Ceiling effect of clomiphene citrate on the testosterone to estradiol ratio in eugonadal infertile men

Yian Liao; Yi-Kai Chang; Shuo-Meng Wang; Hong-Chiang Chang

doi:10.1371/journal.pone.0262924

Peer Review History

Original SubmissionAugust 11, 2021
17 Nov 2021 Decision Letter - Joël R Drevet, Editor PONE-D-21-24474Ceiling Effect of Clomiphene Citrate on the Testosterone to Estradiol Ratio in Eugonadal Infertile MenPLOS ONE Dear Dr. HC Chang, 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 at your earliest convenience. 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, Joël R Drevet, Ph.D. Academic Editor PLOS ONE 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 2. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. "Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. Additional Editor Comments (if provided): Your submission has been evaluated by two independent reviewers who found that it has potential but requires substantial revision. Please revise your manuscript by addressing, to the best of your ability, each of the reviewers' concerns and providing additional material to strengthen your report. [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: No Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: 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 Reviewer #2: No ****** 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: I appreciate the manuscript and the work that went into preparing it. Just a few suggestions to strengthen the product: 1. As aromatase breaks down T to E2, would report on the effect of BMI on results/outcomes. 2. Should the Cirrhosis patient be excluded since liver disease effects sex hormone binding globulin production (effects total Testosterone)? 3. Is the standard to use ng/mL for T? I'm more used to ng/dL...with the normal T:E ratio being 10:1. Best regards. Reviewer #2: Dear Dr. Hong-Chiang Chang Based on my own reading of your paper, I have some issues I want to address. I would like to say right away that the theme “ceiling effect” is quite interesting as a predictor for clomiphene citrate (CC) use. I emphasize that the PDF of the article I received does not have line:numbers, which made the review a bit difficult. 1) Introduction: I did not observe references for all the information contained in the paragraph below. Despite being general domain information, it is important to always add reference sources, even if it is in the introduction. “Selective estrogen receptor modulators (SERMs) are drugs that act on estrogen receptors. Such drugs can be divided into receptor agonists and antagonists (Ref?). These drugs selectively act on specific organs, with effects differing by organs (Ref?). Clomiphene citrate is both a nonsteroidal antiestrogen drug and an SERM that competes with estradiol for estrogen receptors in the hypothalamus and blocks the normal negative feedback of circulating estradiol on the hypothalamus (Ref?). The production of gonadotroptin-releasing hormone (GnRH) may not be limited by estrogen; instead, the pituitary gland releases more follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and causes an increase in sperm and testosterone production by the testes (Ref?). High levels of intratesticular testosterone is a key factor in the nonconventional pathway of testosterone for spermatogenesis (Ref?). 2) Materials and Methods I would like the authors to specify which reference was used to define the diagnosis of eugonadism (total testosterone 2.4 to 8.7 ng/mL). The laboratory diagnosis of hypogonadism is based on the measurement of total testosterone. The American Society of Endocrinology recommends the value of 264 ng/dL (9.2 nmol/L) as the lower limit to define the hypogonadal state; however, the cutoff levels for diagnosis are not a consensus. So I would like the authors to specify the Guideline used. (Testosterone Therapy in Men With Hypogonadism: An Endocrine Society* Clinical Practice Guideline) I would like the authors to write “range of the total testosterone level” instead of just “range of the testosterone level” – here it can be confusing if total or free testosterone was used as a parameter. I would like more detail in the table of 22 patients at baseline with data about BMI, waistline and testicular volume if possible 3) Discussion An important point to be considered is the ability of the CC to induce a response in patients with central or mixed hypogonadism. In the different studies in which its use was evaluated, it was possible to define response (elevation of testosterone) in patients with Leydig cells capable of reacting to the central stimulus restored by CC (that's why I would like to know about testicular volume). It is known that the unbalance of the T:E2 ratio is directly implicated in the pathophysiology of hypogonadism. It is important to emphasize that the increase in estrogen with the use of CC results from the aromatization of testosterone in the adipose tissue, especially the visceral tissue. I would like to see the details of the abdominal fat of these patients in order to understand whether the ceiling effect proposed by the authors is related to patients with larger abdominal fat/waistline and therefore greater visceral fat. ******** 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: No Reviewer #2: Yes:** Andressa Heimbecher Soares [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.0262924.r001
