PONE-D-22-30636

Cholinergic-estrogen interaction underpins the effect of education on attenuating cognitive sex differences in a Thai healthy population

PLOS ONE

Dear Dr. Nudmamud-Thanoi,

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Engineering and Biotechnology, Pathum Thani, Thailand."

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Additional Editor Comments:

The manuscript has been evaluated by two reviewers, and their comments are available below.

The reviewers have raised a number of concerns that need attention, and they request additional information on methodological aspects of the study and analyses. Further consideration will depend on your ability to thoroughly respond to all issues raised. 

Could you please revise the manuscript to carefully address the concerns raised?

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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: Partly

Reviewer #2: Partly

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

Reviewer #1: No

Reviewer #2: 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

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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

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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 study aims at examining the effects of education level on cognitive sex differences. The authors classify participants at different ages to three categories based on their education levels and then examine the differences in various WCST scores between males and females. They have also examined the sex-specific neurotransmission-related protein expression profiles and tried to link it to the sex-related differences in behavioural measurements. Understanding the differences in cognitive abilities between males and females and delineating the effects of environmental factors are important and therefore the main goal of this study has merit. However, there are various uncontrolled factors in this study, which make it inconclusive. The analytical approach also hinders reaching a clear conclusion.

Major points:

1- In this study, the education level (primary, secondary,…) has been considered as an isolated factor, however various other factors (such as socioeconomic status) might be strongly associated with the education level. The authors also cite previous studies indicating that socioeconomic status might significantly affect cognitive development and performance in cognitive tasks. This issue has not been controlled in this study.

2- The analytical approaches should be accompanied by direct comparison of the groups within the same statistical analyses (multi-factorial ANOVA). While assessing the evidence for a particular hypothesis for each separate group is informative, direct comparison of groups appears necessary. In other words, the effects of Age, Sex and Education factors should be examined with analytical approaches (same statistical analysis) to confirm the significance (or absence of significance) for the main effects and their interactions.

3- The examination of differentially expressed proteins between males and females, is informative, however it is simply a correlational analysis and does not establish any causal interaction between the cognitive sex difference and alterations in these proteins. Elsewhere in the manuscript a causative association is claimed “we discovered that cholinergic signalling may be at the root of the effect of education on attenuating cognitive sex differences” or “Cholinergic estrogen interaction underpins the effect of education on attenuating cognitive sex differences in a Thai healthy population”. However, the experimental approaches and results do not establish a causative link and conclusions are not supported.

Minor issues

1- Although they have used a computerized version of the WCST, the participants’ response time (RT) has not been measured/reported. RT could have been a more sensitive measure to show the sex-related differences (Feizpour et al. Psychology of Music 2018).

2- Literature review in the Introduction and Discussion needs to cite and discuss relevant studies in the context of the WCST.

3- Figure 1 can be improved in terms of clarity and quality. Currently, the data points cannot be easily detected.

Reviewer #2: This manuscript hypothesizes that education decreases the cognitive differences between genders. To examine this an experimental design was developed in humans, especially those who studied above primary level of education and those who didn’t. The authors use of Bayes factor as a statistic is impressive and the results that span behavioural, biological and bioinformatical domains showcases the strong experimental design. has

1. These findings could be discussed in light of following papers, as some studies have found marginal improvements with education and gender based changes in cognitive performances. - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608774/ ; Efstathios D. Gennatas, Brian B. Avants, Daniel H. Wolf, Theodore D. Satterthwaite, Kosha Ruparel, Rastko Ciric, Hakon Hakonarson, Raquel E. Gur, Ruben C. Gur. Age-Related Effects and Sex Differences in Gray Matter Density, Volume, Mass, and Cortical Thickness from Childhood to Young Adulthood. The Journal of Neuroscience, 2017; 37 (20): 5065 DOI: 10.1523/JNEUROSCI.3550-16.2017

2. Lines 63-65 are not clear, What is the conclusion of that study?

3. Also, a population with age range of 22-70 was used here, this is a big range for age. As some are young adults and some are aging.

4. WISC is used in set shifting, capability of a person to change his attention based on new rules, it is not a memory test, Although it showcases frontal lobe function, especially the DLPFC, still many other forms of cognitive/memory testing could be done and would have elevated the paper. An explanation to this in the discussion would help, please cite other papers where WCST was used alone and the reliability of WCST for cognitive measurements as a stand-alone test.

5. Line 176-177, the age differences between groups, meaning the tertiary and secondary educated groups may just perform well because they are significantly lower in age compared to primary educated group?

6. The graphs are not consistent, In one graph, males are followed by females, while in others, it’s the reverse.

7. Also, did the authors make a comparison of females in tertiary education to primary education? Was there a difference? Careful examination of data with multiple comparisons are needed in primary vs. secondary educated females, males across all three parameters shown.

8. Also, a discussion on why the other two parameters of WCST were not showing results needs to be discussed? Please refer to any previous publications that showed such discrepancies if any? If not, please consider adding more numbers to the study.

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Reviewer #1: No

Reviewer #2: Yes: Srinivasa P Kommajosyula

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PONE-D-22-30636R1Cholinergic-estrogen interaction is associated with the effect of education on attenuating cognitive sex differences in a Thai healthy populationPLOS ONE

Dear Dr. Nudmamud-Thanoi,

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 Jun 01 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,

Thiago P. Fernandes, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Please respond to each comment AND highlight them.

Also enlist a native English speaker to edit your manuscript.

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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

Reviewer #3: 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

Reviewer #3: Yes

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

Reviewer #2: Yes

Reviewer #3: Yes

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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 #2: Yes

Reviewer #3: 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

Reviewer #3: 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: All the raised queries have been addressed by the authors of this manuscript.

I thank the editor for the opportunity to review this study.

Reviewer #3: The article titled "Sex Differences in Cognitive Performance: Potential Compensatory Mechanisms Revealed by Proteomic Analysis" presents a study investigating sex differences in cognitive performance and potential compensatory mechanisms using proteomic analysis. The article is well-structured, providing detailed information on the study's background, methods, results, and discussion.

The study involved 135 subjects, 70 males, and 65 females, with a mean age of 57.75±10.35 years. The authors explored the effects of education on sex differences in cognitive performance by administering the Wisconsin Card Sorting Test (WCST) and comparing the results between males and females in different education groups. They found that education had potential compensatory effects on three out of the five WCST sub-scores: %Corrects, %Errors, and 1st Category. In the primary education group, males performed better in %Corrects and had lower %Errors, while females completed the first category in fewer trials. However, in the higher education group, sex differences in those scores reversed, with females performing better in %Corrects and males completing the first category in fewer trials. The authors suggest that education may play a role in compensating for sex differences in cognitive performance, although further research is needed to confirm this hypothesis.

In addition to the behavioral tests, the authors used a label-free quantitative proteomics approach to identify differentially expressed proteins (DEPs) between males and females. They found that 11 DEPs were present in both education groups, with all but one upregulated in females. The authors then investigated the protein-protein interactions of these DEPs and their relationship with cognitive function. They found that LRP4, TTR, TRIM38, and PP2B directly interacted with nAChR3, a subunit of nicotinic acetylcholine receptor that positively regulates cognition. On the other hand, CD34, SERPING1, HSD17B1, KNG1, and TTR were indirectly connected to the cholinergic pathways through interaction with estrogen, suggesting that the cholinergic-estrogen interaction may have an influence on cognitive processes.

The significance of this study lies in its exploration of the potential effects of education on sex differences in cognitive performance and their relationship with differentially expressed proteins between men and women. The study provides insights into the complex interactions between education, sex, and cognitive function, and sheds light on potential molecular mechanisms underlying sex differences in cognitive performance.

