A longitudinal study of perceived stress and cortisol responses in an undergraduate student population from India

Anuradha Batabyal; Anindita Bhattacharya; Maria Thaker; Shomen Mukherjee

doi:10.1371/journal.pone.0252579

Peer Review History

Original SubmissionOctober 9, 2020
22 Jan 2021 Decision Letter - Julia Dratva, Editor PONE-D-20-29949 A longitudinal study of perceived stress and cortisol responses in an undergraduate student population from India PLOS ONE Dear Dr. Bhattacharya, 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 Mar 06 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Julia Dratva Academic Editor PLOS ONE Additional Editor Comments: I believe the research was well done and is a first "proof of feasibility"of longitudinal cortisol measures generating first hypotheses in the Indian undergraduate context. The longitudinal study design partly conteracts the small sample size, however, it remains a concern. In addition, to the reviewers' comments please address the following: Throughout the article the term "sex difference" is used. I suggest to use gender-difference, since the authors themselves point to non-biological factors potentially causing the increase in cortisol in women. Or gender-/sex-difference if they which to point to potential biological differences in the stress-response by sex. Impact of "stressors" were assessed in stratified analyses over all time points. As far as I understand they did not adress interactions of sex and stressors. Please adress the limitations in more detail: sample size, missing confounders; as well as the validity of cortisol measures. The authors should also dicuss the generalizability of the results and how unmeasured confounders might impact the results, especially from a gender point of view. 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 [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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 ****** 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 ****** 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: The authors present a very interesting and well written longitudinal study on perceived psychological stress and cortisol levels in a student population. I very much enjoyed to review this manuscript. I have only some minor points which should be addressed by the authors. A. Participants (page 5, lines 91-101): A1. The authors should provide some further information on the selection process, i.e. sampling procedure. How were students recruited? A2. The authors state that 25 students participated in the study and six measurements were available for ~20 individuals. Please indicate whether patients were included and analyzed on an intention-to-treat basis and if so how many observations were present at each time point. If only individuals with complete measurements were included, please justify. What do the authors mean by ~ (approximately) 20 individuals? Please specify precisely for how many subjects measurements were available at all time points. A3. Please justify your sample size. B. Discussion: B1. (page 13, line 285): There is a typo in the suicide incidence rate, i.e. “1,00,000” should probably read “100,000”. Reviewer #2: The study tracked psychological (distress, perceived stress, positive mood) and salivary cortisol responses over an academic year in 25 residential undergraduate students in India. The study reported sex differences in stress (K10; PSS; men decreased over time while women remained stable) and cortisol responses (overall higher for women, and specifically higher at the end of the academic year). Strengths of the study included the longitudinal assessment over the 1-year study period and the recruitment of an Indian undergraduate student cohort. Major limitations are listed below. First and foremost, a major limitation is the small sample size: n = 25 total with nMen = 7, nWomen = 15 across the study period. The uneven and small sample size for men (n= 7) is particularly of concern given that the main results focus on sex differences. My second concern is the measurement of cortisol, which was obtained at 0800-0830 h. Without more detailed information on wake time and sleep, it is unclear what a cortisol value at this specific time means. Differences in wake time need to be taken into account with this data. Other Comments: The authors review evidence for the increased risk for suicide in Asian young adults, but did not include information regarding depressive symptoms or suicidal ideation over the study period. If available, such data could strengthen the paper. How did the authors ensure that no consumption of food/drink or teeth brushing was conducted half an hour before providing the salivary sample? The authors name “self-imposed” stress as a reason for higher overall and maintained stress levels, but also mention high parental pressure. I would suggest rephrasing this word, since self-imposed implies that there is no external/social pressure. I might also add that social/cultural expectations to succeed might be particularly prominent for young women. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes:** Thomas Volken 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 figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0252579.r001
Revision 1
14 Apr 2021 Author Response Dear Editor and Reviewers, Please find responses to your comments below: Responses to Editor’s comments: Throughout the article the term "sex difference" is used. I suggest to use gender-difference, since the authors themselves point to non-biological factors potentially causing the increase in cortisol in women. Or gender-/sex-difference if they which to point to potential biological differences in the stress-response by sex. Point well taken, in the revised manuscript gender/sex difference has been used. Impact of "stressors" were assessed in stratified analyses over all time points. As far as I understand they did not address interactions of sex and stressors. We did look at sex and stressors interaction, but it was not significant. Please see lines 217-226: There were no sex differences in the types of stressors reported (z=-0.66, p=0.509), but different type of stressors significantly differed across time. We thus divided the data across sexes and performed separate mixed effect models to understand how the stressors were different across time points. Total number of stressors reported by both men and women were lowest at time point 4 compared to time point 1 (men: z=-2.5, p=0.012, Fig. 2a; women: z=-2.86, p=0.004, Fig. 2b). Additionally, men also reported a significantly lower number of stressors at time point 5 compared to 1 (z=-2.23, p=0.025, Fig. 2a). Please address the limitations in more detail: sample size, missing confounders; as well as the validity of cortisol measures. The authors should also discuss the generalizability of the results and how unmeasured confounders might impact the results, especially from a gender point of view. These points have been addressed in the concluding paragraph (lines 314-326) of the revised manuscript. Responses to the Reviewers’ comments: Reviewer 1: A. Participants (page 5, lines 91-101): A1. The authors should provide some further information on the selection process, i.e. sampling procedure. How were students recruited?s An initial meeting with participants after their first few weeks of joining the semester was done where the researcher explained the course of the study and a research assistant obtained informed consent. The participants completed demographic and psychological questionnaires and were then instructed about the standardized collection of saliva samples according to the study protocol. Participants were asked not to brush their teeth or eat at least 30 minutes before sampling (lines 98-103) A2. The authors state that 25 students participated in the study and six measurements were available for ~20 individuals. Please indicate whether patients were included and analyzed on an intention-to-treat basis and if so how many observations were present at each time point. If only individuals with complete measurements were included, please justify. What do the authors mean by ~ (approximately) 20 individuals? Please specify precisely for how many subjects measurements were available at all time points. Sample sizes across time points: We had different sample sizes across time points and across physiological and psychological measures. Time 1: Cortisol data: N=23 (F=16, M=7); Psychological data: N=18 (F=11, M=7) Time 2: Cortisol data: N=24 (F=16, M=8); Psychological data: N=25 (F=16, M=9) Time 3: Cortisol data: N=21 (F=16, M=5); Psychological data: N=23 (F=15, M=8) Time 4: Cortisol data: N=25 (F=16, M=9); Psychological data: N=24 (F=17, M=7) Time 5: Cortisol data: N=24 (F=17, M=7); Psychological data: N=24 (F=17, M=7) Time 6: Cortisol data: N=23 (F=16, M=7); Psychological data: N=23 (F=16, M=7) This information has been added in the text (lines – 124-129 ) A3. Please justify your sample size. To the best of our knowledge, this is the first study of its kind in the Indian population and was a preliminary investigation looking at the correlation between physiological and psychological correlates of stress. We started with 34 individuals. However, since the batch had a low number of male students, we could not increase our male sample size. The initial number of males was 9, and even at a later time, 8-9 males continued to participate in the study, indicating that most females dropped out. Though the sample size is small, the total salivary samples were 140. It was also quite challenging to follow up with students for 6 points of time (1 year). The cost and time associated with taking a larger sample, buying salivary cortisol kits, and analysing them in the lab were other factors that determined our sample size. These are some limitations of our current study (which we have now added in detail), but we hope to address these in future studies. B. Discussion: B1. (page 13, line 285): There is a typo in the suicide incidence rate, i.e. “1,00,000” should probably read “100,000”. Thanks for pointing this out, we have made the change in the revised manuscript Reviewer 2: First and foremost, a major limitation is the small sample size: n = 25 total with nMen = 7, nWomen = 15 across the study period. The uneven and small sample size for men (n= 7) is particularly of concern given that the main results focus on sex differences. This concern has been addressed in the response given to the first reviewer justifying the small sample size. Please see lines (51-61) above. My second concern is the measurement of cortisol, which was obtained at 0800-0830 h. Without more detailed information on wake time and sleep, it is unclear what a cortisol value at this specific time means. Differences in wake time need to be taken into account with this data. We had no control over the sleep cycle and whether students experienced a sound sleep or the exact duration of sleep either. Wake time of most students were within an hour before the sample collection (we asked wake time for some students during sample