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Gonadotropin-releasing hormone and gonadotropin-releasing hormone receptor are expressed at tubal ectopic pregnancy implantation sites
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GnRH and GnRH receptor are expressed at tubal ectopic pregnancy implantation sites Q3
Bo Peng, Ph.D.,a Christian Klausen, Ph.D.,a Lisa Campbell, M.B.Ch.B.,b Peter C. K. Leung, Ph.D.,a Andrew W. Horne, Ph.D.,b and Mohamed A. Bedaiwy, M.D., Ph.D.a a Department of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada; and b MRC Centre for Reproductive Health, The Queen's Medical Research Institute, University of Edinburgh, Edinburgh, Scotland, United Kingdom
Objective: To investigate whether GnRH and GnRH receptor (GnRHR) are expressed at tubal ectopic pregnancy sites, and to study the potential role of GnRH signaling in regulating immortalized human trophoblast cell viability. Design: Immunohistochemical and experimental studies. Setting: Academic research laboratory. Patient(s): Fallopian tube implantation sites (n ¼ 25) were collected from women with ectopic pregnancy. First-trimester human placenta biopsies (n ¼ 5) were obtained from elective terminations of pregnancy. Intervention(s): None. Main Outcome Measure(s): GnRH and GnRHR expression was examined by means of immunohistochemistry and histoscoring. Trophoblastic BeWo choriocarcinoma and immortalized extravillous trophoblast (HTR-8/SVneo) cell viability was examined by means of cell counting after incubation with GnRH and/or GnRH antagonist (Antide). Result(s): GnRH and GnRHR immunoreactivity was detected in cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast in all women with tubal pregnancy. GnRH immunoreactivity was higher and GnRHR immunoreactivity lower in syncytiotrophoblast compared with cytotrophoblast. GnRH and GnRHR immunoreactivity was detected in adjacent fallopian tube epithelium. Whereas neither GnRH nor Antide altered HTR-8/SVneo cell viability, treatment with GnRH significantly increased the overall cell viability of BeWo cells at 48 and 72 hours, and these effects were abolished by pretreatment with Antide. Conclusion(s): GnRH and GnRHR are expressed in trophoblast cell populations and fallopian tube epithelium at tubal ectopic pregnancy sites. GnRH increases BeWo cell viability, an effect mediated by the GnRHR. Further Use your smartphone work is required to investigate the potential role of GnRH signaling in ectopic pregnancy. (Fertil to scan this QR code SterilÒ 2016;-:-–-. Ó2016 by American Society for Reproductive Medicine.) and connect to the Key Words: GnRH, GnRHR, ectopic pregnancy, trophoblast, fallopian tube Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/pengb-gnrh-gnrhr-ectopic-pregnancy/
A
n ectopic pregnancy is a pregnancy-related complication that is characterized by aberrant embryo implantation outside the normal endometrial cavity, with 95% occurring within the fallopian tube (1). Ectopic pregnancies occur in 1%–2% of all pregnancies (2), and, in the Western World, they remain the
most common cause of maternal mortality in the first trimester of pregnancy (3). The expressions of several genes are known to be altered in the fallopian tubes of women with tubal ectopic pregnancies (4). In addition, some trophoblast-derived factors have been identified that may contribute to tubal implantation and placentation. For
Received September 16, 2015; revised January 14, 2016; accepted February 1, 2016. B.P. has nothing to disclose. C.K. has nothing to disclose. L.C. has nothing to disclose. P.C.K.L. has nothing to disclose. A.W.H. has nothing to disclose. M.A.B. has nothing to disclose. Reprint requests: Mohamed A. Bedaiwy, M.D., Ph.D., Department of Obstetrics and Gynaecology, University of British Columbia, D415A-4500 Oak Street, Vancouver, British Columbia, V6H 3N1, Canada (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2016 0015-0282/$36.00 Copyright ©2016 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2016.02.003
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example, leukemia inhibitory factor is expressed in trophoblasts from ectopic pregnancies and supports trophoblast adhesion to a fallopian tube cell line (5). Moreover, trophoblast-secreted factors up-regulate galactin-1, a molecule involved in intrauterine implantation, in fallopian tube epithelial cells in vitro (6). Further work characterizing gene expression at the fallopian tube implantation site is important to improve understanding of the etiology of ectopic pregnancy. GnRH and its G-protein–coupled receptor (GnRHR) play a central role in regulating reproductive function (7, 8). Best known for their expression
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and function in the central nervous system, GnRH and GnRHR have also been detected in a variety of other normal and neoplastic tissues, both within and outside the reproductive system (9, 10). Indeed, GnRH and GnRHR have been detected in both the maternal and fetal components of first-trimester human placenta (11, 12). On the fetal side, GnRH and GnRHR are expressed in cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast cells (13, 14). On the maternal side, GnRH and GnRHR mRNA and protein are expressed in the decidua (15). Additionally, GnRHR has been detected in rat oviduct during the postimplantation period (16). However, whether or not GnRH and GnRHR are expressed in tubal ectopic pregnancy is unknown. In first-trimester human placenta, GnRH has been shown to stimulate hCG secretion in placental explants, primary trophoblasts, and choriocarcimoma BeWo cells (17–19). Moreover, GnRH has been shown to increase primary and HTR-8/SVneo immortalized extravillous trophoblast cell invasion (13,20–22). These biologic functions contribute significantly to the establishment of early pregnancy, but the role of GnRH signaling in ectopic pregnancy has not been studied. In the present study, we examined the expression of GnRH and GnRHR in fallopian tube implantation sites from women with ectopic pregnancy and the effect of GnRH and a GnRH antagonist on cell viability in two immortalized trophoblast cell lines.
MATERIALS AND METHODS Reagents and Antibodies Native human GnRH I and the GnRH antagonist, Antide, were purchased from Bachem (Belmont, CA). Rabbit polyclonal antibodies against mouse GnRH (Ab5617; antigen sequence identical to human GnRH) and b-hCG (Ab9376) were purchased from Abcam. Mouse monoclonal antibody against human GnRHR (clone F1G4) was purchased from Thermo Scientific. Mouse monoclonal antibody against human Ki67 (clone MIB-1) was purchased from Dako. Mouse monoclonal human leukocyte antigen G (HLA-G) antibody (clone 4H84) was obtained from Exbio. Mouse monoclonal antibody against human cytokeratin-7 (clone OV-TL 12/30) was purchased from Millipore. Normal rabbit control IgG (sc-2027) and mouse control IgG1 (M5284; clone MOPC21) were purchased from Santa Cruz Biotechnology and Sigma-Aldrich, respectively. Trypan Blue solution (0.4% in phosphatebuffered saline solution [PBS]) was purchased from Life Technologies.
Tissue Collection This study was approved by the Research Ethics Board of the University of British Columbia (H07-01149) as well as the Scotland A Research Ethics Committee (LREC 04/S1103/20), and every patient provided informed written consent. Fallopian tube implantation sites (n ¼ 25) were collected from women undergoing salpingectomy for the treatment of tubal ectopic pregnancy. First-trimester placenta biopsies
(6–12 weeks, n ¼ 5) were obtained from women undergoing elective termination of pregnancy.
Immunohistochemistry and Histoscore Analysis Fallopian tube samples containing ectopic implantation sites and first-trimester human placenta samples were fixed in 4% formaldehyde and embedded in paraffin for sectioning. Sections were deparaffinized in xylene, rehydrated through graded ethanol, and processed for wet heat-induced antigen retrieval in a steamer for 20 minutes with a modified citrate buffer (pH 6.1; Dako). Sections were incubated in 3% H2O2 in PBS for 30 minutes at room temperature to quench endogenous peroxidase, and then blocked with serum-free protein block for 1 hour at room temperature. Sections were incubated with antibodies against GnRH (20 mg/mL), GnRHR (20 mg/mL), Ki67 (4 mg/mL), cytokeratin-7 (5 mg/mL), HLAG (5 mg/mL), and b-hCG (5 mg/mL) overnight at 4 C. Immunoreactivity was detected with the use of the horseradish peroxidase–linked Envision system (Dako) and 3,30 diaminobenzidine chromogen solution. Exposure time to 3,30 -diaminobenzidine chromogen solution for all slides was 5 minutes. Slides were counterstained with Harris hematoxylin (Sigma-Aldrich) for 2 minutes, dehydrated through graded ethanol to xylene, mounted in a xylene-based mounting medium, and observed under a light microscope (Leica). Immunohistochemical scoring (histoscore) was performed as previously described (23, 24) with minor modifications. Briefly, five representative fields containing placental tissue and five representative fields containing fallopian tube were examined per patient (n ¼ 25) with the use of a Zeiss light microscope at 200 magnification. The intensity of GnRH and GnRHR immunostaining was classified into four categories (0 ¼ negative; 1 ¼ weak; 2 ¼ moderate; and 3 ¼ strong). Immunostaining was scored in five cell populations; villous cytotrophoblast, syncytiotrophoblast, extravillous trophoblast, fallopian tube epithelium, and fallopian tube stroma. The percentage of cells in each cell population with negative, weak, moderate, or strong staining was noted. A histoscore for each cell population in each field was calculated as follows: histoscore ¼ 0 percentage of negative staining cells þ 1 percentage of weak staining cells þ 2 percentage of moderate staining cells þ 3 percentage of strong staining cells. The histoscore of each sample was calculated as the mean of the histoscores from five different fields.
Cell Culture The HTR-8/SVneo immortalized extravillous trophoblast cell line was a kind gift from Dr. P. K. Lala (Western University, London, Onatrio) and was cultured in Dulbecco Modified Eagle Medium (DMEM; Life Technologies) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin; Life Technologies). The expression of GnRHR in HTR-8/SVneo cells has been previously demonstrated (13). The human choriocarcinoma BeWo cell line (which expresses GnRH and GnRHR; VOL. - NO. - / - 2016
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Supplemental Fig. 1 [available online at www.fertstert.org]) was purchased from American Type Culture Collection and maintained in a 1:1 mixture of DMEM and Ham F-12K (Sigma-Aldrich) supplemented with 10% FBS and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin). All cells were maintained at 37 C in a humidified atmosphere with 5% CO2.
Cell Viability Assay HTR-8/SVneo cells (1 105) and BeWo cells (1 105) were seeded in 12 well cell culture plates and switched to serumfree medium after 24 hours. Cells were incubated with or without Antide (10 nmol/L) for 1 hour before the addition of a fixed concentration of GnRH I (10 nmol/L). The concentration of GnRH and Antide was optimized to exert the biologic effect in the trophoblast cell culture system (Supplemental Fig. 2, available online at www.fertstert.org). Cells were trypsinized at different time points (0, 24, 48, 72, or 96 hours), mixed 1:1 with Trypan blue (0.4%), and incubated for 2 minutes. The number of viable (nonstained) cells was calculated by counting with the use of a hemocytometer and an inverted light microscope.
Statistical Analysis Histoscore results are presented as the mean SD and were analyzed with the use of nonparametric Kruskal-Wallis statistic followed by Dunn multiple comparison test. Cell viability results are presented as the mean SEM and were analyzed with the use of one-way analysis of variance followed by Tukey multiple-comparison test. All statistical analyses were performed with the use of Graphpad Prism 5.
