Gestational Diabetes Mellitus and Biomarker Profiles: A BMI-Stratified Analysis of Gremlin 1 and BMP 4—A Cross-Sectional Study

Comments 1: Journal Diagnostics (ISSN 2075-4418) Manuscript ID diagnostics-4080941 Type Article Title Gestational Diabetes Mellitus and Biomarker Profiles: A BMI Stratified Analysis of Gremlin 1 and BMP 4— A Cross Sectional Study Dear authors, I have red your cross sectional study concerning measurement of new biomarkers - Gremlin 1 and BMP 4 in gestational diabetes mellitus (GDM).  GDM with constantly rising incidence is becoming highly interesting both for clinicians and investigators, and new biomarkers are investigated. Therefore, any investigation of GDM biomarkers is interesting.  In your study you investigated two biomarkers - Gremlin 1 and BMP 4 in patients with diabetes and controls. Introduction is correct. Aim of the study is concise and clear. Methodology describes investigation. 

Response 1: We appreciate the reviewer's thoughtful evaluation of our manuscript and the recognition of the clinical relevance of investigating novel biomarkers in gestational diabetes mellitus. The reviewer's acknowledgment that our introduction, study aim, and methodology are appropriately structured is encouraging. We have carefully considered all subsequent comments and have made substantial revisions to strengthen the manuscript accordingly.

Comments 2: Could you explain lines 63-66?

Response 2: We sincerely appreciate the reviewer for catching this oversight. Lines 63-66 contained template instructions from the journal's manuscript preparation guidelines that were inadvertently left in the submitted version. This text ("Research manuscripts reporting large datasets that are deposited in a publicly available database should specify where the data have been deposited and provide the relevant accession numbers. If the accession numbers have not yet been obtained at the time of submission, please state that they will be provided during review. They must be provided prior to publication.") was part of the journal's formatting template and should have been removed before submission.

We have now removed this template text entirely from the revised manuscript. This paragraph no longer appears in the document.

Location of change: Original lines 63-66 (page 3) have been deleted.

Comments 3: Results are clearly presented. However, you didn`t find  significance in investigated parameters. I would suggest, in order to complete your survey and improve your study to present data concerning maternal and fetal/neonatal complications and delivery and evaluate whether there was any difference concerning pregnancy outcome and biomarker levels. 

Response 3: We sincerely appreciate this valuable suggestion, which has substantially enhanced the clinical relevance of our manuscript. Following the reviewer's recommendation, we have now incorporated comprehensive analyses of maternal metabolic parameters, delivery outcomes, and fetal parameters, along with their associations with the investigated biomarkers.

Our expanded analysis revealed several important findings: HOMA-IR and TyG was significantly elevated in the GDM group, QUICKI was significantly lower in the GDM group. All three insulin resistance markers confirmed the expected metabolic derangements in GDM.

Cesarean section rates were numerically higher in the GDM group (57.1% vs 40.5%), though this did not reach statistical significance. Mean fetal birth weight showed a trend toward being higher in the GDM group (3456 ± 529 g vs 3235 ± 426 g, p=0.054), approaching statistical significance. Correlation analyses revealed that neither Gremlin-1 nor BMP-4 showed significant associations with: HOMA-IR (Gremlin-1: r=-0.153, p=0.201; BMP-4: r=-0.099, p=0.407), TyG index (Gremlin-1: r=-0.030, p=0.803; BMP-4: r=-0.025, p=0.833), QUICKI (Gremlin-1: r=0.129, p=0.281; BMP-4: r=0.085, p=0.475), Fetal birth weight (Gremlin-1: r=-0.120, p=0.315; BMP-4: r=-0.118, p=0.323).

Location of changes: Abstract section (Page 1, lines 17-21). Results section (Page 4, lines 122-127) (Page 4, lines 116-117). Table 1: Comparative Analysis of Demographic, Fetomaternal, and Biomarker Profiles Between Groups. Discussion section 4.1. Metabolic Characterization and Insulin Resistance Markers (Page 6, lines 181-200)

Comments 4: Discussion is correct explaining that no significance has been found. Limitations of the study have been explained by the authors. 

Response 4: We value the reviewer's positive assessment of our Discussion section. The reviewer's recognition that we have appropriately addressed the non-significant findings and transparently discussed study limitations is encouraging. Following the suggestions from previous comments, we have now substantially expanded the Discussion to incorporate:

Clinical implications of pregnancy outcomes, metabolic marker associations, and lipid metabolism findings.

Comments 5: Conclusion repeats findings and emphasize the need for GDM biomarkers investigations. Although investigated biomarkers didn`t show expected results, this study may be considered inteersting, as it investigates highly interesting topic, and therefore may be published with extended results.

