous ligands, nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) enhances insulin sensitivity, promotes adipocyte differentiation, stimulates adipogenesis, and has the properties of anti-atherosclerosis, anti-inflammation, and anti-oxidation. The Human PPARγ gene ( Being activated by endogenous and exogenous ligands, PPARγ works in concert with retinoid X receptor (RXR) and is able to increase the insulin sensitivity, (PPARG) and CAD, as well as the underlying mechanisms.Coronary artery disease (CAD) is the most common type of cardiovascular disease globally, and is often caused by stenosis of coronary arteries due to atherosclerosis , 2. Undeitivity,  promote itivity,  and stimitivity,  and has itivity, , 28 antiitivity, , 28 and itivity, . This rePPARG is located on the chromosome 3p25.3 and composed of nine exons: exon A1, exon A2, exon B, and exons 1–6 , (Human xons 1–6 . There iARG mRNA . Accordiymorphic . A large649970), , 33 exon649970), , 33 and 649970),  accordinPPARG are associated with CAD . Ding et al. . Our research team recently conducted a case–control study to assess the association between the rs10865710 polymorphism and CAD severity among Chinese patients, and observed that G allele carriers had higher Gensini scores (an indicator of CAD severity) (p < 0.05) and more diseased coronary branches than patients with CC genotype (p < 0.05)  is located in the upstream promoter region of PPARγ3 gene and formed by a transversion from cytosine (C) to guanine (G) –37 Figu. Accordi studies , 36 in C studies  found th < 0.05) , 60, 64.OR, 1.67; 95% CI, 1.03–2.71; p = 0.04). In another Chinese population, T allele of the rs7649970 polymorphism was found to be an independent risk factor for myocardial infarction (MI) after adjustment for traditional risk factors  .The rs7649970 polymorphism  is located in the upstream promoter region of PPARγ2 gene and formed by a transition from C to thymine (T) , 39 Fig. Accordi < 0.01)  observedPPARG such as c.25924C > T and c.26233T > A in PPARγ3 promoter, and c.93640T > C, c.93673T > C, and c.93695C > T in PPARγ4 promoter to CAD were explored in an Italian population, and the explorers found that the c.93695C > T polymorphism was significantly correlated with acute coronary syndrome (ACS); T allele conferred a protective effect against ACS at both univariate  and multivariate  analyses . In a case–control study carried out in Inner Mongolia, China, the investigators concluded that G allele of the rs1801282 polymorphism was an independent risk factor for MI after adjustment for conventional risk factors   among Egyptian patients with type 2 diabetes mellitus (T2DM). Maciejewska-Skrendo et al. . The association between the rs1801282 polymorphism and CAD appeared to be gender-dependent. Vogel et al. , but not among women. Similarly, Schneider et al. (p = 0.001). In a prospective cohort study involving middle-aged French men, the subjects with GG genotype of the rs1801282 polymorphism had a marginally insignificantly higher risk of CAD than those with CC genotype   is located in exon B of titution –51  . Hasan e = 0.04)  demonstro et al.  found thl et al.  demonstr = 0.06) .HR, 4.38; 95% CI, 1.03–18.57; p = 0.05). A few studies involving American and European Caucasians confirmed that G allele of the rs1801282 polymorphism was associated with a lower risk of CAD , and the ACS patients carrying one or two G alleles had lower Gensini scores (p < 0.001) and less number of diseased coronary arteries (p < 0.001) than those with CC genotype.Somehow, several case–control and prospective cohort studies have come to an opposite conclusion, demonstrating that G allele of the rs1801282 polymorphism was significantly associated with a reduced risk of CAD –51. Ho ek of CAD –50. In aMany well-designed studies have failed to detect a significant association between the rs1801282 polymorphism and CAD –76. ThrePPARG polymorphisms with environmental factors on cardiovascular risk factors, i.e., different alleles have different impacts on the expression patterns of PPARG under different environmental conditions. Abaj et al.  and in Caucasians , but not in Asians. However, the results from several other meta-analyses did not support this finding and concluded that the rs1801282 polymorphism was not associated with CAD in overall and subgroup analyses . Zhou and the other two teammates . The protective effect of T allele on CAD was also reported by other explorers in Chinese populations (p = 0.02), but not among patients free of T2DM (p = 0.70).The rs3856806 polymorphism  is located in exon 6 of n PPARγ2 –64 . A significant association between T allele of the rs3856806 polymorphism and higher risk of ACS was detected among Chinese mainland residents  (p = 0.03) ,  (p = 0.03)   and Ital = 0.03) . In a Fr = 0.03) .Some researchers failed to detect any significant association of the rs3856806 polymorphism with CAD , 79–82. OR, 0.69; 95% CI, 0.59–0.82; p < 0.001). Gonzlez-Castro et al. . However, Ding et al. . In addition, the results from several other meta-analyses indicated that the rs3856806 polymorphism was not correlated with CAD at all . Tian et al. . The rs709158 polymorphism is located in intron 5 of PPARG and formed by a transition from A to G. Gallicchio et al. , but G allele carriers had significantly lower levels of triglycerides than the subjects with CC genotype (p < 0.001) , the levels of total cholesterol, LDL-C, and HDL-C were comparable between the rs1801282 genotypes (CG + GG < 0.001) . Koohdan< 0.001)  also repSeveral studies demonstrated that the T allele of the rs3856806 polymorphism was correlated with decreased levels of triglycerides,  total chA few studies have produced conflicting results that the T allele of the rs3856806 polymorphism was correlated with higher levels of atherogenic lipids , 62, 79.A couple of studies , 39 showHypertension is a recognized risk factor for CAD. Ettehad et al.  reportedZ-score) frequency than CC homozygotes (Body mass index (BMI), waist-to-hip ratio (WHR), and waist circumference (WC) are common indicators of obesity and are closely related to CAD –119. Sevozygotes . Zafar eozygotes  found thozygotes , 53.Atherosclerosis is actually an ongoing chronic inflammatory disorder, not just a simple lipid deposition on the intima and media walls of blood vessels , 128. A PPARG polymorphisms and CAD. Two polymorphic loci (rs1801282 and rs3856806) in the exonic region of PPARG were extensively explored in various populations and were reported to be significantly associated with CAD, but the risk alleles of these polymorphic loci are still elusive. In the future, multi-center, multi-ethnic, and large-sample case–control and cohort studies are needed to identify PPARG risk alleles for CAD. In addition, great efforts are required to assess the interactions between PPARG polymorphisms and environmental factors on the expression patterns of PPARG, the function of PPARγ, and the susceptibility to CAD.With the increase of morbidity and mortality in patients with CAD in recent decades, researchers from all over the world have carried out a large number of observational and experimental studies on the associations between All authors retrieved the literature and wrote and organized the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Recent theory on the “gut-brain axis” suggests a close relationship between the dysfunction of the gut and the disorders of the brain.We performed a systemic literature search followed by a multi-step inclusion selection for all studies on the risk of Colorectal cancer (CRC) in Parkinson’s disease (PD) patients using the following databases: PubMed, EMBASE and WOS. Relative risk (RR) and the 95% confidence intervals (CI) were calculated using either the random-effects model or the fixed-effects meta-analysis model, based on the assessment of heterogeneity.Seventeen studies involving a total of 375,964 PD patients and 879,307 cancer patients were included. Independent meta-analyses for cohort studies and case-control studies showed that the overall pooled RR of the cohort studies was 0.78 (0.66–0.91), and that of the case-control studies was 0.78 (0.65–0.94), indicating that patients with PD have a significantly decreased risk for CRC. The significant lower risk is present in both the colon and the rectum subgroups classified by tumor location. Moreover, the risk for CRC is significantly lower in America (RR = 0.58), Europe (RR = 0.82) and Asia (RR = 0.83) compared to the control population.The occurrence of CRC was significantly lower in patients with diagnosis of PD.The online version contains supplementary material available at 10.1186/s12877-021-02497-z. Parkinson’s disease (PD) is one of the most common neurodegenerative diseases characterized by motor dysfunction, such as resting tremor, rigidity, hypokinesia and postural instability , as wellColorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer death worldwide . CRC ranThese evidences from both the PD and CRC research fields have suggested a potential interaction between the pathogenic mechanisms of the brain and the gut. Previous epidemiology studies conducted in China showed inconsistent results, indicating the necessity of our research. The goal of the present study is to use the public database to explore the disease risk association between PD and CRC. In the recent 20 years, accumulating epidemiological studies have revealed that patients with PD may be associated with a lower risk of certain cancers –14, howeRelevant studies from January 2000 to April 2020 were collected from the three major online databases including PubMed, Web of Science and EMBASE by two independent investigators (Hongsheng Fang and Jiayin Tang). We input PARKINSON DISEASE, PARKINSONISM, TUMOR, NEOPLASM, and CANCER as Medical Subject Heading (MeSH) terms and then connected through Boolean operators. We placed no restrictions on the region of residence or the age of the subjects, but we restricted the search to studies including human study participants. Moreover, the relevant reviews and references of articles were also manually screened to identify additional related studies that may supply relevant data. We conducted this meta-analysis according to the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) .Inclusion criteria were defined as follows: (1) Studies are either cohort or case-control studies about the CRC risk of PD patients ; (2) An estimate of association  can be collected from the study; (3) The distribution data could be obtained by contacting the author of a relevant report; (4) Studies contain the risk of CRC after the diagnosis of PD (Studies concerning the risk of CRC before the PD diagnosis were excluded.); (5) When duplicated studies were identified, only the most informative study was included.The quality of the included articles was scored using the Newcastle-Ottawa scale (NOS)  by two i2 statistics [2 is less than 50% and the random-effects meta-analysis model was used when I2 is higher than 50% [P value less than 0.05.STATA 15.0  was used to perform data analyses in this study. The pooled relative risk (RR) at the 95% confidence interval (95% CI) was assessed to evaluate the association between PD and CRC. The RR and the 95% CI was calculated using the “inverse variance” method; and the statistical heterogeneity was evaluated using the Iatistics . The fixthan 50% . The RR than 50% . StatistThe systemic search result and the subsequent eligibility selection workflow is shown in Fig. p<0.001, I2 = 90.4% = in the cohort studies .The pooled RRs of the overall CRC risk in PD patients was 0.78  for case–control studies. In analyses stratified by tumor location, the combined risk for colon tumor with PD is 0.68  (p<0.001) (p<0.001) (p<0.001) (p = 0.442) (2) in the Asia group would reduce from 97 to 0% if excluding the study with OR of 1.47.We carried out a series of subgroup analyses based on the tumor location and the region of population, subgroups are selected only from the cohort studies Table . We founp<0.001) , and thap<0.001) . To segrp<0.001) , the avep<0.001) , followep<0.001) , in the = 0.442) . PD patiIn our meta-analysis, sensitivity analysis was conducted to assess the stability of the results. The persistent inverse association between PD and CRC risk did not change in the sensitivity analysis, which was conducted by omitting one study per iteration. .p = 0.319) showed no significant evidence for bias in the data, Begg’s test (p = 0.951) was also not strongly suggestive of publication bias, thus confirming the absence of obvious publication bias in these studies  It was unclear if the results represented the risk of cancer influenced by PD, or vice versa; (2) The data collected in that study was incomplete; (3) It is methodologically wrong to mix the results from the cohort studies and the case-control studies due to the different design of the two studies.Our study has overcome the aforementioned weaknesses in the previous work and examined specifically the risk of CRC in patients with diagnosis of PD. We analyzed 17 studies involving 375,964 PD patients and 879,307 cancer patients. The pooled results for all populations indicated that PD patients have a decreased risk of CRC in Western population. Subgroup analysis showed that the significant inverse relationship between PD and risk of CRC is not affected by differences in types of study design, tumor location, or different regions of the Western population.2 = 97%) and the significance is not as clear as data from the American and European groups(P = 0.442). We think the reason could be due to the wide variability of the Asian data. Some large-scale studies on the Asian population are unfortunately not completed like the similar studies for the Western population. For the two studies that are completed in Taiwan, China, the results are opposite. The RR of the study completed by Sun [The inverse association between PD and CRC in the Western population