Research ajog.

org

OBSTETRICS
Progesterone inhibits in vitro fetal membrane
weakening
Deepak Kumar, MD; Edward Springel, MD; Robert M. Moore, MS; Brian M. Mercer, MD;
Elliot Philipson, MD; Joseph M. Mansour, PhD; Sam Mesiano, PhD; Fredrick Schatz, PhD;
Charles J. Lockwood, MD; John J. Moore, MD

OBJECTIVE: Inflammation/infection and abruption are leading causes of
preterm premature rupture of the membranes. Recently, we identified
granulocyte-macrophage colony-stimulating factor (GM-CSF) as a crit-
ical mediator of both tumor necrosis factor-ae (TNF; modeling
inflammation) and thrombin-induced (modeling abruption) weakening of
the fetal membranes. We found that (1) TNF and thrombin both induced
GM-CSF in the choriodecidua, (2) blockade of GM-CSF action with
neutralizing antibodies inhibited both TNF- and thrombin-induced fetal
membrane weakening, and (3) GM-CSF alone induced fetal membrane
weakening. GM-CSF is thus part of an overlap of the inflammation and
abruption-induced fetal membrane weakening pathways. The effects of
progesterone analogs on the pathways by which fetal membranes are
weakened have not been investigated. We examined the effects of
progesterone, medroxyprogesterone acetate (MPA) and 17a-hydrox-
yprogesterone (HP) on TNF- and thrombin-induced fetal membrane
weakening.
STUDY DESIGN: Full-thickness fetal membranes from uncomplicated
term repeat cesarean deliveries were mounted in Transwell inserts in
Minimum Essential Medium alpha and incubated at 37
C in 5% CO2.
The choriodecidua side of the fetal membrane fragments were pre-
incubated with progesterone, MPA, HP, or vehicle for 24 hours. Fetal
membranes were then exposed to TNF, thrombin, or GM-CSF on the
choriodecidua side for an additional 48 hours. The fetal membrane
tissues were then strength tested, and medium from the choriodecidua
and amnion compartments was assayed for GM-CSF content.
RESULTS: TNF and thrombin both weakened fetal membranes and
elevated media GM-CSF levels on the choriodecidua side of the
fetal membrane. Pretreatment with progesterone, MPA, or HP in-
hibited both TNF- and thrombin-induced fetal membrane weakening
and also inhibited the induced increase in GM-CSF. GM-CSF de-
creased fetal membrane rupture strength by 68%, which was inhibited
by progestogen pretreatment with a potency order: progesterone
<MPA <HP.
CONCLUSION: Progestogen pretreatment blocks TNF- and thrombin-
induced fetal membrane weakening by inhibiting both the produc-
tion and action of GM-CSF. These findings are consistent with the
administration of progestogens in the prevention of preterm premature
rupture of the membranes.
Key words: fetal membrane, GM-CSF, medroxyprogesterone acetate,
PPROM, progesterone, thrombin, TNF, weakening

Cite this article as: Kumar D, Springel E, Moore RM, et al. Progesterone inhibits in vitro fetal membrane weakening. Am J Obstet Gynecol 2015;213:520.e1-9.

P reterm premature rupture of the fetal

membranes (PPROM) and resultant
premature birth is a major cause of in-
fant morbidity and death.1,2 Infection/
inflammation with cytokine production
and decidual bleeding/abruption with
thrombin production, respectively, in-
crease the risk of PPROM; however, the
mechanisms by which these conditions
affect PPROM remain obscure.2-7
Research into the physiologic condition
of PPROM is limited because animal
models fail to mimic the human condition

From the Departments of Pediatrics (Drs Kumar and J. J. Moore and Mr R. M. Moore), Reproductive Biology (Drs Springel, Mercer, Mesiano, and J. J.
Moore), and Mechanical and Aerospace Engineering (Dr Mansour), Case Western Reserve University, and Women’s Health Institute, Cleveland Clinic (Dr
Philipson), Cleveland, OH, and the Department of Obstetrics and Gynecology, University of South Florida College of Medicine, Tampa, FL (Drs Schatz and
Lockwood).
Received March 6, 2015; revised May 13, 2015; accepted June 2, 2015.
Supported by March of Dimes Prematurity Research Center Ohio Collaborative grant number 22-FY14-470 and March of Dimes grant number 22-FY15-
003. The funding source had no influence on research or the decision to publish.
The authors report no conflict of interest.
Presented at a podium session at the 62nd annual meeting of the Society for Reproductive Investigation, San Francisco, CA, March 26-28, 2015.
Corresponding author: John J. Moore, MD. jmoore@metrohealth.org, jjm6@case.edu
0002-9378/free  ª 2015 Elsevier Inc. All rights reserved.  http://dx.doi.org/10.1016/j.ajog.2015.06.014

See related editorial, page 447

520.e1 American Journal of Obstetrics & Gynecology OCTOBER 2015

ajog.org
and because cell culture studies do not
address adequately the major tissue
changes that are associated with fetal
membrane weakening and rupture. To
address these shortcomings, we developed
an in vitro explant model system in
which human fetal membrane rupture
strength, which is the major parameter
of clinical interest, and concomitant
(and presumably causative) biochemical
changes within the fetal membrane can be
measured accurately.8-10 Using this system,
we found that cytokines such as tumor
necrosis factor-a (TNF) and interleukin
1ß, which induce tissue level inflamma-
tion, and thrombin, which causes changes
that are associated with abruption and
bleeding, markedly weaken full-thickness
fetal membrane.4,9,11,12 Concomitant
with weakening, these agents induce
biochemical and histologic tissue
changes that mimic those seen in the
physiologic weak zone (fetal membrane
rupture initiation site) in the fetal
membrane region overlying the cer-
vix.9,11-18
Studies of the pathways of TNF/
interleukin-1ße and thrombin-induced
fetal membrane weakening have demon-
strated that the initial tissue and cellular
targets of these agents are in the ch-
oriodecidua component of the fetal
membrane rather than the amnion.19,20
This is consistent with the sources of
inflammation or bleeding that orig-
inate from the maternal side of the
fetal membrane. Using an enhanced
version of the model system that mi-
mics the directional aspect of the fetal
membrane weakening pathways, we
recently demonstrated that TNF or th-
rombin, when applied to only the cho-
riodecidua side of fetal membrane,
caused weakening in the same manner
as when both sides of the fetal mem-
brane were exposed. We also identified
granulocyte-macrophage colony-stimu-
lating factor (GM-CSF) as a critical
intermediate in the fetal membrane
weakening pathways that are induced
by both TNF and thrombin.20 As docu-
mented in this previous report, GM-CSF
is induced in the choriodecidua by
TNF and thrombin in a concentration-
dependent manner that is concomi-
tant with TNF- and thrombin-induced
fetal membrane weakening. Impor-
tantly, GM-CSF alone also causes dose-
dependent fetal membrane weakening
and blockade of GM-CSF with neutral-
izing antibody prevents fetal membrane
weakening by TNF or thrombin.20 These
observations suggest that GM-CSF me-
diates fetal membrane weakening that is
induced by both inflammation and
bleeding.20 It is therefore possible to
reduce the fetal membrane weakening
process into those events that are