RESULTS Immunolocalization of GnRH and GnRHR in Trophoblasts from Women with Ectopic Pregnancy We immunolocalized GnRH and GnRHR protein in tubal ectopic pregnancy implantation site biopsies by immunohistochemistry. GnRH protein was immunolocalized in villous and column cytotrophoblasts as well as syncytiotrophoblasts of placental tissues from ectopic pregnancy (Fig. 1A and B). GnRH immunoreactivity was especially abundant in the cytoplasm of the syncytiotrophoblasts. In extravillous trophoblast cells from ectopic pregnancy, GnRH immunoreactivity was observed in the cytoplasm (Fig. 1C and D). GnRHR immunoreactivity was abundant in cytotrophoblasts of ectopic placental tissues (Fig. 1E and F). GnRHR protein was also localized at the apical surface of the syncytiotrophoblasts from ectopic pregnancy biopsies. GnRHR (Fig. 1G and H) were immunolocalized in extravillous trophoblasts from ectopic pregnancies. The cell proliferation marker Ki67 was predominantly expressed by villous and column cytotrophoblasts (Fig. 1I and J), but not in deeply invasive extravillous trophoblasts (Fig. 1L and M). Immunostaining for cytokeratin-7 was performed to label cell populations of epithelial origin, such as extravillous trophoblasts and cytotrophoblasts (Fig. 1M and O). b-hCG
(Fig. 1N and P) and HLA-G (Fig. 1Q and S) were used as markers of syncytiotrophoblasts and extravillous trophoblasts, respectively. No immunoreactivity was observed in sections incubated with a mixture of rabbit and mouse control IgGs (Fig. 1R and T).
Immunolocalization of GnRH and GnRHR in Fallopian Tube Adjacent to Ectopic Pregnancy Implantation Sites Immunostaining for GnRH (Fig. 2A) and GnRHR (Fig. 2B) was observed in the fallopian tube epithelium adjacent to tubal implantation sites. Faint immunostaining was also observed in fallopian tube stroma. Cytokeratin-7 was used as a marker of fallopian tube epithelial cells (Fig. 2C). No immunoreactivity was observed after substitution of primary antibodies with a mixture of rabbit and mouse control IgGs (Fig. 2D).
Immunostaining Intensity of GnRH and GnRHR in Trophoblast and Fallopian Tube Cells from Ectopic Pregnancy Implantation Sites Immunohistochemical staining intensities of GnRH and GnRHR in different trophoblast and fallopian tube cell populations from 25 tubal ectopic pregnancy implantation sites were scored using the histoscore method. The distribution of the GnRH and GnRHR histoscores in trophoblast and fallopian tube cells are listed in Figure 3A. Moderate to strong (histoscore 2–3) GnRH staining was observed in the syncytiotrophoblasts (84.0%) and extravillous trophoblasts (60.0%) from a majority of the tubal ectopic pregnancy specimens. Weak to moderate (histoscore 1 to <2) GnRH immunoreactivity was detected in a majority of cytotrophoblast (76.0%) and fallopian tube epithelium (65.2%) samples. Moderate to strong GnRHR immunoreactivity was observed in cytotrophoblasts and extravillous trophoblasts from 40.0% and 48.0% of the tubal ectopic pregnancy implantation site samples, respectively. The majority of samples demonstrated weak to moderate GnRHR staining in the syncytiotrophoblasts (72.0%) and fallopian tube epithelium (60.9%). Negative to weak (histoscore 0 to <1) GnRH and GnRHR immunostaining was observed in the fallopian tube stroma in 65.2% and 69.6% of the cases, respectively. Among trophoblast cell populations, immunoreactivity for GnRH (Fig. 3B) was significantly higher in the syncytiotrophoblast compared with cytotrophoblasts (P< .001) and extravillous trophoblasts (P< .05). Higher immunoreactivity for GnRH was detected in extravillous trophoblast compared with cytotrophoblast cells (P< .05). Additionally, GnRH immunoreactivity in the fallopian tube epithelium was similar to that of cytotrophoblasts, but was significantly lower compared with syncytiotrophoblasts (P< .001) and extravillous trophoblasts (P< .05). Significantly lower GnRH immunoreactivity was observed in fallopian tube stroma compared with fallopian tube epithelium (P< .01) and all trophoblast cell populations (P< .001 vs. syncytiotrophoblast; P< .01 vs. cytotrophoblasts; and P< .001 vs. extravillous trophoblasts) in ectopic pregnancies.
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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) b-hCG, and (Q and S) HLA-G in placental villi of tubal ectopic pregnancy specimens (n ¼ 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 ¼ 100 mm; 400 scale bar ¼ 25 mm. Peng. GnRH and GnRHR in ectopic pregnancy. Fertil Steril 2016.
GnRHR immunoreactivity (Fig. 3C) was significantly lower in syncytiotrophoblasts than in cytotrophoblasts (P< .001) and extravillous trophoblasts (P< .001). GnRHR immunostaining was not significantly different between cytotrophoblast and extravillous trophoblast cell populations. Immunoreactivity for GnRHR was significantly lower in fallopian tube epithelium than in cytotrophoblasts
(P< .05) and extravillous trophoblasts (P< .01), but did not differ significantly from that in syncytiotrophoblasts. Significantly lower GnRHR immunoreactivity was observed in fallopian tube stroma compared with cytotrophoblasts (P< .001), extravillous trophoblasts (P< .001), and fallopian tube epithelium (P< .01), but not compared with syncytiotrophoblasts. VOL. - NO. - / - 2016
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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 ¼ 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 ¼ 100 mm. Peng. GnRH and GnRHR in ectopic pregnancy. Fertil Steril 2016.
GnRH Increases the Overall Viability of BeWo Cells, and GnRH Antagonist Attenuates this Effect Next, we examined the role of GnRH and GnRH antagonist in regulating trophoblast cell viability with the use of human choriocarcinoma BeWo cells (a villous cytotrophoblast cell model) and HTR-8/SVneo cells (an extravillous trophoblast cell model). Native GnRH at 10 nmol/L significantly increased the overall cell viability of BeWo cells at 48 hours and 72 hours after treatment (Fig. 4A; P< .01 and P< .05, respectively), whereas lower concentration of GnRH (1 nmol/L) did not exert this effect (Supplemental Fig. 3, available online at www.fertstert.org). Treatment with GnRH antagonist Antide (10 nmol/L) alone did not affect the number of viable BeWo cells, but pretreatment with Antide significantly attenuated this effect at 48 hours and 72 hours after treatment (Fig. 4A). In contrast, treatment with either GnRH (10 nmol/L) or Antide (10 nmol/L) did not alter the number of viable HTR-8/SVneo cells from 24 hours to 96 hours (Fig. 4B).
