Dear Dr. Baba,

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 Nov 23 2025 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.. 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.

• 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 . 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,

Sanaz Alaeejahromi

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. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

3. Thank you for stating the following in the Competing Interests section:

[The authors have declared that no competing interests exist.].

We note that one or more of the authors are employed by a commercial company: Sapporo ART Clinic.

1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: 'This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

3. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

[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?

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: 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: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: No

Reviewer #2: Yes

**********

Reviewer #1: Reviewer Comments

General assessment

Dear authors,

I review the manuscript entitled: “Myo-inositol and D-chiro-inositol in murine in vitro follicular development: an experimental study relevant to polycystic ovary syndrome.” The manuscript investigates the comparative effects of myo-inositol (MI) and D-chiro-inositol (DCI) on murine in vitro follicular development, with potential implications for polycystic ovary syndrome (PCOS). The research is timely, relevant, and fills an important gap in terms of the direct impact of MI/DCI on follicular growth, in addition to the known effects on growth due to their metabolic effects. The manuscript is well formatted in general; the methods are clear, and the figures support the findings. There are, however, a few points that require elucidation, analysis, and presentation enhancement.

Major Comments

1. Novelty and Significance

While the topic is relevant, the novelty should be better emphasized in the introduction and discussion. Many prior studies have reported MI/DCI in PCOS, but this work’s unique value (direct follicle culture model) should be more clearly highlighted.

2. Statistical Power and Sample Size

Follicles were obtained from 8 mice in total (n=12 follicles per group). This is a relatively small sample size, and variability in follicle culture is usually high. The authors should justify whether this number provides sufficient statistical power.

3. Interpretation of Results

The authors conclude that DCI promotes the transition of secondary to early antral follicles, but the effect was transient (lost by day 10). This should be discussed more critically - is this effect biologically meaningful or simply a short-term in vitro artifact?

The discussion suggests a possible involvement of the PI3K/AKT signaling pathway, but no experiments were conducted to test this hypothesis. The speculative nature of this statement should be acknowledged more clearly.

4. MI Data Interpretation

MI showed no significant effect. The authors attribute this to a high FSH concentration and a low MI dose; however, they should expand on why the chosen doses were used and whether pilot experiments with higher MI levels were considered.

5. Clinical Relevance

The link to PCOS patients is somewhat overstated. Since this is a murine follicle culture system, the limitations for extrapolating to humans should be described more carefully.

More Comments

1. Language and Style

English is understandable but requires polishing for clarity and conciseness. Example:

Abstract:

In lines 20-23, “This study investigated the effects of myo-inositol and d-chiro-inositol on the development of murine ovarian follicles in vitro, and their potential efficacy in patients with polycystic ovary syndrome.”

- “their potential efficacy in patients” is misleading, because the authors did not test patients, only mice.

- “myo-inositol and d-chiro-inositol”, chemical names should be italicized consistently or abbreviated after first use.

Lines 23-25, “d-chiro-inositol treatment exhibited significantly larger diameters compared to the control group on Day 6 (D=275.20 ± 12.54 μm; p=0.037) and Day 8 (D=277.47 11.47 μm; p=0.048), but this difference was no longer observed by Day 10.”

“exhibited significantly larger diameters”, awkward phrasing; better: “resulted in significantly larger follicle diameters.”

“277.47 11.47 μm” → missing ± sign.

“but this difference was no longer observed” → wordy.

The correct sentence could be: DCI treatment resulted in significantly larger follicle diameters compared to controls on Day 6 (275.20 ± 12.54 μm; p = 0.037) and Day 8 (277.47 ± 11.47 μm; p = 0.048), although this effect was not maintained by Day 10.

Lines 25-27, “The rate of follicular cavity formation was significantly higher in the d-chiro-inositol-treated group on day 6 (p<0.05); however, no significant differences were observed on days 8 and 10.”

“day 6” → should be capitalized consistently (Day 6).

“d-chiro-inositol-treated” → should be abbreviated consistently to DCI-treated.

The correct sentence could be: The rate of follicular cavity formation was significantly higher in the DCI-treated group on Day 6 (p < 0.05); however, no significant differences were observed on Days 8 and 10.

Lines 27-29, In contrast, myo-inositol treatment did not produce significant differences in follicular survival rate, diameter, or cavity formation compared with the control.

Again, use the abbreviation MI for consistency.

“produce significant differences”, better phrasing: “did not affect.”

The correct sentence could be: In contrast, MI treatment did not affect follicular survival, diameter, or cavity formation compared with controls.

Lines 29-31, Estradiol concentrations and relative expression of follicle-stimulating hormone receptor and aromatase genes did not differ significantly between the treatment groups.

