NIT_CH: A Study Protocol for Evaluating the Effect of Inorganic Nitrate Capsules in Chronic Hypertensive Pregnancies – A Triple-Blind Placebo-Controlled Randomized Trial

NIT_CH: A Study Protocol for Evaluating the Effect of Inorganic Nitrate Capsules in Chronic Hypertensive Pregnancies – A Triple-Blind Placebo-Controlled Randomized Trial | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Study protocol NIT_CH: A Study Protocol for Evaluating the Effect of Inorganic Nitrate Capsules in Chronic Hypertensive Pregnancies – A Triple-Blind Placebo-Controlled Randomized Trial Priscila Barbosa, Vinicius Aniceto, Luiz Sergio Lima-Junior, Aline Costa, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8157989/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Jan, 2026 Read the published version in BMC Pregnancy and Childbirth → Version 1 posted 4 You are reading this latest preprint version Abstract Background Hypertensive disorders of pregnancy represent a substantial risk to maternal and fetal health, with increasing prevalence worldwide linked to factors such as advanced maternal age, rising obesity rates, and metabolic syndrome. Effective management of borderline hypertension during pregnancy remains critical. However, given the limited safety data on conventional antihypertensive drugs in this population, alternative therapeutic strategies are needed. Inorganic nitrate supplementation has demonstrated potential in reducing blood pressure and enhancing maternal uterine artery function, primarily due to the production of nitric oxide from nitrate. We will evaluate whether early initiation and sustained supplementation with inorganic nitrate capsules can control blood pressure and improve maternal and perinatal outcomes in women with chronic hypertension during pregnancy. Methods This randomized, triple-blind, placebo-controlled clinical trial intends to recruit an estimated sample size of 144 pregnant women with chronic hypertension. Once included, participants will receive standard antihypertensive treatment and preeclampsia prophylaxis and will be randomized into either the treatment or placebo group. Starting from the 16th week and continuing until delivery, patients will undergo continuous monitoring of blood pressure, maternal and fetal ultrasonography, as well as biochemical and dietary assessments. The primary aim of the study is to evaluate blood pressure control. At the same time, secondary objectives include monitoring the need for additional antihypertensive medications, the occurrence of preeclampsia and fetal growth restriction, Doppler parameters, biochemical and inflammatory markers profiles, and perinatal outcomes. Data analyses will be conducted using both R and Stata software tools for descriptive and analytic statistics, with a significance level of p < 0.05. Discussion Evaluating the efficacy of inorganic nitrate as an adjunctive treatment for chronic hypertension during pregnancy could provide an alternative to conventional antihypertensive therapies. Successful results may offer a safer and natural option to help manage blood pressure, improve outcomes for both mothers and infants, and potentially guide the development of new strategies for managing hypertensive disorders in pregnant patients. Trial registration: ClinicalTrials.gov NCT06105775 (Registration Date: October 27, 2023 – updated on March 26, 2025). chronic hypertension inorganic nitrate nitric oxide pregnancy preeclampsia Background Hypertensive disorders of pregnancy (HDP) have a global prevalence of approximately 5% to 8% in pregnant women and significantly contribute to adverse maternal and fetal complications ( 1 ). Chronic arterial hypertension affects roughly 2 to 5% of pregnant women and has increasing in recent years ( 2 ). The prevalence of pregnancies complicated by chronic hypertension is rising, likely attributed to factors such as increasing maternal age, obesity rates, and metabolic syndrome ( 3 ). Pregnant women diagnosed with chronic hypertension often pose a reduced likelihood of experiencing the physiological cardiovascular adaptations in the first half of pregnancy, which play a vital role in the process of uteroplacental adaptation and fetal development, including the decrease in peripheral vascular resistance and the subsequent drop in blood pressure ( 4 ). The results of the CHIPS trial (Control of Hypertension in Pregnancy Study) demonstrated the significance of blood pressure management in pregnant women with borderline hypertension to prevent the development of severe hypertension and the associated maternal and perinatal complications ( 5 ). Due to the need for maternal-fetal safety data, the exploration of new antihypertensive therapies is constrained, as is the utilization of the current therapeutic options. Inorganic nitrate and beetroot juice supplementation are being recognized as a safe and well-tolerated dietary supplement ( 6 , 7 ). Their mechanism of action is associated with the production of nitric oxide (NO), an endogenous vasodilator, from the bioconversion of nitrate ( 7 , 8 ). The alternative production of NO operates through the recycling of nitrite (NO 2 − ) and nitrate (NO 3 − ) anions, following a pathway that differs from the classical L-arginine-NO-synthase pathway. Furthermore, for efficient enzymatic conversion of nitrate to nitrite, a specific group of nitrate reductase enzymes is required, present in specific bacterial populations in the mouth and intestine of mammals, in addition to the acidic stomach environment. Despite these requirements, inorganic nitrate and beetroot juice supplementation offer the advantage of a wide therapeutic range, allowing the use of high dosages without significant risks of toxicity ( 9 , 10 ). The utilization of inorganic nitrate to increase NO bioavailability can be advantageous, especially in the presence of hypoxia and hypertension ( 11 ). Preliminary data from studies using a mouse model of pregnancy-related hypertension have shown that a 6-day supplementation with nitrate-rich beetroot juice led to a reduction in blood pressure and an enhancement in the endothelial function of the maternal uterine artery ( 12 ). In healthy volunteers, a 3-day dietary supplementation with sodium nitrate resulted in a significant reduction in diastolic pressure by 3.7 mmHg compared to a placebo (sodium chloride) ( 13 ). A study investigating inorganic nitrate supplementation in the form of beetroot juice for pregnant women in the third trimester (from 28 weeks onwards) over 8 days demonstrated a significant increase in plasmatic and salivary nitrate and nitrite concentrations in the treated group when compared to the placebo, with peak concentrations reached approximately 2–4 hours after the juice’s ingestion ( 14 ). Although the study did not establish a reduction in blood pressure in the treated group, there was a correlation between the increase in plasma nitrite concentrations and the decrease in diastolic blood pressure within this group. In another study conducted by the same group, significant reductions in both systolic and diastolic blood pressure were observed in women with hypertension 2.5 hours after consuming nitrate-rich beetroot juice ( 15 ). The authors also discussed the potential influence of oral microbiota on nitrate metabolism and blood pressure response. Therefore, further studies on the long-term use of inorganic nitrate are needed to provide a more comprehensive investigation that might fully assess the potential benefits and mechanisms of action of nitrate supplementation in pregnant patients. The primary aim of this study is to evaluate blood pressure control in pregnant women with chronic hypertension who will be supplemented with inorganic nitrate in comparison to a placebo group. Both groups will receive standard antihypertensive treatment and prophylaxis for preeclampsia, with one of the secondary objectives being the monitoring of additional antihypertensive medications need. Other secondary aims encompass the evaluation of maternal and fetal Doppler analyses, fetal growth, overlapping incidence of superimposed preeclampsia and adverse outcomes, umbilical artery vascular reactivity, hematological and metabolic assessments, concentrations of nitric oxide metabolites, markers of preeclampsia, inflammation and oxidative stress, and the analysis of the relationship between dietary indices and markers of oxidative and inflammatory stress. We hypothesize that supplementation with 400 mg of inorganic nitrate, initiated at the beginning of the second trimester (16th week) and continued over a long period during the pregnancy (22 weeks), may yield promising results in blood pressure control, associated to an enhancement in uteroplacental flow, adequate fetal growth and preserving fetal vitality, improving obstetric results. Methods Trial Design This study is a randomized, placebo-controlled, triple-blind, parallel-group clinical trial. Pregnant women with chronic hypertension will be randomly assigned to the intervention group or the placebo, in blocks of four to ensure balance between the groups. Study Setting The study is a single-site trial conducted at the Clinical Research Unit, a dedicated facility for clinical research linked to the Hospital das Clínicas de Ribeirão Preto, located in Ribeirão Preto, Brazil. Participants will attend the Clinical Research Unit according to the visit schedule outlined in the study protocol, preferably in the morning. All study procedures, including enrolment, disposal of supplementation, ultrasonography, and blood collection, will be performed at the Clinical Research Unit by trained members of the research and clinical team of the study, as well as nurses and pharmacists employed by the Clinical Research Unit. Deliveries will take place at the Hospital das Clínicas de Ribeirão Preto and will be managed by obstetricians affiliated with the hospital. Trial Status The NIT_CH study received funding in 2021 from the Foundation of the state of São Paulo (FAPESP). The Research Ethics Committee of the Hospital das Clínicas, Ribeirão Preto Medical School – São Paulo University, was granted on September 19, 2024, with the last updated approval on February 26, 2025. This paper reflects Protocol Version 3.0. Recruitment began in September 2024, and data collection is ongoing. The first results are expected to be submitted for publication in December 2026. Recruitment Eligible pregnant women with chronic hypertension will be identified and invited to participate through targeted outreach, including posters and direct referrals from healthcare professionals within the municipal health network. To achieve the target of enrolling 144 pregnant women, an average recruitment rate of 6 women per month will be required. Recruitment progress will be continuously monitored, and if necessary, additional measures such as meetings with the municipal health network and active on-site recruitment will be implemented. Participants Inclusion Criteria Pregnant women diagnosed with chronic hypertension as defined by the Brazilian Network for Studies on Hypertension in Pregnancy/Preeclampsia and by the Brazilian Federation of Gynecology and Obstetrics Associations Protocol: Systolic Blood Pressure (SBP) above 140 mmHg and/or Diastolic Blood Pressure (DBP) above 90 mmHg prior to pregnancy or before 20 weeks of gestation. Currently using monotherapy with methyldopa (first-line antihypertensive drug used during pregnancy in Brazil) at the time of inclusion. Below 16 weeks of gestation, confirmed by first-trimester ultrasound. Exclusion Criteria Multiple pregnancies, below 18 years of age, inability to provide informed consent, or a history of poor medication adherence. Patients with uncontrolled chronic hypertension, with pressure measurements above 160x110mmHg and/or using other antihypertensives in addition to methyldopa at the moment of inclusion. Users of illicit drugs, smokers, or alcohol abusers. Patients diagnosed with previous bariatric surgery, coronary artery disease, moderate to severe congestive heart failure, moderate to severe liver failure, chronic kidney failure (with creatinine clearance less than 30 ml/min/1.73 m 2 body surface area), and pre-existing type 1 and type 2 diabetes. Frequent users of non-steroidal anti-inflammatory drugs, nasal decongestants, and anorectics before pregnancy; users of proton pump inhibitors, H2 receptor antagonists, or any other medication that affects stomach pH, starting 2 weeks before and maintained throughout the study. Sample Size An analysis of robustness was conducted to determine the required sample size based on Ambulatory Blood Pressure Monitoring data. To detect a significant difference of 5.0 mmHg in arterial pressure between groups (with a standard deviation of 10 mmHg), a total of 64 pregnant women per group is required to ensure 80% power at 0.05 significance level. Accounting for a potential 12% dropout rate or treatment abandonment, the sample size was adjusted to 72 pregnant women per group, resulting in a total of 144 participants. The sample size was computed using the following formula: \(\:\varvec{n}\varvec{P}=\left(\frac{{\left(\frac{\varvec{Z}\varvec{\alpha\:}}{2}+\varvec{Z}\varvec{\beta\:}\right)}^{2}\times\:\:\varvec{s}.\varvec{d}}{\varvec{Ḏ}}\right)²\) Randomization and Masking Participants will be randomized using a computer-generated sequence in blocks of four to ensure balance between the groups. The on-site pharmacist, who is not involved in the study’s execution, will assign participants based on this sequence. An external pharmacist from an independent company will manufacture both the intervention and placebo capsules. The on-site pharmacist will label each bottle with the participant's name and a unique identification number corresponding to the assigned group, ensuring allocation concealment from the research team, participants, and statistician until the trial’s conclusion. Only the on-site and external pharmacists will have access to the allocation sequence, maintaining the integrity of blinding. At visit 1, the UPC pharmacist will dispense a sufficient quantity of capsules to last until the next visit, including an additional of 7–10 capsules to account for potential delays between visits or emergency use. The research and medical team involved in the study will not know the group assigned to the participant. The trial will employ a triple-blind design, where participants, the research and medical team, and data analysts will remain blinded to treatment allocation from the point of intervention assignment until the completion of the clinical trial and final data analysis. In case of a medical emergency requiring unblinding, a co-principal investigator will have access to the allocation group through a formal request submitted directly to the on-site pharmacist. Interventions Women referred from primary care who meet the inclusion criteria will be contacted by the research team and invited to an appointment at the Clinical Research Unit. During this appointment, the informed consent form will be presented, and all questions will be addressed by members of the research team, who have been trained in the study protocol. Participants will then be asked to review and sign the informed consent form before any study procedures are initiated. Participants will be enrolled by the research team upon obtaining the written informed consent form. From the day of enrollment to the next appointment, the pharmacist will randomize the participants to the intervention or placebo. Placebo is selected as a comparator to provide a comparison point. In the intervention arm, participants will receive sodium nitrate, which includes 400 mg of nitrate and 140 mg of sodium (totaling 540 mg of sodium nitrate). This amount has proven to be safe, as reported in other studies ( 14 , 15 ). The placebo group will be produced using sodium chloride with the same amount of sodium present in nitrate salt (140 mg, which represents 7% of the daily recommendation of sodium). All participants will be instructed to take one capsule each morning after breakfast, continuing this regimen for 22 weeks (approximately 154 days), ending either on the 38th week of pregnancy or childbirth, whichever occurs first. In both cases (intervention and placebo), the usual medications for preeclampsia prophylaxis will be maintained (acetylsalicylic acid 100 mg at night daily, up to 36 weeks of gestation and calcium carbonate 1000 mg per day, until delivery) and usual antihypertensive treatment. The dosage of acetylsalicylic acid specified in the trial (100 mg) represents the available and commonly used presentation in Brazil. The intervention may be discontinued based on the clinical judgment of the medical team in cases of adverse events or upon the participant’s request at any time. To ensure adherence, patients will be provided with additional capsules and asked to return any unused capsules at each visit to verify adherence to the supplementation. Additionally, researchers will apply the Morisky-Green questionnaire to evaluate adherence ( 16 ). Participants will be advised to avoid the use of mouthwash during the study period, as it may eliminate oral microbiota that is crucial for the conversion of nitrate to nitrite, a critical step in the intervention's mechanism of action. Study Protocol and Timeline After the enrolment, participants will be randomized, and another appointment will be scheduled for the 16 + 0+6 weeks of pregnancy with a member of the research team (Visit 1). Participants will be required to arrive fasting for 8 hours for sample collection. The participants will undergo an obstetric and clinical exam, blood pressure measurement, and an obstetric ultrasound, complemented by a Doppler study of the uterine arteries. Blood samples, urine and saliva will be collected for further analysis. For the subsequent visits – Visit 2 through Visit 9 – participants will periodically return to the Clinical Research Unit. Each follow-up visit will include a clinical exam, blood pressure measurement, blood sample, saliva and urine collection, obstetric consultation, and ultrasounds. The Doppler study of uterine arteries will be conducted at each visit, while the Doppler study of the middle cerebral artery and the umbilical artery will begin at 24 weeks of pregnancy and continue at each subsequent visit. The medical team will consistently monitor participants' antihypertensive medications, making necessary adjustments to maintain optimal blood pressure control. Each visit is estimated to last about 30 minutes to 1 hour. The overall expected study duration per participant spans either 154 days or up to the point of childbirth, whichever occurs first. To offset potential travel expenses to the clinic, participants will receive a reimbursement of forty Brazilian Real (R $ 40.00) per visit. The participant timeline as well as the events of the protocol can be found in Table 1. Adverse event reporting and harms We do not anticipate significant side effects from the use of inorganic nitrate, as previous studies in pregnant women have shown no evidence of adverse maternal or fetal risks. However, participants will be closely monitored for any potential adverse events. Any symptoms, diseases, or side effects, whether or not related to the intervention, will be documented, and participants will receive appropriate medical care as needed. All adverse events will be reported and registered in accordance with local regulations and communicated to relevant authorities. Outcomes Primary Outcome The primary outcome, blood pressure control, will be assessed through frequent blood pressure measurements using a standardized method. The measurements will be taken using an automated blood pressure monitor. Simultaneously, the frequency and magnitude of dosage adjustments in antihypertensive medications across both groups during the study period will be closely monitored and documented. Any changes in medication dosage will be recorded to provide a comprehensive evaluation of the primary outcome. Secondary Outcomes Fetal Growth: Fetal growth will be assessed through periodic ultrasound examinations. Measurements of head circumference, abdominal circumference, and femur length will be obtained using standard ultrasound techniques by experienced physicians to estimate the fetal weight using Hadlock’s formula. Preeclampsia and Adverse Maternal Outcomes: The incidence and severity of superimposed preeclampsia and other adverse maternal outcomes will be determined through clinical assessments and regular monitoring of symptoms. Diagnostic criteria for preeclampsia will follow established medical guidelines. Perinatal outcomes: Birthweight and Apgar scores at 1 and 5 minutes will be recorded immediately after delivery by healthcare professionals. Blood Profile Analysis: Blood profile measurements will be conducted through laboratory tests. Hemogram, lipid profile, liver and kidney functionalities, glucose levels, and fasting insulin will be determined using standard laboratory techniques. Nitrate and Nitrite Concentrations: The concentration of nitrate and nitrite in plasma, urine, and saliva will be measured using validated methodology. Specific Serum Indicators of Preeclampsia: Specific serum indicators of preeclampsia (sFLT-1 and PlGF) will be measured through laboratory tests. Inflammatory markers: The levels of inflammatory markers, including adiponectin, ICAM-1, IL-6, IL-8, IL-10, leptin, resistin, TNF-α, VCAM-1, VEGF, and visfatin, will be determined through laboratory assays. Oxidative Stress: Lipid peroxidation levels and total antioxidant capacity of plasma will be assessed using established colorimetric methods. Matrix metalloproteinase: The concentrations of matrix metalloproteinase (MMPs 2 and 9) and their inhibitors (TIMPs 1, 2, 3, and 4) will be quantified through laboratory analyses. Dietary Pattern: Dietary patterns will be assessed using a dietary marker recommended by Brazil’s Ministry of Health (SISVAN). Biological Specimens Blood (plasma and serum), saliva, urine, and umbilical cord samples will be securely stored at -80℃ and will be used exclusively for the analysis outlined in the research protocol and described in the participant consent form. Ancillary and Post-Trial Care There are no specific provisions for post-trial care. However, in the event of any harm resulting directly from participation in the trial, participants will receive all necessary medical care to address the condition until it is resolved or significantly improved. Compensation for trial-related injuries will be provided in accordance with applicable Brazilian ethical guidelines and regulatory requirements. Statistical Methods Initially, the quantitative data obtained in this study will be analyzed using a descriptive statistical approach. Quantitative variables will be presented using central position (mean and median) and dispersion measures (standard deviation, interquartile range, minimum, and maximum values), which will be visualized using box-plot graphs and histograms. Absolute and relative frequencies will be used to summarize all qualitative variables. Socio-demographic variables and other baseline characteristics will undergo statistical analysis based on data distribution. The intervention and placebo groups will be compared using the Student’s t-test or the Wilcoxon test, depending on whether the data are parametric or non-parametric, respectively. Categorical variable analysis will start by calculating absolute and relative frequencies for each category, followed by proportion comparisons using chi-squared or Fisher’s exact tests. A linear regression model with mixed effects will be used to assess the treatment effect over time, comparing groups using orthogonal contrasts and examining model fitting through a residual analysis. There will be per-protocol and intention-to-treat analyses. Because of the study’s prospective design, which includes patient monitoring until delivery, the occurrence of clinical outcomes like preeclampsia, fetal growth restriction, and hemodynamic centralization will be depicted using Kaplan-Meier curves in each group. Cox regression will be used to compare the occurrence of outcomes between groups. Correlation between dietary indices and oxidative stress variables, and inflammatory markers will be shown in a heatmap. For variable pairings, a correlation measure, such as the Pearson or Spearman correlation coefficient, will be produced after organizing data in a matrix format. The heatmap created with these coefficients will show varied color intensities to represent different levels of correlation, providing an intuitive depiction of changeable inter-relationships. Food pattern categories will be identified using exploratory factor analysis. A correlation matrix will be used to analyze the correlation between meal pattern variables after they have been chosen based on theoretical relevance and data availability, followed by an exploratory factor analysis using the principal component analysis technique. All statistical analyses will use a p < 0.05 significance level. Data analyses will be conducted using both R and Stata software tools. Any missing data will be reported, and if more than 5% of primary outcome data is missing, a sensitivity analysis will be performed using multiple imputation techniques to handle the missing data, assuming the missingness is unrelated to the outcome. Methods for additional analyses Additional analyses will include a subgroup analysis based on responsiveness and non-responsiveness, as well as stratification by BMI. Responders will be defined as women in the intervention group who did not develop preeclampsia, while non-responders will be those in the same group who developed preeclampsia. Baseline characteristics will be compared between responders and non-responders using Student’s t-test or the Wilcoxon test for continuous variables, and the chi-square or