Abstract: Author(s) K.A. Zakuraeva¹, M.I. Yarmolinskaya¹, A.Yu. Vinokurov², M.Yu. Pogonyalova² zakuraevak@icloud.com, +7 960 374 12 22 Affiliation(s) ¹ D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology, Saint Petersburg, Russia ² I.S. Turgenev Orel State University, Orel, Russia Introduction / Rationale of the Study Currently, there is no unified approach or effective treatment for premature ovarian insufficiency (POI). The main strategy — hormone replacement therapy — is aimed at eliminating estrogen deficiency and its associated complications but does not restore lost ovarian function or fertility. Therefore, further study of the pathogenesis of POI is needed, along with the development of alternative pathogenetically grounded therapies. A promising direction is the evaluation of the effectiveness of various pharmacological agents for this condition within preclinical trials using cell-based models. Experimental modeling of POI, closely resembling the origin and mechanism of the human disease, can be effectively used to develop promising therapeutic approaches, particularly for the testing of new drugs. Materials and Methods A culture of rat granulosa cells (Wistar strain) after five passages was treated with cyclophosphamide to achieve a working concentration of 0.1 mg/mL in the growth medium, followed by incubation for 6 hours. Results Treatment with cyclophosphamide at 0.1 mg/mL resulted in changes in the morphology of the mitochondrial network (Figure 1), indicating the development of mitochondrial dysfunction. Figure 1. Results of statistical analysis of the fluorescence intensity of tetramethylrhodamine methyl ester. FCCP — carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Given that the formation of ΔΨm is ensured by the coordinated work of the mitochondrial respiratory chain complexes, the use of the F1-F0-ATP synthase inhibitor oligomycin A (2 µg/mL), the complex I inhibitor rotenone (2 µM), and the mitochondrial uncoupler FCCP (2 µM) makes it possible to identify potential causes of impairment. For model development, the optimal cyclophosphamide concentration and incubation duration were determined to induce the required level of mitochondrial functional changes. Incubation of cells with cyclophosphamide in all cases led to a decrease in ΔΨm, with the concentration of 0.1 mg/mL being consistently associated with complex I dysfunction (Figure 2). Figure 2. Experimental plots of tetramethylrhodamine methyl ester (TMRM) fluorescence intensity under the influence of oligomycin A, rotenone, and FCCP in control cells and cells treated with various cyclophosphamide concentrations. As shown by experiments with varying incubation durations, mitochondrial dysfunction develops as early as 2 hours after cyclophosphamide exposure; however, a 6-hour exposure is characterized by stable alterations in mitochondrial metabolism, manifesting as complex I dysfunction (Figure 3). Figure 3. Experimental plots of tetramethylrhodamine methyl ester (TMRM) fluorescence intensity under the influence of oligomycin A, rotenone, and FCCP in control cells and cells treated with cyclophosphamide for 2–8 hours. Conclusion The developed cell model demonstrates high reproducibility and allows, under controlled laboratory conditions, the study of mechanisms leading to POI, as well as the testing of potential therapeutic agents aimed at restoring mitochondrial function in granulosa cells. Keywords premature ovarian insufficiency; experimental model; cyclophosphamide.

Cite: Закураева К.А.
In vitro Model of Premature Ovarian Insufficiency Based on Cyclophosphamide-Induced Mitochondrial Dysfunction in Granulosa Cells
XXVI Всероссийский научно-образовательный форум «Мать и дитя», 24-26 сентября, Москва 24-26 Sep 2025