{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T20:19:44Z","timestamp":1789417184051,"version":"build-2803163510"},"update-to":[{"DOI":"10.12688\/wellcomeopenres.23616.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2025,6,6]],"date-time":"2025-06-06T00:00:00Z","timestamp":1749168000000}}],"reference-count":62,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2025,6,6]],"date-time":"2025-06-06T00:00:00Z","timestamp":1749168000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100010269","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["227204"],"award-info":[{"award-number":["227204"]}],"id":[{"id":"10.13039\/100010269","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["wellcomeopenresearch.org"],"crossmark-restriction":false},"short-container-title":["Wellcome Open Res"],"abstract":"<ns3:p>Introduction Epidemiological evidence linking heat exposure to adverse maternal and child health outcomes is compelling. However, the biological and social mechanisms underlying these associations remain poorly understood. Understanding the pathways explaining these associations is important given rising global temperatures, and the urgent need for developing and testing adaptive interventions. Methods This transdisciplinary study in Johannesburg, South Africa, will monitor a cohort of 200 women from their second trimester until one-year postpartum, alongside their infants. Heat exposure and environmental factors will be tracked using personal, community and facility-level temperature monitors and geospatial data. Data will be collected on social conditions, medical and obstetric history, heat stress and adaptation, hydration, mental wellbeing, and sleep quality. Clinical data includes physical measurements, ultrasound, cardiotocography, and biological specimens (blood, urine, saliva) analysed for inflammatory markers, RNA, metabolic indicators, renal function and hormonal levels. Placental and cord blood analyses will assess foetal stress. Infant data will include medical history, hospital visits, neurodevelopment, anthropometric measurements, vital signs, and urine analysis. Three nested sub-studies (20\u201350 participants) will explore specific aspects: Sub-study 1 will use wearable devices to monitor sleep, activity, and heart rate in high-risk women; Sub-study 2 will involve qualitative interviews; and Sub-study 3 will assess breastmilk composition and volume. Planned analyses Our primary aim is to document linkages between heat exposure and inflammatory pathways that precede preterm birth. The hypothesis that heat exposure triggers maternal inflammation will be tested by analysing epigenetic changes associated with inflammatory cytokine protein and gene expression. We will investigate thermoregulation and hydration during labour. Using isotope techniques, we assess whether heat exposure alters breastmilk composition and volume. Conceptual frameworks and graphical causal models will be developed to delineate pathways of vulnerability and protective mechanisms.<\/ns3:p>","DOI":"10.12688\/wellcomeopenres.23616.2","type":"journal-article","created":{"date-parts":[[2025,6,6]],"date-time":"2025-06-06T12:40:06Z","timestamp":1749213606000},"page":"121","update-policy":"https:\/\/doi.org\/10.12688\/wellcomeopenres.crossmark-policy","source":"Crossref","is-referenced-by-count":3,"title":["Study protocol for the Bio-HEAT study: Investigating the Biological pathways from HEAT exposure to preterm birth and other adverse maternal and child health outcomes in South Africa"],"prefix":"10.12688","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2045-3053","authenticated-orcid":false,"given":"Ijeoma","family":"Solarin","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6114-7477","authenticated-orcid":false,"given":"Darshnika Pemi","family":"Lakhoo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kimberly","family":"Mc Alpine","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5390-8827","authenticated-orcid":false,"given":"Margaret M.","family":"Brennan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Admire","family":"Chikandiwa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6843-6236","authenticated-orcid":false,"given":"Nicholas B.","family":"Brink","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3184-9165","authenticated-orcid":false,"given":"Lebohang","family":"Radebe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mari\u00e9","family":"Landsberg","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Clive","family":"Gray","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3080-1007","authenticated-orcid":false,"given":"G Justus","family":"Hofmeyr","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Howard","family":"Chang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1953-3520","authenticated-orcid":false,"given":"Robyn","family":"Hetem","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sibusisiwe","family":"Makhanya","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8280-8940","authenticated-orcid":false,"given":"Phelelani