The AGE–RAGE Pathway in Endometriosis: A Focused Mechanistic Review and Structured Evidence Map

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Although the review is well written, however, following modifications are suggested to further polish the manuscript:

Elevate this point in the Discussion or a new subsection titled "Limitations and Critical Gaps," explicitly state that the current evidence base is largely one of association with RAGE ligands, not demonstration of RAGE-mediated causality. The field needs "receptor-resolution" experiments (e.g., comparative RAGE vs. TLR4 knockdown/blockade, use of RAGE-specific antagonists). The conclusion's final translational agenda should list this as the top priority for mechanistic studies.

Frame the discussion around a clear hierarchy of evidence ie., Biological Plausibility (AGEs are modifiable, patients seek dietary advice), Current Evidence Gap (No interventional trials in endometriosis), Informed Speculation (Based on related conditions (PCOS, diabetes), what endpoints might be affected (systemic inflammation, oxidative stress)? What endpoints are less likely (complete lesion regression)?

These suggestions will move the discussion from "it's plausible but untested" to "here is how to test it."

In abstract consider adding a sentence to explicitly state the main conclusion (e.g., "Current evidence supports a model where the AGE-RAGE axis functions primarily as a disease-amplifying loop involved in chronic inflammation and fibrosis, rather than an initiating trigger.").

In introduction, the transition from the general problem of endometriosis management to the specific hypothesis about AGE-RAGE is excellent. To further strengthen it, consider briefly mentioning the shared metabolic phenotype (insulin resistance, oxidative stress) as the logical bridge that makes AGE-RAGE a plausible candidate pathway.

Ensure consistent use of "AGE-RAGE axis" vs. "AGE-RAGE pathway" throughout.

Reference 139 in the text (Fujii et al.) seems to be incorrectly numbered, as the final reference list entry 139 is Zhu et al.

Author Response

Dear Reviewers we appreciate your support into revisioning this manuscript. Following you can read the specifications point by points.

#1 Reviewer

Although the review is well written, however, following modifications are suggested to further polish the manuscript:

1.Q: Elevate this point in the Discussion or a new subsection titled "Limitations and Critical Gaps," explicitly state that the current evidence base is largely one of association with RAGE ligands, not demonstration of RAGE-mediated causality. The field needs "receptor-resolution" experiments (e.g., comparative RAGE vs. TLR4 knockdown/blockade, use of RAGE-specific antagonists). The conclusion's final translational agenda should list this as the top priority for mechanistic studies.

A: We thank the Reviewer for this important and constructive recommendation. We agree that, in the current endometriosis literature, much of the mechanistic evidence is ligand- and pathway-associated rather than receptor-resolved and therefore does not consistently establish RAGE-dependent causality. In response, we have elevated and clarified this limitation by adding an explicit statement in the Discussion (and, where appropriate, within a dedicated “Limitations and Critical Gaps” subsection) emphasizing the need for receptor-resolution experiments. We now more clearly distinguish ligand-associated effects from receptor-mediated signaling, noting that major RAGE ligands (e.g., HMGB1, S100 proteins) can also engage alternative receptors such as TLR4. Finally, we have aligned the Conclusion and translational agenda to identify receptor-resolution approaches (e.g., comparative RAGE vs. TLR4 knockdown/blockade and/or use of RAGE-specific antagonists) as a top priority for future mechanistic studies.

 

2.Q:Frame the discussion around a clear hierarchy of evidence ie., Biological Plausibility (AGEs are modifiable, patients seek dietary advice), Current Evidence Gap (No interventional trials in endometriosis), Informed Speculation (Based on related conditions (PCOS, diabetes), what endpoints might be affected (systemic inflammation, oxidative stress)? What endpoints are less likely (complete lesion regression)? These suggestions will move the discussion from "it's plausible but untested" to "here is how to test it."

