Asthma is a common multifactorial disease affecting millions worldwide. The Barker hypothesis postulates an association between later onset disease risk and energy exposure in utero. Birth weight corrected for gestational age is better for measuring the infant size, which reflects energy exposure in utero. Findings on asthma and birth weight corrected for gestational age have been inconclusive. We conducted a meta-analysis to further clarify the relationship between birth weight corrected for gestational age and later onset asthma.A systematic literature search of the PubMed, Web of Science, MEDLINE, and Scopus databases up to January 2021 was conducted. The subject terms were used as follows: “asthma”, “allerg*”, “respiratory”, “birth weight”, “gestational age”, “birth outcomes”, “intrauterine growth retardation”, and “fetal growth restriction”.We included 12 articles with data from a total of 6,713,596 people. Compared with non-SGA infants, infants small for gestation age (SGA) were not associated with an increased risk of asthma . However, in the subgroup analysis, we found an increased risk of later onset asthma among SGA in studies conducted in Asia, with a large sample size, and defined asthma through medical records rather than questionnaires. Large for gestational age (LGA) was not associated with an increased risk of asthma when non-LGA or appropriated for gestational age (AGA) infants were used as the reference .These results indicated that neither SGA nor LGA was associated with an increased risk of asthma. However, considering the limitations of the research, these results should be interpreted with caution. Asthma is a common multifactorial disease affecting millions worldwide . Its etiHowever, most studies assessing risk factors for asthma development have been limited to life events that occur long after birth. Humans are influenced by various environmental factors from the moment of conception . The BarHowever, a major limitation of many of these studies was that they did not consider the gestational age of the infants. LBW may be a proxy for prematurity, which is an independent risk factor for respiratory morbidity in childhood . TherefoWe performed a literature retrieval including the databases PubMed, Web of Science, MEDLINE, and Scopus using the terms “asthma”, “allerg*”, “respiratory”, and “birth weight”, “gestational age”, “birth outcomes”, “intrauterine growth retardation”, and “fetal growth restriction” in the title. The search was conducted through January 2021.The references of the literature selected for the present study were also examined to improve the recall rate.To be eligible, a study must meet the following criteria: it must have been an original document assessing the relationship between BW/GA and asthma, and there were sufficient data to calculate the required results. In addition, BW/GA should be reported in categories within accepted ranges , birth weight between the 10th and 90th percentile for GA; LGA, birth weight above the 90th percentile for GA). The reasons for exclusion were as follows: (1) if the paper was a review or comment; (2) if the full text could not be obtained; (3) it was a duplicate.The primary outcome was asthma and the diagnosis was obtained from medical records or questionnaires in all included studies. The main exposure was BW/GA, including SGA, AGA, LGA.First, two reviewers screened the literature by reading titles and abstracts independently. Then, they read the full text to determine whether the previously selected articles fulfilled the inclusion criteria. They were included in the meta-analysis if they fulfilled the eligibility criteria. If there were discrepancies between the reviewers, the inclusion or exclusion of the article was decided by the third reviewer.The Newcastle–Ottawa instrument recommended by the Cochrane Collaboration was used to assess the quality of the included cohort and case–control studies. It contains eight questions in three areas: selection, comparability, outcome or exposure. The article receives a star when it meets one term criterion, and the criteria for the grades of study quality are (1) low quality—when the article gets no more than 3 stars, (2) moderate quality—gets 4 to 6 stars, and (3) high quality—gets 7 to 9 stars. The cross-sectional study appraisal tool developed by the Joanna Briggs Institute (JBI) was used to assess the cross-sectional studies and it contains eight evaluation items. Studies were assessed based on the subjects, diseases, the measurement of influencing and confounding factors and the data analysis. The reviewers use “yes”, “no”, “unclear” or “unsuitable” for judging each item.Data were extracted from the included studies by two researchers separately and then they were cross-checked. The data contained the following fields: author’s name, year of publication, country of origin, study design, characteristics of the participants, and the outcomes.2) value to quantitatively judge the heterogeneity. A fixed-effects model was applied when there was no significant heterogeneity (I2 ≤ 50%). Otherwise, the random-effects model was used (I2 > 50%). Subgroup analyses and sensitivity analysis were applied to explore the potential sources of heterogeneity, and the latter was also performed to assess the robustness of the results. Publication bias was evaluated by using Egger’s test [Statistical analysis was conducted using STATA 14.0. The odds ratio (OR) was used as the measure for dichotomous outcomes, and the 95% confidence interval (CI) for each outcome was estimated to reflect the uncertainty of the point estimates. The unadjusted risk estimates were calculated only when the adjusted risk estimates were unavailable. The effect model for the statistical calculation was selected according to the heterogeneity. Statistical heterogeneity was assessed by chi-square test and it was combined with the I-squared , and the results from this analysis revealed that infants with SGA were not associated with an increased risk of later onset asthma  , and the pooled estimates revealed no association between LGA and an increased risk of later onset asthma   Table .Subgroup analyses of SGA compared with non-SGA were stratified by study design, study sites, sample size, and methods of obtaining the diagnosis of asthma to investigate the potential sources of heterogeneity. The results of the subgroup analyses revealed that SGA was associated with an increased risk of asthma in studies conducted in Asia, with sample sizes ranging from 50,000 to 100,000 and more than 100,000, and they defined asthma through medical records. The sample size could probably be regarded as the source of the heterogeneity between studies .Examination of the included investigations did not show a significant effect of publication bias. We assessed the potential publication bias by applying Egger’s test, and it was not statistically significant (In the present meta-analysis, 12 articles reporting 14 original studies with 6,713,596 subjects were included. By conducting a systematic review, we estimated that there was no association between BW/GA and an increased risk of later asthma. After removing the studies of Grischkan , Koshy , and MiySGA is defined as infants with a birth weight below the 10th percentile for GA or below 2 S.D. of the reference population mean for BW/GA, which is different from the preterm or LBW population . SGA infSome studies found that SGA was strongly associated with an increased risk of asthma only when subjects were stratified by certain factors, such as maternal smokingSubgroup analysis for the sample size showed low heterogeneity among the subgroups, and SGA became a risk factor for asthma when the sample size was more than 50,000. A large sample size is more representative, and a more accurate estimation can be obtained to improve the accuracy of the results. In the forest plot, the 95% CIs for large samples were narrower. A twin study showed that low birth weight is associated with adult-onset asthma, and the analysis suggested that the findings were unlikely to be confounded by genetic or shared environmental factors . TherefoStudies on the relationship between LGA and asthma were contradictory; some articles found no correlation , 23, whiSeveral limitations of this meta-analysis should be considered. First, half of the included articles were cross-sectional, which could not provide a direct causal link between BW/GA and asthma. However, most of these studies had a large sample size, and the findings in cross-sectional studies were consistent with those in cohorts. Second, many studies did not provide a specific gestational age, which may be a potential source of bias in the present article. Preterm birth is associated with LBW, which is a risk factor for asthma. However, we found that the outcomes in full-term infants with SGA were consistent with those in infants unrestricted gestational months. Furthermore, the diagnosis of asthma in young children is considered less accurate because of its clinical instability in the early years of life . Last, tGenerally, this meta-analysis showed that infants with SGA or LGA were not associated with an increased risk of later asthma. Additional stratified analyses need to be published to explore the effect of perinatal synergistic mechanisms on asthma.


