Our studies were supported by the National Natural Science Foundation of China (82171603), the Foundation of Shanghai Municipal Health Commission (202140341), the Science and Technology Commission of Shanghai Municipality (23JC1403803), and Innovation Promotion Program of NHC and Shanghai Key Labs, SIBPT (RC2023-02).

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 ...

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

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+of+trophoblast+to+uterine+epithelium+as+related+to+the+state+of+trophoblast+differentiation:+in+vitro+studies+using+cell+lines.">Adhesion of trophoblast to uterine epithelium as related to the state of trophoblast differentiation: in vitro studies using cell lines.</a> Mol Reprod Dev. 57 (2), 135-145 (2000).</li><li>Ho, H., Singh, H., Aljofan, M., Nie, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+in+vitro+model+of+human+embryo+attachment.">A high-throughput in vitro model of human embryo attachment.</a> Fertil Steril. 97 (4), 974-978 (2012).</li><li>Karvas, R. 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L., Veiga, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+implantation+in+the+laboratory:+an+update+on+current+techniques.">Embryo implantation in the laboratory: an update on current techniques.</a> Hum Reprod Update. 27 (3), 501-530 (2021).</li><li>Berneau, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+osteopontin+in+an+in+vitro+model+of+embryo+implantation.">Characterisation of osteopontin in an in vitro model of embryo implantation.</a> Cells. 8 (5), 432 (2019).</li><li>Jiang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+HOXA10+sumoylation+inhibits+embryo+implantation+in+women+with+recurrent+implantation+failure.">Enhanced HOXA10 sumoylation inhibits embryo implantation in women with recurrent implantation failure.</a> Cell Death Discov. 3, 17057 (2017).</li><li>Kang, Y. J., Forbes, K., Carver, J., Aplin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+osteopontin-integrin+&#945;v&#946;3+interaction+at+implantation:+functional+analysis+using+three+different+in+vitro+models.">The role of the osteopontin-integrin &#945;v&#946;3 interaction at implantation: functional analysis using three different in vitro models.</a> Hum Reprod. 29 (4), 739-749 (2014).</li><li>Duan, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+exposure+to+supraphysiological+estradiol+during+ovarian+stimulation+increased+the+risk+of+adverse+perinatal+outcomes+after+frozen-thawed+embryo+transfer:+a+retrospective+cohort+study.">Oocyte exposure to supraphysiological estradiol during ovarian stimulation increased the risk of adverse perinatal outcomes after frozen-thawed embryo transfer: a retrospective cohort study.</a> J Dev Orig Health Dis. 11 (4), 392-402 (2020).</li><li>Chen, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+&#946;1+and+&#946;3+are+biomarkers+of+uterine+condition+for+embryo+transfer.">Integrins &#946;1 and &#946;3 are biomarkers of uterine condition for embryo transfer.</a> J Transl Med. 14 (1), 303-312 (2016).</li><li>Liang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgelin+2+is+required+for+embryo+implantation+by+promoting+actin+polymerization.">Transgelin 2 is required for embryo implantation by promoting actin polymerization.</a> FASEB J. 33 (4), 5667-5675 (2019).</li><li>Green, C. J., Fraser, S. T., Day, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-like+growth+factor+1+increases+apical+fibronectin+in+blastocysts+to+increase+blastocyst+attachment+to+endometrial+epithelial+cells+in+vitro.">Insulin-like growth factor 1 increases apical fibronectin in blastocysts to increase blastocyst attachment to endometrial epithelial cells in vitro.</a> Hum Reprod. 30 (2), 284-298 (2015).</li><li>Kang, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiR-145+suppresses+embryo-epithelial+juxtacrine+communication+at+implantation+by+modulating+maternal+IGF1R.">MiR-145 suppresses embryo-epithelial juxtacrine communication at implantation by modulating maternal IGF1R.</a> J Cell Sci. 128 (4), 804-814 (2015).</li><li>Berger, C., Boggavarapu, N. R., Menezes, J., Lalitkumar, P. G., Gemzell-Danielsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+ulipristal+acetate+on+human+embryo+attachment+and+endometrial+cell+gene+expression+in+an+in+vitro+co-culture+system.">Effects of ulipristal acetate on human embryo attachment and endometrial cell gene expression in an in vitro co-culture system.</a> Hum Reprod. 30 (4), 800-811 (2015).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+models+of+human+endometrium+for+studying+trophoblast-endometrium+interaction+during+implantation.">Three-dimensional culture models of human endometrium for studying trophoblast-endometrium interaction during implantation.</a> Reprod Biol Endocrinol. 20 (1), 120-138 (2022).</li><li>Fitzgerald, H. C., Schust, D. J., Spencer, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+the+human+endometrium:+evolution+and+application+for+women's+health.">In vitro models of the human endometrium: evolution and application for women's health.</a> Biol Reprod. 104 (2), 282-293 (2021).</li><li>Ahn, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+microengineered+vascularised+endometrium-on-a-chip.">Three-dimensional microengineered vascularised endometrium-on-a-chip.</a> Hum Reprod. 36 (10), 2720-2731 (2021).</li><li>Sternberg, A. K., Buck, V. U., Classen-Linke, I., Leube, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+mechanical+forces+change+the+human+endometrium+during+the+menstrual+cycle+in+preparation+for+embryo+implantation.">How mechanical forces change the human endometrium during the menstrual cycle in preparation for embryo implantation.</a> Cells. 10 (8), 2008 (2021).</li></ol>

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</sup...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>17-&#946;-estradiol</td>
			<td>SIGMA</td>
			<td>3301</td>
			<td>Most potent mammalian estrogenic hormone</td>
		</tr>
		<tr>
			<td>BD Falcon</td>
			<td>BD</td>
			<td>353001</td>
			<td>Bacteriological Petri Dishes 35 x 10 mm style w/tight lid, crystal-grade virgin polystyrene, sterile</td>
		</tr>
		<tr>
			<td>Biosafety Cabinet</td>
			<td>ESCO</td>
			<td>class <img alt="Equation 1" src="/files/ftp_upload/66873/66873eq01.jpg" style="margin: auto;" />BSC</td>
			<td>Aseptic operations, making culture dishes, aliquoting reagents, etc</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo</td>
			<td>8000DH</td>
			<td>Embryo culture</td>
		</tr>
		<tr>
			<td>Culture plate</td>
			<td>Corning</td>
			<td>3506</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>1133032</td>
			<td>DMEM/F12 (1x), liquid 1:1,Contains L-glutamine and 15 mM HEPES buffer</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10099141</td>
			<td>Fetal Bovine Serum, Qualified, Australia Origin</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>SIGMA</td>
			<td>G9391</td>
			<td>Type B, powder, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>HCG</td>
			<td>Nanjing Aibei</td>
			<td>M2520</td>
			<td>Sterilization reagent, intraperitoneal injection, 50 IU/mL</td>
		</tr>
		<tr>
			<td>Hyaluronidase&#160;</td>
			<td>SIGMA</td>
			<td>V900833</td>
			<td>Reagent grade, powder&#160;</td>
		</tr>
		<tr>
			<td>KSOM</td>
			<td>Merck</td>
			<td>MR-020P-D</td>
			<td>(1x), Powder, w/o Phenol Red, 5 x 10 mL</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030081</td>
			<td>L-glutamine-200 mM (100x), liquid</td>
		</tr>
		<tr>
			<td>M2</td>
			<td>Merck</td>
			<td>MR-015-D</td>
			<td>EmbryoMax M2 Medium (1x), Liquid, with Phenol Red</td>
		</tr>
		<tr>
			<td>Mineral oil&#160;</td>
			<td>SIGMA</td>
			<td>M8410</td>
			<td>Mineral oil is suitable for use as a cover layer to control evaporation and cross-contamination in various molecular biology applications</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, liquid</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>10,000 Units penicillin (base) and 10,000 units streptomycin (base), utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline</td>
		</tr>
		<tr>
			<td>PMSG</td>
			<td>Nanjing Aibei</td>
			<td>M2620</td>
			<td>Sterilization reagent ,intraperitoneal injection, 50 IU/mL</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>SIGMA</td>
			<td>5341</td>
			<td>Steroid hormone secreted by the corpus luteum during the latter half of the menstrual cycle</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360070</td>
			<td>Sodium pyruvate is commonly added to cell culture media as a carbon source in addition to glucose</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Olympus</td>
			<td>SZX7</td>
			<td>Embryo retrieval and observation of embryo development</td>
		</tr>
	</tbody>
</table>

establishment of an embryo implantation model in vitro

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.

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.

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&#160;and P4&#160;on embryo implantat...

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 ...

establishment-of-an-embryo-implantation-model-in-vitro

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Biology

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.

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.

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

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

Summary