Fig 1: EMs combined with iron overload modeling affected mouse pregnancy.A The average body weight of the pregnant mice was calculated from control (CON, n = 10), EMs model (EMs, n = 10), EMs combined with iron overload model (IRON, n = 10), DFO treatment after IRON model (IRON + DFO, n = 10), and VITE treatment after IRON model (IRON + VITE, n = 10). B Representative photographs of the uterus after pregnancy in each of the five groups of female mice. C, D Total litter size and specific number of litters per mouse in five groups (n = 10). E For Prussian blue, H&E, and GPX4 immunohistochemical staining, ovarian tissues were observed from the five groups of mice. Scale bar = 50 µm; Scale bar = 200 µm; Scale bar = 20 µm. F Western blot analysis of TP53 expression in the ovarian tissues of mice in each group. Expression of GAPDH was used as an internal control.
Fig 2: Iron-overloaded EMFF induced ferritinophagy-dependent ferroptosis in granulosa cells.A–C Levels of total iron, hepcidin, and transferrin in EMFF (n = 15) and COFF (n = 15). Data are expressed as means ± SD and analyzed by Student’s t test. D Results of mouse granulosa cells proliferation under different intervention conditions (each group in the figure is compared with COFF group). DFO, iron chelators; FER, ferroptosis inhibitor; NEC, necrosis inhibitor; ZDF, apoptosis inhibitor; ME, autophagy inhibitor. Data are expressed as means ± SD and analyzed by one-way ANOVA. E–H Comparison of ferritinophagy-related proteins FTH1, NCOA4, and ATG5 between human granulosa cells of infertile patients with EMs (EMGC) and of control group (COGC). The expression of ß-actin was used as an internal control. Data are expressed as means ± SD and analyzed by Student’s t test. I–L Detection of ferroptosis-related indicators iron, GSH, GPX4, and MDA in COGC and EMGC. Data are expressed as means ± SD and analyzed by Student’s t test. M Representative images of the mitochondrial morphology of mouse granulosa cells intervened by COFF and EMFF were observed under TEM. Yellow arrows indicate mitochondrion. Scale bar = 1.0 µm. Scale bar = 5.0 µm. N Representative images of ROS and ferrous ion fluorescence staining after COFF and EMFF intervention in mouse granulosa cells. Scale bar = 100 µm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns, no significance.
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