Comparative Analysis of Immune Cell Populations From Two Sampling Techniques of Human Term Decidua Utilizing High-Parameter Full-Spectrum Flow Cytometry

Abstract

Problem: The decidua is the interface between the uterus and the fetus. Studying decidual cells can reveal how healthy pregnancies are supported and mechanisms of pregnancy complications. There are two methods of obtaining decidual tissue following delivery. The placental bed can be suctioned following C-section deliveries, or a thin layer of decidual tissue can be dissected from the placenta. This study aimed to compare immune cell populations obtained using the two methods.

Method of study: From individuals with scheduled C-sections, we collected peripheral blood, decidua via vacuum suction of the placental bed, and decidua via dissection of the uterine-facing side of the placenta. Samples were analyzed using a 22-color full-spectrum flow cytometry panel to identify immune cell subsets and functional markers.

Results: The cellular composition of both decidual tissue collection methods were more similar to each other than to peripheral blood. Decidua collected via vacuum suction (Suc. decidua) had more live CD45+ cells. Decidua collected via dissection of the uterine-facing side of the placenta (Plac. decidua) had significantly higher expression of Helios in CD4+ cells, suggesting more fetal T cells. Both types of decidual samples contained similar levels of Tr1-like regulatory T lymphocytes expressing LAG3 and CD49b, whereas peripheral blood did not have this cell type.

Conclusion: Collecting decidual tissue using either method resulted in largely similar immune cell populations, suggesting studies are largely comparable regardless of whether samples were collected via suction or placental dissection. This will allow for greater flexibility in sample collection methods.

Keywords: decidua; flow cytometry; immune system; leukocytes; placenta.

FIGURE 1

Gating strategy to identify the major immune cell types. Gating shows live leukocytes including T lymphocytes, B lymphocytes, natural killer (NK) cells, innate lymphoid cells (ILCs), regulatory T lymphocytes (Tregs), and type 1 regulatory‐like T lymphocytes.

FIGURE 2

Overview of immune cell types. (A) Uniform Manifold Approximation and Projection (UMAP) and FlowSOM clustering containing data from all participants and all sample types. (B) UMAP for each sample type. (C) Bar graphs showing immune cell type frequencies in the live leukocyte (CD45+) population. Paired samples are represented by the same color/shape. A two‐tailed paired t‐test was used to compare frequencies between sample types. Significance levels are indicated as follows: *p ≤ 0.05 and **p ≤ 0.01.

FIGURE 3

Overview of T lymphocyte populations. (A) Uniform Manifold Approximation and Projection (UMAP) and FlowSOM clustering containing data from all participants and all sample types. Scaling for the heat map is across all markers. (B) Stacked bar graph and UMAPs illustrating the frequencies of clusters in each sample type.

FIGURE 4

Expression pattern of lymphocyte‐associated markers in CD4+ T lymphocytes. (A) Comparison of lymphocyte markers in CD4+ T lymphocytes and (B and C) regulatory CD4+ T lymphocytes. Paired samples are represented by the same color/shape. A two‐tailed paired t‐test was used to compare frequencies between sample types. Significance levels are indicated as follows: *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

FIGURE 5

Subsets of natural killer (NK) cells (CD56+) across sample types. (A) Gating strategy to define tissue‐resident (CD49a+) populations in natural killer cell subpopulations based on expression level of CD56 and CD16. (B) Frequencies of two natural killer cell subpopulations in individual sample types. Stacked bar graph represents composition of the natural killer cell subpopulations in each sample type. (C) Bar graph comparison of tissue‐resident natural killer cells, defined as CD49a+, and expression level of CD94. Paired samples are represented by the same color/shape. A two‐tailed paired t‐test was used to compare frequencies between sample types. Significance levels are indicated as follows: *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.