The authors are grateful to all women who consented and donated samples for this study.

The research content and protocol has been ethically reviewed and approved by the research ethics committee of Shenzhen Zhongshan Urology Hospital. All women (20- 40 years old) involved in the study provided informed consent for sample collection and usage.
1. Acquisition of pathological tissue
<ol>
	<li>Prepare the tools for tissue harvesting namely, measuring ruler, tweezer, embedding cassette, embedding paper, and tissue basket.</li>
	<li>Observe whether the amount of end...

Image Analysis of Immunohistochemically Stained Endometrial Tissue

<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. 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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. 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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. 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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. 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The authors have nothing to disclose.

This protocol established a digital immunohistochemistry image analysis platform to quantitatively analyze endometrial immune cells of RM patients. Here, six&#160;endometrial immune markers were detected to evaluate the endometrial immune microenvironment in RM patients.
A receptive endometrium during the mid-luteal phase is key for successful implantation and pregnancy27,28. Therefore, evaluation of percent endometrial immune cells pl...

Recurrent miscarriage (RM) is the loss of two or more consecutive pregnancies and is a complex disease drawing attention from clinicians in recent years.The incidence rate of RM in women of childbearing age is 1%-5% 1. Results of previous studies show that immune factors are closely associated with the pathogenesis of RM2,3,4,5. Maintaining immune homeostasis at the maternal-fetal interface is required for embryo implantation and development. Endometrial immune cells perform several regulatory roles to maintain this homeostasis, such as promoting trophoblast invasion, remodeling spiral arteries, and contributing to placenta development6,7,8,9.
Aberrant endometrial immune cells in women with RM have previously been reported. Results show a close association between the high density of uterine natural killer cells (uNKs) and the occurrence of RM10,11,12. An increased number of macrophages has been reported in the endometrium of women with RM, compared with those who had a live birth13. Regulatory T cells (Treg) play a role in maternal immune tolerance toward the embryo, and their level and function are decreased in the decidua of RM patients14. Cytotoxicity T cells (CTL) and dendritic cells (DCs) also play a role in the immune regulation of pregnancy15,16. Therefore, a comprehensive quantitative analysis of local endometrial immune cells during the mid-luteal phase could help to better understand the pathogenesis of RM. Some current methods for quantitative analysis of endometrial immune cells use flow cytometry which can accurately label immune cells with multiple markers17,18. However, clinical application of flow cytometry is limited because it can only be performed on fresh tissue. Obtaining fresh tissue is only feasible when a large volume of excess tumor is available, a rare occurrence for endometrium. Immunohistochemistry can observe tissue morphology well in situ and can also label various immune cells, while traditional immunohistochemical techniques cannot perform quantitative analysis of immune cells.
Compared to conventional immunohistochemistry experiments, quantitative immunohistochemical analysis of immune cells in the endometrium has important clinical significance. IHC intensity scoring is usually ranked on a four-point scale or strong and weak in pathological diagnostics and research19,20,21. However, this semi-quantitative technique is subjective, highly inaccurate, and demonstrates significant intra-observer and inter-observer variability22. One possible solution is the application of machine learning, which is valuable indigital image analysis23,24. By providing quantitative measurements, this approach enables a more precise assessment of immune cell infiltration, distribution, and density within the uterine tissue. This quantitative information can help to elucidate the dynamic changes in immune cell populations during the menstrual cycle and in various pathological conditions. Overall, the ability to quantitatively analyze immune cells in the endometrium through immunohistochemistry offers valuable insights into the immune microenvironment of the uterus.
Therefore, the protocol aimed to developed and validated a digital immunohistochemistry image analysis platform to quantitatively analyze endometrial immune cells including uNK cells, Tregs, macrophages, DCs and cytotoxic T cells during the mid-luteal phase in RM patients.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Automated coverslipper</td>
			<td>Sakuraus</td>
			<td>DRS-Prisma-P-JCS&#38;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

To evaluate the endometrial immune microenvironment of patients with recurrent miscarriage (RM), a digital immunohistochemistry image analysis platform was developed and validated to quantitatively analyze endometrial immune cells during the mid-luteal phase. All endometrium samples were collected during the mid-luteal phase of the menstrual cycle. Paraffin-embedded endometrial tissues were sectioned into 4 &#181;m thick slides, and immunohistochemistry (IHC)staining was carried out for detecting endometrial immune cells, including CD56+ uNK cells, Foxp3+ Tregs, CD163+ M2 macrophages, CD1a+ DCs, and CD8+ T cells. The panoramic slides were scanned using a digital slide scanner and a commercial image analysis system was used for quantitative analysis. The percentage of endometrial immune cells was calculated by dividing the number of immune cells in the total endometrial cells. Using the commercial image analysis system, quantitative evaluation of endometrial immune cells, which are difficult or impossible to analyze with conventional image analysis, could be easily, and accurately analyzed. This methodology can be applied to quantitatively characterize the endometrium microenvironment, including interaction between immune cells, and its heterogeneity for different reproductive failure patients. The platform for quantitative evaluation of endometrial immune cells may be of important clinical significance for the diagnosis and treatment of RM patients.

Author Spotlight: Advancing Reproductive Immunology with a Protocol for the Quantitative Evaluation of Endometrial Immune Cells 

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

Currently, it is challenging to stabilize the analysis within a certain range. It is also difficult to maintain the quality control of the experiment. Our research can help the patients with abnormal endometrial sensitivity in immune to find the cause of reproductive failure.The local proportion of immune cells in the endometrial can be stably and repeatedly detected. Our findings may promote the research progress of the endometrial sensitivity evaluation in patients with repeated implantation failure.

In order to evaluate endometrial immune cells quantitatively and reduce the instability caused by man-made operational mistakes, we established a digital quantitative analysis platform for endometrial immune cells by using automatic immunohistochemical detection and digital quantitative evaluation system. Immunohistochemistry image analysis platform was established to quantitatively analyze endometrial immune cells of patients with recurrent miscarriage (RM) in the window of implantation. All endometrium tissues were col...

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

Here, a digital immunohistochemistry image analysis platform was developed and validated to quantitatively analyze the endometrial immune cells of patients with recurrent miscarriages in the window of implantation.

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

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

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JoVE Journal

Biology

Dehydration of Human Endometrial Tissue for Sectioning

To begin, use tweezers to transfer the endometrial tissue from the formalin onto the embedding paper. With a ruler, measure the dimensions of the endometrial tissue. Now wrap the tissue in the embedding paper and place it into an embedding cassette.Transfer the cassette into the tissue basket for dehydration. To dehydrate the endometrial tissue, place the tissue basket in the reaction chamber of the dehydrator and initiate the procedure for routine tissue dehydration. Once the dehydration is complete, open the reaction chamber of the dehydrator and remove the tissue basket.

Human Endometrial Tissue Embedding and Sectioning for Immunohistochemistry and Digital Image Analysis

To begin, select an appropriate embedding mold that matches the size of the dehydrated endometrial specimen. Fill it with paraffin wax, heated at 70 degrees Celsius. Immediately place the dehydrated human endometrial tissue in the mold.Use forceps to carefully adjust the tissue, to ensure it is placed centrally. Now, transfer the mold to the cooling plate and gently press the tissue with tweezers as the paraffin at the bottom solidifies. Place the embedded cassette over the mold, and top it with more wax.Then place the mold back on the cooling plate. Once the paraffin has solidified completely, remove the block and its attached cassette from the mold. For sectioning, insert the block onto the sample clip of the microtome.Place the blade into the holder and adjust the angle between the blocks plane and the blade. Now adjust the thickness of the section to four micrometers. Then turn the hand wheel to start slicing.Cut thin sections from the block to expose the appropriate tissue surface. Use the brush to gather the continuous and complete sections. Once enough sections have been obtained, use tweezers to remove the unqualified sections at the front.With a brush, transfer the sections into a warm water bath, heated to 42 degrees Celsius. When the sections have fully flattened out, use anti detachment slides to lift them. Next, bake them in a slide warmer at 65 degrees Celsius for over 60 minutes before removing.

Tissue Immunohistochemical Staining of Sectioned Human Endometrial Tissue

To begin, transfer the diluted antibody solution into a special reagent bottle. Insert the detection kit into the reagent compartment of an automatic immunohistochemical standing instrument. Next, set the slides with the sectioned endometrial tissue on a slide holder and cover them with a special cover tile.Then insert the holder into the experimental reaction compartment of the instrument. Once the instrument automatically recognizes the information on the reagent and slide, press the start button to initiate the immunohistochemical staining. After staining, take out the slide holder.Put the stained slide into the slide holder to wash off any remaining dye with clean water. Now, place the slide holder onto an automated cover slipper and run the dehydration and sealing procedure. Remove the slides once dehydration and sealing are complete.

Tissue Scanning and Image Analysis of Immunohistochemically Stained Human Endometrium

To start, place the slides with the immunohistochemically stained endometrial sections on the slide rack of a panoramic pathological image scanner. Then, place the rack in the instrument's slide scanning compartment to initiate scanning. Once scanning is complete, launch the image analysis software and create a new folder.Import the images that require analysis. To build a tissue classifier, mark several tissues along with a blank annotation to establish a classifier that identifies the tissue and blank areas. Use realtime tuning to observe the software's recognition capacity during the mark annotation.To build an analysis algorithm, select the standard multiplex IHC algorithm in the software and choose the negative and positive pixels to set the color parameters of cell recognition. Establish the parameters of the nucleus, cytoplasm, and cell membrane until the optimal parameters for the image are found. Now, set the positive cell recognition threshold and optimize it until an appropriate threshold is obtained.Next, select the tissue classifier in the algorithm. Check the tissue part within it to identify cells based on tissues. Select the analysis area and then use the established algorithm to analyze the images.Once the analysis is complete, manually check the accuracy of tissue recognition, negative and positive recognition. If the image recognition is inaccurate, adjust the algorithm and parameter thresholds again. Run the image analysis until it is successful.Then, export the results of the analysis. Set the different analysis parameters according to the expression of different endometrial immune markers. Use the software to calculate the proportion of endometrial immune cells.Following calculation, use the values to assess the levels of various immune cells in the endometrium of patients with recurrent miscarriage. Different immune cells were immunohistochemically stained based on their individual expression of different markers. The brown stain represents the positive immune cells and the blue represents the nucleus.Different immune cells were expressed in women with recurrent miscarriages. CD56 cells appear to be predominant at 16.76%with CD1a and Foxp3 cells making up 0.11 to 0.12%of the immune cell population.

723 Views.  Shenzhen Zhongshan Urology Hospital.  Here, a digital immunohistochemistry image analysis platform was developed and validated to quantitatively analyze the endometrial immune cells of patients with recurrent miscarriages in the window of implantation.

Image Analysis of Immunohistochemically Stained Endometrial Tissue

Summary