P-321 increased expression of TGF-β1 contributes to the downregulation of progesterone receptor expression in the eutopic endometrium of infertile women with minimal/mild endometriosis
Authors: Wu L , Xin H , Wei H 
Published: June 2023

Abstract

Study question
Do immune cells regulate progesterone resistance and endometrial receptivity of eutopic endometrial stromal cells (ESCs) by transforming growth factor-β1 (TGF-β1)?

Summary answer
By activating TGF-β/Smad signaling in eutopic ESCs, elevated TGF-β1 from CD45+ immune cells could attenuate expression of progesterone receptor (PR), and further decrease endometrial receptivity.

What is known already
Endometriosis is a hormone-dependent disease related to impaired immunoregulation. We characterized the trascriptomic transformation of eutopic endometrium from patients with minimal/mild endometriosis and controls across the menstrual cycle. However, the regulatory mechanism of altered immune microenvironment on eutopic ESCs remains unclear.

Study design, size, duration
Single-cell RNA transcriptomes were profiled using 10x Genomics platform. Primary culture of eutopic ESCs was performed to explore the effects of TGF-β1 on the expression of Smad / PR, and the in vitro decidualization. Additionally, co-immunoprecipitation (Co-IP) was used to explore the direct interaction between Smad and PR.

Participants/materials, setting, methods
Endometrial tissue samples were obtained from twenty-four women who were laparoscopically confirmed minimal/mild endometriosis according to the revised American Fertility Society classification. Additionally, four patients who were laparoscopically endometriosis-free were enrolled as controls. All samples were collected in DMEM/F12 medium and processed in one of two ways, either for single-cell sequencing (n = 10) or primary cell culture (n = 14).

Main results and the role of chance
The results of single-cell RNA sequencing revealed that the percentage of CD45+ cells in eutopic endometrium was increase in both proliferative (26% vs 24.1%) and secretory (19.3% vs 8.3%) stages. Overexpression of TGF-β1 of CD45+ cells was observed in eutopic endometrium of endometriosis in both proliferative and secretory stages. We found attenuate expression of PRB (p = 0.026) and PRA (p = 0.678) after using TGF-β1 in eutopic ESCs by western blot. Similarly, qRT-PCR results showed that the mRNA level of PR (p＜0.001), PRB (p = 0.003) and HOXA10 (p＜0.001) decreased significantly after TGF-β1 treatment, but that increased (p＜0.01) after SB431542 treatment in the eutopic ESCs. Moreover, TGF-β1 has a negative effect on the in vitro decidualization of eutopic ESCs (p = 0.003). And the group with treatment of both TGF-β1 and SB435142 in eutopic ESCs showed significant decidual-like changes with increased prolactin level (p = 0.01). Co-IP showed no physical interaction between the PR and p-Smad3/Smad3 proteins.

Limitations, reasons for caution
This study focused on CD45+ immune cells and the cytokine, TGF-β1. Further in vivo and in vitro studies are needed to elucidate the specific types of immune cell and the mechanisms that behind endometrial receptivity decrease in endometriosis.

Wider implications of the findings
Our results demonstrate abnormal activation of CD45+ cells in eutopic endometrium with minimal/mild endometriosis. And overexpression of TGF-β1 was found by CD45+ cells. We found that TGF-β1 suppresses PR expression and decidualization in eutpoic ESCs. These results provide a novel insight into the mechanisms behind progesterone resistance in endometriosis.

Trial registration number
not applicable