Response 5: We are grateful for the reviewer's overall positive evaluation and the recommendation for publication with extended results. The reviewer's acknowledgment that our study addresses a highly interesting and clinically relevant topic is encouraging, particularly given the null findings for the primary biomarkers.

We appreciate the reviewer's recognition that negative results contribute valuable information to the field. The lack of significant differences in Gremlin-1 and BMP-4 levels between GDM and control groups, and their absence of correlation with metabolic and lipid parameters, provides important evidence that helps refine our understanding of these biomarkers' roles in GDM pathophysiology. As the reviewer notes, this underscores the continued need for comprehensive biomarker investigations in GDM. Following all the reviewer's constructive suggestions, we have substantially extended our results to include:

Comprehensive lipid profile analysis (HDL-C, LDL-C, total cholesterol, triglycerides)

Multiple insulin resistance indices (HOMA-IR, TyG, QUICKI)

Pregnancy outcomes (delivery mode, fetal birth weight)

Extensive correlation analyses between biomarkers and all metabolic, lipid, and clinical parameters

Enhanced discussion contextualizing our findings within recent literature

We have also refined the Conclusion section to better balance the presentation of our findings with their implications for future research, while maintaining appropriate emphasis on the ongoing need for GDM biomarker discovery.

3. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Response 1: We appreciate the reviewer's positive assessment of the manuscript's language quality. We have carefully reviewed the entire manuscript to ensure clarity, consistency, and adherence to scientific writing standards throughout all sections, including the newly added content.

4. Additional clarifications

Regarding the expanded analyses:

Following the reviewers' valuable suggestions, we have substantially enhanced our manuscript by incorporating comprehensive metabolic and clinical outcome data.  

We wish to emphasize that while our primary biomarkers (Gremlin-1 and BMP-4) did not show the expected differences between GDM and control groups, we believe these negative results contribute valuable information to the field. The absence of associations between these biomarkers and established metabolic markers, lipid parameters, and clinical outcomes helps refine our understanding of their roles in GDM pathophysiology and highlights the need for: Longitudinal measurements throughout pregnancy, mechanistic studies exploring specific pathways and larger multicenter investigations with diverse populations.

Comments 1: The manuscript entitled "Gestational Diabetes Mellitus and Biomarker Profiles: A BMI-Stratified Analysis of Gremlin 1 and BMP 4— A Cross Sectional Study" explores a relevant topic.

However, the text is not well developed and presents clear points where the use of AI for writing the article can be easily detected, such as in the Materials and Methods section, where it is possible to observe the following paragraph described in lines 62-66:

"Research manuscripts reporting large datasets that are deposited in a publicly available database should specify where the data has been deposited and provide the relevant accession numbers. If the accession numbers have not yet been obtained at the time of submission, please state that they will be provided during review. They must be provided prior to publication."

These questions, even those raised in the text by the authors themselves through the use of detectable AI, are pertinent. 

Response 1: We sincerely appreciate the reviewer's careful reading of our manuscript and for bringing this issue to our attention. We acknowledge the reviewer's concerns regarding both the specific template text and the overall manuscript development.

Regarding lines 62-66: We apologize for this oversight. The paragraph in question was part of the journal's manuscript preparation template and was inadvertently left in the submitted version during the formatting process. This was a simple copy-paste error during manuscript preparation, not related to AI usage. We have now completely removed this template text from the revised manuscript.

Regarding manuscript development and writing style: We want to clarify that while we used AI tools for language polishing and grammar checking (as is increasingly common in scientific writing), all scientific content, study design, data analysis, interpretation, and conclusions are entirely our own work based on our original research.

After considering the reviewer's feedback, we have rewritten sections to ensure more natural, human-centered scientific writing throughout. Technical descriptions have been simplified and made more direct. We also have expanded the Results and Discussion sections substantially (as requested by Reviewer 1) to provide more comprehensive clinical context. We have replaced any overly formal or template-like phrasing with more straightforward scientific prose. We have incorporated more interpretation and discussion that reflects our specific findings and clinical experience. We have been particularly careful to ensure that the revised manuscript maintains an authentic scientific voice while meeting high standards for clarity and precision. All additions and revisions have been marked in red throughout the manuscript for easy identification.

Throughout manuscript: Language revised for more natural scientific writing style

We hope these revisions address the reviewer's concerns and demonstrate the genuine scientific contribution of our work. We remain committed to transparent and authentic scientific communication.

Comments 2: It is essential that research involving human subjects clearly presents ethical aspects, with the proper approval from the ethics committee, which was not presented in this article.

