PONE-D-22-33049Association of refractory Asherman syndrome with pituitary gonadotrophin pulse patterns: A pilot studyPLOS ONE

Dear Dr. Silva Rivas,

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 Feb 09 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Ahmed Mohamed Maged, MD

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. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information.

3. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. [DETAILS AS NEEDED] Please clarify whether this [conference proceeding or publication] was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript.

4. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. [abstract, tables 1 & 2, figures 1 & 2. See annex posterAG.pdf] Please clarify whether this [conference proceeding or publication] was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript.

5. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

6. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

7. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 

Additional Editor Comments:

Please respond to all reviewers comments point by point

Reviewer 1

Thank you for the invitation for reviewing this interesting study , aiming for discussing association of refractory Asherman syndrome with pituitary gonadotrophin pulse Patterns and recommended that a specific clinical protocol may be necessary for estrogen treatment of these patients. I'm asking the authors for clarification of the next items.

1- In abstract sector: you mentioned this phrase (In both groups, blood was collected during the follicular phase of the menstrual cycle). You should revise and readjust to avoid misleading readers, as group one presented with amenorrhea.

2- In abstract sector: you mentioned (During the first hour of monitoring, cumulative FSH pulsatile frequency was lower in the AS group than in the control group within the next 3 h of measurements). Rewrite the phrase more clearly

3- In introduction section, defining Asherman syndrome has about 11 references, what is the rationale for this great number? Can we minimize especially no benefit from the repetition ?.

4- The study was retrospective :

� In methodology section, the statement (These patients had amenorrhea for at least six months after admission to the study). May be understood. You mean before admission to the study?

� Also, I just want to confirm that 8 patients refused to participate?

5- In methodology section, Clarify the meaning of (The same was done for women with Asherman syndrome, exception the analysis of gonadotropins and sex steroids, estrogen, and progesterone.).

6- You have a reference for this blood sampling protocol ?

(For the collection of blood samples, the following protocols were followed: a) patients were fasted and remained in the horizontal supine position; b) intravenous puncture was performed with a size-19 catheter, and the vein was maintenance with a saline solution drip (20 drops per minute); and c) 25 blood samples were collected from each woman)

7- In the phrase (We observed that there was no statistically significant difference between the mean values of serum concentrations of LH, FSH, and progesterone, or in the mean serum concentration levels of estradiol (p>0.05)).

The (p value) equal or more? confirm.

8- In discussion section: what you mean by (The influence of hormonal status at the time of injury and relevant factors on genesis). There are missing words? I don’t understand.

9- I discussion section and at the end of page 10 in (pdf copy) (They observed the occurrence of a pulse every 100-120 minutes in the follicular phase of normal women)

The word they refer to whom???

10- You abbreviated asherman to AS in introduction section , however , I noticed you mention it again with full . Revise that allover the article .

Reviewer 2

1. Why did you choose the term “Asherman syndrome”. It is well known that Asherman syndrome is intrauterine synechia following surgical evacuation. The term “intrauterine synechia” is a more accepted term.

2. In the introduction section, in the last paragraph “correct “his” to “this”.

3. You collected 25 samples from each patient, and this was approved by your IRB. What was your rational for this? I mean what is the benefit that the patient was told to expect? I would be grateful if you would share your IRB. Additionally maintaining the IV line with saline infusion, would this not alter your results? Please explain.

4. What is meant by “The G1 patients were followed by serial ultrasounds, and the ultrasound was performed in the next cycle when there were at least five small cystic images in one or both ovaries, which could suggest an early follicular phase”

5. The discussion is well written but please express your points of strength and limitation.

Reviewer 3

Although the concept is interesting and Asherman syndrome is not frequent BUT this does not justify the very low number of participants in this study

Asherman syndrome has classification of severity : it seems that the authors included only the severe cases with Amenorrhoea : pls explain why not cases with moderate IU adhesions

The following statement in the manuscript is very unusual in research design : "These patients had amenorrhea for at least six months after admission to the study." why to wait six month after recruitment !!!!!!

The authors failed to mention which day of cycle blood samples were taken : is it the same day for all the participants for example cycle day 11

The authors failed to correlate the level of hormones with the follicle size at the day of sampling : what if day of sampling was when follicle 11mm in Asherman cases and follicle 15mm in control group : Hormonal assays differe according to follicular activity

Additional comments to be clarified

Exclusion criteria what about women with endocrinological disorders especially PCOS women

How was the diagnosis of refractory AS established based on what criteria

Was the trial registered and what is the registration number

How was the study retrospective

Clarify the duration of fasting before sampling

Please discuss the effect of saline infused on the results o sampled blood especially its dilutional effect

More details about kits and its sensitivity

Define primary and secondary outcomes with references

Remove amenorrhea duration and menstrual cycle from table 1

In discussion

Physiological explanation of findings , strengths and limitations of the study should be added to discussion

Masterdata lack age

[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

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: I Don't Know

**********

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: Although the concept is interesting and Asherman syndrome is not frequent BUT this does not justify the very low number of participants in this study

Asherman syndrome has classification of severity : it seems that the authors included only the severe cases with Amenorrhoea : pls explain why not cases with moderate IU adhesions

The following statement in the manuscript is very unusual in research design : "These patients had amenorrhea for at least six months after admission to the study." why to wait six month after recruitment !!!!!!

The authors failed to mention which day of cycle blood samples were taken : is it the same day for all the participants for example cycle day 11

The authors failed to correlate the level of hormones with the follicle size at the day of sampling : what if day of sampling was when follicle 11mm in Asherman cases and follicle 15mm in control group : Hormonal assays differe according to follicular activity

Reviewer #2: Thank you for the invitation for reviewing this interesting study , aiming for discussing association of refractory Asherman syndrome with pituitary gonadotrophin pulse Patterns and recommended that a specific clinical protocol may be necessary for estrogen treatment of these patients. I'm asking the authors for clarification of the next items.

1- In abstract sector: you mentioned this phrase (In both groups, blood was collected during the follicular phase of the menstrual cycle). You should revise and readjust to avoid misleading readers, as group one presented with amenorrhea.

2- In abstract sector: you mentioned (During the first hour of monitoring, cumulative FSH pulsatile frequency was lower in the AS group than in the control group within the next 3 h of measurements). Rewrite the phrase more clearly

3- In introduction section, defining Asherman syndrome has about 11 references, what is the rationale for this great number? Can we minimize especially no benefit from the repetition ?.

4- The study was retrospective :

� In methodology section, the statement (These patients had amenorrhea for at least six months after admission to the study). May be understood. You mean before admission to the study?

� Also, I just want to confirm that 8 patients refused to participate?

5- In methodology section, Clarify the meaning of (The same was done for women with Asherman syndrome, exception the analysis of gonadotropins and sex steroids, estrogen, and progesterone.).

6- You have a reference for this blood sampling protocol ?

(For the collection of blood samples, the following protocols were followed: a) patients were fasted and remained in the horizontal supine position; b) intravenous puncture was performed with a size-19 catheter, and the vein was maintenance with a saline solution drip (20 drops per minute); and c) 25 blood samples were collected from each woman)

7- In the phrase (We observed that there was no statistically significant difference between the mean values of serum concentrations of LH, FSH, and progesterone, or in the mean serum concentration levels of estradiol (p>0.05)).

The (p value) equal or more? confirm.

8- In discussion section: what you mean by (The influence of hormonal status at the time of injury and relevant factors on genesis). There are missing words? I don’t understand.

9- I discussion section and at the end of page 10 in (pdf copy) (They observed the occurrence of a pulse every 100-120 minutes in the follicular phase of normal women)

The word they refer to whom???

10- You abbreviated asherman to AS in introduction section , however , I noticed you mention it again with full . Revise that allover the article .

Reviewer #3: I have read with interest the manuscript titled : “Association of refractory Asherman syndrome with pituitary gonadotrophin pulse patterns: A pilot study”. I would like to thank the authors for the effort put until this work came out to light. I just have a few comments,

1. Why did you choose the term “Asherman syndrome”. It is well known that Asherman syndrome is intrauterine synechia following surgical evacuation. The term “intrauterine synechia” is a more accepted term.

2. In the introduction section, in the last paragraph “correct “his” to “this”.

3. You collected 25 samples from each patient, and this was approved by your IRB. What was your rational for this? I mean what is the benefit that the patient was told to expect? I would be grateful if you would share your IRB. Additionally maintaining the IV line with saline infusion, would this not alter your results? Please explain.

4. What is meant by “The G1 patients were followed by serial ultrasounds, and the ultrasound was performed in the next cycle when there were at least five small cystic images in one or both ovaries, which could suggest an early follicular phase”

5. The discussion is well written but please express your points of strength and limitation.

**********

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: Hesham Al-Inany

Reviewer #2: No

Reviewer #3: Yes: Hisham Haggag. Prof Of Ob/gyn Cairo University.

**********

[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.

Attachments

Attachment

Submitted filename: Reviewer comments.docx

PONE-D-22-33049R1Association of intrauterine synechiae with pituitary gonadotrophin pulse patterns: A pilot studyPLOS ONE

Dear Dr. Silva Rivas,

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 respond to all reviewers' comments

Please submit your revised manuscript by Jul 14 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Ahmed Mohamed Maged, MD

Academic Editor

PLOS ONE

Additional Editor Comments:

Please respond to all reviewers' comments

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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: (No Response)

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: (No Response)

Reviewer #2: Dear editor ,

Thank you for your second invitation for revision evaluation and thanks for authors reply .

Actually, the study design (retrospective or prospective )should be clarified in the manuscript with the date of IRP approval

including patient refusal and the duration of amenorrhea before recruitment .

Additionally, make an effort to get details on the appropriate sampling technique to reduce errors as, from what I understand, it was remote .As an example, to ensure for a trial to proceed properly, blood including fluid was aspirated and drawn before the sample was obtained and submitted for analysis and keep that also in the limitation.

**********

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: Hesham Al-Inany

Reviewer #2: Yes: Essamedin M Negm

**********

[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.

Association of intrauterine synechiae with pituitary gonadotrophin pulse patterns: A pilot study

PONE-D-22-33049R2

Dear Dr. Silva Rivas,

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,

Ahmed Mohamed Maged, MD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: