Smooth muscle electromyography for detecting major alterations in the estrus cycle in rats

Kalman F. Szucs; Dora Vigh; Mohsen Mirdamadi; Reza Samavati; Tamara Barna; Annamaria Schaffer; Karmen Alasaad; Robert Gaspar

doi:10.1371/journal.pone.0307932

Peer Review History

Original SubmissionMarch 19, 2024
24 Jun 2024 Decision Letter - Abeer El Wakil, Editor PONE-D-24-11044Smooth muscle electromyography for detecting major alterations in the estrus cycle in ratsPLOS ONE Dear Dr. Gaspar, 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 Aug 08 2024 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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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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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 Reviewer #2: Yes Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: 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: Yes Reviewer #3: 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 Reviewer #3: 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: Some Remarks to the Authors: 1) It would be better to give a brief and appropriate explanation about Cetrorelix and its pharmacological mechanism of action in the Introduction section. 2) In the Methods section, which variance analysis (ANOVA, etc) was performed for multiple comparisons should be stated in the Statistical analysis sub-section. 3) References should be added to the use of Jongh's solution for the uterus, Tyrode's solution for the cecum, and the contractility study method. 4) For the Figure 1: The figure should be adjusted correctly, as if KCl application on uterine contractions begins at the 15th minute rather than the 16th minute. Thanks for your work. Reviewer #2: Comments for the authors: In this paper, the authors investigated smooth muscle electromyography for detecting major alterations in the estrus cycle in rats. This study is well-designed, the language is easy to understand, the style is enjoyable, and it contains scientific novelties. Their research results are innovative, as they have both scientific and health relevance and may have economic (e.g., animal husbandry) implications. Minor Remarks: 1. What is the basis for the dose and duration of cetrorelix acetate treatment? 2. What practical indications are that de Joung solution was used for uterus samples and Tyrode buffer for cecum samples in isolated organ baths? 3. Line 153 of the results section lacks a precise description of the in vivo smooth muscle electromyography result. Supplement the legend of Figure 3 with the Fourier transform FFT notation! 4. I think the bibliographic reference in line 158 (the legend of Figure 3) is redundant. Literature 11 can be found in the methods section and the summary also. 5. In line 175: "Cetrorelix suppressed staining in the uterus (bottom image row)." I suggest instead, for the reader's ease of understanding, "Cetrorelix treatment reduced αvβ3 integrin expression, which caused a significant decrease in intensity." 6. In Figure 5, I propose to mark the uterine horns with a white arrow in each image. 7. Why the effect on KCl-induced contractions was considered to be justified in addition to the spontaneous uterine contractility study. Reviewer #3: The authors have found simultaneous increases in the contraction of the cecum and the non-pregnant uterus when they used smooth muscle electromyographic (SMEMG) in the female rats. They have also observed that fluorescence imaging showed high levels of uterine αvβ3 integrin during the proestrus-estrus phase, but inhibiting the sexual cycle reduced fluorescence activity. Based on the results, the authors have suggested that the SMEMG and IVIS imaging methods are suitable for detecting estrus phase alterations in rats. As the authors mentioned, the SMEMG method might be suitable for the complex measurement of reproductive processes in other mammals or in female patients or healthy volunteers after clinical translation. The one point to be cleared in the present experiment is that whether the animals which were used in the study were observed in terms of regular estrus cycle before they began the experiment. In the manuscript, there is no any explanation about this important point. The authors have to mention that the animals used were chosen among those that show regular estrus cycle. ****** 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: No Reviewer #3: No ******** [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.0307932.r001
Revision 1
6 Jul 2024 Author Response The authors thank the Editor and the Reviewers for their useful questions and remarks which have contributed considerably to improving the quality of our manuscript. Our answers are given below. RESPONSE TO EDITOR 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. We are thankful for this remark; we have made all the necessary changes according to the requirements. 2. Thank you for stating the following financial disclosure. Please state what role the funders took in the study. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. Based on the request of the Editor, the following sentence is inserted in the manuscript: ‘The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.’ 3. In the online submission form, you indicated that [Data are available for request]. All PLOS journals now require all data underlying the findings described in their manuscript to be freely available to other researchers, either 1. In a public repository, 2. Within the manuscript itself, or 3. Uploaded as supplementary information. The data based on the research results are uploaded in table form as supplementary datasets. 4. 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. The reference list has been checked and does not contain retracted papers. However, based on Reviewers' requests, the complete list has been supplemented; we have added 3 additional literature references to the complete list, which are highlighted in the manuscript. RESPONSE TO REVIEWERS REVIEWER 1 1) It would be better to give a brief and appropriate explanation about Cetrorelix and its pharmacological mechanism of action in the Introduction section. The authors thank the Reviewer for this remark. The following has been inserted in the Introduction: ‘The physiological processes induced by the increase in the LH level can be inhibited by drugs. Cetrorelix is a potent and selective competitive antagonist of the GnRH receptor. Reversibly reduces LH and FSH from the anterior pituitary gland, thus preventing ovulation and inhibiting the production of sex hormones E2 and P4 sex hormones [5]. ‘ 2) In the Methods section, which variance analysis (ANOVA, etc) was performed for multiple comparisons should be stated in the Statistical analysis sub-section. In our experiments, we compared the values of two different groups of rats, so after parametric analysis of the data, we conducted unpaired t-tests as statistical analyzes. Therefore, it was impossible to make multiple comparisons. 3) References should be added to the use of Jongh's solution for the uterus, Tyrode's solution for the cecum, and the contractility study method. New references have been inserted into the text and the reference list based on this comment. (Gaddum JH. THE TECHNIQUE OF SUPERFUSION. Br J Pharmacol 1997;120(Suppl 1):82. Szűcs et al. Detection of stress and the effects of central nervous system depressants by gastrointestinal smooth muscle electromyography in wakeful rats. Life Sci. 2018;205:1–8.) 4) For the Figure 1: The figure should be adjusted correctly, as if KCl application on uterine contractions begins at the 15th minute rather than the 16th minute. We apologize for this mistake. The time axis indicating the 30-minute time frame has been modified in Figure 1. REVIEWER 2 1. What is the basis for the dose and duration of cetrorelix acetate treatment? The dose and duration of cetrorelix treatment were established based on data from the previously published literature. Among the treatment protocols followed by the various working groups, the most frequently used dose of 0.5 mg/bwkg was administered over a 7-day period. New references have been inserted into the text and the reference list based on this comment. 2. What practical indications are that de Joung solution was used for uterus samples and Tyrode buffer for cecum samples in isolated organ baths? Based on several decades of laboratory practice, we use two different buffer solutions during the examination of uterus and cecum samples. I have also confirmed this in the manuscript with accepted international publications. New references have been inserted into the text and the reference list based on this comment. (Gaddum JH. THE TECHNIQUE OF SUPERFUSION. Br J Pharmacol 1997;120(Suppl 1):82. Szűcs et al. Detection of stress and the effects of central nervous system depressants by gastrointestinal smooth muscle electromyography in wakeful rats. Life Sci. 2018;205:1–8.) 3. Line 153 of the results section lacks a precise description of the in vivo smooth muscle electromyography result. Supplement the legend of Figure 3 with the Fourier transform FFT notation! We are thankful for this remark, the Reviewer is right; however, line 153 refers to the interpretative diagram shown in Figure 3, the actual changes between the animal groups are described in Figure 4 and lines 159-160. On the other hand, the legend of Figure 3 was supplemented with missing information about the FFT abbreviation. 4. I think the bibliographic reference in line 158 (the legend of Figure 3) is redundant. Literature 11 can be found in the methods section and the summary also. Thank you for your valuable comment; the mentioned reference has been removed from the legend of Figure 3. 5. In line 175: "Cetrorelix suppressed staining in the uterus (bottom image row)." I suggest instead, for the reader's ease of understanding, "Cetrorelix treatment reduced αvβ3 integrin expression, which caused a significant decrease in intensity." Based on the Reviewer's remark, the legend of Figure 5 has been changed as suggested. 6. In Figure 5, I propose to mark the uterine horns with a white arrow in each image. On the basis of this observation, we have modified Figure 5. 7. Why the effect on KCl-induced contractions was considered to be justified in addition to the spontaneous uterine contractility study. In isolated organ contractility studies, the examination of spontaneous contractions can be considered physiological; however, it is necessary to monitor the increase in contraction by a stimulus. This stimulus can be triggered with electrical voltage, a drug or - which we used in our study - KCl solution. From the degree of response to the stimulus, we can draw conclusions about the degree of structural or functional change caused by different treatment protocols. Furthermore, it should be noted that although the uterus is characterized by regular spontaneous contraction (which increases during pregnancy), this cannot be said for the cecum or other gastrointestinal sections. Therefore, KCl-stimulated contractions provide equal circumstances for the comparison of changes in the two different types of smooth muscle. REVIEWER 3 The one point to be cleared in the present experiment is that whether the animals which were used in the study were observed in terms of regular estrus cycle before they began the experiment. In the manuscript, there is no any explanation about this important point. The authors have to mention that the animals used were chosen among those that show regular estrus cycle. We are grateful to the Reviewer for this remark; we have detailed the relevant part in the methodology. The following section has been changed in the manuscript: ‘The estrus cycle of healthy young adult female rats was followed and measured daily using the Estrus Cycle Monitor (IM-01, MSB-MET Ltd., Balatonfüred, Hungary). Rats with vaginal impedance values of 4.5 to 7.5 kΩ were considered to be in the proestrus phase and were included in the experiment.’ Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0307932.r002
16 Jul 2024 Decision Letter - Abeer El Wakil, Editor Smooth muscle electromyography for detecting major alterations in the estrus cycle in rats PONE-D-24-11044R1 Dear Dr. Gaspar, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. 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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: The authors have redacted the minor revisions mentioned; therefore the manuscript is suitable for the publication. Reviewer #2: I accept the answers of the authors and the corrections in the text. I recommend the manuscript for publication. ****** 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: No Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0307932.r003
Formally Accepted
29 Jul 2024 Acceptance Letter - Abeer El Wakil, Editor PONE-D-24-11044R1 PLOS ONE Dear Dr. Gaspar, 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 Professor Abeer El Wakil Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0307932.r004

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