Endometriosis and PMOS Models

Ovarian and uterine weights were assessed in female B6 and BKS-Lepr⁻/⁻ mice at 12 and 16 weeks of age without any additional experimental induction. BKS-Lepr⁻/⁻ mice exhibited significantly reduced ovarian and uterine weights compared with B6 controls, particularly at 16 weeks of age. In contrast, the changes in ovarian and uterine weights in B6-Lepr⁻/⁻ mice were less pronounced and varied with age.

(A) Ovarian weight and (B) uterine weight in B6 and B6-Lepr⁻/⁻ mice at 12 and 16 weeks of age. (C) Ovarian weight and (D) uterine weight in B6 and BKS-Lepr⁻/⁻ mice at 12 and 16 weeks of age. Data are presented as mean ± SEM. ***p < 0.001.

Estrous cyclicity was monitored beginning at 8 weeks of age in female mice without any additional experimental induction. Both B6-Lepr⁻/⁻ and BKS-Lepr⁻/⁻ mice exhibited profound disruption of estrous cyclicity, with virtually no completed estrous cycles observed during the 10-day monitoring period, in contrast to the regularly cycling B6 controls. Individual stage profiles further demonstrated persistent arrest in diestrus in both mutant strains, indicating spontaneous reproductive cyclicity abnormalities.

(A) Total number of completed estrous cycles in B6 and B6-Lepr⁻/⁻ mice during the 10-day monitoring period. (B) Representative individual estrous stage profiles in B6 and B6-Lepr⁻/⁻ mice over 10 consecutive days. (C) Total number of completed estrous cycles in B6 and BKS-Lepr⁻/⁻ mice during the 10-day monitoring period. (D) Representative individual estrous stage profiles in B6 and BKS-Lepr⁻/⁻ mice over 10 consecutive days. P, proestrus; E, estrus; M, metestrus; D, diestrus. Data are presented as mean ± SEM.

Representative ovarian histological sections from female B6, B6-Lepr⁻/⁻, and BKS-Lepr⁻/⁻ mice at 12 and 16 weeks of age revealed strain-dependent differences in ovarian morphology. B6 control mice displayed corpora lutea (CL), consistent with ongoing ovulatory activity. In contrast, B6-Lepr⁻/⁻ ovaries contained multiple cyst-like follicular structures (asterisks), while BKS-Lepr⁻/⁻ ovaries exhibited abnormal ovarian architecture, with cyst-like structures and additional morphological abnormalities (arrows). These findings indicate spontaneous ovarian structural alterations in leptin receptor-deficient mice, with differences between the B6 and BKS genetic backgrounds.

Representative hematoxylin and eosin (H&E)-stained ovarian sections from B6, B6-Lepr⁻/⁻, and BKS-Lepr⁻/⁻ mice at 12 and 16 weeks of age. CL, corpus luteum; asterisks indicate cyst-like follicular structures; arrows indicate regions of abnormal ovarian morphology.

Female SD rats were divided into three groups: sham control, EMT model, and estrogen-supported EMT model. For the EMT model, endometrial tissue was collected during the natural estrus phase, cut into standardized fragments, and surgically transplanted onto the abdominal wall with the endometrial surface exposed to promote ectopic lesion formation. The estrogen-supported EMT group underwent preoperative estrus induction and received postoperative estrogen administration (Q2W). Sham-operated rats underwent the same surgical procedure, with adipose tissue transplanted instead of endometrial tissue. Ectopic lesion establishment, progression, histopathological changes, and pain-related behavioral responses were subsequently evaluated.

Figure 1. Histopathological validation of the rat endometriosis (EMT) model by H&E staining.

Gross examination confirmed the successful establishment of ectopic endometrial lesions, which presented as typical cyst-like structures with visible vascularization at Day 28 after transplantation. Histological evaluation by hematoxylin and eosin (H&E) staining further confirmed the presence of ectopic endometrial tissues, characterized by preserved epithelial components, glandular structures, stromal proliferation, and inflammatory cell infiltration. Compared with sham animals, model animals exhibited well-developed ectopic lesions with characteristic endometrial morphology, confirming the successful establishment of the rat endometriosis model. Continuous estradiol stimulation further maintained lesion development and induced more pronounced histopathological alterations.

(A) Histopathological scores of ectopic lesions evaluated based on epithelial integrity, glandular/cystic structure formation, stromal hyperplasia, and inflammatory cell infiltration. Data are presented as mean ± SEM. *P < 0.05. (B) Representative hematoxylin and eosin (H&E)-stained sections of ectopic lesions from the Sham, EMT Model, and EMT + Estradiol Model groups. No obvious ectopic endometrial structures were observed in the Sham group. The EMT Model group developed typical ectopic endometrial lesions, whereas continuous estradiol stimulation further maintained lesion development and induced more pronounced histopathological alterations in the EMT + Estradiol Model group. Blue arrowheads indicate the monolayer endometrial epithelium; green arrowheads indicate endometrial glands; yellow arrowheads  indicate fibrotic regions; red arrowheads indicate stromal cells; black arrowheads indicate inflammatory cell infiltration; asterisks (*) indicate the uterine lumen. H&E stain; scale bars = 2.5mm or 50 μm.

Figure 2. Representative photograph of the Von Frey assay for assessing mechanical hypersensitivity in the rat endometriosis model.

To evaluate endometriosis-associated pain, multiple behavioral assays were incorporated.Mechanical hypersensitivity was assessed using Von Frey filament testing, including evaluation of lesion-associated abdominal pain and peripheral hypersensitivity.Thermal nociception was evaluated using the tail-flick assay to assess heat sensitivity changes during disease progression.The model showed reproducible pain-related phenotypes, including increased mechanical sensitivity and altered thermal pain responses after model induction.

(A) Mechanical sensitivity of ectopic lesions evaluated using the Von Frey filament test on Days 22 and 28. Each point represents the average of 5 measurements taken by an animal. (B) Mechanical withdrawal threshold of the hind paw measured using the Von Frey filament test on Days 7, 14, 21, 22, and 28. Each point represents the average of 5 measurements taken by an animal. (C) Thermal nociceptive sensitivity assessed using the tail-flick test on Days 7, 14, 21, and 28. Each point represents the average of 3 measurements taken by an animal. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (one-way ANOVA).

Figure 3. Estradiol promotes ectopic lesion development in the rat endometriosis model.

In addition to behavioral assessment, molecular characterization was performed to evaluate pain-related mechanisms.TRPV1 expression analysis in dorsal root ganglia (DRG) demonstrated activation of pain-associated neuronal pathways, supporting the involvement of peripheral sensitization in the model.Combined behavioral and molecular evidence indicates that the rat endometriosis model successfully recapitulates key features of endometriosis-associated pain.

(A) Quantitative analysis of ectopic lesion fluorescence at Day 28. Stable ectopic lesions were detected in the EMT Model group, while estradiol treatment showed a trend toward increasing the fluorescence-positive area and the proportion of positive signals. (B) Representative fluorescence images of dorsal root ganglia (DRG). Green fluorescence indicates TRPV1-positive signals, and blue fluorescence indicates cell nuclei (DAPI). Compared with the EMT Model group, the EMT + Estradiol Model group showed a trend toward increased fluorescence intensity and lesion expansion; However, the differences were not statistically significant. Data are presented as mean ± SEM. Scale bars = 50 μm.