Gonadotropin-releasing hormone and gonadotropin-releasing hormone receptor are expressed at tubal ectopic pregnancy implantation sites  Bo Peng, Ph.D., Christian Klausen, Ph.D., Lisa Campbell, M.B.Ch.B., Peter C.K. Leung, Ph.D., Andrew W. Horne, Ph.D., Mohamed A. Bedaiwy, M.D., Ph.D.  Fertility and Sterility  Volume 105, Issue 6, Pages 1620-1627.e3 (June 2016) DOI: 10.1016/j.fertnstert.2016.02.003 Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Immunolocalization of GnRH and its receptor (GnRHR) in trophoblasts from women with tubal ectopic pregnancy. Representative images showing the immunolocalization of (A, B, C, and D) GnRH, (E, F, G, and H) GnRHR, (I, J, K, and L) Ki67, (M and O) cytokeratin-7, (N and P) β-hCG, and (Q and S) HLA-G in placental villi of tubal ectopic pregnancy specimens (n = 25). (R and T) A lack of staining was observed in adjacent control sections incubated with a mixture of mouse and rabbit control (Ctrl) IgGs. The cytotrophoblast (CTB) and syncytiotrophoblast (STB) monolayers are indicated by red and green arrows, respectively. Column cytotrophoblasts are indicated with CC. Extravillous trophoblast (EVT) cells are indicated with blue arrows. ×100 scale bar = 100 μm; ×400 scale bar = 25 μm. Fertility and Sterility 2016 105, 1620-1627.e3DOI: (10.1016/j.fertnstert.2016.02.003) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Immunolocalization of GnRH and its receptor (GnRHR) in fallopian tube from women with tubal ectopic pregnancies. Representative images showing the immunolocalization of (A) GnRH, (B) GnRHR, and (C) cytokeratin-7 in fallopian tube tissues of tubal ectopic pregnancy specimens (n = 23). (D) A lack of staining was observed in adjacent sections incubated with a mixture of mouse and rabbit control (Ctrl) IgG. Fallopian tube epithelium (FTE) and stroma (FTS) are indicated by red and green arrows, respectively. ×100 scale bar = 100 μm. Fertility and Sterility 2016 105, 1620-1627.e3DOI: (10.1016/j.fertnstert.2016.02.003) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Comparison of GnRH and receptor (GnRHR) histoscores between trophoblast and fallopian tube cells from women with tubal ectopic pregnancies. (A) Immunostaining intensity distribution of GnRH and GnRHR in trophoblast and fallopian tube cells from women with tubal ectopic pregnancies. Dot plots representing the histoscores of (B) GnRH and (C) GnRHR between trophoblast cell subpopulations (cytotrophoblast [CTB], syncytiotrophoblast [STB], and extravillous trophoblast [EVT]) and fallopian tube epithelium (FTE) and stroma (FTS). CTB, STB, and EVT: n = 25; FTE and FTS: n = 23. Fertility and Sterility 2016 105, 1620-1627.e3DOI: (10.1016/j.fertnstert.2016.02.003) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 4 GnRH antagonist (Antide) abolishes GnRH-induced increases in the number of viable BeWo cells, but there is no effect of GnRH or GnRH antagonist in HTR-8/SVneo cells. BeWo cells and HTR-8/SVneo cells were treated with 10 nmol/L GnRH I for 0, 24, 48, 72 or 96 hours. (A) BeWo and (B) HTR-8/SVneo viable (nonstained) cell numbers were measured at each time point by means of Trypan blue cell counting. Results are presented as the mean ± SEM of three independent experiments, and significant differences are indicated by asterisks (n = 3; *P<.05; **P<.01). Fertility and Sterility 2016 105, 1620-1627.e3DOI: (10.1016/j.fertnstert.2016.02.003) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 GnRH and receptor (GnRHR) expressions in BeWo and HTR-8/SVneo cells. (A) mRNA levels of GnRH in BeWo and HTR-8/SVneo cells. (B) mRNA levels of GnRHR in BeWo and HTR-8/SVneo cells. (C) Protein levels of GnRHR in BeWo and HTR-8/SVneo cells. Fertility and Sterility 2016 105, 1620-1627.e3DOI: (10.1016/j.fertnstert.2016.02.003) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Lower concentration of GnRH (1 nmol/L) did not alter the overall cell viability in both BeWo cells and HTR-8/SVneo cells. BeWo cells and HTR-8/SVneo cells were treated with 1 nmol/L GnRH I for 0, 24, 48, 72 or 96 hours. (A) BeWo and (B) HTR-8/SVneo viable cell numbers were measured at each time point by means of Trypan blue cell counting. Results are presented as the mean ± SEM of three independent experiments. GnRHR = GnRH receptor. Fertility and Sterility 2016 105, 1620-1627.e3DOI: (10.1016/j.fertnstert.2016.02.003) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 3 Immunolocalization of GnRH and its receptor (GnRHR) in trophoblast cells from first-trimester human placenta. Representative images showing the immunolocalization of (A and B) GnRH and (C and D) GnRHR in trophoblast cells from first-trimester human placenta. (E) A lack of staining was observed in sections incubated with a mixture of mouse and rabbit control (Ctrl) IgG. The cytotrophoblast (CTB), syncytiotrophoblast (STB), and extravillous trophoblast (EVT) cell populations are indicated with red, green, and black arrows, respectively. ×100 scale bar = 100 μm. Fertility and Sterility 2016 105, 1620-1627.e3DOI: (10.1016/j.fertnstert.2016.02.003) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions