P28-10 Bisphenol Z dysregulates metabolic substrate utilization in human ovarian granulosa cells

Bisphenol Z, a commonly used bisphenol A analogue, has risen concerns due to its potential effects on reproductive health. Although BPZ is thought to be safe, it has been detected in environmental media such as indoor dust and thermal receipt paper, as well as human biological samples 1 , 2 , 3 . Reports are emerging that suggest negative impact of BPZ on follicullogenesis, steroidogenesis and its association with increased risk of PCOS [4] . Ovarian granulosa cells (GCs) are critical for mentioned ovarian functions as they regulate steroid hormone biosynthesis, oocyte maturation and follicle growth [5] . Maintaining those functions requires ongoing metabolic adaption. The interconnected metabolic processes of glycolysis, oxidative phosphorylation, and the TCA cycle – which is powered by glucose, fatty acids, and glutamine – typically fuel the bioenergetic demands [6] . Here, we investigated how BPZ at 10 nM, dose reflecting environmental exposure, influences each metabolic substrate utilization in human ovarian GCs. To investigate the effects of BPZ we used human ovarian GCs line (HGrCl) and GCs isolated from follicular fluid from women undergoing in vitro fertilization (IVF). The study included 80 women undergoing IVF and was approved by The Ethical Committee of the Jagiellonian University (1072.6120.30.2023). The cells were treated with 10nM of BPZ for 24h prior to real-time PCR analysis and for 6h prior to real-time MIto Fuel Flex test performed using Seahorse XF analyzer (Agilent), and glucose (Promega)/fatty acids (Abcam) uptake tests. First, after BPZ treatment, we measured cell reliance on a particular fuel pathway to maintain baseline respiration, where we observed statistically significant increase in dependency of GCs on fatty acids and lowered dependency on glutamine. Then, we established that the ability of a cell's mitochondria to oxidize fatty acids when other fuel pathways are inhibited was lowered post BPZ stimulation, as well as the ability of cells to increase oxidation of fatty acids and glucose to compensate for inhibition of alternative fuel pathways. Those changes where not accompanied with disruption of gene expression of each substrate transporters, nor substrate uptake into the cell. To conclude, the results suggest that BPZ does not disrupt particular substrate bioavailability but strictly affect metabolic substrate utilization. Observed increased dependency on fatty acid oxidation post BPZ treatment is particularly important for GCs as those are highly steroido-genic cells and any disturbances in lipid metabolism can be detrimental for sustaining cell homeostasis. Such changes can further influence steroidogenesis, proliferation and/or apoptosis of GCs disrupting overall ovarian health.