Establishment of an Embryo Implantation Model In Vitro

Summary

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

Abstract

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) 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-β-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.

Introduction

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 µm, 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 testing^8,9,10. 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.

Protocol

Mouse handling and experimental studies were performed under protocols approved by the Animal Committee of the Shanghai Institute for Biomedical and Pharmaceutical Technologies. Ensure Safety Procedures: Always wear appropriate personal protective equipment (PPE) when handling chemicals or biological materials.

1. Acquisition of mouse embryos

NOTE: This section describes the process of obtaining mouse embryos. Adult female mice (6-8 weeks old, hybrid C57BL/6 and DBA/2) were maintained under standard environmental conditions of 12 h light and 12 h dark at 20-25 °C and 40-60% humidity with food and water provided ad libitum.

1. Inject female mice intraperitoneally with 10 IU of pregnant mare's serum gonadotropin (PMSG) and then, injected with 10 IU of human chorionic gonadotropin (HCG) at a 42-48 h interval.
2. Mate each female mouse with a male of comparable age and check for a vaginal plug the next morning.
   NOTE: Vaginal plug is a light yellow lump of suppository formed after semen solidifies at the vaginal opening.
3. Euthanize the female mice with vaginal plugs by CO₂ inhalation.
4. Open the abdomen and locate the female genitalia; stretch the oviduct, ovary, and fat pads with forceps; cut the oviduct and ovary with scissors; and then, cut the oviduct and uterus (Figure 1).
5. Transfer the obtained oviduct to a 35 mm Petri dish prefilled with M2 medium.
   NOTE: M2 medium should be preheated in an incubator (37 °C, 5% CO₂) for 30 min.
6. Puncture the enlarged ampulla of the oviduct with a 25 G needle to release or pinch the cumulus-zygote complex (Figure 2).
7. Use forceps to transfer the cumulus-zygote complex to M2 solution containing 0.5 mg/mL hyaluronidase and incubate for several minutes until the cumulus cells dissociate (Figure 3).
8. Collect the zygotes using an egg transfer pipette and wash them in M2 medium containing 4 mg/mL BSA to remove residual hyaluronidase, cumulus cells, and impurities.

2. In vitro culture of mouse embryo

NOTE: This section details the process of in vitro culturing mouse embryos from zygotes to blastocysts. KSOM culture microdrops should be prepared 24 h ahead to ensure temperature and gas equilibrium.

1. Prepare 12 microdroplets of KSOM medium, each with a volume of 20 µL, at the bottom of a 35 mm Petri dish. Cover the microdroplets with 3-5 mL of mineral oil (Figure 4), and place the Petri dishes in an incubator at 37 °C with 5% CO₂ for equilibration.
2. Transfer the mouse embryos from M2 medium to KSOM medium using a transfer pipette for thorough washing.
   NOTE: This step requires a stereomicroscope with a 37 °C heating stage. Minimize the duration of embryo handling outside the incubator.
3. Place the washed embryos in the prepared KSOM microdroplets and incubate them at 37 °C with 5% CO₂ for 4 days.
   NOTE: To ensure adequate nutrition, culture 5-10 embryos per 20 µL microdrop.
4. Select advantageous blastocysts according to their size (100-130 µm), inner cell mass (the inner cell mass is composed of many cells that are all compacted), number of trophoblast cells (the trophectodermis composed of many cells that are cohesive and packed together), and the degree of expansion (blastocoel occupies 80-100% of the embryo).

3. Preparation of endometrial cells

1. Inside a biosafety cabinet, aspirate 1 mL of 2% gelatin using a pipette and dispense it into a 12-well culture plate. Gently rotate the plate to ensure complete coverage of the bottom with the gelatin. Place the plate in a 37 °C incubator for 30 min. Afterward, aspirate the excess gelatin and allow the bottom of the plate to dry completely.
2. After the coating process is complete, culture human endometrial adenocarcinoma cells (Ishikawa) with DMEM/F12 (see the Table of Materials for details), 1 mM sodium pyruvate, 2 mM L-glutamine, 100 µg/mL streptomycin, and 100 IU/mL penicillin, seed them into the gelatin-coated 12-well plates (2 × 10⁵cells/well), and incubate for 24 h.

