Endometriosis Induction in Murine Model

Syngeneic female donor mice were primed with 5 IU of pregnant mare serum gonadotropin (PMSG; i.p.) to stimulate endometrial growth 48 h before euthanasia. Immediately after euthanasia, uterine horns were isolated and cut into small pieces (< 0.5 mm), washed with sterile phosphate-buffered saline (PBS), and resuspended in 1.0 ml of PBS. A similar model of endometriosis induction has previously been used in mice, albeit with estradiol supplementation [66 (link)]. Recipient mice were anesthetized using ketamine/xylazine (87 mg/kg; 15 mg/kg; i.p.). A small dorsolateral incision (5 mm) was made, and 0.5 ml of tissue fragment suspension containing uterine tissue equal to 1 uterine horn (Endometriosis group; experiment-1 n = 6–11/timepoint, experiment-2 n = 6) or sterile PBS only (Sham group; experiment-1 n = 6/timepoint, experiment-2 n = 6) was carefully injected into the peritoneal cavity of the recipient mice. Closure of the peritoneal cavity was achieved by simple interrupted suturing of the muscle layer with plain gut 5–0 and wound clips for skin incisions. Recipient mice also received buprenorphine (0.05 mg/kg: s.c.) 1 h before administering anesthesia and 6–10 h after the initial dose to reduce pain from surgery. Mice were euthanized in estrus (based on vaginal smears) at ~ 4, ~ 8, ~ 16, and ~ 32 days after induction of endometriosis (Fig. 1). Mice were transcardially perfused with sterile PBS, endometriotic lesions, brains, and spines were collected and placed in 10% neutral buffered formalin for 24 h, then transferred to 70% ethanol until further processing.Fig. 1

Experimental timeline (created with BioRender.com). Syngeneic female donor mice were primed with 5 IU of pregnant mare serum gonadotropin (PMSG; i.p.) to stimulate endometrial growth for 48 h before euthanasia. Immediately after euthanasia, uterine horns were isolated, minced (< 0.5 mm), washed with sterile phosphate-buffered saline (PBS), and resuspended in 1.0 ml of PBS to create a uterine tissue suspension. While recipient mice were anesthetized, a small dorsolateral incision (5 mm) was made, and 0.5 mL of tissue fragment suspension from a single uterine horn (Endometriosis group; experiment-1 n = 6–11/timepoint, experiment-2 n = 6) or sterile PBS only (Sham group; experiment-1 n = 6/timepoint, experiment-2 n = 6) was carefully injected into the peritoneal cavity of the recipient mice. Finally, the lesions, brains, and spinal cords were collected from recipient mice euthanized in estrus (based on vaginal smears) at ~ 4, ~ 8, ~ 16, and ~ 32 days after induction of endometriosis