Association of maternal heavy metal exposure during pregnancy with isolated cleft lip and palate in offspring: Japan Environment and Children’s Study (JECS) cohort study

Masato Takeuchi; Satomi Yoshida; Chihiro Kawakami; Koji Kawakami; Shuichi Ito; Japan Environment and Children’s Study Group

doi:10.1371/journal.pone.0265648

Peer Review History

Original SubmissionSeptember 10, 2021
24 Nov 2021 Decision Letter - Forough Mortazavi, Editor PONE-D-21-29327Association of maternal heavy metal exposure during pregnancy with isolated cleft lip and palate in offspring: Japan Environment and Children’s Study (JECS) cohort studyPLOS ONE Dear Dr. Takeuchi, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jan 08 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information. 3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. Additional Editor Comments (if provided): Dear authors, Thank you for submitting your manuscript to PLOS ONE. Please consider the reviewers’ comments carefully and revise the manuscript accordingly. As the reviewers stated, manganese and selenium are not considered as heavy metals. The justification for considering manganese and selenium as heavy metals is not sufficient and convincing since both of these are among the prescribed minerals during pregnancy. You need to cite at least one more study with regard to this matter in the introduction section in addition to the reference 18. Also, arsenic is not included in your list of five heavy metals without any justification. PLS explain the scientific evidence and rationale for the selection of the five heavy metals. PLS provide the statistics regarding the rate of exposure to heavy metals as well as the rate of cleft lip/palate in Japan in the introduction section to justify conducting this study. This study did not find any relationship between exposure to heavy metals and cleft lip/palate in Japan; therefore, it is not necessary to measure the effects of other risk factors of cleft lip/palate because they would not change the relationship between exposure to heavy metals and anomalies. But if you have collected data regarding other risk factors, I suggest that in addition to heavy metals, you consider other risk factors in relation to cleft lip/palate in your analysis. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: No ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Although the authors pick an important topic on heavy metal exposure and CL/P in Japan, no significant association was found, and there are many reasons, including over matched the controls (1:10), low blood metal levels, although the authors only report the lead level in the abstract, statistical analyses and et al. There are some major concerns that the authors should pay attention, which was not limited to the following: 1. Selenium did not belong to heavy metals, and the correlation of selenium with birth defects was not coincided with other heavy metals which the authors needs to be aware of it. Line 130-131: please check it. 2. Line 75-78: the risk factors was too general and it did not mentioned the heavy metals, i.e., lack of introduction of why did the author conduct the current research? 3. Line 83: needs to clarify that “Because exposure to heavy metals is potentially modifiable” 4. Controversies about the description: Line 117-118 vs Line 219-221 vs Line 222-224. Line 117-118: Unlike this previous study, we used information of children whose birth outcome was miscarriage or stillbirth, with the aim of minimizing “live birth” bias (17). Line 219-221: Fourth, our primary cohort included still births. However, cases of cleft L/P were eventually all live born. As a result, the effect of live birth bias could not be evaluated”. Line 222-224: “A second explanation is that early fetal losses (e.g., before perception of pregnancy) were not included in the JECS dataset” 5. Statistical problem: (1) Mean metal level, median metal level and Median with interquartile range are both used in the study, due to the distribution of metal concentration, the expression needs to be uniformed. (2) Line 165-166: We found that metal concentrations were heavily skewed owing to outliers. How to tackle with “outliers”? (3) The control variables were not enough in the multivariate analysis, which only sex and other metals controlled, how about other risk factors related to CL/P? (4) As there are several metals in the analysis, proper statistical analysis may need, for example, BKMR analysis may be considered. 6. It’s hard to understand the notes under S1 Table: Major congenital anomalies recorded in Dr0m and Dr1m1 (1: Cleft lip, cleft palate, and cleft lip and palate are listed, bur are omitted from this table.) Reviewer #2: The manuscript highlighted the associations of prenatal exposures of heavy metals and selected congenital anomalies. The manuscript is well written. The study design, analysis, results, and interpretation are acceptable. I have the following observation, which needs to be addressed before acceptance of the articles. The introduction needs further elaboration about the burden of metal exposures worldwide. Further, the justification of including manganese and selenium is not sufficient. The authors should specify the health impact of metals on the outcome of interest, including the risk estimates. It is not clear why other cases of congenital anomalies were not considered in the analysis. Did the author graphically observe the associations by scatter plots (with Loess)? Linear associations are not always observed between toxic exposures and health outcomes. Whether there is any effect in the low exposure levels should be checked graphically. Furthermore, the authors should also explore the associations by grouping the exposures into tertile/quartile. One of the weaknesses of the study is that exposure at very early gestation is not available. This weakness should be discussed. Further, are there any other unmeasured metals (such as arsenic) that may influence the observed association? It is better if the authors add a separate table for exposures, including the mean, minimum and maximum values. It is better for a reader if the authors add paragraphs on the strengths and limitations of the study. Minor issues: Page 23, Line 220: missing full-stop; Page 15, Table 1: replace ‘media’ with ‘median’ at the end. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes: Jufen Liu Reviewer #2: Yes:** Anisur Rahman [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0265648.r001
Revision 1
5 Jan 2022 Author Response Editors’ comments Comment E1 Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information. Response to comment E1 The detailed items of questionnaire, including the responses, cannot be shared because of JECS regulations. Comment E2 We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. Response to comment E2 Data formerly described as “not shown” has been added as supplemental information (S3 Table). Comment E3 As the reviewers stated, manganese and selenium are not considered as heavy metals. The justification for considering manganese and selenium as heavy metals is not sufficient and convincing since both of these are among the prescribed minerals during pregnancy. You need to cite at least one more study with regard to this matter in the introduction section in addition to the reference 18. Also, arsenic is not included in your list of five heavy metals without any justification. Please explain the scientific evidence and rationale for the selection of the five heavy metals. Please provide the statistics regarding the rate of exposure to heavy metals as well as the rate of cleft lip/palate in Japan in the introduction section to justify conducting this study. Response to comment E3 We thank the Editor as well as the Reviewers for pointing out this important issue. After discussion among the authors, we removed selenium from the current analysis. The analyses were updated in this revision accordingly, and we found that the results were mostly unchanged. By contrast, we retained manganese because this metal can be broadly classified as a heavy metal (density: 7.2 g/cm3) and high concentrations of manganese may be associated with congenital anomalies (Crit Rev Oncol Hematol. 2002;42(1):25–34). As for arsenic, it is now being measured and data will be available in the future (personal communication with Dr. Nakayama, the author of Ref. 23). According to Dr. Nakayama, arsenic cannot be assessed in tandem with the other metals examined, given the situation in Japan. Residents of Japan ingest organic arsenic (assumed to be less toxic than inorganic arsenic) from seaweed, whereas exposure to inorganic arsenic is thought to be low. Accordingly, the exposure level of organic and inorganic arsenic should be specified separately to evaluate the environmental hazard of each. However, the method of measuring arsenic differs from the measurement of other metals. This was the reason for data on arsenic being unavailable to date. Again, the measurement of arsenic is now ongoing. There was another operational reason for selecting five metals. JECS has several topic groups, such as congenital anomaly or child development, and each group has a variety of research interests. In addition, JECS measured numerous environmental factors other than metals (e.g., household chemicals and allergens). From the initial research themes submitted to the JECS core team, the priority was set for the five metals selected; as noted above, data on other metals, such as copper and zinc, would be subsequently updated. At first, JECS measured the levels of five metals in approximately 20% of samples, then expanded the measurement to the entire cohort. This process required several years, illustrating how time-consuming the analysis of 100,000 blood samples was. Finally, although the Editor requested adding the statistics regarding the rate of exposure to heavy metals to the Introduction, little is known about the overall exposure level to metals in Japan, a situation that partly prompted the JECS. In response to the Reviewer concern, we have instead added a concise description of global metal exposure and possible adverse health effects to improve the flow of the Introduction. Comment E4 This study did not find any relationship between exposure to heavy metals and cleft lip/palate in Japan; therefore, it is not necessary to measure the effects of other risk factors of cleft lip/palate because they would not change the relationship between exposure to heavy metals and anomalies. But if you have collected data regarding other risk factors, I suggest that in addition to heavy metals, you consider other risk factors in relation to cleft lip/palate in your analysis. Response to comment E4 We thank the Editors for this comment. We performed a multivariable logistic regression in the sensitivity analysis, and list the odds ratios for other risks obtained from this analysis in S5 Table. Reviewers' comments: Reviewer #1 Comment 1-1 Selenium did not belong to heavy metals, and the correlation of selenium with birth defects was not coincided with other heavy metals which the authors needs to be aware of it. Line 130-131: please check it. Response to Comment 1-1 We thank the Reviewer for this comment. As we responded to the Editor’s comment above (Comment E3), selenium has been removed from the revised version. Comment 1-2 Line 75-78: the risk factors was too general and it did not mentioned the heavy metals, i.e., lack of introduction of why did the author conduct the current research? Response to Comment 1-2 We thank the Reviewer for commenting this important point; a similar comment was made by the Editor and another Reviewer. To explain the rationale for this study, we added a brief description of the hazards of metals on child health. Comment 1-3 Line 83: needs to clarify that “Because exposure to heavy metals is potentially modifiable” Response to Comment 1-3 We have added the explanation as follows: “At the population level, the source of metal levels has now shifted from the atmosphere to foods in Japan as well as many countries (17). Because such a metal exposure from contaminated food is potentially modifiable by monitoring or education, this association merits further investigation in a large cohort.” Comment 1-4 Controversies about the description: Line 117-118 vs Line 219-221 vs Line 222-224. Line 117-118: Unlike this previous study, we used information of children whose birth outcome was miscarriage or stillbirth, with the aim of minimizing “live birth” bias (17). Line 219-221: Fourth, our primary cohort included still births. However, cases of cleft L/P were eventually all live born. As a result, the effect of live birth bias could not be evaluated”. Line 222-224: “A second explanation is that early fetal losses (e.g., before perception of pregnancy) were not included in the JECS dataset” Response to Comment 1-4 We thank the Reviewer for pointing out inconsistent descriptions. The sentences have been modified as follows: Line 117-118: Unlike this previous study, we used the information of children whose birth outcome was miscarriage or stillbirth in order to attempt minimizing “live birth” bias (17). (To the Reviewer: Please note that we explained the rationale for including miscarriage or stillbirth in the planning phase, in contrast to the cohort profile paper of JECS; the JECS core team is very sensitive to even minor differences in the case numbers between studies.) Line 219-221: Fourth, our primary cohort included still births. However, although there were 172 cases of stillbirth in the control pool, cases of cleft L/P were all live-born. As a result, the effect of live birth bias could not be evaluated.” Line 222-224: “A second explanation is that very early fetal losses (e.g., before perception of pregnancy) were not included in the JECS dataset.” (To the Reviewer: Please also note that this possibility was discussed, but was unlikely as mentioned in the text thereafter.) Comment 1-5 Statistical problem Comment 1-5-1 Mean metal level, median metal level and Median with interquartile range are both used in the study, due to the distribution of metal concentration, the expression needs to be uniformed. Response to Comment 1-5-1 Throughout the manuscript, we uniformly used the median with IQR in this revision. Comment 1-5-2 Line 165-166: We found that metal concentrations were heavily skewed owing to outliers. How to tackle with “outliers”? Response to Comment 1-5-2 We addressed this issue before submission by checking that the primary results were unchanged even when data were log-transformed (and also confirmed that the data were graphically normally distributed after transformation). In addition, following the Editor’s suggestion, we present the results calculated from the log-transformed metal concentrations in this revision (S3 Table). We thank the Reviewer for this comment. Comment 1-5-3 The control variables were not enough in the multivariate analysis, which only sex and other metals controlled, how about other risk factors related to CL/P? Response to Comment 1-5-3 Other maternal risk factors for CL/P (e.g., smoking status or folic acid intake) were used for matching variables. After matching, these variables were well-balanced between cases and controls; thus, we did not perform further adjustment using these variables. Furthermore, in sensitivity analysis 2, we modeled the analysis by incorporating 10 additional maternal factors used for matching, and the results were similar to those of the primary matched analysis. For these reasons, we do not think that the control variables were insufficient. We thank the Reviewer for this comment. Comment 1-5-4 As there are several metals in the analysis, proper statistical analysis may need, for example, BKMR analysis may be considered. Response to Comment 1-5-4 We thank the Reviewer for insightful suggestion. We conducted Bayesian kernel machine regression for matched pairs, using the bkmr package in R, and found that the results were similar to those of the primary analysis (i.e., no association). (Z1, Hg; Z2, Pb; Z3, CD; Z4, Mn) Although Bayesian kernel machine regression is a sophisticated approach, Journal readers who are not statisticians may not be familiar with it. Thus, we decided to retain logistic regression analysis to present the primary results. Comment 1-5-5 It’s hard to understand the notes under S1 Table: Major congenital anomalies recorded in Dr0m and Dr1m1 (1: Cleft lip, cleft palate, and cleft lip and palate are listed, bur are omitted from this table.) Response to Comment 1-5-5 We apologize for the typo and the confused writing in the footnote. We have revised this as “Cleft lip, cleft palate, and cleft lip and palate are also available, but are not listed here for simplicity.” Reviewer #2 Comment 2-1 The introduction needs further elaboration about the burden of metal exposures worldwide. Further, the justification of including manganese and selenium is not sufficient. The authors should specify the health impact of metals on the outcome of interest, including the risk estimates. Response to Comment 2-1 We thank the Reviewer for this important point; the Editor and another Reviewer commented similarly. To explain the rationale for this study, we added a brief description of the hazards of ingesting metals. As for manganese and selenium, we treated manganese as a heavy metal but omitted selenium from this revision; please refer to our detailed response to comment E3. Comment 2-2 It is not clear why other cases of congenital anomalies were not considered in the analysis. Response to Comment 2-2 In the terminology of congenital malformation, syndrome (or syndromic) cases are commonly defined as a patient with malformation of more than one developmental field or body site, with or without identifiable causes such as chromosomal abnormalities (according to Ref. #7). Cleft lip/palate is classified as syndromic or non-syndromic (i.e., without malformation of other body sites) and it is assumed that these two categories have different etiologies. For example, children with Down syndrome (trisomy 21) are at higher risk of cleft lip/palate together with congenital heart defects or gastrointestinal malformation, which constellations are thought to originate from a chromosomal abnormality (although this has not yet been confirmed). We therefore enrolled cases without other malformations in the primary analysis because they were likely to represent non-syndromic cases. However, as mentioned in the text, the classification of syndromic vs. non-syndromic cases may differ between researchers, and it is possible that the environmental factors (heavy metals in our case) may increase the risk of cleft lip/palate conjoint with other potential risk factors in syndromic cases. To account for this, we conducted a sensitivity analysis that involved cases/controls with other anomalies (sensitivity analysis 3), and found that the results were unaffected by case definition. We thank the Reviewer for this comment. Comment 2-3 Did the author graphically observe the associations by scatter plots (with Loess)? Linear associations are not always observed between toxic exposures and health outcomes. Whether there is any effect in the low exposure levels should be checked graphically. Furthermore, the authors should also explore the associations by grouping the exposures into tertile/quartile. Response to Comment 2-3 We thank the Reviewer for this thoughtful comment. Another Reviewer suggested an alternative analytical approach, Bayesian kernel machine regression (Biostatistics. 2015 Jul;16(3):493–508). The advantage of this approach is that it can estimate the health effects of multi-pollutant mixtures (e.g., interactions) and investigate exposure-response functions even when the relationship is non-linear. We found that, by graphical inspection, a dose-dependency between metal exposure level and risk of cleft L/P was unlikely. Please refer to the response to Comment 1-5-4. Comment 2-4 One of the weaknesses of the study is that exposure at very early gestation is not available. This weakness should be discussed. Further, are there any other unmeasured metals (such as arsenic) that may influence the observed association? Response to Comment 2-4 We thank the Reviewer for this critical comment. First, we agree with the Reviewer that the lack of data on exposure at very early gestation is the major limitation. We have added the following text as the first limitation: “However, fetal development of lip and palate begins as early as 4–5 weeks of gestation (2), the period around or before pregnancy perception in most women. Blood sample collection prior to this period is a challenge in a large-scale cohort; it should be acknowledged that there is often a trade-off between research feasibility and scientific basis.” With respect to other metals such as arsenic, data would be updated in the future as mentioned in the response to Comment E3. Comment 2-5 It is better if the authors add a separate table for exposures, including the mean, minimum and maximum values. Response to Comment 2-5 We thank the Reviewer for this comment. As another Reviewer recommended using either the mean or median uniformly, your recommendation was incorporated into the supplemental table (S4 Table). Comment 2-6 It is better for a reader if the authors add paragraphs on the strengths and limitations of the study. Response to Comment 2-6 We have created a paragraph on the strengths and limitations of the study, as suggested. Comment 2-7 Minor issues: Page 23, Line 220: missing full-stop; Page 15, Table 1: replace ‘media’ with ‘median’ at the end. Response to Comment 2-7 We have accordingly corrected these errors. We thank the Reviewer for pointing them out. Attachments Attachment Submitted filename: Rev1_ResponseLetter.docx https://doi.org/10.1371/journal.pone.0265648.r002
27 Jan 2022 Decision Letter - Forough Mortazavi, Editor PONE-D-21-29327R1Association of maternal heavy metal exposure during pregnancy with isolated cleft lip and palate in offspring: Japan Environment and Children’s Study (JECS) cohort studyPLOS ONE Dear Dr. Takeuchi, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Mar 13 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Forough Mortazavi Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: Dear authors, Thank you for submitting your manuscript to PLOS ONE. The manuscript has been revised according to the comments except for a few points which should be addressed. Please kindly check the manuscript to ensure that all the RECORD checklist items are complied with. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Abstract needs to be revised, especially the part of results, results of conditional logistic regression which test the association between heavy metal exposure and isolated cleft L/P needs to be demonstrated。 Reviewer #2: The authors addressed all the issues raised by the reviewer. It is better to attach the revision indicating the changes made. Furthermore, the authors also should add the line and page number of the new texts to make the review easy. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes: Jufen Liu Reviewer #2: Yes:** Anisur Rahman [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0265648.r003
Revision 2
8 Feb 2022 Author Response Reviewer #1 Comment 1-1 Abstract needs to be revised, especially the part of results, results of conditional logistic regression which test the association between heavy metal exposure and isolated cleft L/P needs to be demonstrated. Response 1-1 Following this suggestion, we have added the results of conditional logistic regression analysis to the Abstract (ln. # 56-58). “the adjusted odds ratios (per 1 μg/L increase) with 95% intervals were 0.96 (0.91-1.03), 1.01 (0.94-1.08), 1.00 (0.61-1.63) and 1.00 (0.97-1.03) for Hg, Pb, Cd and Mn, respectively” Reviewer #2 Comment 2-1 It is better to attach the revision indicating the changes made. Furthermore, the authors also should add the line and page number of the new texts to make the review easy. Response 2-1 We apologize for the inconvenience in the previous version. In this revision, we indicated the changes with the line number. Attachments Attachment Submitted filename: Rev2_ResponseLetter.docx https://doi.org/10.1371/journal.pone.0265648.r004
7 Mar 2022 Decision Letter - Forough Mortazavi, Editor Association of maternal heavy metal exposure during pregnancy with isolated cleft lip and palate in offspring: Japan Environment and Children’s Study (JECS) cohort study PONE-D-21-29327R2 Dear Dr. Takeuchi, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Forough Mortazavi Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: Yes:** Anisur Rahman https://doi.org/10.1371/journal.pone.0265648.r005
Formally Accepted
10 Mar 2022 Acceptance Letter - Forough Mortazavi, Editor PONE-D-21-29327R2 Association of maternal heavy metal exposure during pregnancy with isolated cleft lip and palate in offspring: Japan Environment and Children’s Study (JECS) cohort study Dear Dr. Takeuchi: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Forough Mortazavi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0265648.r006

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