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Standardized Modeling Platform for Endometriosis: Recreating Inflammatory Pathology to Accelerate the Validation of New Gynecological Therapeutics

　　Introduction

　　Endometriosis (EMS) is a benign proliferative disorder that is estrogen‑dependent, progesterone‑resistant, and characterized primarily by chronic pelvic inflammation. Its core pathological features include ectopic endometrial implantation, remodeling of the inflammatory microenvironment, enhanced angiogenesis, and aberrant macrophage infiltration. In both basic research and drug development, model stability and phenotypic consistency directly determine the reliability of efficacy assessments.

　　Aniphe BioLab has established a standardized abdominal wall–peritoneal heterotopic transplantation model of endometriosis using female BALB/c mice, and conducted systematic validation through morphological, pathological, immunohistochemical analyses, and measurements of inflammatory cytokines, while also employing this model for the efficacy evaluation of candidate drugs.

　　01 Experimental Design

　　This study comprised four groups: G1, the sham‑operation control group; G2, the EMS model group; G3, the EMS model plus Positive Control 1; and G4, the EMS model plus Positive Control 2, with N = 6 in each group. Following successful model establishment, treatment was initiated and administered continuously for three weeks. Tissue samples were collected after the final dose, and gross morphological, histopathological, immunohistochemical, and inflammatory cytokine analyses were performed.

　　

 

　　02 Model Construction Strategy

　　This model employs an orthotopic implantation technique, whereby endometrial tissue is adhered to the peritoneal surface of the abdominal wall, combined with exogenous estrogen induction, to mimic the survival and proliferation of ectopic endometrium within the peritoneal cavity.

　　| Estrous cycle synchronization, reducing variability in donor sources

　　Seven days prior to model induction (D‑7), donor mice were pre‑treated with subcutaneous injections of estradiol benzoate. For five consecutive days, vaginal smears were collected daily to monitor the estrous cycle; on the day of tissue collection, uteri from donor mice exhibiting proestrus or estrus on the smear were selected, ensuring that all donor uterine endometria were at a consistent physiological stage and minimizing inter‑animal variability in model establishment and lesion size attributable to cyclical fluctuations.

　　| Establishment of a model by heterotopic implantation in the abdominal wall

　　The donor uterus was aseptically isolated, and endometrial fragments were excised. The functional surface was oriented toward the peritoneal cavity and securely fixed to the inner surface of the recipient mouse’s abdominal wall, with precise alignment and suturing. Postoperatively, estradiol benzoate was administered intraperitoneally according to the experimental protocol to mimic a high‑estrogen microenvironment, thereby supporting the engraftment, proliferation, and angiogenesis of ectopic lesions.

　　03 Establishment and Validation of the EMS Mouse Model

　　| Good synchrony of estrous cycles

　　Vaginal smears from donor mice demonstrated synchronization of the estrous cycle, ensuring temporal consistency in the collection of endometrial samples and minimizing the impact of cyclical variability on model establishment.

　　| Typical manifestations of ectopic foci

　　Following model induction, newly formed blood vessels were observed on the surface of the endometrial grafts and in the adjacent areas across all groups. The ectopic lesions appeared as transparent or semi‑transparent vesicles containing clear fluid, with a dark red or brown hue. Their gross morphological features were consistent with those of typical endometriosis lesions, indicating successful model establishment.

　　| HE staining reveals active tissue and inflammatory infiltration

　　Hematoxylin–eosin staining of the transplanted tissue reveals intact endometrial glands and stromal cells, accompanied by inflammatory cell infiltration, suggesting that the ectopic endometrium possesses tissue viability and is associated with an activated inflammatory response.

　　

 

　　04 Efficacy Evaluation System

　　1. Weight changes

　　Following model induction, the body weights of mice in the G2 and G4 groups decreased significantly compared with those in the G1 control group (P<0.05), then gradually recovered to levels approaching normal; no significant difference was observed between the G3 group and the G1 group (P>0.05). Each group n=6. In the figure, *P<0.05, **P<0.01, ***P<0.001.

