BIOLOGY OF REPRODUCTION (2014) 91(3):73, 1–10

Published online before print 13 August 2014.

DOI 10.1095/biolreprod.114.119990

Endocrine Gland-Derived Endothelial Growth Factor (EG-VEGF) Is a Potential Novel

Regulator of Human Parturition1
C. Dunand,3,4,5 P. Hoffmann,3,4,5 V. Sapin,8 L. Blanchon,8 A. Salomon,3,6 F. Sergent,3,6
M. Benharouga,5,6,7 S. Sabra,9 J. Guibourdenche,10 S.J. Lye,9 J.J. Feige,3,5,6 and N. Alfaidy2,3,5,6
3
Institut National de la Santé et de la Recherche Médicale (INSERM U1036), Grenoble, France

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4
Department of Obstetrics and Gynaecology, Grenoble Hospital, Grenoble, France
5
Université Grenoble-Alpes, Grenoble, France
6
Commissariat à l’Energie Atomique (CEA), iRTSV-BCI, Grenoble, France
7
Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5249, Laboratoire de Chimie et Biologie des
Métaux, Grenoble, France
8
EA7281 R2D2, Biochemistry Laboratory, Medicine School, Auvergne University, Clermont-Ferrand, France
9
Lunenfeld-Tanenbaum Research Institute Mount Sinai Hospital, University of Toronto, Toronto, Canada
10
Hôpital Cochin AP-HP, Inserm U767, Faculté de pharmacie, Université Paris Descartes, Paris, France

ABSTRACT INTRODUCTION

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EG-VEGF is an angiogenic factor that we identified as a new
placental growth factor during human pregnancy. EG-VEGF is
also expressed in the mouse fetal membrane (FM) by the end of
gestation, suggesting a local role for this protein in the
mechanism of parturition. However, injection of EG-VEGF to
gravid mice did not induce labor, suggesting a different role for
EG-VEGF in parturition. Here, we searched for its role in the FM
in relation to human parturition. Human pregnant sera and total
FM, chorion, and amnion were collected during the second and
third trimesters from preterm no labor, term no labor, and term
labor patients. Primary human chorion trophoblast and FM
explants cultures were also used. We demonstrate that
circulating EG-VEGF increased toward term and significantly
decreased at the time of labor. EG-VEGF production was higher
in the FM compared to placentas matched for gestational age.
Within the FM, the chorion was the main source of EG-VEGF.
EG-VEGF (endocrine gland-derived endothelial growth
factor), also called prokineticin 1, is the canonical member of
a recently described family of cytokines, the prokineticin
family [1, 2]. EG-VEGF expression was mainly reported in
endocrine tissues, including the placenta [3–8]. Prokineticin
family members exert their effects via two G protein-coupled
prokineticin receptors, PROKR1 and PROKR2 [1, 9].
Recent data from our group showed that 1) EG-VEGF is
abundant in the human placenta during the first trimester of
pregnancy, with the strongest expression found in the
syncytiotrophoblast [6], 2) its expression is up-regulated by
hypoxia [6], 3) it controls trophoblast invasion and placental
angiogenesis [10], and 4) its circulating levels are significantly
increased in placental pathologies such as preeclampsia and
intrauterine growth restriction [10, 11]. Altogether, these

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EG-VEGF receptors, PROKR1 and PROKR2, were differentially
expressed within the FM with increased expression toward term
and an abrupt decrease with the onset of labor. In chorion
trophoblast and FM explants collected from nonlaboring
patients, EG-VEGF decreased metalloproteinase-2 and -9 activ-
ities and increased PGDH (prostaglandin-metabolizing enzyme)
expression. Altogether these data demonstrate that EG-VEGF is a
new cytokine that acts locally to ensure FM protection in late
pregnancy. Its fine contribution to the initiation of human labor
is exhibited by the abrupt decrease in its levels as well as a
reduction in its receptors.
EG-VEGF, fetal membranes, human pregnancy, mechanism of
parturition, preterm birth, trophoblast
1 mechanism of parturition [13]. To date, no studies have
Supported by INSERM U1036, University Joseph-Fourier, Commissar-
iat à l’Energie Atomique (DSV/iRTSV/BCI), the region Rhônes Alpes,
and the Canadian Institutes of Health Research (to S.J.L.). Presented in
part at the 61st Annual Meeting of the Society for Gynecologic
Investigation, Florence, Italy, 25–29 March, 2014.
2
Correspondence: Nadia Alfaidy, HDR, Unité INSERM U1036,
Laboratoire BCI-iRTSV, CEA, Grenoble 17, rue des Martyrs, 38054
Grenoble cedex 9, France. E-mail: nadia.alfaidy-benharouga@cea.fr
Received: 3 April 2014.
First decision: 6 May 2014.
Accepted: 14 July 2014.
Ó 2014 by the Society for the Study of Reproduction, Inc.
eISSN: 1529-7268 http://www.biolreprod.org
ISSN: 0006-3363
1 Article 73
findings suggest that EG-VEGF is a new pregnancy factor
that might play additional roles to those established during
early pregnancy. Former publications that identified EG-VEGF
and its analog, prokineticin 2, reported roles in the regulation of
contractility for these factors in the gastrointestinal smooth
muscle [2], suggesting a potential role for EG-VEGF in the
mechanism of parturition, a multistep process that includes
myometrial contraction, cervical ripening, and fetal membrane
(FM) rupture [12]. In relation to the FM, Lannagan et al. [13]
recently showed that EG-VEGF mRNA expression increased
significantly in murine FM on Day 18 (as compared with Day
16) but not in the uterus or placenta. Nevertheless, intrauterine
injection of EG-VEGF in gravid mice did not result in preterm
delivery, suggesting a different role for EG-VEGF in the
examined the expression of EG-VEGF and its receptors,
PROKR1 and PROKR2, in the human FM during late human
pregnancy and in relation to the labor process. A role in the
mechanism of parturition in humans remains to be investigated.
Human FM represent a complex multilaminate tissue
composed of the amnion and the chorion, two closely adherent
layers that consist of several cell types, including epithelial and
mesenchymal cells for the amnion and cytotrophoblastic and
mesenchymal cells for the chorion. This tissue is characterized
by the presence of abundant local metalloproteinase (MMP)
activities that contribute to their rupture at the onset and
progression of labor [14, 15]. The FMs are also the main
intrauterine site that control prostaglandin (PG) synthesis and
 DUNAND ET AL.
metabolism during the third trimester of pregnancy and at the
time of labor. In women, PG synthesis and metabolism are
discretely compartmentalized in the FM [16]. Human amnion is
a major site of PG synthesis because it expresses the highest
levels of cyclooxygenase (COX-2), the key PG-synthesizing
enzyme. In contrast, the chorion is the major site of PG
metabolism as it expresses the highest levels of 15-hydrox-
yprostaglandin dehydrogenase (PGDH), the key PG-metabo-
lizing enzyme [17, 18]. It is well-established that during the
third trimester of human pregnancy, a fine regulation of PG
synthesis and metabolism occurs to ensure the quiescence of
the uterus until the onset of labor. In the FM, the chorion layer
is a key compartment of the FM that ensures this quiescence by
preventing active PGs from crossing to the decidua and
myometrium before the onset of the labor process [16, 18, 19].
However to date, little is known about local regulators of these
processes in the intrauterine tissues and particularly in the FM.
Because EG-VEGF exhibits high circulating levels in
preterm-associated pregnancies, such as preeclampsia and
intrauterine growth restriction [10, 11], and because its local
levels increase in the FM during late gestation, we hypothe-
sized that this factor might a play a direct role in human
parturition, other than triggering the onset of labor. Here, we
investigated the role of EG-VEGF in human parturition during
late pregnancy and in relation to the labor process. We
compared the levels of circulating EG-VEGF in sera collected
from pregnant women at the second and the third trimesters, in
the absence or the presence of labor, determined the site of
expression of EG-VEGF and its receptors within the human
FM, and determined the ontogeny of these proteins in late
pregnancy and in relation to the initiation of labor. We have
also compared the levels of EG-VEGF in FM versus placental
explant cultures and in chorion versus amnion explant cultures.
More importantly, we characterized the mechanism by which
EG-VEGF contributes to the control of the onset of human
labor; this includes the regulation of the key parturition
enzymes, MMP-2 and -9 and PDGH.
MATERIALS AND METHODS
Tissue and Sera Collection
Tissue collection. Third-trimester human FMs were collected from women
aged 33–39 (n ¼ 23; average age 36 yr) with uncomplicated healthy singleton
pregnancies (no infection, no fetal growth restriction, no preeclampsia, and no
premature rupture of membranes). Second-trimester human FMs (n ¼ 7;
average age 33 yr) were provided by the Mount Sinai Hospital of Toronto,
Canada, with the same sampling conditions. These samples were collected for a
study published in 2003 by N. Alfaidy et al. [20]; the study examined the in situ
ontogeny of the enzyme 11-beta-hydroxysteroid dehydrogenase expression in
the FMs throughout human pregnancy. The indications for the preterm
nonlaboring patients in this study included antepartum hemorrhage, cervical
cancer, and multiple pregnancy. There were no clinical or pathological signs of
preeclampsia or other placental disease. Term human placentas and FMs were
