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 endometrial receptivity.

Our study aims to detect four subtypes of uterine NK cells, using multiplex immunohistochemistry staining. Immunohistochemistry staining has been widely used to mirror 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 begin, obtain the human endometrial tissue section on a glass slide for multiplex immunohistochemistry, and bake the tissue sections at 60 degrees celsius for two hours. Subsequently, immerse the slide sequentially in appropriate solutions for dewaxing, hydration and fixation.

Add 200 milliliters of the respective AR6 or AR9 buffer for each antibody to a heat-resistant antigen retrieval cassette. After placing the sections in the antigen retrieval buffer, heat them in a microwave on high power until boiling. Cover the cassette loosely, and heat on low power for 15 minutes.

After the slide cools down, rinse it with double distilled water followed by PBST. Then remove the excess solution with absorbent paper, and draw a complete circle around the tissue with a hydrophobic barrier pen. Then incubate the sections with 3%hydrogen peroxide for eight minutes at room temperature.

Next, apply a blocking solution and incubate for 10 minutes at room temperature. After removing the blocking solution, apply 150 microliters of the first primary antibody CD49a to each slide at a dilution of one to 1000 in 1X antibody diluent. Before applying the secondary antibody, remove excess moisture using absorbent paper.

Add four to five drops of the 1X anti-mouse and rabbit horseradish peroxidase secondary antibody onto each slide. Then dilute the tyramide signal amplification or the TSA dye one to 100 times in the amplification diluent. And add 150 microliters of this diluted solution to each slide and incubate.

To eliminate unbound fluorophores, boil the sections in 200 milliliters of AR6 buffer within a heat-resistant cassette in a microwave for two minutes. Then turn the microwave from high power to low power for 15 minutes. Add 150 microliters of DPI working solution diluted in PBST dropwise.

Dry the sections entirely in a dark area for approximately 30 minutes. Apply a single drop of the anti-quenching fluorescent mountant, and carefully place a coverslip. To begin, stain the mounted endometrial tissue sections with the desired antibodies.

Turn on the imager, and select appropriate fluorophores based on TSA dyes. Manually focus on an area of the slide that shows a fair exposure of each marker, and use the auto-exposure feature to set the exposure time for that fluorophore. To ensure unbiased sampling, randomly select the initial area for image acquisition with a focus on the luminal epithelial border.

After capturing the initial field, move one field left or right to select subsequent areas, skipping one field between each image captured. Maintain the luminal epithelial border within the frame to capture at least five different areas to include around 10, 000 stromal cells. TSA monoplex and multiplex staining, revealed clear co-localization of NK cell markers, allowing the identification of distinct NK cell populations.

The multiplex strategy used fluorescent dyes with distant wavelengths to avoid spectral overlap and ensure clear background confirming signal specificity. To begin, image the endometrial tissue sections, after staining with appropriate antibodies. To import multi-spectral images, navigate to file, and then open image in the software interface.

Click Select Fluorophores, and choose the appropriate spectral library to unmix the fluorophores and click Okay to confirm. Isolate the autofluorescence spectrum, using the AF eyedropper tool to sample a representative tissue area from the AF image. And label the four-cell surface markers, and assign specific colors for identification.

Click Prepare Image, or prepare all to finalize preparation. For tissue segmentation, manually delineate three to five regions per tissue type, including epithelial, stromal, and blank areas. Adjust regions and parameters as needed to improve the training dataset, enhancing the software's accuracy in identifying tissue structures.

Under segment cells, select Nuclei and Membrane. Choose DPI to identify cell nuclei, and adjust the intensity setting to detect all cell nuclei without background noise. Select CD16, CD49a and CD56 signals to find the membrane and assist in nuclear splitting.

Use the nuclear component splitting feature to distinguish closely located nuclei. In the phenotyping scheme, adopt the CD56, CD49a, CXCR4, and CD16 markers for cell phenotyping. Use the Add button to categorize cells as positively or negatively expressing each marker.

Manually label at least five cells for each phenotype during the training process. After configuring cell phenotyping, the software will indicate the expression of surface markers for each cell. Export the data to a spreadsheet for further analysis.

The novel classification strategy identified for NK cell subtypes based on the expression of CD49a and CXCR4.