作者感谢所有同意并为这项研究捐赠样本的女性。

研究内容和方案已通过深圳市中山泌尿外科医院研究伦理委员会的伦理审查和批准。参与研究的所有女性（20-40 岁）都对样本收集和使用提供了知情同意。
1.病理组织的获取
<ol><li>准备用于组织采集的工具，即测量尺、镊子、包埋盒、包埋纸和纸巾篮。</li>
	<li>观察使用移液管的标准方法收集的子宫内膜组织（比绿豆大）的数量是否足够。</li>
	<li>用镊子将福尔马林的子宫内膜组织转移到包埋纸上，并用尺子测量子宫内膜组织尺寸。</li>
	<li>用包埋纸包裹子宫内膜组织，然后放入包埋盒中。</li>
	<li>将包埋盒放入纸巾篮中进行脱水。</li>
</ol>2.组织脱水
<ol><li>将组织篮放入脱水机的反应室中（参见 材料表），并开始常规组织脱水程序：福尔马林100分钟;福尔马林100分钟;75%酒精60分钟;85% 酒精 60 分钟;95% 酒精 60 分钟;100%酒精60分钟;100%酒精60分钟;100%酒精60分钟;二甲苯35分钟;二甲苯20分钟;二甲苯20分钟;蜡80分钟;蜡80分钟;打蜡 80 分钟。该过程大约需要15小时。</li>
	<li>在组织脱水程序结束时，打开脱水机的反应室，取出组织篮。</li>
</ol>3. 组织包埋
<ol><li>根据试样尺寸取出合适的包埋模具，在70°C下填充石蜡。</li>
	<li>快速将纸巾放入模具中并仔细调整，使纸巾位于模具的中心。</li>
	<li>将模具平稳地移动到冷却板上，轻轻按压纸巾，同时底部的石蜡凝固。</li>
	<li>将包埋盒放在模具顶部，并加满更多的蜡。</li>
	<li>将模具放在冷却板上，当石蜡完全凝固时，将带有连接盒的块从模具上取下。</li>
</ol>4. 组织切片
<ol><li>将块插入切片机的样品夹上，将刀片放入支架中，调整块平面与刀片之间的角度，将切片厚度调整为4μm，转动手轮，开始切片。</li>
	<li>通过从块上切下几片薄片来暴露适当的组织表面。用刷子取出连续和完整的部分。</li>
	<li>当切出足够多的断面时，停止转动手轮，用镊子去除前端不合格的断面，用镊子和刷子将切片漂浮在水浴中的42°C水面上。</li>
	<li>将部分完全压平后，在防分离载玻片上挑选部分，并转移到65°C的载玻片加热器中烘烤60分钟。</li>
	<li>烘烤完成后，将载玻片从载玻片加热器中取出。</li>
</ol>5.免疫组化染色
<ol><li>用抗体稀释剂将一抗稀释到工作溶液中。详见 表1 。</li>
	<li>将稀释后的抗体溶液放入专用试剂瓶中，将检测试剂盒放入全自动IHC染色仪的试剂室中（见 材料表）。</li>
	<li>将载玻片放在载玻片支架上，用特殊的盖子覆盖，然后将它们插入仪器的实验反应室中。</li>
	<li>仪器自动识别载玻片上的试剂和实验信息后，点击开始按钮 开始 免疫组化染色。实验持续约3小时。</li>
</ol>6.载玻片的脱水和密封
<ol><li>免疫组化染色后，取出载玻片支架，取下专用盖板，将染色的载玻片放入载玻片支架中，用清水洗去载玻片上残留的染料。</li>
	<li>将载玻片架转移到自动盖玻片机上（参见 材料表），选择并运行脱水和密封程序。</li>
	<li>脱水密封后取出载玻片。</li>
</ol>7. 扫描载玻片
<ol><li>将载玻片放在全景病理图像扫描仪的载玻片架上（见 材料表），放入仪器载玻片扫描舱进行全景病理图像扫描。扫描需要 2 分钟。请参阅 图 1。</li>
</ol>8. 图像分析
<ol><li>导入图像<ol><li>打开病理图像分析软件（参见 材料表）并创建一个新文件夹。导入要分析的免疫组织化学图像。</li>
	</ol></li>
	<li>构建组织分类器<ol><li>标记多个组织和空白注释，以训练和建立分类器，分别用于识别组织和空白区域。</li>
		<li>使用实时调优，在标记标注过程中实时观察软件的识别能力。如果识别不及时，则备注注释并再次训练，直到组织识别准确。</li>
	</ol></li>
	<li>构建分析算法<ol><li>根据实验类型在软件中选择标准算法：多重 IHC。</li>
		<li>通过选择典型的负像素和正像素来设置单元格识别的颜色参数。 在此基础上，设置细胞核、细胞质、细胞膜的参数，实时观察细胞识别情况，直到找到最适合图像的参数。</li>
		<li>设置阳性细胞识别阈值，实时观察识别情况，直到调整到合适的阈值。</li>
		<li>在算法中选择组织分类器，在组织分类器中检查组织部分，从而在组织的基础上识别细胞。至此，分析算法的建立就完成了。</li>
	</ol></li>
	<li>运行图像分析<ol><li>选择分析区域。使用已建立的算法分析图像。</li>
	</ol></li>
	<li>软件分析完成后，手动检查图像识别是否准确，包括组织识别、阴性和阳性细胞识别。</li>
	<li>如果图像识别不准确，请重新调整算法和参数阈值，重新运行图像分析，直到成功。导出分析结果。请参阅 图 1。</li>
	<li>其他免疫细胞也可以按照上述步骤进行分析（见图1）。根据不同子宫内膜免疫标志物（CD56+uNK细胞、Foxp3+Tregs、CD68+巨噬细胞、CD163+M2巨噬细胞、CD1a+DCs和CD8+T细胞25,26）的实际表达设置不同的分析参数。</li>
	<li>通过软件的计算，得到子宫内膜免疫细胞的比例（见 表2）。用它来评估复发性流产患者子宫内膜中各种免疫细胞的水平。</li>
</ol>