T.","family":"Mpangase","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7124-3788","authenticated-orcid":false,"given":"Shane","family":"Norris","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Urban","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Valerie","family":"Vannevel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5182-9060","authenticated-orcid":false,"given":"Amy","family":"Wise","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew F.","family":"Chersich","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Karl-Gunter","family":"Technau","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9113-6584","authenticated-orcid":false,"given":"Renate","family":"Strehlau","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"2560","published-online":{"date-parts":[[2025,6,6]]},"reference":[{"key":"ref-1","article-title":"Climate change 2023: synthesis report. 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status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.21956\/wellcomeopenres.26054.r121797, Ashish KC, University of Gothenburg, Gothenburg, 23 Apr 2025, version 1, 1 approved, 1 approved with reservations","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-121\/v1#referee-response-121797","order":0,"name":"referee-response-121797","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Ijeoma Solarin<\/b>; \n<i>Posted: 27 May 2025<\/i>; Reviewer:&nbsp;\n<b>Ashish KC,&nbsp;<\/b>University of Gothenburg, Gothenburg, Sweden \n<b>No<\/b> \n<b>Reviewer comment<\/b> \n<b>Author response<\/b> 1.1 \n<i>&nbsp;Thank you for inviting me to review this interesting study protocol on biological pathway on heat exposure to preterm birth and other maternal and child health outcomes. This is a very valuable study to provide the epidemiological explanation on heat events to preterm birth. This study aim to investigate the environmental exposure to high ambient temperature on preterm birth and the causal pathway to exposure.<\/i> &nbsp; &nbsp; Thank you for these comments and careful review. &nbsp; 1.2 \n<i>I have some comments on the hypothesis driven for the study - Non-optimal temperature either lower than 50th centile or higher than 50th centile has been associated to poor obstetric outcomes including preterm birth, still birth and neonatal mortality https:\/\/www.nature.com\/articles\/s41591-024-03245-7 and Ref:1 indicating that non- optimal ambient temperature exposure has a corelation between heat and preterm birth. It would be worth to provide a note to it. <\/i> &nbsp; &nbsp; Thank you for this helpful comment and for sharing the relevant references. We agree that both high and low ambient temperatures have been associated with adverse obstetric outcomes, including preterm birth, stillbirth, and neonatal mortality. While the primary focus of our study is on high temperature exposures\u2014given their growing relevance in the context of anthropogenic climate change, the particular relevance in our study setting in sub-Saharan Africa and the urgent need to understand underlying biological mechanisms\u2014we recognise the importance of non-optimal low temperature exposure as well. While the references shared by the reviewer (Kc et al. (2024) and Hanson et al (2024)) underscore the importance of considering both cold and hot non-optimal temperatures, even these studies report that heat exposure tends to have a more consistent and pronounced association with adverse outcomes than cold exposure, especially in low-resources contexts. We have nevertheless updated the manuscript to acknowledge this broader evidence base and will consider it when interpreting our findings. &nbsp; Added text in section on Global health relevance: \n<i>While both high and low ambient temperatures have been associated with poor obstetric outcomes (Kc et al., 2024, <\/i>Nyadanu et al., 2024\n<i>), this study focuses on heat exposure, which is of particular concern in our African context.<\/i> &nbsp; 1.3 \n<i>Even though there is a cumulative effect of non-optimal temperature to preterm birth, the lag effect i.e harvesting effect might be seen in preterm birth, can the researchers discuss about it <\/i> &nbsp; &nbsp; Thank you for this comment. We appreciate the suggestion to consider a potential lag or \u201charvesting\u201d effect. As we understand it, the harvesting effect typically refers to temporal displacement of events that would have occurred soon regardless, and it is most often applied in the context of mortality, particularly among older or frail individuals. We are not certain that this concept directly translates to preterm birth, as a shift from term to preterm delivery due to heat exposure may represent a clinically meaningful outcome that could have been avoided rather than temporal displacement of an outcome that was likely to occur regardless. Nonetheless, we acknowledge that further discussion on lag structures and potential timing effects is warranted and will consider this in our interpretation of findings. 