Effect of the estrus cycle stage on the establishment of murine endometriosis lesions

Abstract

Background: Establishment of a standardized animal endometriosis model is necessary for evaluation of new drug effects and for explaining different ethological aspects of this disease. For this purpose, we need a model which has more similarity to human endometriosis.

Objective: Our objective was to establish an autologous endometriosis mouse model based on endogenous estrogen level and analyze the influence of estrus cycle on the maintenance of endometriotic lesions.

Materials and methods: In this experimental study, endometriotic lesions were induced in 52 female NMRI mice by suturing uterine tissue samples to the abdominal wall. The transplantation was either performed at proestrus/estrus or at metestrus/diestrus cycles. Urine-soaked beddings from males and also male vasectomized mice were transferred to the cages to synchronize and maintenance of estrus cycle in female mice. The mice were sacrificed after different transplantation periods (2, 4, 6 or 8 wk). The lesions size, macroscopic growth, model success rate, histological and immune-histochemical analyses were assessed at the end.

Results: From a total of 200 tissue samples sutured into the peritoneal cavity, 83 endometriotic lesions were confirmed by histopathology (41.5%). Model success rate for proestrus/estrus mice was 60.7% vs. 79.2% for metestrus/diestrus mice. The endometriotic lesions had similar growth in both groups. Number of caspase-3, Ki67-positive cells and CD31-positive micro vessels were also similar in endometriotic lesions of two groups.

Conclusion: If we maintain the endogenous estrogen levels in mice, we can induce endometriosis mouse model in both proestrus/estrus and metestrus/diestrus cycle without any significant difference.

Keywords: Animal models; Autologous; Endometriosis; Estrus cycle; Transplantation.

Figure 1

Cytological assessment of vaginal smears stained by Crystal Violet (A) Proestrus: epithelial cells-mostly rounded but some cells showing early stages of cornification of approaching estrus (B) Estrus: large cornified cells in clumps (C) Metestrus: leucocytes with smaller numbers of non-nucleated epithelial cells (D) Diestrus: mainly leucocytes with small number of nucleated epithelial and cornified cells. Scale bars: 10 µm

Figure 2

The cystic endometriotic lesion on the right lower quadrant of peritoneum, 6 wk after implantation

Figure 3

H&E stained section of a cystic endometriotic peritoneal lesion, 6 wk after implantation, Magnification: 40×.

Figure 4

(A–F) Immunofluorescent sections of endometriotic lesions at proestrus/estrus (A, C, E) or at metestrus/diestrus stages (B, D, F), by fixation of uterine tissue samples to the peritoneal wall; Sections were stained with DAPI to identify cell nuclei (blue), an antibody against the apoptosis marker Caspase-3 (red) (A-B); and an antibody against the proliferation marker Ki67 (red) (C-D); an antibody against CD31 for the detection of the microvascular endothelium (red) (E-F). Magnification: ×200.

Figure 5

Caspase-3, Ki67 positive cells (%) and CD 31 positive micro vessels (%) in peritoneal endometriotic lesions at proestrus/ estrus (white bars) or metestrus/estrus (Grey bars) stages at operation day. Mean ± SEM.