Causality of anthropometric markers associated with polycystic ovarian syndrome: Findings of a Mendelian randomization study

Kushan De Silva; Ryan T. Demmer; Daniel Jönsson; Aya Mousa; Helena Teede; Andrew Forbes; Joanne Enticott

doi:10.1371/journal.pone.0269191

Peer Review History

Original SubmissionFebruary 11, 2022
31 Mar 2022 Decision Letter - Renato Polimanti, Editor Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. PONE-D-22-04303Both general- and central- obesity are causally associated with polycystic ovarian syndrome: Findings of a Mendelian randomization studyPLOS ONE Dear Dr. De Silva, 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 May 15 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. 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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. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. [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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 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: 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 ****** 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: This study asks an interesting question, if anthropometric traits are causal for PCOS. The study is well planned and the statistical analysis is rigurous. As the authors also mention, the most important concern about this study is the low number of cases for PCOS. I suggest meta-analyzing several cohorts (e.g., the UK Biobank and FinnGen) for future studies to increase the sample size. 1. You state that central obesity has a stronger association with PCOS, than BMI. However, you analyze the anthropometric markers, WC, HC, and WHR separately as exposures. Therefore, you have no evidence on ‘central obesity’ as a whole to be causal for PCOS, but you have evidence on separate factors measured for central obesity. Please explain this in the introduction/discussion sections. I suggest running a multivariable MR analysis to understand the complex relationship between all exposures (two different analyses: only markers for central obesity, and markers for central and general obesity) with PCOS as the outcome. 2. To understand the bidirectional relationship between the anthropometric markers and PCOS, it would be interesting to see if PCOS as an exposure is causal for these traits, as we know that the metabolic and hormonal changes in PCOS can lead to weight gain and eventually obesity. Please explain in the discussion the bidirectional relationship between these traits. 3. Please mention in the methods the software you used for the analysis, what I suppose was the TwoSampleMR package in R. 4. I am not sure about the relevance of ‘3.8. Genetic architectures underlying causality’ for this paper. The fact that these anthropometric traits have shared genetic background is already known. The selected IVs were the SNPs that were associated with the exposure dataset, so this part of the manuscript does not help understanding the causality among anthropometric traits and PCOS. In the discussion, ‘A gene set rather similar to those found in the present study was underlying the causal association between BMI and PCOS in a previous MR study as well’ should be explained more thoroughly. What was similar between these genes? What could be their roles? 5. The novel results of this study are WC and HC being causal for PCOS, however, not much is discussed about these results in the discussion section. You should also explain why you think you did not find any association with WHR, another marker for central obesity. ****** 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:** Dora Koller [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.0269191.r001
Revision 1
15 May 2022 Author Response RESPOSNE TO REVIEWERS We sincerely thank the reviewers for all the comments provided to our initial submission. We have now revised the manuscript accordingly and our point-by-point responses to the comments are given below. Corresponding changes are highlighted in red in the “Revised Manuscript with Track Changes”. Please note that since our initial submission was made, the outcome cohort has been updated on the IEU OpenGWAS project. The original PCOS cohort named “finn-a-E4_POCS” with a sample size of 96391(219 cases; 96172 controls) has now been updated to “finn-b-E4_POCS” with a larger sample size of 118870 (642 cases; 118228 controls). In order to ensure the reproducibility of our findings based on publicly available data, we therefore re-conducted all analyses, using this larger, updated PCOS cohort. All updated results from these re-analyses are presented in the revised manuscript. 1. This study asks an interesting question, if anthropometric traits are causal for PCOS. The study is well planned and the statistical analysis is rigorous. As the authors also mention, the most important concern about this study is the low number of cases for PCOS. I suggest meta-analyzing several cohorts (e.g., the UK Biobank and FinnGen) for future studies to increase the sample size. We have acknowledged this in the revised manuscript. Also note that the PCOS cohort used in the updated version is slightly larger than the one used in our original manuscript. As suggested, we have highlighted the need for meta-analyzing several cohorts in future studies, in Discussion section. Specifically, we added the following: “For future studies, we suggest meta-analyzing several cohorts from sources such as the UK Biobank and FinnGen as a judicious avenue for formidably increasing the sample size.” 2. You state that central obesity has a stronger association with PCOS, than BMI. However, you analyze the anthropometric markers, WC, HC, and WHR separately as exposures. Therefore, you have no evidence on ‘central obesity’ as a whole to be causal for PCOS, but you have evidence on separate factors measured for central obesity. Please explain this in the introduction/discussion sections. I suggest running a multivariable MR analysis to understand the complex relationship between all exposures (two different analyses: only markers for central obesity, and markers for central and general obesity) with PCOS as the outcome. Anthropometric traits are customarily used as surrogate markers for operationalizing obesity. Previous MR studies assessed total causal effects of central- and general obesity using these anthropometric markers. This has now been explained in Introduction as follows: “Anthropometric traits are customarily exploited in MR studies as valid and reliable surrogate markers for operationalizing central- and general obesity [30-32]. In these studies, multiple anthropometric markers were incorporated in univariable MR to gain evidence on causal roles of central- and general obesity [30-32].” Furthermore, as suggested, we conducted multivariable MR analyses to understand the complex relationship between all exposures and findings have been discussed. Findings alluded to the existence of complex causal relationships such as potential causal mediations in the presence of both central- and general obesity and indicated that normal weight-central obesity might have direct causal effects on PCOS. Multivariable analyses are described in Methods under “Multivariable MR analyses” section. Findings of multivariable MR are summarized in Table 6 and described in Results under “Multivariable MR findings” section. We have discussed these findings including their implications in Discussion under “Findings of multivariable MR models and their implications” section. 3. To understand the bidirectional relationship between the anthropometric markers and PCOS, it would be interesting to see if PCOS as an exposure is causal for these traits, as we know that the metabolic and hormonal changes in PCOS can lead to weight gain and eventually obesity. Please explain in the discussion the bidirectional relationship between these traits. This has now been addressed. As described in Methods under “Bidirectional MR analyses”, we conducted MR in the opposite direction, using the same data sources and model specifications and criteria applied to primary univariable MR analyses. However, as described in Results under “Bidirectional MR findings”, sufficient number of functional variants could not be retrieved from existing data. In Discussion, under “Bidirectional associations/Reverse causality”, we have discussed and contextualized findings from previous studies which assessed reverse causality (whether PCOS can cause obesity). All previous bidirectional analyses confirmed that there is no reverse causality. Specifically, we have included the following in Discussion: “For bidirectional 2SMR analyses, sufficient number of IVs could not be retrieved from existing data sources, but, with the gradual expansion of these open-source GWAS summary data, we envisage that it will be possible to conduct bidirectional analyses more robustly, in the future. Stringent eligibility criteria to select only highly uncorrelated and independent SNPs (LD-R2 = 0.001) may also have contributed to the lack of functional variants produced for assessing reverse causality. However, concordant evidence from previous bidirectional analyses provides reassurance that there is no reverse causality i.e. PCOS would not cause obesity [25, 26].” 4. Please mention in the methods the software you used for the analysis, what I suppose was the TwoSampleMR package in R. This information is now included in the manuscript. Specifically, in Methods: “Analyses were conducted in R (version 4.1.2) [44], using “TwoSampleMR” [45], “MRPRESSO” [42], and “RadialMR” [43] packages.” “This was performed using the multivariable inverse variance weighted method deployed through the ‘mr_mvivw’ function in the ‘MendelianRandomization’ R package [49, 50].” 5. I am not sure about the relevance of ‘3.8’. Genetic architectures underlying causality’ for this paper. The fact that these anthropometric traits have shared genetic background is already known. The selected IVs were the SNPs that were associated with the exposure dataset, so this part of the manuscript does not help understanding the causality among anthropometric traits and PCOS. In the discussion, ‘A gene set rather similar to those found in the present study was underlying the causal association between BMI and PCOS in a previous MR study as well’ should be explained more thoroughly. What was similar between these genes? What could be their roles? We underscore that MR studies increasingly explore downstream genetic underpinnings including shared genetic basis. As highlighted by previous MR studies, such downstream analyses could help us gain important etiologic insights and clinically valuable signals such as drug targets via functional annotations. This has now been clarified as follows: “Subsequent to causality testing, MR studies increasingly conduct downstream analyses to explore genetic underpinnings [46, 47]. For instance, previous MR studies showed that functional annotations of highly significant SNPs