Fisher’s exact test for categorical variables. Differences in primary and secondary outcomes between these subgroups will be assessed using regression models adjusted for potential confounders. For the BMI-based analysis, participants will be categorized into different BMI groups, and outcome measures will be compared across these groups to evaluate whether BMI influences treatment response. Interaction terms in regression models will be used to assess the moderating effect of BMI on study outcomes. Additionally, logistic regression will be employed to identify predictors of treatment response, while Cox regression models stratified by BMI will be used to evaluate differences in the time to clinical events, such as preeclampsia and fetal growth restriction. Interim analyses An interim analysis is expected to be carried out when one-third of the sample size is enrolled. This analysis will focus mainly on adherence and reported adverse events, but also on the assessment of the primary outcome. If the intervention proves to be unsafe (which is clearly not expected to be) or ineffective based on this analysis, a premature ending of the trial is planned to avoid further potential damage to the patients, and money spent. Ethics and Dissemination Data collection will be conducted at the Clinical Research Unit throughout the study period. The data collected will include numeric data (weight, blood pressure, biochemical test values, laboratory tests, and measurements obtained from fetal ultrasound), textual data (dietary intake records, medical and family history), categorical data (marital status, type of residence, profession/occupation, and ethnicity), and images and medical reports. Demographic data, dietary intake, and information on medical and family history will be collected at enrolment only. Additional data will be updated throughout the intervention period based on new measurements and assessments obtained during scheduled visits. Data collection will include questionnaires, clinical and laboratory evaluations, and gynecological examinations performed at each visit. No major adverse effects are anticipated with the proposed intervention. However, any potential side effects will be documented, and participants will receive individualized and appropriate medical care as needed. All participants will provide written informed consent prior to enrollment. The study protocol has been reviewed and approved by the Ethics Review Board of the Ribeirão Preto's Clinical Hospital (approval number 7.390.821). Confidentiality Data will be encrypted with a unique identity in a password-protected local database. To ensure patient confidentiality, each participant will be assigned a unique identification code, distinct from any personal identifiers. This code will be used in all research documentation and data entry processes. The primary identifier permitting patient traceability, the medical record number from the Hospital das Clínicas de Ribeirão Preto system, will be utilized only during clinical visits to document care provided by health professionals. In the research database, this identifier will be linked to a numerical code representing the patient. Ultrasonographic data, such as arterial reactivity, fetal weight, and others, along with laboratory test results, will be entered into the research database as numerical data, devoid of any personal identifiers. Amended to the protocol Any changes to the study protocol that may affect its execution, potential participant benefits, or patient safety will necessitate a formal amendment. This includes alterations to the study’s objectives, design, or procedures. These amendments must be submitted and approved by the Ethics Review Board of the Ribeirão Preto's Clinical Hospital before implementation. Data management All data obtained from the analysis and medical records will be recorded and stored in the REDCap software, leveraging its cloud storage capability and robust database support. A specific study form was created by a research team member in collaboration with the REDCap software coordinator at the University of São Paulo. To ensure data quality, all research team members will be trained to use the REDCap software. The system will enforce data integrity by locking fields after entry. To prevent misclassification, numerical and categorical variables will be clearly defined and input into their respective field types, with validation checks in place to further enhance accuracy. Furthermore, in alignment with Open Science principles, de-identified raw research data (excluding any patient-traceable information) will be made publicly available in the University of São Paulo’s online repository for a period of 10 years. A data management plan has been created and registered using the DMPTool ( 17 ), outlining the procedures for data collection, storage, and sharing throughout the project. Dissemination plans We will disseminate our results through multiple channels to ensure broad accessibility and impact. We will publish our findings in an international open-access journal, ensuring free global accessibility to our data for researchers and the general public. Additionally, we will be presenting our findings at both local and international conferences to stimulate scholarly discussion and foster collaborative research opportunities. To translate our research into actionable health guidelines, we will engage with policymakers through briefings, meetings, and workshops. This engagement is essential for the practical application of our findings in health policy and practice. Public awareness is also important; therefore, we will translate our research into accessible formats for public talks, community workshops, and collaboration with media outlets to reach a wider audience. Composition of the coordinating centre and trial steering committee The research and medical team will hold regular meetings to discuss the study’s progression, recruitment status, and any necessary amendments to the protocol. The principal investigator will be responsible for managing data monitoring and maintaining communication with participants. As this study will be conducted at the university level, there will be no formal Trial Steering Committee. The principal investigator will coordinate the study site, ensuring the overall management and execution of the trial. However, regular updates may be requested by the Research Clinical Unity team to ensure the study is being conducted in accordance with ethical and regulatory standards. Discussion This study protocol addresses a significant gap in research on the long-term efficacy of inorganic nitrate as an adjuvant therapy for chronic hypertension in pregnancy, particularly for blood pressure control. The NIT_CH trial is specifically designed to evaluate the therapeutic potential of inorganic nitrate in this high-risk population. Chronic hypertension during pregnancy is a challenging medical condition that significantly impacts both maternal and fetal health ( 18 ). One of the main challenges in managing chronic hypertension in pregnancy is controlling blood pressure while minimizing the need for multiple antihypertensive medications or increasing their dosages, as these drugs have safety and efficacy limitations during pregnancy ( 19 ). Moreover, superimposed preeclampsia is a common complication in pregnant women with chronic hypertension. Although aspirin is used as a first-line treatment, its effectiveness in preventing preeclampsia appears to be lower in this population ( 20 ). Inorganic nitrate has emerged as a promising alternative due to its vasodilatory, antioxidant, and antihypertensive properties, which are particularly beneficial with prolonged use ( 21 ). Furthermore, its low cost could offer a significant advantage in the long-term management of chronic hypertension. We hope, with the present study, to avoid the need for additional anti-hypertensive medications and any increase in the number of cases progressing to preeclampsia. The rationale behind conducting this protocol study is based on prior research in both animal and human models ( 9 , 12 , 14 , 15 , 22 ), which showed that inorganic nitrate may help reduce blood pressure and improve vascular function. Assessing the use of nitrate in pregnant women with chronic hypertension requires a careful analysis of safety and efficacy, particularly given the unique aspects of pregnancy. In this sense, the research team involved in this study has extensive experience in the field of hypertension disorders in pregnancy ( 23 – 27 ). The researchers’ wealth of experience plays a pivotal role in ensuring the success and reliability of the study, emphasizing the importance of having a dedicated and proficient research group conducting this type of investigation. To the best of our knowledge, this is the first randomized controlled trial assessing inorganic nitrate from the second trimester of pregnancy until delivery in pregnant women diagnosed with chronic hypertension. A key strength of the study is the sustained and multifaceted assessment, providing a comprehensive understanding of the effects of inorganic nitrate over an extended period. Although most studies evaluating inorganic nitrate during pregnancy have used beetroot juice, we understand that our long-duration protocol makes supplementation with juice impractical due to the taste aversions common in pregnancy and the inherent difficulty in finding a matched placebo. Therefore, the use of inorganic nitrate in the form of salt was chosen by our team as a more feasible and cost-effective option. To achieve a dose of 400 mg of nitrate, the corresponding amount of sodium in sodium nitrate is 140 mg. Thus, to control this quantity, the placebo will be formulated with an equivalent amount of sodium chloride (140 mg, or 7% of the dietary sodium requirement), thereby isolating the therapeutic effect of the nitrate anion. We strongly believe that the inclusion of 140 mg of sodium chloride in the placebo is unlikely to be problematic for pregnant women with chronic hypertension, given its small contribution to total sodium intake. Moreover, the women will be monitored during all period of pregnancy, and if the intake of the placebo represents to be harmful for this population, the study will be suspended. The NIT_CH project represents a significant endeavor aimed at assessing the therapeutic potential of inorganic nitrate in pregnant women with chronic hypertension, thus filling a critical gap in clinical research. We expect that the results obtained in this study may contribute to advance knowledge and potentially foster the development of more effective therapies during pregnancy, thereby benefiting maternal and fetal health. Abbreviations CHIPS Control of Hypertension in Pregnancy Study DBP diastolic blood pressure DMPTool Data Management Plan Tool ICAM-1 Intracellular Adhesion Molecule 1 IL-6 Interleukin-6 IL-8 Interleukin-8 IL-10 Interleukin-10 HDP Hypertensive Disorders of Pregnancy MMPs metalloproteinases NO nitric oxide PlGF Placenta Growth Factor SBP Systolic Blood Pressure sFLT-1 Soluble Fms-like Tyrosine kinase-1 SISVAN Sistema de Vigilância Alimentar e Nutricional TIMPs Tissue Inhibitors of Metalloproteinases TNF-α Tumor Necrosis Factor alpha VCAM-1 Vascular Cell Adhesion Molecule 1 VEFG Vascular Endothelial Growth Factor. Declarations Ethics approval and consent to participate Ethical approval for this study protocol was granted in August 2024 by the Research Ethics Committee of Hospital das Clínicas, Ribeirão Preto Medical School – São Paulo University (approval number 7.390.821). Written informed consent will be obtained from all participants. A trained researcher will introduce the study to potential participants and provide a comprehensive explanation of the trial’s objectives and procedures. Participants will receive an information document outlining the study details and will have the opportunity to discuss any questions or concerns with the researcher. The informed consent process will be thoroughly documented, with consent forms securely stored for auditing purposes. Consent for publication Not applicable. Availability of data and materials The data collected for the study will be made available as a de-identified dataset on the Open Science Framework (OSF) and the São Paulo University repository after the final clinical study report. Access to the data will be facilitated according to the study's data management plan (17). Competing interests The authors declare that they have no competing interests. Funding This clinical trial is embedded in a project named “Endothelial Dysfunction in Hypertensive Disorders of Pregnancy”, grant number: 2021/12010-7, São Paulo Research Foundation – FAPESP. Priscila O. Barbosa is funded by FAPESP (grant number: 2023/07589-1). Additional support is provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Grant number: 308504/2021-6 and 302614/2025-7). FAPESP is a state agency, and CNPq is a federal funding agency. Neither had any role in the design of the study, data collection, management, analysis, or interpretation. The research team maintains full independence in all aspects of the study. Authors' contributions Study concept and design: V.C.S. and R.C.C. Trial management: P.O.B and R.C.C. Ethics application and acquisition of data: L.S.L.J., P.O.B., R.C.C. and V.A. Drafting of the manuscript: A.S.C., P.OB. and V.A. Critical Revision: L.S.L.J. and J.E.T.S. Project administration and Resources: R.C.C. and V.C.S. All authors contributed to refinement of the study protocol and approved the final manuscript. Acknowledgements We would like to acknowledge the following contributors to this study: Suleimy C Mazin, for her help with the data analysis plan and all statistical aspects; Ricardo Perussi e Silva, for his assistance with the database on REDCap; and Professor Jon Lundberg, from the Karolinska Institute, for his guidance and expertise on inorganic nitrate administration. References Report of the National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy. Am J Obstet Gynecol. 