Ferroptosis-associated oxidative stress in cumulus cells and mitochondrial alterations in oocytes of women with diminished ovarian reserve

Ferroptosis is a regulated form of cell death driven by iron-dependent lipid peroxidation and oxidative stress and has been implicated in ovarian dysfunction. However, its role in the follicular microenvironment of the diminished ovarian reserve (DOR) remains incompletely understood. This study aimed to investigate ferroptosis-associated oxidative stress in cumulus cells and mitochondrial membrane potential-related fluorescence in oocytes from women with DOR. A total of 81 women undergoing intracytoplasmic sperm injection were enrolled, including 46 with DOR and 35 with normal ovarian reserve. The expression of ferroptosis-associated genes (GPX4 and EMP1) and Hippo signalling pathway genes (MST1, LATS2 and YAP1) was assessed in cumulus cells using qRT-PCR. Follicular fluid reactive oxygen species (ROS) and ferritin levels were measured, intracellular ROS levels in cumulus cells were evaluated and mitochondrial membrane potential-related fluorescence in germinal vesicle-stage (GV) oocytes was assessed using MitoTracker™ fluorescence. Control and DOR group cumulus cells were subjected to immunofluorescence staining for YAP, p-YAP, p-LATS1/2, and p-MST1. Nuclei were stained with DAPI. Fluorescence images were captured and quantified to determine protein expression levels and nuclear localization. The DOR group showed significantly lower AMH levels and reduced oocyte yield compared with controls. GPX4 and EMP1 expression levels were significantly increased in cumulus cells from DOR patients, accompanied by elevated follicular fluid ROS concentrations, whereas ferritin levels were unchanged. Mito Tracker fluorescence intensity in GV oocytes was reduced in the DOR group. Intracellular ROS levels in cumulus cells showed a slight, non-significant increase in the DOR group compared with controls. In contrast, the expression of Hippo pathway genes in cumulus cells did not differ between the groups, either in vivo or following ferroptosis modulation in vitro. The cumulus cells from DOR group significantly increased the nuclear p-YAP/YAP ratio ( p < 0.01) and p-LATS1/2 expression compared with the control group ( p < 0.05). p-YAP expression showed a slight increase, while p-MST1 expression showed a decreasing trend. Collectively, these findings indicate an altered oxidative stress environment within the follicular microenvironment of DOR. While Hippo pathway activity did not appear to be altered at the transcriptional level, increased ROS levels together with changes in ferroptosis-related gene expression suggest molecular changes compatible with oxidative stress-associated molecular alterations. These findings warrant further investigation into the potential involvement of ferroptosis-related pathways. Furthermore, the observed changes in YAP phosphorylation and LATS1/2 activation indicate that DOR may modulate Hippo/YAP signaling at the post-translational level.