No abstract

Endometriosis (EMs) occurs in approximately 50% of women with infertility. The main causes of EMs-related infertility are follicle dysplasia and reduced oocyte quality. Iron overload occurs in ovarian follicular fluid (FF) of patients with EMs, and this condition is associated with oocyte maturation disorder. However, the underlying molecular mechanism remains largely unknown. In the present study, we identified the mechanism underlying ferroptosis in ovarian granulosa cells and oocyte maturation failure in EMs based on a retrospective review of in vitro fertilization/intracytoplasmic sperm injection-frozen embryo transfer outcomes in infertile patients with EMs. Mouse granulosa cells were treated with EMs-related infertile patients' follicular fluid (EMFF) in vitro. Western blot analysis, quantitative polymerase chain reaction, fluorescence staining, and transmission electron microscopy were used to assess granulosa cells ferroptosis. The effects of exosomes were examined by nanoparticle tracking analysis, RNA-seq, and Western blot analysis. Finally, the therapeutic values of vitamin E and iron chelator (deferoxamine mesylate) in vivo were evaluated in an EMs-related infertility model. Patients with ovarian EMs experienced poorer oocyte fertility than patients with non-ovarian EMs. We observed that EMFF with iron overload-induced granulosa cell ferroptosis in vitro and in vivo. Mechanically, nuclear receptor coactivator four-dependent ferritinophagy was involved in this process. Notably, granulosa cells undergoing ferroptosis further suppressed oocyte maturation by releasing exosomes from granulosa cells. In therapeutic studies, vitamin E and iron chelators effectively alleviated EMs-related infertility models. Our study indicates a novel mechanism through which EMFF with iron overload induces ferroptosis of granulosa cells and oocyte dysmaturity in EMs-related infertility, providing a potential therapeutic strategy for EMs-related infertility.

H NMR spectra were recorded on a Varian 300 MHz NMR at ambient temperature. Chemical shifts (ppm) were referenced to tetramethylsilane or residual protonated solvent. UV measurements were made using a Jasco V-530 spectrometer. Solvents were purchased from Sigma-Aldrich, Fisher, VWR, and were purified and dried by passing through a PURE SOLV® solvent purification system (Innovative Technology). Deuterated solvents were purchased from Cambridge Isotope Laboratories. All other reagents were purchased from Sigma-Aldrich and were used as received. Surface area and nitrogen adsorption isotherms were calculated using the Brunauer-Emmett-Teller (BET) model. Gas adsorption studies were carried out using Quantachrome Autosorb Automated Gas Sorption System. 1.1 Jobʼs plot of EA9A and MAA Continuous variation analysis (Jobʼs plot) at 8.5 mM in CD 3 CN showed MAA to EA9A stoichiometry between 2:1 and 1:1. To determine the stoichiometry, data points on each side of the maximum chemical shift was fitted to a straight line. The x-value at cross point was analytically determined from the equations from the fitted lines. Measurement of Jobʼs plot was not possible at concentration higher than 8.5 mM due to the solubility limit of EA9A in acetonitrile. Although carboxylic acids such as MAA can form higher order (2:1 and 3:1) complex with adenine guests such as ethyl adenine-9-acetate (EA9A), the first order complex will dominate in CD 3 CN because chances are slim to form higher order complexes based on the probability of placing two or three functional monomer around the template molecule.