Mitochondrial Function Under Combined Oral Contraceptive Exposure in a Neuroblastoma Model: A Preliminary Investigation

Background: Endogenous estradiol/progesterone (E2/P4) shape neuronal mitochondrial bioenergetics and redox. The mechanisms by which combined oral contraceptive (COC) steroids operate within physiologically relevant hormonal milieus remain poorly defined. Methods: Human SH-SY5Y cells (Cytion) were starved 48 h in Cytion medium without FBS and with 1% ITS, then exposed for 48 h to six conditions: vehicle (F0); follicular-like E2/P4 (F1); luteal-like E2/P4 (F2); F1 + dienogest/ethinylestradiol (DNG/EE; F3); F2 + DNG/EE (F4); and DNG/EE alone (F5). Primary endpoints were mitochondrial membrane potential (JC-1 red/green ratio) and ROS (H₂DCFDA/DCF); nitric oxide (DAF-FM) was also recorded. Hoechst 33342 nuclear fluorescence served both as a per-well proxy of cell number and as a proportionality factor for normalization. Results: Follicular- and luteal-like backgrounds produced distinct ΔΨm/ROS set-points. Superimposing DNG/EE generated background-dependent shifts marked by higher DCF and lower JC-1 ratios versus vehicle; DAF-FM indicated concordant NO changes. Hoechst-based normalization preserved these patterns, indicating per-cell functional modulation rather than cell-number artifacts; proliferation differences were modest and background-dependent. Conclusions: Neuroendocrine background matters: physiologic E2/P4 milieus prime mitochondrial ΔΨm/ROS/NO states in SH-SY5Y, onto which COC components impose con-text-dependent modulation at 48 h. These preliminary findings provide a standardized framework for subsequent image-based mitochondrial phenotyping.