OR09-06 The Cholesterol Oxidation Product 7-Ketosterol Reduces Progesterone Production By Human Cytotrophoblast Cells In A Sex Dimorphic Manner

Disclosure: E. Ticiani: None. N. Pascuali: None. P. Danos: None. C. Murga-Zamalloa: None. I. Buhimschi: None. A. Veiga-Lopez: None. Many pregnancy complications including recurrent miscarriage and preterm birth are strongly associated with placental dysfunction and heightened inflammation. Diet-derived cholesterol, especially via consumption of fast foods that have undergone thermal processing or long-term storage, is highly susceptible to oxidation which results in cholesterol oxidation products, known as oxysterols. Oxysterols are known drivers of inflammation and have been implicated in chronic diseases, including atherosclerosis and neurodegeneration. We hypothesized that the prototype oxysterol 7-ketosterol (7-KT) induces reduced steroid synthesis and/or secretion in human placental cells associated with a pro-inflammatory response. To test this hypothesis, we isolated human primary cytotrophoblast cells (hCTB) from healthy term pregnancies with singleton female fetuses (n=4) and exposed them to 7-KT (2.5 µg/ml) or vehicle control (C; 0.1% DMSO) for 96h. Cells were subjected to RNA sequencing using Illumina TruSeq (p<0.05, FDR <0.05 and log2 fold change >1). Pathway analysis discovered that 10 of the most down-regulated genes (CYP51A1, MSMO1, IDI1, HMGCS1, HMGCR, ACAT2, DDX31, DHCR7, SREBF2, NLRP7) were associated with lipid metabolism. Among them, sterol regulatory element binding transcription factor 2 (SREBF2), is a known activator of cholesterol synthesis. Using female (n=4) and male hCTB (n=4), we further evaluated sex specific effects of 7-KT after 96h of exposure using RT-qPCR. We confirmed that 7-KT reduced the expression of SREBF2 in female and in male hCTB, as well as of genes responsible for cholesterol influx (LDLR) and synthesis (HMGCR and MVK), but not of the cholesterol efflux gene ABCA1. Similar gene expression patterns were observed in JEG-3 cells exposed to 7-KT, a choriocarcinoma cell line that retains the ability to synthetize progesterone. This was coupled with a ∼50% reduction in intracellular cholesterol in JEG-3 cells exposed to 7-KT. Considering that cholesterol is the precursor for progesterone, we assessed progesterone secretion in JEG-3 and in male and female-derived hCTBs cells by ELISA. 7-KT reduced progesterone secretion by JEG-3 and female hCTB by ∼40% while male hCTB were not affected. Additionally, a short exposure (24h) to 7-KT increased the expression of pro-inflammatory genes (IL1α, IL6 and TNFα) in female, but not male hCTB. Altogether, our results demonstrate that the oxysterol 7-KT induces early pro-inflammatory gene expression, followed by a reduction in cholesterol metabolism, lower intracellular cholesterol, and progesterone secretion. Future studies should further investigate the mechanism by which 7-KT drives sex-specific changes in placental cells, and the impact of excess oxysterols on pregnancy outcomes. Presentation: Friday, June 16, 2023