In recent decades, forests have expanded from natural resources for conservation and production to health-promoting resources. With the growing body of evidence supporting the therapeutic effects of forests, the number of investigations on the relationship between forest characteristics and therapeutic effects have increased. However, quantitative synthesis of primary studies has rarely been conducted due to a limited number of health studies including forest description and high heterogeneity of forest variables. In this study, we conducted a systematic review and meta-analysis to evaluate the relationship between the forest structure and the therapeutic effect. We systematically searched the studies examining the therapeutic effects of forests with different stand density and canopy density and synthesized the results. As a result of subgroup analysis, we found that stand density modifies the therapeutic effects. Emotional and cognitive restoration showed greatest improvement in low-density forests with a stand density of less than 500/ha and the therapeutic effects diminish as the stand density increases. The impact of canopy density was not found due to a lack of studies reporting canopy density. Although some limitations remain, the findings in this study have great significance in providing the basis for establishing management strategies of forests for therapeutic use. Human use of nature to promote mental and physical health has a long history and continues accumulating plenty of empirical evidence. In the 1980s, two representative theoretical frameworks were proposed that explained how experiencing nature benefits human health. One is the attention restoration theory (ART) which suggests cognitive recovery through experience in nature. According to ART, natural environments are rich in components aiding recovery from mental fatigue . ExposurSince 2007, some East Asian and European countries have started to utilize urban forests, peri-urban forests, and conservation areas to improve public health and well-being ,15,16,17Recently, studies have started to examine the relationship between therapeutic outcomes and various forest variables. Investigations were conducted on a wide range of forest variables such as spatial structure ,60,61, oThe structure of the forest affects the sense of openness, lightness, comfort, and aesthetics that humans feel, so it can be expected that the structural characteristics will change the therapeutic effect of the forest. Further, we focused on the density of trees, a major component of the forest environment. Previous studies revealed that high-density trees limit visual access and ease of movement, and may disturb immersion in nature in that it threatens a sense of security ,74,75. CA systematic review is a research method that summarizes the latest empirical evidence relevant to a specific research question, and meta-analysis is a statistical method that quantitatively synthesizes the data from searched studies. In general, meta-analysis is performed to estimate the effect size more accurately by synthesizing homogeneous studies. It can also be conducted to confirm whether study-level variables change the outcomes of each study . The keyWe followed the guidelines of the Cochrane Handbook for Systematic Reviews of Interventions (Version 6.2) , and we P: All human participants .I: Spending time in the forest or urban forest with the description of forest environment in terms of stand density and canopy density—stand density, tree density, trees/ha, basal area, canopy density, canopy openness, canopy closure, sky view factor etc.C: Studies with a control site or not.O: The studies that contained quantitative outcomes related to cognitive restoration, psychological restoration, physiological relaxation, and stress reduction.In order to address a clearly formulated research question, we set our research question by specifying the population, intervention, comparison, outcome (PICO). We also established eligibility criteria following the PICOS framework for the study selection, displayed in We took 24 pilot searches and finalized search keywords to gather relevant studies and obtain reproducible search results. The search keywords contain keywords relevant to forest-based activities, forest variables, and therapeutic effects . We condFrom the database search, we found a total of 932 studies with 180 on PubMed, 68 on EBSCO, 113 on Web of Science, and 571 on Scopus. The results were exported to EndNote Citation Manager software . After removing 159 duplicates, the titles and abstracts of the 773 publications were reviewed. After removing 504 explicitly irrelevant cases, two investigators independently reviewed the full text for 269 studies based on eligibility criteria (E.K. and Y.C.). Disagreements during the screening process were resolved by two other investigators (G.K. and S.P.). We included ten studies from our database search and added two studies that were manually identified. Thus, in total, 12 studies were included in the review .Two investigators (E.K. and S.K.) independently used the same data extraction form and cross-checked them. Data were extracted from individual studies to identify studies, determine the homogeneity between studies, and estimate the effect size of individual studies. The extracted data included study information, samples, forest characteristics, environmental characteristics, intervention, and outcome measurements as follows.Study information: year of publication, author, country, city, study design, conducted date, time of measurementSamples: sample size, gender, and ageForest characteristics: longitude, latitude, altitude, stand density (trees/ha), canopy density (%), diameter at breast height (cm), height (m), dominant tree speciesEnvironmental characteristics: temperature (°C), relative humidity (%), radiant heat(°C), wind speed (m/s), illuminance (lx), noise level (dB)Intervention: activities, activity duration, and frequencyOutcome: measurement indices, pre-measurement (M ± SD), post-measurement (M ± SD), change in measurement (M ± SD), inter-trial correlation2/ha) and growing stock (m3/ha), we converted it into the number of trees per hectare. In the case of canopy density reported as canopy openness, we reversed and unified it.In the case of stand density reported as basal area  to estimate the effect size. The mean, standard deviation, number of samples, and inter-trial correlation of pre-and post-measured values were used for SMD calculation. If the study did not report the inter-trial correlation, we estimated inter-trial correlation according to the recommendations of the Cochrane Handbook . When seThere are two statistical models for meta-analysis. One is a fixed-effect model, which assumes that studies share a common effect size and that differences in results result from sampling error. The other is a random-effects model, which assumes that the true effect size varies by study and is distributed around the overall mean. As we hypothesized that the therapeutic effect of forest varies by forest structure, we used a random-effects model to estimate effect size.p < 0.10 for statistical significance) and I2 (I2 > 50% used as a threshold for significant heterogeneity). We observed whether the two structural variables affected the healing effect by the significance test of subgroup differences and the comparison of heterogeneity according to the subgroup analysis. We also report the 95% confidence interval of SMD for each subgroup.We conducted a subgroup analysis to investigate the relationship between the structural variable of forest sites and their therapeutic effect. We divided structural variables into three or four subgroups based on their homogeneity and calculated the proper sample size using G*Power 3.1. software to ensure a satisfactory power value. To assess inter-study heterogeneity in subgroup analysis, we used Cochrane’s Q test , depression , anger , confusion , fatigue , vigor , negative and positive affect . Cognitive restoration included restorative experience . Physiological relaxation included lowering blood pressure , heart rate and pulse rate , relaxation effects measured through heart rate variability and stress hormone . Additionally, one study measured the blood concentration of monoterpenes as an indicator of therapeutic effect.Prior to the meta-analysis, we reviewed participant characteristics, species compositions, microclimate, noise, and the timing of measurement to rule out possible confounding factors. As a result, nine studies of twelve included studies were used for meta-analysis. Since thermal discomfort can disrupt the therapeutic experience, we excluded three studies that are difficult to be considered homogeneous in terms of environmental characteristics (temperature and humidity). There were some differences in how each forest was managed. However, it was not thought that there would be a significant difference in the perception of the forest environment among the participants, mainly non-experts, as most of the studies investigated conifer-dominant mixed forest.Methodological quality was assessed using ROBINS-I. Overall, the risk of bias was moderate in most studies . At the We classified stand density into four groups: low for <500/ha, medium for 500–1000/ha, high for 1000–1500/ha, and very high for ≥1500/ha. In the case of emotional restoration, improving anxiety, depression, anger, frustration, and fatigue, enhancing vitality, alleviating negative emotions, and promoting positive emotions were reported. The restorative outcome scale (ROS), which measures restorative experience, was reported regarding cognitive restoration. For physiological relaxation, lowering effects on blood pressure, pulse rate, and heart rate were reported. The results for each outcome indices are described in subsections.p < 0.0001; I2 = 76%; 106 participants). As a result of subgroup analysis, there was a significant difference in the effect size according to the stand density , and “medium” effect in high-density environments % CI −0.96 to −0.59; p < 0.0001; I2 = 9%) “small” effect in extremely high-density environments  appeared. One case in an environment with moderate stand density was excluded from the subgroup analysis.Twelve cases with different stand densities were used for meta-analysis. Anxiety state significantly relieved in the forest environment . As a result of subgroup analysis, there was a marginally significant difference in the effect size according to the stand density  appeared, however, no significant effect was shown in high-density environments  and extremely high-density environments . One case in an environment with moderate stand density was excluded from the subgroup analysis.Twelve cases with different stand densities were used for meta-analysis. Depression state significantly relieved in the forest environment . As a result of subgroup analysis, there was a significant difference in the effect size according to the stand density , and a “small” effect in high-density environments  appeared. While there was no significance effect shown in extremely high-density environments . One case in an environment with moderate stand density was excluded from the subgroup analysis.Twelve cases with different stand densities were used for meta-analysis. Anger state significantly relieved in the forest environment . As a result of subgroup analysis, there was a significant difference in the effect size according to the stand density , and “small” effect in extremely high-density environments  appeared. One case in the low- and moderate-density environment was excluded from the subgroup analysis.Eleven cases with different stand densities were used for meta-analysis. Confusion state was significantly relieved in the forest environment . As a result of subgroup analysis, there was a significant difference in the effect size according to the stand density , and a very “small” effect in extremely high-density environments  appeared. One case in the low- and moderate-density environment was excluded from the subgroup analysis.Eleven cases with different stand densities were used for meta-analysis. Fatigue state significantly relieved in the forest environment . As a result of subgroup analysis, there was a significant difference in the effect size according to the stand density  appeared, however, there were no significant effects shown in high-density environments  and extremely high-density environments . One case in an environment with moderate stand density was excluded from the subgroup analysis.Fifteen cases with different stand densities were used for meta-analysis. Vigor significantly improved in the forest environment . As a result of subgroup analysis, there was a significant difference in the effect size according to the stand density , and a “small” effect in high-density  appeared, one case in an environment with extremely high stand density was excluded from the subgroup analysis, and no case with moderate stand density.Nine cases with different stand densities were used for meta-analysis. Negative affect significantly alleviated in the forest environment . As a result of subgroup analysis, there was a significant difference in the effect size according to the stand density  appeared, however, no significant effect was shown in high-density environments . One case in an environment with extremely high stand density was excluded from the subgroup analysis, and no cases with moderate stand density were excluded.Nine cases with different stand densities were used for meta-analysis. Positive affect significantly improved in the forest environment . As a result of subgroup analysis, there was a significant difference in the effect size according to the stand density , and a “medium” effect in high-density  appeared. One case in an environment with extremely high stand density was excluded from the subgroup analysis, and no cases with moderate stand density were excluded.Nine cases with different stand densities were used for meta-analysis. Restorative experience measured by ROS significantly increased in the forest environment . As a result of subgroup analysis, there was no significant difference in the effect size according to the stand density  and extremely high-density environments . No cases with low- and moderate-density environments were reported.Seven cases with different stand densities were used for meta-analysis. Diastolic blood pressure significantly lowered in the forest environment . As a result of subgroup analysis, there was no significant difference in the effect size according to the stand density , however, no significant effect was shown in extremely high-density environments . No cases with low- and moderate-density environments were reported.Seven cases with different stand densities were used for meta-analysis. Systolic blood pressure significantly lowered in the forest environment . As a result of subgroup analysis, there was no significant difference in the effect size according to the stand density , however, no significant effect was shown in extremely high-density environments . No cases with low- and moderate-density environments were reported.Seven cases with different stand densities were used for meta-analysis. Heart rate or pulse rate significantly lowered in the forest environment . As a result of subgroup analysis, there was a marginally significant difference in the effect size according to the canopy density  while no significant effect was shown in moderate-density environments . No cases with low canopy densities were reported.Five cases with different canopy densities were used for meta-analysis. Diastolic blood pressure significantly lowered in the forest . As a result of subgroup analysis, there was a significant difference in the effect size according to the canopy density  while no significance effect was shown in high-density environments . No cases with low canopy densities were reported.Five cases with different canopy densities were used for meta-analysis. Systolic blood pressure significantly lowered in the forest . As a result of subgroup analysis, there was a significant difference in the effect size according to the canopy density  while no significance effect was shown in moderate-density environments . No cases with low canopy densities were reported.Five cases with different canopy densities were used for meta-analysis. There was no significant change in heart rate or pulse in the forest environment . Forest therapy has been studied in earnest over the past decade, and quantitative forest descriptions have only recently begun to be included in intervention information in clinical trials. Most RCTs were excluded from the review process as the information on the forest environment was insufficient. For this reason, we included not only RCTs but also randomized cross-over studies and NRCTs as the scope of this study. Secondly, we extracted effect size using pre-and post- measurements of each forest site rather than differences in forest sites and controls. The studies used in the meta-analysis did not contain the control group examining pre-and post-outcomes outside the forest environment, rather, they assigned the forest site with different condition as controls or did not assign controls among forest settings. Most of the included studies adopted cross-over designs to investigate the difference in the therapeutic effects in different forest settings. Only Bach et al.  and ElsaThe key challenge of this study was to investigate moderating role of stand density and canopy density. Through this study, we found that stand density modifies the level of therapeutic effects in the forest. Our findings had a great significance in suggesting the differences in therapeutic effects according to the environmental characteristics of nature. However, within the scope of this study, we cannot elucidate the underlying mechanisms. Several previous studies exploring differences in preference or beauty according to the characteristics of forest settings explained the differences using semantic difference measures ,50,57,70In addition, we found an intriguing trend as a result of additional analysis. There was a difference in the distribution of the therapeutic effect by region and forest management. The therapeutic effect of the forest reserve was the highest in all regions, and the order of the therapeutic effect of an urban forest and commercial forest differed by region. Future investigation on the impact of management type, landscape characteristics by country, and public use pattern are required.In this study, a systematic literature review and meta-analysis were conducted to evaluate the relationship between the structural characteristics of forests and the therapeutic effect. We systematically searched, selected, and statistically synthesized the literature examining the therapeutic effects of forests with different stand density and canopy density. Subgroup analysis showed that stand density modifies the therapeutic effects. Emotional and cognitive restoration showed the largest effect size in low-density forests with a stand density of less than 500/ha and the therapeutic effects diminished as the stand density increases. The impact of canopy density has not been identified due to a lack of studies reporting canopy density. In addition, there would be a risk of false positives as we did not control the experimental design and control group. Although some limitations, we expect that our findings that highlight the impact of forest structure on the therapeutic effects of forests will contribute to advances in the therapeutic use of forests. We also expect that our findings may be used as a basis for creating adequate forest sites for future forest therapy.


