Activated glucocorticoid and eicosanoid pathways in endometriosis Diana Monsivais, M.S., Jeffrey D. Bray, Ph.D., Emily Su, M.D., Mary Ellen Pavone, M.D., Matthew T. Dyson, Ph.D., Antonia Navarro, B.S., Toshiyuki Kakinuma, M.D., Ph.D., Serdar E. Bulun, M.D.  Fertility and Sterility  Volume 98, Issue 1, Pages 117-125 (July 2012) DOI: 10.1016/j.fertnstert.2012.03.030 Copyright © 2012 Terms and Conditions

Figure 1 (A) Hierarchical clustering demonstrates a unique gene expression profile for ovarian endometriosis when compared with matched eutopic tissues. Fold change values of twofold or greater for significantly regulated genes were subjected to hierarchical clustering. Red represents the up-regulation of a gene, whereas green denotes a decrease relative to the mean intensity value for each probe. Each row represents a single probe set, and the columns represent the individual tissue samples. (B) Numbers in the overlapping regions indicate similarly regulated genes according to three microarray studies of ovarian endometriosis: the present study, Hever et al. (15), and Eyster et al. (16). The selected genes used to construct the Venn diagram from each study had a twofold or greater change and P<.05 for endometriosis compared with the eutopic endometrium. (C–H) Genes in the prostaglandin pathway are altered in endometriosis (E-OSIS) compared with the eutopic endometrium (E-IUM). Ribonucleic acid was isolated from six matched endometriotic and eutopic tissues and subjected to real-time PCR as described in Materials and Methods. (C, D) Phospholipase enzymes: (C) PLA2G2; (D) PLA2G5; (E) prostacyclin synthase (PTGIS); (F) HPGD; (G, H) PGE2 receptors: (G) PTGER1, (H) PTGER3. Data are represented as fold change ± SEM. Fertility and Sterility 2012 98, 117-125DOI: (10.1016/j.fertnstert.2012.03.030) Copyright © 2012 Terms and Conditions

Figure 2 Genes involved in cortisol synthesis and action are altered in endometriosis. (A–D) Real-time PCR validation of gene expression in stromal cells from six eutopic (E-IUM) and six endometriosis (E-OSIS) samples. (A) HSD11B1 was significantly up-regulated by 7.4-fold and (B) HSD11B2 was significantly down-regulated by 0.46-fold in E-OSIS relative to E-IUM; (C) GR was increased by 2.4-fold in E-OSIS relative to E-IUM; (D) MR was increased but not significantly. (E) Protein expression for HSD11B1, HSD11B2, GR, and β-actin in stromal cells from four normal patients (samples 1–4) and five endometriosis patients (samples 5–9). (F, G) Densitometric quantification of (F) HSD11B1 and (G) GR protein levels in E-IUM vs. E-OSIS. Data are presented as fold change ± SEM. Fertility and Sterility 2012 98, 117-125DOI: (10.1016/j.fertnstert.2012.03.030) Copyright © 2012 Terms and Conditions

Figure 3 Proinflammatory cytokine TNF increases HSD11B1 and GR expression in endometriotic stromal cells. (A–D) Endometriotic stromal cells were cultured and treated for 24 hours with TNF (10 ng/mL). Gene and protein expression analysis was conducted for HSD11B1, HSD11B2, GR, and MR. (A) HSD11B1 gene expression was increased by 4.9-fold; (B) HSD11B2 was significantly decreased by 0.68-fold after exposure to TNF; (C) GR is significantly increased by threefold after treatment; (D) MR gene expression was unchanged after TNF treatment. (E) Protein levels in three endometriosis patients treated with or without TNF for 24 hours. (F, G) Densitometric quantification of (F) HSD11B1 and (G) GR protein shows that HSD11B1 and GR protein levels increase significantly after TNF treatment by 2.5-fold and 1.5-fold, respectively. HSD11B2 was unchanged (densitometric analysis not shown), and β-actin was used as a loading control. Data are presented as fold change ± SEM. Fertility and Sterility 2012 98, 117-125DOI: (10.1016/j.fertnstert.2012.03.030) Copyright © 2012 Terms and Conditions