Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model

All animals were housed in temperature-controlled, high-barrier facilities (Monash University Animal Research Laboratory) with free food and water access and a 12 h light-dark cycle. All the procedures were performed in accordance with approval from the Monash Animal Research Platform Ethics committee (#21908, 17971) and performed in accordance with the National Health and Medical Research Council Code of Practice for the care and use of animals.

1. Surgical preparation

1. Autoclave all the surgical instruments, gauze, and paper towels required for the procedures on a hard/dry goods cycle at 121 °C with a hold time of 30 min and a drying time of 30 min.
2. Lay out a sterile bench pad for the surgical workspace and prepare analgesics.
   1. Dilute carprofen in sterile saline to 1 mg/ml, and dilute bupivacaine in sterile saline to a 0.5% (w/v) solution.
   2. Add 3.5 mL of meloxicam to a 400 mL cage water bottle.
3. Pre-warm the heat pad(s) for the recovery cages, and set up heat lamps to shine indirect light on the recovering animals.
4. Ensure all the appropriate PPE is worn, including a hair net, face mask, gown, and gloves.
5. Practice good sterile technique, including regularly spraying the gloves with ethanol and allowing them to evaporate before handling the animal or surgical tools to avoid ethanol contamination.

2. Performing ovariectomy

1. Using a gas anesthesia machine with isoflurane, prefill the induction box for 3-5 min at 5% isoflurane with the flow rate set at 4 L/min.
2. Place the mouse inside the induction box and, once unconscious, move it to the nose cone and reduce the flow rate to 0.4 L/min, with the isoflurane vaporizer set to ~2.5%.
   NOTE: The percentage of isoflurane used for the remainder of the procedure varies based on mouse strain, age, and exposure to treatments (e.g., chemotherapy) and should be adjusted based on a close assessment of each individual animal's breathing patterns. Breathing patterns should remain as regular abdominal breaths. Rapid, thoracic breaths may indicate that a deep surgical plane has not been reached or maintained; in this case, adjust the isoflurane vaporizer percentage as necessary.
3. Apply eye lubricant generously by squeezing the tube and gently dabbing the eye.
4. Shave a small (2 cm x 2 cm) area at and below the hunch of the spine.
5. Administer 5 mg/kg carprofen from a diluted solution of 1 mg/mL subcutaneously at the scruff of the neck.
6. Test the depth of anesthesia with the toe pinch reflex by pinching the mouse's back toe. If there is no toe pinch reaction, the animal is in the deep surgical plane, and the procedure can continue.
7. Apply betadine to the surgical area, and cover with a surgical drape (a gauze with a 2 cm x 2 cm window cut out).
8. Using rat-toothed forceps, pull the skin at the hunch of the back upward, and make a ~5 mm longitudinal incision.
   NOTE: A skin incision at this height on the animal's back is best for surgical clip placement to reduce the chance of the animal removing the clips and requiring clip repairs.
9. Using blunt forceps, proceed to blunt dissect the skin away from the underlying muscle layer, moving down and to one side toward the kidney.
10. Identify the kidney, ovary, and ovarian fat pad visually through the muscle wall.
    NOTE: The kidney will appear a dark red color, the fat pad will appear bright white, and if visible, the ovary will look like a small pink dot within the fat pad.
11. Using forceps, grab and lift the muscle layer. Make a ~0.5-1 cm incision with sharp surgical scissors. Continue holding the muscle wall with forceps, and change from scissors to blunt forceps to pull the ovarian fat pad through the incision.
12. Using a curved needle holder, clamp underneath the ovary and oviduct at the distal end of the uterine horn.
    NOTE: Alternatively, the ovary alone can be removed, leaving the oviduct intact. However, a dissecting microscope is required to accurately visualize the distinction between the ovary and oviduct.
13. Remove the ovary with scissors or a scalpel. Continue to clamp for 30 s to avoid excessive bleeding.
14. Remove the clamp, and dab it with sterile gauze if necessary.
15. To close the muscle wall incision, use forceps to lift the top of the incision so that the incision naturally pulls together.
16. Use silk sutures (size 3-0) to close the muscle wall incision with a surgeon's knot.
17. Apply two to three drops of bupivacaine topically using a 1 mL syringe without a needle attached, and repeat steps 2.9-2.17 on the other side.
18. To close the skin incision, use gauze to dab the area dry of excess bupivacaine, and press the two sides of the skin together.
19. Apply one to two 7 mm surgical clips, allowing space for swelling as part of the healing process.
20. Move the mouse to a recovery cage, and monitor closely for 15 min.
    NOTE: The animals should wake quickly; be sure to monitor breathing closely for normal thoracic breathing patterns.