involved in the generation of GM-CSF
and those events caused by the action of
GM-CSF. Similarly, inhibitors of
the weakening process can now be cate-
gorized as acting to prevent GM-CSF
generation, acting to inhibit GM-CSF
action, or both. This categorization fa-
cilitates the identification of the point of
action of specific inhibitors and thus
provides information that may allow
their use in inhibition of PPROM.
Currently, progestogens are the only
agents recommended by the American
College of Obstetricians and Gynecolo-
gists for the prevention of preterm
delivery.21 Weekly intramuscular admin-
istration of 17 (OH) progesterone-
caproate or daily vaginal administration
of progesterone have been recommended
for the prevention of recurrent preterm
birth or for short cervical length, respec-
tively.21,22 However, there is no direct ev-
idence that progestogens inhibit fetal
membrane weakening. The cellular tar-
gets for progesterone action in fetal
membrane are thought to be mainly
decidual cells that express the nuclear
progesterone receptor (nPR) isoforms
(PR-A and PR-B).23 In a previous study,
we found that the progesterone analog
medroxyprogesterone acetate (MPA) in-
hibited TNF-induced GM-CSF induction
in decidual cells.24 It thus seemed appro-
priate to hypothesize that progesterone
would inhibit fetal membrane weakening
by blocking GM-CSF production. How-
ever, GM-CSF is proposed to act, in part,
by the activation of resident decidual
mononuclear cells, which express mem-
brane progesterone receptors. Thus, it is
also possible that progesterone could
block fetal membrane weakening by
inhibiting GM-CSF action.25,26 The
studies herein were undertaken to
OCTOBER 2015 American Journal of Obstetrics & Gynecology
Obstetrics Research
determine whether progestogens inhibit
TNF- and thrombin-induced fetal mem-
brane weakening.
M ATERIALS AND M ETHODS
Materials
Humankine TNF, Humankine GM-CSF
(both produced in HEK 293 cells), pro-
gesterone, MPA, 17a-hydroxyproges-
terone (HP), and other miscellaneous
reagents, unless otherwise stated, were
obtained from Sigma-Aldrich, St. Louis,
MO. Thrombin (from bovine plasma,
1500NIH U/mg protein) was obtai-
ned from Thermo Fisher Scientific,
Pittsburgh, PA. RU486 (mifepristone)
was obtained from Cayman Chemical
Co, Ann Arbor, MI.
Fetal membrane collection and
preparation
Full-thickness fetal membrane fragments
from term uncomplicated repeat cesarean
deliveries were collected after patient
consent and approval by MetroHealth
Medical Center’s Institutional Review
Board (# IRB10-00861; Cleveland, OH).
None of the patients had a history of
preterm birth. Fetal membrane tissue was
taken from areas remote from the weak
zone region, washed in 2  250emL
changes of phosphate-buffered saline so-
lution (pH 7.2) and mounted, choriode-
cidua side down, in 24-mm Transwell
(Costar, Corning, NY) inserts that were
secured with an O-ring to separate the
choriodecidua and amnion sides. The
inserts were placed in 6-well culture plates
and 2 mL MEMþ (Minimum Essential
Medium alpha with Earle’s salts, supple-
mented with 1 mmol/L L-glutamine, 2.24
g/L sodium bicarbonate [Mediatech,
Manassas, VA], 10 ml/L Antibiotic-
Antimycotic (Sigma Chemical Co, St.
Louis, MO), and 50 mg/L gentamicin
sulfate) was added to the upper (amnion
side) and the lower (choriodecidua side)
chambers.20 Progestogens (progesterone,
HP, MPA [each 10e7mmol]) or vehicle
(0.01% ethanol final concentration) were
added to the choriodecidua side 24 hours
before the addition of weakening agents.
Where indicated, RU486 (10e8 mmol)
was also added to the choriodecidua side
medium 1 hour before the addition of
progestogens. Weakening agents (TNF
520.e2
Research Obstetrics ajog.org

[20 ng/mL], thrombin [10 U/mL], GM-

CSF [200 ng/mL]) were then added to FIGURE 1
the choriodecidua side, and the cultures Progestogens inhibit thrombin- and TNF aeinduced fetal membrane
were incubated at 37
C in 5% CO2 and weakening
100% relative humidity for an additional
48 hours. After culture, medium from
each compartment was collected, centri-
fuged for 15 minutes at 12,000g/10
C, and
the supernatants were stored at e70
C.
Doses for TNF, thrombin, and GM-
CSF that were used in these studies
were determined from dose-response
studies in our previous publication,20
and they were added to the choriodeci-
dua side only, because our previous
studies have shown that the choriodeci-
dua (rather than the amnion) is the
initial target tissue or the tissue of origin
of each.4,19
Progestogens were chosen for study
with the following rationale: progester-
one was selected as the natural proges-
togen of major focus. MPA was chosen
because it is known to be an effective
inhibitor of TNF-induced GM-CSF
production.24 It also metabolizes less
rapidly in tissue culture than does pro-
gesterone.27 HP was selected as a natural
progestogen that has a strong affinity for
the membranous progesterone receptor
(mPR),28-32 but not the nPR. 17-OH
progesterone caproate was not used in
this study. The concentration of
Preincubation with A, progesterone (10e7 mmol), B, MPA (10e7 mmol), or C, 17a-hydrox-
progestogens used (10e7M) was within
yprogesterone (10e7 mmol) for 24 hours inhibited fetal membrane weakening by either TNF
the range that is seen in pregnant serum
(20 ng/mL) or Thr (10 U/mL) applied for 48 additional hours. In all studies, all agents were applied to
at term. Progestogens were added to the
only the choriodecidual side of the fetal membranes. Strength testing was done at 72 hours for all
choriodecidua side to ensure that they
fetal membrane fragments. The data shown in each panel are for 1 representative experiment that
reached the target tissue concomitant
was performed in triplicate with each condition. Each experiment was repeated 3 times with 3
with the weakening agents.
different placentas (data are presented as mean  SD). Symbols designate pairs of columns with
significant differences (A, the asterisk and plus symbols indicate P < .01, and the circumflex accent
Strength testing of fetal membrane
and number symbols indicate P < .05; B, the asterisk, plus, and circumflex accent symbols indicate
Fetal membrane fragments were
P ¼ .01, and the number symbol indicates P ¼ .02; C, all of the symbols indicate P < .01).
strength-tested within the Transwell in- C, control; HP, 17a-hydroxyprogesterone; MPA, medroxyprogesterone acetate; P, progesterone; Thr, thrombin; TNF, tumor necrosis
serts with the use of our rupture testing factor a.
apparatus, as previously reported.8,13,20 Kumar. Progesterone analogs inhibit fetal membrane weakening. Am J Obstet Gynecol 2015.
Briefly, Transwell-mounted fetal mem-
brane were secured in a 2.5-cm
diameter fixture between the aligned rupture. From these data, force (rupture the choriodecidua and amnion sides of
horizontal plates of the rupture testing strength in newtons) and maximum Transwell cultured fetal membrane were
equipment. A motor-driven 1-cm dia- displacement (centimeters) that were determined with the use of the Human
meter spherical-head plunger that was needed to cause fetal membrane rupture GM-CSF Quantikine ELISA Kit (R&D
aligned perpendicular to the fetal were determined. Systems, Minneapolis, MN), according
membrane was then forced against the to the manufacturer’s protocol. Intra-
amnion side. Fetal membrane dis- GM-CSF determination and interassay precision of the assay was
placement and concomitant plunger GM-CSF levels in thawed supernatants 2.7% and 5.3% coefficient of variation,
force were recorded continuously until from the fluid compartments adjacent to respectively, for cell culture supernatants

520.e3 American Journal of Obstetrics & Gynecology OCTOBER 2015

ajog.org Obstetrics Research
at a sensitivity level of 3 pg/mL with
FIGURE 2 Escherichia coli expressed, recombinant
Effect of progestogens on TNF ae and thrombin-induced GM-CSF human GM-CSF as standard.

Statistical analysis
All experiments were performed at least
in triplicate. Data were analyzed by
analysis of variance followed by post-
hoc pair-wise comparisons (Holm-
Sidak method) with the use of Sigmaplot
software (Systat Software, Inc, Chi-
cago, IL). Differences were considered
significant when at a probability value
of < .05.

R ESULTS
Progestogens inhibit thrombin- and
TNF-induced fetal membrane
weakening
Thrombin or TNF, with or without pro-
gestogens, were each added only to the
choriodecidua side of the fetal membr-
ane to mimic the physiologic conditions
of pregnancy in which the choriodeci-
dua is exposed to the maternal circulation
and the amnion is in contact with the
amniotic fluid.4,19,20 Compared with
control membrane strength, thrombin or
TNF each markedly decreased fetal
membrane rupture strength in all studies

=
GM-CSF released on the choriodecidua side of
the fetal membranes (left) and the amnion side
of the fetal membranes (right) in the experiment
shown in Figure 1 is shown for A, progesterone,
B, MPA, and C, 17a-hydroxyprogesterone. The
data shown in each panel are for 1 representa-
tive experiment that was performed in triplicate
with each condition. Each experiment was
repeated 3 times using 3 different placentas
(data are presented as mean  SD). Symbols
designate pairs of columns with significant dif-
ferences (A, the asterisk and number symbols
indicate P < .01, and the plus symbol indicates
P < .05; B, all of the symbols indicate P < .01;
C, the asterisk, number, and plus symbols
indicate P < .01, and the circumflex accent
symbol indicates P < .05).
C, control; GM-CSF, granulocyte-macrophage colony-stimulating
factor; HP, 17a-hydroxyprogesterone; MPA, medroxyprogester-
one acetate; P, progesterone; Thr, thrombin; TNF, tumor necrosis
factor a.
Kumar. Progesterone analogs inhibit fetal membrane weak-
ening. Am J Obstet Gynecol 2015.

OCTOBER 2015 American Journal of Obstetrics & Gynecology 520.e4

Research Obstetrics
(both P < .01). Preincubation with
progesterone (10e7mmol) inhibited both
thrombin- and TNF-induced fetal mem-
brane weakening (both P < .05;
Figure 1, A). MPA (10e7mmol) pre-
incubation also inhibited fetal membrane
weakening that was induced by thrombin
(P ¼.01) and by TNF (P ¼.02; Figure 1, B).
Finally, HP (10e7mmol) preincubation
inhibited fetal membrane weakening
that was induced by thrombin and
TNF (both P < .01; Figure 1, C).
Preincubation with progesterone, MPA,
or HP alone did not affect rupture
strength (Figure 1).
Effect of progestogens on
GM-CSF production
Because GM-CSF is a critical interme-
diate in both the TNF- and thrombin-
induced fetal membrane weakening
pathways,20 further studies were done
to determine whether progestogens
inhibit TNF- or thrombin-induced fetal
membrane weakening by blocking GM-
CSF production. Consistent with our
previous report, TNF and thrombin
each increased GM-CSF release on the
choriodecidua side concomitant with
fetal membrane weakening (Figure 2;
P < .01).20 MPA markedly inhibited
GM-CSF production by both TNF and
thrombin to control levels (P < .01). HP
markedly inhibited the GM-CSF in-
crease that was produced by TNF
(P < .01) but only partially inhibited the
GM-CSF increase that was produced
by thrombin (P < .05). Finally, proges-
terone partially inhibited the GM-CSF
increase produced by TNF (P < .05).
The progesterone effect on thrombin-
induced GM-CSF production was not
significant. Only small amounts of GM-
CSF were detected on the amnion side of
the fetal membrane (Figure 2).
Progestogens inhibit
GM-CSFeinduced fetal
membrane weakening
Studies were then performed to deter-
mine whether progestogens inhibit
TNF- and thrombin-induced fetal mem-
brane weakening by inhibiting the ac-
tion of GM-CSF. Consistent with our
previous report,20 GM-CSF incubation
on the choriodecidua side of fetal
520.e5 American Journal of Obstetrics & Gynecology OCTOBER 2015
membrane induced significant (P < .01)
fetal membrane weakening (Figure 3).
This GM-CSFeinduced fetal membrane
weakening was almost completely in-
hibited by preincubation with either
MPA (10e7 mmol) or HP (10e7 mmol;
both P < .01). Although preincubation
with progesterone (10e7 mmol) also
blunted GM-CSFeinduced fetal mem-
brane weakening, its effect was not sta-
tistically significant. Rupture strength
after incubation with progesterone, HP,
or MPA alone was not different from
control membranes (Figure 3).
RU486 blocks HP inhibition
of GM-CSFeinduced fetal
membrane weakening
In the experiment with RU486, consistent
with the previously presented experi-
ments, GM-CSF significantly decreased
fetal membrane rupture strength, and
preincubation with HP inhibited the
GM-CSFeinduced fetal membrane we-
akening. RU486 (10e8 mmol) that was
applied 1 hour before HP blocked HP
inhibition of GM-CSFeinduced weak-
ening (P < .01; Figure 4).
C OMMENT
The results presented here demonstrate
for the first time that the natural pro-
gestogens (progesterone and HP) and
the synthetic progestogen (MPA) inhibit
both TNF- and thrombin-induced we-
akening of human fetal membrane
in vitro. These observations suggest that
progesterone blocks both inflammation-
and bleeding-initiated fetal membrane
weakening. Previously, we found that
GM-CSF is a critical intermediate in
TNF- and thrombin-induced weakening
of human fetal membrane. In these ex-
periments, HP and MPA each inhibited
both GM-CSF production by choriode-
cidua- and GM-CSF-induced fetal
membrane weakening. Although pro-
gesterone also decreased both GM-CSF
production and GM-CSFeinduced we-
akening, its inhibitory effect did not
attain statistical significance. Taken
together, these data suggest that pro-
gestogens act at multiple points in the
pathway to fetal membrane weakening,
evidenced by their effect on both GM-
CSF production and action.
ajog.org
FIGURE 3
Progestogens inhibit GM-CSFe
induced fetal membrane
weakening
Preincubation with progesterone, MPA, or 17a-
hydroxyprogesterone (all 10e7 mmol) for 24
hours inhibited fetal membrane weakening by
GM-CSF (200 ng/mL) applied for 48 additional
hours. In all studies, all agents were applied to
only the choriodecidual side of the fetal mem-
branes. Strength testing was done at 72 hours for
all fetal membrane fragments. The data shown
are for 1 representative experiment that was
performed in triplicate with each condition. The
experiment was repeated 3 times with 3 different
placentas (data are presented as mean  SD).
Symbols designate pairs of columns with signifi-
cant differences (all symbols indicate P < .01).
C, control; G, GM-CSF; GM-CSF, granulocyte-macrophage
colony-stimulating factor; HP, 17a-hydroxyprogesterone; MPA,
medroxyprogesterone acetate; P, progesterone.
Kumar. Progesterone analogs inhibit fetal membrane weak-
ening. Am J Obstet Gynecol 2015.
GM-CSF is a pro-inflammatory me-
diator that we have previously shown
to be produced by choriodecidua in
response to either TNF or thrombin
and mediates the fetal membrane
weakening action of both agents.20,24
Decidual stromal cells have been shown
to produce GM-CSF in response to TNF
stimulation that is inhibited by MPA.24
Our present findings confirm progesto-
gen inhibition of TNF-induced GM-CSF
production in choriodecidua by MPA.
They also demonstrate inhibition of
TNF-induced GM-CSF production by
progesterone, and HP and inhibition of
thrombin-induced GM-CSF production
by HP and MPA (Figure 2).
ajog.org
FIGURE 4
RU486 blocks 17a-
hydroxyprogesterone inhibition
of GM-CSFeinduced fetal
membrane weakening
Preincubation initially with RU486 (10e8 mmol)
for 1 hour and then with 17a-hydroxypro-
gesterone (10e7 mmol) for 24 hours blocked the
inhibition of GM-CSF (200 ng/mL applied for 48
additional hours) induced fetal membrane
weakening by 17a-hydroxyprogesterone. In all
studies, all agents were applied only to the
choriodecidual side of the fetal membrane.
Strength testing was done at 72 hours for all fetal
membrane fragments. The data shown are for 1
representative experiment that was performed in
triplicate with each condition. The experiment
was repeated 3 times with 3 different placentas
(data are presented as mean  SD). Symbols
designate pairs of columns with significant
differences (all symbols indicate P < .01).
C, control; G, GM-CSF; GM-CSF, granulocyte-macrophage
colony-stimulating factor; HP, 17a-hydroxyprogesterone;
R, RU486 (mifepristone).
Kumar. Progesterone analogs inhibit fetal membrane weak-
ening. Am J Obstet Gynecol 2015.
GM-CSF is postulated to recruit
mononuclear cells, to stimulate their
conversion to macrophages, and then to
activate macrophages resulting in
increased protease production and fetal
membrane weakening (Figure 5). Nu-
clear progesterone receptors have not
been found in the amnion or chorion or
in the mononuclear cells of the decidua.
In fetal membranes, they have been
described only in decidual cell nuclei.23
Nuclear progesterone receptors have also
not been detected in murine macro-
phages or normal human mononuclear
cells.25,33 Decidual monocytes/macro-
phages and amnion and chorion cells do
express mPRs that make progestogen
modulation of monocytic cell activation
possible.31-33 Peripheral blood mono-
nuclear cells that were obtained from
pregnant women who had been exposed
to sustained progesterone supplementa-
tion demonstrate decreased immunore-
activity in vitro to gram-positive and
gram-negative bacterial infections.34
Progesterone also inhibits proinfla-
mmatory cytokines in a fetoplacental
artery explant model, inhibits TNF- and
interleukin-1einduced MMP-1 and
MMP3 in decidua, and inhibits TNF-
induced apoptosis in fetal mem-
branes.35-37 Our results demonstrate
that progestogens inhibit GM-CSFe
induced fetal membrane weakening
(Figure 3).
In most viviparous species, the process
of parturition is initiated by progesterone
withdrawal.38 Human parturition, how-
ever, occurs without systemic progester-
one withdrawal. Maternal, fetal, and
amniotic fluid concentrations of proges-
terone and HP remain high throughout
pregnancy and labor and delivery.39-41
Term and preterm labor onset in humans
is thus postulated to result from a func-
tional progesterone withdrawal, whereby
the gestation tissue becomes refractory
to progesterone signaling. Several mech-
anisms for functional progesterone
withdrawal have been proposed. One
mechanism likely active in the choriode-
cidua decidual cells is mediated via
changes in the relative levels of the
nPR isoforms, PR-A and PR-B, coupled
with changes in progesterone receptor
coregulator levels, which leads to
decreased progesterone receptor tran-
scriptional activity.42,43 Total progester-
one receptor expression in human
decidual cells is decreased with the onset
of term and preterm labor.44 In a recent
study, decidual cell TNF immunostain-
ing, and protein levels, the PR-A/PR-B
ratio and type II cyclooxygenase were
increased significantly in association with
the onset of labor.45 Interestingly, in
quiescent (ie, not in labor) decidual cells,
TNF induced a concentration-dependent
increase in NF-kB activity was associated
with increased levels of PR-A and type II
OCTOBER 2015 American Journal of Obstetrics & Gynecology
Obstetrics Research
FIGURE 5
Proposed mechanism for fetal
membrane weakening
TNF a (modeling inflammation) and thrombin
(modeling decidual bleeding/abruption) act on
decidua cells in the choriodecidua to produce
GM-CSF. Blocking GM-CSF with neutralizing
antibody blocks both thrombin- and TNF ae
induced weakening.20 GM-CSF is postulated to
recruit mononuclear cells, stimulate their con-
version to macrophages, and then activate mac-
rophages, which results in protease production
and fetal membrane weakening. Progestogens
act on both GM-CSF production and action.
GM-CSF, granulocyte-macrophage colony-stimulating factor;
MMP, matrix metaloproteinase; TNF, tumor necrosis factor a.
Kumar. Progesterone analogs inhibit fetal membrane weak-
ening. Am J Obstet Gynecol 2015.
cyclooxygenase, which suggests a role for
TNF in regulating functional progester-
one withdrawal.45 Similarly, abruption-
associated preterm delivery is associated
with thrombin-mediated functional
progesterone withdrawal that involves
PR-A and PR-B in decidual cells.46 There
is no information on how mPRs change
at the end of gestation.
Whatever the mechanism, pro-
gestogens should not be effective in
mediating any response in term tissue
once functional progesterone with-
drawal has occurred. The experiments
presented here were performed on term
(37-39 weeks of gestation) fetal mem-
branes that were obtained after elective
cesarean deliveries in women who were
not in clinical labor. We must assume
that the robust protective effects of
progesterone (and other progestogens)
that has been demonstrated in these data
were mediated either through the nPRs
in cells in the choriodecidua and that the
520.e6
Research Obstetrics
progesterone receptor isoform ratio in
the cells was conducive to mediating
progestational effects of progesterone or
they were mediated through mPR for
which changes around labor remain
unclear. Because it is not clear at what
point in gestation or how suddenly
the postulated changes in nPR subty-
pes occur, further studies are needed
to determine whether the protective ef-
fects of progestogens remain in fetal
membranes that are obtained from
women in active labor, in whom a
functional progesterone withdrawal
mechanism likely would be activated.
The reported effectiveness of exoge-
nous progesterone in the prevention of
preterm birth suggests a persistence of
tissue responsiveness to progesterone
that is consistent with the results pre-
sented here. Clinical trials suggest that
progestogen prophylaxis reduces the risk
of preterm birth in at-risk women.22,44-50
However, observations regarding the
capacity for progestogen therapy to
prevent PPROM specifically are lack-
ing.22,47-50 In most studies, either
PPROM rates were not reported or pa-
tients with PPROM were excluded from
analysis.48-52 Our results suggest that
progestogen therapy may be effective
in the prevention of PPROM, provided
that it is initiated before the onset
of functional progesterone withdrawal
in the choriodecidua. Studies in myo-
metrial cells suggest that progesterone,
via PR-B, exerts antiinflammatory ac-
tions by desensitizing the cells to pro-
inflammatory stimuli.53,54 Thus, if
progesterone exerts similar effects on
decidual cells, consistent with this scen-
ario, progestogen supplementation may
enhance the capacity of endogenous
progesterone to exert antiinflammatory
actions and thus prevent inflammation-
induced fetal membrane weakening.
There are data that suggest the effec-
tiveness of progestogens in the preven-
tion of preterm delivery might be a
function of the dose and the specific
progestogen that is administered.55 Stu-
dies with primary human chorion cells,
and in a mouse inflammatory-mediated
parturition model, suggest that MPA
may be more promising than 17-OH
progesterone caproate for prevention
520.e7 American Journal of Obstetrics & Gynecology OCTOBER 2015
of preterm birth.56-59 However, the
only randomized clinical trial to use
MPA to prevent prematurity in at-risk
pregnancies failed to demonstrate
benefit.60
Although progesterone, MPA, and HP
each inhibited both TNF- and thrombin-
induced fetal membrane weakening, their
effects on GM-CSF production and ac-
tion were different. This may be the result
of several factors. Progesterone has been
reported to undergo rapid metabolism in
cell and tissue studies.27 MPA acts at the
glucocorticoid receptor in addition to
acting on both nPRs and mPRs. HP has
much less efficacy at the nPR than the
mPR.28-32 The blockade of HP effect by
RU486 may not be informative because
RU486 can act as an antagonist at mPR
and at nPR.30 Additional studies will be
necessary to determine whether the dif-
ferences in inhibition of fetal membrane
weakening that is seen with different
progestogens are the result of differences
in their activity at the progesterone nu-
clear or membranous receptors, differ-
ences in activity at other receptors, or
differences in metabolism.
In summary, our data show that pro-
gestogens inhibit TNF- (modeling in-
flammation) and thrombin- (modeling
bleeding) induced fetal membrane weak-
ening. Progestogens also inhibit fetal
membrane weakening that is induced by
GM-CSF, which is a cytokine that has
been shown previously to be a critical
intermediate in the pathways of both
TNF- and thrombin-induced fetal mem-
brane weakening. Progestogens also
inhibit TNF- and thrombin-induced GM-
CSF production by choriodecidua. Inter-
estingly, HP, a progestogen with much
greater mPR than nPR activity, markedly
inhibited both TNF- and thrombin-
induced fetal membrane weakening.
Based on these findings, we propose that
progesterone inhibits inflammation- and
bleeding-induced fetal membrane weak-
ening through (1) interaction with nPRs
in decidual cells that leads to the inhi-
bition of GM-CSF induction and (2) in-
teraction with mPRs in inhibition of
GM-CSF-mediated fetal membrane
weakening action. In the context of our
in vitro model system, the data also clearly
indicate that progestogens are effective on
ajog.org
late gestation fetal membrane tissue,
which may have implications for current
therapeutic regimens for progestogen use
in the prevention of PPROM that is
associated preterm birth. -
ACKNOWLEDGMENTS
The investigators thank the medical and nur-
sing staffs of MetroHealth Medical Center,
Cleveland, OH and Hillcrest Hospital, Mayfield
Heights, OH.
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