Selective progesterone receptor modulator (SPRM) action on the human endometrium

Abstract

INTRODUCTION: Selective Progesterone Receptor Modulators (SPRMs), of which Ulipristal Acetate (UPA) is a class member, have shown utility for gynaecological therapies including the symptom of heavy menstrual bleeding (HMB). UPA exhibits stronger antagonistic than agonistic action on the progesterone receptor (PR).

During the normal menstrual cycle oestradiol stimulates endometrial proliferation and progesterone stimulates endometrial cell differentiation and opposes oestradiol-induced proliferation. SPRMs such as UPA, with antagonistic action to the PR, create the potential for unopposed endometrial proliferation alongside an endocrine environment which continues to expose the endometrium to circulating oestradiol. Contrary to expectation, SPRM UPA decreases endometrial cell proliferation as assessed by cell proliferation marker Ki-67. This observation presents an unexplained paradox that requires deeper understanding of the mechanism of action of SPRM UPA within the endometrium.

HYPOTHESIS AND OBJECTIVES: The anti-proliferative effect of SPRMs is dependent on cell-cell communication between the two main endometrial cell types - stromal and epithelial cells. To test this hypothesis, four objectives were set:

1. Identify the SPRM-associated changes related to cell cycle in whole tissue endometrial transcriptome before, during, and after SPRM administration. 2. Determine the effect of SPRM on selected genes and proteins involved in cell cycle and progesterone-dependent downstream signalling in isolated human endometrial stromal cells

3. Establish endometrial epithelial organoid cultures and determine the effect of SPRM on selected genes and proteins involved in cell cycle and progesterone-dependent downstream signalling.

4. Develop a co-culture model with human endometrial stromal cells and epithelial organoids grown in a novel 3-dimensional (3D) gel microenvironment to determine the effect of SPRM upon cell cycle and progesterone-dependent downstream signalling.

METHODS AND RESULTS: To address Objective 1, RNA-Sequencing and pathway analysis were employed. Over 2000 differentially expressed genes (DEG) were identified when comparing pre-UPA-treated samples to UPA-treated samples of proliferative phase endometrium tissue biopsies. DEG were involved in suppression of canonical pathways of the oestrogen-mediated S-phase entry and cell-cycle progression. Genes encoding for cell cycle regulators cyclins A, B and E, E2F2, and CDC25A were also significantly downregulated when validation experiments were conducted using standard RT-qPCR. Notably, there were no enriched pathways between pre-treatment and post-UPA treated endometrium both in proliferative and secretory phase samples, indicating the altered gene expression was reversible.

To address Objectives 2 and 3, a protocol for isolation of endometrial stromal and epithelial cells was established and optimised. Endometrial epithelial cells were grown in Matrigel to form epithelial organoids. Using standard immunostaining and RT-qPCR protocols, the progesterone receptor was identified in both cell cultures and evidence of progesterone action was established by the detection of elevated mRNA levels of the progesterone-dependent genes Prolactin and IGFBP1. Despite this, no SPRM UPA-dependent (progesterone antagonist) effect was evident in either stromal cell or epithelial organoid cultures for genes and proteins of the cell cycle (Cyclin A1, Cyclin E2, E2F2, CDC25A), progesterone, androgen, glucocorticoid, and oestrogen receptors, nor for progesterone-dependent HOXA10 and FOXO1. Reverse Phase Protein Array (RPPA) using 120 pre-selected antibodies did not reveal UPA-dependent changes at the post-translational level in either stromal cell or epithelial organoid cultures. To address Objective 4, isolated endometrial stromal cells and epithelial organoids were incorporated into either Matrigel or functionalised cross-linked polymer of polyethylene glycol (PEG) gel. RT-qPCR did not reveal SPRM-associated changes in either Matrigel or PEG co-cultures. In contrast, RPPA results showed dose-dependent differential abundance of protein and phosphorylated epitopes in response to SPRM treatment in PEG co-cultures only. The SPRM UPA modulated proteins that were identified represent known mediators of cell cycle, MAPK/AKT, and chromatin regulation pathways.

SUMMARY: In these studies, endometrial epithelial organoid, and stromal-epithelial co-culture models have been established. Through RNA sequencing studies in whole endometrial tissue exposed to the SPRM, UPA, novel targets of SPRM mechanism of action were identified (Cyclin A1, Cyclin E2, E2F2, and CDC25A). These targets were tested in isolated monoculture stromal cell, epithelial organoid, and stromal-epithelial co-culture models. Results from isolated stromal and organoid cultures did not replicate observations in whole endometrial biopsies. Only co-cultures of isolated endometrial stromal cells with epithelial organoids in PEG gel matrix demonstrated comparable results to observations seen in intact endometrium, confirming the hypothesis that the anti-proliferative effect of SPRMs is dependent on cell-cell communication. Furthermore, an appropriate choice of 3D scaffold for in vitro experiments is critical for determining the functional response of primary cells to exogenous treatments.