The findings of the study suggest that education may have compensatory effects on sex differences in cognitive performance, particularly in the WCST sub-scores of %Corrects, %Errors, and 1st Category. The study also identified 11 differentially expressed proteins between men and women, most of which were upregulated in females, and highlighted the potential roles of cholinergic-estrogen interactions in cognitive processes.

These results have important implications for understanding sex differences in cognitive function and developing interventions to improve cognitive performance in both sexes. By identifying the molecular mechanisms underlying these differences, future research may be able to develop targeted interventions that leverage the compensatory effects of education and modulate the cholinergic-estrogen pathway to improve cognitive function in both men and women.

Overall, the article provides valuable insights into the potential compensatory mechanisms underlying sex differences in cognitive performance. The use of proteomics analysis adds a novel dimension to the study, providing a more detailed understanding of the biological mechanisms underlying cognitive performance. However, the study has some limitations, including the relatively small sample size, the cross-sectional design, and the lack of information on other potential confounding factors such as lifestyle factors or medical conditions. Nevertheless, the study provides a valuable contribution to the literature on sex differences in cognitive performance and sets the stage for further research in this area.

Major concern

1. Sample size: The sample size of the study is relatively small, which limits the generalizability of the findings. Larger and more diverse samples would provide a more robust assessment of the relationships among cognitive performance, sex, education, and protein expression.

2. Methodological limitations: The study used a cross-sectional design, which makes it challenging to draw causal inferences about the relationships among the variables under investigation. Longitudinal studies would be useful for tracking changes in cognitive performance and protein expression over time and for evaluating potential causal relationships.

3. Statistical analysis: Although the study used Bayesian statistical methods, the presentation of the results could be improved by providing more detailed information on the Bayesian analyses, such as prior distributions and sensitivity analyses.

Minor concerns

1. Clearer headings and subheadings: The headings and subheadings in this article are a bit confusing and could be made clearer. For example, the section on demographic data could be titled "Demographic Characteristics of the Study Population" to better convey what the section is about.

2. Improved organization: The article would benefit from a clearer organization of ideas. For instance, the section on the effects of education on sex differences in cognitive performance could be restructured to present the findings in a more coherent manner. The section could start with an introduction explaining why the effects of education on cognitive performance were investigated, followed by a clear description of the results and a discussion of the findings.

3. Improved clarity: Some of the language used in the article is a bit difficult to understand. For example, the phrase "age differences between males and females in primary, secondary and tertiary education groups were supported by weak and substantial evidence, respectively" is confusing. A clearer way to say this might be "The study found that there were significant age differences between males and females in the secondary and tertiary education groups, but not in the primary education group."

4. More detailed discussion: The discussion section of the article is quite short and could benefit from more detailed analysis of the findings. For example, the article could explore why females in the higher education group performed better on certain cognitive tasks than males, whereas males in the lower education group performed better.

5. Address limitations: The limitations of the study should be addressed in the discussion section, and the authors should acknowledge any potential sources of bias or error in their study.

6. Clearer conclusions: The article's conclusions could be made clearer and more concise. The article could end with a summary of the main findings and their implications, along with suggestions for future research.

7. Consider alternative explanations: The authors should consider alternative explanations for their findings, and discuss other factors that may influence cognitive function, such as lifestyle factors, socio-economic status, and cultural differences.

Overall, the article contains valuable findings, but could benefit from some improvements in organization, clarity, and depth of analysis.

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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

Reviewer #3: Yes: NAVEEN JAYAPRAKASH

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Cholinergic-estrogen interaction is associated with the effect of education on attenuating cognitive sex differences in a Thai healthy population

PONE-D-22-30636R2

Dear Dr. Nudmamud-Thanoi,

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.

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Kind regards,

Thiago P. Fernandes, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

By my own reading, the authors addressed all the raised concerns.

Reviewers' comments: