EET Reduction Contributes to Granulosa Cell Senescence And Endometriosis-Associated Infertility Via the PI3K/AKT/mTOR Signaling Pathway

Oxidative stress is involved in endometriosis (EM)-related infertility via various mechanisms, such as accelerating ovarian granulosa cell (GC) senescence, inhibiting ovulation, and interfering with lipid metabolism. In this study, we aimed to examine abnormal oxidative lipid levels and their related mechanisms in EM-associated infertility. Through liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis, we observed that epoxyeicosatrienoic acids (EETs), which have antioxidant and anti-senescence effects, had decreased levels in follicular fluid samples from EM patients. The EET levels were positively correlated with in vitro fertilization outcomes. Using qPCR and western blot experiments, we found elevated soluble epoxide hydrolase (EPHX2) expression levels in EM-GCs. This enzyme metabolizes EETs into dihydroxyeicosatrienoic acids (DHETs), explaining the decreased 14, 15-EET levels in EM patients. Lower 14, 15-EET concentrations led to decreased GC antioxidant capacity, reduced ATP production, reactive oxygen species (ROS) accumulation in oocytes, and abnormal cumulus-oocyte complex (COC) expansion, which ultimately resulted in decreased fertility. Inhibiting EPHX2 in vivo or in vitro could reverse these observed abnormalities by upregulating 14, 15-EET. Mechanistic studies showed that 14, 15-EET alleviated GC senescence and improved fertility by inhibiting excessive PI3K/AKT/mTOR signaling pathway activation in EM-GCs. Moreover, the PI3K/AKT/mTOR pathway inhibitor BEZ-235 significantly alleviated ROS-induced cell senescence and abnormal COC expansion. Further work showed that an oxidative stress-induced decrease in EZH2/H3K27Me3 histone methylation led to the elevated EPHX2 expression patterns in EM-GCs. In addition, the decreased 14, 15-EET levels led to ROS accumulation, decreased EZH2 enzymatic activity, less EPHX2/H3K27Me3 histone methylation, and increased EPHX2 protein expression levels, which further reduced 14, 15-EET levels as part of a vicious feedback loop. Using LC-MS/MS, viral infection, transcriptomic sequencing, knockout mice, and EM mouse model experiments, we elucidated the molecular mechanisms of 14, 15-EET in EM-associated infertility and the mechanism controlling its decreased levels.