“between the treatment groups” could be simplified to “among groups.”

Long phrase could be smoothed.

The correct sentence could be: Estradiol concentrations and expression levels of follicle-stimulating hormone receptor and aromatase genes did not differ significantly among groups.

Lines 31-33, Our findings indicate that d-chiro-inositol promotes the transition of preantral to early antral follicles. The decreased myo-inositol/d-chiro-inositol ratio observed in polycystic ovary syndrome may facilitate early follicular development.

“Our findings indicate”, better as “These findings suggest” (more cautious wording for science).

Consistency: use MI/DCI abbreviations instead of repeating full terms.

The correct sentence could be: These findings suggest that DCI promotes the transition from preantral to early antral follicles. The decreased MI/DCI ratio observed in PCOS may facilitate early follicular development.

Lines 34-35, Further research is needed to clarify its potential role in clinical applications for polycystic ovary syndrome treatment.

“its potential role”, unclear pronoun (refers to DCI or ratio?).

“clinical applications for PCOS treatment”, better phrasing possible.

The correct sentence could be: Further studies are required to clarify the potential clinical relevance of these findings for PCOS treatment.

Introduction:

Line 41, “proportion” is singular; so, the verb should be “manifests”.

Line 42, considered to be two-fold, “two-fold” is acceptable, but in scientific writing, “twofold” (without hyphen) is preferred.

The correct form is: “considered twofold”.

Line 44-46, "in theca cells (and follicle-stimulating hormone (FSH) receptor and insulin-like growth factor (IGF)-1 receptor expression in granulosa cells)"

The problem is that the nested parentheses are confusing.

The correct form could be: "in theca cells and increase the expression of follicle-stimulating hormone (FSH) and insulin-like growth factor 1 (IGF-1) receptors in granulosa cells"

Line 51-52, "Although the pathophysiology of PCOS is complex and not yet fully understood, insulin resistance is believed to be the underlying cause."

The correct or smother form could be: "The pathophysiology of PCOS is complex and not fully understood; however, insulin resistance is believed to be a major contributing factor."

Line 63-66, "This is because insulin resistance causes low epimerase activity in normal tissues, resulting in systemic DCI deficiency, whereas in the ovaries, insulin sensitivity is preserved, and hyperinsulinemia increases epimerase activity."

This sentence could be split into two sentences to improve clarity.

The correct or smother form could be: "Insulin resistance causes low epimerase activity in normal tissues, resulting in systemic DCI deficiency. In contrast, insulin sensitivity in the ovaries is preserved, and hyperinsulinemia increases epimerase activity."

Line 78-80,"Although the efficacy of MI and DCI in the treatment of PCOS has been previously reported, most studies have focused on their metabolic effects, and the direct effects of MI/DCI on follicular development remain insufficiently understood."

The correct form could be: "Although MI and DCI have shown efficacy in treating PCOS, most studies have focused on their metabolic effects. The direct impact of MI/DCI on follicular development remains poorly understood."

Some more minor suggestions:

- Avoid repeating “in patients with PCOS” multiple times; vary phrasing.

- Avoid using “this study focused on” repeatedly; consider synonyms like “we investigated” or “we examined”.

- For conciseness, consider combining shorter sentences with semicolons or by rephrasing.

Materials and methods:

Some questions:

1- How many animals were used in total across all experiments (not just per group)?

2- Were animals randomized to groups before follicle collection, or were follicles pooled?

3- Were both ovaries from each mouse used, and if so, were they treated as independent samples or paired?

4- Was the culture medium serum-containing (5% FBS) throughout, or was serum-free medium also tested?

5- How was the vehicle control for DMSO matched to experimental groups (same final concentration in all groups)?

6- Was osmolarity or pH of the culture medium checked after MI/DCI supplementation?

7- How was the culture medium collected for E2 measurement-pooled from wells or from individual follicles?

8- Were E2 values normalized to follicle number per well?

9- Was a power calculation done to justify the sample size (n = 8 mice)?

10- Were the data tested for normality before applying ANOVA?

11- Were multiple comparisons corrected for (beyond Student-Newman-Keuls)?

Lines 95-97, "The method of euthanasia followed handling was performed in accordance with the guidelines…"

Problem: Unclear and ungrammatical.

The correct form could be: "Euthanasia and subsequent handling were performed in accordance with the guidelines of Sapporo Medical University and the Center for Animal Protection Scientists."

Lines 99-100, "sacrificed through intraperitoneal administration of pentobarbital"

Comment: sacrificed is less precise and discouraged. Euthanized is preferred.

The correct form could be: "euthanized via intraperitoneal administration of pentobarbital (120 mg/kg)"

Lines 112-114, "…the culture medium was refreshed… and the culture was continued for a total …"

The correct form could be: "The culture medium was refreshed every other day by replacing half of the volume with fresh medium, and culture was continued for a total of 10 days."

Lines 133-134, "when the granulosa cells appeared dissolved black"

The problem is using awkward/unclear phrasing.

The correct form could be: "when the granulosa cells appeared degenerated or darkened"

Line 139, "E2 concentrations in the culture medium were measured on days…"

The correct form could be: "Measurement of estradiol (E2) concentrations in the culture medium was performed on days 5 and 9 using an ELISA kit (R&D Systems, Minneapolis, MN, USA; detection range: ~0–3.0 ng/mL)."

Also, there are some inconsistent units formatting (μM vs uM, spacing in “p<0.001” vs “p < 0.001”), inconsistent gene names (italicize Fshr, Cyp19a1), and missing italics for species (Mus musculus).

Results:

Question:

For follicle diameters: are you reporting mean ± SE consistently, or sometimes using SD? The text says SE, but the numbers look like SD ranges.

Comments:

Lines 170-172, that” refers to something singular; follicle diameter is plural.

The correct form could be: “were significantly larger than those in the CTRL group”

Lines 216-217, “The E2 concentration in the culture medium on day 5 of the DCI experiment in CTRL, DCI 20 μM, and DCI 50 μM groups were …”

Correction: “…was …”

Discussion:

Lines 266-267, we found that the addition of DCI promoted the development of secondary follicles from early antral follicles.”

Problem: The phrasing suggests secondary follicles come from antral follicles (biologically confusing).

Correction: “…we found that the addition of DCI promoted the transition of secondary follicles to the early antral stage.”

Lines 268-269, “This implies that a reduction in the MI/DCI ratio is closely related to the etiology of PCOS.”

Problem: Too strong for in vitro evidence.

Correction: “…This suggests that a reduction in the MI/DCI ratio may contribute to the pathophysiology of PCOS.”

Lines 273-275, “…follicles cultured without DCI, which were initially delayed in development, eventually caught up with those cultured in the presence of DCI.”

Correction: “…Follicles cultured without DCI, although initially delayed, eventually reached similar developmental stages as those exposed to DCI.”

Lines 310-313, “…humans are monovulatory animals, whereas mice are polyovulatory animals that develop several follicles in one cycle. Therefore, it is normal for murine ovaries to show a multifollicular morphology, and do not fully mimic…”

Correction: “…Therefore, murine ovaries normally show multifollicular morphology, which does not fully mimic human follicle development, especially in PCOS.”

2. Figures

Figure legends are descriptive but could be streamlined. Ensure all abbreviations are defined at first use in each figure.

3. References

Recent reviews/meta-analyses on inositol and PCOS (2023-2024) are cited, which is good. However, authors might expand citations regarding in vitro follicle culture systems beyond their own group’s work.

Recommendation

Given the issues above, I recommend a Major Revision.

The experimental work is solid and interesting, but the interpretation, discussion depth, and contextualization of findings need significant strengthening.

Language and presentation also need editing before acceptance.

Best regards,

Reviewer #2: The statement "d-chiro-inositol treatment exhibited significantly larger diameters" is grammatically awkward; it should be rephrased to "follicles treated with d-chiro-inositol exhibited significantly larger diameters..."

The p-values for the diameter on Day 6 (p=0.037) and Day 8 (p=0.048) are very close to the significance threshold of 0.05, indicating a marginal effect.

The field for keywords is blank in the provided document. This is a major omission.

The literature review could more sharply define the specific knowledge gap this study aims to fill. It could be more explicit about the lack of in vitro studies isolating the effects of these inositols on specific stages of folliculogenesis.

The text referencing Unfer et al. [14] contains a formatting error with plus signs ("3.00 + 1.22 μM") that should be "±".

The description of the euthanasia method is slightly repetitive and contains a grammatical error in the sentence: "The method of euthanasia followed handling was performed in accordance with..." This should be corrected for clarity.

The use of a high, maximally effective FSH concentration likely masked potential subtle effects of MI.

The y-axis label in Figure 5, "Change relative to CTRL (log2 transformed)," is ambiguous. It should specify that it represents the log2 of the fold-change.

The authors acknowledge the limitation that their model did not fully replicate the arrested development seen in later-stage PCOS follicles.

The section on the mechanism is speculative, as the study did not include experiments to test the proposed PI3K/AKT pathway hypothesis. This should be framed more explicitly as a hypothesis for future research, not a finding.

The conclusion could be strengthened by more directly stating that the study provides in vitro evidence for a specific, stage-dependent role of DCI, rather than a general "promotion" of development.

