This study was supported by Hong Kong Obstetrical and Gynecological Trust Fund in 2018 and Hong Kong Health and Medical Research Fund (06170186, 07180226).

The study was approved by the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee. Informed consent was obtained from the participants before collecting the endometrial biopsies. Refer to the introduction section for inclusion criteria of the control and RM groups.
1. Sample preparation
<ol>
	<li>Ensure that all the women in this study undergo a daily urine dipstick test from day 9 of the menstrual cycle onwards to identify the luteinizing ho...

<ol>
	<li>ESHRE Guideline Group on RPL 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=ESHRE+guideline:+recurrent+pregnancy+loss.">ESHRE guideline: recurrent pregnancy loss.</a> Human Reproduction Open. 2018 (2), 004 (2018).</li><li>Stirrat, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+miscarriage.">Recurrent miscarriage.</a> Lancet. 336 (8716), 673-675 (1990).</li><li>Rai, R., 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=Recurrent+miscarriage.">Recurrent miscarriage.</a> Lancet. 368 (9535), 601-611 (2006).</li><li>Royal College of Obstetricians &amp; Gynaecologists. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+investigation+and+treatment+of+couples+with+recurrent+first-trimester+and+second-trimester+miscarriage.+Green-top+Guideline+No.+17.">The investigation and treatment of couples with recurrent first-trimester and second-trimester miscarriage. Green-top Guideline No. 17.</a> Royal College of Obstetricians &amp; Gynaecologists. , (2011).</li><li>King, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uterine+leukocytes+and+decidualization.">Uterine leukocytes and decidualization.</a> Human Reproduction Update. 6 (1), 28-36 (2000).</li><li>Le Bouteiller, P., Piccinni, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+NK+cells+in+pregnant+uterus:+why+there.">Human NK cells in pregnant uterus: why there.</a> American Journal of Reproductive Immunology. 59 (5), 401-406 (2008).</li><li>Lash, G. 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> Journal of Reproductive Immunology. 116, 50-59 (2016).</li><li>Yang, Y., 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=HOXA-10+and+E-cadherin+expression+in+the+endometrium+of+women+with+recurrent+implantation+failure+and+recurrent+miscarriage.">HOXA-10 and E-cadherin expression in the endometrium of women with recurrent implantation failure and recurrent miscarriage.</a> Fertility and Sterility. 107 (1), 136-143 (2017).</li><li>Tuckerman, E., Laird, S. M., Prakash, A., Li, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+value+of+the+measurement+of+uterine+natural+killer+cells+in+the+endometrium+of+women+with+recurrent+miscarriage.">Prognostic value of the measurement of uterine natural killer cells in the endometrium of women with recurrent miscarriage.</a> Human Reproduction. 22 (8), 2208-2213 (2007).</li><li>Jasper, M. 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=Macrophage-derived+LIF+and+IL1B+regulate+alpha(1,2)fucosyltransferase+2+(Fut2)+expression+in+mouse+uterine+epithelial+cells+during+early+pregnancy.">Macrophage-derived LIF and IL1B regulate alpha(1,2)fucosyltransferase 2 (Fut2) expression in mouse uterine epithelial cells during early pregnancy.</a> Biology of Reproduction. 84 (1), 179-188 (2011).</li><li>Kopcow, H. 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=T+cell+apoptosis+at+the+maternal-fetal+interface+in+early+human+pregnancy,+involvement+of+galectin-1.">T cell apoptosis at the maternal-fetal interface in early human pregnancy, involvement of galectin-1.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (47), 18472-18477 (2008).</li><li>Johnson, P. M., Christmas, S. E., Vince, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+aspects+of+implantation+and+implantation+failure.">Immunological aspects of implantation and implantation failure.</a> Human Reproduction. 14, 26-36 (1999).</li><li>Hong, 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=Multiplexed+fluorescent+immunohistochemical+staining,+imaging,+and+analysis+in+histological+samples+of+lymphoma.">Multiplexed fluorescent immunohistochemical staining, imaging, and analysis in histological samples of lymphoma.</a> Journal of Visualized Experiments: JoVE. (143), e58711 (2019).</li><li>Carstens, J. 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=Spatial+computation+of+intratumoral+T+cells+correlates+with+survival+of+patients+with+pancreatic+cancer.">Spatial computation of intratumoral T cells correlates with survival of patients with pancreatic cancer.</a> Nature Communications. 8, 15095 (2017).</li><li>Zhao, Y., 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+use+of+multiplex+staining+to+measure+the+density+and+clustering+of+four+endometrial+immune+cells+around+the+implantation+period+in+women+with+recurrent+miscarriage:+comparison+with+fertile+controls.">The use of multiplex staining to measure the density and clustering of four endometrial immune cells around the implantation period in women with recurrent miscarriage: comparison with fertile controls.</a> Journal of Molecular Histology. 51 (5), 593-603 (2020).</li></ol>

The authors declare that they have no conflicts of interest to disclose.