is statistically significant in our study. However, the association in the Asian population remains obscure. We found contradictory conclusions in published studies. In our meta-analysis, we identified a modest lower risk of CRC in patients with PD (RR = 0.83) with a high level of heterogeneity  of available datasets. It is worth noting that publication bias and other forms of bias may still exist, and a more detailed subgroup analysis is incomplete due to the insufficient data from the primary articles. Nonetheless, our study has several strengths, including its comprehensive literature search for the latest data, large number of cases, careful assessment of the quality of evidence, which altogether made the results more reliable compared to earlier studies.In conclusion, our research suggests that patients with PD predict a lower risk of CRC. Further studies are warranted to explore the underlying mechanisms of this correlation and to prevention and treatment of both diseases.Additional file 1: Fig. S1 Forest plot of subgroup analysis (colon). Fig. S2 Forest plot of subgroup analysis (rectum). Fig. S3 Forest plot of subgroup analysis (America). Fig. S4 Forest plot of subgroup analysis (Europe). Fig. S5 Forest plot of subgroup analysis (Asia). Fig. S6 Sensitivity analysis of cohort study. Fig. S7 Funnel plot of cohort study. Fig. S8 Egger’s tests of cohort study.Additional file 2: Table S1 The quality of the included studies assessed by NOS.


Folic acid (FA), as a synthetic form of folate, has been widely used for dietary supplementation in pregnant women. The preventive effect of FA supplementation on the occurrence and recurrence of fetal neural tube defects (NTD) has been confirmed. Incidence of congenital heart diseases (CHD), however, has been parallelly increasing worldwide. The present study aimed to evaluate whether FA supplementation is associated with a decreased risk of CHD.Q and I2 statistics were used to test for the heterogeneity.We searched the literature using PubMed, Web of Science and Google Scholar, for the peer-reviewed studies which reported CHD and FA and followed with a meta-analysis. The study-specific relative risks were used as summary statistics for the association between maternal FA supplementation and CHD risk. Cochran's P < 0.001, I2 = 92.7%). FA supplementation within 1 month before and after pregnancy correlated positively with CHD , and high-dose FA intake is positively associated with atrial septal defect . Pregnant women with irrational FA use may be at increased risk for CHD.Maternal FA supplementation was found to be associated with a decreased risk of CHD . However, the heterogeneity of the association was high (Data from the present study indicate that the heterogeneity of the association between maternal FA supplementation and CHD is high and suggest that the real relationship between maternal FA supplementation and CHD may need to be further investigated with well-designed clinical studies and biological experiments.The online version contains supplementary material available at 10.1186/s12937-022-00772-2. Folic acid (FA) is a synthetic  form of folate. Clinical and epidemiologic studies have demonstrated that folate deficiency during pregnancy can lead to birth defects, such as fetal neural tube defects (NTD) –3. To prIn China, the public health policy of FA supplementation for pregnant women originated from the US-China joint research project, which successfully reduced the incidence of NTD by 41–79% . From 19We searched the literature with a cut-off date of June 30, 2021, using PubMed, Web of Science and Google Scholar, for the peer-reviewed studies with English abstracts which reported CHD and FA. The main search terms used were  and . In addition, we searched for the studies that using the key words of coronary artery disease and birth defects and examined the relevant references. We further followed published quality standards for conducting the meta-analyses .We selected the articles that (1) were original epidemiologic studies and clinical control studies , (2) examined the association between periconceptional FA use and either CHD overall or ASD  subtypes in infants, (3) were published in the English language, (4) either the results reported as risk ratios or odds ratios (OR) and 95% confidence intervals (CI) or provided raw data from which these measures could be calculated, (5) defined CHD or ASD  subtypes as an outcome. We also excluded the results of research in the pregnant women with diseases . In addition, non-peer reviewed articles and the studies with experimental animals, concerning ecological assessments, and mechanisms were excluded. Articles that reported results contained multiple populations were considered to consist of separate studies, with one study for each population investigated. Only first published article or the largest number of cases was included in our study, when multiple articles were found to examine the same study.Data extraction and quality assessment were completed independently by two researchers to reduce the bias and errors of in the date extraction process. Disagreements between investigators were resolved through discussions until a consensus was reached. The following study characteristics were recorded: publication year, geographic region, the sample size, case classification information, exposure, and outcome assessments, adjusted estimates and their corresponding 95% Cl and confounding factors that were controlled for by adjustments in the data analysis. Because a proportion of women might take a multivitamin containing FA during pregnancy, in the current study, we thus analyzed the data separately and found similar results when taking FA alone or taking a multivitamin , geographical region , study quality  and relevant confounder . In addition, we have made statistics on the time and dose of FA intake. We evaluated heterogeneity between subgroups by meta-regression analysis. A P-value of < 0.05. All statistical analyses were performed using STATA software .Publication bias was assessed by via visual inspection of a funnel plot for asymmetry using Egger's linear regression  and BeggThe initial search returned a total of 1,824 potentially eligible publications from databases. Finally, a total of 21 studies involving 106,920 CHD individuals were included for the analysis. The process of identification and selection in the studies for the current meta-analysis is summarized in Fig. I2 = 0%, P = 0.429); the high-dose group was 1.06 , with no significant heterogeneity between studies .Figure As presented in Fig. As seen in Fig. The initiation of FA supplementation was represented by three variables: within 1 month before pregnancy, within 1 month after pregnancy, after 1 month of pregnancy, the ORs of ASD in offspring was 1.09 , 1.11 , 1.02  compared with the reference group with no FA intake Fig. C. The inP < 0.001, I2 = 92.7%), with no publication bias , with no publication bias , we found that the heterogeneity of studies for possible association between FA supplementation and CHD was significant (I2 did not decrease substantially when any individual study was removed. Subsequently, a subgroup analysis was carried out on the studies of CHD and maternal FA supplementation. In the subgroup analyses, the corresponding pooled OR was not materially altered in any stratification (Please see Supplemental Figs. To clarify the sources of heterogeneity, we conducted a sensitivity analysis. However, Table We have conducted a meta-analysis of the recent 21 studies concerning maternal FA supplementation and CHD. Although the data from our analysis implicate that maternal FA supplementation is associated with the reduced risk of CHD, the heterogeneity of this association is high. First, the association is presented to be geographically different. The reports from China suggest that FA supplementation is associated with the decreased incidence of CHD –34, whilAccording to clinical phenotypes, ASD is classified as one of subtypes of CHD . In the In epidemiological studies, there are many factors that may affect the analysis of the results and the results of the analysis. Differences in methods used for studies are probably the most common factor contributing to heterogeneity. Some scholars have proposed that the addition of multivitamins containing folic acid may improve the effect of primary prevention of CHD compared with the use of FA alone . To exclSeveral studies have demonstrated that the continued FA supplementation after pregnancy may increase the risk of large-for-gestational-age birth , childhoData from the current meta-analysis suggest that although the maternal FA supplementation seems associated with a decreased risk of CHD, the heterogeneity of this association is significantly high. The heterogeneity may be caused by the confounders such as timing and dose of FA administration, and the heterogeneity may subsequently influence the outcome on actual effect of FA supplementation on CHD. On this basis, we believe it is necessary to correctly assess the association of FA supplementation with CHD. Further experiments designed to study the association between FA and CHD and its molecular mechanisms have been taken into consideration.Additional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.