Iron-overloaded follicular fluid increases the risk of endometriosis-related infertility by triggering granulosa cell ferroptosis and oocyte dysmaturity [miRNA-seq]

To explore the differences of miRNAs from exosomes of granulosa cell in different groups, we extracted exosomes from mouse granulosa cells intervened by different reagents. Mouse granulosa cells were acclimated in DMEM / F12 without exosomes for 24 h, and then subjected to different interventions. No special treatment was provided to the control group (CON). The ferric citrate (FAC) group was treated with 100 μM ferric citrate. The deferoxamine mesylate (DFO) group was treated with ferric citrate (100 μM) and deferoxamine mesylate (100 μM). VE group was treated with ferric citrate (100 μM) and VITE (200 μM). All groups were cultured for 48 h. After termination of incubation, centrifugation was carried out in steps at 4 °C and 300 ×g for 10 min to remove cells and 3 000 ×g for 10 min to remove large cell debris. After centrifugation at 10 000 ×g for 40 min, the supernatant was filtered through a 0.22 μM PVDF filter (Millipore, USA). Finally, centrifugation at 100 000 ×g for 90 min, the supernatant was removed and resuspended in 200 μL of PBS to obtain the cell supernatant exosomes precipitation.The integrity was confirmed using Agilent 2100 TapeStation (Agilent). Small RNA sequencing was conducted using Illumina HiSeq 2500 with read lengths of 15–41 bp.