STUDY QUESTION Does fibrin promote trophoblast growth in human and mouse blastocysts during early embryo implantation? SUMMARY ANSWER Mouse blastocysts were unaffected by fibrin; however, human blastocysts were significantly suppressed by fibrin in trophoblast growth and then switched to growth promotion through increased fibrinolysis with urokinase-type plasminogen activator (uPA) activity. WHAT IS KNOWN ALREADY Fibrin(ogen) plays an important role in various physiological processes and is also critical for maintaining feto-maternal attachment during pregnancy. The addition of fibrin to embryo transfer media has been used to increase implantation rates in human ART; however, its mechanism of action’ in vitro has not yet been characterized. STUDY DESIGN, SIZE, DURATION Vitrified mouse and human blastocysts were warmed and individually cultured in vitro for up to 120 and 168 h, respectively, on a fibrin substrate. Blastocysts were cultured at 37°C in 6% CO2, 5% O2 and 89% N2. Blastocyst development and related fibrinolytic factors were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS ICR strain mouse embryos were purchased from a commercial supplier. Human blastocysts were donated with informed consent from two fertility centers. Mouse and human blastocysts cultured on fibrin-coated plates were compared to those on non-coated and collagen-coated plates in vitro. Trophoblast growth and fibrin degradation were assessed based on the cell area and fibrin-free area, respectively. Fibrinolytic factors were detected in supernatants using plasminogen-casein zymography. The fibrinolytic activity of blastocysts was investigated using a selective uPA inhibitor, exogenous uPA, plasminogen activator inhibitor-1 (PAI-1) inhibitor and fibrin degradation products (FDPs). Fibrinolysis-related mRNA expression level was detected using quantitative real-time PCR. MAIN RESULTS AND THE ROLE OF CHANCE Fibrin did not affect the developmental speed or morphology of mouse blastocysts, and a large fibrin-degrading region was observed in the attachment stage. In contrast, fibrin significantly suppressed the outgrowth of trophoblasts in human blastocysts, and trophoblasts grew after the appearance of small fibrin-degrading regions. uPA was identified as a fibrinolytic factor in the conditioned medium, and uPA activity was significantly weaker in human blastocysts than in mouse blastocysts. The inhibition of uPA significantly reduced the outgrowth of trophoblasts in mouse and human blastocysts. Human blastocysts expressed PLAU (uPA), PLAUR (uPA receptor), SERPINE1 (PAI-1) and SERPINB2 (PAI-2), whereas mouse blastocysts were limited to Plau, Plaur and Serpine1. In a subsequent experiment on human blastocysts, the addition of exogenous uPA and the PAI-1 inhibitor promoted trophoblast growth in the presence of fibrin, as did the addition of FDPs. LIMITATIONS, REASONS FOR CAUTION This model excludes maternal factors and may not be fully reproduced in vivo. Donated human embryos are surplus embryos that may inherently exhibit reduced embryonic development. In addition, donated ART-derived embryos may exhibit weak uPA activity, because women with sufficient uPA-active embryos may not originally require ART. The present study used orthodox culture methods, and results may change with the application of recently developed protocols for culture blastocysts beyond the implantation stage. WIDER IMPLICATIONS OF THE FINDINGS The present results suggest that the distinct features of trophoblast outgrowth in human blastocysts observed in the presence of fibrin are regulated by a phenotypic conversion induced by contact with fibrin and FDPs. Mouse embryos did not exhibit the human phenomenon, indicating that the present results may be limited to humans. STUDY FUNDING/COMPETING INTEREST(S) The present study was supported by the Department of Obstetrics and Gynecology at the Hamamatsu University School of Medicine and Kishokai Medical Corporation. None of the authors have any conflicts of interest to declare. TRIAL REGISTRATION NUMBER N/A.

