Review Reports - NT-proBNP as a Predictive and Prognostic Biomarker for Complications in Hypertensive Pregnancy Disorders

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript reports a prospective, multicenter observational study with 180 patients of the utility of NT-proBNP as a diagnostic adjuvant, or substitute for the sFlt-1/PLGF assay, for the diagnosis of preeclampsia in Romania. The present study complements a previous meta-analysis (reference 10) demonstrating the efficacy of NT-proBNP in the diagnosis of suspected preeclampsia with a real-time prospective study that compares NT-proBNP to the more conventional sFlt-1/PLGF assay. Patients were grouped into 3 categories for analysis: preeclampsia (Group A), gestational hypertension or chronic hypertension (HDP) (Group B), and normotensive controls (Group C). The major findings from the study were that: 1. NT-proBNP levels were significantly higher than in HDP or normotensive controls, and NT-proBNP concentrations correlated with disease severity; 2. An elevated NT-proBNP yielded an AUC of 0.78 (95% CI 0.71-0.84), comparable to sFlt-1/PLGF (AUC 0.84); combining NT-proBNP with sFlt-1/PLGF improved the diagnostic efficacy to an AUC of 0.88 ((95% CI 0.82-0.92).

The manuscript is very well written, with a thoughtful discussion and recognition of limitations and suggests further studies where sFlt-1/PLGF may not be readily available. The statical analysis is appropriate and the results are clearly presented. However, there are several minor criticisms that merit consideration:

Thue authors suggest NT-proBNP as an alternative to sFlt-1/PLGF as a less cumbersome technique that might be advantage in low resource settings. However, the sFlt-1/PLGF assay is now widely available as a single use assay for point-of-care testing and is already in wide use in low resource settings
- The NT-proBNP kit used in the current study still requires a 96 well plate and an OD reader, diminishing it’s appeal in low resource settings
Women in Group A already had been diagnosed as having preeclampsia. Thus, NT-proBNP may be useful as a diagnostic adjuvant or predictor of severity, but may not be useful in preeclampsia screening algorithms. This should be recognized by the authors.
As noted in the discussion, many studies have already correlated NT-proBNP with preeclampsia and severe preeclampsia and with other angiogenic markers like sFlt-1/PLGF (reference 21), reducing the novelty of the present study
Table 3, Group A. it is not clear in the table what the p-value is comparing. Presumably, it is comparing Group A to the combined Groups B and C, as indicated in the text (lines 202-204) but this should also be made clear in the Table

Overall, this is a well written manuscript, with analysis and results clearly presented that provides useful information and support for a role for NT-proBNP in the diagnosis of preeclampsia.

Author Response

The authors acknowledge the valuable observations and suggestions of the reviewer’s as concerns the manuscript entitled

NT-proBNP as a Predictive and Prognostic Biomarker for Complications in Hypertensive Pregnancy Disorders

Diana Mocuta ¹, Cristina Aur ^1*, Ioana Alexandra Zaha^1,2, Carmen Delia Nistor Cseppento³, Liliana Sachelarie ⁴^*, Anca Huniadi^1,²

According to the reviewer’s recommendations, all the suggestions were taken into account, as follows:

Thue authors suggest NT-proBNP as an alternative to sFlt-1/PLGF as a less cumbersome technique that might be advantage in low resource settings. However, the sFlt-1/PLGF assay is now widely available as a single use assay for point-of-care testing and is already in wide use in low resource settings

The NT-proBNP kit used in the current study still requires a 96 well plate and an OD reader, diminishing it’s appeal in low resource settings

We fully agree that the sFlt-1/PlGF ratio is currently available as a single-use point-of-care assay and is increasingly implemented in a wide range of healthcare settings, including those with limited resources.

Our intention was not to suggest that NT-proBNP is inherently less technically demanding than sFlt-1/PlGF in its current laboratory implementation, nor to compare assay platforms. Instead, our focus was on the biomarker concept and its potential clinical applicability, particularly in settings where angiogenic testing may still be unavailable, delayed, or financially constrained.

Although ELISA was used to measure NT-proBNP in the present study, we acknowledge that this approach requires laboratory infrastructure. Importantly, however, NT-proBNP is routinely available worldwide on automated immunoassay platforms commonly used in cardiology and internal medicine, often with lower per-test costs and broader laboratory penetration than angiogenic assays. We have now clarified this distinction and revised our statements regarding applicability in low-resource settings accordingly.

Women in Group A already had been diagnosed as having preeclampsia. Thus, NT-proBNP may be useful as a diagnostic adjuvant or predictor of severity, but may not be useful in preeclampsia screening algorithms. This should be recognized by the authors.

We thank the reviewer for this necessary clarification. Our findings support the use of NT-proBNP primarily as a prognostic marker and predictor of disease severity and maternal–fetal complications, rather than as a tool for population-level screening. The study aimed to evaluate the ability of NT-proBNP to refine risk stratification among women with established hypertensive disorders of pregnancy, particularly in identifying those at higher risk of adverse outcomes.

We have now explicitly acknowledged this distinction in the manuscript and clarified that the potential role of NT-proBNP in early screening algorithms would require dedicated prospective studies with first-trimester sampling and unselected populations.

This distinction has now been explicitly clarified in the Introduction and Discussion, and acknowledged as a limitation of the study.

As noted in the discussion, many studies have already correlated NT-proBNP with preeclampsia and severe preeclampsia and with other angiogenic markers like sFlt-1/PLGF (reference 21), reducing the novelty of the present study

We fully acknowledge that previous studies have demonstrated associations between NT-proBNP levels, preeclampsia severity, and angiogenic markers such as the sFlt-1/PlGF ratio. The present study does not aim to replicate these associations per se. The novelty of our work lies in several complementary aspects. First, we identified a clinically actionable NT-proBNP threshold (200 pg/mL) that was independently associated with maternal–fetal complications, supported by ROC analysis and multivariable regression. Second, we formally evaluated the incremental prognostic value of NT-proBNP beyond the sFlt-1/PlGF ratio, using discrimination and reclassification metrics (ΔAUC and NRI), rather than simple correlation analyses. Third, this study provides prospective multicenter data from a Romanian cohort, a population underrepresented in the existing literature, where access to angiogenic testing remains heterogeneous.

We have revised the manuscript to clarify the original contributions and avoid overstating novelty based solely on biomarker associations.

In the Discussion section, a clarifying paragraph was added after the review of previous studies to explicitly distinguish previously reported associations from the original contributions of the present work, emphasizing threshold definition, prognostic modeling, and reclassification analyses.

Table 3, Group A. it is not clear in the table what the p-value is comparing. Presumably, it is comparing Group A to the combined Groups B and C, as indicated in the text (lines 202-204) but this should also be made clear in the Table

To improve clarity and ensure that the table is self-explanatory, we have revised Table 3 to indicate the comparisons underlying the reported p-values explicitly. In Table 3, a clarifying footnote was added specifying that p-values represent comparisons between Group A and the combined Groups B and C.

Overall, this is a well written manuscript, with analysis and results clearly presented that provides useful information and support for a role for NT-proBNP in the diagnosis of preeclampsia.

We are grateful for the insightful and constructive comments, which significantly contributed to improving the clarity, rigor, and presentation of the manuscript.

Thank you,

Prof. Dr. Liliana Sachelarie

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Congratulations to the authors for addressing this important topic.

Please elaborate more on NT pro BNP, the pathophysiology, in which conditions is elevated, especially the pathway that may play a causal role in HDP pathogenesis.

Which was the exact period of the study, which 2 years since the Ethical approval is from 11/2024?

Figure 1. The total number of pregnancies was 210 during 2 years?

Measuring NT pro BNP from 20 weeks of gestation is a long period, since NT pro BNP changes throughout trimesters. Could you specify in which week/trimester you measured this marker in patients? Also, a table of normal ranges during pregnancy should be implemented.

Author Response

The authors acknowledge the valuable observations and suggestions of the reviewer’s as concerns the manuscript entitled

NT-proBNP as a Predictive and Prognostic Biomarker for Complications in Hypertensive Pregnancy Disorders

Diana Mocuta ¹, Cristina Aur ^1*, Ioana Alexandra Zaha^1,2, Carmen Delia Nistor Cseppento³, Liliana Sachelarie ⁴^*, Anca Huniadi^1,²

According to the reviewer’s recommendations, all the suggestions were taken into account, as follows:

Congratulations to the authors for addressing this important topic.

Please elaborate more on NT pro BNP, the pathophysiology, in which conditions is elevated, especially the pathway that may play a causal role in HDP pathogenesis.

The Discussion section was expanded to elaborate further the pathophysiological context of NT-proBNP in hypertensive disorders of pregnancy, focusing on its integrative role in cardiovascular, vascular, and renal stress responses.

NT-proBNP is released from ventricular cardiomyocytes in response to increased myocardial wall stress and volume overload and is generally regarded as an integra-tive marker of cardio–renal strain. In pregnancy, elevated NT-proBNP levels may re-flect maladaptive maternal cardiovascular responses, particularly in the setting of in-creased afterload, endothelial dysfunction, and altered plasma volume regulation that characterize hypertensive disorders of pregnancy. Angiogenic imbalance, a hallmark of preeclampsia, may further amplify myocardial stress through systemic vascular re-sistance and microvascular dysfunction. From a pathophysiological perspective, na-triuretic peptide signaling is therefore thought to integrate hemodynamic, vascular, and renal stress pathways rather than acting as a disease-specific trigger. In this con-text, NT-proBNP may serve as an integrative biomarker reflecting the complex cardio–vascular–renal interactions underlying HDP severity.

Which was the exact period of the study, which 2 years since the Ethical approval is from 11/2024?

The manuscript has been revised to clearly specify that this was a prospective multicenter observational study conducted between November 2024 and October 2025. Ethical approval was obtained prior to participant enrollment (Ethics Committee approval No. 35973/21.11.2024). All participants provided written informed consent before inclusion in the study. The study period and design are now described consistently throughout the manuscript to avoid any ambiguity.

This prospective, multicenter observational study was conducted between November 2024 and October 2025 at the Bihor County Emergency Clinical Hospital and the Pelican Clinical Hospital–Medicover Oradea, Romania, to investigate the prognostic value of NT-proBNP in hypertensive disorders of pregnancy and its complementary role relative to the angiogenic sFlt-1/PlGF ratio. The study protocol was approved by the Ethics Committee of the Bihor County Emergency Clinical Hospital (No. 35973/21.11.2024), and all procedures were conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants in accordance with institutional requirements.

Figure 1. The total number of pregnancies was 210 during 2 years?

The Methods section has been revised to specify the study period clearly and to clarify that 210 pregnant women were assessed for eligibility, of whom 180 were included in the final analysis.

Measuring NT pro BNP from 20 weeks of gestation is a long period, since NT pro BNP changes throughout trimesters. Could you specify in which week/trimester you measured this marker in patients? Also, a table of normal ranges during pregnancy should be implemented.

NT-proBNP was measured at a single time point at study enrollment, after 20 weeks of gestation, predominantly during the second and third trimesters of pregnancy. Gestational age at sampling was recorded for all participants. This information has now been explicitly added to the Methods section and summarized in the Abstract to improve clarity. As the objective of the present study was to evaluate the prognostic value of NT-proBNP in hypertensive disorders of pregnancy rather than to establish trimester-specific reference intervals, NT-proBNP was not measured longitudinally across pregnancy. Therefore, a table of normal ranges during pregnancy was not included.

We are grateful for the insightful and constructive comments, which significantly contributed to improving the clarity, rigor, and presentation of the manuscript.

Thank you,

Prof. Dr. Liliana Sachelarie

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “NT-proBNP as a Predictive and Prognostic Biomarker for Complications in Hypertensive Pregnancy Disorders” investigates the association between NT-proBNP and hypertensive disorders of pregnancy in a Romanian cohort. The authors report significantly higher NT-proBNP levels in preeclampsia compared with gestational hypertension, chronic hypertension, and healthy controls. In addition, NT-proBNP showed a moderate correlation with the sFlt-1/PlGF ratio, suggesting that it reflects complementary pathophysiological mechanisms beyond angiogenic imbalance. Overall, the study is well designed and includes appropriate control groups. However, there is a marked imbalance in sample sizes between study groups, which may affect statistical power and should be more clearly acknowledged. The findings are clinically relevant and contribute to the identification of novel biomarkers for hypertensive disorders of pregnancy. Nevertheless, several points should be addressed to improve clarity and presentation of the manuscript.

Comments

The introduction would benefit from elaborating on why angiogenic biomarkers (sFlt-1 and PlGF) are currently the most validated predictors of preeclampsia would help contextualize the rationale for comparing NT-proBNP with these markers.
The term hypertensive disorders of pregnancy is used interchangeably in full and as the abbreviation HDP. Please choose one format and use it consistently throughout the manuscript. In addition, several other abbreviations appear to be introduced more than once. A careful review of the entire text to ensure consistent and single time introduction of all abbreviations is recommended.
In Tables 1 and 2, p-values are reported for some rows but omitted for others. It appears that only statistically significant p-values are shown if I am not wrong. For transparency, all p-values should be reported, with statistically significant values clearly highlighted (e.g., bold font).
For Tables 1 and 2, it is unclear whether the reported p-values reflect comparisons among all three groups simultaneously or pairwise comparisons. Please clarify the statistical approach used. If global comparisons were performed, it may also be appropriate to include pairwise comparisons versus the control group, particularly for clinically relevant variables
The current organization and formatting of the tables make interpretation difficult. Improving table structure, alignment, and grouping of related variables would enhance readability and clarity.
The inclusion criterion specifies gestational age above 20 weeks. Did the authors assess whether NT-proBNP levels differed between earlier versus later gestational ages within the cohort? An analysis or brief discussion addressing potential gestational age–dependent effects would strengthen the manuscript.
NT-proBNP is described as an inactive cleavage product of proBNP. Could the authors clarify whether NT-proBNP itself has defined target tissues or receptor-mediated signaling pathways.

Author Response

The authors acknowledge the valuable observations and suggestions of the reviewer’s as concerns the manuscript entitled

NT-proBNP as a Predictive and Prognostic Biomarker for Complications in Hypertensive Pregnancy Disorders

Diana Mocuta ¹, Cristina Aur ^1*, Ioana Alexandra Zaha^1,2, Carmen Delia Nistor Cseppento³, Liliana Sachelarie ⁴^*, Anca Huniadi^1,²

According to the reviewer’s recommendations, all the suggestions were taken into account, as follows:

Comments

The introduction would benefit from elaborating on why angiogenic biomarkers (sFlt-1 and PlGF) are currently the most validated predictors of preeclampsia would help contextualize the rationale for comparing NT-proBNP with these markers.

The Introduction has been revised to further elaborate on the pathophysiological basis and clinical validation of angiogenic biomarkers. We now explicitly describe the central role of sFlt-1–mediated angiogenic imbalance in preeclampsia and highlight why the sFlt-1/PlGF ratio is considered the most validated biomarker and incorporated into international guidelines. This clarification better contextualizes the rationale for comparing NT-proBNP with angiogenic markers as a complementary, rather than competing, biomarker.

The term hypertensive disorders of pregnancy is used interchangeably in full and as the abbreviation HDP. Please choose one format and use it consistently throughout the manuscript. In addition, several other abbreviations appear to be introduced more than once. A careful review of the entire text to ensure consistent and single time introduction of all abbreviations is recommended.

The manuscript has been carefully revised to ensure consistent use of terminology. The term “hypertensive disorders of pregnancy” is now introduced in full at first mention and subsequently used consistently as the abbreviation “HDP” throughout the text.

The entire manuscript was carefully reviewed to ensure that all abbreviations are introduced only once at first appearance and used consistently throughout the text.

In Tables 1 and 2, p-values are reported for some rows but omitted for others. It appears that only statistically significant p-values are shown if I am not wrong. For transparency, all p-values should be reported, with statistically significant values clearly highlighted (e.g., bold font).

Tables 1 and 2 have been revised to report p-values for all variables, including non-significant comparisons. Statistically significant p-values are now consistently highlighted in bold to improve transparency and readability.

For Tables 1 and 2, it is unclear whether the reported p-values reflect comparisons among all three groups simultaneously or pairwise comparisons. Please clarify the statistical approach used. If global comparisons were performed, it may also be appropriate to include pairwise comparisons versus the control group, particularly for clinically relevant variables

p-values reported in Tables 1 and 2 reflect global comparisons among the three study groups, performed using one-way ANOVA or Kruskal–Wallis tests for continuous variables and χ² or Fisher’s exact tests for categorical variables, as appropriate.

Pairwise comparisons versus the control group were not included in the tables, as the primary objective of the study was to evaluate the independent and complementary prognostic value of NT-proBNP using multivariable regression models, rather than to perform multiple unadjusted subgroup comparisons. This approach was chosen to avoid inflation of type I error and to maintain clarity of presentation.

The current organization and formatting of the tables make interpretation difficult. Improving table structure, alignment, and grouping of related variables would enhance readability and clarity.

Tables 1 and 2 were reorganized to improve structure, alignment, and readability. Related variables were grouped more clearly, p-values were uniformly reported and highlighted where appropriate, and section headers were added to distinguish clinically associated outcomes. These changes were implemented to enhance clarity and facilitate the interpretation of the data.

The inclusion criterion specifies gestational age above 20 weeks. Did the authors assess whether NT-proBNP levels differed between earlier versus later gestational ages within the cohort? An analysis or brief discussion addressing potential gestational age–dependent effects would strengthen the manuscript.

This aspect has now been addressed in the Discussion section as a limitation of the study.

NT-proBNP is described as an inactive cleavage product of proBNP. Could the authors clarify whether NT-proBNP itself has defined target tissues or receptor-mediated signaling pathways.

We thank the reviewer for this necessary clarification. NT-proBNP is a biologically inactive cleavage fragment of proBNP and does not have known target tissues or receptor-mediated signaling pathways. Its clinical utility derives from its stability and its role as a circulating biomarker reflecting myocardial stress and neurohormonal activation rather than direct biological effects. This clarification has now been added to the manuscript to avoid potential ambiguity.

We thank you for the constructive and insightful comments, which helped us improve the clarity, methodological transparency, and overall quality of the manuscript.