Prospective cohort studies on coffee, tea and caffeine in relation to the risk of rheumatoid arthritis (RA) have shown conflicting results. The aim of this study was to conduct a dose–response meta-analysis of cohort studies on the association between dietary caffeine, different types of coffee and tea consumption and the risk of RA.PubMed/Medline, Scopus and EMBASE were searched up to July 2021 to identify relevant studies that had considered different types of coffee (caffeinated or decaffeinated), tea or caffeine exposure with RA as the main, or one of the, outcome(s). Two authors independently screened 742 publications. Finally, five prospective cohort studies were included in our meta-analysis. Pooled relative risks (RRs) were calculated by using a fixed-effects model. We also performed linear and non-linear dose-response analyses to examine the dose-response relations.I2 = 0%, n = 5) and decaffeinated coffee  consumption and risk of RA. One additional cup of coffee consumed per day was associated with an increased risk of RA by 6% . This increase in the risk of RA for one cup/d of decaffeinated coffee was 11% . No significant association was observed between caffeinated coffee, tea or caffeine intake and the risk of RA.Comparing extreme categories, we found a positive, significant association between coffee (RR: 1.30; 95% CI: 1.04–1.62; We found that a higher intake of coffee and decaffeinated coffee was associated with increased risk of RA. No significant association between caffeinated coffee, tea or caffeine intake and the risk of RA was observed.https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=227665, identifier: CRD42021227665. Rheumatoid arthritis (RA) is a prevalent autoimmune disease diagnosed by joint function deterioration, erosion of cartilage and bone distraction, which can lead to daily activity dysfunction and decreased quality of life . This coThe etiology of RA is not clear; nevertheless, studies have demonstrated that genetic and environmental factors are the most common determining factors in the pathogenesis of RA . EnvironWe reported the current meta-analysis by following the Meta-analysis of Observational Studies in Epidemiology (MOOSE) guidelines .https://www.crd.york.ac.uk/PROSPERO (registration number CRD 42021227665). A systematic search of available studies was performed up to July 2021 from the online databases of PubMed/Medline, EMBASE and Scopus. The following text and MeSH heading search key words were used in our search strategy:  AND (“Camellia sinensis”) OR “Tea” OR “Coffee” OR “caffeine” OR “caffeinated”). There was no language or time restriction. We did not include published abstracts in the present systematic review and meta-analysis. More information related to search strategy are presented in The study protocol is available at The title and abstract of each study were reviewed by two independent authors (F. A and F. D) to identify potentially relevant studies. Studies with the following criteria were included: (i) all human prospective cohort studies that considered coffee, tea, caffeine or types of coffee (caffeinated or decaffeinated) as the exposure and RA as the main or one of the secondary outcomes; (ii) publications in which effect sizes were reported in the form of odds ratio, rate or risk ratios, relative risk (RRs), or hazard ratios. Letters, comments, reviews, meta-analyses, ecological studies, animal studies and studies that were conducted on children, were excluded from our systematic review and meta-analysis. The detailed information of PICOS  for inclusion and exclusion of studies is available at Two independent investigators (F. A and F. D) performed the literature research and data extraction, including the name of the first author, publication year, country, age range, sex, follow-up duration, number of cases or cohort size, exposure type, exposure assessment method, outcome assessment, comparison categories, relevant adjusted effect sizes with 95% confidence intervals and confounding variables adjusted for in the statistical analysis. When eligible studies reported several risk estimates, we extracted the fully adjusted effect sizes. Disagreements were resolved through discussion by third author (HM).n ≤ 5), a fixed-effects meta-analysis was performed for combining study-specific results  and a p-value < 0.05 was considered statistically significant.Linear dose–response meta-analysis was also performed to estimate the RRs per increment of one cup/d of coffee, caffeinated coffee, decaffeinated coffee and tea consumption, as well as per 200-mg/d increment of caffeine intake according to previous meta-analyses , 30, by The Newcastle-Ottawa Scale (NOS) was used to evaluate the quality of the included studies . With thn = 1), the study outcome was not relevant (n = 2), review articles (n = 2), and irrelevant study design (n = 6)  . Finally (n = 6) –42 with  (n = 6) –41, one  (n = 6) . Age at  (n = 6) –42 and o (n = 6) . All theI2= 0.0%; P = 0.61). Sensitivity analysis showed that removing each particular study at a time, did not affect the summary effects. We observed some evidence of publication bias using Begg's test (P = 0.05) and Egger's test (P = 0.02). There was a significant positive association such that an additional cup of coffee per day was correlated with a 6% increase in the risk of RA  and Egger's test (P = 0.87). There was no significant association between one cup of caffeinated coffee in a day and risk of RA . Sensitivity analysis showed that removal of the study by Mikuls et al. changed the significant summary effects to non-significant . There was no evidence of publication bias by Begg's test (P = 0.18) and Egger's test (P = 0.17). We found a positive association between one cup of decaffeinated coffee and risk of RA. Each additional cup of decaffeinated coffee per day was associated with an 11% increased risk of RA . Sensitivity analysis indicated that exclusion of any particular study from the analysis did not alter the pooled effect sizes. No publication bias was found based on Begg's test (P = 0.12) and Egger's test (P = 0.58). There was no significant association between a 200 mg increment of caffeine intake in a day and the risk of RA . Furthermore, sensitivity analysis found that the overall effect size did not depend on a single study. There was no evidence of publication bias by Begg's test (P = 0.60) and Egger's test (P = 0.70). There was no statistically significant association between one cup of tea per day and risk of RA  with RA which revealed their different effects on RA. Moreover, the included studies were a cohort with a relatively large sample size which increased the generalizability of the findings and statistical inferences.In spite of these strengths, this study has some limitations which should be taken into account when interpreting the results. First, our study included a small number of publications that weakened the interpretation of the relationships in the meta-analyses. Second, the results are subject to residual confounding because of the cohort design of the included studies. Third, most of the included studies used a food frequency questionnaire to evaluate dietary intake and, therefore, measurement error and misclassification of the study participants in terms of exposure were inevitable. Finally, the size of the cup of coffee and tea in the entire study was not determined, which could influence the observed associations.Subject to the limitations such as low number of studies and weak statistical power, we observed a positive association between coffee, decaffeinated coffee intake and the risk of RA. However, no statistically significant associations were observed between caffeinated coffee, caffeine or tea consumption with the incidence of RA. More experimental and observational studies are needed to substantiate such findings.The original contributions presented in the study are included in the article/FA, FD, and AH contributed to the conception, design literature search, and manuscript drafting. AJ and AF contributed to the design, statistical analyses, and interpretation of data. HM contributed to the conception, design, data interpretation, drafting of the manuscript, and supervised the study. Final manuscript was approved by all authors approved prior to submission.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.


We also found that maternal high folate status may be associated with increased risk of GDM . Compared with non‐GDM group, women with GDM are prone to higher folate level. Moreover, high maternal folate status may predict a higher risk of GDM. As the number of included studies was limited, further large population studies are needed in the future.Studies on the association between maternal folate status and gestational diabetes mellitus (GDM) have yielded inconsistent results. This meta‐analysis was performed to determine whether there may exist some association between maternal folate status and GDM. Unrestricted searches of PubMed, Web of Science, Cochrane, and Embase were conducted. All relevant studies on the association between maternal folat status and GDM risk were screened. The standardized mean difference (SMD) with 95% CIs was used to determine the association between maternal folate and GDM. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated using random‐effects models to assess the impact of maternal folate status on GDM risk. 12 studies were included. The overall data revealed that compared with the non‐GDM group, women with GDM had higher level of folate (SMD 0.41, 95% CI 0.07 to 0.21,  High folate status may predict a higher risk of GDM in pregnant women. However, more large studies are needed to confirm. Accumuln = 3,747) demonstrated a positive association between daily FA supplementation in the first trimester and the risk of GDM   indicated that prepregnancy habitual intake of FA supplementation was negatively associated with GDM risk in the USA , a water‐soluble B vitamin, is now widely used to prevent neural tube defects (NTDs) in offspring  statement as described previously  independently reviewed all the included studies, and the determination of eligible studies was finalized. Disagreements were settled through consensus or the help of a third reviewer (JZ). All the articles to be included in this meta‐analysis met the following criteria: (a) Studies with information on pregnancy folate status and GDM; (b) Studies with two groups, including a GDM and control group; and (c) Studies published in English. Articles were excluded if they met the following criteria: (a) Participants with multiple pregnancies or pre‐existing diabetes (type 1 diabetes or type 2 diabetes); (b) Meeting abstracts that contained insufficient information for assessment; or (c) Studies that did not have available information or usable data for the purpose of this meta‐analysis.2.3All relevant articles were guided in EndNote X9 software and reviewed independently by two authors (YY and ZC). Discrepancies between authors were settled with the help of a third reviewer (JZ). The following information was extracted by two independent investigators from the finalized study: author, year, country, study design, sample size, number of GDM, age (year), number of GDM cases, GDM criteria, and Newcastle–Ottawa Scale (NOS). All the extracted data were then imported into Excel software.2.4The quality of the included studies was assessed by the NOS Stang, . We asse2.5So far, there is no accepted definition of high folate status. The World Health Organization (WHO) has recommended that RBC folate level greater than or equal to 906 nmol/L be the appropriate concentration for the prevention of neural tube defects . The OR and 95% CI were used for discontinuous data. The heterogeneity among all studies was assessed by Q test and I2 statistics. All results were analyzed by a random‐effects model. Sensitivity analysis was performed by excluding each study one by one to evaluate the credibility of pooled results. Begg's and Egger's tests and funnel plots were used to detect potential publication bias, with a 33.1n = 996) on maternal folate status and GDM included searches in PubMed (n = 118), Web of Science (n = 195), Embase (n = 638), and Cochrane Library (n = 45)  Figure  used FA ) Figure . Therefo) Figure  for qualAmong the 12 studies included in this analysis, there were 5,362 control subjects and 1,114 GDM patients. Three of the included studies were performed in China , Begg's test (p = .511), and funnel plot showed no publication bias , Egger's test (p = .192) and a symmetric distribution funnel plots  in blood is associated with the dysfunction of NK cells (Bayer & Fraker, Additionally, vitamin B12 along with folate is essential for one‐carbon metabolism, including the synthesis of lipids, protein, and DNA (Finer et al., Another possible mechanism is that low vitamin B12 during pregnancy can contribute to the increased levels of adipose‐derived circulating microRNAs, which may participate in the development of insulin resistance (Adaikalakoteswari et al., 4.3This meta‐analysis may have both important clinical and research implications. First, we demonstrated that maternal folate status was higher in GDM pregnant women than normal, and high folate status was associated with increased GDM risk especially in B12‐insufficient conditions, indicating that understanding the role of abnormally high folate levels in pregnant women is necessary. On the research side, our meta‐analysis indicated that low B12 level may be the critical factor for the negative influence of high folate status. More research is needed to identify the synergistic effect of high folate and low B12 status on GDM. The optimal maternal folate levels should also be the focus of future research.4.4The limitations of our meta‐analysis are as follows. First, due to the limited number of included studies, the conclusion that high folate status may increase the risk of GDM should be interpreted with caution. Second, considering the crucial role of B12 in GDM development, the effect of high folate status on GDM risk may depend on low B12 level. More studies are needed to determine the effect of high folate and low B12 status in GDM. Third, there were variations in the measurement of folate concentration within the included studies. Fourth, most of the maternal folate levels were measured in the second or third trimester among all included studies, which cannot prove a causal relationship between high folate status and GDM. Finally, high‐quality studies are needed to determine whether high maternal folate status may be an independent risk factor of GDM.5In summary, our meta‐analysis shows that a high folate concentration may be related to an increased GDM risk, especially when combined with vitamin B12 deficiency, indicating that high FA concentrations combined with low vitamin B12 may be a novel marker for predicting GDM risk. However, more large and well‐designed studies are needed to prove this finding and explore the synergistic effect of high folate and low B12 status on GDM development.6Although the study involves human subjects, it is a meta‐analysis based on evaluating published research data. Therefore, no ethical issues are involved.The authors declare no conflict of interest.Y. Y. and Z. C. involved in conceptualization and formal analysis. Z. C., J. Z., and Y. Y. involved in methodology and validation. Y. Y. involved in software, investigation, and writing—original draft preparation. J. Z. involved in resources, writing—review and editing, visualization, project administration, funding acquisition. Z. C. involved in data curation. Z. C. and J. Z. supervised the study. All authors have read and agreed to the published version of the manuscript.Fig S1aClick here for additional data file.Fig S1bClick here for additional data file.Fig S2aClick here for additional data file.Fig S2bClick here for additional data file.Table S1Click here for additional data file.


MTHFR gene with maternal risk for DS.Down syndrome (DS) is one of the most common chromosomal abnormalities among live-born babies and one of the best-known intellectual disability disorders in humans. Errors leading to trisomy 21 are primarily arising from defects in chromosomal segregation during maternal meiosis (about 88% of cases), and the focus of many investigations has been to identify maternal risk factors favoring chromosome 21 malsegregation during oogenesis. Maternal polymorphisms of genes required for folate metabolism are the most investigated risk factors for the birth of children with DS. Through this review, we sought to investigate the association of the polymorphisms “C677T” and “A1298C” of the We will use the databases PubMed, Embase, Scopus and Web of Science to search for case-control studies published from 1999 up to September 2021 without language restriction. Results will be presented as relative risks and 95% confidence intervals for dichotomous outcomes and mean differences, or standardized mean differences along with 95% confidence intervals, for continuous outcomes. The all data synthesis will be analyzed on the Review Manager 5.2 version software.This study will be able to clarify all the doubts we seek and that it will be able to provide accurate data that will be able to describe how these polymorphisms can act to increase the predisposition for the birth of children with DS in different populations and under different dietary conditions.MTHFR gene with increased the maternal risk for Down syndrome.This study will clarify the relationship between C677T and A1298C polymorphisms This systematic review and meta-analysis protocol has been registered on the Prospective Registry of International Systematic Review and Meta-analyses: CRD42021269338. Several subsequent studies carried out for more than 15 years support a potential contribution of polymorphisms of genes required for folate metabolism as maternal risk factors for the birth of a child with DS.Folate metabolism provides one-carbon units required for DNA synthesis and methylation, thus playing a pivotal role in maintaining genome stability during cell divisions.MTHFR) is one of the most important enzymes in folate metabolism. It converts 5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate, an