collection). We understand the data limitation here as we would not know where in the cortisol daily cycle we are actually sampling. However, we think that the repeated sampling design might take care of this limitation to a large extent. The authors review evidence for the increased risk for suicide in Asian young adults but did not include information regarding depressive symptoms or suicidal ideation over the study period. If available, such data could strengthen the paper. In the current sample we did not have any students who showed depressive symptoms or suicidal ideation over the study period. (lines- 188-189) in the revised manuscript. How did the authors ensure that no consumption of food/drink or teeth brushing was conducted half an hour before providing the salivary sample? We instructed students specifically on this criterion and confirmed it before sample collection. This has been added in the methods ( lines 98-103) in the revised manuscript. The authors name “self-imposed” stress as a reason for higher overall and maintained stress levels, but also mention high parental pressure. I would suggest rephrasing this word, since self-imposed implies that there is no external/social pressure. I might also add that social/cultural expectations to succeed might be particularly prominent for young women. This is a relevant point; the word has been rephrased and a relevant research study has been added in the revised manuscript. (please see lines 263-266) Attachments Attachment Submitted filename: Response to Reviewers comments final.docx https://doi.org/10.1371/journal.pone.0252579.r002
11 May 2021 Decision Letter - Julia Dratva, Editor PONE-D-20-29949R1 A longitudinal study of perceived stress and cortisol responses in an undergraduate student population from India PLOS ONE Dear Dr. Bhattacharya, 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 25 2021 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: http://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, Julia Dratva 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 (if provided): Thank you for addressing the reviewer's points and requests for improvement. One last point, should still be considered, you answered the reviewer 2 point on cortisol measurement (see below), however, you did not take it up in the discussion. It seems a limitation worth mentioning, especially as you did ask for wake time (add to methods): "We had no control over the sleep cycle and whether students experienced a sound sleep or the exact duration of sleep either. Wake time of most students were within an hour before the sample collection (we asked wake time for some students during sample collection). We understand the data limitation here as we would not know where in the cortisol daily cycle we are actually sampling. However, we think that the repeated sampling design might take care of this limitation to a large extent. " Please introduce this limitation and your arguments into the manuscript. [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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: Thank you for addressing my comments. The manuscript has been substantially improved. Especially study limitations are now much clearer. ****** 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:** Thomas Volken [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0252579.r003
Revision 2
13 May 2021 Author Response Dear Editor and Reviewers, Please find responses to your comments below: Responses to Editor’s comments: Editor: Thank you for addressing the reviewer's points and requests for improvement. One last point, should still be considered, you answered the reviewer 2 point on cortisol measurement (see below), however, you did not take it up in the discussion. It seems a limitation worth mentioning, especially as you did ask for wake time (add to methods): "We had no control over the sleep cycle and whether students experienced a sound sleep or the exact duration of sleep either. Wake time of most students were within an hour before the sample collection (we asked wake time for some students during sample collection). We understand the data limitation here as we would not know where in the cortisol daily cycle we are actually sampling. However, we think that the repeated sampling design might take care of this limitation to a large extent. " Please introduce this limitation and your arguments into the manuscript. Response: a line [ lines 103- 104] on wake time has been added to the method section in the main manuscript. Data limitation about cortisol measurement not knowing the wake time has been included in the discussion section as suggested [lines 318-324] in the manuscript. Attachments Attachment Submitted filename: Response to Reviewers 12521.docx https://doi.org/10.1371/journal.pone.0252579.r004
19 May 2021 Decision Letter - Julia Dratva, Editor A longitudinal study of perceived stress and cortisol responses in an undergraduate student population from India PONE-D-20-29949R2 Dear Dr. Bhattacharya, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Julia Dratva Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0252579.r005
Formally Accepted
26 May 2021 Acceptance Letter - Julia Dratva, Editor PONE-D-20-29949R2 A longitudinal study of perceived stress and cortisol responses in an undergraduate student population from India Dear Dr. Bhattacharya: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Julia Dratva Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0252579.r006

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