Revision 1
30 Nov 2021 Author Response To editor: We really appreciate for reminding us all additional journal requirements. The article has now met PLOS ONE's style requirements. As for the Data Availability statement, we stated that there are no supporting information in the main body of our article. To reviewer 1: Q: 1. As aromatase breaks down T to E2, would report on the effect of BMI on results/outcomes. Answer: Different BMI levels may probably affect T/E2 ratio. However, we did not include patients’ BMI in our data base. Your suggestion is good for further study of this topic and data of BMI would make a more precise analysis. Q: 2. Should the Cirrhosis patient be excluded since liver disease effects sex hormone binding globulin production (effects total Testosterone)? Answer: Cirrhosis surely affect sex hormone binding globulin production, indirectly influencing the bioavailability of testosterone. As for the patient included in our study with liver cirrhosis, his Child-Pugh score is only grade A with normal bilirubin, PT/APTT and albumin level, which may hardly have an impact on sex hormone binding globulin production. Q: 3. Is the standard to use ng/mL for T? I'm more used to ng/dL...with the normal T:E ratio being 10:1. Answer: There may not be a standard unit of testosterone level. According to EAU(European Association of Urology) guideline, ng/mL and nmol/L have both been used as the unit of testosterone level. To reviewer 2: 1) Introduction: Q: I did not observe references for all the information contained in the paragraph below. Despite being general domain information, it is important to always add reference sources, even if it is in the introduction. “Selective estrogen receptor modulators (SERMs) are drugs that act on estrogen receptors. Such drugs can be divided into receptor agonists and antagonists (Ref?). These drugs selectively act on specific organs, with effects differing by organs (Ref?). Clomiphene citrate is both a nonsteroidal antiestrogen drug and an SERM that competes with estradiol for estrogen receptors in the hypothalamus and blocks the normal negative feedback of circulating estradiol on the hypothalamus (Ref?). The production of gonadotroptin-releasing hormone (GnRH) may not be limited by estrogen; instead, the pituitary gland releases more follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and causes an increase in sperm and testosterone production by the testes (Ref?). High levels of intratesticular testosterone is a key factor in the nonconventional pathway of testosterone for spermatogenesis (Ref?). Answer: We really appreciate the advice of adding references. Three references have been added to the introduction. The following paragraph is the revised introduction. “Selective estrogen receptor modulators (SERMs) are drugs that act on estrogen receptors and such drugs can be divided into receptor agonists and antagonists [1]. These drugs selectively act on specific organs, with effects differing by organs [1]. Clomiphene citrate is both a nonsteroidal antiestrogen drug and an SERM that competes with estradiol for estrogen receptors in the hypothalamus and blocks the normal negative feedback of circulating estradiol on the hypothalamus [2]. The production of gonadotroptin-releasing hormone (GnRH) may not be limited by estrogen; instead, the pituitary gland releases more follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and causes an increase in sperm and testosterone production by the testes [2]. High levels of intratesticular testosterone is a key factor in the nonconventional pathway of testosterone for spermatogenesis [3].” Reference: 1. Chua, M.E., et al. Revisiting oestrogen antagonists (clomiphene or tamoxifen) as medical empiric therapy for idiopathic male infertility: a meta-analysis. Andrology, 2013. 1: 749. 2. Ribeiro, R.S., et al. Clomiphene fails to revert hypogonadism in most male patients with conventionally treated nonfunctioning pituitary adenomas. Arq Bras Endocrinol Metabol, 2011. 55: 266. 3. Hussein, A., et al. Clomiphene administration for cases of nonobstructive azoospermia: a multicenter study. J Androl, 2005. 26: 787. 2) Materials and Methods Q: I would like the authors to specify which reference was used to define the diagnosis of eugonadism (total testosterone 2.4 to 8.7 ng/mL). The laboratory diagnosis of hypogonadism is based on the measurement of total testosterone. The American Society of Endocrinology recommends the value of 264 ng/dL (9.2 nmol/L) as the lower limit to define the hypogonadal state; however, the cutoff levels for diagnosis are not a consensus. So I would like the authors to specify the Guideline used. (Testosterone Therapy in Men With Hypogonadism: An Endocrine Society Clinical Practice Guideline) Answer: Exactly I agree that the cutoff level of hypogonadism and eugonadism has not reached a consensus. Different hospital may have different lab reference level with regard to their laboratory department. All patient included in our study have done blood examination in National Taiwan University Hospital. The reference range of total testosterone(2.4 to 8.7 ng/mL) is defined by Department of Laboratory Medicine of National Taiwan University Hospital. Q: I would like the authors to write “range of the total testosterone level” instead of just “range of the testosterone level” – here it can be confusing if total or free testosterone was used as a parameter. Answer: We really appreciate the advice of modifying “testosterone” to “total testosterone” not to be confusing. Adjustment have been made throughout the whole manuscript(changing the testosterone level to the total testosterone level) Q: I would like more detail in the table of 22 patients at baseline with data about BMI, waistline and testicular volume if possible Answer: BMI level, waistline and testicular volume are surely the possible factor that may affect total testosterone and estradiol level, indirectly influencing the T/E2 ratio. Unfortunately, these parameters were not included in our study. Due to the retrospective nature of our study, these factors were not recorded in our data base. Further prospective study including these factors may be needed. 3) Discussion Q: An important point to be considered is the ability of the CC to induce a response in patients with central or mixed hypogonadism. In the different studies in which its use was evaluated, it was possible to define response (elevation of testosterone) in patients with Leydig cells capable of reacting to the central stimulus restored by CC (that's why I would like to know about testicular volume). It is known that the unbalance of the T:E2 ratio is directly implicated in the pathophysiology of hypogonadism. It is important to emphasize that the increase in estrogen with the use of CC results from the aromatization of testosterone in the adipose tissue, especially the visceral tissue. I would like to see the details of the abdominal fat of these patients in order to understand whether the ceiling effect proposed by the authors is related to patients with larger abdominal fat/waistline and therefore greater visceral fat. Answer: According to EAU (European Association of Urology) guideline, larger abdominal fat and greater visceral fat has now been established to be related to hypogonadism. Increase in estrogen by aromatase in visceral fat has also been established in several studies. Unfortunately, these factors were also not included in our study and may not be achieved due to the retrospective nature of our study. We will work our best on the further prospective study of relevant topics. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0262924.r002
30 Dec 2021 Decision Letter - Joël R Drevet, Editor PONE-D-21-24474R1Ceiling Effect of Clomiphene Citrate on the Testosterone to Estradiol Ratio in Eugonadal Infertile MenPLOS ONE Dear Dr. Hong-Chiang Chang, 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 March 2022. 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, Joël R Drevet, Ph.D. Academic Editor PLOS ONE Additional Editor Comments: Although your manuscript has been improved, there are still aspects that require the authors' attention. [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 #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: Dear Dr Hong-Chiang Chang Based on my own reading of your Manuscript Draft - Revision 1, I have some issues in particular I wish to address. 1) In this part of the manuscript, I would like to correct “A small population study by Shabsigh [5] also reported an increased T/E ratio after clomiphene use in patients with hypogonadism” to “A small population study by Shabsigh [5] also reported an increased T/E2 ratio after clomiphene use in patients with hypogonadism”. The number 2 on E was missing. 2) I would like the description of the laboratory kits used for the analysis of total testosterone level, estradiol level, LH, FSH and prolactin level to be detailed. 3) I would like to know if the dosage of sex hormone binding globulin (SHBG) levels was measured via morning lab draws. 4) To make it easy in terms of standardization, I would like to put the total testosterone level in ng/dL and estradiol levels in ng/dL (https://doi.org/10.1016/j.juro.2017.02.2652) 5) In the “limitations” part of the study, I would like it to be detailed that there wasn't possible to get BMI level, waistline and testicular volume data. I suggest that the authors should proceed with the study to evaluate obese men versus men with normal weight and analyse if there are differences in the ceiling effect. 6) If I may suggest, a greater number of participants would improve the strength of the results 7) The study generates an interesting hypothesis about which subgroup of patients may benefit most from the use of clomiphene citrate ****** 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:** Andressa Heimbecher Soares [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.0262924.r003
Revision 2
3 Jan 2022 Author Response Reviewer #2: Q: 1) In this part of the manuscript, I would like to correct “A small population study by Shabsigh [5] also reported an increased T/E ratio after clomiphene use in patients with hypogonadism” to “A small population study by Shabsigh [5] also reported an increased T/E2 ratio after clomiphene use in patients with hypogonadism”. The number 2 on E was missing. A: We really appreciate for checking through the manuscript for any missing word or number. “Number 2” has now been added to T/E ratio. Q: 2) I would like the description of the laboratory kits used for the analysis of total testosterone level, estradiol level, LH, FSH and prolactin level to be detailed. A: We add the following description about the measurement methods in my document. All hormone test(testosterone, estradiol, LH, FSH and prolactin) included in our study are sent to the central lab in “Department of Laboratory Medicine of National Taiwan University Hospital”. FSH, LH, E2 and prolactin were measured by using IMMULITE® 2000 immunoassay system and Testosterone was measured by the ARCHITECT® 2nd Generation Testosterone assay which is a chemiluminescent microparticle immunoassay for the quantitative determination of testosterone. Q:3) I would like to know if the dosage of sex hormone binding globulin (SHBG) levels was measured via morning lab draws. A: Sex hormone binding globulin (SHBG) levels is surely a possible factor that may affect bioavailable testosterone, indirectly influencing the T/E2 ratio. Unfortunately, this parameter was not included in our study. Due to the retrospective nature of our study, this factor was not recorded in our data base. Further prospective study including this factor may be needed. Q:4) To make it easy in terms of standardization, I would like to put the total testosterone level in ng/dL and estradiol levels in ng/dL (https://doi.org/10.1016/j.juro.2017.02.2652) A: We really appreciate for this suggestion. All testosterone level and estradiol level have been changed to ng/dL. T/E2 ratio has also been adjusted due to the unit change of testosterone and estradiol. Q:5) In the “limitations” part of the study, I would like it to be detailed that there wasn't possible to get BMI level, waistline and testicular volume data. I suggest that the authors should proceed with the study to evaluate obese men versus men with normal weight and analyse if there are differences in the ceiling effect. A: We appreciate for the advice of adding this part to out limitation. We surely will proceed with the study to include these parameters for analysis to see if there are differences in the ceiling effect. Q:6) If I may suggest, a greater number of participants would improve the strength of the results A: Thanks for reviewer’s nice suggestion. Small study population is surely a limitation of our study. Further studies will include more participants to strengthen our result. Q:7) The study generates an interesting hypothesis about which subgroup of patients may benefit most from the use of clomiphene citrate A: We are glad that this study leads to an interesting result of “Clomiphene citrate significantly increase the T/E2 ratio in eugonadal infertile men under the premise of its ceiling effect (T/E2 ratio < 200)”. We would work hard on increasing the sample size and including more important parameters for further analysis. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0262924.r004
10 Jan 2022 Decision Letter - Joël R Drevet, Editor Ceiling Effect of Clomiphene Citrate on the Testosterone to Estradiol Ratio in Eugonadal Infertile Men PONE-D-21-24474R2 Dear Dr. HC Chang, 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, Joël R Drevet, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0262924.r005
Formally Accepted
21 Jan 2022 Acceptance Letter - Joël R Drevet, Editor PONE-D-21-24474R2 Ceiling Effect of Clomiphene Citrate on the Testosterone to Estradiol Ratio in Eugonadal Infertile Men Dear Dr. Chang: 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 Prof. Joël R Drevet Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0262924.r006

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .