Progestins inhibit estradiol-induced vascular endothelial growth factor and stromal cell– derived factor 1 in human endometrial stromal cells  Hidetaka Okada, M.D., Rika Okamoto, M.T., Tomoko Tsuzuki, M.D., Shoko Tsuji, M.D., Katsuhiko Yasuda, M.D., Hideharu Kanzaki, M.D.  Fertility and Sterility  Volume 96, Issue 3, Pages 786-791 (September 2011) DOI: 10.1016/j.fertnstert.2011.06.048 Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Expression of vascular endothelial growth factor (VEGF) and stromal cell–derived factor 1 (SDF-1) mRNA in endometrial stromal cells (ESCs). ESCs were grown for 12 days with E2 (E; 10−8 mol/L) and/or medroxyprogesterone acetate (MPA; 10−7 mol/L). (A) Agarose gel electrophoresis of polymerase chain reaction (PCR) products, showing a pure single band in each amplication. Marker = molecular weight marker; RT(−) = reverse transcriptase (−) as negative control. (B) PRL, (C) VEGF, and (D) SDF-1 mRNA levels were analyzed by real-time PCR analysis and calculated after normalization to elongation factor 1α mRNA expression. Results are representative data of three experiments with different cell preparations, and each value represents mean and SEM in triplicate wells. Values significantly different versus control (∗P<.01; ∗∗P<.05). Fertility and Sterility 2011 96, 786-791DOI: (10.1016/j.fertnstert.2011.06.048) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effect of steroid hormone on VEGF and SDF-1 production in ESCs. ESCs were cultured for 12 days with either control, E2 (E; 10−8 mol/L), MPA (10−7 mol/L), E2 + MPA, or E2 + MPA + RU-486 (RU; 10−7 mol/L) with medium changes every 3 days. The ELISA measured (A) VEGF and (B) SDF-1 concentrations in the culture medium during the last 3 days of a 12-day culture period. Data are given for the four individual cell preparations with treatments. The effect of vehicle (control) was assigned a potency of 100% (C and D). Columns and vertical bars represent the mean and SEM for combined data of four separate experiments. Values significantly different versus control (∗P<.01). Abbreviations as in Figure 1. Fertility and Sterility 2011 96, 786-791DOI: (10.1016/j.fertnstert.2011.06.048) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Time course of (A) VEGF and (B) SDF-1 secretion from ESCs by steroid hormones. ESCs were cultured for 12 days with either control, E2 (E; 10−8 mol/L), or E2 + MPA (10−7 mol/L) with medium changes every 3 days. The ELISA measured VEGF and SDF-1 concentrations in the culture medium. Results are representative data of three experiments with different cell preparations, and each value represents the mean and SEM in triplicate wells. Abbreviations as in Figure 1. Fertility and Sterility 2011 96, 786-791DOI: (10.1016/j.fertnstert.2011.06.048) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effects of various progestins on E2-stimulated (A) VEGF and (B) SDF-1 secretion from ESCs. ESCs were cultured for 12 days in the presence of E2 (10−8 mol/L) with either vehicle, progesterone (P), MPA, norethisterone (NET), levonorgestrel (LNG), or dienogest (DNG) with medium changes every 3 days. The ELISA measured VEGF and SDF-1 concentrations in the culture medium during the last 3 days of a 12-day culture period. The effect of E2 + vehicle (control) was assigned a potency of 100%. Columns and vertical bars represent the mean and SEM for combined data of four separate experiments. Values significantly different versus control (∗P<.01; ∗∗P<.05). Abbreviations as in Figure 1. Fertility and Sterility 2011 96, 786-791DOI: (10.1016/j.fertnstert.2011.06.048) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions