Saideh Mikaeili1, Batool Hossein Rashidi2, Majid Safa3,4, Atefeh Najafi1, Aligholi Sobhani1, Ebrahim Asadi1, Mehdi Abbasi5. 1. Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. 2. Vali-e-Asr Reproductive Health Research Centre, Tehran University of Medical Sciences, Tehran, Iran. 3. Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran. 4. Department of Hematology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran. 5. Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. abbasima@tums.ac.ir.
Abstract
PURPOSE: To determine the level of apoptosis, and alteration of FoxO3 (forkhead box O3 transcription factor) expression and phosphorylation in human granulosa cells amongst polycystic ovary syndrome (PCOS) patients and control group. METHODS: We recruited infertile women with PCOS (n = 14) and compared them with infertile women due to tubal blockage or male factor infertility (n = 14, controls). GnRH agonist and gonadotropins were used for ovarian stimulation. Follicular fluids from large follicles (>16 mm) were pooled and granulosa cells (GCs) were isolated using cell strainer methodology. Apoptosis of purified GCs was measured by flow cytometry using Annexin V and propidium iodide. Quantitative real-time PCR and western blotting were performed to assess alteration of FoxO3 expression and phosphorylation in GCs. RESULTS: There were higher percentages of early and late apoptosis in GCs of PCOS patients than in the control group. FoxO3 mRNA level and total FoxO3 protein were significantly higher in PCOS group than in the control group. The ratio of p-FoxO3/total FoxO3 decreased significantly in PCOS than in the control group. It was inferred that unphosphorylated (active form) FoxO3 was higher in GCs of PCOS patients. Apoptosis was significantly and positively correlated with the total FoxO3 and negatively correlated with the p-FoxO3 protein levels in PCOS patients. CONCLUSIONS: Activation and overexpression of FoxO3 in granulosa cells of PCOS women correlated with higher apoptosis levels in these cells suggesting that FoxO3 may be a candidate for the higher apoptosis in granulosa cells from women with PCOS.
PURPOSE: To determine the level of apoptosis, and alteration of FoxO3 (forkhead box O3 transcription factor) expression and phosphorylation in human granulosa cells amongst polycystic ovary syndrome (PCOS) patients and control group. METHODS: We recruited infertilewomen with PCOS (n = 14) and compared them with infertilewomen due to tubal blockage or male factor infertility (n = 14, controls). GnRH agonist and gonadotropins were used for ovarian stimulation. Follicular fluids from large follicles (>16 mm) were pooled and granulosa cells (GCs) were isolated using cell strainer methodology. Apoptosis of purified GCs was measured by flow cytometry using Annexin V and propidium iodide. Quantitative real-time PCR and western blotting were performed to assess alteration of FoxO3 expression and phosphorylation in GCs. RESULTS: There were higher percentages of early and late apoptosis in GCs of PCOSpatients than in the control group. FoxO3 mRNA level and total FoxO3 protein were significantly higher in PCOS group than in the control group. The ratio of p-FoxO3/total FoxO3 decreased significantly in PCOS than in the control group. It was inferred that unphosphorylated (active form) FoxO3 was higher in GCs of PCOSpatients. Apoptosis was significantly and positively correlated with the total FoxO3 and negatively correlated with the p-FoxO3 protein levels in PCOSpatients. CONCLUSIONS: Activation and overexpression of FoxO3 in granulosa cells of PCOSwomen correlated with higher apoptosis levels in these cells suggesting that FoxO3 may be a candidate for the higher apoptosis in granulosa cells from women with PCOS.