obtained from uncomplicated pregnancies after elective cesarean section
(nonlaboring women, n ¼ 6) and after spontaneous labor (n ¼ 6). To compare
EG-VEGF expression in amnion and chorion layers, FMs collected from
nonlaboring women were further dissected to separate the amnion (n ¼ 3) and
the chorion (n ¼ 3); those samples were used in quantitative RT-PCR
experiments. Samples were either stored at 808C shortly after collection or
fixed in paraformaldehyde 4% for 24 h at 48C and then stored in 70% alcohol.
Sera collection. The sera were obtained from another group of pregnant
women during their routine visits at the second and third trimesters of
pregnancy at Mount Sinai Hospital of Toronto, Canada. The blood samples
were divided into three groups according to the term and type of delivery: term
deliveries not in labor (n ¼ 11, average age and term: 35 yr and 39 wk of
gestation [wg], respectively), term deliveries in labor (n ¼ 12, average age and
term: 32 yr and 39 wg, respectively), and preterm deliveries not in labor (n ¼
14, average age and term: 30 yr and 26 wg, respectively). There were no
clinical or pathological signs of preeclampsia or other placental diseases.
2 Article 73
Ethics. Tissues and sera collections were approved by the Ethics
Committees of the University Hospital of Grenoble and Mount Sinai Hospital,
respectively. Written informed consents were obtained from patients before
collection.
Immunohistochemistry
FMs were fixed for 24 h at 48C in 4% (vol/vol) paraformaldehyde, then in
70% alcohol. They were embedded in paraffin and cut into 5 lm sections as
described previously [21]. FM sections were deparafinized and rehydrated.
Endogenous peroxidase activity was quenched using 40 ml of pure methanol
mixed with 1.3 ml of 30% hydrogen peroxide solution (Sigma-Aldrich) for 30
min at room temperature (RT). After two washes using PBS, nonspecific
protein binding was blocked by 5% normal goat serum for 1 h at RT (Dako).
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Sections were then incubated with rabbit anti-human EG-VEGF, PROKR1, or
PROKR2 polyclonal antibodies for 24 h at 48C (45 lg/ml anti-EG-VEGF, 33
lg/ml anti-PROKR1, 69 lg/ml anti-PROKR2) (Covalab). Anti-cytokeratin 7
antibody, a marker of trophoblast cells, was also used to identify the chorion
leaf in first-trimester specimens. After two washes with PBS, a biotinylated
secondary antibody (1.1 g/L polyclonal goat anti-rabbit biotinylated immuno-
globulin) (Dako) was applied for 1 h at 1:400 dilution followed by peroxidase-
avidin-biotin complex for 1 h (Vectastain ABC Kit; Vector Laboratories).
Peroxidase activity was detected by incubation with 3,3 0 -diaminobenzidine for
30 sec to 1 min (liquid 3,3 0 -diaminobenzidine and substrate chromogen system)
(Dako); the reaction was stopped in deionized water. The sections were then
washed, counterstained with hematoxylin, dehydrated, and mounted with
Merckoglass. Negative controls were performed using PBS 13 buffer instead of
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the primary antibody. Photographs were taken using a Zeiss AxioCam HRc
coupled to a Zeiss Axioplan microscope.
Immunofluorescence
Tissue sections were rehydrated in serial dilutions of alcohol (100%, 90%,
70%, and 50%) and washed in PBS. Nonspecific binding of antibodies was
blocked with 1% bovine serum albumin in PBS for 2 h at RT. The samples
were then incubated with rabbit anti-human cytokeratin 7, anti-PROKR1 (33
lg/ml), and anti-PROKR2 (69 lg/ml). Tissue sections were incubated
overnight (18–24 h) with the primary antibodies at 48C. After the incubation
period, the sections were washed three times in 0.1 M PBS. The secondary
antibodies were added and incubated at RT for 2 h. The secondary antibodies
used were fluorophore-labeled Alexa Fluor 568 goat anti-rabbit immunoglob-
ulin G (IgG) and Alexa Fluor 488 goat anti-mouse IgG (1:200 dilution in 1%
bovine serum albumin; Molecular Probes). Samples were washed again in PBS
and then dehydrated in serial dilutions of alcohol (50%, 70%, 90%, and 100%).
Anti-fading reagent, 1 mg/ml p-phenylendiamine, in 50% glycerol and PBS
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was added to the tissue sections, and coverslips were applied before
observation.
Western Blot Analysis
Frozen placental samples were homogenized on ice with a Polytron 1200
mixer (Bioblock Scientific) in 1 ml of RIPA lysis buffer (Sigma-Aldrich) and
10 ll of protease inhibitor cocktail (Sigma-Aldrich). The homogenates were
centrifuged (15 000 3 g at 48C) for 15 min, and the supernatants were collected.
Protein concentrations were determined using the Bradford assay with a
Multiscan EX (Thermo LabSystems) and Accent Software 2.6. Fifty
micrograms of proteins extracted from total FM, amnion or chorion were
electorophoretically separated in 12% acrylamide gels, and electrically
transferred onto 0.45 lm nitrocellulose membranes (BioRad). Appropriate
transfer was confirmed by protein staining with Ponceau S (Sigma-Aldrich).
The blots were washed with PBS and 0.1% Tween (PBS-T) and incubated 1 h
in blocking solution (5% skimmed milk in PBS-T). Membranes were
immunoblotted overnight with a rabbit antibody against PROKR1 or PROKR2
(1:400 of 33 lg/ml anti-PROKR1 or 1:300 of 69 lg/ml anti-PROKR2)
(Covalab). Blots were then rinsed three times with PBS-T and incubated with
biotinylated goat anti-rabbit IgG (450 ng/ml, 1:2000 dilution in blocking
solution; Dako) for 30 min. After three PBS-T washes, the membranes were
incubated with a peroxidase-conjugated extravidin (1:2000 dilution in blocking
solution; Sigma-Aldrich) for 30 min. Blots were washed four times with PBS-
T, and the antibody-antigen complex was detected using the ECL detection
system (GE Healthcare Life Science). The membranes were then exposed to
radiographic films (Kodak Scientific Imaging Products). The intensities of
immunoreactive bands were measured by scanning the photographic film and
analyzing the image on a desktop computer using Image J software. The
PROKR1 and PROKR2 protein were detected between 47–50 kDa. The mean
pixel density for each band was analyzed to obtain relative optical density units
for PROKR1 and PROKR2 protein expression. To standardize for sample
 EG-VEGF CONTROLS HUMAN PARTURITION

