image acquisition after multiplex immuno-staining for distinguishing unk cell subtypes in endometrial tissues

<meta charset="utf8"></head><body>使用多重 IHC 探索子宫内膜中子宫 NK 细胞的异质性

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 cycle, reaching 30%-50% in women less than 40 years old and less than 30% in women older than 40 years2. However, despite these advancements, over half of transferred embryos still fail to implant. Repeated implantation failure (RIF), typically defined as failure after three or more consecutive attempts of transferring high-quality embryos, affects 15% of women who undergo IVF-ET3. Couples with RIF are extremely vulnerable and more prone to undergo expensive and unnecessary procedures that can expose them to undue risks4. Therefore, understanding the causes of RIF and improving embryo implantation is crucial to enhance the success of IVF-ET, particularly for women with RIF. The preparation of endometrium is critical for successful embryo implantation. This process is characterized by a significant accumulation of uterine natural killer (uNK) cells, which transition from constituting 30% of total lymphocytes in the endometrium during mid-secretory phase to 70%-80% in the decidua during early pregnancy5. Notably, uNK cells differ from peripheral NK cells, which are cytotoxic lymphocytes critical to the innate immune system for causing the death of the infected cells through lysis or apoptosis. While the exact functions of uNK cells are not yet fully understood, several lines of evidence suggest that they are involved in angiogenesis remodeling, trophoblast invasion, and fetal development6. The association between the percentage of uNK cells over stromal cells and RIF has garnered extensive attraction over the past 20 years. A recent meta-analysis, which included 8 studies involving 604 women, demonstrated that the density of CD56+uNK cells during the mid-luteal phase is significantly increased in women with RIF compared to fertile controls7. However, it is important to note that the characteristics of uNK cells during the mid-luteal phase differ significantly from those of decidual NK (dNK) cells. Although uNK cells may further differentiate into various subsets of dNK cells post-pregnancy, measuring uNK cells alone does not accurately represent dNK cells8. uNK cells undergo dynamic differentiation and play different roles in the menstrual cycle and decidualization processes, making them more complex than can be identified by CD56 alone. Multiple markers are required to achieve a comprehensive understanding of uNK cell behavior during endometrial preparation. Our recent study employed single-cell RNA sequencing to identify the diversity of uNK cells throughout menstrual cycles. The results, validated using flow cytometry, have shown the presence of four distinct subtypes of uNK cells, each exhibiting dynamic changes during the menstrual cycle9. Gene enrichment analysis and gene ontology functional enrichment indicate these uNK subsets fulfill different functions at various stages of menstruation. Nevertheless, flow cytometry is not universally accessible in clinical laboratories, and the immediate processing of fresh endometrial tissue for enzyme digestion renders it impossible to repeat experimental steps upon errors.
The aim of this study was, therefore, to investigate the measurement of these four subpopulations of NK cells using a multiplex staining assay, which provides a more practical diagnostic approach. In multiple staining, different specific antibodies against each target are linked to different fluorophore labels that emit different wavelengths of light when excited by a specific wavelength of light. Compared to the traditional IHC staining method, this method can quantitatively compare the relative abundance and distribution of multiple targets in a sample and provide information on the interaction and co-localization of different targets, enabling us to identify different subtypes of uNK cells. This approach will not only deepen our understanding of the relationship between uNK cells and RIF but will also provide insights for investigating other immune cell subpopulations in endometrial-related diseases.

<meta charset="utf8"></head><body>作者聚焦：多重免疫组织化学了解妊娠期子宫 NK 细胞的免疫调节

使用多重荧光免疫组织化学染色测量反复植入失败女性的四种子宫 NK 细胞亚型

多重免疫染色后的图像采集以区分子宫内膜组织中 uNK 细胞亚型

image-acquisition-after-multiplex-immuno-staining-for-distinguishing

Research

JoVE Journal

Medicine

Image Acquisition After Multiplex Immuno-Staining for Distinguishing uNK Cell Subtypes in Endometrial Tissues

81 次观看. 首先，用所需的抗体对封固的子宫内膜组织切片进行染色。打开成像仪并根据 TSA 染料选择合适的荧光基团。手动聚焦在玻片上显示每个标记物曝光均匀的区域，并使用自动曝光功能设置该荧光基团的曝光时间。为确保无偏采样，随机选择用于图像采集的初始区域，重点是管腔上皮边界。捕获初始字段后，向左或向右移动一个字段以选择后续区?...

视频: 多重免疫染色后的图像采集以区分子宫内膜组织中 uNK 细胞亚型

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

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.

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.

Immunohistochemistry (IHC) plays a crucial role in biological research and clinical diagnosis, serving as the most commonly used method for identifying ...