我们的研究得到了国家自然科学基金 （82171603）、上海市卫生健康委员会基金 （202140341）、上海市科学技术委员会 （23JC1403803） 以及国家卫健委和上海市重点实验室创新促进计划（RC2023-02）的支持。

小鼠处理和实验研究根据上海生物医学与制药技术研究所动物委员会批准的方案进行。确保安全程序： 处理化学品或生物材料时，请始终佩戴适当的个人防护设备 （PPE）。
1. 小鼠胚胎的获取
注意：本节介绍获取小鼠胚胎的过程。成年雌性小鼠 （6-8 周龄，杂交 C57BL/6 和 DBA/2） 在 20-25 °C 和 40-60% 湿度下维持 12 小时光照和 12 小时黑暗的标准环境条件下， 随意提供食物和水。
<ol><li>腹膜内注射雌性小鼠 10 IU 妊娠母马血清促性腺激素 （PMSG），然后以 42-48 小时的间隔注射 10 IU 人绒毛膜促性腺激素 （HCG）。</li>
	<li>将每只雌性小鼠与同等年龄的雄性小鼠交配，并在第二天早上检查阴道塞。 注意：阴道栓是精液在阴道口凝固后形成的浅黄色栓剂块。</li>
	<li>通过吸入 CO2 对带有阴道塞的雌性小鼠实施安乐死。</li>
	<li>打开腹部，找到女性生殖器;用镊子拉伸输卵管、卵巢和脂肪垫;用剪刀剪断输卵管和卵巢;然后，切开输卵管和子宫（图 1）。</li>
	<li>将获得的输卵管转移到预装有 M2 培养基的 35 mm 培养皿中。 注：M2 培养基应在培养箱（37 °C，5% CO2 ）中预热 30 分钟。</li>
	<li>用 25 G 针刺穿扩大的输卵管壶腹，以释放或捏住卵丘-受精卵复合物（图 2）。</li>
	<li>使用镊子将卵丘-合子复合物转移到含有 0.5 mg/mL 透明质酸酶的 M2 溶液中，并孵育几分钟直至卵丘细胞解离（图 3）。</li>
	<li>使用鸡蛋转移移液管收集受精卵，并在含有 4 mg/mL BSA 的 M2 培养基中洗涤，以去除残留的透明质酸酶、卵丘细胞和杂质。</li>
</ol>2. 小鼠胚胎的 体外 培养
注：本节详细介绍了从受精卵到囊胚的 小鼠 胚胎体外培养的过程。KSOM 培养微滴应提前 24 小时制备，以确保温度和气体平衡。
<ol><li>在 35 mm 培养皿的底部准备 12 个 KSOM 培养基微滴，每个微滴的体积为 20 μL。用 3-5 mL 矿物油覆盖微滴（图 4），并将培养皿放入 37 °C 和 5% CO2 的培养箱中以平衡。</li>
	<li>使用移液管将小鼠胚胎从 M2 培养基转移到 KSOM 培养基中，以进行彻底洗涤。 注意：此步骤需要具有 37 °C 加热台的立体显微镜。尽量减少在培养箱外处理胚胎的时间。</li>
	<li>将洗涤过的胚胎放入准备好的 KSOM 微滴中，并在 37 °C 和 5% CO2 下孵育 4 天。 注：为确保营养充足，每 20 μL 微滴培养 5-10 个胚胎。</li>
	<li>根据它们的大小 （100-130 μm）、内细胞质量（内细胞团由许多全部压实的细胞组成）、滋养层细胞的数量（滋养外胚层由许多紧密结合并堆积在一起的细胞组成）和扩张程度（囊胚腔占据胚胎的 80-100%）选择有利的囊胚。</li>
</ol>3. 子宫内膜细胞的制备
<ol><li>在生物安全柜内，使用移液管吸出 1 mL 2% 明胶，并将其分配到 12 孔培养板中。轻轻旋转板以确保明胶完全覆盖底部。将板置于 37 °C 培养箱中 30 分钟。然后，吸出多余的明胶，让板底完全干燥。</li>
	<li>包被过程完成后，用 DMEM/F12（详见 材料表 ）、1 mM 丙酮酸钠、2 mM L-谷氨酰胺、100 μg/mL 链霉素和 100 IU/mL 青霉素培养人子宫内膜腺癌细胞（Ishikawa），将它们接种到明胶包被的 12 孔板（2 × 105个细胞/孔）中，并孵育 24 小时。</li>
</ol>4. 胚胎附着
<ol><li>轻轻地将 5-15 个新鲜囊胚放入含有上一步制备用于共培养的 Ishikawa 细胞的 12 孔板的每个孔中（图 5）。</li>
	<li>孵育 48 小时后，在显微镜下观察胚胎以评估其附着状态。要观察胚胎附着，请快速移动板 3 次。未附着的胚胎将在 Ishikawa 细胞表面表现出漂浮或滚动运动。</li>
	<li>使用等式 （1） 计算胚胎着床率;使用胚胎粘附率表示植入效果。 胚胎着床率 = （附着的胚胎数/植入的胚胎总数） × 100% （1）</li>
</ol>

在与 Ishikawa 细胞的体外共培养模型中评估胚胎粘附

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作者无需声明任何财务或其他利益冲突。

所提出的胚胎植入体外模型的简单性和快速性为早期药物筛选和其他研究应用提供了显着的优势。简单的方案，加上 Ishikawa 细胞系的高通量特性，使其成为大规模筛选的理想选择，尤其是在药物发现的早期阶段。Liang13 发现 TAGLN2 敲低降低了滋养层细胞的侵袭能力。Green14 表明胰岛素样生长因子提高了基于小鼠胚胎-Ishikawa 细胞系统的小鼠囊胚的粘附能力?...

胚胎植入是成功怀孕的第一步，对于了解其生物学基础和应对不孕症的挑战至关重要。然而，伦理限制对收集人类临床胚胎样本构成了重大限制，阻碍了对怀孕早期人类胚胎与子宫内膜之间复杂相互作用的研究1。对这些复杂机制的深刻理解对于推进基础生物学研究、药物发现和生殖健康至关重要。
以前的体外模型采用粘附模型，其中单层人子宫内膜上皮细胞 （RL95-2）2 与胚胎替代品（如 BeWo、JAr 或 Jeg-33 绒毛膜癌细胞）共培养。然而，球体大小的选择（范围为 70-100 μm）至关重要，因为更大或更小的细胞聚集体可能会影响实验的准确性。值得注意的是，每种制剂中只有 25% 的球体符合合适大小4 的标准，并且与囊胚的生理条件相比存在显着差异。
此外，子宫内膜和囊胚的三维 （3D） 培养系统提供了更具生理相关性的环境5，但它们需要高技术专长、细胞生长缓慢、资源和维护成本高、标准化困难和可重复性低6。
在该协议中，我们提出了一种经济高效且快速的胚胎植入体外模型，该模型可以在大多数实验室中开发和使用。该方法的总体目标是为在受控环境中研究胚胎和子宫内膜之间的相互作用提供一个可靠且可重复的平台。通过模拟 体外胚胎植入的关键事件，该模型旨在弥合动物模型和临床环境之间的差距，从而实现更精确和有针对性的研究。
与胚胎替代品相比，小鼠囊胚的使用提供了体内胚胎植入过程的更生理相关性的表示7。此外，源自人子宫内膜腺癌的 Ishikawa 细胞系具有腺上皮和子宫腔上皮1 的混合特性，使其能够表达类似于正常子宫内膜中的酶、结构蛋白和功能性类固醇受体，从而为胚胎植入提供生理相关的底物。共培养系统的简单性和快速性可实现高通量筛选和药物检测 8,9,10。通过提供一个强大且可重复的平台，这种方法为促进我们对胚胎植入及其对人类健康影响的理解提供了机会。

<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

胚胎植入是成功怀孕的第一步。胚胎植入的 体外 模型对于基础生物学研究、药物开发和筛选至关重要。本文提出了一种简单、快速、高效的 胚胎 植入体外模型。在该方案中，我们首先介绍小鼠囊胚获取和人子宫内膜腺癌细胞 （Ishikawa） 植入制剂，然后是小鼠胚胎和 Ishikawa 细胞的共培养方法。最后，我们进行了一项研究，以评估不同浓度的 17-β-雌二醇 （E2） 和孕酮 （P4） 对基于该模型的胚胎粘附率的影响。我们的研究结果表明，高浓度的 E2 显着降低了胚胎粘附，而添加黄体酮可以恢复粘附率。该模型提供了一个简单快速的平台，用于评估和筛选参与粘附过程的分子，例如细胞因子、药物和控制着床和子宫内膜容受性的转录因子。

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.

作者聚焦：通过分析胚胎-子宫内膜相互作用推进提高妊娠率的治疗策略

体外胚胎植入模型的建立

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.

在辅助生殖技术过程中，受控卵巢过度刺激 （COH） 是关键步骤，与自然周期相比，导致患者的雌激素水平显着升高 10-20 倍以上11。鉴于此，我们询问了高血清雌激素浓度是否会影响新鲜胚胎移植过程中的胚胎着床。基于这个胚胎植入模型，我们研究了不同浓度的 E2 和 P4 对胚胎着床率的影响12。饥饿处理后，将 Ishikawa 细胞与 E2 （0、0.1、 1、10 ?...

该协议描述了建立简单有效的胚胎植入 体外 模型的方法，用于评估影响胚胎植入过程的相关分子。

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

Research

JoVE Journal

Biology

Establishment of an Embryo Implantation Model In Vitro

338 次观看.  Fudan University. 胚胎植入是成功怀孕的第一步，对于了解其生物学基础和应对不孕症的挑战至关重要。本研究的目的是建立一个一致的平台，用于在受控条件下分析胚胎子宫内膜相互作用。基于这个体外模型，我们发现超高浓度的雌激素显着降低了胚胎着床的风险，而黄体酮补充剂可以依赖地减轻这些负面影响。本研究为改善 IVF 控制性卵巢刺激期间新鲜胚胎?...