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. 2023 Feb 23;18(2):e0281910. doi: 10.1371/journal.pone.0281910

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

Gaerolwe Masheto 1,2,*, Sikhulile Moyo 1,2,3, Terence Mohammed 1, Christine Banda 1, Charlene Raphaka 1, Gloria Mayondi 1, Joseph Makhema 1,2, Roger Shapiro 1,2,4, Mosepele Mosepele 1,2,5, Rebecca Zash 1,6,7, Shahin Lockman 1,2,6,7
Editor: Ashish K C8
PMCID: PMC9949641  PMID: 36821530

Abstract

Background

Women living with HIV-1 (WLHIV) are at higher risk of having an adverse birth outcome, but the underlying mechanism(s) are unknown. We hypothesized that HIV-associated endothelial activation could adversely impact placental function and lead to impaired fetal growth or stillbirth.

Methods

We used stored samples from WLHIV and HIV-negative women who had enrolled during pregnancy in the observational Botswana Tshipidi cohort. Written informed consent was obtained from the participants. We measured plasma levels of markers of endothelial activation (soluble vascular adhesion molecule 1 [VCAM-1], intercellular adhesion molecule 1 [ICAM-1] and E-selectin) from samples taken during pregnancy. We compared log10 biomarker levels by maternal HIV status and by the timing of ART initiation (ART prior to conception vs. during pregnancy; ART prior to sample collection vs. no ART prior to sampling) using t-tests and the Kruskal-Wallis rank test. We evaluated the association between these biomarkers and adverse birth outcomes (composite of stillbirth or small for gestational age [SGA]) using univariate and multivariate log-binomial regression controlling for maternal age (continuous) and timing of ART start. We also used generalized linear models (GLM) to evaluate the association between continuous birthweight (in grams) and gestational age (in weeks) and markers of endothelial dysfunction, adjusting for maternal age (continuous) and timing of ART relative to sample collection.

Results

Specimens collected before delivery were available for 414 women (372 WLHIV and 42 HIV-negative women), with a median age of 28 years and median gestational age at sample collection of 30 weeks (range 26, 35 weeks). WLHIV had significantly higher median VCAM1 (p = 0.002) than HIV-negative women, but HIV-negative women had higher median ICAM1 (p = 0.01); e-Selectin levels did not differ by maternal HIV status. Women starting ART during pregnancy had higher log10 VCAM1 levels than those on ART before conception, regardless of whether the sample was collected before (p = 0.02) or after (p = 0.03) ART initiation. However, ICAM1 and e-Selectin did not differ significantly by ART status or ART timing. Ninety-eight women (91 WLHIV and 7 HIV-negative), or 9 (2%) and 89 (22%) included in this study, had a stillborn or SGA baby respectively. Univariate and adjusted analyses did not show significant associations between levels of any of the biomarkers with these adverse birth outcomes. However, lower birthweight (p = 0.03) and lower gestational age at delivery were associated e-Selectin and ICAM (p = 0.008), respectively.

Conclusion

Maternal HIV infection and lack of ART (or recent ART initiation) were associated with one marker of greater endothelial activation (VCAM-1), but not with other markers (ICAM-1 nor E-selectin) in pregnancy. e-Selectin was associated with lower birthweight and every unit increase in log ICAM-1 at delivery was associated with lower gestation age at delivery.

Introduction

Increased access to 3-drug antiretroviral therapy (ART) during pregnancy has dramatically reduced new pediatric HIV infections globally and has improved maternal health [1–4]. However, women living with HIV (WLHIV) who take ART are at significantly higher risk of having an adverse pregnancy outcome (stillbirth, preterm delivery, and small for gestational age [SGA]) than women without HIV, particularly if ART is started prior to conception [5–14]. The underlying mechanisms for this increased risk are unknown. Given that more than 1.3 million WLHIV deliver annually [11] it is, therefore, crucial that we study the mechanism(s) underlying adverse birth outcomes in WLHIV, to help inform interventions to improve maternal and child outcomes.

