Thrombin inhibits GnRH receptor (GnRHR) gene expression in first trimester decidual stromal cells

TRIMESTER DECIDUAL STROMAL CELLS ERROL NORWITZ, FREDERICK SCHATZ, HEE JOONG LEE, VICTORIA SNEGOVSKIKH, MICHAEL PAIDAS, CATALIN BUHIMSCHI, IRINA BUHIMSCHI, CHARLES LOCKWOOD, JOSEPH HUANG, Yale University, Ob/Gyn, New Haven, Connecticut OBJECTIVE: Hypothalamic GnRH (GnRH-I) is critical in regulating mammalian reproduction. GnRH-I, a second isoform (GnRH-II), and their cognate receptor (GnRHR) are produced also by extra-hypothalamic tissues (placenta and decidua) and have local autocrine/paracrine functions. Decidual GnRH appears to regulate trophoblast invasion. Decidual hemorrhage (abrupion) is associated with adverse pregnancy outcome. This study investigates the effects of thrombin on GnRH-I, GnRH-II, and GnRHR gene expression in first trimester decidual stromal cells (DSCs). STUDY DESIGN: First trimester decidua was identified from pregnancy termination tissues (n=3). DSCs were isolated by enzymic digestion, purified, and depleted of leukocytes. Cells were pretreated with estradiol (10-8 M [E2]) and medroxyprogesterone acetate (10-7 M [MPA]) for 7 days. After 6h incubation with or without thrombin (2.5 U/mL), RNA was extracted, cDNA synthesized and purified, and cRNAs prepared and hybridized on Affymetrix HG_U133 Plus 2.0 human chips (which screens for 47,400 human genes and ESTs) followed by fluorescence labeling and optical scanning. Raw data of 3 separate experiments without normalization were generated using Affymetrix GeneChip Operating Software v. 1.1.1 (Santa Clara, CA) and analyzed by GeneSpring software 6.1 (Silicon Genetics, Redwood City, CA). Gene readouts were normalized to 50th percentile of the distribution of all measurements in each chip. Fold ratios were derived by comparing normalized data between different groups. Data are expressed as mean (SEM). RESULTS: DSCs expressed mRNA for GnRH-I, GnRH-II, and GnRHR. Thrombin did not alter GnRH-I or -II mRNA, but significantly downregulated GnRHR gene expression by 5.5-fold (P!0.01) (Figure).