Response 2: We appreciate the reviewer raising this critical point. We would like to respectfully clarify that ethical approval information was included in the original manuscript submission. The manuscript contains dedicated sections addressing ethical aspects:

Institutional Review Board Statement: "The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board (Ethics Committee) of Recep Tayyip Erdoğan University Faculty of Medicine (Approval number: 2024/200; approval date: July 23, 2024)."

Informed Consent Statement: "Informed consent was obtained from all subjects involved in the study."

These statements were included in the manuscript following the journal's formatting guidelines. However, we thought that the reviewer may have found the presentation insufficient or the location of these statements unclear. To address this concern and enhance clarity, we have now expanded the ethics information within the Materials and Methods section with additional details: "This study was conducted in full compliance with the Declaration of Helsinki and received approval from the Institutional Review Board (Ethics Committee) of Recep Tay-yip Erdoğan University Faculty of Medicine (Approval number: 2024/200; approval date: July 23, 2024) at the Department of Obstetrics and Gynecology, Recep Tayyip Erdoğan University Training and Research Hospital. Pregnant women diagnosed with GDM or newly diagnosed during routine antenatal follow-up, as well as healthy pregnant women, who provided written informed consent after receiving detailed information about the study objectives, procedures, potential risks, and benefits were recruited. Participants were informed of their right to withdraw from the study at any time without affecting their clinical care. Patient confidentiality was strictly maintained throughout the study, and all data were anonymized prior to analysis."

Location of changes: Original ethics statements remain in their designated sections per journal format. Enhanced ethics information added to Materials and Methods section, 2.1. Study Population and Ethnicity (Page 3, lines 66-69).

We hope this clarification and the additional detail address the reviewer's concern regarding the ethical aspects of our research.

Comments 3: The review results are presented in figures of quality that could be improved; submission in JPEG or PNG format is suggested for better quality.

Response 3: We appreciate the reviewer's attention to figure quality. Following this recommendation, we have converted the figure to high-resolution (1646 × 962 pixels, 300 dpi) PNG format.

Location of change: Figure 1 (Page 6, line 161).

Comments 4: In the conclusion section, "Limitations" presents significant concerns that diminish the quality of the work to the point of failing to engage the reader and distrust the study. I suggest rewriting it to avoid these problems.

Response 4: We genuinely appreciate this constructive feedback. The reviewer raises an important point about how we presented our study limitations. Upon reflection, we recognize that our original limitations section may have been overly critical and potentially undermined confidence in our findings. We have now completely rewritten the limitations section to present a more balanced perspective that acknowledges methodological constraints while maintaining appropriate confidence in our results.

Location of change: Conclusion/Discussion section, "Limitations" subsection rewritten.

Comments 5: Overall, the text presents an interesting topic, but with these important ethical concerns. I recommend a careful review of the language throughout the manuscript, especially regarding the English and the use of AI.

Response 5: We appreciate the reviewer's overall positive assessment of our topic and take the ethical and language concerns seriously.

Regarding ethical concerns: As clarified in Response 2, ethical approval was included in the original manuscript and has now been expanded in the Materials and Methods section for greater clarity and transparency.

Regarding language and writing: We have conducted a comprehensive review of the entire manuscript with specific attention to natural scientific writing, clarity, directness, and consistency.

While we used AI tools for grammar checking and language polishing (a common practice in scientific writing), all scientific content, analysis, interpretation, and conclusions are entirely our original work. The revised manuscript has been carefully edited to ensure it maintains a professional scientific voice.

Comments 6: I also suggest updating the information in Reference 10 of the American Diabetes Association. The most recent version of the ADA Standards of Care (2026) has already been published online.

Response 6: Thank you for this important update. We have now updated Reference 10 to cite the most recent ADA Standards of Care (2026).

Location of change: Reference list, Reference 10 updated to ADA Standards of Care 2026 (Page 12, lines 467,468).

Comments 7: I recommend a careful review of the language throughout the manuscript, especially regarding English.

Response 7: We have conducted a thorough language review of the entire manuscript, focusing on grammar and syntax, scientific terminology, flow and coherence. The revised manuscript has been carefully edited to meet high standards for scientific English.  

Location of changes: Language improvements throughout all sections of the manuscript (Introduction, Materials and Methods, Results, Discussion, Conclusion).

Comments 1: Markers, various proteins, adipokines, genetic factors, etc. that are involved in the etiopathogenesis and evolution of DG have different values ​​and influence in different populations. It was necessary to specify which racial groups the patients in the study groups belong to. In this context, the study confirms or not the conclusions of other studies conducted on various population groups.

Response 1: We sincerely appreciate this important observation. The reviewer correctly highlights that biomarker profiles can vary significantly across different ethnic and racial populations due to genetic, environmental, and lifestyle factors. We acknowledge that we should have explicitly stated the ethnic composition of our study population in the original manuscript.

Our study was conducted at Recep Tayyip Erdoğan University Faculty of Medicine in Rize, Turkey. All participants were of Turkish ethnicity from the Eastern Black Sea region of Turkey. This population is relatively homogeneous in terms of ethnic background, which reduces confounding from population stratification but also limits generalizability to other ethnic groups. We have now added this important demographic information to the manuscript in the following locations: Materials and Methods section 2.1. Study Population and Ethnicity (Page 3, Lines 70-71) and Discussion section 4.3. Gremlin 1 and BMP 4 (Page 8 Lines 251-267) and 4.7. Limitations (Page 10, Lines 356-358).

Comments 2: Since Gremlin-1 changes are associated with altered lipid metabolism in pregnant women, it was useful to also take these aspects into consideration. Highlighting the changes in lipid biological samples in pregnant women with DG in correlation with Gremlin-1 values ​​would increase the value of the study.

Response 2: We greatly appreciate this insightful comment, which has significantly enhanced the clinical depth of our manuscript. The reviewer correctly points out the established relationship between Gremlin-1 and lipid metabolism in pregnancy, as demonstrated by recent studies (Deischinger et al., 2023). Following this valuable suggestion, we have now incorporated comprehensive lipid profile analyses and their correlations with Gremlin-1 and BMP-4 levels.

Our expanded analysis includes the following lipid parameters: TC, LDL-C, HDL-C, TG, and TyG index (triglyceride-glucose index, a composite marker of insulin resistance and lipid-glucose metabolism).

Key findings from lipid profile analysis: TG levels were significantly elevated in the GDM group compared to controls (121.77 ± 83.80 mg/dL vs 87.16 ± 39.30 mg/dL, p=0.015), TC showed a numerical increase in the GDM group (172.49 ± 35.10 mg/dL vs 163.14 ± 34.78 mg/dL, p=0.130), though this did not reach statistical significance. HDL-C and LDL-C levels did not differ significantly between groups. Spearman correlation analyses revealed that neither Gremlin-1 nor BMP-4 showed significant associations with any lipid parameters: Gremlin-1 vs HDL-C: r=-0.011, p=0.929; Gremlin-1 vs TC: r=-0.099, p=0.407; Gremlin-1 vs LDL-C: r=-0.043, p=0.719; Gremlin-1 vs TG: r=-0.046, p=0.700; BMP-4 vs HDL-C: r=-0.024, p=0.842; BMP-4 vs TC: r=-0.092, p=0.442; BMP-4 vs LDL-C: r=-0.017, p=0.890; BMP-4 vs TG: r=-0.048, p=0.686.

Location of changes: Abstract section (Page 1, line 17-18), Results section 3.1. Comparison between the GDM and control groups (Page 4, lines 127-130) and 3.3. Correlation analysis (Page 5, lines 156-169), Table 1 (revised): Now includes lipid parameters, Materials and Methods section 2.2. Sample Collection and Biochemical Analysis (Page 3, lines 97-99), Discussion section “4.2. Lipid Metabolism and Dyslipidemia” (Page 7, lines 202-220)

Comments 3: It was necessary to specify in the introduction that there are several forms of BMP, some with negative effects (4) and others with protective effects (7) in DG.

Response 3: We thank the reviewer for this important suggestion. The reviewer correctly points out that the BMP family comprises multiple members with distinct and sometimes opposing metabolic effects. We have now expanded the Introduction to clarify the differential roles of BMP family members, particularly contrasting BMP-4 and BMP-7.

We have added the following paragraph to the Introduction section:

Location of change: Introduction section (Page 2, lines 49-56)

Comments 4: BMP 4 may determine, along with other factors, the occurrence of hypertension in pregnancy. It was interesting to study this potential effect in the studied groups.

Response 4: We appreciate the reviewer's suggestion regarding the potential relationship between BMP-4 and hypertensive disorders of pregnancy. This represents an interesting research direction worth exploring in future studies.

However, we were unable to evaluate this relationship in our current study because hypertensive disorders (chronic hypertension, gestational hypertension, and preeclampsia) were among our exclusion criteria, as stated in the Materials and Methods section. Our study population consisted exclusively of normotensive pregnant women to focus specifically on GDM-related metabolic effects without confounding from hypertensive disorders.

4. Response to Comments on the Quality of English Language

Point 1: (x) The English is fine and does not require any improvement.

Response 1: We appreciate the reviewer's positive assessment of the manuscript's language quality. Throughout the revision process, we have maintained careful attention to clarity, precision, and adherence to scientific writing standards. All newly added sections (metabolic parameters, lipid profiling, pregnancy outcomes, ethnic considerations, and BMP family diversity) have been written with the same attention to language quality and have been thoroughly reviewed to ensure consistency with the rest of the manuscript.