Positive HIV status and ART use have both been independently associated with manifestations of endothelial dysfunction, including cardiovascular disease (CVD) [15–21]. Endothelial dysfunction is a term that covers diminished production/availability of nitric oxide and/or an imbalance in the relative contribution of endothelium-derived relaxing and contracting factors [22]. In the general population, high levels of endothelial adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1), intracellular adhesion molecule 1 (ICAM-1) and selectins (P-selectin or E-selectin) have been consistently associated with endothelial dysfunction [23–26]. These molecules work in concert to promote the development of atherosclerotic disease, with selectins facilitating activated leukocyte rolling and the adhesion molecules permitting adhesion and passage of leukocytes into the sub-endothelial space [26,27]. During pregnancy, endothelial function is critical for placentation, maternal volume expansion, and placental perfusion. Markers of endothelial dysfunction are associated with adverse birth outcomes in pregnant women without HIV [28], but similar data for pregnant women with HIV do not exist. We hypothesized that vascular endothelial dysfunction due to maternal HIV infection (and/or due to ART taken in pregnancy) adversely impacts placental perfusion during pregnancy, leading to an increased risk of placenta-mediated pregnancy complications such as fetal growth restriction (small for gestational age) and stillbirth. We, therefore, assessed the relationship between (1) HIV, ART and the biomarkers VCAM-1, ICAM-1 and e-selectin, and (2) these biomarkers and adverse birth outcomes.

Methods

Study population

Our study utilized stored plasma samples and existing data collected in pregnant women who previously participated in the “Tshipidi” study in Botswana [29]. The Tshipidi study enrolled WLHIV and women without HIV (≥18 years of age) during pregnancy or shortly after delivery from 2010 to 2012 and followed mothers and babies for 2 years postpartum to evaluate pediatric neurodevelopmental outcomes. The majority (88%) of women were enrolled before delivery at a median gestational age of 27 weeks. Maternal and infant blood samples were collected at enrollment, delivery, and 1, 6 and 24 months postpartum. Demographic information, HIV history (including the date of diagnosis, antiretroviral [ARV] regimen and date of ARV initiation, and most recent CD4 count and viral load), general medical history, and pregnancy outcome were recorded for all participants. Gestational age was estimated by the last menstrual period (which was ascertained using a combination of maternal self-report and written obstetric records that are completed for all pregnant women during antenatal care and labor/delivery, which nearly universally occur during admission to maternity wards). Written obstetric records also served as the source of birthweight and stillbirth data. Small for gestational age was calculated using estimated gestational age and birthweight.

Women received antiretroviral regimens according to Botswana national guidelines at the time. From 2008 to 2012, pregnant women with baseline CD4 count >250 cells/mm3 and no WHO Clinical Stage 3 or 4 HIV illness received zidovudine. Women with baseline CD4 <250 cells/mm3 or with WHO Clinical Stage 3 or 4 received 3-drug ART (generally zidovudine + lamivudine + nevirapine); the CD4 threshold was changed to <350 cells/mm3 in 2012. Multiple gestations were excluded from this analysis.

The Tshipidi study (from which the de-identified data for this analysis came from) was approved by the Botswana Health Research Development Committee and the Office of Human Research Administration at the Harvard T.H. Chan School of Public Health. Mothers provided written informed consent for study participation. The work summarized in this paper was consistent with original study aims, and was also approved by IRBs as an amendment.

Laboratory testing

One of the secondary objectives of the Tshipidi study was to investigate potential immune-mediated mechanisms that might explain adverse birth outcomes in HIV-exposed children. We evaluated this objective using stored maternal plasma samples from all participants who enrolled prior to delivery and who had known infant birth weight, gender, and gestational age. More samples were available for WLHIV than women without HIV at the time we conducted sample testing for this study because most plasma samples for women without HIV had been used by other studies. We measured plasma concentrations of endothelial dysfunction biomarkers ICAM-1 (detection range 0.3–20 ng/mL and sensitivity 0.057) ng/mL, VCAM-1(detection range 6.3–200 ng/mL and sensitivity 1.26 ng/mL) and E-selectin (0.1–8 ng/mL and sensitivity 0.027 ng/mL) using commercial ELISA kits (R&D Inc, Minneapolis, USA), following the manufacturer’s instructions [30]. Testing was done at the ISO 15189 accredited Botswana Harvard HIV reference laboratory.

Statistical analysis

Biomarkers were analyzed as continuous variables. Timing of ART relative to sample collection was defined as 1) initiated pre-conception, 2) initiated post-conception, did not start ART by the time of sampling and, 3) initiated during pregnancy sample collection after ART start.

We compared maternal baseline characteristics using the two-sample t-test for continuous variables and Fisher’s exact test for categorical variables. Log biomarker levels were compared by maternal HIV status and by the timing of ART initiation and sample collection using t-tests and the Kruskal-Wallis rank test. We also evaluated the association between these biomarkers and the adverse birth outcomes that are more likely to be related to endothelial dysfunction, namely stillbirth or small for gestational age (defined as <10th percentile weight-for-gestational age according to Intergrowth-21 standards) [31,32] using univariate and multivariate log-binomial regression controlling for maternal age (continuous) and timing of ART initiation. We also used the generalized linear models (GLM) to evaluate the association between continuous birthweight and gestational age and markers of endothelial dysfunction, adjusting for maternal age (continuous) and timing of ART relative to sample collection.

Results

Among 454 women living with HIV and 458 women without HIV who were enrolled in Tshipidi, this analysis includes 372 women with HIV (82%) and 42 women without HIV (9%) who were enrolled in antepartum and had birth outcomes data and stored plasma collected before delivery available. Among women without HIV: women who had samples available for our analysis had slightly lower median gestation age [(39.5 vs 41 weeks; p = 0.001) but were similar in median age (p = 0.50), educational status (p = 0.90) and the number of stillbirths (p = 0.46) compared with HIV-negative women who did not have samples available. Among 372 WLHIV in our study sample, 346 (93%) had a known time of ARV/ART initiation of whom 39 started ART prior to conception, 181 started during pregnancy prior to enrollment (29 on ART and 152 on ZDV monotherapy) and 126 started after study enrollment/sample collection. Among the 26 women with unknown timing of ART start, 3 were on 3-drug ART, 3 on ZDV, 4 ARV naïve and 16 had no records. Table 1 below shows demographics and clinical characteristics of pregnant WLHIV and pregnant women without HIV. Women living with HIV were older, less educated and had lower income.

Table 1. Demographics and clinical characteristics of pregnant WLHIV and pregnant women without HIV (N = 414).

Variables All women Women living with HIV Women without HIV p-value
  N = 414 N = 372 N = 42  
Age (years)  
Median (Q1, Q3) 28 (23, 32) 23 (21, 30) 28 (24, 33) <0.001
Highest Education level, n (%)  
    Junior Secondary school or lower 278 (67) 262 (71) 16 (38) <0.001
    Senior Secondary school 99 (24) 81 (21) 18 (43)
    Tertiary 36 (9) 28 (8) 8 (19)
Income per month (in US $), n, (%)  
<20 215 (55%) 192 (54) 23 (60) 0.045
20–50 30 (8%) 30 (8) 0
51–100 76 (19%) 72 (20) 4 (11)
> 100 73 (18%) 62 (18) 11 (29)
Delivery type, n, (%)  
     Vaginal delivery 376 (95) 338 (95) 38 (100) 0.154
    Caesarean section 18 (5) 18 (5) 0
Gestational Age at the time of blood collection (weeks), median (Q1, Q3) 30 (26, 35) 30 (26, 35) 34 (26, 37) 0.044
Gestational age at delivery (weeks), median (Q1, Q3) 40 (38, 41) 39 (38, 41) 41(39, 42) <0.001
Stillbirth, n, (%) 9 (2) 8 (2) 1 (3) 0.063
Preterm delivery, n, (%) 53 (13) 51 (14) 2 (5) 0.087
Small for Gestational Age (SGA), n, (%) 89 (23) 83 (23) 6 (16) 0.52
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Markers of endothelial dysfunction by maternal HIV status and timing of ART initiation

Fig 1 shows the levels of VCAM-1, ICAM-1 and e-selectin by maternal HIV status. WLHIV had a statistically significantly higher mean VCAM1 (2.90 ng/mL; 95% Confidence Interval (CI):2.88–2.93) than women without HIV (2.77 ng/mL; 95%CI: 2.72–2.83), Fig 1A. Women without HIV had a statistically significantly higher mean ICAM1 (2.42 ng/mL; 95%CI:2.40–2.44) compared to WLHIV (2.50 ng/mL;95% CI: 2.44–2.55), Fig 1B. No difference was observed in e-selectin levels by maternal HIV status (Fig 1C).

Fig 1.

Fig 1

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Endothelial biomarker level by receipt and timing of ART initiation and sample collection in relation to pregnancy is shown in Fig 2 (initiated pre-conception, initiated post-conception, or did not start ART by the time of sampling). ICAM-1 and e-selectin levels were not significantly different by the timing of ART relative to sample collection (Fig 2B and 2C). However, women starting ART during pregnancy had statistically significantly higher mean log10 VCAM1 levels (2.92 ng/mL; 95%CI:2.88–2.95) than women who were on ART before conception, in both the samples that were collected before (2.83 ng/mL; 95%CI: 2.77–2.88;p = 0.02) or after (2.91 ng/mL; 95%CI: 2.87–2.944;p = 0.03) ART initiation, Fig 2A.

Fig 2.

Fig 2

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Markers of endothelial dysfunction and adverse birth outcomes

Univariate and adjusted analyses did not show significant associations between levels of any of these biomarkers and the adverse birth outcomes of interest (stillbirth or SGA). Overall, 9 (2%) pregnancies ended in stillbirth and 89 (22%) with infants born SGA. Median ICAM-1 was 292 ng/mL [Q1,Q3: 214, 346] among stillbirths and 267 ng/mL [Q1,Q3: 209, 347] among live births (p = 0.89), median VCAM-1 was 868 ng/mL [Q1,Q3: 706, 1132]] among stillbirths and 780 ng/mL [Q1,Q3: 565, 1048] among live births (p = 0.36), and median e-Selectin was 24.2 ng/mL [Q1,Q3: 18.7, 27.3] among stillbirths and 22.7 ng/mL [Q1,Q3: 16.1, 33.3] among live births (p = 0.88). Median ICAM-1 was similar among SGA (276 ng/mL, Q1,Q3:199,349] and non-SGA (261 ng/mL, (Q1,Q3:199,349)]. Median VCAM was similar among SGA (740 ng/mL, Q1,Q3:569, 1104] and non-SGA (804 ng/mL, (Q1,Q3:586,1062)]. Median e-Selectin was also similar among SGA (24.6 ng/mL, Q1,Q3:17.5, 36.3] and non-SGA (22.5 ng/mL, (Q1,Q3:15.9,31.2)]. Every 1 unit increase in log e-Selectin (ng/mL) was associated with lower birthweight (β: -0.07, p = 0.03) and every 1 unit increase in log ICAM-1 (ng/mL) at delivery was associated with lower gestational age (weeks) at delivery (β: -0.06, p = 0.008), regardless of HIV status.

Discussion

We evaluated whether maternal HIV status or timing of ART initiation was associated with biomarkers of vascular endothelial dysfunction during pregnancy and whether levels of these biomarkers were associated with SGA or stillbirth. We found that positive maternal HIV status was associated with significantly higher VCAM-1 levels, an indication of greater endothelial activation; however, positive maternal HIV status was not associated with increased levels of ICAM-1 or e-selectin. None of these three biomarkers were associated with the occurrence of stillbirth or SGA (although there we did observe a non-significant trend toward a higher risk of these adverse pregnancy outcomes with higher VCAM-1 levels, and we had limited power to evaluate stillbirth as an outcome).

Consistent with other studies for non-pregnant adults, median VCAM-1 levels were elevated in pregnant WLHIV compared with pregnant women without HIV [26–28], and there was no difference in e-selectin level by HIV status [26]. In the general population, elevated VCAM-1 has been consistently associated with endothelial dysfunction. Unlike prior data from non-pregnant adults [23,26], we found that median ICAM1 levels were higher in women without HIV compared with WLHIV. The reasons for this difference are not clear. ICAM-1 is an important molecule in immune-mediated and inflammatory processes and functions as a co-stimulatory signal which is important for the trans-endothelial migration of leukocytes and the activation of T cells [33,34]. Studies of ICAM-1 in women with HIV show no difference in the levels of ICAM-1 in normal pregnancy compared to non-pregnant women, but significantly increased levels of ICAM-1 in women with pregnancy-induced hypertension, especially preeclampsia [35,36].

We hypothesized that HIV-associated endothelial dysfunction would lead to a poorly functioning placenta and that there would be an increased prevalence of adverse birth outcomes associated with abnormal placentas, including SGA and stillbirth. However, we found no association between these outcomes and biomarkers of endothelial dysfunction. This could indicate that the biomarkers we chose are not the most specific for endothelial dysfunction in pregnancy, or we may have lacked the power to detect differences given the relatively small study sample size.

Our study had several limitations. Our sample size was small for detecting associations with infrequent outcomes such as stillbirth. We could not measure all biomarkers of endothelial dysfunction and could not determine the causal relationship between endothelial dysfunction and adverse birth outcomes. However, our data suggest a possible role of e-selectin and ICAM-1 in lower birthweight and gestation age at delivery, respectively. This possible role of e-selectin and ICAM-1 in lower birthweight and gestation age at delivery is regardless of HIV status because there were no differences in these parameters by HIV status.

Not all stillbirths or SGA would result from placental dysfunction. Nevertheless, we believe that this study generated valuable pilot data in an area that has not been investigated thoroughly.

Conclusion

Our study did not support endothelial activation as a mechanism for adverse birth outcomes in WLHIV, but further research is needed given the limitations and small sample size in this study.

Supporting information

S1 File

(XLS)

Click here for additional data file. (605KB, xls)

Acknowledgments

We thank and acknowledge the Tshipidi Study participants and the investigators as well as the Botswana Harvard AIDS Institute Partnership leadership for their support.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

Dr. Gaerolwe Masheto was supported by 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. Dr. Sikhulile Moyo was partially supported through the Sub-Saharan African Network for TB/HIV Research Excellence (SANTHE 2.0), by Bill and Melinda Gates Foundation (INV-033558) and the National Institutes of Health NIH Fogarty International Center K43 TW012350-01. Dr. Shahin Lockman was supported by the National Institutes of Health NIH/ National Institute of Allergy and Infectious Diseases K24 mentoring grant - NIH K24 AI131928.

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Decision Letter 0

Emily Chenette

18 Jul 2022

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.

Please submit your revised manuscript by Aug 29 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.

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We look forward to receiving your revised manuscript.

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Emily Chenette

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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

**********

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: 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 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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Decision Letter 1

Ashish KC

9 Nov 2022

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

**********

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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: 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

Reviewer #2: (No Response)

**********

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?

**********

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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Decision Letter 2

Ashish KC

5 Feb 2023

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:

Acceptance letter

Ashish KC

14 Feb 2023

PONE-D-21-20063R2

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

Dear Dr. Masheto:

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. Ashish KC

Academic Editor

PLOS ONE

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