Burrowing behavior is a potential non-invasive proxy for lesion development in a syngeneic murine model of endometriosis

Background: Endometriosis (EM) is a persistent, chronic inflammatory condition associated with excruciating pelvic pain and infertility. The absence of a pre-clinical model that reliably replicates the clinical and functional hallmarks of human EM continues to limit progress in the domain. Furthermore, no rodent model developed to date has achieved a 100% incidence rate, compromising the reproducibility of existing models. Further, the inability to detect lesion development without sacrificing the animal presents a significant barrier for preclinical interventional trials designed to improve the management of EM. Methods: We employed a non-invasive method based on the altered burrowing behavior of the animal to predict lesion development in the syngeneic mice EM model. We used the burrowing assay (BA) before dissection as a non-invasive behavioral marker to evaluate lesion progression across three distinct laboratory strains: C57BL/6j, BALB/c, and Swiss albino to account for variation due to genetic, immunological, and strain specificity. Results: EM mice displayed a significant decline in burrowing activity compared to controls across all three strains. Based on BA performance, recipient mice were stratified into two groups: Recipients with a low burrow score (LB) and those with a high burrow score (HB). Additionally, LB mice exhibited decreased exploratory behavior and increased sensitivity to thermal pain. In contrast, HB mice had exploratory and thermal responses comparable to those of the control group. Post-dissection, LB mice were presented with ectopic lesions (LB+), whereas HB mice were lesion-negative (HB-). BA performance correlated strongly with lesion presence via ROC analysis, with a combined AUC of 0.883 (and an AUC of 1 for C57BL/6j), indicating excellent diagnostic accuracy of BA in predicting EM incidence. The combined approach of correlating burrowing behavior with other evoked behavioral responses and non-evoked provided a comprehensive assessment of EM disease progression. Conclusion: To our knowledge, our research provides evidence for the feasibility of ethologically valid burrowing behaviour as a non-invasive predictor of EM incidence in the syngeneic mice model. Future preclinical drug research for EM management could leverage BA to identify and select only lesion-positive animals for intervention trials. This approach will enhance the translational value of EM research.