Critical steps within the protocol 
It is important to note that multiplex staining requires diligent optimization. Antigen retrieval, using citrate buffer and microwave technology, requires optimization to ensure complete antibody stripping and maintain tissue viability. As TSA reagents covalently bind to sites surrounding the antigen, they can potentially inhibit the binding of a subsequent primary antibody through steric hindrance (also known as the &#34;umbrella effect&#34;). This tends to occur...

Recurrent miscarriage (RM) can be defined as the loss of two or more pregnancies before 24 weeks of gestation1. This frequent reproductive condition affects up to 1% of couples worldwide2,3. The pathophysiology is multifactorial and can be divided into embryologically driven causes (mainly due to an abnormal embryonic karyotype) and maternally driven causes that affect the endometrium and/or placental development. This manifestation can result from parental genetic abnormalities, uterine anomalies, prothrombotic conditions, endocrinology factors, and immunological disorders4.
In recent years, immune effector cell dysfunction has been implicated in the pathogenesis of early pregnancy loss5. This has inspired many investigations into elucidating the specific populations of immune cells in the endometrium during the menstrual cycle, implantation, and early pregnancy, with specific roles in early pregnancy. Among these immune cells, uterine natural killer (uNK) cells play a critical role during embryo implantation and pregnancy, particularly in the processes of trophoblastic invasion and angiogenesis6. Studies have shown an increased uNK cell density in the endometrium of women with RM7,8, although this finding was not associated with an increased risk of miscarriage9. However, this stimulated research evaluating the density of other immune cell types (such as macrophages, uterine dendritic cells) in the endometrium in women with RM10, 11. Nevertheless, it remains uncertain whether there is a significant alteration in the immune cell density in the peri-implantation endometrium in women with RM.
One possible explanation for the uncertainty is that evaluation of the endometrial immune cell density might be difficult due to the rapid changes in the endometrium during the window of implantation. During the 24 h timeframe, significant changes in the endometrium alter immune cell density and cytokine secretion, introducing a source of variation in these results12. In addition, most reports mainly rely on the use of single-cell staining (e.g., traditional IHC methods) that could not examine multiple markers on the same tissue section. Although flow cytometry can be used for detecting multiple cell populations in a single sample, the large amounts of cells required and the time-consuming optimization hinder the popularity and efficiency of this method. Hence, the recent advancement in multiplex IHC staining could solve this problem by immunostaining multiple markers on the same slide to evaluate multiple parameters, including cell lineage and histological localization of individual immune subpopulations. Further, this technology can maximize the information obtained in case of limited tissue availability. Ultimately, this technique can help elucidate the differences in immune cell interactions in the endometrium between fertile women and women with RM.
Two groups of women were recruited from the Prince of Wales Hospital, including fertile control women (FC) and women with unexplained recurrent miscarriage (RM). Fertile control was defined as women who had at least one live birth without any history of spontaneous miscarriage, and RM women were defined as those who had a history of &#8805;2 consecutive miscarriages before 20 weeks gestation. The subjects from the two groups met the following inclusion criteria: (a) age between 20 to 42 years old, (b) non-smoker, (c) regular menstrual cycle (25-35 days) and normal uterine structure, (d) no use of any hormonal regimen for at least 3 months before the endometrial biopsy, (e) no hydrosalpinx by hystero-salpingogram. In addition, all the subjects recruited had normal karyotyping, normal 3-dimensional ultrasonography hysterosalpingogram, day 2 follicle-stimulating hormone &#60; 10 IU/L, mid-luteal progesterone &#62; 30 nmol/L, normal thyroid function, and tested negative for lupus anticoagulant and anticardiolipin IgG and IgM antibodies.
To better understand the immunological basis of RM, it would be most desirable to simultaneously quantify and localize the major immune cell types present in the endometrium at the time of implantation. This paper describes the entire protocol from sample preparation, the 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. Moreover, this paper describes how to determine the density and clustering of the immune cell types simultaneously in the endometrium.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amplification Diluent</td>
			<td>Perkin Elmer</td>
			<td>FP1498</td>
			<td>Fluorophore diluent buffer</td>
		</tr>
		<tr>
			<td>Antibody diluent</td>
			<td>Perkin Elmer</td>
			<td>ARD1001EA</td>
			<td>Diluting the antibody</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>Spring Bioscience</td>
			<td>M3072</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>CD20</td>
			<td>Biocare Medical</td>
			<td>CM004B</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>CD56</td>
			<td>Leica</td>
			<td>NCL-CD56-504</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>CD68</td>
			<td>Spring Bioscience</td>
			<td>M5510</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Citrate Buffer Solution, pH 6.0 (10x)</td>
			<td>Abcam</td>
			<td>AB64214</td>
			<td>Antigen retrieval solution</td>
		</tr>
		<tr>
			<td>EMSURE Xylene (isomeric mixture)</td>
			<td>Merck</td>
			<td>108297</td>
			<td>Dewaxing</td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Merck</td>
			<td>107017</td>
			<td>Ethyl alcohol for rehydration</td>
		</tr>
		<tr>
			<td>HistoCore BIOCUT Manual Rotary Leica Microtome</td>
			<td>Leica</td>
			<td>RM2125RTS</td>
			<td>Sectioning of paraffin-embedded tissue</td>
		</tr>
		<tr>
			<td>inForm Advanced Image Analysis Software</td>
			<td>Perkin Elmer</td>
			<td>inForm&#174; Tissue Finder Software 2.2.1 (version 14.0)</td>
			<td>Data Analysis software</td>
		</tr>
		<tr>
			<td>Mantra&#174; Workstation</td>
			<td>Akoya Biosciences</td>
			<td>CLS140089</td>
			<td>Spectral imaging</td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>Panasonic</td>
			<td>Inverter</td>
			<td>Microwave stripping</td>
		</tr>
		<tr>
			<td>Opal 520</td>
			<td>Perkin Elmer</td>
			<td>FP1487A</td>
			<td>Appropriate tyramide based fluorescent reagent</td>
		</tr>
		<tr>
			<td>Opal 620</td>
			<td>Perkin Elmer</td>
			<td>FP1495A</td>
			<td>Appropriate tyramide based fluorescent reagent</td>
		</tr>
		<tr>
			<td>Opal 650</td>
			<td>Perkin Elmer</td>
			<td>FP1496A</td>
			<td>Appropriate tyramide based fluorescent reagent</td>
		</tr>
		<tr>
			<td>Opal 690</td>
			<td>Perkin Elmer</td>
			<td>FP1497A</td>
			<td>Appropriate tyramide based fluorescent reagent</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Memmert</td>
			<td>U10</td>
			<td>Dewaxing</td>
		</tr>
		<tr>
			<td>Peroxidase Blocking Solution</td>
			<td>DAKO</td>
			<td>S2023</td>
			<td>Removal of tissue peroxidase activities</td>
		</tr>
		<tr>
			<td>Poly-L-lysine coated slide</td>
			<td>FISHER SCIENTIFIC</td>
			<td>120-550-15</td>
			<td>Slide for routine histological use</td>
		</tr>
		<tr>
			<td>PolyHRP Broad Spectrum</td>
			<td>Perkin Elmer</td>
			<td>ARH1001EA</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>ProLong&#8482; Gold Antifade Mountant</td>
			<td>ThemoFisher Scientific</td>
			<td>P36930</td>
			<td>Mounting</td>
		</tr>
		<tr>
			<td>Spatstat</td>
			<td>/</td>
			<td>Version 2.1-0</td>
			<td>Spatial point pattern analysis</td>
		</tr>
		<tr>
			<td>Spectral DAPI</td>
			<td>Perkin Elmer</td>
			<td>FP1490A</td>
			<td>Nucleic acid staining</td>
		</tr>
		<tr>
			<td>Tissue Processor</td>
			<td>Thermo Fischer</td>
			<td>Excelsior ES</td>
			<td>Tissue processing for dehydration and paraffination</td>
		</tr>
		<tr>
			<td>Tris Buffer Saline (TBS), 10x</td>
			<td>Cell Signaling Technology</td>
			<td>12498S</td>
			<td>Washing solution</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1370-1L</td>
			<td>Nonionic detergent</td>
		</tr>
	</tbody>
</table>

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.

The overall schematic process of performing a 4-color multiplex assay for the detection of 4 endometrial immune cell types is shown in Figure 1. In brief, the protocol for this multiplex immunofluorescence staining required 8 key steps: 1. Slide preparation, 2. Epitope retrieval, 3. Blocking, 4. Primary antibody application, 5. Secondary antibody application, 6. Signal amplification, 7. Removal of antibody, and 8. Counterstain and mount. Image rendering and analysis were then conducted using...

Watch this Scientific Journal Video about Multiplexed Fluorescent Immunohistochemical Staining of Four Endometrial Immune Cell Types in Recurrent Miscarriage at JoVE.com

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

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.

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

The present technology allows the use of antibody raised in the same species for detecting up to seven markers. Demonstrating the procedure will be Loucia Chan, a technician from the laboratory. Begin by preparing the formalin fixed paraffin embedded glass slides mounted with the tissue sample, by placing the slides flat in an oven, to bake at 60 degrees celsius with the tissue facing upwards.After one hour, de-wax and rehydrate the slides per 10 minutes each with the series of chemicals, as described in the manuscript. Submerge the slides in tris-buffered saline, and then in a plastic slide box filled with a mixture of formaldehyde diluted in methanol, to fix the samples. Next, wash the slides twice in deionized water for two minutes.Proceed to epitope retrieval, by placing the rack of slides in a heat resistance box filled with citric acid buffer at pH 6.0. And then placing the box in the microwave, to heat the slides first at 100%power for 50 seconds, followed by 20 minutes at 20%power, to maintain the same temperature. After rinsing the slides in distilled water, and washing with TBST, immerse the slide into a jar containing a peroxidation blocking solution for 10 minutes.After the incubation, rinse the slides in distilled water and wash with TBST, then use a hydrophobic barrier pen to mark a boundary around the tissue section on the slide. Then rinse the slide in TBST, cover the tissue sections with the blocking buffer and incubate the slides in a humidified chamber for 15 minutes. To remove the blocking reagents, first incubate the slides with the primary antibody of interest, and then remove the primary antibody by washing the slides thrice with TBST, before incubation with the polymer horseradish peroxidase labeled, secondary antibody.Following the incubation, wash the slides before applying the Opal flora for TSA working solution, and then rinse the slide with the antigen retrieval buffer. Perform the microwave-based stripping by heating the slides in antigen retrieval buffer in a microwave at 100%power, for 50 seconds, followed by 20%power for 20 minutes in the microwave safe containers. After cooling the samples at room temperature for 15 minutes, rinse the slides in distilled water and TBST and later incubate with dappy solution for five minutes.Then air dry the slide before mounting with the appropriate mounting medium. Before imaging of the slides preset the appropriate filters in the workstation. Capture a minimum of 10 fields for analysis under 200 times magnification.To count the cells, click on the count objects button in the step bar, to display the object counting settings panel. If the tissue has been segmented, select the tissue category in which the objects are to be found. Then select the desired signal scaling from auto-scale or fixed scale.For object segmentation, select the primary component from the drop-down list. To detect objects touching other objects as individual and not combined objects, check the maximum size and pixels box and then opt for the refined splitting. Next, count all stromal cells in the immune cells separately, to express the data as the percentages of the immune cells relative to the total number of stromal cells for each captured image.Later, report the final cell count as an average of all fields. Using the view editor, examine the resulting data tables post-processing and then export the count data table. In the representative analysis, four immune cell types in the endometrium sample we're differentiated.The processed image shows tissue segmentation as epithelial, and stromal compartments. The multiplex immunostaining was performed on the endometrial biopsies from a fertile control woman and a woman with unexplained recurrent miscarriage, single floor for us representing CD3, CD56, CD68, and CD20 were revealed in the immunostaining. Once the immune sound has been identified, further staining under the cytokine can help to address their interaction and mechanism for implantation success.

multiplexed-fluorescent-immunohistochemical-staining-four-endometrial

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Biology

Unit 1

The Endocrine System

SCIENTISTS IN ACTION

3.1K vistas.  The Chinese University of Hong Kong, Prince of Wales Hospital. La tecnología actual permite el uso de anticuerpos criados en la misma especie para detectar hasta siete marcadores. La demostración del procedimiento estará a cargo de Loucia Chan, técnica del laboratorio. Comience preparando los portaobjetos de vidrio incrustados de parafina fija de formalina montados con la muestra de tejido, colocando los portaobjetos planos en un horno, para hornear a 60 grados centígrados con el tejido hacia arriba.Después de una hora, descere y rehidrate l...