Running title: GnRH and GnRHR in ectopic pregnancy

GnRH and GnRHR are Expressed at Tubal Ectopic Pregnancy Implantation Sites

Bo Peng, PhD1, Christian Klausen, PhD1, Lisa Campbell, MBChB2, Peter C.K. Leung, PhD1,

Andrew W. Horne, PhD,2 and Mohamed A. Bedaiwy MD, PhD1

1Department of Obstetrics & Gynaecology, Child & Family Research Institute, The University of

British Columbia, Vancouver, Canada.

2MRC Centre for Reproductive Health, The Queen's Medical Research Institute, The University

of Edinburgh, Edinburgh, United Kingdom.

Corresponding author and person to whom reprint requests should be addressed:

Mohamed A. Bedaiwy, MD, PhD

Department of Obstetrics & Gynaecology,

The University of British Columbia,

D415A-4500 Oak Street, Vancouver, BC, V6H 3N1, Canada

Phone: +1-604-875-2000 ext 4310, Fax: +1-604-875-2725

Email: mohamed.bedaiwy@cw.bc.ca

Disclosure: The authors have nothing to disclose.

Capsule: This study discussed the expression of GnRH and its receptor at tubal ectopic

pregnancy implantation sites, and investigated the role of GnRH and a GnRH antagonist in

regulating trophoblastic BeWo cell viability.

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ABSTRACT

OBJECTIVE: To investigate whether GnRH and 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.

PATIENTS: Fallopian tube implantation sites (n=25) were collected from women with ectopic

pregnancy. First-trimester human placenta biopsies (n=5) were obtained from elective

terminations of pregnancy.

INTERVENTIONS: None

MEASURES: GnRH and GnRHR expression was examined by immunohistochemistry and

Histoscoring. Trophoblastic BeWo choriocarcinoma and immortalized extravillous trophoblast

(HTR-8/SVneo) cell viability was examined by cell counting following incubation with GnRH

and/or GnRH antagonist (Antide).

RESULTS: GnRH and GnRHR immunoreactivity was detected in cytotrophoblasts,

syncytiotrophoblast and extravillous trophoblasts in all women with tubal pregnancy. GnRH

immunoreactivity was higher, whereas GnRHR immunoreactivity was lower, in the

syncytiotrophoblast compared to cytotrophoblasts. 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.

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CONCLUSIONS: 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 work is required to investigate the potential role of

GnRH signaling in ectopic pregnancy.

Key words: GnRH, GnRHR, ectopic pregnancy, trophoblast, Fallopian tube

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INTRODUCTION

An 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 expression 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 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 upregulate

Galactin 1 (a molecule involved in intrauterine implantation) in Fallopian tube epithelial cells in

vitro (6). Further work characterising gene expression at the fallopian tube implantation site is

important to improve understanding of the aetiology of ectopic pregnancy.

Gonadotropin-releasing hormone (GnRH) and its G-protein coupled receptor (GnRHR) play a

central role in regulating reproductive function (7, 8). Best known for their expression 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

cytotrophoblasts, 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 post-implantation 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 human chorionic

gonadotrophin (hCG) secretion in placental explants, primary trophoblasts and choriocarcimoma

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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 biological functions

contribute significantly to the establishment of early pregnancy, but the role of GnRH signaling in

ectopic pregnancy has not been studied.

In this 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.

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MATERIALS AND METHODS

Reagents and antibodies

Native human GnRH I and the GnRH receptor antagonist, Antide, were purchased from

Bachem (Belmont, CA). Rabbit polyclonal antibodies against mouse GnRH (Ab5617, antigen

sequence identical to human GnRH) and beta human chorionic gonadotropin (βhCG; Ab9376)

were purchased from Abcam (Cambridge, MA). Mouse monoclonal antibody against human

GnRHR (Clone F1G4) was purchased from Thermo Scientific (Waltham, MA). Mouse

monoclonal antibody against human Ki67 (clone MIB-1) was purchased from Dako (Burlington,

ON). Mouse monoclonal human leukocyte antigen G (HLA-G) antibody (Clone 4H84) was

obtained from Exbio (Vestec, Czech Republic). Mouse monoclonal antibody against human

cytokeratin 7 (clone OV-TL 12/30) was purchased from Millipore (Billerica, MA). Normal rabbit

control IgG (sc-2027) and mouse control IgG1 (M5284, clone MOPC21) were purchased from

Santa Cruz Biotechnology (Santa Cruz, CA) and Sigma-Aldrich (St. Louis, MO), respectively.

