Stromal cell senescence contributes to impaired endometrial decidualization and defective interaction with trophoblast cells

Abstract

Study question: What are the consequences of endometrial stromal cell (EnSC) senescence for endometrial function?

Summary answer: Senescence of EnSC contributes to impaired endometrial decidualization and impaired interaction with trophoblast cells but application of senomorphics diminishes the adverse effects of senescent EnSC on decidualization and implantation.

What is known already: A prolonged and highly disordered pro-inflammatory secretory profile of EnSC, which resembles the senescence-associated secretory phenotype, is associated with implantation failure. Furthermore, it has been suggested that implantation failure may be associated with increased EnSC senescence during the proliferative phase of the menstrual cycle.

Study design, size, duration: Primary EnSC cell cultures were isolated from endometrial biopsies taken from four patients without any endometrial complications planning to undergo IVF. EnSC senescence was induced by oxidative stress (1 h exposure to 200 µM H2O2) followed by 14 days culture but some results were confirmed in a replicative senescence model (after 25 passages). The decidual reaction was evaluated with routine methods and a genetic tool previously designed by us that estimates integral decidual response by fluorescence of a reporter protein. Time-course RNA-sequencing of control and senescent EnSC before and during decidualization was performed using four replicates for each state. To extend our findings, we applied several publicly available datasets. To model implantation in vitro, the choriocarcinoma cell line BeWo b30 was used. To reduce the senescent phenotype of EnSC, two classical senomorphics were applied-rapamycin and metformin.

Participants/materials, setting, methods: EnSC cultures were used to investigate the effects of senescence on decidualization and on an in vitro implantation model using spheroids derived from BeWo cells. Co-culture models (2D and 3D) were used to explore the effect of senescent cells on neighbouring control cells. The following methods were used to assess cell function, RNA-sequencing, bioinformatic analysis, CRISPR/Cas9 genome editing, FACS, western blotting, RT-PCR, immunofluorescence, molecular cloning, lentiviral transduction and ELISA.

Main results and the role of chance: Premature senescence of EnSC could be a cause of impaired decidualization. Hormone-induced decidual transformation of EnSC cultures was negatively affected by senescence. Bioinformatics revealed crucial disturbances in the decidual reaction of senescent EnSC which could affect embryo invasion, alter the 'meta-signature' of human endometrial receptivity, disturb the emergence of mature and senescent decidual cells subpopulations, impair ligand-receptor interaction with trophoblasts and modify the architecture of extracellular matrix. These predictions were functionally validated using an in vitro implantation model. Moreover, we observed that senescent EnSC, likely via the altered secretome, caused 'bystander' quenching of the decidual reaction in adjacent cells, reinforcing dysfunction of the stromal compartment. Application of senomorphics that reduced the senescence phenotype diminished adverse effects of senescent EnSC on decidualization and implantation.

Large scale data: The data used in this study are available in the GEO database (GEO identifier GSE160702).

Limitations, reasons for caution: The present study was based on in vitro cell cultures derived from only four women. Further studies involving patients with impaired implantation are needed to confirm our findings.

Wider implications of the findings: The presence of senescent EnSC within the stromal compartment of the endometrium may be a risk-factor for the failure of embryo implantation. Application of senomorphics during the proliferative phase of the menstrual cycle is a promising strategy to alleviate negative effects of senescent EnSC and to improve embryo implantation rates.

Study funding/competing interest(s): This study was funded by the Russian Science Foundation (# 19-74-10038). The authors do not have any competing interests to declare.

Keywords: decidualization; embryo implantation; endometrial stromal cells; senescence; senomorphics.