To begin, obtain embryos from freshly mated female mouse. Prepare 12 microdroplets of Potassium Simplex Optimized Medium at the bottom of a 35-millimeter Petri dish. Cover the microdroplets with three to five milliliters of mineral oil, and place the Petri dishes in an incubator at 37 degrees Celsius with 5%carbon dioxide for equilibration.
Using a transfer pipette, transfer the mouse embryos from M2 medium to KSOM medium. Place the washed embryos in the prepared KSOM microdroplets, and incubate them at 37 degrees Celsius with 5%carbon dioxide for four days. Select advantageous blastocysts according to their size, inner cell mass, number of trophoblast cells, and degree of expansion.
Add one milliliter of 2%gelatin to a 12-well culture plate, and incubate at 37 degrees Celsius for 30 minutes. Then aspirate the excess gelatin, and let the bottom of the plate dry completely. Seed human endometrial adenocarcinoma cells or Ishikawa cells into the gelatin-coated 12-well plate, and incubate the plate for 24 hours.
Gently place five to 15 fresh blastocysts into each well of the 12-well plate containing the cells. After 48 hours, observe the embryos under a microscope to assess their attachment status after moving the plate three times. Unattached embryos will float or roll over the surface of cells.
Finally, calculate the embryo implantation rate in the presence of various estrogen concentrations. The results demonstrated that near physiological concentrations of 17 beta-estradiol increased embryo implantation rates compared to the control without 17 beta-estradiol. However, ultra-high concentrations of 17 beta-estradiol around 100 nanomolar significantly reduced embryo implantation rates.
