Platform for Quantitative Detection of Endometrial Immune Cells Based on Immunohistochemistry and Digital Image Analysis

Summary

Here, a digital immunohistochemistry image analysis platform was developed and validated to quantitatively analyze the endometrial immune cells of patients with recurrent miscarriages in the window of implantation.

Abstract

To evaluate the endometrial immune microenvironment of patients with recurrent miscarriage (RM), a digital immunohistochemistry image analysis platform was developed and validated to quantitatively analyze endometrial immune cells during the mid-luteal phase. All endometrium samples were collected during the mid-luteal phase of the menstrual cycle. Paraffin-embedded endometrial tissues were sectioned into 4 µm thick slides, and immunohistochemistry (IHC)staining was carried out for detecting endometrial immune cells, including CD56+ uNK cells, Foxp3+ Tregs, CD163+ M2 macrophages, CD1a+ DCs, and CD8+ T cells. The panoramic slides were scanned using a digital slide scanner and a commercial image analysis system was used for quantitative analysis. The percentage of endometrial immune cells was calculated by dividing the number of immune cells in the total endometrial cells. Using the commercial image analysis system, quantitative evaluation of endometrial immune cells, which are difficult or impossible to analyze with conventional image analysis, could be easily, and accurately analyzed. This methodology can be applied to quantitatively characterize the endometrium microenvironment, including interaction between immune cells, and its heterogeneity for different reproductive failure patients. The platform for quantitative evaluation of endometrial immune cells may be of important clinical significance for the diagnosis and treatment of RM patients.

Introduction

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 RM^2,3,4,5. 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 h....

Protocol

The research content and protocol has been ethically reviewed and approved by the research ethics committee of Shenzhen Zhongshan Urology Hospital. All women (20- 40 years old) involved in the study provided informed consent for sample collection and usage.

1. Acquisition of pathological tissue

1. Prepare the tools for tissue harvesting namely, measuring ruler, tweezer, embedding cassette, embedding paper, and tissue basket.
2. Observe whether the amount of endometrial tissue (bigger than a mung bean), collected using a standard approach with a pipelle catheter, is sufficient.
3. Transfer the endometr....

Results

In order to evaluate endometrial immune cells quantitatively and reduce the instability caused by man-made operational mistakes, we established a digital quantitative analysis platform for endometrial immune cells by using automatic immunohistochemical detection and digital quantitative evaluation system. Immunohistochemistry image analysis platform was established to quantitatively analyze endometrial immune cells of patients with recurrent miscarriage (RM) in the window of implantation. All endometrium tissues were col.......

Discussion

This protocol established a digital immunohistochemistry image analysis platform to quantitatively analyze endometrial immune cells of RM patients. Here, six endometrial immune markers were detected to evaluate the endometrial immune microenvironment in RM patients.

A receptive endometrium during the mid-luteal phase is key for successful implantation and pregnancy^27,28. Therefore, evaluation of percent endometrial immune cells pl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Automated coverslipper	Sakuraus	DRS-Prisma-P-JCS&Film-JC2
CD163	GrowGn Biotechnology	NCL-L-CD163
CD1a	Gene Tech	GM357129
CD56	Gene Tech	GT200529
CD8	Novocastra	NCL-L-CD8-4B11
Dehydrator	Thermo Fisher	Excelsior ES
Digital pathology and	Indica labs	HALO
Foxp3	YILIFANG biological	14-477-82
IHC stainer	Leica	BOND III
Image analysis platform	Indica labs	HALO
Slide Scanner	Olympus life science	VS200