active form of folic acid acting as methyl donor for the remethylation of homocysteine to methionine in a reaction catalyzed by methionine synthase. The methionine is then converted into S-adenosyl-methionine (SAM), which is the main intracellular methyl donor compound for methylation reactions.,8Methylenetetrahydrofolate reductase  is the result of a transition mutation from C to T at nucleotide 677, causing an amino acid substitution  in the catalytic domain of the protein.The most well known polymorphisms of the MTHFR enzymatic activity. The MTHFR A1298C polymorphism (rs1801131) is another common missense mutation leading to a Glu429Ala aminoacidic change in the regulatory domain of the protein and the homozygous genotype (C/C) results in about 40% reduction in enzymatic activity. Noteworthy, the MTHFR C677T and A1298C polymorphisms are in strong linkage disequilibrium (LD), and the 677T allele has been nearly always observed in cis with the 1298A one. LD is not complete, but very low frequencies have generally been reported for the rare 677T-1298C haplotype.The homozygous genotype (T/T) is responsible for the reduction in up to 70% in MTHFR polymorphisms was proposed considering the fact that MTHFR works as a dimer protein in which monomers associate head to tail, and the stability of the dimer depends on what aminoacid is present at position 222 and what at position 429, resulting from the combined MTHFR 677/1298 genotype. Consequently, the overall activity of the MTHFR protein is better explained by the combined MTHFR 677/1298 genotype, rather than by each single polymorphism alone.A biological explanation for the LD existing between the two MTHFR 677T allele renders the enzyme thermolabile, particularly in homozygous (T/T) individuals that are prone to dimer destabilization under conditions of reduced folate bioavailability. In summary, combinations of genetic and dietary factors contribute to MTHFR protein stability and activity. In addition, dietary folates and related metabolic factors can also regulate MTHFR gene methylation and expression levels.,13Physiological levels of folate stabilize the MTHFR dimer, but the  supporting the original hypothesis by James et al and suggesting that MTHFR polymorphisms promote changes in global DNA methylation during maternal meiosis leading to chromosomal non-disjunction and increasing the maternal risk to have children with DS.A recent study revealed that the fission yeast methylenetetrahydrofolate reductase protein (Met11) is important for maintaining the pericentromeric heterochromatin structure to ensure mitotic and meiotic chromosome segregation fidelity,MTHFR C677T and A1298C polymorphisms have been linked to an increased risk of chromosome malsegregation in lymphocytes of mothers of DS children. Following the original study by James and coworkers, several investigators performed case–control studies to evaluate MTHFR C677T and/or A1298C polymorphisms as maternal risk factors for the birth of a child with DS, but the small sample-size of these studies coupled to differences in allele frequencies, lifestyles and dietary habits among different populations, yielded conflicting results.–22In addition, both MTHFR C677T and/or A1298C polymorphisms are associated with the maternal risk for having a child with DS, and despite that significant associations emerged for the C677T one in the overall population, the different inclusion criteria selected by the authors yielded conflicting or inconclusive results after subgroup stratification according to ethnicity.–25At least three different meta-analyses of those studies, performed between 2013 and 2014, addressed whether MTHFR C677T/A1298C polymorphisms.,26–28 Moreover, congenital heart defects (CHD) are frequently observed in DS babies and owing to the role of folate metabolism in cell division and methylation, there is increasing interest in searching whether maternal MTHFR polymorphisms could represent risk factors for CHD in DS newborns.,17,21More recent case–control studies have highlighted a different effect size of these polymorphisms in the maternal risk for a DS birth according to ethnicity, maternal age at conception, errors occurring at maternal meiosis I or II, gene-nutrient interactions, and combined MTHFR C677T and/or A1298C polymorphisms as maternal risk factors for DS addressing not only the overall contribution of each of the two polymorphisms to DS risk, but performed in such a way to extract data on combined genotypes, ethnicity, serum folate levels, maternal age at conception, errors occurred at maternal meiosis I or II, as well as data on CHD in the DS children, in order to shed light on the different role of the MTHFR genotype in the various subgroups. Therefore, the aim this review is to evaluate the association between frequency of the C677T and/or A1298C polymorphisms of the MTHFR gene with maternal risk for DS.What is missing is an updated systematic review and meta-analysis of the studies investigating 2MTHFR gene and the maternal risk for DS.The aim of this systematic review and meta-analysis is to clarify the relationship between the C677T and A1298C polymorphisms 3 This protocol was registered in the prospective international registry of systematic reviews and the trail registration is number CRD42021269338 (https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=269338).The elaboration of this study will be conducted according to Preferred Reporting Items for Systematic Review and Meta-analysis Protocol Guidelines.3.13.1.1Will be included studies have to meet the following criteria:(1)articles should be published from 1999 up to September 2021 without language restriction;(2)article should have sufficient data on both allele and genotype frequencies to calculate the odds ratio with 95% confidence intervals (CI);(3)article should be case–control studies; and(4)authors should describe the genotyping protocols.MTHFR C677T and/or A1298C allele and genotype frequencies will be excluded as well as Review articles, editorials and/or commentaries, meeting abstracts and any other kind of article not published in a peer-reviewed journal.Case–control studies not showing tabular data with 3.1.2Studies shall include women who gave birth to at least one child with DS in this systematic review and meta-analyses. Women/mothers of children who do not have DS or other diagnosed syndromes, and without history of spontaneous miscarriages will be the control group.3.1.3Maternal risk for the birth of a child with DS.3.1.4The role of polymorsphisms as risk factors for CHD in DS newborns.3.23.2.1We will use four databases: PubMed, EMBASE, Scopus and Web of Science to search for articles of studies of case control published from 1999 up to September 2021 without language restriction.3.2.2The search terms (MESH) that will be used to conduct the research are: (mothers OR pregnancy) AND (methylenetetrahydrofolate reductase OR MTHFR OR genetic polymorphism OR gene polymorphism) AND (down syndrome OR trisomy 21). The studies considered eligible will also be selected from the reference lists of the retrieved articles.3.33.3.1Two authors, CTAG and JRDL, will independently examine the results of the research using the titles and abstracts. Duplicate studies and reviews will be excluded, and the studies by the same authors will be controlled for a possible overlapping of included patients. The reviewers will review the full text to determine whether the studies meet the inclusion criteria. Various characteristics of the eligible studies will be extracted: title, surnames of the first authors, year of publication, study location (country), study design, primary objective, population, sample size, follow-up period, inclusion/exclusion criteria, genotyping method, type of control and primary results. The process of selection of the studies is summarized in Figure 3.3.2The assess the risk of bias in eligible studies using the Newcastle-Ottawa Scale 9-star tool. The quality of each study will be judged in three broad categories – namely: selection of the study population, comparability of the groups and determination of the exposure or the result of interest. The Newcastle-Ottawa Scale score vary from 0 to 9, in which (score 0–5) was low quality study and (score 6–9) was high quality study.3.42 ≥ 50%), the random effects model will be used to combine the studies to calculate the OR and 95% CI, using the DerSimonian-Laird algorithm in the meta for package, which provides functions to conduct meta-analysis in R. Other characteristics and results of the study will be summarized narratively if the meta-analysis cannot be performed for all or for some of the included studies. Sensitivity analyses will be used to explore the robustness of the results in relation to the quality of the study and the sample size. Sensitivity analyses will be shown in a summary table. If meta-analysis cannot be performed for all or some of the included studies, the results will be presented qualitatively.The data will be entered into the Review Manager 5.2 version software . For dichotomous results, the odds ratio (OR) and 95% CI for each study will be extracted or calculated. In case of heterogeneity (I3.52 test with a significance level of P < .1. To assess heterogeneity, we plan to calculate the I2 statistic, which is a quantitative measure of inconsistency between studies. A value of 0% indicates no heterogeneity, whereas I2 values ≥50% indicate a substantial level of heterogeneity; however, heterogeneity will be assessed only if it is appropriate to conduct a meta-analysis.The heterogeneity between the results of the study will be assessed using a standard χ3.6MTHFR C677T and A1298C polymorphism. Standardized data extraction forms will be created specifically for this review and the results will be later inserted into a database. All data entries will be double rechecked.Will be perform subgroup analyses to investigate the effect of ethnic differences in allele frequencies, combined genotypes, circulating folate levels and maternal age at conception to analyze the type of meiotic error in modulating the maternal risk for DS resulting from 3.7The analysis of the evidence for all outcomes will be assessed using the Grading of Recommendations Assessment, Development and Evaluation Working Group methodology by classifying the evidence as high, moderate, low, or very low.3.8Ethical approval is not necessary because it is a systematic review article that will use data published in the literature. The findings of this manuscript will be published in a peer-reviewed journal and will be updated if there is enough new evidence to alter our conclusions.4 Folate is an important nutrient required for DNA synthesis and methylation, and several in vitro studies have shown that its depletion causes chromosome damage and malsegregation in cultured cells.,33 The folic acid, nothing else is a synthetic form of folate, a water-soluble vitamin whose active form is 5-methyltetrahydrofolate.Studies published since 1999 support a relationship between genetic polymorphisms of folate metabolic genes and the maternal risk for having a child with DS, albeit with varying effect size in different populations and under different nutrient bioavailability. MTHFR is one of the major enzymes in folate metabolism, linking the folate and the methionine cycles, thus providing one-carbon moieties for DNA synthesis and methylation reactions.Indeed, the rationale for the association between maternal polymorphisms of folate metabolic genes and chromosome 21 non-disjunction is an altered chromatin structure of chromosome 21 favoring its malsegregation during meiosis and resulting from the impaired folate metabolism caused by the polymorphisms.MTHFR polymorphisms, namely C677T and A1298C, impair MTHFR stability and activity overall leading to hyperhomocysteinemia and reduced SAM production, and have been largely investigated as maternal risk factors for spontaneous abortion and DS risk.,16–28,34Common MTHFR C677T polymorphism and the risk of birth of a child with DS,–25 but many questions are still unsolved. Particularly, it is becoming evident that combined MTHFR C677T/A1298C genotypes give rise to more or less stable MTHFR dimers, and that intracellular levels of folate, SAM, and metabolic cofactors regulate MTHFR protein stability and activity, as well as the promoter methylation and expression levels of the MTHFR gene itself,,12,28 also the frequency of MTHFR polymorphisms and their combinations varies among different populations, likely as a result of complex gene-nutrient interactions over time that might have favored certain haplotypes.Previous literature meta-analyses support an association between the maternal  It is therefore time to perform an updated systematic review and meta-analysis of the literature concerning the association of the polymorphisms C677T and A1298C of the maternal MTHFR gene with DS risk in attempt to address all these points.There is also evidence that genetic factors predisposing to chromosome 21 non-disjunction in maternal meiosis I might be different from those predisposing to errors occurring at maternal meiosis II.Studies conducted over the years have some limitations regarding the sample size of these studies, the differences in allele frequencies and how the lifestyle and eating habits of mothers of children with DS could influence the measurement of this risk. Furthermore, data resulting from the analysis of ethnicity in these groups, when not properly conducted, generate conflicting and inconclusive results.With this protocol of systematic review and meta-analysis, we wish to identify the association of the polymorphisms C677T and A1298C of the MTHFR gene with the maternal risk for DS, as well as to perform subgroup analyses to investigate the effect of ethnic differences in allele frequencies, combined genotypes, circulating folate levels, maternal age at conception and type of meiotic error in modulating the maternal risk for DS resulting from these polymorphisms, and investigate their role as risk factors for CHD in DS newborns.We hope that our study will be able to clarify all the doubts we seek and that it will be able to provide accurate data that will be able to describe how these polymorphisms can act to increase the predisposition for the birth of children with DS in different populations and under different dietary conditions. This could help to identify individuals and populations at higher risk and seek nutritional strategies that can be implemented to reduce the maternal risk for DS and associated congenital complications.We would like to thank to doctoral student, Medeiros, KS and Sarmento, ACA from Post-graduation Program in Health Sciences, University Federal of Rio Grande do Norte for the contribution for the preparing this protocol systematic review and meta-analysis.Conceptualization: Carla Talita Azevedo Ginani, Jefferson Romáryo Duarte da Luz, Saulo Victor e Silva.Methodology: Carla Talita Azevedo Ginani, Jefferson Romáryo Duarte da Luz, Fabio Coppedè, Maria das Graças Almeida.Resources: Carla Talita Azevedo Ginani, Jefferson Romáryo Duarte da Luz.Supervision: Fabio Coppedè, Maria das Graças Almeida.Writing – original draft: Carla Talita Azevedo Ginani, Jefferson Romáryo Duarte da Luz.Writing – review & editing: Fabio Coppedè, Maria das Graças Almeida.


With the general population in Taiwan as a reference, a decreased standardized mortality ratio  from all causes combined, between 1 January 1985 and 31 December 1996, was observed, indicating a healthy worker effect. When we reanalyzed the data using standardized PMR, elevated risks were observed for all cancers combined  and cancers of the digestive organs and peritoneum , especially stomach cancer . The results showed that PMR can detect increases in mortality when a study population is generally healthier than the comparison population and call for further studies on the possible carcinogenic effects of low-level acid mist exposures on the stomach.When a study population is relatively healthy, such as an occupational population, epidemiological studies are likely to underestimate risk. We used a case study on the cancer risk of workers with exposure to acid mists, a well-documented carcinogen, to demonstrate that using proportional mortality ratios (PMRs) is more appropriate than mortality ratios in assessing risk in terms of mortality. The study included 10,229 employees of a telecommunication company who worked in buildings with battery rooms. In these buildings, the battery rooms had the highest levels of sulfuric acid in the air (geometric mean = 10.7 μg/m In epidemiological studies, when the study population is relatively healthier than the comparison population, risk is likely to be underestimated. A typical scenario is that an occupational population is likely to be healthier than the general population because it comprises individuals necessarily healthy enough to be employable. Consequently, when a study evaluates effects of a health hazard using mortality as the indicator of outcome, if an occupational population with exposure to that health hazard is compared to the general population, the risk associated with the health hazard is likely to be underestimated. This phenomenon is often called the “healthy worker effect” . Standardized mortality ratio (SMR) is a frequently used measure of the relative risk of mortality, expressing the mortality experience of the study population relative to that of a comparison (standard) population. With the general population as the standard, the SMR for a study population that is relatively healthier  will underestimate the mortality experience of the study population, because the baseline mortality risk is initially lower than that of the standard population. We hereby use a case study on the cancer risk of workers with exposure to acid mists, a well-documented carcinogen, to demonstrate that using proportional mortality ratios (PMRs) is more appropriate than SMR in such cases.3. While this level was below the 1000 μg/m3 standard proposed by the Occupational Safety and Health Administration of the United States, excess risks of cancer were still observed. To assess the risk associated with even lower exposure levels, a study was conducted on the workers of a large telecommunication company in Taiwan, who had worked in buildings with battery rooms [Strong inorganic acids, including sulfuric acid, hydrogen acid, and nitric acid, are commonly used in industry. Long term exposure to high-levels of strong inorganic acid mists is a well-documented risk factor for developing cancer, especially in the upper respiratory tract, such as lung and laryngeal cancers among steel workers, while the International Agency for Research on Cancer (IARC) classified it as a Class I carcinogen (carcinogenic to humans) . An incrry rooms . The batThe study population consisted of employees of a large telecommunication company in Taiwan, who had worked in buildings with battery rooms. In Taiwan, before 1996, telecommunication was a monopoly business run by the government, and the company offered very good benefits, including the salary and health insurance, in comparison with other companies in general. Therefore, its employees constituted a very healthy population compared to the general population and thus provide a good example for demonstrating the healthy worker effect.The batteries are used as back-up power supplies in case of power shortage, and they are generally placed in battery rooms, which no workers occupied on a regular basis. These large batteries contain strong acids, mainly sulfuric acid. During the charging process, both hydrogen and oxygen bubbles can be generated, and the bursting of gas bubbles on top of the acid solution may lead to the