中文翻译：

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早期人类滋养细胞的纤维蛋白介导的生长限制通过纤维蛋白溶解转换为促进生长

研究问题 在早期胚胎植入过程中，纤维蛋白是否促进人和小鼠囊胚中滋养层的生长？总结 答案 小鼠囊胚不受纤维蛋白的影响。然而，人类胚泡在滋养层生长中受到纤维蛋白的显着抑制，然后通过尿激酶型纤溶酶原激活物 (uPA) 活性增加的纤维蛋白溶解转为促进生长。什么是已知的 纤维蛋白（原）在各种生理过程中发挥重要作用，并且对于在怀孕期间维持胎儿与母体的附着也至关重要。在胚胎移植培养基中添加纤维蛋白已被用于提高人类 ART 的植入率；然而，其在体外的作用机制尚未得到表征。研究设计，尺寸，持续时间 玻璃化的小鼠和人类胚泡被加热并在纤维蛋白基质上分别体外培养长达 120 和 168 小时。囊胚在 37°C、6% CO2、5% O2 和 89% N2 中培养。分析囊胚发育及相关纤溶因素。参与者/材料、设置、方法 ICR 品系小鼠胚胎购自商业供应商。人类囊胚是在两个生育中心的知情同意下捐赠的。在体外将在纤维蛋白包被板上培养的小鼠和人类胚泡与未包被和胶原包被板上的胚泡进行比较。分别根据细胞面积和无纤维蛋白面积评估滋养层生长和纤维蛋白降解。使用纤溶酶原-酪蛋白酶谱法在上清液中检测纤溶因子。使用选择性 uPA 抑制剂、外源性 uPA、纤溶酶原激活物抑制剂-1 (PAI-1) 抑制剂和纤维蛋白降解产物 (FDP) 研究了囊胚的纤维蛋白溶解活性。使用定量实时 PCR 检测纤溶相关的 mRNA 表达水平。主要结果及CHANCE的作用纤维蛋白不影响小鼠囊胚的发育速度或形态，在附着期观察到较大的纤维蛋白降解区。相比之下，纤维蛋白显着抑制了人类囊胚中滋养层的生长，并且滋养层在出现小的纤维蛋白降解区域后生长。uPA 被鉴定为条件培养基中的纤溶因子，并且在人胚泡中的 uPA 活性显着弱于在小鼠胚泡中的活性。uPA 的抑制显着降低了小鼠和人类囊胚中滋养层的生长。人类囊胚表达 PLAU (uPA)、PLAUR (uPA 受体)、SERPINE1 (PAI-1) 和 SERPINB2 (PAI-2)，而小鼠囊胚仅限于 Plau、Plaur 和 Serpine1。在随后的人类胚泡实验中，添加外源性 uPA 和 PAI-1 抑制剂促进了纤维蛋白存在下的滋养层生长，添加 FDP 也是如此。限制、注意原因 该模型不包括母体因素，可能无法在体内完全复制。捐赠的人类胚胎是多余的胚胎，可能固有地表现出胚胎发育减少。此外，捐赠的 ART 衍生胚胎可能表现出较弱的 uPA 活性，因为具有足够 uPA 活性胚胎的女性最初可能不需要 ART。本研究使用正统的培养方法，结果可能会随着最近开发的用于培养胚泡的方案的应用而改变。研究结果的更广泛意义 目前的结果表明，在存在纤维蛋白的情况下观察到的人胚泡中滋养层生长的独特特征受与纤维蛋白和 FDP 接触诱导的表型转换的调节。小鼠胚胎没有表现出人类现象，表明目前的结果可能仅限于人类。研究资金/竞争利益 本研究得到了滨松大学医学院妇产科和 Kishokai 医疗公司的支持。没有一个作者有任何利益冲突需要声明。试用注册号 不适用。并且结果可能会随着最近开发的用于培养囊胚的方案在植入阶段之后的应用而改变。研究结果的更广泛意义 目前的结果表明，在存在纤维蛋白的情况下观察到的人胚泡中滋养层生长的独特特征受与纤维蛋白和 FDP 接触诱导的表型转换的调节。小鼠胚胎没有表现出人类现象，表明目前的结果可能仅限于人类。研究经费/竞争兴趣 本研究得到了滨松大学医学院妇产科和纪所会医疗公司的支持。没有一个作者有任何利益冲突需要声明。试用注册号 不适用。并且结果可能会随着最近开发的用于培养囊胚的方案在植入阶段之后的应用而改变。研究结果的更广泛意义 目前的结果表明，在存在纤维蛋白的情况下观察到的人胚泡中滋养层生长的独特特征受与纤维蛋白和 FDP 接触诱导的表型转换的调节。小鼠胚胎没有表现出人类现象，表明目前的结果可能仅限于人类。研究经费/竞争兴趣 本研究得到了滨松大学医学院妇产科和纪所会医疗公司的支持。没有一个作者有任何利益冲突需要声明。试用注册号 不适用。