A: We appreciate this helpful suggestion and have revised the Discussion to adopt a clearer hierarchy of evidence. Specifically, we now (i) summarize the biological plausibility for targeting AGE burden and AGE–RAGE signaling, including the modifiability of AGEs and the clinical relevance of patient-driven interest in dietary and lifestyle interventions; (ii) explicitly delineate the key evidence gap, namely the absence of interventional trials in endometriosis directly testing AGE-lowering strategies or RAGE-targeted approaches; and (iii) provide an “informed speculation” framework grounded in adjacent conditions (e.g., PCOS and diabetes) to outline tractable endpoints that are most plausibly affected (e.g., systemic inflammatory markers, oxidative stress indices, metabolic parameters, and fibrosis-related readouts), while also clarifying endpoints that are less likely to be achievable in the near term (e.g., complete lesion regression). This restructuring is intended to move the narrative from plausibility to a more operational agenda for hypothesis-driven study design.

 

3.Q: In abstract consider adding a sentence to explicitly state the main conclusion (e.g., "Current evidence supports a model where the AGE-RAGE axis functions primarily as a disease-amplifying loop involved in chronic inflammation and fibrosis, rather than an initiating trigger."). thank you for the suggestions we followed the advice and make the modification properly.

A: We thank the Reviewer for this suggestion. We have revised the Abstract to include a concise sentence stating the central conclusion of the review, emphasizing that the current evidence is most consistent with an AGE–RAGE-driven disease-amplifying loop contributing to chronic inflammation and fibrosis, rather than a primary initiating trigger.

 

4. Q: In introduction, the transition from the general problem of endometriosis management to the specific hypothesis about AGE-RAGE is excellent. To further strengthen it, consider briefly mentioning the shared metabolic phenotype (insulin resistance, oxidative stress) as the logical bridge that makes AGE-RAGE a plausible candidate pathway.

A: We thank the Reviewer for this insightful recommendation. We have revised the Introduction to make the mechanistic bridge more explicit by briefly highlighting the convergence between endometriosis and a metabolic/oxidative-stress phenotype (including insulin resistance, oxidative stress, and chronic inflammatory stress), which are recognized upstream conditions that promote AGE formation and RAGE-ligand signaling. This addition strengthens the logical transition from unmet clinical need and systemic comorbidities to the specific focus of this review. We have also retained appropriately cautious wording to avoid overgeneralization and to acknowledge heterogeneity across patient phenotypes.

 

5.Q:Ensure consistent use of "AGE-RAGE axis" vs. "AGE-RAGE pathway" throughout.

A: We thank the Reviewer for noting this point. We have standardized terminology throughout the manuscript to ensure consistent use of “AGE–RAGE axis” (or “AGE–RAGE pathway,” as selected) across the Abstract, main text, and figure legends.

 

6. Q: Reference 139 in the text (Fujii et al.) seems to be incorrectly numbered, as the final reference list entry 139 is Zhu et al.

A: We thank the Reviewer for identifying this discrepancy. We have checked the citation numbering and reference list and corrected the in-text citation and corresponding reference numbering to ensure that Fujii et al. is accurately matched to the appropriate entry in the final reference list.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

This is a well-structured and timely review on the AGE–RAGE axis in endometriosis, with strong integration of literature from inflammatory and fibrotic diseases. I suggest clarifying distinctions between ligand-mediated effects and receptor-specific RAGE signaling, elaborating the evidence-mapping framework criteria, and separating structural AGE effects from RAGE-mediated signaling. Expanding the discussion to include broader literature on metabolic and environmental stressors, endothelial dysfunction, EndoMT, and relevant reviews on AGE–RAGE stress responses would provide additional context. Aligning the tone of earlier sections with the cautious conclusions, standardizing abbreviations, and clarifying figure content will further strengthen clarity and coherence. Overall, the manuscript is informative, and addressing these points would enhance its interpretive depth and readability.

Comments for author File: Comments.pdf

Author Response

Dear Reviewers we appreciate your support into revisioning this manuscript. Following you can read the specifications point by points.