DISCUSSION Although the presence of the GnRH-GnRHR system has previously been reported at the maternal-fetal interface in early pregnancy, this study is the first to describe the expression of
GnRH and GnRHR in ectopic pregnancy. A limitation of this study was the inability to control for trophoblast number/ function between implantation sites, because serum hCG levels were not available for the women who donated their tissues. However, even accounting for the difference in GnRH/ GnRHR expression that might be expected between patients of different gestations, we were able to detect differences in GnRH/GnRHR expression between different trophoblast subtypes at tubal implantation sites. In general, our findings are in agreement with our previous observations about the abundance of GnRH and GnRHR in first-trimester human placenta (13, 14), and these results have been further validated in the present study (Supplemental Fig. 3). However, equivalent or elevated levels of GnRH transcripts in cytotrophoblasts compared with syncytiotrophoblasts have been previously reported in intrauterine pregnancies (25), and we observed a similar staining pattern for GnRH in control first-trimester human placental samples (Supplemental Fig. 3). In contrast, reduced expression of GnRH was observed in cytotrophoblasts compared with syncytiotrophoblast and extravillous trophoblast cell populations at ectopic pregnancy implantation sites. GnRH signaling has been shown to stimulate extravillous trophoblast invasion (13, 20–22), and we found that GnRH increases the number of viable BeWo cells (cytotrophoblast-
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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 ¼ 25; FTE and FTS: n ¼ 23. Peng. GnRH and GnRHR in ectopic pregnancy. Fertil Steril 2016.
like cells). In light of these findings it will be interesting to discover if the discrepancy in GnRH expression between intrauterine and ectopic pregnancies reflects an alteration in GnRH regulation of trophoblast function in ectopic pregnancies, contributing to the aberrant placentation observed in this condition (26). Additionally, inflammation in the pelvic region is reported to be associated with ectopic pregnancy (27). Inflammation-related signaling pathways, such as IKK-b and NF-kB, have been reported to inhibit GnRH secretion in the hypothalamus (28). Whether inflammation alters GnRH expression in trophoblasts from ectopic pregnancies is still unknown and will be an interesting area for further study. Here we found that GnRHR was localized in all trophoblast cell populations from ectopic pregnancies. Coexpression of GnRH and GnRHR in trophoblast cell populations supports the hypothesis that autocrine and/or paracrine GnRH-GnRHR signaling may contribute to the regulation of trophoblast cell behavior in ectopic pregnancies, similarly to that observed in intrauterine pregnancies (29). In particular, high levels of GnRH secreted from the syncytiotrophoblast cell layer may elicit regulatory effects primarily on cytotrophoblast and extravillous trophoblast cells which express the highest levels of GnRHR.
Previous studies have detected GnRH and GnRHR in the oviducts of cows and pregnant rats (16, 30, 31). Here we reported that both GnRH and GnRHR are localized predominantly in the fallopian tube epithelium from ectopic pregnancies, with weak expression in the fallopian tube stroma. These findings are in agreement with similar studies on the expression of GnRHR in the pregnant rat oviduct (16). At present, the role of the GnRH-GnRHR system in fallopian tube function is still poorly understood. Many endometrial receptivity markers such as integrin avb3, fibronectin, osteopontin, and leukemia inhibitory factor have been detected in fallopian tube epithelium (32–34). Administration of GnRH agonist has been reported to restore the endometrial expression of integrin b3 subunit and leukemia inhibitory factor after ovarian stimulation in mice (35), suggesting that GnRH may regulate the expression of receptivity markers. However, further studies will be required to investigate if GnRH regulates receptivity markers in the fallopian tube epithelium. GnRH and its analogues are known to regulate the proliferation of several types of cancer cells (9), peripheral lymphocytes (36), and biliary tract cells (37). To the best of our knowledge, the present study provides the first evidence that GnRH positively regulates trophoblastic BeWo VOL. - NO. - / - 2016
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Fertility and Sterility® Acknowledgments: The authors thank the women who donated fallopian tube samples and first-trimester placenta samples used in this study.
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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 ¼ 3; *P<.05; **P<.01). Peng. GnRH and GnRHR in ectopic pregnancy. Fertil Steril 2016.
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cell viability via GnRHR, because preadministration of GnRH antagonist Antide abolished this effect. This effect would most likely be exerted on villous and column cytotrophoblasts, given their high proliferative rate (frequent staining for Ki67) and abundant GnRHR expression. In contrast, GnRH does not appear to regulate HTR-8/SVneo cell viability. This may due to the characteristic of the two different trophoblast cell types, because GnRH signaling regulates viability in cytotrophoblast-like cells (BeWo) but invasiveness in extravillous trophoblast cells (13, 20–22). Thus, the functions of GnRH may be distinct in cytotrophoblasts and extravillous trophoblasts; however, each appears to be positively involved in placental development. In summary, we have demonstrated the expression of GnRH and GnRHR in trophoblasts and fallopian tubes from women with ectopic pregnancy. Additionally, we have shown that GnRH promotes the viability of trophoblastic BeWo cells, and that this effect is mediated by GnRHR, because it can be inhibited by pretreatment with GnRH antagonist. Further work is required to elucidate the role of GnRH signaling in ectopic pregnancy.
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expression of its mRNA in the oviduct during pregnancy in rats. J Histochem Cytochem 2007;55:525–34. Singh R, Graves ML, Roskelley CD, Giritharan G, Rajamahendran R. Gonadotropin releasing hormone receptor gene and protein expression and immunohistochemical localization in bovine uterus and oviducts. Domest Anim Endocrinol 2008;34:319–26. Makrigiannakis A, Karamouti M, Petsas G, Makris N, Nikas G, Antsaklis A. The expression of receptivity markers in the fallopian tube epithelium. Histochem Cell Biol 2009;132:159–67. Brown JK, Shaw JL, Critchley HO, Horne AW. Human fallopian tube epithelium constitutively expresses integrin endometrial receptivity markers: no evidence for a tubal implantation window. Mol Hum Reprod 2012;18:111–20. Guney M, Erdemoglu E, Oral B, Karahan N, Mungan T. Leukemia inhibitory factor (LIF) is immunohistochemically localized in tubal ectopic pregnancy. Acta Histochem 2008;110:319–23. Ruan HC, Zhu XM, Luo Q, Liu AX, Qian YL, Zhou CY, et al. Ovarian stimulation with GnRH agonist, but not GnRH antagonist, partially restores the expression of endometrial integrin beta3 and leukaemia-inhibitory factor and improves uterine receptivity in mice. Hum Reprod 2006;21:2521–9. Tanriverdi F, Gonzalez-Martinez D, Hu Y, Kelestimur F, Bouloux PM. GnRH-I and GnRH-II have differential modulatory effects on human peripheral blood mononuclear cell proliferation and interleukin-2 receptor gamma-chain mRNA expression in healthy males. Clin Exp Immunol 2005;142:103–10. Ray D, Han Y, Franchitto A, DeMorrow S, Meng F, Venter J, et al. Gonadotropin-releasing hormone stimulates biliary proliferation by paracrine/autocrine mechanisms. Am J Pathol 2015;185:1061–72.
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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. Peng. GnRH and GnRHR in ectopic pregnancy. Fertil Steril 2016.
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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 ¼ GnRH receptor. Peng. GnRH and GnRHR in ectopic pregnancy. Fertil Steril 2016.
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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 ¼ 100 mm. Peng. GnRH and GnRHR in ectopic pregnancy. Fertil Steril 2016.
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GnRH and GnRH receptor are expressed at tubal ectopic pregnancy implantation sites Q3
Bo Peng, Ph.D.,a Christian Klausen, Ph.D.,a Lisa Campbell, M.B.Ch.B.,b Peter C. K. Leung, Ph.D.,a Andrew W. Horne, Ph.D.,b and Mohamed A. Bedaiwy, M.D., Ph.D.a a Department of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada; and b MRC Centre for Reproductive Health, The Queen's Medical Research Institute, University of Edinburgh, Edinburgh, Scotland, United Kingdom
Objective: To investigate whether GnRH and GnRH receptor (GnRHR) are expressed at tubal ectopic pregnancy sites, and to study the potential role of GnRH signaling in regulating immortalized human trophoblast cell viability. Design: Immunohistochemical and experimental studies. Setting: Academic research laboratory. Patient(s): Fallopian tube implantation sites (n ¼ 25) were collected from women with ectopic pregnancy. First-trimester human placenta biopsies (n ¼ 5) were obtained from elective terminations of pregnancy. Intervention(s): None. Main Outcome Measure(s): GnRH and GnRHR expression was examined by means of immunohistochemistry and histoscoring. Trophoblastic BeWo choriocarcinoma and immortalized extravillous trophoblast (HTR-8/SVneo) cell viability was examined by means of cell counting after incubation with GnRH and/or GnRH antagonist (Antide). Result(s): GnRH and GnRHR immunoreactivity was detected in cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast in all women with tubal pregnancy. GnRH immunoreactivity was higher and GnRHR immunoreactivity lower in syncytiotrophoblast compared with cytotrophoblast. GnRH and GnRHR immunoreactivity was detected in adjacent fallopian tube epithelium. Whereas neither GnRH nor Antide altered HTR-8/SVneo cell viability, treatment with GnRH significantly increased the overall cell viability of BeWo cells at 48 and 72 hours, and these effects were abolished by pretreatment with Antide. Conclusion(s): GnRH and GnRHR are expressed in trophoblast cell populations and fallopian tube epithelium at tubal ectopic pregnancy sites. GnRH increases BeWo cell viability, an effect mediated by the GnRHR. Further Use your smartphone work is required to investigate the potential role of GnRH signaling in ectopic pregnancy. (Fertil to scan this QR code SterilÒ 2016;-:-–-. Ó2016 by American Society for Reproductive Medicine.) and connect to the Key Words: GnRH, GnRHR, ectopic pregnancy, trophoblast, fallopian tube Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/pengb-gnrh-gnrhr-ectopic-pregnancy/
A
n ectopic pregnancy is a pregnancy-related complication that is characterized by aberrant embryo implantation outside the normal endometrial cavity, with 95% occurring within the fallopian tube (1). Ectopic pregnancies occur in 1%–2% of all pregnancies (2), and, in the Western World, they remain the
most common cause of maternal mortality in the first trimester of pregnancy (3). The expressions of several genes are known to be altered in the fallopian tubes of women with tubal ectopic pregnancies (4). In addition, some trophoblast-derived factors have been identified that may contribute to tubal implantation and placentation. For
Received September 16, 2015; revised January 14, 2016; accepted February 1, 2016. B.P. has nothing to disclose. C.K. has nothing to disclose. L.C. has nothing to disclose. P.C.K.L. has nothing to disclose. A.W.H. has nothing to disclose. M.A.B. has nothing to disclose. Reprint requests: Mohamed A. Bedaiwy, M.D., Ph.D., Department of Obstetrics and Gynaecology, University of British Columbia, D415A-4500 Oak Street, Vancouver, British Columbia, V6H 3N1, Canada (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2016 0015-0282/$36.00 Copyright ©2016 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2016.02.003
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example, leukemia inhibitory factor is expressed in trophoblasts from ectopic pregnancies and supports trophoblast adhesion to a fallopian tube cell line (5). Moreover, trophoblast-secreted factors up-regulate galactin-1, a molecule involved in intrauterine implantation, in fallopian tube epithelial cells in vitro (6). Further work characterizing gene expression at the fallopian tube implantation site is important to improve understanding of the etiology of ectopic pregnancy. GnRH and its G-protein–coupled receptor (GnRHR) play a central role in regulating reproductive function (7, 8). Best known for their expression
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and function in the central nervous system, GnRH and GnRHR have also been detected in a variety of other normal and neoplastic tissues, both within and outside the reproductive system (9, 10). Indeed, GnRH and GnRHR have been detected in both the maternal and fetal components of first-trimester human placenta (11, 12). On the fetal side, GnRH and GnRHR are expressed in cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast cells (13, 14). On the maternal side, GnRH and GnRHR mRNA and protein are expressed in the decidua (15). Additionally, GnRHR has been detected in rat oviduct during the postimplantation period (16). However, whether or not GnRH and GnRHR are expressed in tubal ectopic pregnancy is unknown. In first-trimester human placenta, GnRH has been shown to stimulate hCG secretion in placental explants, primary trophoblasts, and choriocarcimoma BeWo cells (17–19). Moreover, GnRH has been shown to increase primary and HTR-8/SVneo immortalized extravillous trophoblast cell invasion (13,20–22). These biologic functions contribute significantly to the establishment of early pregnancy, but the role of GnRH signaling in ectopic pregnancy has not been studied. In the present study, we examined the expression of GnRH and GnRHR in fallopian tube implantation sites from women with ectopic pregnancy and the effect of GnRH and a GnRH antagonist on cell viability in two immortalized trophoblast cell lines.
MATERIALS AND METHODS Reagents and Antibodies Native human GnRH I and the GnRH antagonist, Antide, were purchased from Bachem (Belmont, CA). Rabbit polyclonal antibodies against mouse GnRH (Ab5617; antigen sequence identical to human GnRH) and b-hCG (Ab9376) were purchased from Abcam. Mouse monoclonal antibody against human GnRHR (clone F1G4) was purchased from Thermo Scientific. Mouse monoclonal antibody against human Ki67 (clone MIB-1) was purchased from Dako. Mouse monoclonal human leukocyte antigen G (HLA-G) antibody (clone 4H84) was obtained from Exbio. Mouse monoclonal antibody against human cytokeratin-7 (clone OV-TL 12/30) was purchased from Millipore. Normal rabbit control IgG (sc-2027) and mouse control IgG1 (M5284; clone MOPC21) were purchased from Santa Cruz Biotechnology and Sigma-Aldrich, respectively. Trypan Blue solution (0.4% in phosphatebuffered saline solution [PBS]) was purchased from Life Technologies.
Tissue Collection This study was approved by the Research Ethics Board of the University of British Columbia (H07-01149) as well as the Scotland A Research Ethics Committee (LREC 04/S1103/20), and every patient provided informed written consent. Fallopian tube implantation sites (n ¼ 25) were collected from women undergoing salpingectomy for the treatment of tubal ectopic pregnancy. First-trimester placenta biopsies
(6–12 weeks, n ¼ 5) were obtained from women undergoing elective termination of pregnancy.
Immunohistochemistry and Histoscore Analysis Fallopian tube samples containing ectopic implantation sites and first-trimester human placenta samples were fixed in 4% formaldehyde and embedded in paraffin for sectioning. Sections were deparaffinized in xylene, rehydrated through graded ethanol, and processed for wet heat-induced antigen retrieval in a steamer for 20 minutes with a modified citrate buffer (pH 6.1; Dako). Sections were incubated in 3% H2O2 in PBS for 30 minutes at room temperature to quench endogenous peroxidase, and then blocked with serum-free protein block for 1 hour at room temperature. Sections were incubated with antibodies against GnRH (20 mg/mL), GnRHR (20 mg/mL), Ki67 (4 mg/mL), cytokeratin-7 (5 mg/mL), HLAG (5 mg/mL), and b-hCG (5 mg/mL) overnight at 4 C. Immunoreactivity was detected with the use of the horseradish peroxidase–linked Envision system (Dako) and 3,30 diaminobenzidine chromogen solution. Exposure time to 3,30 -diaminobenzidine chromogen solution for all slides was 5 minutes. Slides were counterstained with Harris hematoxylin (Sigma-Aldrich) for 2 minutes, dehydrated through graded ethanol to xylene, mounted in a xylene-based mounting medium, and observed under a light microscope (Leica). Immunohistochemical scoring (histoscore) was performed as previously described (23, 24) with minor modifications. Briefly, five representative fields containing placental tissue and five representative fields containing fallopian tube were examined per patient (n ¼ 25) with the use of a Zeiss light microscope at 200 magnification. The intensity of GnRH and GnRHR immunostaining was classified into four categories (0 ¼ negative; 1 ¼ weak; 2 ¼ moderate; and 3 ¼ strong). Immunostaining was scored in five cell populations; villous cytotrophoblast, syncytiotrophoblast, extravillous trophoblast, fallopian tube epithelium, and fallopian tube stroma. The percentage of cells in each cell population with negative, weak, moderate, or strong staining was noted. A histoscore for each cell population in each field was calculated as follows: histoscore ¼ 0 percentage of negative staining cells þ 1 percentage of weak staining cells þ 2 percentage of moderate staining cells þ 3 percentage of strong staining cells. The histoscore of each sample was calculated as the mean of the histoscores from five different fields.
Cell Culture The HTR-8/SVneo immortalized extravillous trophoblast cell line was a kind gift from Dr. P. K. Lala (Western University, London, Onatrio) and was cultured in Dulbecco Modified Eagle Medium (DMEM; Life Technologies) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin; Life Technologies). The expression of GnRHR in HTR-8/SVneo cells has been previously demonstrated (13). The human choriocarcinoma BeWo cell line (which expresses GnRH and GnRHR; VOL. - NO. - / - 2016
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Supplemental Fig. 1 [available online at www.fertstert.org]) was purchased from American Type Culture Collection and maintained in a 1:1 mixture of DMEM and Ham F-12K (Sigma-Aldrich) supplemented with 10% FBS and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin). All cells were maintained at 37 C in a humidified atmosphere with 5% CO2.
Cell Viability Assay HTR-8/SVneo cells (1 105) and BeWo cells (1 105) were seeded in 12 well cell culture plates and switched to serumfree medium after 24 hours. Cells were incubated with or without Antide (10 nmol/L) for 1 hour before the addition of a fixed concentration of GnRH I (10 nmol/L). The concentration of GnRH and Antide was optimized to exert the biologic effect in the trophoblast cell culture system (Supplemental Fig. 2, available online at www.fertstert.org). Cells were trypsinized at different time points (0, 24, 48, 72, or 96 hours), mixed 1:1 with Trypan blue (0.4%), and incubated for 2 minutes. The number of viable (nonstained) cells was calculated by counting with the use of a hemocytometer and an inverted light microscope.
Statistical Analysis Histoscore results are presented as the mean SD and were analyzed with the use of nonparametric Kruskal-Wallis statistic followed by Dunn multiple comparison test. Cell viability results are presented as the mean SEM and were analyzed with the use of one-way analysis of variance followed by Tukey multiple-comparison test. All statistical analyses were performed with the use of Graphpad Prism 5.
RESULTS Immunolocalization of GnRH and GnRHR in Trophoblasts from Women with Ectopic Pregnancy We immunolocalized GnRH and GnRHR protein in tubal ectopic pregnancy implantation site biopsies by immunohistochemistry. GnRH protein was immunolocalized in villous and column cytotrophoblasts as well as syncytiotrophoblasts of placental tissues from ectopic pregnancy (Fig. 1A and B). GnRH immunoreactivity was especially abundant in the cytoplasm of the syncytiotrophoblasts. In extravillous trophoblast cells from ectopic pregnancy, GnRH immunoreactivity was observed in the cytoplasm (Fig. 1C and D). GnRHR immunoreactivity was abundant in cytotrophoblasts of ectopic placental tissues (Fig. 1E and F). GnRHR protein was also localized at the apical surface of the syncytiotrophoblasts from ectopic pregnancy biopsies. GnRHR (Fig. 1G and H) were immunolocalized in extravillous trophoblasts from ectopic pregnancies. The cell proliferation marker Ki67 was predominantly expressed by villous and column cytotrophoblasts (Fig. 1I and J), but not in deeply invasive extravillous trophoblasts (Fig. 1L and M). Immunostaining for cytokeratin-7 was performed to label cell populations of epithelial origin, such as extravillous trophoblasts and cytotrophoblasts (Fig. 1M and O). b-hCG
(Fig. 1N and P) and HLA-G (Fig. 1Q and S) were used as markers of syncytiotrophoblasts and extravillous trophoblasts, respectively. No immunoreactivity was observed in sections incubated with a mixture of rabbit and mouse control IgGs (Fig. 1R and T).
Immunolocalization of GnRH and GnRHR in Fallopian Tube Adjacent to Ectopic Pregnancy Implantation Sites Immunostaining for GnRH (Fig. 2A) and GnRHR (Fig. 2B) was observed in the fallopian tube epithelium adjacent to tubal implantation sites. Faint immunostaining was also observed in fallopian tube stroma. Cytokeratin-7 was used as a marker of fallopian tube epithelial cells (Fig. 2C). No immunoreactivity was observed after substitution of primary antibodies with a mixture of rabbit and mouse control IgGs (Fig. 2D).
Immunostaining Intensity of GnRH and GnRHR in Trophoblast and Fallopian Tube Cells from Ectopic Pregnancy Implantation Sites Immunohistochemical staining intensities of GnRH and GnRHR in different trophoblast and fallopian tube cell populations from 25 tubal ectopic pregnancy implantation sites were scored using the histoscore method. The distribution of the GnRH and GnRHR histoscores in trophoblast and fallopian tube cells are listed in Figure 3A. Moderate to strong (histoscore 2–3) GnRH staining was observed in the syncytiotrophoblasts (84.0%) and extravillous trophoblasts (60.0%) from a majority of the tubal ectopic pregnancy specimens. Weak to moderate (histoscore 1 to <2) GnRH immunoreactivity was detected in a majority of cytotrophoblast (76.0%) and fallopian tube epithelium (65.2%) samples. Moderate to strong GnRHR immunoreactivity was observed in cytotrophoblasts and extravillous trophoblasts from 40.0% and 48.0% of the tubal ectopic pregnancy implantation site samples, respectively. The majority of samples demonstrated weak to moderate GnRHR staining in the syncytiotrophoblasts (72.0%) and fallopian tube epithelium (60.9%). Negative to weak (histoscore 0 to <1) GnRH and GnRHR immunostaining was observed in the fallopian tube stroma in 65.2% and 69.6% of the cases, respectively. Among trophoblast cell populations, immunoreactivity for GnRH (Fig. 3B) was significantly higher in the syncytiotrophoblast compared with cytotrophoblasts (P< .001) and extravillous trophoblasts (P< .05). Higher immunoreactivity for GnRH was detected in extravillous trophoblast compared with cytotrophoblast cells (P< .05). Additionally, GnRH immunoreactivity in the fallopian tube epithelium was similar to that of cytotrophoblasts, but was significantly lower compared with syncytiotrophoblasts (P< .001) and extravillous trophoblasts (P< .05). Significantly lower GnRH immunoreactivity was observed in fallopian tube stroma compared with fallopian tube epithelium (P< .01) and all trophoblast cell populations (P< .001 vs. syncytiotrophoblast; P< .01 vs. cytotrophoblasts; and P< .001 vs. extravillous trophoblasts) in ectopic pregnancies.
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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) b-hCG, and (Q and S) HLA-G in placental villi of tubal ectopic pregnancy specimens (n ¼ 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 ¼ 100 mm; 400 scale bar ¼ 25 mm. Peng. GnRH and GnRHR in ectopic pregnancy. Fertil Steril 2016.
GnRHR immunoreactivity (Fig. 3C) was significantly lower in syncytiotrophoblasts than in cytotrophoblasts (P< .001) and extravillous trophoblasts (P< .001). GnRHR immunostaining was not significantly different between cytotrophoblast and extravillous trophoblast cell populations. Immunoreactivity for GnRHR was significantly lower in fallopian tube epithelium than in cytotrophoblasts
(P< .05) and extravillous trophoblasts (P< .01), but did not differ significantly from that in syncytiotrophoblasts. Significantly lower GnRHR immunoreactivity was observed in fallopian tube stroma compared with cytotrophoblasts (P< .001), extravillous trophoblasts (P< .001), and fallopian tube epithelium (P< .01), but not compared with syncytiotrophoblasts. VOL. - NO. - / - 2016
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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 ¼ 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 ¼ 100 mm. Peng. GnRH and GnRHR in ectopic pregnancy. Fertil Steril 2016.
GnRH Increases the Overall Viability of BeWo Cells, and GnRH Antagonist Attenuates this Effect Next, we examined the role of GnRH and GnRH antagonist in regulating trophoblast cell viability with the use of human choriocarcinoma BeWo cells (a villous cytotrophoblast cell model) and HTR-8/SVneo cells (an extravillous trophoblast cell model). Native GnRH at 10 nmol/L significantly increased the overall cell viability of BeWo cells at 48 hours and 72 hours after treatment (Fig. 4A; P< .01 and P< .05, respectively), whereas lower concentration of GnRH (1 nmol/L) did not exert this effect (Supplemental Fig. 3, available online at www.fertstert.org). Treatment with GnRH antagonist Antide (10 nmol/L) alone did not affect the number of viable BeWo cells, but pretreatment with Antide significantly attenuated this effect at 48 hours and 72 hours after treatment (Fig. 4A). In contrast, treatment with either GnRH (10 nmol/L) or Antide (10 nmol/L) did not alter the number of viable HTR-8/SVneo cells from 24 hours to 96 hours (Fig. 4B).
DISCUSSION Although the presence of the GnRH-GnRHR system has previously been reported at the maternal-fetal interface in early pregnancy, this study is the first to describe the expression of
GnRH and GnRHR in ectopic pregnancy. A limitation of this study was the inability to control for trophoblast number/ function between implantation sites, because serum hCG levels were not available for the women who donated their tissues. However, even accounting for the difference in GnRH/ GnRHR expression that might be expected between patients of different gestations, we were able to detect differences in GnRH/GnRHR expression between different trophoblast subtypes at tubal implantation sites. In general, our findings are in agreement with our previous observations about the abundance of GnRH and GnRHR in first-trimester human placenta (13, 14), and these results have been further validated in the present study (Supplemental Fig. 3). However, equivalent or elevated levels of GnRH transcripts in cytotrophoblasts compared with syncytiotrophoblasts have been previously reported in intrauterine pregnancies (25), and we observed a similar staining pattern for GnRH in control first-trimester human placental samples (Supplemental Fig. 3). In contrast, reduced expression of GnRH was observed in cytotrophoblasts compared with syncytiotrophoblast and extravillous trophoblast cell populations at ectopic pregnancy implantation sites. GnRH signaling has been shown to stimulate extravillous trophoblast invasion (13, 20–22), and we found that GnRH increases the number of viable BeWo cells (cytotrophoblast-
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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 ¼ 25; FTE and FTS: n ¼ 23. Peng. GnRH and GnRHR in ectopic pregnancy. Fertil Steril 2016.
like cells). In light of these findings it will be interesting to discover if the discrepancy in GnRH expression between intrauterine and ectopic pregnancies reflects an alteration in GnRH regulation of trophoblast function in ectopic pregnancies, contributing to the aberrant placentation observed in this condition (26). Additionally, inflammation in the pelvic region is reported to be associated with ectopic pregnancy (27). Inflammation-related signaling pathways, such as IKK-b and NF-kB, have been reported to inhibit GnRH secretion in the hypothalamus (28). Whether inflammation alters GnRH expression in trophoblasts from ectopic pregnancies is still unknown and will be an interesting area for further study. Here we found that GnRHR was localized in all trophoblast cell populations from ectopic pregnancies. Coexpression of GnRH and GnRHR in trophoblast cell populations supports the hypothesis that autocrine and/or paracrine GnRH-GnRHR signaling may contribute to the regulation of trophoblast cell behavior in ectopic pregnancies, similarly to that observed in intrauterine pregnancies (29). In particular, high levels of GnRH secreted from the syncytiotrophoblast cell layer may elicit regulatory effects primarily on cytotrophoblast and extravillous trophoblast cells which express the highest levels of GnRHR.
Previous studies have detected GnRH and GnRHR in the oviducts of cows and pregnant rats (16, 30, 31). Here we reported that both GnRH and GnRHR are localized predominantly in the fallopian tube epithelium from ectopic pregnancies, with weak expression in the fallopian tube stroma. These findings are in agreement with similar studies on the expression of GnRHR in the pregnant rat oviduct (16). At present, the role of the GnRH-GnRHR system in fallopian tube function is still poorly understood. Many endometrial receptivity markers such as integrin avb3, fibronectin, osteopontin, and leukemia inhibitory factor have been detected in fallopian tube epithelium (32–34). Administration of GnRH agonist has been reported to restore the endometrial expression of integrin b3 subunit and leukemia inhibitory factor after ovarian stimulation in mice (35), suggesting that GnRH may regulate the expression of receptivity markers. However, further studies will be required to investigate if GnRH regulates receptivity markers in the fallopian tube epithelium. GnRH and its analogues are known to regulate the proliferation of several types of cancer cells (9), peripheral lymphocytes (36), and biliary tract cells (37). To the best of our knowledge, the present study provides the first evidence that GnRH positively regulates trophoblastic BeWo VOL. - NO. - / - 2016
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Fertility and Sterility® Acknowledgments: The authors thank the women who donated fallopian tube samples and first-trimester placenta samples used in this study.
FIGURE 4
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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 ¼ 3; *P<.05; **P<.01). Peng. GnRH and GnRHR in ectopic pregnancy. Fertil Steril 2016.
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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. Peng. GnRH and GnRHR in ectopic pregnancy. Fertil Steril 2016.
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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 ¼ GnRH receptor. Peng. GnRH and GnRHR in ectopic pregnancy. Fertil Steril 2016.
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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 ¼ 100 mm. Peng. GnRH and GnRHR in ectopic pregnancy. Fertil Steril 2016.
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