Trypan Blue Solution (0.4% in phosphate-buffered saline) was purchased from Life Technologies

(Carlsbad, CA).

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 all

patients provided informed written consent. Fallopian tube implantation sites (n=25) were

collected from women undergoing salpingectomy for the treatment of tubal ectopic pregnancy.

First-trimester placenta biopsies (6-12 weeks, n=5) were obtained from women undergoing

elective termination of pregnancy.

Immunohistochemistry and Histoscore analysis

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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 min with a modified citrate buffer (pH 6.1;

Dako). Sections were incubated in 3% H2O2 in phosphate-buffered saline (PBS) for 30 min 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

(20μg/ml), GnRHR (20 μg/ml), Ki67 (4μg/ml), cytokeratin 7 (5μg/ml), HLA-G (5μg/ml) and

βhCG (5μg/ml) overnight at 4°C. Immunoreactivity was detected using the horseradish

peroxidase-linked EnvisionTM system (Dako, EnvisionTM + Dual link) and 3,3′-diaminobenzidine

chromogen solution. Exposure time to 3,3′-diaminobenzidine chromogen solution for all slides

were 5 min. Slides were counterstained with Harris hematoxylin (Sigma-Aldrich, St. Louis, MO)

for 2 min, dehydrated through graded ethanol to xylene, mounted in a xylene-based mounting

medium, and observed under a light microscope (Leica, Wetzlar, Germany).

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=25) with a Zeiss

light microscope at 200× magnification. The intensity of GnRH and GnRHR immunostaining was

classified into four categories (0 = negative, 1 = weak, 2 = moderate, and 3 = 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 = 0 ×

percentage of negative staining cells + 1 × percentage of weak staining cells + 2 × percentage of

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moderate staining cells + 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, ON) and was cultured in Dulbecco's Modified Eagle

Medium (DMEM, Life Technologies) supplemented with 10% FBS and antibiotics (100 U/ml

penicillin and 100 μg/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 – Supplemental Fig 1) was purchased from

American Type Culture Collection (Manassas, VA) and was maintained in a 1:1 mixture of

DMEM and Ham’s F-12K (Sigma-Aldrich) supplemented with 10% FBS and antibiotics (100

U/ml penicillin and 100 μg/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 serum-free medium after 24 h. Cells were incubated with or without Antide

(10nM) for 1h prior to the addition of a fixed concentration of GnRH I (10nM). The

concentration of GnRH and Antide was optimized to exert the biological effect in the trophoblast

cell culture system (see Supplemental Fig 2). Cells were trypsinized at different time points (0,

24, 48, 72 or 96 h), mixed 1:1 with Trypan blue (0.4%, Life Technologies), and incubated for 2

minutes. The number of viable (non-stained) cells was calculated by counting with a

hemocytometer and an inverted light microscope.

Statistical Analysis

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Histoscore results are presented as the mean ± SD and were analyzed by non-parametric

Kruskal-Wallis statistic followed by Dunn's Multiple Comparison Test. Cell viability results are

presented as the mean ± SEM and were analyzed by one-way ANOVA followed by Tukey's

multiple comparison test. All statistical analyses were performed using GraphPad Prism 5

(GraphPad Software, San Diego, CA).

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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 syncytiotrophoblast of placental tissues from ectopic

pregnancy (Fig 1. A and B). GnRH immunoreactivity was especially abundant in the cytoplasm

of the syncytiotrophoblast. In extravillous trophoblast cells from ectopic pregnancy, GnRH

immunoreactivity was observed in the cytoplasm (Fig 1. C and D). GnRHR immunoreactivity

was abundant in cytotrophoblasts of ectopic placental tissues (Fig 1. E and F). GnRHR protein

was also localized at the apical surface of the syncytiotrophoblast from ectopic pregnancy

biopsies. GnRHR (Fig 1. G and H) were immunolocalized in extravillous trophoblasts from

ectopic pregnancies. The cell proliferation marker Ki67 was predominantly expressed by villous

and column cytotrophoblasts (Fig 1. I and J), but not in deeply invasive extravillous trophoblasts

(Fig 1. L and M).

Immunostaining for cytokeratin 7 was performed to label cell populations of epithelial

origin, such as extravillous trophoblasts and cytotrophoblasts (Fig 1. M and O). βhCG (Fig 1. N

and P) and HLA-G (Fig 1. Q and S) were used as markers of syncytiotrophoblast and extravillous

trophoblasts, respectively. No immunoreactivity was observed in sections incubated with a

mixture of rabbit and mouse control IgGs (Fig. 1. R and T).

Immunolocalization of GnRH and GnRHR in Fallopian tube adjacent to ectopic pregnancy

implantation sites

Immunostaining for GnRH (Fig 2. A) and GnRHR (Fig 2. B) was observed in the Fallopian

tube epithelium adjacent to tubal implantation sites. Faint immunostaining was also observed in

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Fallopian tube stroma. Cytokeratin 7 was used as a marker of Fallopian tube epithelial cells (Fig

2. C). No immunoreactivity was observed after substitution of primary antibodies with a mixture

of rabbit and mouse control IgGs (Fig 2. D).

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 3. A. Moderate to strong

(2≤Histoscore≤3) GnRH staining was observed in the syncytiotrophoblast (84.0%) and

extravillous trophoblasts (60.0%) from a majority of the tubal ectopic pregnancy specimens.

Weak to moderate (1≤Histoscore<2) GnRH immunoreactivity was detected in a majority of

cytotrophoblasts (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 syncytiotrophoblast

(72.0%) and Fallopian tube epithelium (60.9%). Negative to weak (0≤Histoscore<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. 3. B) was

significantly higher in the syncytiotrophoblast compared to cytotrophoblasts (P<0.001) and

extravillous trophoblasts (P<0.05). Higher immunoreactivity for GnRH was detected in

extravillous trophoblast compared to cytotrophoblast cells (P<0.05). Additionally, GnRH

immunoreactivity in the Fallopian tube epithelium was similar to that of cytotrophoblasts, but was

significantly lower compared to the syncytiotrophoblast (P<0.001) and extravillous trophoblasts

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(P<0.05). Significantly lower GnRH immunoreactivity was observed in Fallopian tube stroma

compared to Fallopian tube epithelium (P<0.01) and all trophoblast cell populations (P<0.001 vs.

syncytiotrophoblast, P<0.01 vs. cytotrophoblasts, and P<0.001 vs. extravillous trophoblasts) in

ectopic pregnancies.

GnRHR immunoreactivity (Fig. 3. C) was significantly lower in the syncytiotrophoblast

than cytotrophoblasts (P<0.001) and extravillous trophoblasts (P<0.001). GnRHR

immunostaining was not significantly different between cytotrophoblast and extravillous

trophoblast cell populations. Immunoreactivity for GnRHR was significantly lower in Fallopian

tube epithelium than cytotrophoblasts (P<0.05) and extravillous trophoblasts (P<0.01), but did not

differ significantly from that in the syncytiotrophoblast. Significantly lower GnRHR

immunoreactivity was observed in Fallopian tube stroma compared to cytotrophoblasts

(P<0.001), extravillous trophoblasts (P<0.001) and Fallopian tube epithelium (P<0.01), but not

the syncytiotrophoblast.

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 using human choriocarcinoma BeWo cells (a villous cytotrophoblast cell model) and

HTR-8/SVneo cells (an extravillous trophoblast cell model). Native GnRH at 10 nM significantly

increased the overall cell viability of BeWo cells at 48h and 72h after treatment (Fig. 4. A,

P<0.01 and P<0.05, respectively), whereas lower concentration of GnRH (1nM) did not exert this

effect (Supplemental Figure 3). Treatment with GnRH antagonist Antide (10 nM) alone did not

affect the number of viable BeWo cells, however pre-treatment with Antide significantly

attenuated this effect at 48h and 72h after treatment (Fig. 4. A). In contrast, treatment with either

GnRH (10nM) or Antide (10nM) did not alter the number of viable HTR-8/SVneo cells from 24h

to 96h (Fig. 4. B).

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DISCUSSION

Although the presence of the GnRH-GnRHR system has previously been reported at the

maternal-fetal interface in early pregnancy, our 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, as 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 our current study (Supplemental Figure 3).

However, equivalent or elevated levels of GnRH transcripts in cytotrophoblasts compared to

syncytiotrophoblast in intrauterine pregnancies (25), and we observed a similar staining pattern

for GnRH in control first-trimester human placental samples (Supplemental Figure 3). In contrast,

reduced expression of GnRH was observed in cytotrophoblasts compared to 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 have found that GnRH increases the number of viable BeWo cells (cytotrophoblast-like

cells). In light of these findings it will be interesting to discover if the discrepancy in GnRH

expression between intra-uterine 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 signalling pathways such as IKK-β

and NF-κB have been reported to inhibit GnRH secretion in the hypothalamus (28). Whether

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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, similar to that observed in intra-

uterine 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 oviduct 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 pregnancy, 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 αvβ3,

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 β3 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 analogs 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, our

study provides the first evidence that GnRH positively regulates trophoblastic BeWo cells

viability via GnRHR, as pre-administration of GnRH antagonist Antide abolished this effect. This

effect would most likely be exerted on villous and column cytotrophoblasts given their high

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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, as GnRH signalling 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 the

GnRHR, as it can be inhibited by pre-treatment with GnRH antagonist. Further work is required

to elucidate the role of GnRH signalling in ectopic pregnancy.

ACKNOWLEDGEMENTS

We thank the women who donated Fallopian tube samples and first-trimester placenta

samples used in this study.

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Figure 1. Immunolocalization of GnRH and GnRHR in trophoblasts from women with

tubal ectopic pregnancy. Representative images showing the immunolocalization of GnRH (A,

B, C and D), GnRHR (E, F, G and H), Ki67 (I, J, K and L), cytokeratin 7 (M and O), β-hCG (N

and P) and HLA-G (Q and S) in placental villi of tubal ectopic pregnancy specimens (n=25). A

lack of staining was observed in adjacent control sections incubated with a mixture of mouse and

rabbit control (Ctrl) IgGs (R and T). The cytotrophoblast (CTB) and syncytiotrophoblast (STB)

monolayers are indicated with red and green arrows, respectively. Column cytotrophoblasts are

indicated with CC. EVT cells are indicated with blue arrows. 100× scale bar = 100 µM, 400×

scale bar = 25 µM.

Figure 2. Immunolocalization of GnRHR and GnRH in Fallopian tube from women with

tubal ectopic pregnancy. Representative images showing the immunolocalization of GnRH (A),

GnRHR (B) and cytokeratin 7 (C) in Fallopian tube tissues of tubal ectopic pregnancy specimens

(n=23) . A lack of staining was observed in adjacent sections incubated with a mixture of mouse

and rabbit control (Ctrl) IgG (D). Fallopian tube epithelium (FTE) and stroma (FTS) are indicated

by red and green arrows, respectively. 100× scale bar = 100 µM.

Figure 3. Comparison of GnRH and GnRHR Histoscores between trophoblast and

Fallopian tube cells from women with tubal ectopic pregnancy. Immunostaining intensity

distribution of GnRH and GnRHR in trophoblast and Fallopian tube cells from women with tubal

ectopic pregnancy (A). Dotplot representing the Histoscores of GnRH (B) and GnRHR (C)

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.

Figure 4. GnRH antagonist abolishes GnRH-induced increases in the number of viable

BeWo cells, but no effect of GnRH or GnRH antagonist in HTR-8/SVneo cells. BeWo cells

and HTR-8/SVneo cells were treated with 10 nM GnRH I for 0, 24, 48, 72 or 96h. BeWo (A) and

HTR-8/SVneo (B) viable (non-stained) cell numbers were measured at each time point by Trypan

blue cell counting. Results are presented as the mean ± S.E.M. of three independent experiments,

and significant differences are indicated by asterisks (n=3, *=p<0.05, **=P<0.01).

Supplemental Figure 1. GnRH and GnRHR expression in BeWo and HTR-8/SVneo cells.

The mRNA levels of GnRH in BeWo and HTR-8/SVneo cells are presented in bargraph (A). The

mRNA levels of GnRHR in BeWo and HTR-8/SVneo cells are presented in bargraph (B). The

protein levels of GnRHR in BeWo and HTR-8/SVneo cells are presented in (C).

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Supplemental Figure 2. Lower concentration of GnRH (1nM) does 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 nM GnRH I for 0, 24, 48, 72 or 96h. BeWo (A) and HTR-8/SVneo (B) viable

cell numbers were measured at each time point by Trypan blue cell counting. Results are

presented as the mean ± S.E.M. of three independent experiments.

Supplemental Figure 3. Immunolocalization of GnRHR and GnRH in trophoblast cells

from first-trimester human placenta. Representative images showing the immunolocalization

of GnRH (A and B) and GnRHR (C and D) in trophoblast cells from first-trimester human

placenta. A lack of staining was observed in sections incubated with a mixture of mouse and

rabbit control (Ctrl) IgG (E). 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.

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