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PONE-D-23-30079THC and sperm: impact on fertilization capability, pre-implantation in vitro development and epigenetic modificationsPLOS ONE

Dear Dr. Favetta,

Thank you for submitting your manuscript to PLOS ONE. After a thorough review, we think it has merit, but could be improved if you followed the reviewers' advice and answered their questions to the best of your ability. Therefore, we invite you to submit a revised version of the manuscript that addresses the minor points raised during the review process.

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Joël R Drevet, Ph.D.

Academic Editor

PLOS ONE

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Additional Editor Comments:

Three reviewers with good expertise in the field evaluated this manuscript rather positively. They suggested minor revisions that could further improve this already good contribution. The authors are encouraged to follow these comments and advice and to respond to the various queries.

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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

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #2: Yes

Reviewer #3: Yes

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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

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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: I am sincerely appreciative of the opportunity I've been given to assess the manuscript authored by Laura A. Favetta and her colleagues, which is currently under consideration for publication in PLOS ONE. The primary objective of this research is to investigate the effects of in vitro exposure to THC on both the physical and functional aspects of sperm, as well as its inherent functions, particularly its role in embryo development subsequent to IVF. On the whole, I find the manuscript to be exceptionally well-structured and proficiently written. The data analysis is executed with precision, and the experimental methods are clearly explained, offering valuable insights into the impact of THC on male reproductive function. The Discussion section, while extensive, effectively delves into the physiological implications of the study's findings. It provides substantial evidence to support these implications and also acknowledges and addresses any limitations. Notably, the use of bovine sperm and oocytes represents a significant limitation in this study, but it has been thoroughly elucidated and acknowledged. Consequently, I recommend that the manuscript, in its current form, be accepted for publication.

Reviewer #2: Dear Authors,

The manuscript PONE-D-23-30079, titled "THC and sperm: impact on fertilization capability, pre-implantation in vitro development and epigenetic modifications," presents a compelling investigation into the effects of THC exposure on both morpho-physiological and intrinsic sperm functions. The study's findings provide a comprehensive understanding of how such exposure affects embryo development following in vitro fertilization. The manuscript is an original research article that uses a suitable methodology and is well-written in English. Moreover, it meets all adequate standards for the ethics of preclinical research and data availability, making it an impeccable report. Thus, I recommend this manuscript for publication in this journal. Nonetheless, this paper needs some prior minor modifications:

1) To improve your Introduction, I recommend that, in the sentence where you report the environmental and lifestyle factors (page 9, lines 74-7), you read and include the following article that discusses the association of cannabis among other several habits in male infertility (DOI: 10.3389/frph.2022.820451);

2) What are the references to the statement on page 10, lines 94-95? Are they the same as in the previous sentence (references 25 and 26)? Make this clear in the text;

3) Also in the Introduction, please include and explain the antagonist role of THC at CB receptors, as you reported on page 16, line 222;

4) I suggest the authors remove the sentence on page 13, lines 158-60, where they express the results of their research in the Introduction. My opinion is that it is better placed at the beginning of the discussion section if they want to maintain this information;

5) Please add the reference to the ethical principles for animal studies of the Canadian Council on Animal Care (page 13, line 166);

6) In the statistical analysis, specify the software versions and specifications, as usual.

Reviewer #3: Dear Authors, this manuscript is timely and appropriate. Congratulations on the initiative to provide the scientific and the public in general with additional valuable information on the matter of Marijuana and THC.

To further expedite the manuscript, please provide a few corrections and explanations:

Line 69: Male infertility accounts for approximately 20% of cases and contributes to another 50%. If that were the case, 20+50% would contribute to the male side of 70% overall! Did you mean to say that 30-35% is solely the male component, plus 20% of the couple's problem combined and the total contribution of the male component to the overall infertility is around 50%? Please correct.

Lines 147 - 148: Could you please provide further information on the choice of bovine sperm? We know that humans have the lowest sperm quality among mammals (the only exception for the Orangotangos) and that bovine sperm has a better quality as males have been selected through the years. So, the question is: Is there an ideal animal model to study bad-quality sperm? Another question is: if THC can affect a species with high-quality sperm, what to expect for humans? Even worst! Could you please add insights into the discussion?

Line 176: sperm was obtained through cryopreserved samples. It is known that cryopreservation can affect overall sperm quality. Both processes, cryopreservation and, thing can potentially have lethal or sublethal damage to the spermatozoa. Why have you not performed a group of fresh semen samples? Or, in the bovine model, is it not necessary to use fresh sperm?

Line 258: Please substitute 18 for Eighteen.

Dear authors, it may sound challenging to extrapolate the low-THC and high-THC concentrations. However, it would be nice if you could assume how many marijuana cigars these concentrations would mean in the human setting. We know that a marijuana cigar has around 0.3 grams of the Cannabis plant and that the THC concentrations vary from 9 to 30%. However, it can reach as much as 40 to 50% THC concentrations, depending on the plant's variety and breeding conditions. In the discussion section, if you believe that you could translate this research into practical information for users who use it rarely and for those who smoke regularly, believe it would be helpful in the clinical setting.

Discussion:

Line: 461. I believe that you have to include in the first phase: cryopreserved and thawed sperm.

It may sound too much or even unrealistic, but do you think it possible to include a figure with potential mechanistic and physiopathological pathways proposed here? If not, it is entirely understandable. Again, congratulations on the excellent piece of work.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Jorge Hallak, M.D.;Ph.D.

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THC and sperm: impact on fertilization capability, pre-implantation in vitro development and epigenetic modifications

PONE-D-23-30079R1

Dear Dr. Laura Favetta,

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.

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Kind regards,

Joël R Drevet, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for the changes made to your manuscript in response to the requests/requirements of the reviewers.

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

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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 #2: Yes

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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

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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 #2: Yes

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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 authors,

You have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication.

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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 #2: No

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