The present study was supported by the Health and Medical Research Fund (10210956).

The study was approved by the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee (CREC ref no.: 2022.581). Women with RIF were recruited from the Assisted Reproductive Technology Center, Prince of Wales Hospital, Chinese University of Hong Kong. RIF was defined as the failure to achieve a clinical pregnancy after the transfer of at least 4 good-quality embryos in a minimum of 3 fresh or frozen cycles in a woman under the age of 40 years10. Informe...

Exploring Uterine NK Cell Heterogeneity in the Endometrium with Multiplex IHC

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	<li>De Geyter, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assisted+reproductive+technology:+Impact+on+society+and+need+for+surveillance.">Assisted reproductive technology: Impact on society and need for surveillance.</a> Best Pract Res Clin Endocrinol Metab. 33 (1), 3-8 (2019).</li><li>. State-Specific Assisted Reproductive Technology Surveillance. CDC Available from: <a target="_blank" href="https://www.cdc.gov/art/state-specific-surveillance/2021/index.html">https://www.cdc.gov/art/state-specific-surveillance/2021/index.html</a> (2024)</li><li>Busnelli, 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=How+common+is+real+repeated+implantation+failure?+An+indirect+estimate+of+the+prevalence.">How common is real repeated implantation failure? 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Z., 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=Single-cell+transcriptome+profiling+of+the+human+endometrium+of+patients+with+recurrent+implantation+failure.">Single-cell transcriptome profiling of the human endometrium of patients with recurrent implantation failure.</a> Theranostics. 12 (15), 6527-6547 (2022).</li><li>Coughlan, 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=Recurrent+implantation+failure:+definition+and+management.">Recurrent implantation failure: definition and management.</a> Reprod BioMed Onl. 28 (1), 14-38 (2014).</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> Am J Obst Gyneco. 217 (6), 680.e1-680.e6 (2017).</li><li>Yang, W. 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E., 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=Standardisation+of+uterine+natural+killer+(uNK)+cell+measurements+in+the+endometrium+of+women+with+recurrent+reproductive+failure.">Standardisation of uterine natural killer (uNK) cell measurements in the endometrium of women with recurrent reproductive failure.</a> J Reprod Immunol. 116, 50-59 (2016).</li><li>Mansfield, J. R., Gossage, K. W., Hoyt, C. C., Levenson, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autofluorescence+removal,+multiplexing,+and+automated+analysis+methods+for+in+vivo+fluorescence+imaging.">Autofluorescence removal, multiplexing, and automated analysis methods for in vivo fluorescence imaging.</a> J Biomed Optics. 10 (4), 41207 (2005).</li><li>Hoyt, C. 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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=Exploring+natural+killer+cell+testing+in+embryo+implantation+and+reproductive+failure:+An+overview+of+techniques+and+controversies.">Exploring natural killer cell testing in embryo implantation and reproductive failure: An overview of techniques and controversies.</a> Reprod Sci. 31 (3), 603-632 (2024).</li><li>Russell, P., 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=The+distribution+of+immune+cells+and+macrophages+in+the+endometrium+of+women+with+recurrent+reproductive+failure:+I:+Techniques.">The distribution of immune cells and macrophages in the endometrium of women with recurrent reproductive failure: I: Techniques.</a> J Reprod Immunol. 91 (1), 90-102 (2011).</li><li>Wang, W., Sung, N., Gilman-Sachs, A., Kwak-Kim, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helper+(Th)+cell+profiles+in+pregnancy+and+recurrent+pregnancy+losses:+Th1/Th2/Th9/Th17/Th22/Tfh+cells.">Helper (Th) cell profiles in pregnancy and recurrent pregnancy losses: Th1/Th2/Th9/Th17/Th22/Tfh cells.</a> Front Immunol. 11, 2025 (2020).</li><li>Nagamatsu, T., Schust, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+macrophages+to+normal+and+pathological+pregnancies.">The contribution of macrophages to normal and pathological pregnancies.</a> Am J Reprod Immunol. 63 (6), 460-471 (2010).</li></ol>

Embryo implantation involves a complex interaction between the embryo and the endometrium. The immunological status of endometrial homeostasis plays a pivotal role in determining endometrial receptivity. During WOI, the predominant leukocyte population in the endometrium is NK cells. Approximately 90% of uNK cells exhibit high CD56 expression but lack CD16. However, a minor subset of uNK cells resembles peripheral blood NK cells, displaying low CD56 expression but positive CD16 expression18. These...

The first documented live birth after in vitro fertilization-embryo transfer (IVF-ET) was reported in 1978. Over the past 40 years, there has been a high demand for the assistance of IVF-ET among infertile couples1. In 2021, 238,126 patients initiated a total of 413,776 IVF cycles in the United States. This marks a 25% increase in cycles from 2 years prior and a 135% increase from 20122. This surge is mainly attributed to the rising prevalence of infertility and delayed planning for pregnancy. Advancements in embryo culture techniques and superovulation protocols have led to an increased live birth rate per ET cycle, reaching 30%-50% in women less than 40 years old and less than 30% in women older than 40 years2. However, despite these advancements, over half of transferred embryos still fail to implant. Repeated implantation failure (RIF), typically defined as failure after three or more consecutive attempts of transferring high-quality embryos, affects 15% of women who undergo IVF-ET3. Couples with RIF are extremely vulnerable and more prone to undergo expensive and unnecessary procedures that can expose them to undue risks4. Therefore, understanding the causes of RIF and improving embryo implantation is crucial to enhance the success of IVF-ET, particularly for women with RIF. The preparation of endometrium is critical for successful embryo implantation. This process is characterized by a significant accumulation of uterine natural killer (uNK) cells, which transition from constituting 30% of total lymphocytes in the endometrium during mid-secretory phase to 70%-80% in the decidua during early pregnancy5. Notably, uNK cells differ from peripheral NK cells, which are cytotoxic lymphocytes critical to the innate immune system for causing the death of the infected cells through lysis or apoptosis. While the exact functions of uNK cells are not yet fully understood, several lines of evidence suggest that they are involved in angiogenesis remodeling, trophoblast invasion, and fetal development6. The association between the percentage of uNK cells over stromal cells and RIF has garnered extensive attraction over the past 20 years. A recent meta-analysis, which included 8 studies involving 604 women, demonstrated that the density of CD56+uNK cells during the mid-luteal phase is significantly increased in women with RIF compared to fertile controls7. However, it is important to note that the characteristics of uNK cells during the mid-luteal phase differ significantly from those of decidual NK (dNK) cells. Although uNK cells may further differentiate into various subsets of dNK cells post-pregnancy, measuring uNK cells alone does not accurately represent dNK cells8. uNK cells undergo dynamic differentiation and play different roles in the menstrual cycle and decidualization processes, making them more complex than can be identified by CD56 alone. Multiple markers are required to achieve a comprehensive understanding of uNK cell behavior during endometrial preparation. Our recent study employed single-cell RNA sequencing to identify the diversity of uNK cells throughout menstrual cycles. The results, validated using flow cytometry, have shown the presence of four distinct subtypes of uNK cells, each exhibiting dynamic changes during the menstrual cycle9. Gene enrichment analysis and gene ontology functional enrichment indicate these uNK subsets fulfill different functions at various stages of menstruation. Nevertheless, flow cytometry is not universally accessible in clinical laboratories, and the immediate processing of fresh endometrial tissue for enzyme digestion renders it impossible to repeat experimental steps upon errors.
The aim of this study was, therefore, to investigate the measurement of these four subpopulations of NK cells using a multiplex staining assay, which provides a more practical diagnostic approach. In multiple staining, different specific antibodies against each target are linked to different fluorophore labels that emit different wavelengths of light when excited by a specific wavelength of light. Compared to the traditional IHC staining method, this method can quantitatively compare the relative abundance and distribution of multiple targets in a sample and provide information on the interaction and co-localization of different targets, enabling us to identify different subtypes of uNK cells. This approach will not only deepen our understanding of the relationship between uNK cells and RIF but will also provide insights for investigating other immune cell subpopulations in endometrial-related diseases.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CD49a</td>
			<td>Novus Biologicals</td>
			<td>NBP2-76478</td>
			<td>Primary antibodies</td>
		</tr>
		<tr>
			<td>CD56</td>
			<td>Leica</td>
			<td>NCL-L-CD56-504</td>
			<td>Primary antibodies</td>
		</tr>
		<tr>
			<td>CD16</td>
			<td>abcam</td>
			<td>ab183354</td>
			<td>Primary antibodies</td>
		</tr>
		<tr>
			<td>CXCR4</td>
			<td>R&#38;D</td>
			<td>MAB172</td>
			<td>Primary antibodies</td>
		</tr>
		<tr>
			<td>Amplification diluent</td>
			<td>Akoya Biosciences</td>
			<td>FP1498 series</td>
			<td>Fluorophore dilution buffer</td>
		</tr>
		<tr>
			<td>Antibody diluents</td>
			<td>Akoya Biosciences</td>
			<td>ARD1001EA</td>
			<td>Dilute the antibody</td>
		</tr>
		<tr>
			<td>Citrate buffered solution, pH 6.0 /9.0(10x)</td>
			<td>Akoya Biosciences</td>
			<td>A6001/A9001</td>
			<td>Antigen retrieval solution</td>
		</tr>
		<tr>
			<td>inForm advanced image analysis software</td>
			<td>Akoya Biosciences</td>
			<td>inForm Tissue Finder Software 2.2.6</td>
			<td>Data analysis software</td>
		</tr>
		<tr>
			<td>Mantra Workstations</td>
			<td>Akoya Biosciences</td>
			<td>CLS140089</td>
			<td>Spectral imaging</td>
		</tr>
		<tr>
			<td>microwave</td>
			<td>Akoya Biosciences</td>
			<td>inverter</td>
			<td>Microwave stripping</td>
		</tr>
		<tr>
			<td>TSA 520</td>
			<td>Akoya Biosciences</td>
			<td>FP1487001K</td>
			<td>Suitable tyramide-based fluorescent reagents</td>
		</tr>
		<tr>
			<td>TSA 620</td>
			<td>Akoya Biosciences</td>
			<td>FP1495001K</td>
			<td>Suitable tyramide-based fluorescent reagents</td>
		</tr>
		<tr>
			<td>TSA 650</td>
			<td>Akoya Biosciences</td>
			<td>FP1496001K</td>
			<td>Suitable tyramide-based fluorescent reagents</td>
		</tr>
		<tr>
			<td>TSA 570</td>
			<td>Akoya Biosciences</td>
			<td>FP1488001K</td>
			<td>Suitable tyramide-based fluorescent reagents</td>
		</tr>
		<tr>
			<td>Poly-L-lysine coated slides</td>
			<td>Fisher Technologies</td>
			<td>120-550-15</td>
			<td>Slides for routine histological use</td>
		</tr>
		<tr>
			<td>PolyHRP Broad Spectrum</td>
			<td>Perkin Elmer</td>
			<td>ARH1001EA</td>
			<td>Secondary antibodies</td>
		</tr>
		<tr>
			<td>Invitrogen&#8482;&#160;Fluoromount-G&#8482; Mounting Medium</td>
			<td>ThemoFisher Science</td>
			<td>495802</td>
			<td>Installation</td>
		</tr>
		<tr>
			<td>Spectral DAPI</td>
			<td>Akoya Biosciences</td>
			<td>FP1490A</td>
			<td>Nucleic acid staining</td>
		</tr>
	</tbody>
</table>

measurement of four uterine nk cell subtypes using multiplexed fluorescent immunohistochemical staining in women with repeated implantation failure

Immunohistochemistry (IHC) plays a crucial role in biological research and clinical diagnosis, serving as the most commonly used method for identifying and visualizing tissue antigens. However, traditional IHC staining methods have limitations in distinguishing various subtypes of immune cells. This challenge has driven scientists to explore new technologies and methodologies for precise identification and differentiation of immune cell subtypes. In recent years, multiplex IHC has emerged as a solution, enabling the simultaneous detection of multiple antigens and their visualization within the same tissue sample. Uterine natural killer (uNK) cells play a pivotal role in early pregnancy processes, including decidualization, remodeling of uterine spiral arteries, and embryo implantation. Different subtypes of uNK cells exhibit different functions, allowing them to coordinate various biological events for successful embryo development and pregnancy. Therefore, in-depth research on uNK cell subtypes is essential for elucidating immune regulation mechanisms during pregnancy. Such studies provide valuable insights and novel approaches for addressing related conditions such as infertility and recurrent reproductive failure. This paper introduces a detailed multiplex IHC staining protocol for studying the density of four subtypes of uNK cells in endometrial specimens during the window of implantation (WOI). The protocol includes sample preparation, optimization of subtype markers, microscopic imaging, and data analyses. This multiplex IHC staining protocol offers high specificity and sensitivity, enabling simultaneous detection of different uNK cell subtypes, thus providing researchers with a powerful tool to explore the intricacies and mechanisms of immune regulation during pregnancy.

Author Spotlight: Multiplex Immunohistochemistry for Understanding Immune Regulation by Uterine NK Cells in Pregnancy

Measurement of Four Uterine NK Cell Subtypes Using Multiplexed Fluorescent Immunohistochemical Staining in Women with Repeated Implantation Failure

Uterine NK cells dramatically accumulate during the mid-luteal phase, prepared for embryo implantation. Notably, these cells are heterogeneous. Accurately identifying their phenotypes is crucial for understanding their roles in endometrial receptivity.Our study aims to detect four subtypes of uterine NK cells using multiplex immunohistochemistry staining. Immunohistochemistry staining has been widely used to measure uterine NK cell density in women with reproductive failure. However, this traditional technology does not effectively distinguish between different subtypes of uterine NK cells.Our protocol, using multiplex immunohistochemistry staining, offers an advanced approach to identify four distinct subtypes of uterine NK cells. With the implementation of this protocol, we will evaluate the differences in uterine NK cell subtypes between women with repeated implantation failure and the fertile controls. If significant differences are identified, we will proceed to investigate whether these uterine NK cell subtypes can predict the risk of implantation failure.Ultimately, our long-term goal is to develop a diagnostic test to assess the immune status of uterine NK cells for endometrial receptivity.

To maintain consistency in the timing of endometrial sample collection for women undergoing the natural cycle, a urine test was performed to precisely detect their luteinizing hormone (LH) surge, with endometrial biopsies conducted 7 days after the LH surge. For women undergoing HRT cycles, samples were scheduled precisely 5 days after progesterone supplementation commenced. To quantify different subtypes of NK cells in the endometrium, m-IHC staining was employed. A schematic outlining of the experimental procedure is d...

This study presents a pioneering method for quantifying uterine natural killer cell subsets during the window of implantation using advanced multiplexed fluorescent immunohistochemical staining techniques.

Immunohistochemistry (IHC) plays a crucial role in biological research and clinical diagnosis, serving as the most commonly used method for identifying ...