TABLE 1. Primers used for real-time RT-PCR.

Genes Forward, 5 0 to 3 0 Reverse, 5 0 to 3 0

EG-VEGF TG AGG AAA CGC CAA CAC CAT CC GGG AAC CTG GAG CAC
GAPDH ACC CAG AAG ACT GTG GAT GG TTC TAG ACG GCA GGT CAG GT

loading, the blots were stripped in 5 ml of PBS-T mixed with 500 ll of Re-blot
Plus Mild solution 103 (Millipore) and reprobed with an anti-b-actin antibody
(Sigma-Aldrich) as an internal control for protein loading. The b-actin antibody
detects a 37 kDa protein.
Zymography
Twenty microliters of harvested explant culture medium were electropho-
resed under nonreducing conditions in a 10% acrylamide gel containing 1 mg/
ml gelatin (Sigma) as previously described, [23]. After electrophoresis, the gels

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EG-VEGF Quantification by ELISA
EG-VEGF in the collected sera and in conditioned media collected from
explant cultures was measured by ELISA (PeproTech). Two separate standard
curves were constructed to allow accurate readings of samples at upper and
lower ranges of the assay. All the samples were in the linear range of the
standard curves. The detection limit of the assay was 16 pg/ml. Data were
expressed as pg/ml of sera or as pg/mg of protein extracted from total FM or
from separated chorion and amnion tissues.
Real-Time RT-PCR Analysis
Total RNAs were extracted from the placenta, FM, chorion, and amnion
using the Nucleospin RNA kit (Macherey-Nagel). First-strand cDNAs were
were washed at RT for 1 h in 2.5% Triton X-100 and 50 mM Tris-HCl, pH 7.5,
and then incubated at 378C overnight in buffer containing 150 mM NaCl, 5 mM
CaCl2, and 50 mM Tris-HCl, pH 7.6. Thereafter, gels were stained with 0.1%
(wt/vol) Coomassie brilliant blue R-250 in 30% (vol/vol) isopropyl alcohol and
10% glacial acetic acid for 60 min and destained in 10% (vol/vol) methanol and
5% (vol/vol) glacial acetic acid. Semiquantification of the bands corresponding
to 72- and 92-kDa gelatinases was performed with a densitometer.
Statistical Analysis
Statistical comparisons were made using Student t-test and one-way
ANOVA (both parametric and nonparametric) followed by Dunn or Bonferroni
tests. All the data were checked for normality and equal variance. (SigmaPlot

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synthesized from 1 lg total RNA by reverse transcription using the iScript
system (Bio-Rad) according to the manufacturer’s instructions. Quantitative
real-time RT-PCR, using SYBER-green real-time PCR assay, was performed
on a Bio-Rad CFX96 apparatus and quantitative PCR Master Mix (Promega).
Relative quantification of gene expression was normalized to GAPDH mRNA
expression level. Sequences of the PCR primers used are listed in Table 1.
Placental and FM Explant Cultures
Samples were collected and stored at 48C in culture medium (DMEM:Ham
F-12; 1:1) with an addition of gentalline (1:1000) and penicillin and
streptomycin (1:100). Forty-eight-well plates were coated with 150 ll/well of
an equal mix of Matrigel (BD) and culture medium and preincubated at 378C
for 30 min. Pieces of placenta or FM tissue were dissected, weighed, and placed
on Matrigel for 3 h at 378C to ensure their adhesiveness. Culture medium (300
ll) with or without EG-VEGF (25 and 50 ng/ml) was then added. Each
condition was done in triplicate. We incubated the samples at 378C for 1, 3, 8,
12, or 24 h. The supernatant was collected and stored at 208C. Tissue samples
and SigmaStat; Jandel Scientific Software). All the data are expressed as mean
6 SEM (*P , 0.05 and **P , 0.001).
RESULTS
EG-VEGF Circulating Levels Increase During the Third
Trimester of Pregnancy
To get more insight into the role of EG-VEGF during late
pregnancy, we compared its levels in second- and third-
trimesters sera collected from nonlaboring women only.
Figure1 shows that circulating EG-VEGF levels significantly
increased toward the end of pregnancy, suggesting a potential
role of this factor in the late processes of parturition. The
source of EG-VEGF could not be of placental origin because
both our group and Lannagan et al. [7, 13] showed that

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were homogenized in RIPA and stored at 808C. placental EG-VEGF production does not increase toward the
Primary Chorion Trophoblast Culture
Trophoblast cells from choriodecidual tissue were isolated and cultured using
a modification of the technique described by Kliman et al. [22]. Briefly, human
placentas were obtained from uncomplicated term pregnancies (38–40 wk, n ¼ 3)
after elective cesarean section delivery. The chorion with adherent decidua was
peeled off the amnion and digested three times for 60 min each with 0.125%
trypsin (Gibco), 0.02% deoxyribonuclease I (Sigma), and 0.2% collagenase
(Sigma) in DMEM (Gibco). The dispersed choriodecidual cells were filtered
through 100 lM nylon gauze and loaded onto a continuous Percoll (Sigma)
gradient (5%–70% in 5% steps of 3 ml each) and then centrifuged at 1200 3 g for
20 min at RT to separate different cell types. Cytotrophoblast cells between the
density markers of 1.049 and 1.062 g/ml were collected and plated in 24-well
plates (Corning) at a density of 106 cells/ml. Cells were also plated in 8-well
chamber slides (Lab-Tek) (0.3 3 106 cells/well) for immunostaining. The cells
were cultured for 3 days at 378C in 5% CO2/95% air before experimental
treatments. Under these conditions, the chorionic trophoblast cells formed small
clumps or remained as single cells. The purity of the cell preparation was
assessed at the end of the experiment by histochemical staining for cytokeratin,
an epithelial cell lineage marker (DAKO Corp.) or vimentin, a mesenchymal cell
lineage marker (DAKO Corp.). Cells were counterstained with Carrazzi
hematoxylin. After 72 h, representative cultures were stained as previously
described [21]. Briefly, the cells were fixed with 4% paraformaldehyde and
incubated with 10% normal goat serum that served as a blocking agent for
nonspecific binding. Commercial antibodies were used to detect immunoreactive FIG. 1. Comparison of EG-VEGF serum levels in nonlaboring pregnant
PGDH (Thermo Scientific), cytokeratin (Dako), and vimentin (Dako). The cells women during the second and third trimester of pregnancy. A total of 25
were incubated at 48C for 18 h. For detection, we used the avidin-biotin- serum samples were analyzed (second-trimester samples, n ¼ 14; third-
peroxidase technique and Vectastain ABC kit (Vector Laboratories). Immuno- trimester samples, n ¼ 11). EG-VEGF levels were measured by ELISA. The
reactive proteins were visualized after the addition of 3,30 -diaminobenzidine box plot demonstrates 10th, 25th, 50th, 75th, and 90th percentiles. One-
(Sigma) for 2 min. Cells were counterstained with hematoxylin. way ANOVA was used followed by the Dunn method (*P , 0.05).

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FIG. 2. EG-VEGF secretion from total fetal membrane (FM), placenta, chorion, and amnion. A) A comparison of EG-VEGF secreted levels between FM
explants and their age-matched placentas from 1 to 24 h of culture. EG-VEGF levels were measured by ELISA. One-way ANOVA was used followed by the
Bonferroni test (*P , 0.05; **P , 0.001). B) A comparison of EG-VEGF secreted levels between the chorion and amnion of same placentas. EG-VEGF
levels were measured by ELISA. One-way ANOVA was used followed by the Bonferroni test (*P , 0.05). C) A comparison of EG-VEGF mRNA levels in
placenta (n ¼ 5), FM (n ¼ 3), chorion (n ¼ 3), and amnion (n ¼ 3) samples collected from nonlaboring pregnant women at term of their pregnancy. Student t-
test was used to compare values (*P , 0.05). Different letters are significantly different from each other.

end of pregnancy. These data suggested that another source of
EG-VEGF might exist during late pregnancy.
EG-VEGF Is Produced by Human FMs
Using our established explant culture model, we compared
EG-VEGF production in human FMs and in their age-matched
control placentas. Figure 2A reports a time course secretion of
EG-VEGF from total FM and their respective placentas. EG-
VEGF production was significantly higher in the FM than in
the placenta. More importantly, the chorion layer within the
FM was the main source of EG-VEGF (Fig. 2B). Further
comparisons of EG-VEGF mRNA content in the placenta, total
FM, chorion, and amnion is reported in Figure 2C. The data
show that the chorion layer expresses significantly more EG-
VEGF mRNA compared to the placenta and to the amnion.
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EG-VEGF and PROKRs Expression in Human FM
Here, we determined the localization of EG-VEGF and its
receptors, PROKR1 and PROKR2, within the FMs in
nonlaboring women at term pregnancy. Figure 3Aa shows
that EG-VEGF is mainly expressed in the chorio-decidua layer
with an intense signal in the chorion layer. A strong expression
was also observed in the amnion epithelial layer. Figure 3, Ab
and Ac, show PROKR1 and PROKR2 expression, respective-
ly. PROKR1 was localized to all layers of the FM. The
strongest expression was observed in the chorion mesenchymal
layer. PROKR2 was expressed in the chorion layer, with faint
expression in the amnion epithelial cells. Figure 3Ad is a
negative control that illustrates FM layers. To further
characterize PROKR1 and PROKR2 expression, we used the
immunofluorescence technique to search for colocalization of
PROKR1 and PROKR2 with cytokeratin 7, a marker of
trophoblast and epithelial cells. Figure 3Ba shows that
PROKR1 is mainly expressed in the chorion mesenchymal
layer, while PROKR2 is mainly expressed in the trophoblast
 EG-VEGF CONTROLS HUMAN PARTURITION

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FIG. 3. EG-VEGF, PROKR1, and PROKR2 expression in human fetal membranes (FMs). FMs were collected from nonlaboring women during the third
trimester of pregnancy. A) Immunohistochemistry of term FM immunostained (Brown staining) with anti-EG-VEGF (a); anti-PROKR1 (b), and anti-PROKR2
(c); d is a negative control of the staining. All the sections were counterstained with hematoxylin. Photographs in a 0 , b 0 , and c 0 are higher magnifications of
a, b, and c, respectively. Bars ¼ 50 lm. B) Immunofluorescence of term FMs immunostained with anti-PROKR1 (a); anti-cytokeratin (b and e), and anti-
PROKR2 (d). Colocalization of PROKR1 and cytokeratin is shown in c and colocalization of PROKR2 and cytokeratin is shown in d. Amnion epithelial
cells (Ae), amnion mesenchymal cells (Am), decidua (De), and cytotrophoblast (Ct). Bars ¼ 50 lm.

layer of the chorion (Fig. 3Bd). Figure 3, Bb and Be, show
cytokeratin staining in the same sections. An obvious
colocalization of cytokeratin and PROKR2 could be observed
in the merged photographs shown in Figure 3Bf. A faint
colocalization of PROKR1 and cytokeratin was observed in the
chorion trophoblast, confirming that in the chorion, the
mesenchymal layer is the major site for PROKR1 expression.
We then compared EG-VEGF, PROKR1, and PROKR2
expression in the second- and third trimester FMs collected
from nonlaboring women. Figure 4A–C show a significant
increase in the expression of EG-VEGF and PROKR1. The
increase in PROKR2 expression did not reach significance.
EG-VEGF, PROKR1, and PROKR2 Expression Decreased
with the Onset of Labor
The increase in EG-VEGF levels during late pregnancy
suggested a potential role of the EG-VEGF/PROKRs system in
the mechanism of parturition. Figure 5A shows a comparison
of EG-VEGF local expression in the FMs collected from
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nonlaboring and laboring women. There was an important
decrease in the EG-VEGF protein expression with labor. Figure
5B shows a representative Western blot analysis that compared
EG-VEGF protein levels in both conditions in the FMs, and
Figure 5C shows the quantification of EG-VEGF protein
levels. There was a significant decrease in EG-VEGF protein
levels with the labor process. We then compared circulating
levels of EG-VEGF between laboring and nonlaboring sera.
The data show that EG-VEGF circulating levels significantly
decreased with the labor process (Fig. 5D).
Using our explant culture model, we conducted a time
course comparison of EG-VEGF production in nonlaboring
versus laboring FMs. Figure 5E shows that EG-VEGF
production from laboring FM explants was significantly lower
compared to the production by FM explants collected from
nonlaboring women.
To further characterize the role of EG-VEGF/PROKRs
system in the labor process, we compared the levels of
expression of PROKR1 and PROKR2 in the same conditions.
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FIG. 4. EG-VEGF, PROKR1, and PROKR2 expression during late pregnancy. A) A comparison of the levels of EG-VEGF mRNA expression in total FM
collected from the second and third trimesters of pregnancy. B and C) Comparisons of PROKR1 and PROKR2 protein levels in total FM collected from the
second and third trimesters of pregnancy. Student t-test was used to compare the value in each graph (*P , 0.05); ns, not significant.

Figure 6A shows that PROKR1 and PROKR2 expression
significantly decreased in the FM collected from laboring
women compared to the nonlaboring ones. These observations
were substantiated using Western blot analyses (Fig. 6, B and
C).
Evidence for EG-VEGF Direct Involvement in FM Protection
in Late Pregnancy
Chorion trophoblast cells are the main site of PG
metabolism, playing a major role in the quiescence of the
intrauterine tissues during late pregnancy. This role is due to
the high level of expression of the PG-metabolizing enzyme,
PGDH. To determine the effect of EG-VEGF on the expression
of PGDH, we isolated primary chorion trophoblast cells.
Supplemental Figure S1A (available online at www.biolreprod.
org) shows representative photographs of isolated primary
chorion trophoblast cells. The preparation contained mainly
trophoblast cells, as illustrated by the cytokeratin 7 staining
6 Article 73
(Supplemental Fig. S1Aa). Vimentin-positive cells could also
be observed as seen by the presence of some chorion
mesenchymal and/or decidual cells (Supplementary Figure
S1Ab). Confirmation of EG-VEGF, PROKR1, and PROKR2
expression by isolated chorion trophoblast cells is reported in
photographs (Supplemental Fig. S1, Bd–Bf).
Isolated chorion cells were used to assess EG-VEGF effect
on PGDH expression. Figure 7A shows that treatment of
chorion cells with EG-VEGF (25 and 50 ng/ml) significantly
increased the expression of PGDH. These data were substan-
tiated by Western blot analysis (Fig. 7B).
Besides their role in the regulation of PG metabolism,
chorion cells are also involved in the control of membrane
rupture in late pregnancy via fine regulation of the enzymes
MMP-2 and -9, key MMPs that are abundantly present in the
chorion layer. MMP-2 and -9 expression and activity have been
shown to increase with the onset of labor [23]. Using
zymography, we determined the effect of EG-VEGF on the
activity of MMP-2 and -9 in the FMs. Figure 7C reports a
 EG-VEGF CONTROLS HUMAN PARTURITION

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FIG. 5. Effect of the labor process on EG-VEGF circulating and FM levels. A) Representative photographs of EG-VEGF in term FMs collected from
nonlaboring (n ¼ 6) and laboring women (n ¼ 6). B) A Western blot analysis of EG-VEGF protein expression in nonlaboring versus laboring samples. C) The
quantification of the Western blot analysis. Student t-test was used to compare the values in the graph (*P , 0.05). D) A comparison of circulating EG-
VEGF levels in nonlaboring versus laboring women. A total of 23 serum samples were analyzed (labor samples, n ¼ 12; nonlabor samples, n ¼ 11). EG-
VEGF levels were measured by ELISA. One-way ANOVA test was used followed by Kruskal-Wallis analysis of variance on ranks (*P , 0.05). Amnion
epithelial cells (Ae), amnion mesenchymal cells (Am), decidua (De), cytotrophoblast (Ct), Bars ¼ 50 lm. E) A time course comparison of EG-VEGF
secretion from FM explants collected from nonlaboring and laboring women. Data were obtained from four independent experiments performed in
triplicate and represent the mean 6 SEM. The Student t-test was used to compare the values for each time point (**P , 0.001).

representative zymogram that shows that EG-VEGF decreased
the activities of MMP-2 and -9 in chorion cells isolated from
nonlaboring women. Quantification of three independent
experiments showed a significant decrease in MMP-2 and -9
activities upon EG-VEGF treatment (Fig. 7D).
DISCUSSION
An increasing number of reports in the literature suggest that
EG-VEGF is involved in the reproduction processes, particu-
larly those associated with pregnancy [3–5, 8, 10]. Our group
has shown that EG-VEGF is highly expressed in human
placenta during the first trimester of pregnancy and demon-
7 Article 73
strated its roles in 1) placental angiogenesis, 2) trophoblast
proliferation and survival, and 3) the control of premature
trophoblast invasion of the maternal decidua [3, 10].
Furthermore, we showed that EG-VEGF circulating levels
were elevated in preeclampsia and in intrauterine growth
restriction, raising the possibility that EG-VEGF might
contribute to these disorders [3, 10].
In term pregnancy, we and others have shown that EG-
VEGF expression decreased locally in the placenta with stable
circulating levels found toward term (i.e., measuring both
laboring and nonlaboring specimens). However, when consid-
ering only nonlaboring second- and third-trimester samples, we
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FIG. 6. Effect of labor on PROKR1 and PROKR2 expression. A) Representative photographs PROKR1 (a and b) and PROKR2 (c and d) expression in the
FMs of nonlaboring and laboring women at term. Photographs in e and f are negative controls. Amnion epithelial cells (Ae), amnion mesenchymal cells
(Am), decidua (De), and cytotrophoblast (Ct). Bars ¼ 50 lm. B and C) Western blot analysis of PROKR1 and PROKR2 expression in the FMs of nonlaboring
and laboring women at term. Data represent the mean 6 SEM of at least four independent FMs collected from different women. The Student t-test was
used to compare the values for each time point (*P , 0.05).

found a significant increase in EG-VEGF levels, suggesting a
role for this factor in late pregnancy. Based on this observation,
we demonstrated a new source for EG-VEGF in the chorion
layer of the FMs as well as a local role for this cytokine during
late pregnancy. The present data demonstrate a significant fall
in EG-VEGF expression in FM that correlates with the
initiation of labor. Hence, we propose that EG-VEGF is a
local intrauterine factor that is highly produced by the chorion
trophoblast cells and acts in an autocrine and paracrine manner
to ensure the quiescence of the intrauterine system during late
pregnancy. Our assumptions are based on three main
observations: 1) EG-VEGF and its receptors levels increased
toward the end of pregnancy in a site where quiescence is
required during this gestational period; 2) their levels
significantly decreased in the FMs with the onset of labor,
8 Article 73
which might allow parturition processes to occur; and 3) EG-
VEGF directly controls the levels of expression of three key
enzymes known to govern the mechanisms of parturition and
are reportedly strongly associated with premature membrane
rupture [12, 24]. More importantly, the observed local profile
of expression of EG-VEGF during late pregnancy was
correlated with its circulating levels in agreement with our
observation of a significant increase throughout late pregnancy
and an abrupt decrease with the onset of labor.
These data suggest that deregulations in the levels of
angiogenic factors such as EG-VEGF might well be associated
with preterm deliveries during human pregnancy. An imbal-
ance between angiogenic and antiangiogenic factors is well-
known to be associated with pregnancy pathologies such as
preeclampsia [25–27], fetal growth restriction [28, 29],
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FIG. 7. Effects of EG-VEGF on PGDH expression and on MMP-2 and -9 activities in primary chorion trophoblast cultures. A) Immunoreactive PGDH in
chorion trophoblast culture incubated in the absence or presence of EG-VEGF (25 and 50 ng/ml) for 24 h. Bars ¼ 20 lm. B) Representative Western blot
analysis of EG-VEGF effects on PGDH expression in chorion trophoblast cells for 24 h. The histogram represents the mean 6 SEM of PGDH levels in the
control and 50 ng/ml EG-VEGF. Three independent experiments were performed. The histogram represents the mean 6 SEM of PGDH levels in the control

1-10/2434122 by guest on 07 November 2018

and 25 and 50 ng/ml EG-VEGF. Three independent experiments were performed (*P , 0.05). C) The effect of EG-VEGF on MMP-2 and -9 activities in
chorion trophoblast cells. Student t-test was used to compare the treated conditions to the control (*P , 0.05). Upper zymogram shows a representative
zymography of cells treated or not with EG-VEGF for 24 h at indicated concentrations. D) The lower histogram shows densitometric analysis of
zymographs of three independent experiments. Each bar represents the amount of MMP-2 or -9 as arbitrary units. Data are presented as the mean 6 SEM.
Student t-test was used to compare the values for each time point (*P , 0.05 and **P , 0.001). CTL, control.

placental abruption [30], and unexplained fetal death [31].
However, there is a paucity of information regarding the
changes of these factors in patients destined to develop
spontaneous preterm labor. Further studies are needed to
determine whether the angiogenic factor EG-VEGF could be
used as a biomarker of prematurity.
EG-VEGF and its receptors were mainly localized to the
chorion layer with a privileged expression of EG-VEGF and
PROKR2 to the trophoblast layer and PROKR1 to the
mesenchymal layer. These findings are in line with our first
publication reporting the same localization of EG-VEGF and
PROKR2 to the same cell types, that is, the syncytiotrophoblast
in the placenta, and PROKR1 to another cell type, the
cytotrophoblast [6]. Nevertheless, the immunohistochemistry
staining showed that EG-VEGF is also present in the
juxtaposed decidua. The presence of EG-VEGF in the decidua
is not surprising because this was previously described at the
mRNA level, especially during early pregnancy [4, 5, 8]. In the
present study, we do not exclude the presence of EG-VEGF in
the decidua because EG-VEGF could be both produced by this
tissue and accumulated within it before getting into the
9 Article 73
circulation. The decidua is a vascularized tissue and the FMs
are not [32]. Hence, even if the FMs and especially the chorion
constitute an important source of EG-VEGF, this molecule
must accumulate in the decidua before getting into the
circulation. The decidua might then be both a source and a
niche for EG-VEGF protein.
Previous established studies have demonstrated that PGDH
plays a major role in the control of active PGs that reach to the
decidua to trigger contractions. A decrease in PGDH
expression is associated with premature rupture of membranes
[33]. It has also been demonstrated that the activity of this
enzyme is controlled by glucocorticoids and by inflammatory
cytokines such as IL8 and IL10 [34]. Nevertheless, while these
factors strongly regulate PGDH expression and activity, they
act in paracrine manner because they are both produced by the
placenta. In contrast, EG-VEGF appears to be a new local
regulator of PGDH that is produced by the same cell type.
More importantly, this is the first time where a regulation of
PGDH by a placental angiogenic factor is reported and that the
onset of labor is controlled by an angiogenic factor.
 DUNAND ET AL.
A confirmation of the proposed role for EG-VEGF in FM
protection is its ability to inhibit the activities of MMP-2 and -9
in the FM explants. The inhibition of MMPs activity in the FM
substantiates our previous observation of an inhibition of these
enzymes by EG-VEGF in the placenta during early pregnancy
[10].
Because EG-VEGF belongs to the prokineticin family, with
a described role in intestinal contraction [2], it was suggested
that EG-VEGF might play a role in the contraction of the
decidua at the time of labor. However, repeated injections of
EG-VEGF to gravid mice during late gestation did not trigger
any contractions, and mice proceeded normally to term [13].
These findings confirm the notion that EG-VEGF is rather a
protecting than a constrictor factor during late pregnancy.
In conclusion, we report for the first time a specific and
local expression of EG-VEGF and its receptors in human FM,
with a local increase toward the end of gestation. We have also
reported an increase of its circulating levels toward term and
proposed a role and a mechanism of action for this protein
within the FM. These data are of enormous clinical importance
because treatments using recombinant EG-VEGF during late
pregnancy could be proposed to maintain suspected premature
deliveries.
ACKNOWLEDGMENT
We thank Ms. Vanessa Garnier for her technical help.
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