PONE-D-21-20063

Maternal biomarkers of endothelial dysfunction and pregnancy outcomes in women with and without HIV in Botswana

PLOS ONE

Dear Dr. Masheto,

Thank you for submitting your manuscript to PLOS ONE; I sincerely apologise for the unusually delayed review timeframe.

Your manuscript has been assessed by two reviewers, whose comments are appended below. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Although the reviewers find the topic of this study to be important, they raise concerns regarding whether elevated VCAM-1 in HIV-positive women was clinically or physiologically relevant, and the power of the study to detect significant differences between populations. Although the latter is discussed as a limitation, any revised manuscript should very clearly indicate the limitations of the current dataset and avoid drawing any conclusions (positive or negative) that cannot be fully supported by the analysis. We invite you to submit a revised version of the manuscript that addresses theses and other points raised during the review process.

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Emily Chenette

Editor in Chief

PLOS ONE

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3. Thank you for stating in your Funding Statement: GM received award from the Harvard University Center for AIDS Research (CFAR), an NIH funded program (P30 AI060354), which is supported by the following NIH Co-Funding and Participating Institutes and Centers: National Institute of Allergy and Infectious Diseases, National Cancer Institute, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Heart, Lung, and Blood Institute, National Institute on Drug Abuse, National Institute of Mental Health, National Institute on Aging, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of General Medical Sciences, National Institute on Minority Health and Health Disparities, National Institute of Dental and Craniofacial Research, Office of AIDS Research, and Fogarty International Center. 

SM was partially supported through the Sub-Saharan African Network for TB/HIV Research Excellence (SANTHE), a DELTAS Africa Initiative [grant # DEL-15-006]. The DELTAS Africa Initiative is an independent funding scheme of the African Academy of Sciences (AAS)’s Alliance for Accelerating Excellence in Science in Africa (AESA) and supported by the New Partnership for Africa’s Development Planning and Coordinating Agency (NEPAD Agency) with funding from the Wellcome Trust [grant # 107752/Z/15/Z] and the UK government. 

SL was supported by the National Institutes of Health NIH/ National Institute of Allergy and Infectious Diseases K24 mentoring grant - NIH K24 AI131928. 

NO - All funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript."

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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: Yes

Reviewer #2: I Don't Know

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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: The authors have taken advantage of stored plasma samples and data collected from an observational study (Tshipidi) that enrolled in HIV-positive and HIV-negative pregnant women in Botswana 2010-2012 (a time when ART regimen in pregnancy was determined by CD4 count), following the participants for 2 years after enrollment, to try to assess whether elevation in three biomarkers of endothelial dysfunction may be in the causal pathway for increased risk of adverse pregnancy outcomes in HIV-infected women. This short report found only one marker of endothelial activation, VCAM-1, was elevated in HIV infection and with no/recent ART, and none of the markers were associated with birth outcome (stillbirth, SGA). Unfortunately, there were only limited samples remaining on the comparative HIV-negative women (9% vs 82% for HIV positive women), which could potentially restrict the power of the study to determine differences by HIV status.

Page 13, line 156: Table 1: Consider adding the “N” in the first row under the column titles so it is clear to reader.

Page 15, top paragraph lines 162-165, 170-172: Consider putting in the actual median values for the various markers (log ng/mL) as opposed to just listing the p values. Figure 1 on page 23 does not provide values, leaving the reader to guess what the median is in the comparative groups. A comment that while statistically significantly different by HIV status for VCAM-1 and ICAM-1 , visually the values do not look extremely different. For example, in figure 1 for VCAM-1, the median value looks like ~2.8 in HIV+ vs 2.6 log10 ng/mL in HIV- women. The clinical significance of such differences is not clear to me.

Discussion:

As noted above, the clinical significance of the differences observed is not clear. The authors note that “elevated VCAM-1” in the general population is associated with endothelial dysfunction. However, what levels define “elevated” that are associated with endothelial dysfunction is not discussed. Whether a decimal point difference in levels of the marker between groups (from visual inspection of the graphs) means that the marker is elevated enough to be associated with endothelial dysfunction is not clear. Additionally, references 23, 24 (referred to in line 202) report that while ICAM-1 was associated with risk of MI, levels of VCAM-1 were actually not, with VCAM-1 levels actually similar with and without MI (levels 638 vs 634 ng/mL). I did find a different reference suggesting a potential association (Wallen NH, Eur Heart J 1999, but p value for association was only 0.05).

Have these markers previously been associated with adverse pregnancy outcomes, or is this the first study that has looked at this association? There was only one study cited (ref 28 Chen X et al. PLosOne 2014) that did suggest an association of preterm delivery with elevated sCAM-1 and sVCAM-1 but that was not the adverse outcome being evaluated in this study. Are there associations with stillbirth and SGA?

Reviewer #2: This manuscript presents a prospective cohort study that evaluated differences in biomarkers of endothelial function in pregnancy by HIV and ART status as well as the relationship of the endothelial function biomarkers and birth outcomes. Overall, the research question is novel, and the study was well conducted; however, I think the manuscript could be improved in terms of the clarity of the reporting of the methods and the birth outcomes analyzed. My primary concern, which is also acknowledged as a limitation by the authors, is limited statistical power for the adverse birth outcomes outcomes examined.

1) Introduction – The introduction does a nice job laying out the current literature, but the research question is not entirely clear in the last sentence of the section. Based on the methods and results, I think the study had two components to the research question (1) assess the relationship of HIV, ART with the biomarkers and then (2) the relationship of the biomarkers with adverse birth outcomes. It seems part 1 – which takes up a significant proportion of results and discussion is currently missing from the research question.

2) Methods - Lab section –Were there limits of detection per the kits for each biomarker? If yes, please report and also how in the analysis how those beyond limits of detection contributed to the analysis.

3) Methods – it would be important for the methods for stillbirth determination, gestational age dating and birthweight data collection be defined. The exposure methods for biomarkers are clear but how outcome data was collected is not clear.

4) Preterm birth and Low birthweight –Why were preterm birth, birth weight, low birthweight and other commonly defined pregnancy outcomes not reported? There is certainly existing research and potential links between endothelial dysfunction and preterm birth.

5) Gestation duration, birthweight –Given the sample size, statistical power is a concern as noted by the study team. An analysis of continuous gestational age and birthweight which are commonly analyzed in pregnancy cohorts should be considered. Continuous birthweight-for-gestational age from Intergrowth, which is not as common as birthweight and gestation duration, could also be examined.

6) Stillbirth - There were 9 stillbirths in the cohort and as a result this cohort does not seem well positioned to evaluate the relationship between the biomarkers and stillbirth as a ‘primary’ outcome. While acknowledged in the discussion, it seems making no association with stillbirth a key message when there was almost no possibility of finding a difference is challenging and may be misinterpreted. This is in contrast to SGA, for which the sample size is not large, but there was certainly a chance of finding a moderate to large magnitude of effect if there was one.

Minor

1) Abstract – it would be helpful to report stillbirth and SGA rates separately to match analysis to be clear it was not a composite endpoint

2) Abstract and Discussion conclusion – The abstract conclusion focuses on difference by HIV and then the discussion conclusion focuses on birth outcomes. It seems both components are part of the research question and would be important to address in both sections.

3) Methods – the order of the analyses presented in the statistical analysis is confusing given the relationship of maternal factors with biomarkers is at the beginning and end of the section with the adverse birth outcomes in the middle.

4) Figure 1 – Please check labels – “ARV before pregnancy sampled before ARV start” – I believe this is women who started ARVs in pregnancy but the sample was collected before they started

5) Multiple gestation - where there any twins in the cohort examined? If so, how was this issue addressed in the statistical analysis.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-21-20063R1Maternal biomarkers of endothelial dysfunction and pregnancy outcomes in women with and without HIV in BotswanaPLOS ONE

Dear Dr. Masheto,

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 Dec 24 2022 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,

Ashish KC

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:

Dear Dr. Masheto

Thank you for the revision and proving point to point response to the reviewers' comment. There are some further recommendation made by the reviewers, we look forward to the updated manuscript with response to the reviewer's comment.

warm regards, ashish

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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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: 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 #1: Yes

Reviewer #2: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: (No Response)

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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 #1: The reviewer comments have been generally addressed adequately.

The authors now newly note a possible role of e-Selection and ICAM in lower birthweight and gestation age at delivery at several points in the manuscript. It might be clearly noted that this was regardless of HIV status, given that there were no differences in these parameters by HIV status. So the relevance of these findings are really not HIV-specific but more relate to potential mechanisms of low birth weight and gestational age in general - think this might be made clearer as the paper is focused on differences due to HIV infection.

Reviewer #2: Thank you to the team for your thoughtful response to my comments.

I only have one small comment on the continuous analysis of birth weight and delivery. The Beta is given as -0.07 for example but as a reader I am not clear what the unit is in terms of weight same for the gestation age. As a result the magnitude of the association is not clear.

Is the birthweight kg or grams - is it 70 grams per log increase in log e-selectin? I assume for gestational age its weeks -0.06 weeks per log increase ICAM?

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Christopher Sudfeld

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[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.]

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Maternal biomarkers of endothelial dysfunction and pregnancy outcomes in women with and without HIV in Botswana

PONE-D-21-20063R2

Dear Dr. Masheto,

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,

Ashish KC

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: