mouse embryo culture and attachment on ishikawa cells to explore endometrial receptivity

Evaluating Embryo Adhesion in an In Vitro Co-Culture Model with Ishikawa Cells

Embryo implantation, the initial step of successful pregnancy, is crucial for understanding its biological basis and addressing the challenges of infertility. However, ethical constraints pose significant limitations in collecting human clinical embryo samples, hampering research into the intricate interactions between human embryos and the endometrium during early pregnancy1. A profound comprehension of these complex mechanisms is vital for advancing fundamental biological research, drug discovery, and reproductive health.
Previous in vitro models employed adhesion models where monolayer human endometrial epithelial cells (RL95-2)2 were co-cultured with embryonic substitutes, such as BeWo, JAr, or Jeg-33 choriocarcinoma cells. Nevertheless, the selection of the size of the spheroids, ranging from 70-100 &#181;m, was crucial as larger or smaller cell aggregates could compromise the accuracy of the experiment. Notably, only 25% of the spheroids in each preparation met the standard for the suitable size4, and there were significant differences compared to the physiological conditions of blastocysts.
Furthermore, three-dimensional (3D) culture systems for the endometrium and blastocyst offer a more physiologically relevant environment5, but they require high technical expertise, slow cell growth, high resource and maintenance costs, difficulty in standardization, and low reproducibility6.
In this protocol, we present a cost-effective and rapid in vitro model of embryo implantation that can be developed and utilized in most laboratories. The overall objective of this method is to provide a reliable and reproducible platform for studying the interactions between embryos and the endometrium in a controlled environment. By simulating key events of embryo implantation in vitro, this model aims to bridge the gap between animal models and clinical settings, enabling more precise and targeted research.
Compared to embryonic substitutes, the use of mouse blastocysts offers a more physiologically relevant representation of the in vivo embryo implantation process7. In addition, the Ishikawa cell line, derived from human endometrial adenocarcinoma, possesses mixed characteristics of glandular epithelium and uterine lumen epithelium1, enabling it to express enzymes, structural proteins, and functional steroid receptors similar to those found in normal endometrium, thus providing a physiologically relevant substrate for embryo implantation. The simplicity and rapidity of the co-culture system enable high-throughput screening and drug testing8,9,10. By providing a robust and reproducible platform, this method offers an opportunity to advance our understanding of embryo implantation and its impact on human health.

Author Spotlight: Advancing Therapeutic Strategies for Improving Pregnancy Rates by Analyzing Embryo-Endometrium Interactions

Establishment of an Embryo Implantation Model In Vitro

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 risk whereas progesterone supplementation could dependently alleviated these negative effects.This study offers new insights and therapeutic strategies for improving pregnancy risks in fresh embryo transfer during controlled ovarian stimulation for IVF. Compared to 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.

Mouse Embryo Culture and Attachment on Ishikawa Cells to Explore Endometrial Receptivity

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.

mouse-embryo-culture-attachment-on-ishikawa-cells-to-explore

Research

JoVE Journal

Biology

Embryo implantation is the first step in the establishment of a successful pregnancy. An in vitro model for embryo implantation is critical for basic biological research, drug development, and screening. This paper presents a simple, rapid, and highly efficient in vitro model for embryo implantation. In this protocol, we first introduce mouse blastocyst acquisition and human endometrial adenocarcinoma cells (Ishikawa)&#160;preparation for implantation, followed by the co-culture method for mouse embryos and Ishikawa cells. Finally, we conducted a study to assess the impact of varying concentrations of 17-&#946;-estradiol (E2) and progesterone (P4) on embryo adhesion rates based on this model. Our findings revealed that high concentrations of E2 significantly reduced embryo adhesion, whereas the addition of progesterone could restore the adhesion rate. This model offers a simple and fast platform for evaluating and screening molecules involved in the adhesion process, such as cytokines, drugs, and transcription factors controlling implantation and endometrial receptivity.

This protocol describes methodologies to establish simple and efficient embryo implantation in vitro model for evaluating the relevant molecules affecting the embryo implantation process.

Embryo implantation is the first step in the establishment of a successful pregnancy. An in vitro model for embryo implantation is critical for basic ...

Acquisition of Mouse Embryos for In Vitro Implantation Studies

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.