(fatemeh.masjedikand@gmail.com & masjedi_f@sums.ac.ir) Dual Effect of Vitamin D on Apoptosis Frequency and Its Correlation with Reactive Oxygen Species Production in Human Granulosa Cells of Normal and Polycystic Ovaries Dr. Fatemeh Masjedi1, Dr. Sara Keshtgar1 1- Department of Physiology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran (fatemeh.masjedikand@gmail.com & masjedi_f@sums.ac.ir) Objectives Results Despite the increasing number of growing follicles in PCOS patients, pathways and frequency of apoptosis in cystic follicles is controversial. Vitamin D directly or indirectly influence on genes involved in cell cycling, proliferation, and apoptosis. Therefore, we investigated apoptosis frequency in normal and cystic granulosa cells (GCs) in a basal state and under vitamin D treatment and its correlation with the amount of ROS production. The basal ROS generation by polycystic ovarian GCs was markedly greater than normal ones (P<0.0001). Vitamin D significantly reduced ROS production in both groups. However, the ROS generation by treated GCs of patients was still significantly higher than treated normal cells (Fig. A). The percent of necrotic, early and late apoptotic, and live cells were similar in normal and PCOS groups. The frequency of the apoptotic cells (MFI) significantly (P<0.03) decreased in vitamin D treated normal GCs, whereas it increased in treated cells of PCOS patients (P<0.007) (Table). There were significant direct correlations between RLU and rate of cell apoptosis (MFI) in normal women (P=0.03) and PCOS patients (P=0.0003). Although ROS levels in PCOS group was higher than the controls, there was no significant difference between apoptotic rate of GCs of normal and PCOS women (Fig. B). Materials & Methods Geranulosa cells were obtained from 20 women with PCOS and 20 healthy controls. Ovarian GCs were cultured in presence or absence of vitamin D (100 nM), for 48 hours. ROS production (RLU) was measured by chemiluminescence assay. The apoptotic cells were identified by Annexin-V/Propidium iodide detection kit and mean Annexin fluorescent intensity (MFI) was calculated. The comparisons were undertaken using independent t-test and Pearson correlation coefficient. All statistical analysis performed using GraphPad Prism software v. 6.0. Flowcytometric analysis of apoptosis and viability of GCs from normal and PCOS groups.   Groups (n = 8) untreated normal treated normal P-value untreated PCOS treated PCOS necrotic GCs (Annexin– / PI+) 2.02±0.79 1.07±0.25 N.S 0.96±0.17 0.58±0.11 0.023 late apoptotic GCs (Annexin+ / PI+) 19.87±2.49 14.44±2.23 0.031 23.86±2.01 22.00±2.14 early apoptotic GCs (Annexin+ / PI–) 35.23±3.39 29.20±4.04 35.93±1.04 38.93±1.58* 0.015 viable GCs (Annexin– / PI–) 42.87±2.38 55.25±4.74 39.24±2.36 38.50±2.52* MFI 689.7 527.5 768.4 864.0* 0.007 Data are presented as mean ± SEM. *P< .05 represent significant differences between vit.D-treated normal and PCOS groups. MFI = Mean Fluorescent Intensity, GCs = granulosa cells, vit.D = vitamin D. Conclusion Dysregulation of apoptosis-related genes may result in an attenuated atresia, consequently polycystic ovaries. Vitamin D reduced ROS level in both groups, whereas this vitamin accelerated apoptosis in GCs of PCOS group and maintained cell viability in normal cells. It can be assumed that this vitamin affects gene expression of apoptotic and anti-apoptotic factors. However, future researches are required to elucidate the exact mechanism of vitamin D action. References: Das M, Djahanbakhch O, Hacihanefioglu B, Saridogan E, Ikram M, Ghali L, et al. Granulosa cell survival and proliferation are altered in polycystic ovary syndrome. J Clin Endocrinol Metab 2008;93(3):881–7. Murri M, Luque-Ramirez M, Insenser M, Ojeda-Ojeda M, Escobar-Morreale HF. Circulating markers of oxidative stress and polycystic ovary syndrome (PCOS): a systematic review and meta-analysis. Hum Reprod Update 2013;19(3):268–88. Zuo T, Zhu M, Xu W. Roles of Oxidative Stress in Polycystic Ovary Syndrome and Cancers. Oxid Med Cell Longev 2016;2016:8589318. PCOS, Apoptosis, ROS, Geranulosa cells