1.Q: Throughout the manuscript, the role of RAGE signaling is discussed in detail; however, in some instances the wording may imply a level of receptor-specific mechanistic evidence that is not always directly supported by the underlying studies. Several of the cited works focus on RAGE ligands (e.g., HMGB1, S100 proteins), downstream inflammatory pathways, or associated phenotypes without directly demonstrating RAGE dependency through receptor inhibition or genetic approaches. Given that these ligands can also signal through alternative receptors, a more consistent distinction between ligand-associated effects and receptor-mediated signaling would further strengthen the mechanistic precision of the review.

A: We thank the Reviewer for this important observation and fully agree with the need for greater mechanistic precision. We have revised the manuscript to more consistently distinguish ligand-associated observations (e.g., measurements or perturbations of HMGB1/S100 proteins/AGEs and shared downstream inflammatory nodes such as NF‑κB) from receptor-resolved evidence demonstrating RAGE-dependent causality. In particular, we have refined language in sections describing ligand biology to avoid implying receptor specificity when direct receptor inhibition/genetic approaches were not employed. These changes are reflected in the Discussion (including the paragraph addressing “receptor attribution” as a limitation and the limitations section) and in targeted terminology edits throughout the text where ligand perturbations are discussed.

 

2 Q: The proposed evidence-mapping framework is a useful conceptual tool, but its

methodological basis could be described more explicitly. Studies with heterogeneous

designs, including observational human data, in vitro experiments, and animal models, are

synthesized together, and the criteria used to classify AGE–RAGE roles as drivers,

amplifiers, consequences, or parallel processes are not always clearly defined. Clarifying

these criteria, or explicitly framing the map as a descriptive and hypothesis-generating

framework, would enhance transparency and guide interpretation.

A: We appreciate this constructive recommendation. In response, we have strengthened the description of the methodological basis underpinning the evidence-mapping framework and clarified how heterogeneous study designs were integrated. Specifically, we now (i) explicitly frame the map as a descriptive, hypothesis-generating synthesis intended to organize the current evidence base rather than to infer definitive causality, and (ii) provide clearer operational criteria used to classify observations as “drivers,” “amplifiers,” “consequences,” or “parallel processes,” with explicit attention to the level of experimental control (human observational vs. ex vivo/in vitro vs. animal/interventional data). To enhance transparency, we also direct readers to Table 1 as the primary “traceability” resource, where study design, model system, and key readouts are presented so that the evidentiary basis for each mapped inference can be readily verified.

 

3 Q: The association between RAGE expression and fibrotic features of endometriosis lesions is an important aspect of the manuscript and is generally well supported. Nevertheless, some sections may benefit from a clearer separation between the structural effects of AGEs on extracellular matrix properties and receptor-mediated inflammatory signaling through RAGE. Emphasizing this distinction would help ensure that correlative findings are not unintentionally interpreted as direct causative mechanisms.

A: We thank the Reviewer for this insightful point and agree that a clearer conceptual separation is valuable for interpretation. We have revised the relevant sections to more explicitly differentiate (i) the structural/biophysical effects of AGEs on extracellular matrix properties (e.g., crosslinking, stiffness, altered matrix turnover) from (ii) receptor-mediated inflammatory and pro-fibrotic signaling attributed to RAGE activation. At the same time, we also acknowledge that the available literature does not always permit a strict “either/or” separation, as structural remodeling and inflammatory signaling can be mechanistically intertwined and often co-occur in disease contexts. Accordingly, we have refined the narrative to avoid over-interpreting correlative associations as causative RAGE-mediated mechanisms and have added clarifying language where inference is necessarily limited by study design.

 

4 Q: The discussion of systemic versus local AGE burden is informative, but additional

contextualization would improve interpretative balance. Measurements derived from

distinct biological compartments, such as circulating markers, skin autofluorescence, and

tissue-level expression, are sometimes discussed within a unified mechanistic narrative

despite limited paired or longitudinal data. Explicitly acknowledging the associative and

hypothesis-generating nature of these findings would further strengthen the discussion.

A: We agree with the Reviewer and appreciate the emphasis on interpretative balance across compartments. We have revised the Discussion to more explicitly contextualize that systemic measures (e.g., circulating AGE-related markers, skin autofluorescence) and local tissue-level readouts reflect distinct biological compartments and are not interchangeable, particularly in the absence of paired sampling or longitudinal designs. We now state more clearly that many of these findings remain associative and are best interpreted as hypothesis-generating signals that warrant future studies with paired systemic–local assessments and longitudinal follow-up. In addition, Table 1 is explicitly referenced in this context to enable readers to track compartment, specimen type, and study design for each included report.

 

5 Q: In addition, the authors may consider briefly expanding the contextual framework by

acknowledging broader literature linking metabolic and environmental stressors to

endothelial dysfunction, inflammation, and endothelial-to-mesenchymal transition

(EndoMT), processes that are mechanistically relevant to AGE–RAGE–associated

pathology. A growing body of evidence indicates that diverse non-glycation-related insults,

including endocrine disruptors and uremic toxins, can induce endothelial injury,

inflammatory signaling, cellular senescence, and fibrotic remodeling through pathways

overlapping with those discussed in the manuscript, such as NF-κB activation, oxidative

stress, and EndoMT. Incorporating representative studies from this wider field—for

example, work describing bisphenol-induced endothelial necroptosis and accelerated

endothelial aging (Reventún et al., Sci Rep 2020; Moreno-Gómez-Toledano et al.,

Biomolecules 2021), toxin-driven EndoMT and endothelial calcific remodeling (Delgado-

Marín et al., Cells 2024), as well as seminal and recent studies on EndoMT and fibrosis in

chronic inflammatory disease, and reviews specifically discussing AGE–RAGE signaling

2 and its role in stress responses and coronary artery disease (Prasad & Mishra, Int J Angiol.

2018; Prasad, Int J Angiol. 2021) would help place the AGE–RAGE axis within a more

comprehensive landscape of chronic inflammatory and fibrotic disease mechanisms.

A: We thank the Reviewer for this valuable suggestion and agree that positioning AGE–RAGE within a broader endothelial stress and EndoMT context can enhance interpretability. At the same time, we aimed to keep the review tightly aligned with the predefined scope and screening strategy of our scoping review to avoid diluting the central focus.

 

6 Q: While the Conclusions appropriately frame the AGE–RAGE axis as a potential amplifier

rather than an initiator of endometriosis-associated inflammation and fibrosis, this nuanced positioning could be more consistently reflected across earlier sections of the manuscript. Aligning the tone of the Results and Discussion with the cautious and balanced conclusions would improve internal coherence.

A: We appreciate this recommendation and agree that internal coherence is essential. We have revised Discussion to more consistently reflect the nuanced conclusion that the AGE–RAGE axis is currently best supported as a disease-amplifying loop, particularly in sustaining inflammation and fibrotic remodeling, rather than as a definitive initiating trigger. Specifically, we refined phrasing in sections where earlier wording could be read as implying initiation, and we strengthened signposting regarding the limits of causal inference where evidence is primarily associative. These edits align the tone across the manuscript with the cautious, balanced framing presented in the Conclusions.

 

7 Q: Finally, a few minor issues related to presentation and clarity should be addressed.

Abbreviations such as RAGE and related terms should be consistently defined at first

mention, figures should clearly distinguish between proposed and experimentally

supported mechanisms, and the predominance of ovarian endometrioma studies, and its

implications for generalizability, could be highlighted earlier in the text.

In summary, this is a well-conceived and informative review addressing an important emerging area in endometriosis research. The suggested revisions are primarily aimed at refining interpretation, improving clarity, and enhancing contextualization, and they can be addressed without major restructuring of the manuscript. With these minor revisions, the manuscript would be further strengthened and well suited for publication.

A: We thank the Reviewer for these detailed presentation-focused recommendations. We have implemented the requested clarifications by ensuring that key abbreviations (including RAGE and related terms) are consistently defined at first mention throughout the Abstract and main text.

 

We appreciate the Reviewer’s overall positive assessment and believe that these refinements improve clarity, strengthen interpretative caution, and enhance the manuscript’s overall utility to the field.