release of aerosols containing acids. Whereas it is difficult to estimate the particle sizes of these aerosols, a laboratory study on aerosols generated from chromic acid solutions with various gas (bubble) flow rates indicated that the distribution of particle sizes was bimodal . The mas3, geometric mean = 10.7 μg/m3), but they were all within the regulatory standard of 1000 μg/m3. Although we did not measure acid mist levels in all the buildings covered in this study, because all the building belonged to the same company, who had built most of them, the levels were generally within this range. In addition to acid mists, the levels of electromagnetic field and heavy metals in the buildings with battery rooms were assessed. The mean air level was 0.844 μg/m3 for chromium, 0.002 μg/m3 for cadmium, 1.21 μg/m3 for lead, and 0.804 μg/m3 for nickel, and were all far below the regulatory standards in Taiwan . Sampling was conducted in 23 typical buildings, using a sampling train consisting of one inhalable aerosol sampler and followed by one sorbent tube, for collecting acidic aerosols and vapors, respectively. The obtained exposure levels were generally lower than those studied in other occupational populations. The highest environmental levels of sulfuric acid were measured in the battery rooms , and township of resident of each case. In Taiwan, the prompt reporting of deaths is mandated by law, which ensures the completeness of the registry. The SMR of a given cause of death for the study cohort was calculated using the observed number of deaths as the numerator and the expected number of deaths as the denominator . The perIn addition to SMR, we used standardized PMR (SPMR) to assess the cancer risk. While the SPMR is also obtained by dividing the observed number of cases by the expected number of cases, the expected number of cases is calculated on the basis of the proportion of death due to a specific cause among all deaths in the standard population . Again, All statistical analyses were performed using statistical analysis software , and the statistical significance of SMRs and SPMRs was evaluated by exact tests based on the Poisson distribution at a significant level of 0.05. A total of 10,229 workers that fit our cohort definition were identified, including 9551 men (93.4%) and 678 women (6.6%). The total person-years at follow-up were 106,725, and 155 deaths were observed between 1 January 1985 and 31 December 1996. Among those, 52 died of cancers, including 31 of cancers of the “digestive organs and peritoneum” . There wp < 0.01, p = 0.01), indicating a lower than expected (decreased) risk. For the other three leading causes of death in Taiwan, the SMR was 0.55 for cerebrovascular diseases (p = 0.01), 0.28 for accidents (p < 0.01), and 0.65 for cardiovascular diseases (p = 0.08). The SMR for all causes combined was 0.42, and that for all cancers combined was 0.64; both were statistically significant (both with p < 0.01), and that for all cancers combined was 0.69 (p = 0.01). However, changes in the SMR of specific cancers varied. For example, the SMR for cancers of “lip, oral cavity, and pharynx” and “respiratory and intrathoracic organs” increased, but that for cancers of the “digestive organs and peritoneum” decreased. Even among cancers of the “digestive organs and peritoneum,” whereas the SMR for cancers of the “liver and intrahepatic bile ducts” decreased, the SMR for stomach cancer increased (p = 0.02), indicating a decreased risk. When person-years within the first five years of employment were excluded, a total of 122 deaths, including 40 due to cancers, were observed among the 70,266 person-years at risk. As a result, the SMR generally increased . The SMRncreased . Howeverp = 0.01). Among cancers with a specific three-digit ICD-9 code and with observed deaths, “gallbladder and extrahepatic bile ducts” cancer had the highest SPMR , while “liver and intrahepatic bile ducts” cancers had the lowest SPMR . With eight deaths, stomach cancer had the only elevated SPMR (2.94) with statistical significance (p = 0.01), indicating a higher than expected (increased) risk. As a lower than expected all-cause SMR is regarded as an indicator of a healthy worker effect , we calcp = 0.03) (p = 0.01), indicating an increased risk.When person-years within the first five years of employment and the 12 cancer deaths that occurred with these periods were excluded, the adjusted SPMR for all cancer combined was 1.47 ( = 0.03) . However = 0.03) . Again, 3 was determined on the basis of symptoms (stimulation), not the carcinogenic effect. Therefore, it is not surprising that carcinogenic effects were observed below this level [In the 1980s, several studies on steel workers observed increased risks of laryngeal cancer in those who were exposed to acid mists, mainly sulfuric acid from pickling operations ,10,11,12is level ,5, and ip = 0.01), which indicated a healthy worker effect, because the overall mortality of the cohort members was lower than the general population of the same sex and age group in Taiwan [p = 0.01), the SPMR, which is much less affected by the healthy worker effect [p = 0.01). This observation demonstrates that the health worker effect might lead to underestimation of the risk. In most previous studies on the association between acid mists and cancer, the relative risk of all cancers combined ranged from 0.7 to 1.0 [The SMR for all causes combined of the study cohort was significantly smaller than 1 . Using “telecommunication” or “telephone” combined with “stomach neoplasm,” “stomach cancer,” or “gastric cancer” as key words to search the literature through PubMed, we failed to find any literature on the association between working in the telecommunication industry and the occurrence of stomach cancer since 1966. Studies on the association between occupational exposure to acid mists and the occurrence of stomach cancer were quite limited. Some of the studies on associations between acid mists and cancers that we reviewed did not include stomach cancer, and others observed only one or two cases [p < 0.01 for test for trend) and probability  of exposure to sulfuric acid mists, with a two-fold excess associated with a high probability of high-intensity exposure [p ≈ 0.05) was also observed. In a follow-up of that study, which studied gastric cancers as a whole with four more years of data (from 1984 to 1996), an increasing trend of risk with probability of exposure was observed in workers who had high-intensity of exposure only (p < 0.01), and an increasing trend of risk with intensity of exposure was observed in all exposed workers combined (p < 0.01) and in those who had a medium probability of exposure (p < 0.05) [p > 0.05). The lower consistency in the trends of risk associated with exposure observed in the follow-up study might indicate that the association between acid mists and stomach cancer was more specific to cancer of the gastric cardia. However, the death registry we used did not provide enough information on diagnoses for us to test this hypothesis. A study in the U.S. observed increased risks of stomach cancer in 4518 workers of lead battery plants  and 2300 workers of lead smelters  [p < 0.01) in lead battery plant workers and 1.334 (p > 0.05) in lead smelter workers [An increased risk of stomach cancer was observed in our study cohort. When the first five years of employment were excluded, the SPMR was 3.24  . Workers > 0.05) . A follo workers . No dose workers , the resIn addition to tobacco smoking, Helicobacter pylori, X-radiation, and gamma-radiation were also recognized as Class I carcinogens of the stomach, as well as the rubber production industry. In Taiwan, ionizing radiation in the work place is under strict surveillance by the government, and no excess exposure to ionizing radiation had been reported in the buildings we studied. Regarding Helicobacter pylori, there was no evidence in the literature showing that working in the telecommunication industry or being in buildings with battery rooms is associated with a higher prevalence of Helicobacter pylori infection. Therefore, there was no evidence supporting that the higher risk of stomach cancer observed in our analysis was attributable to these potential confounders. On the other hand, we do not have certainty about the cause of the observed excess mortality due to stomach cancer in this working population, and the finding must be confirmed by further studies. Such studies may include internal comparisons within the study population (including dose-response assessments), which is also an approach commonly applied to minimize the impact of healthy worker effects.In epidemiological studies, it is not uncommon that the study population is relatively healthier than the comparison population, especially when the study population is an occupational population and the comparison population is a sample of the general population. Using a study on the mortality of telecommunication workers as an example, we have demonstrated that the risk associated with the exposure would be underestimated due to the healthy worker effect and that using SPMR is more appropriate than SMR in terms of risk assessment. In this case study, working in buildings with battery rooms was found to be associated with increased mortalities due to all cancer combined and stomach cancer. Whereas exposure to acid mist might have contributed to the increased risks, no personal exposure data were obtained, and no control of other occupational risk factors was made. Furthermore, because the study population was relatively healthy, we did not identify a large number of cancer deaths, which made comparisons among subgroups within the study cohort not feasible. Therefore, further studies should be conducted to evaluate whether the increased risks are related to low-level exposure to acid mists or other factors in the work environment.