免疫组织化学染色的子宫内膜组织的图像分析

<ol>
	<li>Practice Committee of the American Society for Reproductive. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+treatment+of+recurrent+pregnancy+loss:+a+committee+opinion.">Evaluation and treatment of recurrent pregnancy loss: a committee opinion.</a> Fertility and Sterility. 98 (5), 1103-1111 (2012).</li><li>Dimitriadis, E., Menkhorst, E., Saito, S., Kutteh, W. H., Brosens, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+pregnancy+loss.">Recurrent pregnancy loss.</a> Nature Reviews. Disease Primers. 6 (1), 98 (2020).</li><li>Kavvadas, D., 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=Immunohistochemical+Evaluation+of+CD3,+CD4,+CD8,+and+CD20+in+Decidual+and+Trophoblastic+Tissue+Specimens+of+Patients+with+Recurrent+Pregnancy+Loss.">Immunohistochemical Evaluation of CD3, CD4, CD8, and CD20 in Decidual and Trophoblastic Tissue Specimens of Patients with Recurrent Pregnancy Loss.</a> 12 (2), 177-193 (2022).</li><li>Arora, R., Rathee, A., Sachdeva, M., Agrawal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexplained+repeated+pregnancy+loss+and+T+helper+cells.">Unexplained repeated pregnancy loss and T helper cells.</a> European Journal of Obstetrics, Gynecology, and Reproductive Biology. 254, 277-283 (2020).</li><li>Du, 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=Elevated+percentage+of+CD3(+)T+cells+and+pregnancy+outcome+in+women+with+recurrent+pregnancy+loss.">Elevated percentage of CD3(+)T cells and pregnancy outcome in women with recurrent pregnancy loss.</a> Clinica Chimica Acta. 486, 341-346 (2018).</li><li>Faas, M. M., de Vos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uterine+NK+cells+and+macrophages+in+pregnancy.">Uterine NK cells and macrophages in pregnancy.</a> Placenta. 56, 44-52 (2017).</li><li>Huppertz, B., Berghold, V. M., Kawaguchi, R., Gauster, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variety+of+opportunities+for+immune+interactions+during+trophoblast+development+and+invasion.">A variety of opportunities for immune interactions during trophoblast development and invasion.</a> American Journal of Reproductive Immunology. 67 (5), 349-357 (2012).</li><li>Meyer, N., 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=Chymase-producing+cells+of+the+innate+immune+system+are+required+for+decidual+vascular+remodeling+and+fetal+growth.">Chymase-producing cells of the innate immune system are required for decidual vascular remodeling and fetal growth.</a> Scientific Reports. 7, 45106 (2017).</li><li>Smith, S. D., Dunk, C. E., Aplin, J. D., Harris, L. K., Jones, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+immune+cell+involvement+in+decidual+spiral+arteriole+remodeling+in+early+human+pregnancy.">Evidence for immune cell involvement in decidual spiral arteriole remodeling in early human pregnancy.</a> American Journal of Pathology. 174 (5), 1959-1971 (2009).</li><li>Clifford, K., Flanagan, A. M., Regan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endometrial+CD56++natural+killer+cells+in+women+with+recurrent+miscarriage:+a+histomorphometric+study.">Endometrial CD56+ natural killer cells in women with recurrent miscarriage: a histomorphometric study.</a> Human Reproduction. 14 (11), 2727-2730 (1999).</li><li>Chen, 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=Measurement+of+uterine+natural+killer+cell+percentage+in+the+periimplantation+endometrium+from+fertile+women+and+women+with+recurrent+reproductive+failure:+establishment+of+a+reference+range.">Measurement of uterine natural killer cell percentage in the periimplantation endometrium from fertile women and women with recurrent reproductive failure: establishment of a reference range.</a> American Journal of Obstetrics and Gynecology. 217 (6), 680 e1-680 e6 (2017).</li><li>Tuckerman, E., Mariee, N., Prakash, A., Li, T. C., Laird, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uterine+natural+killer+cells+in+peri-implantation+endometrium+from+women+with+repeated+implantation+failure+after+IVF.">Uterine natural killer cells in peri-implantation endometrium from women with repeated implantation failure after IVF.</a> Journal of Reproductive Immunology. 87 (1-2), 60-66 (2010).</li><li>Laird, S. 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=A+review+of+immune+cells+and+molecules+in+women+with+recurrent+miscarriage.">A review of immune cells and molecules in women with recurrent miscarriage.</a> Human Reproduction Update. 9 (2), 163-174 (2003).</li><li>Keller, C. C., Eikmans, M., van der Hoorn, M. P., Lashley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+miscarriages+and+the+association+with+regulatory+T+cells;+A+systematic+review.">Recurrent miscarriages and the association with regulatory T cells; A systematic review.</a> Journal of Reproductive Immunology. 139, 103105 (2020).</li><li>Vallv&#233;-Juanico, J., Houshdaran, S., Giudice, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endometrial+immune+environment+of+women+with+endometriosis.">The endometrial immune environment of women with endometriosis.</a> Human Reproduction Update. 25 (5), 564-591 (2019).</li><li>Yang, F., Zheng, Q., Jin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Function+and+Composition+Changes+of+Immune+Cells+During+Normal+and+Pathological+Pregnancy+at+the+Maternal-Fetal+Interface.">Dynamic Function and Composition Changes of Immune Cells During Normal and Pathological Pregnancy at the Maternal-Fetal Interface.</a> Frontiers in Immunology. 10, 2317 (2019).</li><li>Hey-Cunningham, A. 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=Comprehensive+analysis+utilizing+flow+cytometry+and+immunohistochemistry+reveals+inflammatory+changes+in+local+endometrial+and+systemic+dendritic+cell+populations+in+endometriosis.">Comprehensive analysis utilizing flow cytometry and immunohistochemistry reveals inflammatory changes in local endometrial and systemic dendritic cell populations in endometriosis.</a> Human Reproduction. 36 (2), 415-428 (2021).</li><li>Zhong, Q., 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=Patterns+of+Immune+Infiltration+in+Endometriosis+and+Their+Relationship+to+r-AFS+Stages.">Patterns of Immune Infiltration in Endometriosis and Their Relationship to r-AFS Stages.</a> Frontiers in Genetics. 12, 631715 (2021).</li><li>Attems, 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=Neuropathological+consensus+criteria+for+the+evaluation+of+Lewy+pathology+in+post-mortem+brains:+a+multi-centre+study.">Neuropathological consensus criteria for the evaluation of Lewy pathology in post-mortem brains: a multi-centre study.</a> Acta Neuropathologic. 141 (2), 159-172 (2021).</li><li>Kovacs, G. 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=Multisite+Assessment+of+Aging-Related+Tau+Astrogliopathy+(ARTAG).">Multisite Assessment of Aging-Related Tau Astrogliopathy (ARTAG).</a> Journal of Neuropathology and Experimental Neurology. 76 (7), 605-619 (2017).</li><li>Modis, L. V., 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=Extracellular+matrix+changes+in+corneal+opacification+vary+depending+on+etiology.">Extracellular matrix changes in corneal opacification vary depending on etiology.</a> Molecular Vision. 27, 26-36 (2021).</li><li>Walker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+immunohistochemistry--issues+concerning+methods,+utility+and+semiquantitative+assessment+I.">Quantification of immunohistochemistry--issues concerning methods, utility and semiquantitative assessment I.</a> Histopathology. 49 (4), 406-410 (2006).</li><li>Jensen, K., Krusenstjerna-Hafstrom, R., Lohse, J., Petersen, K. H., Derand, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+quantitative+immunohistochemistry+method+for+precise+protein+measurements+directly+in+formalin-fixed,+paraffin-embedded+specimens:+analytical+performance+measuring+HER2.">A novel quantitative immunohistochemistry method for precise protein measurements directly in formalin-fixed, paraffin-embedded specimens: analytical performance measuring HER2.</a> Modern Pathology. 30 (2), 180-193 (2017).</li><li>Moreno-Ruiz, P., Wik Leiss, L., Mezheyeuski, A., Ehnman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+Immunohistochemistry+and+Digital+Image+Analysis.">Double Immunohistochemistry and Digital Image Analysis.</a> Methods in Molecular Biology. 1913, 3-11 (2019).</li><li>Li, D., Zheng, L., Zhao, D., Xu, Y., Wang, Y. <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+Immune+Cells+in+Recurrent+Spontaneous+Abortion.">The Role of Immune Cells in Recurrent Spontaneous Abortion.</a> Reproductive Sciences. 28 (12), 3303-3315 (2021).</li><li>Diao, 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=New+endometrial+immune+cell-based+score+(EI-score)+for+the+prediction+of+implantation+success+for+patients+undergoing+IVF/ICSI.">New endometrial immune cell-based score (EI-score) for the prediction of implantation success for patients undergoing IVF/ICSI.</a> Placenta. 99, 180-188 (2020).</li><li>Hewitt, S. C., Korach, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology.+A+hand+to+support+the+implantation+window.">Cell biology. A hand to support the implantation window.</a> Science. 331 (6019), 863-864 (2011).</li><li>Afshar, Y., Stanculescu, A., Miele, L., Fazleabas, A. T. <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+chorionic+gonadotropin+and+Notch1+in+implantation.">The role of chorionic gonadotropin and Notch1 in implantation.</a> Journal of Assisted Reproduction and Genetics. 24 (7), 296-302 (2007).</li><li>Tan, W. 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=Overview+of+multiplex+immunohistochemistry/immunofluorescence+techniques+in+the+era+of+cancer+immunotherapy.">Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy.</a> Cancer Communication (London,England). 40 (4), 135-153 (2020).</li><li>Algars, A., 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=Type+and+location+of+tumor-infiltrating+macrophages+and+lymphatic+vessels+predict+survival+of+colorectal+cancer+patients.">Type and location of tumor-infiltrating macrophages and lymphatic vessels predict survival of colorectal cancer patients.</a> International Journal of Cancer. 131 (4), 864-873 (2012).</li><li>Carey, C. D., 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=Topological+analysis+reveals+a+PD-L1-associated+microenvironmental+niche+for+Reed-Sternberg+cells+in+Hodgkin+lymphoma.">Topological analysis reveals a PD-L1-associated microenvironmental niche for Reed-Sternberg cells in Hodgkin lymphoma.</a> Blood. 130 (22), 2420-2430 (2017).</li><li>Ascierto, M. 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=Transcriptional+Mechanisms+of+Resistance+to+Anti-PD-1+Therapy.">Transcriptional Mechanisms of Resistance to Anti-PD-1 Therapy.</a> Clinical Cancer Research. 23 (12), 3168-3180 (2017).</li><li>O'Rourke, D. 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=A+single+dose+of+peripherally+infused+EGFRvIII-directed+CAR+T+cells+mediates+antigen+loss+and+induces+adaptive+resistance+in+patients+with+recurrent+glioblastoma.">A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma.</a> Science Translational Medicine. 9 (399), eaaa0984 (2017).</li><li>Canesin, 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=Treatment+with+the+WNT5A-mimicking+peptide+Foxy-5+effectively+reduces+the+metastatic+spread+of+WNT5A-low+prostate+cancer+cells+in+an+orthotopic+mouse+model.">Treatment with the WNT5A-mimicking peptide Foxy-5 effectively reduces the metastatic spread of WNT5A-low prostate cancer cells in an orthotopic mouse model.</a> PLoS One. 12 (9), e0184418 (2017).</li></ol>

作者没有什么可透露的。

该协议建立了数字免疫组化图像分析平台，以定量分析RM患者的子宫内膜免疫细胞。在这里，检测了六种子宫内膜免疫标志物，以评估RM患者的子宫内膜免疫微环境。
黄体中期的接受性子宫内膜是成功植入和怀孕的关键27,28。因此，评估子宫内膜免疫细胞百分比在估计子宫内膜容受性方面起着重要作用。通过传统的病理学方法进行子...

复发性流产（RM）是指连续两次或两次以上妊娠的流产，是一种复杂的疾病，近年来引起了临床医生的关注。育龄妇女RM的发病率为1%-5%1。既往研究结果表明，免疫因子与RM 2,3,4,5的发病机制密切相关。维持母胎界面的免疫稳态是胚胎植入和发育所必需的。子宫内膜免疫细胞在维持这种稳态方面发挥多种调节作用，例如促进滋养层侵袭、重塑螺旋动脉和促进胎盘发育 6,7,8,9。
RM 女性的子宫内膜免疫细胞异常既往已有报道。结果显示，高密度子宫自然杀伤细胞 （uNK） 与 RM10、11、12 的发生密切相关。据报道，与活产女性相比，RM女性子宫内膜中的巨噬细胞数量有所增加13。调节性T细胞（Treg）在母体对胚胎的免疫耐受中发挥作用，其水平和功能在RM患者的蜕膜中降低14。细胞毒性 T 细胞 （CTL） 和树突状细胞 （DC） 也在妊娠的免疫调节中发挥作用15,16。因此，对黄体中期局部子宫内膜免疫细胞进行全面的定量分析有助于更好地了解RM的发病机制。目前一些用于子宫内膜免疫细胞定量分析的方法使用流式细胞术，它可以准确地标记具有多种标记物的免疫细胞17,18。然而，流式细胞术的临床应用受到限制，因为它只能在新鲜组织上进行。只有当存在大量多余肿瘤时，获得新鲜组织才可行，这在子宫内膜中很少见。免疫组化可以很好地原位观察组织形态，也可以标记各种免疫细胞，而传统的免疫组化技术无法对免疫细胞进行定量分析。
与常规免疫组化实验相比，子宫内膜免疫细胞的定量免疫组化分析具有重要的临床意义。IHC强度评分通常采用四分制或病理诊断和研究中的强弱排序19,20,21。然而，这种半定量技术是主观的、高度不准确的，并显示出显著的观察者内部和观察者间变异性22。一种可能的解决方案是机器学习的应用，这在数字图像分析中很有价值23,24。通过提供定量测量，这种方法可以更精确地评估子宫组织内的免疫细胞浸润、分布和密度。这种定量信息可以帮助阐明月经周期和各种病理条件下免疫细胞群的动态变化。总体而言，通过免疫组化定量分析子宫内膜中免疫细胞的能力为了解子宫的免疫微环境提供了有价值的见解。
因此，该方案旨在开发和验证一种数字免疫组化图像分析平台，以定量分析RM患者黄体中期的子宫内膜免疫细胞，包括uNK细胞、Tregs、巨噬细胞、DC和细胞毒性T细胞。

<table><tbody><tr><td>Automated coverslipper</td><td>Sakuraus</td><td>DRS-Prisma-P-JCS&amp;amp;Film-JC2</td><td></td></tr><tr><td>CD163</td><td>GrowGn Biotechnology</td><td>NCL-L-CD163</td><td></td></tr><tr><td>CD1a</td><td>Gene Tech</td><td>GM357129</td><td></td></tr><tr><td>CD56</td><td>Gene Tech</td><td>GT200529</td><td></td></tr><tr><td>CD8</td><td>Novocastra</td><td>NCL-L-CD8-4B11</td><td></td></tr><tr><td>Dehydrator</td><td>Thermo Fisher</td><td>Excelsior ES</td><td></td></tr><tr><td>Digital pathology and</td><td>Indica labs</td><td>HALO</td><td></td></tr><tr><td>Foxp3</td><td>YILIFANG biological</td><td>14-477-82</td><td></td></tr><tr><td>IHC stainer</td><td>Leica</td><td>BOND III</td><td></td></tr><tr><td>Image analysis platform</td><td>Indica labs</td><td>HALO</td><td></td></tr><tr><td>Slide Scanner</td><td>Olympus life science</td><td>VS200</td><td></td></tr></tbody></table>

platform for quantitative detection of endometrial immune cells based on immunohistochemistry and digital image analysis

为评估复发性流产（RM）患者的子宫内膜免疫微环境，开发并验证了数字免疫组化图像分析平台，用于定量分析黄体中期子宫内膜免疫细胞。所有子宫内膜样本均在月经周期的黄体中期采集。将石蜡包埋的子宫内膜组织切片成4 μm厚的载玻片，进行免疫组化（IHC）染色检测子宫内膜免疫细胞，包括CD56+ uNK细胞、Foxp3+ Tregs、CD163+ M2巨噬细胞、CD1a+ DCs和CD8+ T细胞。使用数字玻片扫描仪对全景玻片进行扫描，并使用商业图像分析系统进行定量分析。子宫内膜免疫细胞的百分比是通过除以总子宫内膜细胞中的免疫细胞数来计算的。使用商用图像分析系统，可以轻松准确地分析传统图像分析难以或不可能分析的子宫内膜免疫细胞的定量评估。该方法可用于定量表征子宫内膜微环境，包括免疫细胞之间的相互作用，以及不同生殖失败患者的异质性。子宫内膜免疫细胞定量评估平台对RM患者的诊断和治疗具有重要的临床意义。

为了定量评估子宫内膜免疫细胞，减少人为操作失误带来的不稳定性，利用免疫组化自动检测和数字定量评价系统，建立了子宫内膜免疫细胞数字化定量分析平台。建立免疫组化图像分析平台，定量分析复发性流产（RM）患者植入窗口期的子宫内膜免疫细胞。所有子宫内膜组织均在月经周期的黄体中期收集。将石蜡包埋的子宫内膜组织切片成4 μm厚的载玻片，进行IHC染色检测子宫内膜免疫细胞，包...

Watch this Scientific Journal Video about Advancing Reproductive Immunology with a Protocol for the Quantitative Evaluation of Endometrial Immune Cells at JoVE.com

作者聚焦：通过子宫内膜免疫细胞定量评估方案推进生殖免疫学 

本文开发并验证了数字免疫组化图像分析平台，用于定量分析复发性流产患者在植入窗口期的子宫内膜免疫细胞。

基于免疫组化和数字图像分析的子宫内膜免疫细胞定量检测平台

To evaluate the endometrial immune microenvironment of patients with recurrent miscarriage (RM), a digital immunohistochemistry image analysis platform ...

目前，将分析稳定在一定范围内具有挑战性。保持实验的质量控制也很困难。我们的研究可以帮助免疫内膜敏感性异常的患者找到生殖衰竭的原因。子宫内膜内免疫细胞的局部比例可以稳定、反复地检测。本研究结果可促进反复着床失败患者子宫内膜敏感性评估的研究进展。

platform-for-quantitative-detection-endometrial-immune-cells-based-on

Research

JoVE Journal

Biology

Platform for Quantitative Detection of Endometrial Immune Cells Based on Immunohistochemistry and Digital Image Analysis

1.1K 次观看.  Shenzhen Zhongshan Urology Hospital. 目前，将分析稳定在一定范围内具有挑战性。保持实验的质量控制也很困难。我们的研究可以帮助免疫内膜敏感性异常的患者找到生殖衰竭的原因。子宫内膜内免疫细胞的局部比例可以稳定、反复地检测。本研究结果可促进反复着床失败患者子宫内膜敏感性评估的研究进展。

视频: 作者聚焦：通过子宫内膜免疫细胞定量评估方案推进生殖免疫学 

Sentinel Lymph Node Mapping and Biopsy for Endometrial Cancer at Early Stage with Laparoscopy

Sentinel lymph node (SLN) mapping and biopsy is a promising technique for visualizing and evaluating lymph node status in cancer. This approach has been recommended for low-risk endometrial cancer (EC) patients by authoritative international guidelines, but it has not been performed broadly in China and worldwide. This work aims to describe detailed SLN mapping and biopsy procedures to promote the clinical application. SLN mapping and postoperative pathologic ultrastaging were conducted in a patient with low-risk EC using indocyanine green (ICG) dye to track the SLNs under laparoscopy and resecting them completely for ultrastaging. In conclusion, this protocol describes details of ICG injection, and SLN mapping and biopsy in EC patients based on the experiences gained during clinical practice.

This protocol describes the identification and resection of sentinel lymph nodes to make the operation as easy and minimally invasive as possible.

腹腔镜检查早期子宫内膜癌的前哨淋巴结定位和活检

Sentinel lymph node (SLN) mapping and biopsy is a promising technique for visualizing and evaluating lymph node status in cancer. This approach has been ...

科研

癌症研究

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

两种方法从子宫切除标本小学建立人子宫内膜基质细胞

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

医学

绘制肿瘤免疫景观图谱：深入的多重 IHC 分析方案

Multiplex Immunohistochemical Analysis of the Spatial Immune Cell Landscape of the Tumor Microenvironment

The immune cell landscape of the tumor microenvironment potentially contains information for the discovery of prognostic and predictive biomarkers. Multiplex immunohistochemistry is a valuable tool to visualize and identify different types of immune cells in tumor tissues while retaining its spatial information. Here we provide detailed protocols to analyze lymphocyte, myeloid, and dendritic cell populations in tissue sections. Starting from cutting formalin-fixed paraffin-embedded sections, automatic multiplex staining procedures on an automated platform, scanning of the slides on a multispectral imaging microscope, to the analysis of images using an in-house-developed machine learning algorithm ImmuNet. These protocols can be applied to a variety of tumor specimens by simply switching tumor markers to analyze immune cells in different compartments of the sample (tumor versus invasive margin) and apply nearest-neighbor analysis. This analysis is not limited to tumor samples but can also be applied to other (non-)pathogenic tissues. Improvements to the equipment and workflow over the past few years have significantly shortened throughput times, which facilitates the future application of this procedure in the diagnostic setting.

作者聚焦：解锁对肿瘤免疫细胞景观的见解

This protocol describes in detail how immune cell characterization of the tumor microenvironment using multiplex immunohistochemistry is carried out.

肿瘤微环境空间免疫细胞景观的多重免疫组织化学分析

The immune cell landscape of the tumor microenvironment potentially contains information for the discovery of prognostic and predictive biomarkers. ...

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans.
Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function.

Here, we present an optimized protocol for imaging entire ovaries for quantitative and qualitative analyses using whole-mount immunostaining, multiphoton microscopy, and 3D visualization and analysis. This protocol accommodates high-throughput, reliable, and repeatable processing that is applicable for toxicology, clinical diagnostics, and genomic assays of ovarian function.

全卵巢免疫荧光、清除和多光子显微镜用于小鼠发育中卵巢储备的定量3D分析

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells ...

发育生物学

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

定量健身分析工作流程

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

生物学

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

肿瘤微环境下免疫细胞种群的可视化、定量和映射

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

免疫与感染

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

多蛋白复合物的比较分析新方法基于<sup&gt; 15</sup&gt;Ñ代谢标记和定量质谱

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

Multiplexed Fluorescent Immunohistochemical Staining of Four Endometrial Immune Cell Types in Recurrent Miscarriage

Immunohistochemistry is the most commonly used method for the identification and visualization of tissue antigens in biological research and clinical diagnostics. It can be used to characterize various biological processes or pathologies, such as wound-healing, immune response, tissue rejection, and tissue-biomaterial interactions. However, the visualization and quantification of multiple antigens (especially for immune cells) in a single tissue section using conventional immunohistochemical (IHC) staining remains unsatisfactory. Hence, multiplexed technologies were introduced in recent years to identify multiple biological markers in a single tissue sample or an ensemble of different tissue samples. 
These technologies can be especially useful in differentiating the changes in immune cell-to-cell interactions within the endometrium between fertile women and women with recurrent miscarriages during implantation. This paper describes a detailed protocol for multiplexed fluorescence IHC staining to investigate the density and clustering of four major immune cell types simultaneously in precisely timed endometrial specimens during embryo implantation. The method includes sample preparation, multiplex optimization with markers for immune cell subtypes, and the scanning of the slides, followed by data analysis, with specific reference to detecting endometrial immune cells. 
Using this method, the density and clustering of four major immune cell types in the endometrium can be simultaneously analyzed in a single tissue section. In addition, this paper will discuss the critical factors and troubleshooting to overcome possible fluorophore interference between the fluorescent probes being applied. Importantly, the results from this multiplex staining technique can help provide an in-depth understanding of the immunologic interaction and regulation during embryo implantation.

Despite the advancements in multiplex immunohistochemistry and multispectral imaging, characterizing the density and clustering of major immune cells simultaneously in the endometrium remains a challenge. This paper describes a detailed multiplex staining protocol and imaging for the simultaneous localization of four immune cell types in the endometrium.

复发性流产中4种子宫内膜免疫细胞类型的多重荧光免疫组织化学染色

Immunohistochemistry is the most commonly used method for the identification and visualization of tissue antigens in biological research and clinical ...

基于免疫组化和数字图像分析的子宫内膜免疫细胞定量检测平台

副本

摘要

探索更多视频

此视频中的章节

腹腔镜检查早期子宫内膜癌的前哨淋巴结定位和活检

两种方法从子宫切除标本小学建立人子宫内膜基质细胞

肿瘤微环境空间免疫细胞景观的多重免疫组织化学分析

肿瘤微环境空间免疫细胞景观的多重免疫组织化学分析

肿瘤微环境空间免疫细胞景观的多重免疫组织化学分析

全卵巢免疫荧光、清除和多光子显微镜用于小鼠发育中卵巢储备的定量3D分析

定量健身分析工作流程

肿瘤微环境下免疫细胞种群的可视化、定量和映射

多蛋白复合物的比较分析新方法基于

复发性流产中4种子宫内膜免疫细胞类型的多重荧光免疫组织化学染色