Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research

All of the animal experiments described were approved by the Affiliated Drum Tower Hospital of Nanjing University Medical School's Committee on the Use and Care of Animals (No. 20171202). All operations follow appropriate animal care and use agency and national guidelines.

NOTE: Mice were raised in a specific pathogen-free (SPF) environment, with a temperature of 22 °C ± 1 °C, relative humidity of 50% ± 1%, a light/dark cycle of 12 h/12 h, and free access to food and water.

1. Mouse ovariectomy

1. Sterilize the surgical instruments with a high-temperature and high-pressure sterilizer 1 day before the operation.
2. Weigh 8-week-old C57BL/6 mice and intraperitoneally (i.p) inject 1% sodium pentobarbital accordingly to anesthetize the mice. The dose used is 40 mg/kg¹⁷. This dose provides 40-60 minutes of sedation, which meets the requirements of an ovariectomy. For pre-operative analgesia, inject mice with 5 mg/kg meloxicam (s.c).
   NOTE: Successful anesthesia in mice can be indicated by the disappearance of deep muscle reflexes and steady breathing. Signs of successful anesthesia include no blinking when touching the inner canthus, no swallowing when pulling the tongue, no leg bending when pinching the skin between the toes, and no bouncing when needling the tail.
3. Apply the protective eye ointment to the eyeballs to prevent dryness during the anesthesia.
4. Start the operation when mice are completely anesthetized. Place and fix the mice in the prone position on the operating surface, and shave the hair on the back after wetting them with soapy water. Disinfect the skin with 70% ethanol after shaving.
5. Find the operation incision site at 0.5 cm (or 1.0 cm below the rib) on the upper edge of the thigh root of both hind limbs parallel to the spine (Figure 1A).
   NOTE: A correct incision location is essential for quickly localizing the ovary.
6. Take the incision site as the center and disinfect the incision area with iodophor 3x, then place a surgical drape around the incision area.
7. Make a longitudinal incision of about 0.5-1.0 cm using scissors. Cut the fascia and passively separate the muscles with tweezers.
8. Find a piece of the white fat pad in the incision field close to the lower pole of the kidney through the thin muscle layer (Figure 1B).
   NOTE: The white fat pad is the most obvious indicator of the location of the ovary.
9. Clamp the fat mass with hemostatic clips, and pull the ovary wrapped by the fat pad out of the incision.
10. Clamp the junction of the ovary and fallopian tube with curved tweezers, and remove the ovary along the ovarian pedicle with scissors. Stop the bleeding using an electrocoagulation pen or a needle of a 1 mL syringe heated on the alcohol lamp.
    NOTE: Be sure to preserve the fallopian tube, which is the anatomical landmark for the subsequent injection of sesame oil into the uterine horns.
11. Rinse the surgical incision with normal saline to prevent tissue adhesion, use a 4-0 suture to sew the muscle and skin respectively, and disinfect the surgical incision with iodophor again.
12. Place the mice in the prone position in the cage and resuscitate them in a 25 °C incubator equipped with a light source and a ventilation system for about 2 h.
13. Pay attention to the mice after the operation, put them back in the original feeding place alone until they recover completely, and give them enough water and food.

2. Postoperative rest and estrogen and progesterone formulation

1. Make sure the mice rest for 2 weeks after ovariectomy.
2. In an ultra-clean cabinet, add estrogen (2 ng/µL) and progesterone (0.2 ng/µL) into sesame oil in RNA enzyme-free centrifuge tubes.
3. Place the centrifuge tubes in a thermostatic water bath at 37 °C and shake the tubes continuously to accelerate dissolution.
4. After complete dissolution, divide the sesame oil solution into 100 µL aliqouts for convenient use. Store the prepared estrogen and progesterone solutions in a refrigerator at 4 °C.

3. Induced artificial decidualization model

1. Subcutaneously (s.c) inject 100 ng of estrogen in 50 µL of sesame oil into each mouse for 3 days, followed by 2 days of rest¹⁵.
   NOTE: Ensure that the estrogen and progesterone have been formulated and placed in different places. Any contamination of estrogen by progesterone may lead to failure.
2. Inject (s.c.) 1 mg of progesterone and 10 ng of estrogen in 50 µL of sesame oil into each mouse for another 3 days¹⁵.
3. Operate the mice to induce the artificial decidualization model¹⁵ 6 h after the third estrogen-progesterone combined injection.
4. Find the uterus horn at the lower end of the fallopian tubes and inject 20 µL of sesame oil slowly along with the uterine horn, push the tissue back, suture the incision, and allow the mouse to recover. The approach to operation is the same as the mouse ovariectomy¹⁵. 
   NOTE: Only proceed with the second surgery in the event of a successful ovariectomy (skin incision is well healed, the ovary is completely excised, and the residual end of the fallopian tube is not adhered to the surrounding tissues).
   NOTE: For inducing the AD model, 20 µL of sesame oil is appropriate; more than 20 µL of sesame oil will easily penetrate the other side.
5. Inject (s.c.) 50 µL of sesame oil containing 1 mg of progesterone and 10 ng of estrogen (H.) for another 4 days. See details in Figure 2^15,18.

4. Sample collection

1. Euthanize the mice (n = 6) by carbon dioxide asphyxiation and collect the uteri. Observe the shape of the bilateral uteri, take photos, and weigh the uterine horns (Figure 3A, B).
2. Fix 3-5 mm of the uterus tissue with 10% formalin, embed in paraffin, and section them. Stain the sections with hematoxylin and eosin to observe the morphological changes¹⁹ (Figure 4A).
3. Embed another 3-5 mm of the uterus tissue in optimal cutting temperature compound (OCT), freeze in liquid nitrogen, and store in a −80 °C refrigerator. Frozen section the tissue to 10 µm and detect alkaline phosphatase activity in the uterine tissue by the azo coupling method²⁰ (Figure 4B).
   NOTE: Satisfactory results can be obtained using frozen sections to detect ALP content, not paraffin sections.
4. Extract the total RNAs of the uterus with Trizol reagent and perform quantitative real-time PCR (qPCR) on a real-time PCR system (Table of Materials) with qPCR master mix (Table of Materials) to detect the relative mRNA expression of prolactin family 8 subfamily member 2 (Prl8a2), alkaline phosphatase liver/bone/kidney (Alpl), and insulin-like growth factor-binding protein 1 (Igfbp1)¹⁵ by normalization to 18S mRNA levels (Figure 4C-E). Follow the PCR conditions: Stage 1, 95 °C for 30 s; Stage 2, 95 °C for 10 s and 60 °C for 30 s; repeat the cycle 40x. A complete list of primer sequences is provided in Table 1.
5. Analyze qPCR data using the 2^-ΔΔCT method and a t-test method for statistical analysis in the appropriate software. In this study, p < 0.05 was considered statistically significant.