1.4 \n<i>In the abstract researchers mention that &quot;maternal inflammation will be tested by analysis the epigenetic changes associated with inflammatory cytokines protein and gene expression&quot;. Based on the studies from Genome wide association study and Mendenlian law, any genetic variant is always constant since conception, so the expression of cytokine is based on the dysfunction of the cytokine receptor dysfunction. <\/i>\n<i>https:\/\/doi.org\/10.3390\/ijms252011332<\/i>\n<i>.<\/i> &nbsp; &nbsp; Our approaches to examining heat exposure and inflammation includes assessing maternal transcriptomic and epigenetic signatures, using serial RNA-Seq data, to track up-and down-regulated genes before-and- after heat extremes. These are not Mendelian changes, but the differential gene expression and pathway analyses assessed using RNA Seq data. We can visualise the significantly differentiated genes between the different time points and see which genes are being expressed. We don\u2019t do GWAS or similar genetic testing in this study, as the reviewer points out. &nbsp; 1.5 \n<i>2.5. further not all peripheral inflammation will cause the trigger preterm labour, until the inflammation cross the bloodbrain barrier, the inflammatory chain of reaction is required.<\/i>\n<i>\u2028<\/i>\n<i>Further, why is the focus on IL-6, there are a range of pro-inflammatory and anti-inflammatory cytokines which can cause BBB inflammation Ref:2<\/i> &nbsp; We appreciate your important point that not all peripheral inflammation will trigger PTB, and that specific inflammatory pathways, including those involving the blood-brain barrier, may play a role. However, our study is primarily focused on inflammatory pathways at the maternal-foetal interface (such as placenta, myometrium), which are more directly implicated in the initiation of labour than the BBB. We also acknowledge that IL-6 is a non-specific marker and that multiple cytokines, both pro- and anti-inflammatory, may contribute to the broader inflammatory response. Our focus on IL-6 is based on several considerations: 1). Evidence supporting its role in PTB; 2). its relative ease of measurement and low cost of testing; and 3). its widespread use in research, which facilitates comparison across studies. Nevertheless, we do plan on conducting a more comprehensive biomarker analysis using a panel of 96 inflammatory markers. This will allow us to investigate a range of cytokines implicated in both peripheral and central inflammation and inform more complex analysis. Biomarker analysis will be conducted using multivariate regression models to examine the association between heat exposure and inflammatory markers, adjusting for potential confounders. Inferential analysis of longitudinal data will include mixed-effects linear models. Much of this analysis is done using visualisations such as heatmaps and volcano plots. Bonferroni corrections will be applied to counteract potential multiple testing biases. We recognise that much remains unknown about the precise pathways - whether systemic, BBB, or at the level of the cervix and membranes - and which pathways are most important. This study will try address some of those questions. We have added reference to a recent review on heat and preterm birth Mushimiyimana 2025, which discusses this in more detail. &nbsp; 1.6 \n<i>&nbsp;<\/i>\n<i>Sample size estimation- I still did not get the basis of the sample size based on IL-6, has there been a study to estimate IL-6 &gt;1000pg\/ml is linked with preterm birth.<\/i> &nbsp; &nbsp; &nbsp; \n<b>Author response:<\/b> Many thanks for this query, the point made here is valid. We should have noted the rationale for selecting Il-6 as the outcome of interest. IL-6 is one of the most widely studied inflammatory markers and indeed, several studies have linked Il-6 with preterm birth, as in the papers we now cite for this: (Prairie et al. 2021, Hatzidaki et al., 2005, Karampitsakos et al., 2025). In addition, IL-6 has been shown to be raised by heat (Maloney &amp; Duffy, 2024). &nbsp; &nbsp; We do however acknowledge that the originally specified IL-6 threshold of &gt;1000 pg\/mL was not appropriate for maternal serum. This was an oversight, and we appreciate the opportunity to correct it. We have revised this threshold in the manuscript to reflect more clinically and biologically relevant values for maternal serum which are much lower depending on presence of infection or specific population group being studied. While there is no universally accepted threshold for IL-6 in predicting PTB, and some variability exists across studies, we have revised our threshold to 10pg\/ml as a conservative measure based on other studies which identify maternal serum IL-6 levels around this value as predictive of preterm birth (e.g., Murtha et al., 1998) and which was our original intent. We also acknowledge that we have made some assumptions due to the scarcity of existing environmental exposure literature in this area. Nevertheless, our assumptions are based on&nbsp; conservative and pragmatic estimates to ensure sufficient power. One of our aims is to contribute new evidence on the potential relationship between environmental temperature and maternal systemic inflammation. We have clarified this in the revised manuscript. &nbsp; Updated text: \n<i>The primary inflammatory outcome interleukin-6 is strongly linked with PTB (Prairie et al., 2021, Hatzidaki et al., 2005, Karampitsakos et al., 2025). However, there is currently no universally accepted threshold for IL-6 in predicting PTB, with high levels of variability across studies. We have based our sample size on IL&gt;10pg\/ml as a conservative measure in keeping with other studies which identify maternal serum levels around this value as predictive of PTB (Murtha et al., 1998).<\/i> 1.7 \n<i>In hypotheses, the researchers mention that psycho-social stressor is a mediator of preterm birth, but does it not also link with the increase in pro-inflammatory or neuro-hormonal disturbance. Can it be that there is a reversal phenomenon that due to increased pro-inflammatory genetic expression and disturbance in neuro-hormonal gene expression, that the social stress can change the pyschological response and thus preterm birth. <\/i> &nbsp; &nbsp; &nbsp; &nbsp; Thank you for this thoughtful comment. We agree that psychosocial stress may not only act as a mediator but may also be shaped by underlying biological processes, such as pro-inflammatory and neuro-hormonal disturbances. We acknowledge the possibility of a bidirectional, feedback loops or reverse relationship, whereby heightened inflammatory or neuroendocrine activity\u2014potentially influenced by genetic expression\u2014could amplify psychological vulnerability or stress perception. In our conceptual model, we view psychosocial stress, inflammatory pathways, and neuro-hormonal disturbances as potentially interacting and reinforcing components. While our study is not designed to fully disentangle these causal directions, our analyses will explore how these variables may individually and collectively mediate the association between heat exposure and preterm birth. We will also consider how these relationships may evolve over the course of pregnancy and be modulated by environmental stressors such as heat. 1.8 \n<i>Thank you once again for inviting me to review the protocol<\/i> Thank you for your valuable feedback.","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-121\/v2#referee-comment-6129","order":1,"name":"referee-comment-6129","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Ijeoma Solarin<\/b>; \n<i>Posted: 27 May 2025<\/i>; Apologies, I tried to submit responses in a table format but the layout&nbsp;did not work. I am therefore reposting so the comments and responses display properly.&nbsp; Response to reviewer:&nbsp;\n<b>Ashish KC&nbsp;<\/b>University of Gothenburg, Gothenburg, Sweden 1.1.&nbsp;\n<i>Thank you for inviting me to review this interesting study protocol on biological pathway on heat exposure to preterm birth and other maternal and child health outcomes. This is a very valuable study to provide the epidemiological explanation on heat events to preterm birth. This study aim to investigate the environmental exposure to high ambient temperature on preterm birth and the causal pathway to exposure.<\/i> Author response:&nbsp;Thank you for these comments and careful review. 1.2.&nbsp;I have some comments on the hypothesis driven for the study - Non-optimal temperature either lower than 50th centile or higher than 50th centile has been associated to poor obstetric outcomes including preterm birth, still birth and neonatal mortality https:\/\/www.nature.com\/articles\/s41591-024-03245-7 and Ref:1 indicating that non- optimal ambient temperature exposure has a corelation between heat and preterm birth. It would be worth to provide a note to it. Author response:&nbsp;Thank you for this helpful comment and for sharing the relevant references. We agree that both high and low ambient temperatures have been associated with adverse obstetric outcomes, including preterm birth, stillbirth, and neonatal mortality. While the primary focus of our study is on high temperature exposures\u2014given their growing relevance in the context of anthropogenic climate change, the particular relevance in our study setting in sub-Saharan Africa and the urgent need to understand underlying biological mechanisms\u2014we recognise the importance of non-optimal low temperature exposure as well. While the references shared by the reviewer (Kc et al. (2024) and Hanson et al (2024)) underscore the importance of considering both cold and hot non-optimal temperatures, even these studies report that heat exposure tends to have a more consistent and pronounced association with adverse outcomes than cold exposure, especially in low-resources contexts. We have nevertheless updated the manuscript to acknowledge this broader evidence base and will consider it when interpreting our findings. Added text in section on Global health relevance: While both high and low ambient temperatures have been associated with poor obstetric outcomes (Kc et al., 2024, Nyadanu et al., 2024), this study focuses on heat exposure, which is of particular concern in our African context. 1.3 Even though there is a cumulative effect of non-optimal temperature to preterm birth, the lag effect i.e harvesting effect might be seen in preterm birth, can the researchers discuss about it Author response:&nbsp;Thank you for this comment. We appreciate the suggestion to consider a potential lag or \u201charvesting\u201d effect. As we understand it, the harvesting effect typically refers to temporal displacement of events that would have occurred soon regardless, and it is most often applied in the context of mortality, particularly among older or frail individuals. We are not certain that this concept directly translates to preterm birth, as a shift from term to preterm delivery due to heat exposure may represent a clinically meaningful outcome that could have been avoided rather than temporal displacement of an outcome that was likely to occur regardless. Nonetheless, we acknowledge that further discussion on lag structures and potential timing effects is warranted and will consider this in our interpretation of findings.&nbsp; 1.4&nbsp;In the abstract researchers mention that &quot;maternal inflammation will be tested by analysis the epigenetic changes associated with inflammatory cytokines protein and gene expression&quot;. Based on the studies from Genome wide association study and Mendenlian law, any genetic variant is always constant since conception, so the expression of cytokine is based on the dysfunction of the cytokine receptor dysfunction. https:\/\/doi.org\/10.3390\/ijms252011332. Author Response:&nbsp;Our approaches to examining heat exposure and inflammation includes assessing maternal transcriptomic and epigenetic signatures, using serial RNA-Seq data, to track up-and down-regulated genes before-and- after heat extremes. These are not Mendelian changes, but the differential gene expression and pathway analyses assessed using RNA Seq data. We can visualise the significantly differentiated genes between the different time points and see which genes are being expressed. We don\u2019t do GWAS or similar genetic testing in this study, as the reviewer points out. 1.5&nbsp;\n<i>further not all peripheral inflammation will cause the trigger preterm labour, until the inflammation cross the bloodbrain barrier, the inflammatory chain of reaction is required.\u2028Further, why is the focus on IL-6, there are a range of pro-inflammatory and anti-inflammatory cytokines which can cause BBB inflammation Ref:2<\/i> Author reponse:&nbsp;We appreciate your important point that not all peripheral inflammation will trigger PTB, and that specific inflammatory pathways, including those involving the blood-brain barrier, may play a role. However, our study is primarily focused on inflammatory pathways at the maternal-foetal interface (such as placenta, myometrium), which are more directly implicated in the initiation of labour than the BBB. We also acknowledge that IL-6 is a non-specific marker and that multiple cytokines, both pro- and anti-inflammatory, may contribute to the broader inflammatory response. Our focus on IL-6 is based on several considerations: 1). Evidence supporting its role in PTB; 2). its relative ease of measurement and low cost of testing; and 3). its widespread use in research, which facilitates comparison across studies. Nevertheless, we do plan on conducting a more comprehensive biomarker analysis using a panel of 96 inflammatory markers. This will allow us to investigate a range of cytokines implicated in both peripheral and central inflammation and inform more complex analysis. Biomarker analysis will be conducted using multivariate regression models to examine the association between heat exposure and inflammatory markers, adjusting for potential confounders. Inferential analysis of longitudinal data will include mixed-effects linear models. Much of this analysis is done using visualisations such as heatmaps and volcano plots. Bonferroni corrections will be applied to counteract potential multiple testing biases. We recognise that much remains unknown about the precise pathways - whether systemic, BBB, or at the level of the cervix and membranes - and which pathways are most important. This study will try address some of those questions. We have added reference to a recent review on heat and preterm birth Mushimiyimana 2025, which discusses this in more detail. 2.6&nbsp;\n<i>Sample size estimation- I still did not get the basis of the sample size based on IL-6, has there been a study to estimate IL-6 &gt;1000pg\/ml is linked with preterm birth.<\/i> Author response: Many thanks for this query, the point made here is valid. We should have noted the rationale for selecting Il-6 as the outcome of interest. IL-6 is one of the most widely studied inflammatory markers and indeed, several studies have linked Il-6 with preterm birth, as in the papers we now cite for this: (Prairie et al. 2021, Hatzidaki et al., 2005, Karampitsakos et al., 2025). In addition, IL-6 has been shown to be raised by heat (Maloney &amp; Duffy, 2024). We do however acknowledge that the originally specified IL-6 threshold of &gt;1000 pg\/mL was not appropriate for maternal serum. This was an oversight, and we appreciate the opportunity to correct it. We have revised this threshold in the manuscript to reflect more clinically and biologically relevant values for maternal serum which are much lower depending on presence of infection or specific population group being studied. While there is no universally accepted threshold for IL-6 in predicting PTB, and some variability exists across studies, we have revised our threshold to 10pg\/ml as a conservative measure based on other studies which identify maternal serum IL-6 levels around this value as predictive of preterm birth (e.g., Murtha et al., 1998) and which was our original intent. We also acknowledge that we have made some assumptions due to the scarcity of existing environmental exposure literature in this area. Nevertheless, our assumptions are based on&nbsp;conservative and pragmatic estimates to ensure sufficient power. One of our aims is to contribute new evidence on the potential relationship between environmental temperature and maternal systemic inflammation. We have clarified this in the revised manuscript. Updated text in sample size calculation: The primary inflammatory outcome interleukin-6 is strongly linked with PTB (Prairie et al., 2021, Hatzidaki et al., 2005, Karampitsakos et al., 2025). However, there is currently no universally accepted threshold for IL-6 in predicting PTB, with high levels of variability across studies. We have based our sample size on IL&gt;10pg\/ml as a conservative measure in keeping with other studies which identify maternal serum levels around this value as predictive of PTB (Murtha et al., 1998). 2.7&nbsp;In hypotheses, the researchers mention that psycho-social stressor is a mediator of preterm birth, but does it not also link with the increase in pro-inflammatory or neuro-hormonal disturbance. Can it be that there is a reversal phenomenon that due to increased pro-inflammatory genetic expression and disturbance in neuro-hormonal gene expression, that the social stress can change the pyschological response and thus preterm birth. Author response:&nbsp;Thank you for this thoughtful comment. We agree that psychosocial stress may not only act as a mediator but may also be shaped by underlying biological processes, such as pro-inflammatory and neuro-hormonal disturbances. We acknowledge the possibility of bidirectional, feedback loops or reverse relationships, whereby heightened inflammatory or neuroendocrine activity\u2014potentially influenced by genetic expression\u2014could amplify psychological vulnerability or stress perception. In our conceptual model, we view psychosocial stress, inflammatory pathways, and neuro-hormonal disturbances as potentially interacting and reinforcing components. While our study is not designed to fully disentangle these causal directions, our analyses will explore how these variables may individually and collectively mediate the association between heat exposure and preterm birth. We will also consider how these relationships may evolve over the course of pregnancy and be modulated by environmental stressors such as heat. 2.8&nbsp;\n<i>Thank you once again for inviting me to review the protocol<\/i> Author response:&nbsp;Thank you for your valuable feedback.","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-121\/v2#referee-comment-6130","order":2,"name":"referee-comment-6130","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.26054.r121795, Claudia Hanson, Karolinska Institutet, Stockholm, 24 Apr 2025, version 1, 1 approved, 1 approved with reservations","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-121\/v1#referee-response-121795","order":3,"name":"referee-response-121795","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Ijeoma Solarin<\/b>; \n<i>Posted: 27 May 2025<\/i>; Response to reviewer:&nbsp;\n<b>Claudia Hanson, Karolinska Institutet, Stockholm, Sweden<\/b> \n<b>2.1&nbsp;<\/b>\n<i>Thank you very much proving me with the opportunity to review this important protocol. The hypothesis is clearly stated and the sub-hypothesis as well. Understanding physiological mechanisms will be of major importance to develop the best interventions for mitigation.<\/i> \n<b>Author&nbsp;response:<\/b>&nbsp;Thank you for these comments. &nbsp;  \n<b>2.2<\/b>&nbsp;\n<i>I am missing a bit a consideration of why which measurements. For example, why which markers in pregnancy, cord blood (which measurement).<\/i> &nbsp; \n<b>Author response:&nbsp;<\/b>We thank the reviewer for this thoughtful comment. While we have described the individual measures used throughout the manuscript, we appreciate that we may have overlooked providing specific rational for selecting certain measurements. Our selection of measurements is driven by our primary and secondary hypotheses and in particular focusses on the plausible pathways that we describe in Figure 1. These include several hypothesised pathways linking heat exposure to preterm birth including immune activation, epigenetic changes, endocrine and stress response, dehydration, placental changes and maternal mental health. Measures such as inflammatory markers, RNA Sequencing and gene regulation may help to answer questions about the effect of heat on the immune system. Urine analysis, including osmolarity and urine specific gravity give us insights into the role of dehydration, while the mental wellbeing tools we use in combination with our qualitative sub-study may help us to understand the role of mental health in the pathways. We have now included a Supplementary Table (Appendix 1) that summarises each measurement, the physiological or psychological process it represents and its relevance to our study. We hope this addition enhances the clarity of our measurement rationale. We have also added the following sentence to the manuscript under the section on Global health relevance to reference the supplementary table. Added text: \n<i>A list of study measures, along with the rational for each, is provided in a supplemental appendix (Appendix 1:&nbsp;Overview of study measurements and rationale).<\/i> &nbsp;  \n<b>2.3<\/b>&nbsp;\n<i>Further, I am missing a consideration of pregnancy complications including pre- \/ eclampsia, vaginal infection other then group B-streptococcus. Also, will you include information of complications at birth?<\/i> \n<b>Author response:<\/b>&nbsp;Thank you for your comment. In the protocol, we specify that women will be clinically examined at each visit which includes taking blood pressure measurements, checking blood sugar levels and testing for infections, specifically HIV and syphilis. These procedures are designed to detect common antenatal complications including pre-eclampsia and gestational diabetes. We acknowledge that other infections, such as malaria or additional vaginal infections could be relevant however, investigating this goes beyond the scope of our study, and we are limited by available resources. Therefore, we do not plan on testing for other infections and acknowledge this a potential limitation. The relevant text in the manuscript is as follows: &nbsp; Updated text in section on Enrolment visit: \n<i>\u201cThe participant will be examined clinically, including blood pressure measurements, weight and height, and a general and system-specific examination to assess overall maternal health. A foetal ultrasound will also be conducted to confirm an intrauterine, viable pregnancy, the number of foetuses, and to confirm gestational age. If not already conducted at the antenatal clinic, blood will be drawn to test for renal function, thyroid function, HBA1c, a full blood count, and infections (HIV and syphilis).\u201d<\/i> &nbsp; We have also clarified that participants will be clinically examined at each antenatal and postnatal visit. Updated text in section on Antepartum study visit: \n<i>Participants will be clinically examined at each visit and blood samples will be taken to test for HIV and syphilis if not already done at the antenatal clinic.<\/i> Updated text in section on Postpartum study visits: \n<i>Study staff will clinically examine participants and collect measurements (e.g., height, weight, waist, hip, and mid-upper arm circumference) and vital signs.<\/i> Regarding complications at birth, we do plan to collect this information by extracting data &nbsp;from the maternity records. We hope we have made this clearer in the manuscript. Updated text in section on Intrapartum study visit: \n<i>Labour progress, mode of delivery, maternal and foetal morbidity, Apgar scores, foetal weight, length, and head circumference, and additional relevant information will be extracted from the maternity case records. Cardiotocography data will also be documented<\/i>. In addition, we hope that the addition of the supplementary table, which lists study measures, helps clarify the scope of data being collected. &nbsp;  \n<b>2.4&nbsp;<\/b>\n<i>The protocol is not well referenced, for example, there is no reference to the Amsterdam classification for placenta pathology. <\/i> &nbsp; \n<b>Author response:&nbsp;<\/b>Thank you for this comment. We have included a reference for the Amsterdam classification as suggested and additional references in several other places.&nbsp; &nbsp;  \n<b>2.5&nbsp;<\/b>\n<i>The quality assurance part does not go into sufficient details. You are collecting a large variety of variable, and different quality assurance mechanisms and SOPs will need to be applied. A similar concern is for the data processes and management. Probably a table on data management for the very different types of data may have been used. <\/i> &nbsp; \n<b>Author response:&nbsp;<\/b>Thank you for your comment. We agree that we are collecting a large variety of data which needs to be carefully managed. A key aspect of quality assurance is training and the availability of standard operating procedures (SOPs). &nbsp;All staff within our study undergo comprehensive training on all study procedures and re-training will be conducted on a regular basis as required. &nbsp;In addition to site specific SOPs, we have developed several study specific SOPs for specimen collection and sampling, device management, qualitative data management. &nbsp;While we do mention staff training under the section about participant safety, we have now expanded the quality assurance section to also include these details. Updated text is provided below. &nbsp; Updated text: \n<i>High quality standards will be reinforced through comprehensive training and retraining of staff as required, along with oversight of adherence to SOPs. Several study-specific SOPs will be developed, covering areas such as specimen collection, wearable device data collection and management and qualitative data handling.<\/i> &nbsp; Additionally, as part of our internal data management plan, we have a table which describes data collection methods and storage locations (Table 1). We have updated the data management section to include the following: \n<i>Data management in the study will follow rigorous protocols across all study components to uphold data accuracy, security, and confidentiality. A wide range of data types will be collected from multiple sources, including questionnaires, physical examinations, radiological procedures, laboratory measurements, and environmental exposure assessments. A detailed Data Management Plan will be developed to guide the handling of data throughout the study. The data types, collection methods and storage locations are summarised in Table 1 below.<\/i> Table 1: Description of data types, collection methods and storage location  \u200b\u200b\u200b\u200b\u200b\u200b\u200b \n<b>2.6<\/b>&nbsp;\n<i>I am also not clear about patient involvement. I am happy to hear about the involvement of the labour room staff in respect to cooling, but I am less clear about patient involvement in the design of the study, eg. patients may raise additional important factors which are not yet included, such as work and commuting.<\/i> &nbsp; \n<b>Author response:&nbsp;<\/b>Thank you for this important comment. While patients were not directly involved in the initial design of the study, we recognise the value of patient and community research partners. To support meaningful engagement and knowledge translation, we plan to work closely with an established Community Advisory Board (CAB) that the research site team has long collaborated with. The CAB is engaged from the start of the project and provide inputs on components of the study including, for example, the participant information sheets and other participant facing documents. We anticipate that their contributions to this research will ensure that the interpretation and dissemination of findings are grounded in the lived experiences of the communities we aim to serve. In addition, we will draw on insights gained through the qualitative aspect of our study. We have added more detail &nbsp;to the manuscript as follows: &nbsp; \n<i><b>Dissemination and public involvement<\/b><\/i> \n<i>We are committed to making the results of this research accessible to all relevant stakeholders, including the scientific community, study participants, the general public, healthcare providers, and policymakers. To support this, findings will be published in open-access, peer-reviewed journals and presented at scientific conferences and expert meetings to advance academic discourse and professional education, as well as public forums. To enhance policy and practice impact, we will actively engage other key stakeholders, including community leaders, advocacy groups, and professional organisations, to ensure the findings inform policy development, advocacy efforts, and intervention strategies at local, national, and international levels.<\/i> \n<i>To reach broader audiences, public communications, such as press releases issued through the University of the Witwatersrand and other media channels will be used to promote &nbsp;awareness and understanding of the findings. Study participants and local communities will receive simple, non-technical summaries of the findings. These will be translated into appropriate languages and disseminated in accessible formats, including leaflets, posters, and online content.<\/i> \n<i>We will work closely with the Community Advisory Board (CAB), who the research site team has long collaborated with. The CAB will be engaged from the start of the project and provide input on participant information sheets and other participant facing documents. At the dissemination stage, we will collaborate with the CAB to ensure the interpretation and dissemination of findings are grounded in the lived experiences of the communities we aim to serve.<\/i>","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-121\/v2#referee-comment-6131","order":4,"name":"referee-comment-6131","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.26873.r126576, Santosh Pandipati, Obstetrix of San Jose, e-L\u014dvu Health, e-L\u014dvu Health, 30 Jun 2025, version 2, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-121\/v2#referee-response-126576","order":5,"name":"referee-response-126576","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This work was supported by Wellcome [227204].\r\nThe funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":6,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}