Measurement of Four Uterine NK Cell Subtypes Using Multiplexed Fluorescent Immunohistochemical Staining in Women with Repeated Implantation Failure

Summary

This study presents a pioneering method for quantifying uterine natural killer cell subsets during the window of implantation using advanced multiplexed fluorescent immunohistochemical staining techniques.

Abstract

Immunohistochemistry (IHC) plays a crucial role in biological research and clinical diagnosis, serving as the most commonly used method for identifying and visualizing tissue antigens. However, traditional IHC staining methods have limitations in distinguishing various subtypes of immune cells. This challenge has driven scientists to explore new technologies and methodologies for precise identification and differentiation of immune cell subtypes. In recent years, multiplex IHC has emerged as a solution, enabling the simultaneous detection of multiple antigens and their visualization within the same tissue sample. Uterine natural killer (uNK) cells play a pivotal role in early pregnancy processes, including decidualization, remodeling of uterine spiral arteries, and embryo implantation. Different subtypes of uNK cells exhibit different functions, allowing them to coordinate various biological events for successful embryo development and pregnancy. Therefore, in-depth research on uNK cell subtypes is essential for elucidating immune regulation mechanisms during pregnancy. Such studies provide valuable insights and novel approaches for addressing related conditions such as infertility and recurrent reproductive failure. This paper introduces a detailed multiplex IHC staining protocol for studying the density of four subtypes of uNK cells in endometrial specimens during the window of implantation (WOI). The protocol includes sample preparation, optimization of subtype markers, microscopic imaging, and data analyses. This multiplex IHC staining protocol offers high specificity and sensitivity, enabling simultaneous detection of different uNK cell subtypes, thus providing researchers with a powerful tool to explore the intricacies and mechanisms of immune regulation during pregnancy.

Introduction

The first documented live birth after in vitro fertilization-embryo transfer (IVF-ET) was reported in 1978. Over the past 40 years, there has been a high demand for the assistance of IVF-ET among infertile couples¹. In 2021, 238,126 patients initiated a total of 413,776 IVF cycles in the United States. This marks a 25% increase in cycles from 2 years prior and a 135% increase from 2012². This surge is mainly attributed to the rising prevalence of infertility and delayed planning for pregnancy. Advancements in embryo culture techniques and superovulation protocols have led to an increased live birth rate per ET c....

Protocol

The study was approved by the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee (CREC ref no.: 2022.581). Women with RIF were recruited from the Assisted Reproductive Technology Center, Prince of Wales Hospital, Chinese University of Hong Kong. RIF was defined as the failure to achieve a clinical pregnancy after the transfer of at least 4 good-quality embryos in a minimum of 3 fresh or frozen cycles in a woman under the age of 40 years¹⁰. Informe.......

Discussion

Embryo implantation involves a complex interaction between the embryo and the endometrium. The immunological status of endometrial homeostasis plays a pivotal role in determining endometrial receptivity. During WOI, the predominant leukocyte population in the endometrium is NK cells. Approximately 90% of uNK cells exhibit high CD56 expression but lack CD16. However, a minor subset of uNK cells resembles peripheral blood NK cells, displaying low CD56 expression but positive CD16 expression¹⁸. These.......

Materials

Name	Company	Catalog Number	Comments
CD49a	Novus Biologicals	NBP2-76478	Primary antibodies
CD56	Leica	NCL-L-CD56-504	Primary antibodies
CD16	abcam	ab183354	Primary antibodies
CXCR4	R&D	MAB172	Primary antibodies
Amplification diluent	Akoya Biosciences	FP1498 series	Fluorophore dilution buffer
Antibody diluents	Akoya Biosciences	ARD1001EA	Dilute the antibody
Citrate buffered solution, pH 6.0 /9.0(10x)	Akoya Biosciences	A6001/A9001	Antigen retrieval solution
inForm advanced image analysis software	Akoya Biosciences	inForm Tissue Finder Software 2.2.6	Data analysis software
Mantra Workstations	Akoya Biosciences	CLS140089	Spectral imaging
microwave	Akoya Biosciences	inverter	Microwave stripping
TSA 520	Akoya Biosciences	FP1487001K	Suitable tyramide-based fluorescent reagents
TSA 620	Akoya Biosciences	FP1495001K	Suitable tyramide-based fluorescent reagents
TSA 650	Akoya Biosciences	FP1496001K	Suitable tyramide-based fluorescent reagents
TSA 570	Akoya Biosciences	FP1488001K	Suitable tyramide-based fluorescent reagents
Poly-L-lysine coated slides	Fisher Technologies	120-550-15	Slides for routine histological use
PolyHRP Broad Spectrum	Perkin Elmer	ARH1001EA	Secondary antibodies
Invitrogen™ Fluoromount-G™ Mounting Medium	ThemoFisher Science	495802	Installation
Spectral DAPI	Akoya Biosciences	FP1490A	Nucleic acid staining