4. Embryo attachment

1. Gently place 5-15 fresh blastocysts into each well of the 12-well plate containing Ishikawa cells prepared in the previous step for co-culture (Figure 5).
2. After 48 h incubation, observe the embryos under a microscope to assess their attachment status. To observe embryo attachment, quickly move the plate three times. Unattached embryos will exhibit floating or rolling motion over the surface of the Ishikawa cells.
3. Calculate the embryo implantation rate using equation (1); use the embryo adhesion rate to represent the implantation effect.
   Embryo Implantation Rate = (Attached Embryos/Total Number of Implanted Embryos) × 100%    (1)

Results

In the process of assisted reproductive techniques, controlled ovarian hyperstimulation (COH) is a crucial step, leading to significantly elevated estrogen levels in patients by more than 10-20 times compared to natural cycles¹¹. Given this context, we asked whether high serum estrogen concentrations impact embryo implantation during fresh embryo transfer. Based on this embryo implantation model, we studied the effects of various concentrations of E₂ and P4 on embryo implantat...

Discussion

The simplicity and rapidity of the proposed in vitro model for embryo implantation offer remarkable advantages for early-stage drug screening and other research applications. The straightforward protocol, coupled with the high-throughput nature of Ishikawa cell lines, makes it an ideal candidate for large-scale screens, particularly in the early stages of drug discovery. Liang¹³ found that TAGLN2 knockdown decreased the invasion ability of trophoblast cells. Green¹⁴

Materials

Name	Company	Catalog Number	Comments
17-β-estradiol	SIGMA	3301	Most potent mammalian estrogenic hormone
BD Falcon	BD	353001	Bacteriological Petri Dishes 35 x 10 mm style w/tight lid, crystal-grade virgin polystyrene, sterile
Biosafety Cabinet	ESCO	class BSC	Aseptic operations, making culture dishes, aliquoting reagents, etc
CO2 Incubator	Thermo	8000DH	Embryo culture
Culture plate	Corning	3506	Cell culture
DMEM/F12	Gibco	1133032	DMEM/F12 (1x), liquid 1:1,Contains L-glutamine and 15 mM HEPES buffer
Fetal Bovine Serum	Gibco	10099141	Fetal Bovine Serum, Qualified, Australia Origin
Gelatin	SIGMA	G9391	Type B, powder, BioReagent, suitable for cell culture
HCG	Nanjing Aibei	M2520	Sterilization reagent, intraperitoneal injection, 50 IU/mL
Hyaluronidase	SIGMA	V900833	Reagent grade, powder
KSOM	Merck	MR-020P-D	(1x), Powder, w/o Phenol Red, 5 x 10 mL
L-glutamine	Gibco	25030081	L-glutamine-200 mM (100x), liquid
M2	Merck	MR-015-D	EmbryoMax M2 Medium (1x), Liquid, with Phenol Red
Mineral oil	SIGMA	M8410	Mineral oil is suitable for use as a cover layer to control evaporation and cross-contamination in various molecular biology applications
Penicillin-Streptomycin, liquid	Gibco	15140122	10,000 Units penicillin (base) and 10,000 units streptomycin (base), utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline
PMSG	Nanjing Aibei	M2620	Sterilization reagent ,intraperitoneal injection, 50 IU/mL
Progesterone	SIGMA	5341	Steroid hormone secreted by the corpus luteum during the latter half of the menstrual cycle
Sodium pyruvate	Gibco	11360070	Sodium pyruvate is commonly added to cell culture media as a carbon source in addition to glucose
Stereomicroscope	Olympus	SZX7	Embryo retrieval and observation of embryo development