P-282 Decreased 14,15-EET in follicle induced ovary granulosa cells senescence and contributes to endometriosis-associated infertility
Authors: Lin X , Yongdong D , Songying Z 
Published: July 2024

Abstract

Study question
What is the expression profile of lipid peroxides in endometriosis (EM) follicle? What is the function of differentially expressed lipid peroxides in endometriosis-associated infertility?

Summary answer
14,15-EET concentration is decreased in EM granulosa cells (GCs) and follicular fluid (FF), decreased 14,15-EET in EM induced GCs senescence by activating PI3K-AKT-mTOR signaling pathway.

What is known already
Oxidative stress (OS) induced EM GCs senescence and intensified peroxidation in the follicle compromise the development of oocyte. Lipid peroxidation products generated in follicle cyclically and can be used as OS markers to evaluate Assisted Reproductive Technology (ART) outcomes. Epoxyeicosatrienoic acids (EETs) is one of the mostly studied lipid peroxides that derived from arachidonic acid (AA) and metabolized to less potent DHETs by soluble epoxide hydrolase (EPHX2). 14,15-EET was considered to be a active molecule that exert anti-reactive oxygen species, anti-inflammatory, anti-migratory, and pro-fibrinolytic effects in cardiovascular disease, kidney injury and metabolic syndrome. But no reports were found in endometriosis.

Study design, size, duration
This study was started on January 1, 2022. We enrolled 101 infertility patients who underwent IVF-ET at our reproductive center to explore the profile of lipid peroxides in follicle. The mechanism of decreased EETs in endometriosis follicle was studied on human GCs and mouse GCs via quantitative reverse transcriptase PCR, western blot, enzyme-linked immunosorbent assay, liquid chromatography-tandem mass spectrometry (LC-MS/MS). Endometriosis mouse model and mouse GCs were used to verify the anti-senescence role of 14,15-EET.

Participants/materials, setting, methods
141 lipid peroxides were measured in FF from 56 EM patients and 45 infertility women with salpingectomy (Control) by LC-MS/MS. Linear regression analysis was conduct to explore the correlations between EETs and ART outcomes. TPPU, an EPHX2 enzyme inhibitor, was used in vitro and in vivo to explore the function of 14, 15-EET in GCs. RNA-Sequencing was performed to find the mechanism by which reduced 14,15-EET leads to abnormal folliculogenesis.

Main results and the role of chance
LC-MS/MS results showed that 15 lipid peroxides differentially expressed in EM-FF, and all of them were downregulated in EM-FF when compared with control FF. The levels of 4 different regioisomers of EETs (5,6-EET, 8,9-EET, 11,12-EET and 14,15-EET) in EM patients were significant lower than that in control women (P &lt; 0.05). Linear regression analysis showed positive correlations between EETs scores and ART outcomes. Decreased EETs in endometriosis follicular fluid indicates lower numbers of retrieved oocytes, mature oocytes, total embryos and high-quality embryos. 14,15-EET addition alleviated OS-induced GCs senescence as well as mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) reduction in GCs. Moreover, TPPU administration lead to increased 14,15-EET expression and rescued cellular senescence, MMP and ATP abnormities of endometriosis GCs in vitro and in vivo. RNA-Sequencing results revealed significantly suppression of PI3K-AKT-mTOR signaling pathway in GCs after TPPU administration. Western blot results indicated that 14,15-EET alleviate GCs senescence via inhibiting PI3K-AKT-mTOR signaling pathway.

Limitations, reasons for caution
14,15-EET is just one of the lipid peroxides in FF and GCs. A variety of lipid peroxides expressed in follicle, and more research were needed to explore the roles of them.

Wider implications of the findings
This research makes the supplement of EETs during IVF-ET become meaningful and TPPU may represent a novel adjuvant therapy strategy for endometriosis-associated infertility.

Trial registration number
Not applied