The manuscript requires careful proofreading for several instances of grammatical errors and awkward phrasing.

The flow in some parts of the Methods and Discussion could be improved with minor editorial revisions.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.). 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 For information about this choice, including consent withdrawal, please see our Privacy Policy .-->

Reviewer #1: Yes: Nima Azari-DolatabadNima Azari-Dolatabad

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.]

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 . 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.. Please note that Supporting Information files do not need this step.

Dear Dr. Baba,

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 note that this decision is rather exceptional as it will be a second round of modification. I thus urge you to adequately answer the various concerns raised by both external reviewers.

Please submit your revised manuscript by Mar 20 2026 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.. 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.

• A 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 . 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,

Jean-Marc A Lobaccaro, PhD

Academic Editor

PLOS One

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

**********

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

Reviewer #3: No

**********

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

Reviewer #3: 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 #3: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #3: Yes

**********

Reviewer #3: Myo-inositol versus D-chiro-inositol in murine in vitro follicular development: an experimental study relevant to polycystic ovary syndrome by Korai et al.

The text has been significantly improved, even if two main concerns remain. First, the little sample size (but probably acceptable for an experimental study). Second, the impossibility to generalize the finding to patients with PCOS. The two points should be more underlined. In addition, the focus of the paper is the PCOS. However, the authors detailed the experimental procedure on mice. It should be interesting to study the effect of inositols on animal model of PCOS.

In the text (see for example the abstract section), the statement “Follicles treated with DCI exhibited..” is confusing.

The authors statement “These observations are similar to the decreased MI/DCI ratio observed in PCOS, which may facilitate early follicular development.” should be avoided in the abstract because it has no sense, and in the text should be better supported.

Similarly, the conclusion on the PCOS has again no sense.

The introduction is focused on PCOS. The mechanism of the anovulation in PCOS should be referenced and, probably, is more complex of those reported.

Data on inositols reported in the introduction are not balanced. The authors should underline that inositols have no evidence-based role for the treatment of infertility in PCOS (see new guidelines on PCOS).

The authors stated that “In healthy individuals, the MI/DCI ratio in the follicular fluid is 100:1”. This means that the “normal” ovary needs of high concentrations of myo-inositol. How can the authors explain this discrepancy?

The discussion is short and superficial. The study limitations are totally lacking as well as the future researches.

Reviewer #4: Myo-inositol versus D-chiro-inositol in murine in vitro follicular development: an experimental study relevant to polycystic ovary syndrome by Korai et al.

The statement "d-chiro-inositol treatment exhibited significantly larger diameters" is grammatically awkward; it should be rephrased to "follicles treated with d-chiro-inositol exhibited significantly larger diameters..."

The p-values for the diameter on Day 6 (p=0.037) and Day 8 (p=0.048) are very close to the significance threshold of 0.05, indicating a marginal effect.

The field for keywords is blank in the provided document. This is a major omission.

The literature review could more sharply define the specific knowledge gap this study aims to fill. It could be more explicit about the lack of in vitro studies isolating the effects of these inositols on specific stages of folliculogenesis.

The text referencing Unfer et al. [14] contains a formatting error with plus signs ("3.00 + 1.22 μM") that should be "±".

The description of the euthanasia method is slightly repetitive and contains a grammatical error in the sentence: "The method of euthanasia followed handling was performed in accordance with..." This should be corrected for clarity.

The use of a high, maximally effective FSH concentration likely masked potential subtle effects of MI.

The y-axis label in Figure 5, "Change relative to CTRL (log2 transformed)," is ambiguous. It should specify that it represents the log2 of the fold-change.

The authors acknowledge the limitation that their model did not fully replicate the arrested development seen in later-stage PCOS follicles.

The section on the mechanism is speculative, as the study did not include experiments to test the proposed PI3K/AKT pathway hypothesis. This should be framed more explicitly as a hypothesis for future research, not a finding.

The conclusion could be strengthened by more directly stating that the study provides in vitro evidence for a specific, stage-dependent role of DCI, rather than a general "promotion" of development.

The manuscript requires careful proofreading for several instances of grammatical errors and awkward phrasing.

The flow in some parts of the Methods and Discussion could be improved with minor editorial revisions.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.). 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 For information about this choice, including consent withdrawal, please see our Privacy Policy .-->

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.]

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

Myo-inositol versus D-chiro-inositol in murine in vitro follicular development: an experimental study relevant to polycystic ovary syndrome

PONE-D-25-44774R2

Dear Dr. Baba,

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. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact  and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support ..

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,

Jean-Marc A Lobaccaro, PhD

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments: