Role of GnRH–GnRH receptor signaling at the maternal-fetal interface Hee Joong Lee, M.D., Ph.D., Victoria V. Snegovskikh, M.D., Joong Shin Park, M.D., Ph.D., Nastaran Foyouzi, M.D., Ku Taek Han, M.D., Ph.D., Eric J. Hodgson, M.D., Seth Guller, Ph.D., Errol R. Norwitz, M.D., Ph.D.  Fertility and Sterility  Volume 94, Issue 7, Pages 2680-2687 (December 2010) DOI: 10.1016/j.fertnstert.2010.03.016 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Effect of GnRH-I and GnRH-II on hCG production by human first trimester cytotrophoblast-derived HTR-8/SV neo cells. HTR-8/SV neo cells were cultured to confluence and stimulated with GnRH-I (1–1,000 nmol/L) or GnRH-II (1–1,000 nmol/L) for 24 hours. hCG was measured in the conditioned supernatant by specific ELISA. Results are presented as mean ± SEM from four separate experiments performed in triplicate. Statistically significant differences are indicated. Fertility and Sterility 2010 94, 2680-2687DOI: (10.1016/j.fertnstert.2010.03.016) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effect of GnRH-I and GnRH-II on the production of select cytokines and chemokines by human first-trimester cytotrophoblast-derived HTR-8/SV neo cells. HTR-8/SV neo cells were cultured to confluence and stimulated with GnRH-I (10, 100, or 1,000 nmol/L) or GnRH-II (10, 100, or 1,000 nmol/L) for 24 hours. IL-1β (1-ng/mL) was used as a positive control. Select growth factors were measured in the conditioned supernatant by specific ELISA, including (A) interleukin-8 (IL-8), (B) IL-6, (C) matrix metalloproteinase 3 (MMP-3), (D) monocyte chemoattractant protein 1 (MCP-1), (E) vascular endothelial growth factor (VEGF), (F) soluble Fms-like tyrosine kinase-1 (sFlt-1), (G) urokinase-type plasminogen activator (uPA), and (H) plasminogen activator inhibitor-1 (PAI-I). Results shown are mean ± SEM from a minimum of three separate experiments performed in triplicate. Statistical differences are indicated. NS = not significant. Fertility and Sterility 2010 94, 2680-2687DOI: (10.1016/j.fertnstert.2010.03.016) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effect of GnRH-I and GnRH-II on the production of select cytokines and chemokines by human first-trimester decidual stromal cells. Human first-trimester DSCs were isolated, purified, grown to confluence with E2 (10−8 mol/L), medroxyprogesterone acetate (10−7 mol/L), both, or vehicle for 7 days, and then stimulated with GnRH-I (1–1,000 nmol/L) or GnRH-II (1–1,000 nmol/L) for 24 hours. Interleukin-1β (1 ng/mL) or thrombin (2.5 U/mL) was used as positive control. Data are shown for 100 nmol/L GnRH-I and -II only. Select growth factors were measured in the conditioned supernatant by specific ELISA, including (A) IL-8, (B) IL-6, (C) MMP-3, (D) MCP-1, (E) VEGF, (F) sFlt-1, (G) uPA, and (H) PAI-I. Results shown are mean ± SEM from a minimum of three separate experiments performed in triplicate. Statistical differences are indicated. Abbreviations as in Figure 2. *P<.05 compared with control with no hormonal stimulation (vehicle only) and control with estrogen only. Fertility and Sterility 2010 94, 2680-2687DOI: (10.1016/j.fertnstert.2010.03.016) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effect of GnRH-I and GnRH-II on the production of select cytokines and chemokines by human term decidual stromal cells. Human term DSCs were isolated, purified, growth to confluence with E2 (10−8 mol/L), medroxyprogesterone acetate (MPA; 10−7 mol/L), both, or vehicle for 7 days, and then stimulated with GnRH-I (1–1,000 nmol/L) or GnRH-II (1–1,000 nmol/L) for 24 hours. Interleukin-1β (1 ng/mL) or thrombin (2.5 U/mL) was used as a positive control. Data are shown for 100 nmol/L GnRH-I and -II only. Select growth factors were measured in the conditioned supernatant by specific ELISA, including (A) IL-8, (B) IL-6, (C) MMP-3, (D) MCP-1, (E) VEGF, (F) sFlt-1, (G) uPA, and (H) PAI-I. Results shown are mean ± SEM from a minimum of three separate experiments performed in triplicate. Statistical differences are indicated. Abbreviations as in Figure 2. *P<.05 compared with control with no hormonal stimulation (vehicle only) and control with estrogen only. Fertility and Sterility 2010 94, 2680-2687DOI: (10.1016/j.fertnstert.2010.03.016) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions