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 C57BL/6 and DBA/2) were maintained under standard environmental conditions of 12 h light and 12 h dark at 20-25 &#176;C and 40-60% humidity with food and water provided ad libitum.
<ol>
	<li>Inject female mice intraperitoneally with 10 IU of pregnant mare&#39;s serum gonadotropin (PMSG) and then, injected with 10 IU of human chorionic gonadotropin (HCG) at a 42-48 h interval.</li>
	<li>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.</li>
	<li>Euthanize the female mice with vaginal plugs by CO2 inhalation.</li>
	<li>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).</li>
	<li>Transfer the obtained oviduct to a 35 mm Petri dish prefilled with M2 medium. 
	NOTE: M2 medium should be preheated in an incubator (37 &#176;C, 5% CO2) for 30 min.</li>
	<li>Puncture the enlarged ampulla of the oviduct with a 25 G needle to release or pinch the cumulus-zygote complex (Figure 2).</li>
	<li>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).</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>Prepare 12 microdroplets of KSOM medium, each with a volume of 20 &#181;L, 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 &#176;C with 5% CO2 for equilibration.</li>
	<li>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 &#176;C heating stage. Minimize the duration of embryo handling outside the incubator.</li>
	<li>Place the washed embryos in the prepared KSOM microdroplets and incubate them at 37 &#176;C with 5% CO2 for 4 days. 
	NOTE: To ensure adequate nutrition, culture 5-10 embryos per 20 &#181;L microdrop.</li>
	<li>Select advantageous blastocysts according to their size (100-130 &#181;m), 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).</li>
</ol>
3. Preparation of endometrial cells
<ol>
	<li>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 &#176;C incubator for 30 min. Afterward, aspirate the excess gelatin and allow the bottom of the plate to dry completely.</li>
	<li>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 &#181;g/mL streptomycin, and 100 IU/mL penicillin, seed them into the gelatin-coated 12-well plates (2 &#215; 105cells/well), and incubate for 24 h.</li>
</ol>
4. Embryo attachment
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>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) &#215; 100%&#160; &#160; (1)</li>
</ol>

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

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	<li>Hannan, N. J., Paiva, P., Dimitriadis, E., Salamonsen, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+for+study+of+human+embryo+implantation:+choice+of+cell+lines.">Models for study of human embryo implantation: choice of cell lines.</a> Biol Reprod. 82 (2), 235-245 (2010).</li><li>Li, H. Y., 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=Establishment+of+an+efficient+method+to+quantify+embryo+attachment+to+endometrial+epithelial+cell+monolayers.">Establishment of an efficient method to quantify embryo attachment to endometrial epithelial cell monolayers.</a> In Vitro Cell Dev Biol Anim. 38 (9), 505-511 (2002).</li><li>Hohn, H. P., Linke, M., Denker, H. 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. M., 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=Stem-cell-derived+trophoblast+organoids+model+human+placental+development+and+susceptibility+to+emerging+pathogens.">Stem-cell-derived trophoblast organoids model human placental development and susceptibility to emerging pathogens.</a> Cell Stem Cell. 29 (5), 810-825 (2022).</li><li>Xiang, L., 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=A+developmental+landscape+of+3D-cultured+human+pre-gastrulation+embryos.">A developmental landscape of 3D-cultured human pre-gastrulation embryos.</a> Nature. 577 (7791), 537-542 (2020).</li><li>Ojosnegros, S., Seriola, A., Godeau, A. 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. 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The authors have no conflicts of financial or other interests to declare.

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><tbody><tr><td>17-&amp;beta;-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 &lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/66873/66873eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt;BSC</td><td>Aseptic operations, making culture dishes, aliquoting reagents, etc</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; 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&amp;nbsp;</td><td>SIGMA</td><td>V900833</td><td>Reagent grade, powder&amp;nbsp;</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&amp;nbsp;</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.

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...

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

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

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 ...

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

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JoVE Journal

Biology

719 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.

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

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Developmental Biology

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.

We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo ...

Cancer Research

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Human Blastocyst Biopsy and Vitrification

Blastocyst biopsy is performed to obtain a reliable genetic diagnosis during IVF cycles with preimplantation genetic testing. Then, the ideal workflow entails a safe and efficient vitrification protocol, due to the turnaround time of the diagnostic techniques and to transfer the selected embryo(s) on a physiological endometrium in a following natural cycle. A biopsy approach encompassing the sequential opening of the zona pellucida and retrieval of 5-10 trophectoderm cells (ideally 7-8) limits both the number of manipulations required and the exposure of the embryo to sub-optimal environmental conditions. After proper training, the technique was reproducible across different operators in terms of timing of biopsy (~8 min, ranging 3-22 min based on the number of embryos to biopsy per dish), conclusive diagnoses obtained (~97.5%) and live birth rates after vitrified-warmed euploid blastocyst transfer (&#62;40%). The survival rate after biopsy, vitrification and warming was as high as 99.8%. The re-expansion rate at 1.5 h from warming was as high as 97%, largely dependent on the timing between biopsy and vitrification (ideally &#8804;30 min), blastocyst morphological quality and day of biopsy. In general, it is better to vitrify a collapsed blastocyst; therefore, in non-PGT cycles, laser-assisted artificial shrinkage might be performed to induce embryo collapse prior to cryopreservation. The most promising future perspective is the non-invasive analysis of the IVF culture media after blastocyst culture as a putative source of embryonic DNA. However, this potential avant-garde is still under investigation and a reliable protocol yet needs to be defined and validated.

Blastocyst biopsy and vitrification are required to efficiently conduct preimplantation genetic testing. An approach entailing the sequential opening of the zona pellucida and retrieval of 7-8 trophectoderm cells in day 5-7 post-insemination limits both the number of manipulations required and the exposure of the embryo to sub-optimal environmental conditions.

Blastocyst biopsy is performed to obtain a reliable genetic diagnosis during IVF cycles with preimplantation genetic testing. Then, the ideal workflow ...

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. ...

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...

3D-Printed Scaffold Model to Investigate Embryo Implantation Mechanisms

Co-culture Model Using Two Types of Adherent Cell Lines

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications between various cell types within the decidua are crucial for regulating endometrial receptivity; however, the molecular mechanisms mediating this interaction are still unclear. In this regard, a model to study the implantation process is needed to establish a comprehensive in vitro model that can recapitulate the biology of endometrial epithelium-stroma interaction. This model is composed of regular cell-culture plates and a matching scaffold, which is generated by three-dimensional (3D) printing from low-cost materials. Here, we detail a set of protocols for model construction, cell preparation, cell seeding, cell culture, observation, and evaluation. Furthermore, we have included representative results with cells exhibiting good growth conditions under the microscope. This study aimed to develop in vitro models that would mimic the interaction between endometrial stromal cells and epithelial cells, as well as between trophoblast cells and endometrial cells.

Author Spotlight: In Vitro Endometrial Models to Study Epithelial-Stroma and Embryo Interactions

The protocol shows a novel in vitro experimental model that can recapitulate the biology of two kinds of adherent cell lines with a three-dimensional (3D)-printed scaffold. The construction of this model and operating procedures, from cell preparation and cell culture to analysis and evaluation, are described.

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications ...

Chicken Chorioallantoic Membrane-based Cancer Modeling: An In Ovo Model to Study Cancer Cell Tumorigenesis and Metastasis

Source: Sharrow, A. C. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/60651">Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers.</a> J. Vis. Exp.&#160;(2020)
In this video, we describe the implantation of cancer cells onto a chicken chorioallantoic membrane to generate an in ovo model. The model successfully engrafts the ovarian cancer cells to study the progression of gynecological cancer.

Modelowanie raka błony moczowo-omoczniowej kurczaka: model in ovo do badania nowotworzenia i przerzutów komórek rakowych

Source: Sharrow, A. C. et al. Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers. J. Vis. ...

JoVE Encyclopedia of Experiments

Gynecologic Cancer

Animal model studies

Chick Chorioallantoic Membrane or CAM Tumor Model: An In Ovo Method to Simulate Perineural Invasion of Tumor Cells

Source: Schmidt, L. B. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/59296" target="_blank">The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer.</a> J. Vis. Exp. (2019)
In this video, we describe the method to co-culture dorsal root ganglia and cancer cells on chick chorioallantoic membrane or CAM to study perineural invasion.

Błona kosmówkowo-omocznaniowa piskląt lub model guza CAM: metoda in ovo do symulacji okołonerwowej inwazji komórek nowotworowych

Source: Schmidt, L. B. et al. The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer. J. Vis. Exp. (2019)
...

Head and Neck Cancer

In vivo studies

In Vitro Fertilization

In vitro fertilization (IVF) is a form of assisted reproductive technology where an egg is fertilized with sperm in a controlled laboratory environment before transferring the resulting embryo into the uterus. This process is designed to help individuals and couples experiencing difficulties conceiving.
The IVF process begins with ovarian stimulation, during which reproductive endocrinologists prescribe hormonal medications to stimulate the ovaries to produce multiple eggs instead of the single egg released during a natural cycle. This increases the likelihood of successful fertilization and embryo development.
Once the ovaries have been sufficiently stimulated, the next step is oocyte retrieval. This procedure typically occurs approximately 36 hours after the triggering injection to induce final egg maturation. A transvaginal ultrasound is performed to visualize the ovaries. Using a needle guide, a physician carefully inserts a thin needle through the vaginal wall and into the ovarian follicles, where the mature eggs reside. The needle aspirates the follicles, collecting the oocytes and the follicular fluid. This process is done under sedation or anesthesia to minimize discomfort for the patient.
After retrieval, the oocytes are placed in a specialized culture dish, where they will be fertilized. In conventional IVF, the retrieved oocytes are incubated with a prepared sperm sample (containing approximately 50,000 to 100,000 sperm cells for 12 to 18 hours, allowing natural fertilization.
In cases where male infertility is a factor, a more specialized technique called intracytoplasmic sperm injection (ICSI) may be utilized. This method involves selecting a single, healthy sperm and directly injecting it into the cytoplasm of a mature egg using a fine glass needle. ICSI improves the likelihood of fertilization, particularly in situations involving low sperm count or poor sperm quality.
After fertilization, the resulting embryos are monitored for development over several days. Embryologists assess their growth and quality, determining the optimal time for transfer. The embryos can be transferred at different developmental stages, such as the cleavage stage (typically 3 days post-fertilization) or the blastocyst stage (about 5 days post-fertilization). Blastocyst transfers are often preferred due to higher implantation rates.
The final step in the IVF process is the embryo transfer. This procedure is performed in a sterile environment, usually done in conjunction with a transabdominal ultrasound for guidance. A thin catheter is inserted through the cervix into the uterus, and the selected embryos are gently placed into the uterine cavity, ideally within 1 to 2 centimeters of the uterine fundus, the upper part of the uterus. Following the embryo transfer, a pregnancy test is typically conducted around two weeks later to determine if implantation has occurred and if the woman is pregnant.

Zapłodnienie in vitro

In vitro fertilization, or IVF, is a process where eggs are fertilized with sperm in a lab and then implanted in the uterus.
The process begins with ...

Establishment of an Embryo Implantation Model In Vitro

Podsumowanie

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Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Human Blastocyst Biopsy and Vitrification

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Co-culture Model Using Two Types of Adherent Cell Lines

Modelowanie raka błony moczowo-omoczniowej kurczaka: model in ovo do badania nowotworzenia i przerzutów komórek rakowych

Błona kosmówkowo-omocznaniowa piskląt lub model guza CAM: metoda in ovo do symulacji okołonerwowej inwazji komórek nowotworowych

Zapłodnienie in vitro