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Endometrium Undergoes Gene Expression Shifts During Menstrual Cycle, Single-Cell Study Finds

NEW YORK – The window during which human embryos can implant into the endometrium opens with an abrupt change in gene expression of endometrial cells, a new single-cell transcriptomic study has found.

During the human menstrual cycle, the endometrium goes through a process of remodeling, shedding, and regeneration. Because of its important function in human fertility and regenerative medicine, researchers led by Stanford University's Stephen Quake sought to characterize its gene expression at the single-cell level.

The researchers performed RNA sequencing on cells from endometrial biopsies obtained from multiple healthy individuals at various points in their menstrual cycle. As they reported on Monday in Nature Medicine, they profiled numerous endometrial cell types and found a shift in gene expression, particularly among unciliated epithelial cells, that corresponded with the opening of the window of implantation (WOI), the brief period during which the endometrium is primed for embryo implantation.

"By studying both the static and dynamic aspects of the tissue, we discovered molecular characterizations of hallmark events such as the WOI, and provide a systematic single-cell transcriptomic delineation of endometrial transformation at the levels of cell type, state, proliferation, and differentiation across the human menstrual cycle," Quake and his colleagues wrote in their paper.

The researchers obtained endometrial biopsies from 19 healthy individuals between four and 27 days after the start of their last menstrual period. They captured single cells for RNA-seq analysis using Fluidigm C1 medium chips and, to validate their findings, processed additional samples from 10 other healthy individuals using the 10x Chromium system.

An analysis of the genes and markers expressed by the endometrial cells pointed to the presence of six cell types, four of which — stromal fibroblasts, endothelium, macrophages, and lymphocytes — could readily be identified. The two other cell types both expressed epithelium-linked markers as well as other genes, leading the researchers to characterize one cell type as 'ciliated epithelium' and the other as 'unciliated epithelium.' 

They noted that the presence of ciliated epithelium in the endometrium had been postulated in the 1890s but that there has been little data regarding this cell type. Here, the researchers found ciliated epithelial cells were consistently present in the endometrium, though their numbers changed throughout the course of the menstrual cycle.

They also examined the transcriptomes of endometrial cells over time to uncover four major phases of endometrial transformation. For instance, the global transcriptome of unciliated epithelial cells was fairly consistent from phase one through phase three, but it changed abruptly with the start of phase four. At that time, those cells began to express genes like PAEP, GPX3, and CXCL14, whose expression has previously been linked to the WOI.

At the same time, stromal fibroblasts upregulated genes like DKK1 and CRYAB during phase four, as well as the decidualization-initiating transcription factor FOXO1 and the decidualization stromal marker IL5. The change in gene expression among stromal fibroblasts upon entering the WOI was not quite as abrupt as it was among the epithelial cells.

The researchers suggested that decidualization — the transformation of the endothelium that includes fibroblasts changing from elongated cells to enlarged round cells — begins before the WOI in a small number of stromal fibroblasts in a more gradual process, while the initiation of WOI is more marked among unciliated epithelial cells.

The WOI closes gradually, the researchers noted. One group of genes, marked by PAEP and GPX3, are expressed throughout phase four and into phase one of the next cycle, while another group of genes that include CXC14, MAOA, and DPP4 decline in expression toward the end of phase four.

The researchers also compared decidualizing stromal fibroblasts in the endometrium to stromal fibroblasts from early pregnancy to find that the former are less heterogeneous. They also lack a subtype otherwise found among stromal fibroblasts from pregnancy, which suggests an implanting embryo triggers the development of stromal fibroblast subtypes.

The researchers noted that their dataset gives additional insight into the healthy endometrium and could be used as a jumping-off point to study not only endometrial disease, but also human fertility.