PONE-D-19-23244

Unexplained infertility treatment, should we change our approach? - An eight-year follow-up study

PLOS ONE

Dear Dr. asali,

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.

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Marco Aurélio Gouveia Alves

Academic Editor

PLOS ONE

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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

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:I have read the journal's policy and the author, Dr. Berkovitz, has the following competing interests: Dr. Berkovitz is the Medical Director of the Male Fertility Clinic where the MSOME was performed. The other authors have no conflicts to declare.:

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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: Partly

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

Reviewer #1: Yes

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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

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Reviewer #1: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This case-control study aims to examine the usefulness of a diagnostic test (MSOME) and therapeutic approach (IMSI), by comparing the outcomes of pregnancy and delivery rates, in a population of couples with unexplained infertility and at least one failed IVF cycle. The cases underwent diagnostic testing and therapeutic approach, the controls did not receive testing and received standard of care. The cases were divided based on MSOME into 3 groups: A (0% normal cells), B (1% normal cells), C (2+% normal cells). When IMSI was used to select the sperm for injection, pregnancy and delivery rates were highest for those groups with the least normal cells by MSOME A>B>C. Controls had pregnancy and delivery rates similar to group C.

Overall, there is a need for additional study in the utility of IMSI, though I would argue, at least at its present level of detail, it’s not clear that this study investigates a population distinct from the male infertility and prior ICSI failure populations that have previously been presented (MSOME and IMSI Reviewed in 2013, PMID 23948449). The study acknowledges the limitations of it being retrospective, with some detail unobtainable. The study does admirably study cumulative pregnancy and delivery rate, including fresh and frozen transfers.

1. Major: Criticism of many unexplained infertility studies stems from differences in inclusion/exclusion criteria for ‘unexplained’ couples. Additional detail in the materials and methods on page 4 (or supplement) regarding the ovulatory, male factor, uterine/tubal/endometriosis, and ovarian reserve workup of the cases and controls would strengthen the ability of the reader to interpret generalizability to their unexplained infertility/mild male factor population. In the discussion it is recognized as a limitation that this level of detail appears unavailable in the control group. But is the case group all unexplained infertility or would some of them qualify as male factor fertility? The novelty of this study is using the technique in an unexplained infertility population rather than male factor infertility, so an ability to differentiate between them is critical to the claim of novelty.

2. Major: studying the pregnancy rate per couple has some advantages, but additional detail regarding the representation of fresh/frozen embryos/numbers of transfers would be appreciated. Additionally, the length of follow-up per couple is inherently different over an overall 8 year timespan. Consider reporting average length of treatment in couples? Do I assume correctly that only the first pregnancy represented by the embryos generated in an IMSI cycle would be represented in the data? Additionally, how was pregnancy defined in your electronic database? Home test, serum?

3. Major: I would presume the comparison of outcomes between controls collected 2010-2018 and cases 2015-2018 would generally find that controls had lower pregnancy/delivery rates, at least with US trends; I don’t know if the same can be extrapolated to other countries. What would a sensitivity analysis comparing the controls from 2015-2018 to cases from 2015-2018 show? On a related question, how was the decision made to include the number of cases you did? Was a power analysis done, or was it a convenience sample based on introduction of an EMR in 2010 and including all cycles that met criteria?

4. Major: The title does not inform the reader of the study design, population, or intervention of this study, and the eight-year follow-up was only for the controls, not the cases. Title revision strongly recommended (consider consulting the STROBE guideline).

5. Minor: I’m not sure I understand/agree with the logic behind the ‘explanation for this apparent contradiction…that a lower percentage of normal cells in MSOME necessitates an extensive search under ultra-magnification to find the highest quality spermatozoa.’ The criteria by which ‘highest quality’ are judged would not seem to be different in group A and B, but the extensiveness of the search has been a major criticism of the IMSI technique and a concern with the time the oocytes wait to be injected.

6. Minor: Bonferroni corrections used for multiple comparisons is great, but it’s not clear what alpha was used to judge significance in the different cases; I like to be able to see how close the p-value is to the alpha.

7. Minor: is the IMSI technique success related to the embryologist/andrologist examining the sperm? I can’t imagine a single embryologist/andrologist performed all the studies, was there a bias in the most experienced performing those for Group A where difficulty in finding normal sperm was anticipated?

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Reviewer #1: No

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PONE-D-19-23244R1

The possibility of integrating motile sperm organelle morphology examination (MSOME) with intracytoplasmic morphologically-selected sperm injection (IMSI) when treating couples with unexplained infertility: a historical, case-control study

PLOS ONE

Dear Dr. asali,

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.

We would appreciate receiving your revised manuscript by Mar 29 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Marco Aurélio Gouveia Alves

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The revised version of the manuscript has somewhat improved but there are still several issues to be addressed, particularly concerning the experimental approach.

[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: (No Response)

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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: Partly

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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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

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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: Comments from prior review have been addressed sufficiently. Limitations of the study design are appropriately acknowledged, details sufficient for the reader to evaluate outcomes.

Reviewer #2: In this manuscript entitled: “The possibility of integrating motile sperm organelle morphology examination (MSOME) with intracytoplasmic morphologically-selected sperm injection (IMSI) when treating couples with unexplained infertility: a historical, case-control study”. Authors asked themselves if the IMSI technique can be used successful on cases of couples with unexplained infertility.

In general, authors don´t explain very well their results and the manuscript is not easy to read. First at all, a general advice for following submission is to include line number. This will not be only beneficial for the reviewers; authors can follow recommendation easier.

One of my concerns is why authors make a difference between MSOME and IMSI. MSOME is not a previous procedure that is part of the process of IMSI? When you read the title and the manuscript it seems that there are 2 different procedures.

Is control group a real control? I am concern about this topic. The only information that we have about them is the reproductive outcome, but we don´t know nothing about the % or normal spermatozoa or the rest of seminogram values. Authors should consider delete this group and built the manuscript discussion making the comparison between groups A, B and C. As they stated: group C is the normal/average group and further comparisons should be done with this group. What we know about the proposed control group?

Authors show beneficial effect of IMSI versus ICSI. This expected result agrees with previous reports that highlight the beneficial outcome of IMSI. Nevertheless, it is hard to understand why the group with higher percentage of anormal spermatozoa had the higher reproductive outcome. The only reason that could explain this contradiction is that the physician made bigger effort to find a normal cell and performed later the ICSI. Once you finish to read the manuscript the conclusion that the reader made is that IMSI is beneficial for those patients with severe morphological sperm problems. But since physician are selecting normal spermatozoa why the rest of group are not improving? Bigger effort should be made to explain this contradictory result.

Minor comments:

Introduction Section:

Please delete this sentence “One study found that ICSI resulted in a similar, cumulative live birth rate as compared with IVF for couples with non-male factor infertility [7]” It doesn´t apport information.

Material and Methods:

Please rephrase it: All couples underwent MSOME when IMSI was done within 6 months of the MSOME. What this means you performed a MSOME analysis and later before 6 months you performed an IMSI. Or maybe did you make a MSOME and you freeze the spermatozoa and later or you performed an IMSI. I am sure this is not the message that you want to send but it is rather confusing. Please clarify.

When you wrote “IVF/ICSI cycles. Male subfertility was defined as a total motile sperm count >1 × 106” What you really mean is less than a million?

Please defined what do you consider “Chemical pregnancy”

When you described the sperm selection “The upper liquid was then removed and the pellet was re-suspended in Quinn's Sperm Washing Medium (Sage In-Vitro Fertilization, Inc.) and centrifuged again for 5 minutes at 600 rpm at room temperature. “What do you mean for the pellet? The spermatozoa that can be found on the bottom of the tube? Do you take the spermatozoa and semen contaminants that can be found on the 40% fraction? Please specify this point.

It is possible to insert in table-3 information about the concentration of the ejaculates. It is the only reason that can explain why different groups that have the same % of motile spermatozoa and same ejaculate volume have different values of total motile count.

Statistical analysis:

Please explain the assumption of a 12% difference in pregnancy rates between groups. How did you calculate this? There is a big gap between the number of couples included in each group. Did the author have this in mind when they performed the statistic? Did you balance statistically this difference?

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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.

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Reviewer #1: Yes: Jamie Peregrine, MD, MS

Reviewer #2: 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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-19-23244R2

The possibility of integrating motile sperm organelle morphology examination (MSOME) with intracytoplasmic morphologically-selected sperm injection (IMSI) when treating couples with unexplained infertility

PLOS ONE

Dear Dr. asali,

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.

We would appreciate receiving your revised manuscript by Apr 30 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Marco Aurélio Gouveia Alves

Academic Editor

PLOS ONE

[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: (No Response)

Reviewer #2: All comments have been addressed

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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: 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: I agree with reviewer 2’s recommendation and the authors’ decision to remove the ‘control’ group.

A few other minor comments:

Page 2: ‘2010-2018’. Now that the control group is removed, isn’t the study only 2015-2018?

Page 3: ‘Using this technique together with a micromanipulation system has allowed the introduction of a modified intracytoplasmic sperm injection (ICSI) procedure, known as intracytoplasmic morphologically-selected sperm injection (IMSI)’. Can you say something about ICSI in the second paragraph to introduce this more familiar acronym sooner? Introducing it in the current state is confusing.

Page 4: ‘All couples underwent MSOME when IMSI was within 6 months of the initial MSOME’. This is unclear.

Page 4: ‘at least one failed IVF-ICSI cycle’ vs ‘several, previous failed IVF/ICSI cycles’. To whom was MSOME offered, and who was included in the study? Also if IVF/ICSI and IVF-ICSI the same, recommend consistency.

Page 10: ‘Group A 10 included 55 cases in which no normal cells were found at MSOME, but normal cells were found during IMSI at ovum pick-up for 49 (89.1%). Group B included 184 couples and Group C 32 couples.’ Did all Group B and Group C patients have normal cells found for IMSI? If not, an argument could be made that maybe MSOME is just not predictive of IMSI, even if <6 months, so I would include the information if you have it.

Page 12: consider modifying as bolded: ‘Thus, MSOME can detect subtle organellar malformations in motile spermatozoa that an embryologist might consider normal for fertilization at 200× to 400× magnification [5], especially important when a normal sperm nucleus is a significant factor for successful implantation and pregnancy in ICSI procedures [13,14].

Page 13: the argument is strengthened, but make sure formatting (IVF-ICSI or IVF/ICSI or IVF ICSI) is consistent.

Page 13: It is confusing to have 2 definitions for ‘male subfertility’: both >1 million cells and Group B.

Reviewer #2: Authors adressed correctly all my questions and concerns. Moreover, modifications were performed on the manuscript.

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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: Jamie Peregrine

Reviewer #2: 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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

The possibility of integrating motile sperm organelle morphology examination (MSOME) with intracytoplasmic morphologically-selected sperm injection (IMSI) when treating couples with unexplained infertility

PONE-D-19-23244R3

Dear Dr. asali,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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.

With kind regards,

Marco Aurélio Gouveia Alves

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: