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>
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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. 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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