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

Bildanalys av immunohistokemiskt färgad endometrievävnad

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.

Författare i fokus: Främja reproduktiv immunologi med ett protokoll för kvantitativ utvärdering av endometrieimmunceller 

Plattform för kvantitativ detektion av endometrieimmunceller baserad på immunhistokemi och digital bildanalys

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  

Human endometrievävnadsinbäddning och snittning för immunhistokemi och digital bildanalys

Till att börja med, välj en lämplig inbäddningsform som matchar storleken på det dehydrerade endometrieprovet. Fyll den med paraffinvax, uppvärmd till 70 grader Celsius. Placera omedelbart den uttorkade mänskliga endometrievävnaden i formen.Använd en pincett för att försiktigt justera vävnaden för att säkerställa att den placeras centralt. Överför nu formen till kylplattan och tryck försiktigt på vävnaden med en pincett när paraffinet i botten stelnar. Placera den inbäddade kassetten över formen och fyll den med mer vax.Sätt sedan tillbaka formen på kylplattan. När paraffinet har stelnat helt, ta bort blocket och dess bifogade kassett från formen. För sektionering, sätt in blocket på sampklämman på mikrotomen.Placera bladet i hållaren och justera vinkeln mellan blockplanet och bladet. Justera nu sektionens tjocklek till fyra mikrometer. Vrid sedan på handhjulet för att börja skiva.Skär tunna sektioner från blocket för att exponera lämplig vävnadsyta. Använd borsten för att samla ihop de kontinuerliga och kompletta sektionerna. När tillräckligt många sektioner har erhållits, använd en pincett för att ta bort de okvalificerade sektionerna på framsidan.Överför sektionerna till ett varmt vattenbad med en borste, uppvärmt till 42 grader Celsius. När sektionerna har planat ut helt, använd anti-lossningsrutschbanor för att lyfta dem. Grädda dem sedan i en bildvärmare vid 65 grader Celsius i över 60 minuter innan du tar bort dem.

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 ...