2000 July;183(1):S1–22. Leonard SA, Siadat S, Main EK, Huybrechts KF, El-Sayed YY, Hlatky MA, et al. Chronic Hypertension During Pregnancy: Prevalence and Treatment in the United States, 2008–2021. Hypertension. 2024;81(8):1716–23. Kent L, McGirr M, Eastwood KA. Global trends in prevalence of maternal overweight and obesity: A systematic review and meta-analysis of routinely collected data retrospective cohorts. Int J Popul Data Sci. 2024;9(2):2401. Poon LC, Shennan A, Hyett JA, Kapur A, Hadar E, Divakar H, et al. The International Federation of Gynecology and Obstetrics (FIGO) initiative on pre-eclampsia: A pragmatic guide for first-trimester screening and prevention. Int J Gynaecol Obstet. 2019;145(Suppl 1Suppl 1):1–33. Magee LA, von Dadelszen P, Rey E, Ross S, Asztalos E, Murphy KE, et al. Less-tight versus tight control of hypertension in pregnancy. N Engl J Med. 2015;372(5):407–17. Lara J, Ashor AW, Oggioni C, Ahluwalia A, Mathers JC, Siervo M. Effects of inorganic nitrate and beetroot supplementation on endothelial function: a systematic review and meta-analysis. Eur J Nutr. 2016;55(2):451–9. Barbosa PO, Tanus-Santos JE, Cavalli R, de Bengtsson C, Montenegro T, Sandrim MF. The Nitrate-Nitrite-Nitric Oxide Pathway: Potential Role in Mitigating Oxidative Stress in Hypertensive Disorders of Pregnancy. Nutrients. 2024;16(10):1475. Lundberg JO, Weitzberg E, Gladwin MT. The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov. 2008;7(2):156–67. Kapil V, Milsom AB, Okorie M, Maleki-Toyserkani S, Akram F, Rehman F, et al. Inorganic nitrate supplementation lowers blood pressure in humans: role for nitrite-derived NO. Hypertension. 2010;56(2):274–81. Ml S, Fj L, M C, Ml MBJP et al. H,. A randomized clinical trial of the effects of leafy green vegetables and inorganic nitrate on blood pressure. The American journal of clinical nutrition [Internet]. 2020 Jan 4 [cited 2025 Nov 19];111(4). Available from: https://pubmed.ncbi.nlm.nih.gov/32091599/ Weitzberg E, Lundberg JO. Novel aspects of dietary nitrate and human health. Annu Rev Nutr. 2013;33:129–59. Tropea T, Renshall LJ, Nihlen C, Weitzberg E, Lundberg JO, David AL, et al. Beetroot juice lowers blood pressure and improves endothelial function in pregnant eNOS-/- mice: importance of nitrate-independent effects. J Physiol. 2020 Sept;598(18):4079–92. Larsen FJ, Ekblom B, Sahlin K, Lundberg JO, Weitzberg E. Effects of dietary nitrate on blood pressure in healthy volunteers. N Engl J Med. 2006;355(26):2792–3. Ormesher L, Myers JE, Chmiel C, Wareing M, Greenwood SL, Tropea T, et al. Effects of dietary nitrate supplementation, from beetroot juice, on blood pressure in hypertensive pregnant women: A randomised, double-blind, placebo-controlled feasibility trial. Nitric Oxide. 2018;80:37–44. Willmott T, Ormesher L, McBain AJ, Humphreys GJ, Myers JE, Singh G, et al. Altered Oral Nitrate Reduction and Bacterial Profiles in Hypertensive Women Predict Blood Pressure Lowering Following Acute Dietary Nitrate Supplementation. Hypertension. 2023;80(11):2397–406. Ben AJ, Neumann CR, Mengue SS. The Brief Medication Questionnaire and Morisky-Green test to evaluate medication adherence. Rev Saude Publica. 2012;46(2):279–89. Cavalli RC, Barbosa PO, Sandrim VC. Inorganic Nitrate Supplementation for Blood Pressure Control in Chronic Hypertensive Pregnancies from the 2nd Trimester (NIT_CH): a Triple-Blind Randomized Placebo-Controlled Trial — Data Management Plan [Internet]. DMPTool; 2025 [cited 2025 Nov 19]. Available from: https://doi.org/10.48321/D10A59C0F7 Battarbee AN, Sinkey RG, Harper LM, Oparil S, Tita ATN. Chronic hypertension in pregnancy. Am J Obstet Gynecol. 2020 June;222(6):532–41. Malha L, August P. Safety of Antihypertensive Medications in Pregnancy: Living With Uncertainty. J Am Heart Assoc. 2019;8(15):e013495. Richards EMF, Giorgione V, Stevens O, Thilaganathan B. Low-dose aspirin for the prevention of superimposed preeclampsia in women with chronic hypertension: a systematic review and meta-analysis. Am J Obstet Gynecol. 2023;228(4):395–408. Bahadoran Z, Mirmiran P, Kabir A, Azizi F, Ghasemi A. The Nitrate-Independent Blood Pressure-Lowering Effect of Beetroot Juice: A Systematic Review and Meta-Analysis. Adv Nutr. 2017;8(6):830–8. Kapil V, Khambata RS, Robertson A, Caulfield MJ, Ahluwalia A. Dietary nitrate provides sustained blood pressure lowering in hypertensive patients: a randomized, phase 2, double-blind, placebo-controlled study. Hypertension. 2015;65(2):320–7. Sandrim VC, Palei ACT, Metzger IF, Cavalli RC, Duarte G, Tanus-Santos JE. Interethnic differences in ADMA concentrations and negative association with nitric oxide formation in preeclampsia. Clin Chim Acta. 2010;411(19–20):1457–60. Gomes HF, Palei ACT, Machado JSR, da Silva LM, Montenegro MF, Jordão AA, et al. Assessment of oxidative status markers and NO bioavailability in hypertensive disorders of pregnancy. J Hum Hypertens. 2013 June;27(6):345–8. Caldeira-Dias M, Viana-Mattioli S, de Souza Rangel Machado J, Carlström M, de Carvalho Cavalli R, Sandrim VC. Resveratrol and grape juice: Effects on redox status and nitric oxide production of endothelial cells in in vitro preeclampsia model. Pregnancy Hypertens. 2021;23:205–10. Brandão TO, Veiga EC, de Levy A, Damaso RF, Sandrim EL, Cavalli VC. Assessment by ABPM verified the presence of hypertension in patients with self-reported hypertension, pregnant women, as well as differences between ethnicities in women aged 38–39 years in the Ribeirão Preto cohort. Front Pharmacol. 2022;13:992595. Nunes PR, Cavalli RC, Belo VA, Sandrim VC, Luizon MR. Longitudinal Study of Plasma Visfatin/Nicotinamide Phosphoribosyltransferase (NAMPT) Levels in Healthy Pregnant Women. Reprod Sci. 2023;30(10):2893–5. Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx GraphicalAbstract.png Legend of the Graphical Abstract:Overview of the NIT_CH triple-blind, randomized, placebo-controlled clinical trial evaluating the effect of inorganic nitrate supplementation in chronic hypertensive pregnancies. Cite Share Download PDF Status: Published Journal Publication published 09 Jan, 2026 Read the published version in BMC Pregnancy and Childbirth → Version 1 posted Editorial decision: Accepted 12 Dec, 2025 Editor assigned by journal 22 Nov, 2025 Submission checks completed at journal 22 Nov, 2025 First submitted to journal 19 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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08:29:38","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":47466,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8157989/v1/36fd78c5ec298d61a2c7317d.docx"},{"id":98432788,"identity":"19025ae8-a03c-4527-b9e7-0db1bc390ca2","added_by":"auto","created_at":"2025-12-17 16:49:58","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":97178,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLegend of the Graphical Abstract:\u003c/strong\u003eOverview of the NIT_CH triple-blind, randomized, placebo-controlled clinical trial evaluating the effect of inorganic nitrate supplementation in chronic hypertensive pregnancies.\u003c/p\u003e","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-8157989/v1/cab6da720e376d9fd960e8b9.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"NIT_CH: A Study Protocol for Evaluating the Effect of Inorganic Nitrate Capsules in Chronic Hypertensive Pregnancies – A Triple-Blind Placebo-Controlled Randomized Trial","fulltext":[{"header":"Background","content":"\u003cp\u003eHypertensive disorders of pregnancy (HDP) have a global prevalence of approximately 5% to 8% in pregnant women and significantly contribute to adverse maternal and fetal complications (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Chronic arterial hypertension affects roughly 2 to 5% of pregnant women and has increasing in recent years (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The prevalence of pregnancies complicated by chronic hypertension is rising, likely attributed to factors such as increasing maternal age, obesity rates, and metabolic syndrome (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePregnant women diagnosed with chronic hypertension often pose a reduced likelihood of experiencing the physiological cardiovascular adaptations in the first half of pregnancy, which play a vital role in the process of uteroplacental adaptation and fetal development, including the decrease in peripheral vascular resistance and the subsequent drop in blood pressure (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe results of the CHIPS trial (Control of Hypertension in Pregnancy Study) demonstrated the significance of blood pressure management in pregnant women with borderline hypertension to prevent the development of severe hypertension and the associated maternal and perinatal complications (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Due to the need for maternal-fetal safety data, the exploration of new antihypertensive therapies is constrained, as is the utilization of the current therapeutic options.\u003c/p\u003e \u003cp\u003eInorganic nitrate and beetroot juice supplementation are being recognized as a safe and well-tolerated dietary supplement (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Their mechanism of action is associated with the production of nitric oxide (NO), an endogenous vasodilator, from the bioconversion of nitrate (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). The alternative production of NO operates through the recycling of nitrite (NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) and nitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) anions, following a pathway that differs from the classical L-arginine-NO-synthase pathway. Furthermore, for efficient enzymatic conversion of nitrate to nitrite, a specific group of nitrate reductase enzymes is required, present in specific bacterial populations in the mouth and intestine of mammals, in addition to the acidic stomach environment. Despite these requirements, inorganic nitrate and beetroot juice supplementation offer the advantage of a wide therapeutic range, allowing the use of high dosages without significant risks of toxicity (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe utilization of inorganic nitrate to increase NO bioavailability can be advantageous, especially in the presence of hypoxia and hypertension (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Preliminary data from studies using a mouse model of pregnancy-related hypertension have shown that a 6-day supplementation with nitrate-rich beetroot juice led to a reduction in blood pressure and an enhancement in the endothelial function of the maternal uterine artery (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In healthy volunteers, a 3-day dietary supplementation with sodium nitrate resulted in a significant reduction in diastolic pressure by 3.7 mmHg compared to a placebo (sodium chloride) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA study investigating inorganic nitrate supplementation in the form of beetroot juice for pregnant women in the third trimester (from 28 weeks onwards) over 8 days demonstrated a significant increase in plasmatic and salivary nitrate and nitrite concentrations in the treated group when compared to the placebo, with peak concentrations reached approximately 2\u0026ndash;4 hours after the juice\u0026rsquo;s ingestion (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Although the study did not establish a reduction in blood pressure in the treated group, there was a correlation between the increase in plasma nitrite concentrations and the decrease in diastolic blood pressure within this group. In another study conducted by the same group, significant reductions in both systolic and diastolic blood pressure were observed in women with hypertension 2.5 hours after consuming nitrate-rich beetroot juice (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The authors also discussed the potential influence of oral microbiota on nitrate metabolism and blood pressure response. Therefore, further studies on the long-term use of inorganic nitrate are needed to provide a more comprehensive investigation that might fully assess the potential benefits and mechanisms of action of nitrate supplementation in pregnant patients.\u003c/p\u003e \u003cp\u003eThe primary aim of this study is to evaluate blood pressure control in pregnant women with chronic hypertension who will be supplemented with inorganic nitrate in comparison to a placebo group. Both groups will receive standard antihypertensive treatment and prophylaxis for preeclampsia, with one of the secondary objectives being the monitoring of additional antihypertensive medications need. Other secondary aims encompass the evaluation of maternal and fetal Doppler analyses, fetal growth, overlapping incidence of superimposed preeclampsia and adverse outcomes, umbilical artery vascular reactivity, hematological and metabolic assessments, concentrations of nitric oxide metabolites, markers of preeclampsia, inflammation and oxidative stress, and the analysis of the relationship between dietary indices and markers of oxidative and inflammatory stress. We hypothesize that supplementation with 400 mg of inorganic nitrate, initiated at the beginning of the second trimester (16th week) and continued over a long period during the pregnancy (22 weeks), may yield promising results in blood pressure control, associated to an enhancement in uteroplacental flow, adequate fetal growth and preserving fetal vitality, improving obstetric results.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTrial Design\u003c/h2\u003e \u003cp\u003eThis study is a randomized, placebo-controlled, triple-blind, parallel-group clinical trial. Pregnant women with chronic hypertension will be randomly assigned to the intervention group or the placebo, in blocks of four to ensure balance between the groups.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy Setting\u003c/h3\u003e\n\u003cp\u003eThe study is a single-site trial conducted at the Clinical Research Unit, a dedicated facility for clinical research linked to the Hospital das Cl\u0026iacute;nicas de Ribeir\u0026atilde;o Preto, located in Ribeir\u0026atilde;o Preto, Brazil. Participants will attend the Clinical Research Unit according to the visit schedule outlined in the study protocol, preferably in the morning. All study procedures, including enrolment, disposal of supplementation, ultrasonography, and blood collection, will be performed at the Clinical Research Unit by trained members of the research and clinical team of the study, as well as nurses and pharmacists employed by the Clinical Research Unit. Deliveries will take place at the Hospital das Cl\u0026iacute;nicas de Ribeir\u0026atilde;o Preto and will be managed by obstetricians affiliated with the hospital.\u003c/p\u003e\n\u003ch3\u003eTrial Status\u003c/h3\u003e\n\u003cp\u003eThe NIT_CH study received funding in 2021 from the Foundation of the state of S\u0026atilde;o Paulo (FAPESP). The Research Ethics Committee of the Hospital das Cl\u0026iacute;nicas, Ribeir\u0026atilde;o Preto Medical School \u0026ndash; S\u0026atilde;o Paulo University, was granted on September 19, 2024, with the last updated approval on February 26, 2025. This paper reflects Protocol Version 3.0. Recruitment began in September 2024, and data collection is ongoing. The first results are expected to be submitted for publication in December 2026.\u003c/p\u003e\n\u003ch3\u003eRecruitment\u003c/h3\u003e\n\u003cp\u003eEligible pregnant women with chronic hypertension will be identified and invited to participate through targeted outreach, including posters and direct referrals from healthcare professionals within the municipal health network. To achieve the target of enrolling 144 pregnant women, an average recruitment rate of 6 women per month will be required. Recruitment progress will be continuously monitored, and if necessary, additional measures such as meetings with the municipal health network and active on-site recruitment will be implemented.\u003c/p\u003e\n\u003ch3\u003eParticipants\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eInclusion Criteria\u003c/h2\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003ePregnant women diagnosed with chronic hypertension as defined by the Brazilian Network for Studies on Hypertension in Pregnancy/Preeclampsia and by the Brazilian Federation of Gynecology and Obstetrics Associations Protocol: Systolic Blood Pressure (SBP) above 140 mmHg and/or Diastolic Blood Pressure (DBP) above 90 mmHg prior to pregnancy or before 20 weeks of gestation.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eCurrently using monotherapy with methyldopa (first-line antihypertensive drug used during pregnancy in Brazil) at the time of inclusion.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eBelow 16 weeks of gestation, confirmed by first-trimester ultrasound.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExclusion Criteria\u003c/h3\u003e\n\u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eMultiple pregnancies, below 18 years of age, inability to provide informed consent, or a history of poor medication adherence.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003ePatients with uncontrolled chronic hypertension, with pressure measurements above 160x110mmHg and/or using other antihypertensives in addition to methyldopa at the moment of inclusion.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eUsers of illicit drugs, smokers, or alcohol abusers.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003ePatients diagnosed with previous bariatric surgery, coronary artery disease, moderate to severe congestive heart failure, moderate to severe liver failure, chronic kidney failure (with creatinine clearance less than 30 ml/min/1.73 m\u003csup\u003e2\u003c/sup\u003e body surface area), and pre-existing type 1 and type 2 diabetes.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eFrequent users of non-steroidal anti-inflammatory drugs, nasal decongestants, and anorectics before pregnancy; users of proton pump inhibitors, H2 receptor antagonists, or any other medication that affects stomach pH, starting 2 weeks before and maintained throughout the study.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e\n\u003ch3\u003eSample Size\u003c/h3\u003e\n\u003cp\u003eAn analysis of robustness was conducted to determine the required sample size based on Ambulatory Blood Pressure Monitoring data. To detect a significant difference of 5.0 mmHg in arterial pressure between groups (with a standard deviation of 10 mmHg), a total of 64 pregnant women per group is required to ensure 80% power at 0.05 significance level. Accounting for a potential 12% dropout rate or treatment abandonment, the sample size was adjusted to 72 pregnant women per group, resulting in a total of 144 participants.\u003c/p\u003e \u003cp\u003eThe sample size was computed using the following formula: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\varvec{n}\\varvec{P}=\\left(\\frac{{\\left(\\frac{\\varvec{Z}\\varvec{\\alpha\\:}}{2}+\\varvec{Z}\\varvec{\\beta\\:}\\right)}^{2}\\times\\:\\:\\varvec{s}.\\varvec{d}}{\\varvec{Ḏ}}\\right)\u0026sup2;\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRandomization and Masking\u003c/h2\u003e \u003cp\u003eParticipants will be randomized using a computer-generated sequence in blocks of four to ensure balance between the groups. The on-site pharmacist, who is not involved in the study\u0026rsquo;s execution, will assign participants based on this sequence. An external pharmacist from an independent company will manufacture both the intervention and placebo capsules. The on-site pharmacist will label each bottle with the participant's name and a unique identification number corresponding to the assigned group, ensuring allocation concealment from the research team, participants, and statistician until the trial\u0026rsquo;s conclusion. Only the on-site and external pharmacists will have access to the allocation sequence, maintaining the integrity of blinding.\u003c/p\u003e \u003cp\u003eAt visit 1, the UPC pharmacist will dispense a sufficient quantity of capsules to last until the next visit, including an additional of 7\u0026ndash;10 capsules to account for potential delays between visits or emergency use. The research and medical team involved in the study will not know the group assigned to the participant.\u003c/p\u003e \u003cp\u003eThe trial will employ a triple-blind design, where participants, the research and medical team, and data analysts will remain blinded to treatment allocation from the point of intervention assignment until the completion of the clinical trial and final data analysis. In case of a medical emergency requiring unblinding, a co-principal investigator will have access to the allocation group through a formal request submitted directly to the on-site pharmacist.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eInterventions\u003c/h2\u003e \u003cp\u003eWomen referred from primary care who meet the inclusion criteria will be contacted by the research team and invited to an appointment at the Clinical Research Unit. During this appointment, the informed consent form will be presented, and all questions will be addressed by members of the research team, who have been trained in the study protocol. Participants will then be asked to review and sign the informed consent form before any study procedures are initiated.\u003c/p\u003e \u003cp\u003e Participants will be enrolled by the research team upon obtaining the written informed consent form. From the day of enrollment to the next appointment, the pharmacist will randomize the participants to the intervention or placebo. Placebo is selected as a comparator to provide a comparison point.\u003c/p\u003e \u003cp\u003e In the intervention arm, participants will receive sodium nitrate, which includes 400 mg of nitrate and 140 mg of sodium (totaling 540 mg of sodium nitrate). This amount has proven to be safe, as reported in other studies (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The placebo group will be produced using sodium chloride with the same amount of sodium present in nitrate salt (140 mg, which represents 7% of the daily recommendation of sodium). All participants will be instructed to take one capsule each morning after breakfast, continuing this regimen for 22 weeks (approximately 154 days), ending either on the 38th week of pregnancy or childbirth, whichever occurs first. In both cases (intervention and placebo), the usual medications for preeclampsia prophylaxis will be maintained (acetylsalicylic acid 100 mg at night daily, up to 36 weeks of gestation and calcium carbonate 1000 mg per day, until delivery) and usual antihypertensive treatment. The dosage of acetylsalicylic acid specified in the trial (100 mg) represents the available and commonly used presentation in Brazil.\u003c/p\u003e \u003cp\u003eThe intervention may be discontinued based on the clinical judgment of the medical team in cases of adverse events or upon the participant\u0026rsquo;s request at any time.\u003c/p\u003e \u003cp\u003eTo ensure adherence, patients will be provided with additional capsules and asked to return any unused capsules at each visit to verify adherence to the supplementation. Additionally, researchers will apply the Morisky-Green questionnaire to evaluate adherence (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e Participants will be advised to avoid the use of mouthwash during the study period, as it may eliminate oral microbiota that is crucial for the conversion of nitrate to nitrite, a critical step in the intervention's mechanism of action.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStudy Protocol and Timeline\u003c/h2\u003e \u003cp\u003eAfter the enrolment, participants will be randomized, and another appointment will be scheduled for the 16\u003csup\u003e+\u0026thinsp;0+6\u003c/sup\u003e weeks of pregnancy with a member of the research team (Visit 1). Participants will be required to arrive fasting for 8 hours for sample collection. The participants will undergo an obstetric and clinical exam, blood pressure measurement, and an obstetric ultrasound, complemented by a Doppler study of the uterine arteries. Blood samples, urine and saliva will be collected for further analysis.\u003c/p\u003e \u003cp\u003eFor the subsequent visits \u0026ndash; Visit 2 through Visit 9 \u0026ndash; participants will periodically return to the Clinical Research Unit. Each follow-up visit will include a clinical exam, blood pressure measurement, blood sample, saliva and urine collection, obstetric consultation, and ultrasounds. The Doppler study of uterine arteries will be conducted at each visit, while the Doppler study of the middle cerebral artery and the umbilical artery will begin at 24 weeks of pregnancy and continue at each subsequent visit.\u003c/p\u003e \u003cp\u003eThe medical team will consistently monitor participants' antihypertensive medications, making necessary adjustments to maintain optimal blood pressure control. Each visit is estimated to last about 30 minutes to 1 hour. The overall expected study duration per participant spans either 154 days or up to the point of childbirth, whichever occurs first. To offset potential travel expenses to the clinic, participants will receive a reimbursement of forty Brazilian Real (R\u003cspan\u003e$\u003c/span\u003e 40.00) per visit. The participant timeline as well as the events of the protocol can be found in Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAdverse event reporting and harms\u003c/h2\u003e \u003cp\u003eWe do not anticipate significant side effects from the use of inorganic nitrate, as previous studies in pregnant women have shown no evidence of adverse maternal or fetal risks. However, participants will be closely monitored for any potential adverse events. Any symptoms, diseases, or side effects, whether or not related to the intervention, will be documented, and participants will receive appropriate medical care as needed. All adverse events will be reported and registered in accordance with local regulations and communicated to relevant authorities.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eOutcomes\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003ePrimary Outcome\u003c/h2\u003e \u003cp\u003eThe primary outcome, blood pressure control, will be assessed through frequent blood pressure measurements using a standardized method. The measurements will be taken using an automated blood pressure monitor. Simultaneously, the frequency and magnitude of dosage adjustments in antihypertensive medications across both groups during the study period will be closely monitored and documented. Any changes in medication dosage will be recorded to provide a comprehensive evaluation of the primary outcome.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSecondary Outcomes\u003c/h2\u003e \u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eFetal Growth: Fetal growth will be assessed through periodic ultrasound examinations. Measurements of head circumference, abdominal circumference, and femur length will be obtained using standard ultrasound techniques by experienced physicians to estimate the fetal weight using Hadlock\u0026rsquo;s formula.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003ePreeclampsia and Adverse Maternal Outcomes: The incidence and severity of superimposed preeclampsia and other adverse maternal outcomes will be determined through clinical assessments and regular monitoring of symptoms. Diagnostic criteria for preeclampsia will follow established medical guidelines.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003ePerinatal outcomes: Birthweight and Apgar scores at 1 and 5 minutes will be recorded immediately after delivery by healthcare professionals.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eBlood Profile Analysis: Blood profile measurements will be conducted through laboratory tests. Hemogram, lipid profile, liver and kidney functionalities, glucose levels, and fasting insulin will be determined using standard laboratory techniques.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eNitrate and Nitrite Concentrations: The concentration of nitrate and nitrite in plasma, urine, and saliva will be measured using validated methodology.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eSpecific Serum Indicators of Preeclampsia: Specific serum indicators of preeclampsia (sFLT-1 and PlGF) will be measured through laboratory tests.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eInflammatory markers: The levels of inflammatory markers, including adiponectin, ICAM-1, IL-6, IL-8, IL-10, leptin, resistin, TNF-α, VCAM-1, VEGF, and visfatin, will be determined through laboratory assays.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eOxidative Stress: Lipid peroxidation levels and total antioxidant capacity of plasma will be assessed using established colorimetric methods.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eMatrix metalloproteinase: The concentrations of matrix metalloproteinase (MMPs 2 and 9) and their inhibitors (TIMPs 1, 2, 3, and 4) will be quantified through laboratory analyses.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eDietary Pattern: Dietary patterns will be assessed using a dietary marker recommended by Brazil\u0026rsquo;s Ministry of Health (SISVAN).\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eBiological Specimens\u003c/h2\u003e \u003cp\u003eBlood (plasma and serum), saliva, urine, and umbilical cord samples will be securely stored at -80℃ and will be used exclusively for the analysis outlined in the research protocol and described in the participant consent form.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAncillary and Post-Trial Care\u003c/h2\u003e \u003cp\u003eThere are no specific provisions for post-trial care. However, in the event of any harm resulting directly from participation in the trial, participants will receive all necessary medical care to address the condition until it is resolved or significantly improved. Compensation for trial-related injuries will be provided in accordance with applicable Brazilian ethical guidelines and regulatory requirements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Methods\u003c/h2\u003e \u003cp\u003eInitially, the quantitative data obtained in this study will be analyzed using a descriptive statistical approach. Quantitative variables will be presented using central position (mean and median) and dispersion measures (standard deviation, interquartile range, minimum, and maximum values), which will be visualized using box-plot graphs and histograms. Absolute and relative frequencies will be used to summarize all qualitative variables. Socio-demographic variables and other baseline characteristics will undergo statistical analysis based on data distribution. The intervention and placebo groups will be compared using the Student\u0026rsquo;s t-test or the Wilcoxon test, depending on whether the data are parametric or non-parametric, respectively. Categorical variable analysis will start by calculating absolute and relative frequencies for each category, followed by proportion comparisons using chi-squared or Fisher\u0026rsquo;s exact tests. A linear regression model with mixed effects will be used to assess the treatment effect over time, comparing groups using orthogonal contrasts and examining model fitting through a residual analysis. There will be per-protocol and intention-to-treat analyses. Because of the study\u0026rsquo;s prospective design, which includes patient monitoring until delivery, the occurrence of clinical outcomes like preeclampsia, fetal growth restriction, and hemodynamic centralization will be depicted using Kaplan-Meier curves in each group. Cox regression will be used to compare the occurrence of outcomes between groups. Correlation between dietary indices and oxidative stress variables, and inflammatory markers will be shown in a heatmap. For variable pairings, a correlation measure, such as the Pearson or Spearman correlation coefficient, will be produced after organizing data in a matrix format. The heatmap created with these coefficients will show varied color intensities to represent different levels of correlation, providing an intuitive depiction of changeable inter-relationships. Food pattern categories will be identified using exploratory factor analysis. A correlation matrix will be used to analyze the correlation between meal pattern variables after they have been chosen based on theoretical relevance and data availability, followed by an exploratory factor analysis using the principal component analysis technique. All statistical analyses will use a p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 significance level. Data analyses will be conducted using both R and Stata software tools.\u003c/p\u003e \u003cp\u003eAny missing data will be reported, and if more than 5% of primary outcome data is missing, a sensitivity analysis will be performed using multiple imputation techniques to handle the missing data, assuming the missingness is unrelated to the outcome.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMethods for additional analyses\u003c/h2\u003e \u003cp\u003eAdditional analyses will include a subgroup analysis based on responsiveness and non-responsiveness, as well as stratification by BMI. Responders will be defined as women in the intervention group who did not develop preeclampsia, while non-responders will be those in the same group who developed preeclampsia. Baseline characteristics will be compared between responders and non-responders using Student\u0026rsquo;s t-test or the Wilcoxon test for continuous variables, and the chi-square or Fisher\u0026rsquo;s exact test for categorical variables. Differences in primary and secondary outcomes between these subgroups will be assessed using regression models adjusted for potential confounders.\u003c/p\u003e \u003cp\u003eFor the BMI-based analysis, participants will be categorized into different BMI groups, and outcome measures will be compared across these groups to evaluate whether BMI influences treatment response. Interaction terms in regression models will be used to assess the moderating effect of BMI on study outcomes. Additionally, logistic regression will be employed to identify predictors of treatment response, while Cox regression models stratified by BMI will be used to evaluate differences in the time to clinical events, such as preeclampsia and fetal growth restriction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eInterim analyses\u003c/h2\u003e \u003cp\u003eAn interim analysis is expected to be carried out when one-third of the sample size is enrolled. This analysis will focus mainly on adherence and reported adverse events, but also on the assessment of the primary outcome. If the intervention proves to be unsafe (which is clearly not expected to be) or ineffective based on this analysis, a premature ending of the trial is planned to avoid further potential damage to the patients, and money spent.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eEthics and Dissemination\u003c/h2\u003e \u003cp\u003eData collection will be conducted at the Clinical Research Unit throughout the study period. The data collected will include numeric data (weight, blood pressure, biochemical test values, laboratory tests, and measurements obtained from fetal ultrasound), textual data (dietary intake records, medical and family history), categorical data (marital status, type of residence, profession/occupation, and ethnicity), and images and medical reports. Demographic data, dietary intake, and information on medical and family history will be collected at enrolment only. Additional data will be updated throughout the intervention period based on new measurements and assessments obtained during scheduled visits. Data collection will include questionnaires, clinical and laboratory evaluations, and gynecological examinations performed at each visit.\u003c/p\u003e \u003cp\u003eNo major adverse effects are anticipated with the proposed intervention. However, any potential side effects will be documented, and participants will receive individualized and appropriate medical care as needed. All participants will provide written informed consent prior to enrollment. The study protocol has been reviewed and approved by the Ethics Review Board of the Ribeir\u0026atilde;o Preto's Clinical Hospital (approval number 7.390.821).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eConfidentiality\u003c/h2\u003e \u003cp\u003eData will be encrypted with a unique identity in a password-protected local database. To ensure patient confidentiality, each participant will be assigned a unique identification code, distinct from any personal identifiers. This code will be used in all research documentation and data entry processes. The primary identifier permitting patient traceability, the medical record number from the Hospital das Cl\u0026iacute;nicas de Ribeir\u0026atilde;o Preto system, will be utilized only during clinical visits to document care provided by health professionals. In the research database, this identifier will be linked to a numerical code representing the patient.\u003c/p\u003e \u003cp\u003eUltrasonographic data, such as arterial reactivity, fetal weight, and others, along with laboratory test results, will be entered into the research database as numerical data, devoid of any personal identifiers.