3. Hormone preparation: Subcutaneous injection

1. Make a 1 mg/mL stock solution of estradiol.
   1. Weigh out 0.001 g (1 mg) of estradiol powder into a 1.5 mL sterile tube.
   2. Add 1 mL of 100% ethanol to the tube, and vortex for a few seconds.
      NOTE: The ethanol will remain clear with visible flecks of estradiol powder.
   3. Wrap the tube with film to prevent any ethanol evaporation.
   4. Wrap the tube in aluminum foil, and place it on a rocker overnight to completely dissolve the estradiol powder.
   5. Dilute this 1 mg/mL stock in sesame oil to the desired final concentration.
      NOTE: Doses of 100 ng for 3 days are required for priming prior to artificial decidualization, and additional low doses of 25 ng are required when progesterone is given. This is to combat the feedback loop controlling progesterone receptor expression. For embryo transfer, two doses of 100 ng on day 1 and day 3 prior to embryo transfer on day 4 are required. At the time of embryo transfer, a low dose of 25 ng is also required.
   6. Draw up the required amount of estradiol in oil into a 1 mL syringe, and then attach a 26 G needle tip.
   7. Inject the appropriate dose subcutaneously (either at the scruff or flank; 100 ng/100 µL or 25 ng/100 µL for priming prior to embryo transfer or artificial decidualization or at the time of embryo transfer) at the required frequency.
      NOTE: Oil is very viscous, so be sure to inject slowly and pause for a few seconds before removing the needle. This will minimize the amount of oil that leaks out of the injection site.
2. Make a 200 mg/mL stock solution of progesterone.
   1. Weigh out 0.4 g (400 mg) of progesterone powder into a 5 mL sterile tube.
   2. Add 2 mL of 100% ethanol to the tube, and vortex for a few seconds.
      NOTE: The ethanol will become white in color.
   3. Repeat steps 3.1.3-3.1.4.
   4. Dilute the 200 mg/mL stock in sesame oil to the desired final concentration.
      NOTE: Doses of 2 mg daily are required for supporting embryo transfer.
   5. Inject the appropriate dose (e.g., 2 mg/100 µL daily for supporting pregnancy) subcutaneously as in steps 3.1.6-3.1.7.

4. Hormone preparation: Slow-release pellets

1. Lay a foil across the surface of a laminar flow or class II biosafety hood.
2. Place all the equipment (gloves, Petri dishes, 1 mL syringes, fine forceps) in the hood, and switch on the UV for 20 min.
   NOTE: Do not switch on the UV with the sealant inside the hood as it will set.
3. Wash the silastic tubing in 100% ethanol, and allow it to air-dry in the hood. Once dry, mark ~1 cm lengths along the tubing, and cut with a scalpel.
4. Remove the plunger from a syringe, and squeeze in ~200 μL of sealant. Replace the plunger, and depress a small amount of sealant out of the syringe.
5. Apply a small amount of sealant to one end of the tubing, and smooth it over with a gloved finger.
6. Allow to dry overnight or for 20-30 min in UV light inside the hood.
7. Pour an appropriate amount of progesterone into a sterilized Petri dish. Using forceps, scoop pellets into the progesterone powder to fill the pellet.
   1. Tap the sealed end of the pellet on the hood surface to condense the progesterone down. Alternatively, use the end of sterilized forceps to stuff the progesterone down. Allow enough room for more sealant.
8. Seal the open end with sealant, as described in steps 3.4-3.5.
9. Wrap the Petri dish containing the progesterone pellets in foil to protect it from light.
10. Activate the pellets for a minimum of 72 h before subcutaneous insertion by incubating in 1% charcoal-stripped FCS (cs-FCS: PBS) at 37 °C.
    NOTE: Pellets can be made in bulk with a single sealed end in advance. However, fresh progesterone should be used to fill them each time. Ensure the pre-made pellets are UV-sterilized prior to filling with progesterone. The pellets will secrete ~500 µg/day for 6-10 days, which is sufficient support for artificial decidualization and embryo transfer procedures, though an additional low-dose estrogen injection may be required to maintain progesterone receptor activity beyond 4-5 days. Beyond 10 days, a replacement progesterone pellet may be required.

5. Hormone preparation: Osmotic mini pumps

1. Prepare progesterone at the desired concentration in an aqueous solution, and select the appropriate mini osmotic pump model (see Table of Materials).
   NOTE: For section 7 (experimental procedure: embryo transfer), the delivery of 2 mg/day for 12 days is required. Therefore, dissolve 28 mg of progesterone in ~100 µL of sterile water per animal (follow the manufacturer's instructions for the specific volume). Serial dilutions may be required. For section 8 (experimental procedure: artificial decidualization), the delivery of 500 µg per day for 3 days is required. Therefore, dissolve 1,500 µg of progesterone in ~100 µL of sterile water per animal. Prepare extra solution to account for the volume lost during the filling procedure.
2. Set up the equipment (gloves, low-lint wipes, Petri dishes, sterile saline, 1 mL syringes, small weigh boats, foil, and scales accurate to 0.01 g) within a class II biosafety hood, and then switch on the UV for 20 min.
3. Draw up the hormone solution into a 1 mL syringe, and then attach a sterile filling tube, carefully ensuring that there are no air bubbles.
4. Weigh the pump and its flow moderator within a sterile weigh boat.
5. Insert the filling tube through the opening at the top of the pump until it can go no further.
6. Holding the pump upright, slowly push the plunger of the syringe to fill the tube.
   NOTE: Rapid filling should be avoided as this can introduce air bubbles into the pump.
7. When the solution overflows from the top of the pump, gently remove the filling tube, and wipe off the excess solution with a sterile low-lint wipe.
8. Insert the flow moderator through the opening at the top of the pump until it can go no further. Once fully in, firmly press the pump and flow moderator together.
9. Weigh the filled pump with the flow moderator in place.
   NOTE: The difference in weight obtained from step 5.3 and step 5.8 will give the net weight of the solution loaded (i.e., a 0.1 g increase = 100 µL of solution added).
10. Place the filled pump into a sterile Petri dish filled with sterile saline.
11. Once all the pumps are filled, wrap the Petri dish in foil, and place it inside a 37 °C incubator to prime for at least 4-6 h (or until ready for use).

6. Surgical procedure: Insertion of subcutaneous hormone pellets and mini pumps

1. Prepare the area as per section 1 (surgical preparation).
2. Anesthetize the animals as per steps 2.1-2.3.
3. Shave a small area at the scruff of the neck (~1 cm x 1 cm).
4. Administer 5 mg/kg carprofen from a diluted solution of 1 mg/mL subcutaneously in the leg flank.
5. Test for the toe pinch reflex. If there is no reflex, the animal is in the deep surgical plane, and the procedure can begin.
6. Apply betadine to the surgical area, and cover it with a surgical drape (a gauze with a 2 cm x 2 cm window cut out).
7. Using rat-toothed forceps, pull the skin at the scruff of the neck (halfway between the true scruff and the hunch of the back) upward, and make a ~5 mm longitudinal incision.
8. Using blunt forceps, blunt dissect the skin away from the underlying muscle layer in a downward direction.
   NOTE: For osmotic mini pump insertion, create a pocket along one side of the animal so that the pump does not restrict the animal's movement or press up against the incision site.
9. Once sufficient space has been made for the hormone pellet or mini pump, use sterile forceps to pick up the pellet or mini pump, and insert it into the subcutaneous pocket made with blunt dissection.
10. To close the skin incision, ensure the pellet or mini pump is far enough into the pocket that surgical clips will not damage it.
11. Topically apply bupivacaine as per step 2.17.
12. Close the wound with one surgical clip. Move the mouse to a recovery cage, and monitor closely for 15 min. As this is a short procedure, the animals should be ambulatory within minutes.

7. Experimental procedure: Embryo transfer

1. For ovariectomized animals, hormone-prime 3 days prior to embryo transfer by a subcutaneous injection of 100 ng/100 µL estradiol (step 3.1).
2. One day prior to the embryo transfer, hormone-prime the animals with a subcutaneous injection of 2 mg/100 µL medroxyprogesterone acetate (step 3.2).
3. Prepare the area as per section 1 (surgical preparation).
4. Anesthetize the animals as per steps 2.1-2.3.
5. Begin the procedure as per steps 2.4-2.10.
6. Under a dissecting microscope, create an intrauterine injection point with a 26 G needle tip.
7. Pipette five blastocysts into an M2 media drop, and then transfer these into the uterine horn.
8. To close the muscle wall incision, lift the top of the incision using forceps so that the incision naturally pulls together.
9. Using silk sutures, close the muscle wall site with a surgeon's knot. Apply drops of bupivacaine topically.
10. Repeat steps 7.5-7.8 on the other side.
11. To close the skin incision, use gauze to dab the area dry of excess bupivacaine, and press the two sides of the skin together.
12. Apply one to two surgical clips, allowing space for swelling as part of the healing process.
    NOTE: If the animals are ovariectomized, exogenous hormones are required at the time of embryo transfer. Either subcutaneously inject progesterone (2 mg) or insert a subcutaneous progesterone pellet or osmotic mini pump. To combat excess progesterone, a subcutaneous injection of low dose (25 ng/100 µL) estrogen is required at the time of embryo transfer.
13. Carefully carry the mouse to a recovery cage, and monitor closely for 15 min.
    NOTE: The animals should wake quickly; be sure to monitor breathing closely for normal thoracic breathing patterns.

8. Experimental procedure: Artificial decidualization

1. Hormone-prime the animals with 100 ng/100 µL estradiol on day 1, day 2, and day 3, as per section 3 (hormone preparation: subcutaneous injection) 8 days prior to artificial decidualization.
2. Hormone-prime the animals with 5 ng/100 µL estradiol on day 7, day 8, and day 9, as per section 3 (hormone preparation: subcutaneous injection) 2 days prior to artificial decidualization.
   NOTE: The final injection must occur a minimum of 3 h (and a maximum of 4 h) before the artificial decidualization procedure.
3. Hormone-prime animals with a subcutaneous progesterone pellet or mini osmotic pump (500 µg/day), as per section 4, section 5, and section 8, 2 days prior to artificial decidualization.
4. Prepare the area as per section 1 (surgical preparation).
5. Anesthetize the animals as per steps 2.1-2.2.
6. Test the depth of anesthesia with the toe pinch reflex. If there is no toe pinch reaction, the animal is in the deep surgical plane, and the procedure can continue.
7. Place the mouse in a prone position, lift the tail, and slowly insert a 6 mm diameter speculum into the vagina.
8. Whilst keeping the nose of the animal in the anesthetic nose cone, place the lower body of the animal between the first and second fingers of the non-dominant hand. Use the thumb to gently push the tail upward to keep the vaginal opening in view.
9. Transfer 20 µL of sesame oil into one uterine horn using a non-surgical embryo transfer tip (attached to a 20 µL pipette).
   1. Keeping the pipette level with the vaginal opening, insert the tip into the vagina and through the cervix into the uterine horn. Once the tip is in the uterine horn, gently press the tip against the endometrial surface (if using the handling technique above, this movement will be felt against the second finger), and slowly expel the oil.
      NOTE: Keep the pipette plunger depressed, wait for 10 s to ensure all the oil has dispersed, and slowly remove the transfer tip whilst keeping the plunger depressed.
10. Remove the speculum from the vagina.
11. Carefully carry the mouse to the recovery cage, and monitor closely for 15 min.
    NOTE: The animals should wake quickly. Monitor their breathing closely for normal thoracic breathing patterns.
12. Limit the handling of the animals, and keep them in a quiet environment for 96 h after the procedure.
    NOTE: Loud noises or abrupt changes to their light and dark cycle will impact the success of the procedure. At the time of tissue dissection, the extent of decidualization success can be measured as a ratio of uterine weight to body weight. The artificial decidualization procedure has an 80% success rate. Therefore, exclude animals that fail to decidualize, and factor this in when selecting sample sizes for the experiments.

9. Surgical procedure: Post-surgical recovery, monitoring, and clip repairs

1. Allow the animals to recover half-on, half-off heat pads overnight before returning them to their home cage.
2. Monitor the animals daily for 5 days post-surgery, paying close attention to the wound site for signs of infection.
3. Perform clip repair if necessary.
   1. Prepare the area as per section 1 (surgical preparation).
   2. Anesthetize the animals as per steps 2.1-2.3.
   3. Remove the existing clip using a clip remover if the clip is still present.
   4. Apply a new surgical clip, as per steps 2.19-2.21.
4. Remove the surgical clips 7 days post-surgery as per steps 9.3.1-9.3.3 or when the animal is next under anesthesia (such as for an embryo transfer, the artificial decidualization procedure, or a subcutaneous injection following surgery).