　　2. Morphological Comparison of Lesions

　　Overall, the results of the gross observations indicate that interventions with both G3 (Positive Control 1) and G4 (Positive Control 2) reduced the volume of ectopic lesions; notably, in the G4 group, both the number of lesions and the degree of intra-abdominal adhesions were significantly less pronounced than in the model group.

　　3. Histological differences in HE (endometrium vs. ectopic lesions)

　　Ectopic lesions: In the G2 model group, the lesion tissue architecture was disorganized, with numerous abnormal cavities and scattered hemorrhagic foci; in the G3 intervention group, the lesion centers frequently contained large vacuoles filled with clear fluid, accompanied by some hemorrhage; in the G4 intervention group, the number of cavities was reduced and their extent was diminished, with hemorrhage markedly alleviated compared to the model group.

　　Endometrium: Intervention with Positive Control 1 reduces the incidence of endometrial bleeding and decreases the number of glands with abnormal morphology; intervention with Positive Control 2 significantly improves glandular morphology, bringing it closer to the normal endometrial architecture.

　　

 

　　4. Immunohistochemistry (Iba-1, a marker of macrophage infiltration)

　　Iba-1 (ionized calcium-binding adapter molecule 1) is a commonly used marker for macrophages, reflecting the degree of macrophage infiltration within tissues.

　　Ectopic lesions: In the G2 model group, the proportion of Iba‑1‑positive cells was significantly higher than in the G3 (P<0.001) and G4 (P<0.001) intervention groups.

　　Endometrium: The proportion of positive cells in the G2 model group was significantly higher than that in the G1 control group (P<0.001); the G3 intervention group and the G4 intervention group showed significant reductions compared with the G2 group (G3: P<0.01; G4: P<0.001).

　　5. Inflammatory cytokine assay (ELISA)

　　ELISA analysis of peritoneal lavage fluid revealed that the levels of IL-6, TNF-α, and IL-1β in the G3 and G4 intervention groups were significantly lower than those in the G2 model group (P<0.001).

　　

 

　　05 Model Features

　　This model possesses the following core advantages:

  1. High stability and homogeneity: synchronization of the estrous cycle and maintenance of estrogen levels jointly ensure consistency in lesion size and growth status.
  2. Controllable cycle: The entire process lasts approximately 6 weeks, and pharmacological efficacy assessments can be conducted steadily once the model is established.
  3. Complete pathological phenotypes: encompassing ectopic implantation, angiogenesis, peritoneal adhesions, inflammatory infiltration, and abnormal tissue architecture—key phenotypes of endometriosis.
  4. Multilevel evaluation: Integrates gross morphology, histopathology, immunohistochemistry, and inflammatory cytokine assays, facilitating cross-validation and traceability of results.

　　06 Summary of Experience

  1. Donor synchronization and the stable maintenance of exogenous estrogen are central to the uniformity of the model.
  2. The stability of the surgical procedure determines the consistency of the implantation site and size of ectopic lesions.
  3. Strict alignment of dosing parameters (timing, dose, and route) is a prerequisite for the comparability of pharmacological effects.
  4. Maintaining low temperatures throughout the entire sample-processing workflow is fundamental to ensuring the reliability of downstream data in assays such as ELISA and immunohistochemistry.
  5. End-to-end documentation—including estrous cycle smears, surgical records, medication administration logs, and schematic diagrams of tissue sampling sites—serves as a critical safeguard for the model’s reproducibility.

　　07 Conclusion

　　The standardized EMS mouse model developed by Aniphe BioLab demonstrates excellent performance in terms of low heterogeneity, high stability, a complete pathological phenotype, and a well-defined pharmacological evaluation window, making it suitable for preclinical research applications such as investigating the pathogenesis of endometriosis, elucidating the peritoneal inflammatory microenvironment, screening innovative drugs, conducting pharmacodynamic evaluations, and validating biomarkers.

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