Oxidative stress and metformin: An in-vitro study on serum and primary human granulosa cell cultures

Abstract

Objective: To observe the effect of metformin on the oxidative stress (OS) levels and SIRT1 expression in primary human granulosa cell cultures from infertile females. Methods: This cross-sectional study was conducted from August 2017-July 2019 on ten infertile patients. After written informed consent, the follicular fluid of these subjects was collected for establishing primary human granulosa cell cultures (HGCs), to test the capacity of metformin to reduce OS. The cultured HGCs were divided into (i) control: HGCs without any treatment, (ii) test1: HGCs induced with H2O2 (metformin absent), and (iii) test2: HGCs treated with metformin after H2O2 induction. Baseline OS (control group) was estimated, it was induced and measured by an increase in optical density (OD) (test1 & test 2). Finally, test2 was incubated with metformin (1 ml of 100 mmol/l concentration) and it effect on the levels of OS were determined by Mishra OS assay. Effect of metformin modulating SIRT1 expression in OS-induced cells was analyzed using quantitative-PCR. Results: Age of female subjects was 32.04±2.29years and BMI was 27.61±2.15 kg/m2. Test (OS induced) samples gave an OD of 0.28 (0.16-0.40) while control HGC samples gave an OD; 0.153 (0.09-0.23). Test cells showed significant reduction in ODs after metformin treatment. The relative expression of SIRT1 in metformin untreated and treated cells was found to be 61.5% and 80%, respectively. Conclusion: Metformin was found to suppress OS in HGCs and increases the expression of SIRT1 in OS induced environment of primary cell cultures, suggesting a relationship between metformin, SIRT1 expression, and reduction of OS.