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

免疫組織化学的に染色された子宮内膜組織の画像解析

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.

著者スポットライト:子宮内膜免疫細胞の定量的評価のためのプロトコルによる生殖免疫学の進歩 

免疫組織化学とデジタル画像解析に基づく子宮内膜免疫細胞の定量的検出のためのプラットフォーム

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  

ヒト子宮内膜組織の包埋と切片化による免疫組織化学およびデジタル画像解析

まず、脱水された子宮内膜標本のサイズに一致する適切な包埋型を選択します。摂氏70度に加熱したパラフィンワックスで満たします。すぐに脱水したヒト子宮内膜組織を型に入れます。鉗子を使用して組織を慎重に調整し、中央に配置されるようにします。次に、型を冷却プレートに移し、下部のパラフィンが固まるとピンセットでティッシュをそっと押します。埋め込まれたカセットを型の上に置き、その上にさらにワックスを塗ります。次に、金型を冷却プレートに戻します。パラフィンが完全に固まったら、ブロックとそれに付属するカセットを型から取り出します。切片化のために、ブロックをミクロトームのサンプルクリップに挿入します。ブレードをホルダーに入れ、ブロック平面とブレードの間の角度を調整します。次に、セクションの厚さを4マイクロメートルに調整します。次に、ハンドホイールを回してスライスを開始します。ブロックから薄い切片を切り取り、適切な組織表面を露出させます。ブラシを使用して、連続したセクションと完全なセクションを収集します。十分な切片が得られたら、ピンセットを使用して、前面の不適格な切片を取り除きます。ブラシで、摂氏42度に加熱した温水浴に切片を移します。セクションが完全に平らになったら、剥離防止スライドを使用して持ち上げます。次に、65°Cのスライドウォーマーで60分以上焼いてから取り出します。

Research

JoVE Journal

Biology

Human Endometrial Tissue Embedding and Sectioning for Immunohistochemistry and Digital Image Analysis

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 ...

生物学

Dehydration of Human Endometrial Tissue for Sectioning

切片作成のためのヒト子宮内膜組織の脱水

Tissue Immunohistochemical Staining of Sectioned Human Endometrial Tissue

切片化されたヒト子宮内膜組織の組織免疫組織化学的染色

Tissue Scanning and Image Analysis of Immunohistochemically Stained Human Endometrium

免疫組織化学的に染色されたヒト子宮内膜の組織スキャンと画像解析