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

Визуальный анализ иммуногистохимически окрашенной ткани эндометрия

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.

В центре внимания автора: Развитие репродуктивной иммунологии с помощью протокола количественной оценки иммунных клеток эндометрия 

Платформа для количественного обнаружения иммунных клеток эндометрия на основе иммуногистохимии и анализа цифровых изображений

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  

Встраивание и секционирование тканей эндометрия человека для иммуногистохимии и анализа цифровых изображений

Для начала выберите подходящую форму для встраивания, которая соответствует размеру обезвоженного образца эндометрия. Залейте его парафином, нагретым до 70 градусов по Цельсию. Немедленно поместите обезвоженную ткань эндометрия человека в форму.Используйте щипцы, чтобы аккуратно отрегулировать ткань, чтобы убедиться, что она расположена по центру. Теперь перенесите форму на охлаждающую пластину и аккуратно прижмите ткань пинцетом по мере застывания парафина на дне. Поместите встроенную кассету на форму и полейте ее большим количеством воска.Затем поместите форму обратно на охлаждающую пластину. Как только парафин полностью застынет, извлеките блок и прикрепленную к нему кассету из формы. Для секционирования вставьте блок в клип образца микротома.Поместите лезвие в держатель и отрегулируйте угол между плоскостью блока и лезвием. Теперь отрегулируйте толщину сечения до четырех микрометров. Затем поверните маховик, чтобы начать нарезку.Отрежьте тонкие срезы от блока, чтобы обнажить соответствующую поверхность ткани. Используйте кисть, чтобы собрать непрерывные и полные участки. После того, как будет получено достаточное количество секций, используйте пинцет, чтобы удалить неквалифицированные секции спереди.Щеткой перенесите срезы на теплую водяную баню, нагретую до 42 градусов по Цельсию. Когда секции полностью расплющутся, используйте противоотрывные направляющие, чтобы поднять их. Затем запекайте их в грелке при температуре 65 градусов Цельсия более 60 минут, прежде чем вынуть.

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 ...