Impact of selective progesterone receptor modulators (SPRMs) on the human endometrium

Abstract

INTRODUCTION: Heavy menstrual bleeding (HMB) is a common, chronic, debilitating and often underreported condition affecting at least 1 in 4 women of reproductive age. There is an unmet for managing the symptom of HMB. Selective Progesterone Receptor Modulators (SPRMs) are a class of drugs that interact with the progesterone receptor (PR) in a mixed agonist and antagonist fashion and may present an alternate medical solution. However, there is limited evidence examining the endometrial impact of SPRMs, their mechanism of action at a molecular and cellular level and the reversibility of the molecular signature of these drugs. This study examined the effect of two SPRMs on the human endometrium: Ulipristal acetate (UPA) and Vilaprisan (VPR). Studies presented in this thesis propose the overarching hypotheses that SPRMs would alter the structure and function of the endometrium and that the endometrial impact would be reversible on discontinuation of the drug.

MATERIALS AND METHODS: Ulipristal acetate (UPA): Endometrial biopsies (n=46) were available from 16 participants with HMB (with and without benign structural uterine abnormalities) in an embedded mechanism of action component of a large multicentre study, the UCON trial (EudraCT 2014-003408-65; REC14/LO/1602). Endometrial biopsies were obtained before UPA treatment, at six months of UPA treatment, and at the end of UPA treatment following a withdrawal bleed (twelve months). Reverse transcription quantitative real time polymerase chain reaction (RTqPCR), immunohistochemistry (IHC) and digital image analysis (DIA) using Qupath were undertaken. Vilaprisan (VPR): From 4 women with HMB and associated fibroids, where eight endometrial biopsies (fresh frozen endometrium) were utilised from the BAYER 15791 clinical trial (EudraCT Number: 2017-000468-13). Participants received treatment with VPR 2 milligrams once daily (OD) for 8-12 weeks. The participants had 2 endometrial biopsies performed (one pre-treatment and one on VPR treatment).

From 13 women with HMB and associated fibroids who were recruited as a part of the ASTEROID 6 (EudraCT Number: 2016-004822-41) study. Participants received treatment with VPR 2 milligrams OD for a duration of > 12 weeks. Participants had 2 endometrial biopsies performed (one pre-treatment and one on VPR treatment). RNA for the experiment was obtained from Formalin-Fixed Paraffin-Embedded (FFPE) blocks. In addition to RTqPCR, IHC and DIA using Qupath were also performed specifically for Ki67. Lexogen QuantSeq 3” mRNA sequencing method was used for RNA sequencing followed by detailed bioinformatic analyses using the Ingenuity Pathway Analysis (IPA).

For both SPRM studies, statistical analysis was undertaken using GraphPad Prism 8.0. p <0.05 was considered statistically significant.

RESULTS: UPA treatment resulted in PAEC, in up to a third of participants. UPA treatment resulted in significant changes in mRNA levels of endometrial steroid receptors ESR1, PR, PRB, AR, GR, and local endometrial steroid metabolising 17βHSD2, 17βHSD5 and 11βHSD2 versus the pre-treatment endometrium with a good temporal and spatial agreement. IHC revealed a unique endometrial phenotype of PR, PRB, AR and GR, which did not conform to that seen in the normal proliferative and secretory endometrium. A statistically significant reduction in cell proliferation assessed by Ki67 versus the pre-treatment proliferative endometrium was identified. UPA also impacted progesterone regulated genes; a statistically significant change was observed in the mRNA levels of FOXM1, IHH and HOXA10. UPA also affected the endometrial immune response; statistically significant alterations in the mRNA levels of IL-15, CD56, CCL2, CD68 and IL-8 were noted. Most importantly, through serial examination of the endometrium of the same participants throughout the study, it was observed that all the changes with UPA treatment described above were completely reversed following discontinuation of the drug and a menstrual withdrawal bleed. VPR treatment also resulted in PAEC, in over half (56.25%) of the participants in this study. VPR treatment resulted in significant changes in mRNA levels of endometrial steroid receptors ESR1, PR, PRB. VPR treatment also resulted in a statistically significant reduction in the mRNA levels of Ki67 and this effect was confirmed at a protein level. No impact on apoptotic markers with was identified. RNA sequencing and Bioinformatic analyses results were obtained using a strict threshold (FDRadjusted P < 0.05; fold change in expression ≥ 2), 636 differentially expressed genes (DEGs) were identified between pre-treatment proliferative samples and VPR treatment. Functional enrichment analysis was undertaken, and Reactome pathways and GO terms associated with cell cycle, and more specifically the mitotic phase, were significantly enriched. Using IPA, VPR was observed to have a profound negative impact on various stages of the cell cycle; G1/S, G2/M and mitotic progress. IPA identified that these effects were mediated through the action of two key regulators, CDK4 and FOXM1. RTqPCR further confirmed a statistically significant reduction in FOXM1 mRNA levels with VPR treatment.

CONCLUSION: In summary, the experimental data from this thesis derived from the examination of two SPRMs extends the knowledge with regards to understanding the impact of SPRMs on the human endometrium. Using UPA, a lack of a long-term impact on the endometrial molecular and cellular signature has been demonstrated, and using VPR, the antiproliferative effect has been shown to be likely mediated by a negative impact on cell cycle progression.