Name: Human Endometrial Tissue Embedding and Sectioning for Immunohistochemistry and Digital Image Analysis
Uploaded: 2023-10-13T13:50:00
Description: Watch this Scientific Journal Video about Human Endometrial Tissue Embedding and Sectioning for Immunohistochemistry and Digital Image Analysis at JoVE.com

human endometrial tissue embedding and sectioning for immunohistochemistry and digital image analysis

Image Analysis of Immunohistochemically Stained Endometrial Tissue

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.

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.

Watch this Scientific Journal Video about Human Endometrial Tissue Embedding and Sectioning for Immunohistochemistry and Digital Image Analysis at JoVE.com

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.

Research

JoVE Journal

Biology

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