Unraveling the causal association between leukocyte telomere length and infertility: A two-sample Mendelian randomization study

Gaole An; Xingnan Zhao; Chenghui Zhao

doi:10.1371/journal.pone.0298997

Peer Review History

Original SubmissionOctober 10, 2023
11 Dec 2023 Decision Letter - Arthur J. Lustig, Editor PONE-D-23-32653Unraveling the Causal Association Between Leukocyte Telomere Length and infertility: A Two-Sample Mendelian Randomization StudyPLOS ONE Dear Dr. Zhao, 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. The statistical analysis associated with MR analysis requires the confounding variates that were included in the studies. The authors need to provide clarification of this issue. Also, please respond to each of the issues raised by the Reviewer. I look forward to your revision. Please submit your revised manuscript by Jan 25 2024 11:59PM. 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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: In this investigation the author’s conduct a bidirectional MR study on the association between LTL and infertility. Overall, the report is concise and is careful to not overstate conclusions beyond the findings presented. However, key details of the statistical analysis and instrumentation are not well described and should be clarified in a revised submission. I suggest the following considerations: MAJOR COMMENTS • Did the authors perform adjustment for any important covariates in their analyses such as age and sex? Although MR operates under the assumption that instrumental variables do not act on confounders, it does not preclude the existence of those confounders. Important confounds of interest (age, sex, study site, BMI) should be included in the first stage regression to generate IVs for SNP-LTL and SNP-infertility related traits or as covariates in the second stage regression. For further details see Statistical Analysis of Kuo et al., 2019 (https://doi.org/10.1111/acel.13017) and Discussion of Burgess et al., 2011; https://doi.org/10.1093/ije/dyr036). • Although the authors provide a supplementary table with a full list of SNPs utilized to generate IVs for LTL and infertility related traits, it would be beneficial to also include a summary table of the total number of SNPs associated with each factor and how much variance in each factor was explained by the overall instrument. MINOR COMMENTS • Results: it should be made clear that the data used for analyses of male and female infertility was derived from the FinnGen datasets in a similar manner as is described for the analyses on LTL and endometriosis. • Line 164-168: It would help to contextualize contrasting results between TL and male infertility and their possible relationship to sample size by including the sample size of Ref #7 to contrast it with the “larger study” in Ref #20. That said, it is unclear if sample size contributes to the discrepancy of findings at all since the study on nonobstructive azoospermia (Ref #19) had a sample size of 866. Instead it might be a difference of outcome and how male infertility is defined. ****** 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. 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Revision 1
11 Jan 2024 Author Response MAJOR COMMENTS Reviewer #1: Did the authors perform adjustment for any important covariates in their analyses such as age and sex? Although MR operates under the assumption that instrumental variables do not act on confounders, it does not preclude the existence of those confounders. Important confounds of interest (age, sex, study site, BMI) should be included in the first stage regression to generate IVs for SNP-LTL and SNP-infertility related traits or as covariates in the second stage regression. For further details see Statistical Analysis of Kuo et al., 2019 (https://doi.org/10.1111/acel.13017) and Discussion of Burgess et al., 2011; https://doi.org/10.1093/ije/dyr036). Response 1: Thanks for your valuable advice. We have thoroughly reviewed the recommended articles, specifically focusing on the statistical analysis conducted by Kuo. In their study, he used SNPs on mean LTL was previously estimated with adjustment for age, sex, body mass index (BMI), and smoking history (Haycock et al., 2017; https://doi.org/10.1001/jamaoncol.2016.5945). However, we were unable to obtain the same GWAS summary data used in their analysis. In our study, the summary GWAS data of LTL was adjusted for age, sex, array, and the first ten principal components. To account for the potential confounding effects of BMI and smoking history, we employed multivariable Mendelian randomization (MVMR) methods. The MVMR results have been included in Tables1-Table3, and further detailed information can be found in S7 Table. In summary, even after excluding the effects of confounding factors such as BMI and smoking history, we did not observe a significant causal relationship between LTL and infertility, while a statistically significant association between LTL and endometriosis was still observed. As suggested, we have provided a detailed description in the Exposure data section of the Methods and highlighted the changes in yellow. Modified (LINE 75-83): The genetic association data for LTL were obtained from a genome-wide association study (GWAS) involving 472,174 European participants (10), adjusting for age, sex, and the first ten principal components (PCs). These participants were aged 40-69 years and had an equal distribution of males (45.8%) and females (54.2%). LTL measurements were determined using a quantitative polymerase chain reaction (qPCR) assay. To mitigate any potential biases stemming from body mass index (BMI) and smoking, we procured comprehensive BMI GWAS summary data from the UK Biobank and smoking GWAS sunmmary data from the GWAS & Sequencing Consortium of Alcohol and Nicotine use (GSCAN). Leveraging these datasets, we conducted multivariable Mendelian Randomization (MVMR) to ensure a robust analysis. Reviewer #2: Although the authors provide a supplementary table with a full list of SNPs utilized to generate IVs for LTL and infertility related traits, it would be beneficial to also include a summary table of the total number of SNPs associated with each factor and how much variance in each factor was explained by the overall instrument. Response 2: Thank you for your valuable suggestions. We acknowledge the significance of providing a comprehensive summary of the single nucleotide polymorphisms (SNPs) utilized as instrumental variables (IVs) for both telomere length (LTL) and infertility-related traits. To address this, we have included a summary table, namely S3 Table, in our revised manuscript. This table presents detailed information regarding the total number of SNPs associated with each factor, as well as the mean F-statistics and the sum of R2 values. In our study, we utilized a total of 135 instrumental variables. The mean F-statistics for these instrumental variables is approximately 120, indicating strong instrument strength. Furthermore, the sum of R2 values for the SNPs is approximately 0.01, suggesting that the SNPs explain a small proportion of the variance in the traits of interest. MINOR COMMENTS Reviewer #3: It should be made clear that the data used for analyses of male and female infertility was derived from the FinnGen datasets in a similar manner as is described for the analyses on LTL and endometriosis. Response 3: Thanks for your valuable advice. We have added the data description in the results parts of male and female infertility. Added (LINE 131-133): We conducted a comprehensive analysis using the male infertility and sperm abnormalities datasets from FinnGen to evaluate the causal association between leukocyte telomere length (LTL) and the occurrence of male infertility and sperm abnormalities. Added (LINE 142-144): To investigate the potential causal relationship between leukocyte telomere length (LTL) and female infertility, we conducted a rigorous analysis utilizing the comprehensive collection of five datasets obtained from FinnGen. Reviewer #4: It would help to contextualize contrasting results between TL and male infertility and their possible relationship to sample size by including the sample size of Ref #7 to contrast it with the “larger study” in Ref #20. That said, it is unclear if sample size contributes to the discrepancy of findings at all since the study on nonobstructive azoospermia (Ref #19) had a sample size of 866. Instead it might be a difference of outcome and how male infertility is defined. Response 4: We think this is an excellent suggestion. Upon further examination of the relevant literature, it was discovered that Reference #19 also supports the absence of a correlation between telomere length (LTL) and obstructive azoospermia (OA), while indicating a correlation between LTL and nonobstructive azoospermia (NOA). OA and NOA are two subtypes of oligozoospermia. Both Reference #7 and Reference #20 conducted research on male infertility populations, but they did not analyze the specific subtypes of oligozoospermia as explored in Reference #19. Reference #7 did not account for confounding factors such as age in their statistical analysis, whereas Reference #20 had a substantial sample size and effectively controlled for confounding factors including age and smoking, rendering its findings more reliable. Considering these factors, we have revised the discussion section regarding the association between LTL and male infertility. Revised paragraph (LINE 200-209): Nevertheless, upon meticulous exclusion of the confounding influences of age, BMI, and smoking, our study findings robustly challenge the prevailing belief that a causal link exists between LTL and male infertility or abnormal spermatogenesis. Importantly, these results align with a comprehensive investigation encompassing 599 individuals [21], lending further credibility to our conclusion. The discrepancy in findings between our study and Yang's study [20] may be attributed to differences in the definition of sperm abnormalities. Specifically, Yang’s study found a significant relationship between LTL and NOA, whereas other types of sperm abnormalities were not significant. Furthermore, it is important to note that Darmishonnejad's study [7] did not account for the confounding effects of age, BMI, and smoking, and had a relatively small sample size, which increases the likelihood of false positive results. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0298997.r002
5 Feb 2024 Decision Letter - Arthur J. Lustig, Editor Unraveling the causal association between leukocyte telomere length and infertility: a two-sample Mendelian randomization study PONE-D-23-32653R1 Dear Dr. Zhao, 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, Arthur J. Lustig, PhD 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: I appreciate the rigor and open-mindedness with which the authors approached their revision. I look forward to seeing this work provide further contribution to the literature on relationships between telomere length and infertility. ****** 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: Waylon James Hastings ******** https://doi.org/10.1371/journal.pone.0298997.r003
Formally Accepted
13 Mar 2024 Acceptance Letter - Arthur J. Lustig, Editor PONE-D-23-32653R1 PLOS ONE Dear Dr. Zhao, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Arthur J. Lustig Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0298997.r004

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