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eAmended to the protocol\u003c/h2\u003e \u003cp\u003eAny changes to the study protocol that may affect its execution, potential participant benefits, or patient safety will necessitate a formal amendment. This includes alterations to the study\u0026rsquo;s objectives, design, or procedures. These amendments must be submitted and approved by the Ethics Review Board of the Ribeir\u0026atilde;o Preto's Clinical Hospital before implementation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eData management\u003c/h2\u003e \u003cp\u003eAll data obtained from the analysis and medical records will be recorded and stored in the REDCap software, leveraging its cloud storage capability and robust database support. A specific study form was created by a research team member in collaboration with the REDCap software coordinator at the University of S\u0026atilde;o Paulo.\u003c/p\u003e \u003cp\u003eTo ensure data quality, all research team members will be trained to use the REDCap software. The system will enforce data integrity by locking fields after entry. To prevent misclassification, numerical and categorical variables will be clearly defined and input into their respective field types, with validation checks in place to further enhance accuracy.\u003c/p\u003e \u003cp\u003eFurthermore, in alignment with Open Science principles, de-identified raw research data (excluding any patient-traceable information) will be made publicly available in the University of S\u0026atilde;o Paulo\u0026rsquo;s online repository for a period of 10 years. A data management plan has been created and registered using the DMPTool (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), outlining the procedures for data collection, storage, and sharing throughout the project.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eDissemination plans\u003c/h2\u003e \u003cp\u003eWe will disseminate our results through multiple channels to ensure broad accessibility and impact. We will publish our findings in an international open-access journal, ensuring free global accessibility to our data for researchers and the general public. Additionally, we will be presenting our findings at both local and international conferences to stimulate scholarly discussion and foster collaborative research opportunities.\u003c/p\u003e \u003cp\u003e To translate our research into actionable health guidelines, we will engage with policymakers through briefings, meetings, and workshops. This engagement is essential for the practical application of our findings in health policy and practice.\u003c/p\u003e \u003cp\u003ePublic awareness is also important; therefore, we will translate our research into accessible formats for public talks, community workshops, and collaboration with media outlets to reach a wider audience.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eComposition of the coordinating centre and trial steering committee\u003c/h2\u003e \u003cp\u003eThe research and medical team will hold regular meetings to discuss the study\u0026rsquo;s progression, recruitment status, and any necessary amendments to the protocol. The principal investigator will be responsible for managing data monitoring and maintaining communication with participants.\u003c/p\u003e \u003cp\u003eAs this study will be conducted at the university level, there will be no formal Trial Steering Committee. The principal investigator will coordinate the study site, ensuring the overall management and execution of the trial. However, regular updates may be requested by the Research Clinical Unity team to ensure the study is being conducted in accordance with ethical and regulatory standards.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study protocol addresses a significant gap in research on the long-term efficacy of inorganic nitrate as an adjuvant therapy for chronic hypertension in pregnancy, particularly for blood pressure control. The NIT_CH trial is specifically designed to evaluate the therapeutic potential of inorganic nitrate in this high-risk population.\u003c/p\u003e \u003cp\u003eChronic hypertension during pregnancy is a challenging medical condition that significantly impacts both maternal and fetal health (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). One of the main challenges in managing chronic hypertension in pregnancy is controlling blood pressure while minimizing the need for multiple antihypertensive medications or increasing their dosages, as these drugs have safety and efficacy limitations during pregnancy (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Moreover, superimposed preeclampsia is a common complication in pregnant women with chronic hypertension. Although aspirin is used as a first-line treatment, its effectiveness in preventing preeclampsia appears to be lower in this population (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Inorganic nitrate has emerged as a promising alternative due to its vasodilatory, antioxidant, and antihypertensive properties, which are particularly beneficial with prolonged use (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Furthermore, its low cost could offer a significant advantage in the long-term management of chronic hypertension. We hope, with the present study, to avoid the need for additional anti-hypertensive medications and any increase in the number of cases progressing to preeclampsia.\u003c/p\u003e \u003cp\u003eThe rationale behind conducting this protocol study is based on prior research in both animal and human models (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), which showed that inorganic nitrate may help reduce blood pressure and improve vascular function. Assessing the use of nitrate in pregnant women with chronic hypertension requires a careful analysis of safety and efficacy, particularly given the unique aspects of pregnancy. In this sense, the research team involved in this study has extensive experience in the field of hypertension disorders in pregnancy (\u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). The researchers\u0026rsquo; wealth of experience plays a pivotal role in ensuring the success and reliability of the study, emphasizing the importance of having a dedicated and proficient research group conducting this type of investigation.\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, this is the first randomized controlled trial assessing inorganic nitrate from the second trimester of pregnancy until delivery in pregnant women diagnosed with chronic hypertension. A key strength of the study is the sustained and multifaceted assessment, providing a comprehensive understanding of the effects of inorganic nitrate over an extended period.\u003c/p\u003e \u003cp\u003eAlthough most studies evaluating inorganic nitrate during pregnancy have used beetroot juice, we understand that our long-duration protocol makes supplementation with juice impractical due to the taste aversions common in pregnancy and the inherent difficulty in finding a matched placebo. Therefore, the use of inorganic nitrate in the form of salt was chosen by our team as a more feasible and cost-effective option. To achieve a dose of 400 mg of nitrate, the corresponding amount of sodium in sodium nitrate is 140 mg. Thus, to control this quantity, the placebo will be formulated with an equivalent amount of sodium chloride (140 mg, or 7% of the dietary sodium requirement), thereby isolating the therapeutic effect of the nitrate anion. We strongly believe that the inclusion of 140 mg of sodium chloride in the placebo is unlikely to be problematic for pregnant women with chronic hypertension, given its small contribution to total sodium intake. Moreover, the women will be monitored during all period of pregnancy, and if the intake of the placebo represents to be harmful for this population, the study will be suspended.\u003c/p\u003e \u003cp\u003eThe NIT_CH project represents a significant endeavor aimed at assessing the therapeutic potential of inorganic nitrate in pregnant women with chronic hypertension, thus filling a critical gap in clinical research. We expect that the results obtained in this study may contribute to advance knowledge and potentially foster the development of more effective therapies during pregnancy, thereby benefiting maternal and fetal health.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCHIPS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eControl of Hypertension in Pregnancy Study\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDBP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ediastolic blood pressure\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMPTool\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eData Management Plan Tool\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eICAM-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIntracellular Adhesion Molecule 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIL-6\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterleukin-6\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIL-8\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterleukin-8\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIL-10\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterleukin-10\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHDP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHypertensive Disorders of Pregnancy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMMPs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emetalloproteinases\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enitric oxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePlGF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePlacenta Growth Factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSBP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSystolic Blood Pressure\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003esFLT-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSoluble Fms-like Tyrosine kinase-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSISVAN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSistema de Vigil\u0026acirc;ncia Alimentar e Nutricional\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTIMPs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTissue Inhibitors of Metalloproteinases\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNF-α\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor Necrosis Factor alpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVCAM-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVascular Cell Adhesion Molecule 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVEFG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVascular Endothelial Growth Factor.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eEthical approval for this study protocol was granted in August 2024 by the Research Ethics Committee of Hospital das Cl\u0026iacute;nicas, Ribeir\u0026atilde;o Preto Medical School \u0026ndash; S\u0026atilde;o Paulo University (approval number 7.390.821). Written informed consent will be obtained from all participants. A trained researcher will introduce the study to potential participants and provide a comprehensive explanation of the trial\u0026rsquo;s objectives and procedures. Participants will receive an information document outlining the study details and will have the opportunity to discuss any questions or concerns with the researcher. The informed consent process will be thoroughly documented, with consent forms securely stored for auditing purposes.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable. \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe data collected for the study will be made available as a de-identified dataset on the Open Science Framework (OSF) and the S\u0026atilde;o Paulo University repository after the final clinical study report. Access to the data will be facilitated according to the study\u0026apos;s data management plan (17).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis clinical trial is embedded in a project named \u0026ldquo;Endothelial Dysfunction in Hypertensive Disorders of Pregnancy\u0026rdquo;, grant number: 2021/12010-7, S\u0026atilde;o Paulo Research Foundation \u0026ndash; FAPESP. Priscila O. Barbosa is funded by FAPESP (grant number: 2023/07589-1). Additional support is provided by Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq) (Grant number: 308504/2021-6 and 302614/2025-7).\u003c/p\u003e\n\u003cp\u003eFAPESP is a state agency, and CNPq is a federal funding agency. Neither had any role in the design of the study, data collection, management, analysis, or interpretation. The research team maintains full independence in all aspects of the study.\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026apos; contributions\u003c/h2\u003e\n\u003cp\u003eStudy concept and design: V.C.S. and R.C.C. Trial management: P.O.B and R.C.C. Ethics application and acquisition of data: L.S.L.J., P.O.B., R.C.C. and V.A. Drafting of the manuscript: A.S.C., P.OB. and V.A. Critical Revision: L.S.L.J. and J.E.T.S. Project administration and Resources: R.C.C. and V.C.S. All authors contributed to refinement of the study protocol and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe would like to acknowledge the following contributors to this study: Suleimy C Mazin, for her help with the data analysis plan and all statistical aspects; Ricardo Perussi e Silva, for his assistance with the database on REDCap; and Professor Jon Lundberg, from the Karolinska Institute, for his guidance and expertise on inorganic nitrate administration.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eReport of the National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy. Am J Obstet Gynecol. 2000 July;183(1):S1\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeonard SA, Siadat S, Main EK, Huybrechts KF, El-Sayed YY, Hlatky MA, et al. Chronic Hypertension During Pregnancy: Prevalence and Treatment in the United States, 2008\u0026ndash;2021. Hypertension. 2024;81(8):1716\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKent L, McGirr M, Eastwood KA. Global trends in prevalence of maternal overweight and obesity: A systematic review and meta-analysis of routinely collected data retrospective cohorts. Int J Popul Data Sci. 2024;9(2):2401.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoon LC, Shennan A, Hyett JA, Kapur A, Hadar E, Divakar H, et al. The International Federation of Gynecology and Obstetrics (FIGO) initiative on pre-eclampsia: A pragmatic guide for first-trimester screening and prevention. Int J Gynaecol Obstet. 2019;145(Suppl 1Suppl 1):1\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMagee LA, von Dadelszen P, Rey E, Ross S, Asztalos E, Murphy KE, et al. Less-tight versus tight control of hypertension in pregnancy. N Engl J Med. 2015;372(5):407\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLara J, Ashor AW, Oggioni C, Ahluwalia A, Mathers JC, Siervo M. Effects of inorganic nitrate and beetroot supplementation on endothelial function: a systematic review and meta-analysis. Eur J Nutr. 2016;55(2):451\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarbosa PO, Tanus-Santos JE, Cavalli R, de Bengtsson C, Montenegro T, Sandrim MF. The Nitrate-Nitrite-Nitric Oxide Pathway: Potential Role in Mitigating Oxidative Stress in Hypertensive Disorders of Pregnancy. Nutrients. 2024;16(10):1475.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLundberg JO, Weitzberg E, Gladwin MT. The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov. 2008;7(2):156\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKapil V, Milsom AB, Okorie M, Maleki-Toyserkani S, Akram F, Rehman F, et al. Inorganic nitrate supplementation lowers blood pressure in humans: role for nitrite-derived NO. Hypertension. 2010;56(2):274\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMl S, Fj L, M C, Ml MBJP et al. H,. A randomized clinical trial of the effects of leafy green vegetables and inorganic nitrate on blood pressure. The American journal of clinical nutrition [Internet]. 2020 Jan 4 [cited 2025 Nov 19];111(4). Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmed.ncbi.nlm.nih.gov/32091599/\u003c/span\u003e\u003cspan address=\"https://pubmed.ncbi.nlm.nih.gov/32091599/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeitzberg E, Lundberg JO. Novel aspects of dietary nitrate and human health. Annu Rev Nutr. 2013;33:129\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTropea T, Renshall LJ, Nihlen C, Weitzberg E, Lundberg JO, David AL, et al. Beetroot juice lowers blood pressure and improves endothelial function in pregnant eNOS-/- mice: importance of nitrate-independent effects. J Physiol. 2020 Sept;598(18):4079\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLarsen FJ, Ekblom B, Sahlin K, Lundberg JO, Weitzberg E. Effects of dietary nitrate on blood pressure in healthy volunteers. N Engl J Med. 2006;355(26):2792\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrmesher L, Myers JE, Chmiel C, Wareing M, Greenwood SL, Tropea T, et al. Effects of dietary nitrate supplementation, from beetroot juice, on blood pressure in hypertensive pregnant women: A randomised, double-blind, placebo-controlled feasibility trial. Nitric Oxide. 2018;80:37\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWillmott T, Ormesher L, McBain AJ, Humphreys GJ, Myers JE, Singh G, et al. Altered Oral Nitrate Reduction and Bacterial Profiles in Hypertensive Women Predict Blood Pressure Lowering Following Acute Dietary Nitrate Supplementation. Hypertension. 2023;80(11):2397\u0026ndash;406.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBen AJ, Neumann CR, Mengue SS. The Brief Medication Questionnaire and Morisky-Green test to evaluate medication adherence. Rev Saude Publica. 2012;46(2):279\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCavalli RC, Barbosa PO, Sandrim VC. Inorganic Nitrate Supplementation for Blood Pressure Control in Chronic Hypertensive Pregnancies from the 2nd Trimester (NIT_CH): a Triple-Blind Randomized Placebo-Controlled Trial \u0026mdash; Data Management Plan [Internet]. DMPTool; 2025 [cited 2025 Nov 19]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.48321/D10A59C0F7\u003c/span\u003e\u003cspan address=\"10.48321/D10A59C0F7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBattarbee AN, Sinkey RG, Harper LM, Oparil S, Tita ATN. Chronic hypertension in pregnancy. Am J Obstet Gynecol. 2020 June;222(6):532\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalha L, August P. Safety of Antihypertensive Medications in Pregnancy: Living With Uncertainty. J Am Heart Assoc. 2019;8(15):e013495.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRichards EMF, Giorgione V, Stevens O, Thilaganathan B. Low-dose aspirin for the prevention of superimposed preeclampsia in women with chronic hypertension: a systematic review and meta-analysis. Am J Obstet Gynecol. 2023;228(4):395\u0026ndash;408.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBahadoran Z, Mirmiran P, Kabir A, Azizi F, Ghasemi A. The Nitrate-Independent Blood Pressure-Lowering Effect of Beetroot Juice: A Systematic Review and Meta-Analysis. Adv Nutr. 2017;8(6):830\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKapil V, Khambata RS, Robertson A, Caulfield MJ, Ahluwalia A. Dietary nitrate provides sustained blood pressure lowering in hypertensive patients: a randomized, phase 2, double-blind, placebo-controlled study. Hypertension. 2015;65(2):320\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSandrim VC, Palei ACT, Metzger IF, Cavalli RC, Duarte G, Tanus-Santos JE. Interethnic differences in ADMA concentrations and negative association with nitric oxide formation in preeclampsia. Clin Chim Acta. 2010;411(19\u0026ndash;20):1457\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGomes HF, Palei ACT, Machado JSR, da Silva LM, Montenegro MF, Jord\u0026atilde;o AA, et al. Assessment of oxidative status markers and NO bioavailability in hypertensive disorders of pregnancy. J Hum Hypertens. 2013 June;27(6):345\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaldeira-Dias M, Viana-Mattioli S, de Souza Rangel Machado J, Carlstr\u0026ouml;m M, de Carvalho Cavalli R, Sandrim VC. Resveratrol and grape juice: Effects on redox status and nitric oxide production of endothelial cells in \u003cem\u003ein vitro\u003c/em\u003e preeclampsia model. Pregnancy Hypertens. 2021;23:205\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrand\u0026atilde;o TO, Veiga EC, de Levy A, Damaso RF, Sandrim EL, Cavalli VC. Assessment by ABPM verified the presence of hypertension in patients with self-reported hypertension, pregnant women, as well as differences between ethnicities in women aged 38\u0026ndash;39 years in the Ribeir\u0026atilde;o Preto cohort. Front Pharmacol. 2022;13:992595.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNunes PR, Cavalli RC, Belo VA, Sandrim VC, Luizon MR. Longitudinal Study of Plasma Visfatin/Nicotinamide Phosphoribosyltransferase (NAMPT) Levels in Healthy Pregnant Women. Reprod Sci. 2023;30(10):2893\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-pregnancy-and-childbirth","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prch","sideBox":"Learn more about [BMC Pregnancy and Childbirth](http://bmcpregnancychildbirth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/prch/default.aspx","title":"BMC Pregnancy and Childbirth","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"chronic hypertension, inorganic nitrate, nitric oxide, pregnancy, preeclampsia","lastPublishedDoi":"10.21203/rs.3.rs-8157989/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8157989/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eHypertensive disorders of pregnancy represent a substantial risk to maternal and fetal health, with increasing prevalence worldwide linked to factors such as advanced maternal age, rising obesity rates, and metabolic syndrome. Effective management of borderline hypertension during pregnancy remains critical. However, given the limited safety data on conventional antihypertensive drugs in this population, alternative therapeutic strategies are needed. Inorganic nitrate supplementation has demonstrated potential in reducing blood pressure and enhancing maternal uterine artery function, primarily due to the production of nitric oxide from nitrate. We will evaluate whether early initiation and sustained supplementation with inorganic nitrate capsules can control blood pressure and improve maternal and perinatal outcomes in women with chronic hypertension during pregnancy.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis randomized, triple-blind, placebo-controlled clinical trial intends to recruit an estimated sample size of 144 pregnant women with chronic hypertension. Once included, participants will receive standard antihypertensive treatment and preeclampsia prophylaxis and will be randomized into either the treatment or placebo group. Starting from the 16th week and continuing until delivery, patients will undergo continuous monitoring of blood pressure, maternal and fetal ultrasonography, as well as biochemical and dietary assessments. The primary aim of the study is to evaluate blood pressure control. At the same time, secondary objectives include monitoring the need for additional antihypertensive medications, the occurrence of preeclampsia and fetal growth restriction, Doppler parameters, biochemical and inflammatory markers profiles, and perinatal outcomes. Data analyses will be conducted using both R and Stata software tools for descriptive and analytic statistics, with a significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003ch2\u003eDiscussion\u003c/h2\u003e \u003cp\u003eEvaluating the efficacy of inorganic nitrate as an adjunctive treatment for chronic hypertension during pregnancy could provide an alternative to conventional antihypertensive therapies. Successful results may offer a safer and natural option to help manage blood pressure, improve outcomes for both mothers and infants, and potentially guide the development of new strategies for managing hypertensive disorders in pregnant patients.\u003c/p\u003e\u003ch2\u003eTrial registration:\u003c/h2\u003e \u003cp\u003eClinicalTrials.gov NCT06105775 (Registration Date: October 27, 2023 \u0026ndash; updated on March 26, 2025).\u003c/p\u003e","manuscriptTitle":"NIT_CH: A Study Protocol for Evaluating the Effect of Inorganic Nitrate Capsules in Chronic Hypertensive Pregnancies – A Triple-Blind Placebo-Controlled Randomized Trial","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-15 08:29:33","doi":"10.21203/rs.3.rs-8157989/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2025-12-12T16:19:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-22T10:06:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-22T10:05:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pregnancy and Childbirth","date":"2025-11-19T18:13:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pregnancy-and-childbirth","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prch","sideBox":"Learn more about [BMC Pregnancy and Childbirth](http://bmcpregnancychildbirth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/prch/default.aspx","title":"BMC Pregnancy and Childbirth","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b8f80b8f-64f6-4a8c-ad1a-68f5a1e1f8dc","owner":[],"postedDate":"December 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T16:07:16+00:00","versionOfRecord":{"articleIdentity":"rs-8157989","link":"https://doi.org/10.1186/s12884-025-08596-8","journal":{"identity":"bmc-pregnancy-and-childbirth","isVorOnly":false,"title":"BMC Pregnancy and Childbirth"},"publishedOn":"2026-01-09 15:57:38","publishedOnDateReadable":"January 9th, 2026"},"versionCreatedAt":"2025-12-15 08:29:33","video":"","vorDoi":"10.1186/s12884-025-08596-8","vorDoiUrl":"https://doi.org/10.1186/s12884-025-08596-8","workflowStages":[]},"version":"v1","identity":"rs-8157989","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8157989","identity":"rs-8157989","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}