Induction of Endometriosis in a Menstruating Mouse Model (Mus musculus): A Translational Animal Disease Model
Improved animal models of endometriosis are needed to accurately represent the pathophysiology of human disease and identify new therapeutic targets that do not compromise fertility. There is tremendous heterogeneity among published rodent models of endometriosis, and the etiology and pathogenesis of endometriosis remain undetermined. The vast majority of endometriosis is found in menstruating women; however, no published mouse models have induced endometriosis in a menstruating mouse, further limiting our understanding of the disease. Our goal was to develop a novel, translationally relevant mouse model of endometriosis in a menstruating mouse by transplanting donor menstrual endometrium into the peritoneal cavity of menstruating, immunocompetent, intact recipients. We initially compared 4 different experimental groups to optimize implanted menstrual tissue type and method of implantation into intact, normally cycling recipient mice. To further optimize this model, a novel fifth experimental group was compared in which discrete pieces of menstrual donor endometrium were implanted via laparoscopy into menstruating recipient mice. Lesions were confirmed to be endometriosis based on histopathology. The use of laparoscopy to place discrete fragments of menstrual phase endometrium intraabdominally was the most effective method for induction of endometriosis. This method was just as effective when used to induce endometriosis in menstruating recipient mice. Menstruating mice returned to normal estrus cyclicity after induction of disease, which can allow for assessment of therapeutic interventions on fertility. This is a novel translationally relevant mouse model of endometriosis in a menstruating mouse that can be used to explore and elucidate the etiology and pathogenesis of this disease.
(A) Overview of experimental design. Groups 1 to 4 compared implantation of 2 different types of menstrual endometrium and 2 different implantation techniques into cycling recipient mice. Group 5 implanted discrete biopsies of menstrual endometrium into menstruating recipient mice. (B) Timeline of procedures and data collections. Donor mice are color-coded pink and recipient mice are color-coded blue. For recipient mice, cycling means sexually intact with a natural estrus cycle. The visual presence of vaginal plug was considered day 0.5. Decidualization was performed on day 4.5. Menstrual tissue was harvested on the same day of implantation into recipient mice. For groups 1 to 4 this was between day 7.5 to 9.5 after the vaginal plug, and for group 5 this occurred on day 9.5 after the vaginal plug. Discrete biopsies are sectioned from menstrual endometrium into ∼2 mm3. Slurry was made from 10 discrete biopsies, minced and mixed with 0.2 mL of sterile saline for an average wet weight of 94.16 mg. Endometrial tissue was implanted via laparoscopy or laparotomy in discrete pieces or via injection of slurry. Recipient mice were sacrificed in proestrus, ∼30 d after resuming the estrus cycle after the procedure.
Images of menstruating mouse with bloody vaginal discharge (A), blood-tinged vaginal cytology from menstruating mouse (B), bicornuate menstrual phase uterus (C), and histomorphology of cross-section of menstrual endometrium with myometrium removed showing expansion of endometrial stroma (arrows) and loss of epithelium and lumen (circle) (D).
Representative histopathology of confirmed endometriotic lesions (solid arrows) showing hemosiderin and stroma on abdominal organs and tissues identified with open arrows. (A) Bladder wall. (B) Abdominal adipose tissue with inset showing endometrial stroma and hemosiderin (golden brown) at 20× original magnification. (C) Peritoneal body wall. (D) Higher original magnification (20×) of endometriotic lesion with hemosiderin (golden brown) and endometrial stroma shown by arrowheads.
Gross endometriotic lesions in situ with brown pigmentation. Arrows point to lesions on (A) bifurcation of uterus, (B) urinary bladder and body wall, and (C) inguinal fat.
Mean (±95% CI) counts of confirmed endometriotic lesions across the 5 groups. The letters a, b, and c are used to denote which groups have significant differences between the mean number of confirmed lesions. The numbers at the top of the bars are the mean counts of confirmed lesions per group. Bars with no letters in common are significantly (P < 0.05) different based on post hoc tests from the Poisson generalized linear model of the log difference between groups. The numbers alongside each group description on the x-axis correlate to experimental group number. Figures were created in ggplot2 using R version 4.2.2.100
Endometriotic lesions with immunohistochemical staining. Sections were stained using primary antibodies for (A) the endothelial cell marker CD31 and (B) the cell proliferation marker Ki67, demonstrating angiogenesis and cell replication, respectively. The presence of macrophages and mast cells was also confirmed using (C) the macrophage marker CD68 and (D) toluidine blue. Arrows indicate antigen-positive cells. Original magnification 20×.
Timing of menstrual cycle phases in women compared with pseudopregnant decidualized menstruating mice. Red mice are mCherry donor mice (D), and gray mice are B6 recipient mice (R). Menstrual endometrium was harvested from donor mice and implanted into menstruating recipient mice to mimic retrograde menstruation. Menstruating mice mimic the hormonal milieu and peritoneal microenvironment of menstruating women.101 Proliferation of ectopic lesions extended for at least 30 d past confirmed return to normal estrus cyclicity in the recipient mouse. Estrus cycles in mice are ∼4 to 5 d. The ectopic lesions would be exposed to the cyclical nature of estrogen and progesterone ∼6 to 7 times during their implantation and growth.
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