that act as major drivers of causality in MR models could assist in gaining etiologic insights [46] and identifying putative drug targets [47].” As shown in S2 Table (Harmonized Data), selected IVs/functional variants associate with both the exposure and the outcome. As summarized in Table 5 and described in Results under “Genetic architectures underlying causality”, we have now explored genetics underlying causality more thoroughly including shared and non-shared SNPs/genes. Furthermore, we have provided evidence on their putative mechanistic roles in S6 Table. The motivation behind this downstream exploration of SNPs/genes underlying causal associations was that it could provide important insights into genetic etiology of PCOS and might provide some directions for future studies aimed at unravelling complex genetic mechanisms of PCOS pathogenesis. This has been discussed in detail in Discussion under “Genetics underlying causality”. 6. The novel results of this study are WC and HC being causal for PCOS, however, not much is discussed about these results in the discussion section. You should also explain why you think you did not find any association with WHR, another marker for central obesity. We have now discussed the novel findings of this study including WC and HC being causal for PCOS, interpretations and implications of findings on direct causal effects emanating from multivariable MR models, and clinical and public health relevance of these results. Specifically, regarding the importance of causal roles of central obesity demarcated by models comprised of WC and HC, we included following in Discussion: “In addition, this study offered novel results such as the total causal effects of central obesity on PCOS, potentially direct causal effects of normal weight-central obesity on PCOS, and that complex causal mechanisms in the presence of both general- and central obesity might lead to PCOS. Of note, although our analyses indicated possibly direct causal effects of normal weight-central obesity on PCOS, people with central obesity but of normal weight are generally ignored in clinical guidelines [69]. Therefore, women with normal weight-central obesity will require an equally important focus as attributed to general obesity phenotype, in clinical guidelines for PCOS management. To this end, Mediterranean style dietary patterns and physical activity both are evidence-based interventions to reduce central obesity and ensure weight stabilization [70]. Causality of central obesity/adiposity demarcated by WC/HC in the present study corroborate findings from mechanistic studies focused on PCOS etiology. Truncal/visceral obesity promotes insulin resistance which in turn aggravates metabolic and reproductive functions in PCOS such as hyperandrogenism, dyslipidemia, and anovulation [71]. Evidence suggests the formation of a vicious circle by central obesity which involves hyperandrogenemia and truncal adiposity in PCOS [72]. On the whole, findings of this study indicating complex causality of central- and general obesity on PCOS, highlight the need to address the prevention of both forms of obesity at a population level through successful fiscal policies and statutory regulations [73].” We have also discussed possible reasons for the non-significance of waist-to-hip ratio. Specifically, we included following in Discussion: “Non-significance of WHR might be owing to its lesser validity and reliability as an anthropometric marker of central obesity, compared to WC/HC, as concluded by previous studies [73, 74]. Lack of adequate statistical power in MR models to uncover causal associations from open-source data might be another contributory factor. This may be verified in future studies using larger cohorts.” RESPONSE TO OTHER COMMENTS/FORMATTING REQUIREMENTS Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. 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 The entire manuscript, including all figures and supporting information, has now been formatted according to the stipulated PLOS requirements. 2. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. Ethics statement has been included in Methods section. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0269191.r002
17 May 2022 Decision Letter - Renato Polimanti, Editor Causality of anthropometric markers associated with polycystic ovarian syndrome: Findings of a Mendelian randomization study PONE-D-22-04303R1 Dear Dr. De Silva, 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, Renato Polimanti, Ph.D. 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 #1: 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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: 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 ****** 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: The authors addressed all my comments and added all necessary discussion points that were missing in the first submission. I recommend accepting the manuscript. ****** 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:** Dora Koller https://doi.org/10.1371/journal.pone.0269191.r003
Formally Accepted
24 May 2022 Acceptance Letter - Renato Polimanti, Editor PONE-D-22-04303R1 Causality of anthropometric markers associated with polycystic ovarian syndrome: Findings of a Mendelian randomization study Dear Dr. De Silva: 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. Renato Polimanti Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0269191.r004

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