Embryo implantation. The initial step of a successful pregnancy is crucial for understanding its biological basis and addressing the challenges of infertility. The aim of this study is to establish a consistent platform for analyzing embryo-endometrium interactions under controlled conditions.

Based on this in vitro model, we found that ultra-high concentrations of estrogen significantly reduce the embryo implantation rates, whereas progesterone supplementation could dose-dependently alleviate these negative effects. This study offers new insights and therapeutic strategies for improving pregnancy rates in fresh embryo transfer during controlled ovarian stimulation for IVF. Compared to a three-dimensional culture system, this in vitro model offers a simple, rapid, and cost-effective platform that can be established in most laboratories and exhibits high reproducibility and reliability, making it an advantageous tool for research and development in the field of reproductive biology.

To begin, obtain a six to eight-week-old female mouse and inject the PMSG followed by HCG at a 42 to 48-hour interval. Mate the female mouse with a male of comparable age. After euthanizing the female mouse, open the abdomen and locate its genitalia.

Using forceps, stretch the oviduct, ovary, and fat pads. With scissors, cut the oviduct and ovary, followed by the uterus. Transfer the obtained oviduct to a 35-millimeter Petri dish prefilled with M2 medium.

Puncture the enlarged ampulla of the oviduct to release or pinch the cumulus zygote complex. Using forceps, transfer the complex to M2 solution containing 0.5 milligrams per milliliter of hyaluronidase and incubate for several minutes. With an egg transfer pipette, collect the zygotes and wash them in M2 medium containing BSA to remove residual hyaluronidase, cumulus cells, and impurities.

To begin, obtain embryos from freshly mated female mouse. Prepare 12 micro droplets 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 5 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.