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Endocrine Reviews 21(5): 514 –550
Copyright © 2000 by The Endocrine Society
Printed in U.S.A.
Endocrine and Paracrine Regulation of Birth at Term
and Preterm*
JOHN R.G. CHALLIS, STEPHEN G. MATTHEWS, WILLIAM GIBB,
STEPHEN J. LYE
AND
Departments of Physiology (J.R.G.C., S.G.M., W.G., S.J.L.) and of Obstetrics and Gynaecology
(J.R.G.C., S.G.M., S.J.L.), University of Toronto, Toronto, Ontario, Canada M55 1A8; Program in
Development and Fetal Health (J.R.G.C., S.J.L.), Samuel Lunenfeld Research Institute, Mount Sinai
Hospital, Toronto, Ontario, Canada M5G 1X5; MRC Group in Fetal and Neonatal Health and
Development (J.R.G.C., S.J.L.); Department of Obstetrics and Gynaecology, and Cellular and
Molecular Medicine (W.G.), University of Ottawa, Ottawa, Ontario, Canada K1H 8L6
ABSTRACT
We have examined factors concerned with the maintenance of
uterine quiescence during pregnancy and the onset of uterine activity at term in an animal model, the sheep, and in primate
species. We suggest that in both species the fetus exerts a critical
role in the processes leading to birth, and that activation of the fetal
hypothalamic-pituitary-adrenal axis is a central mechanism by
which the fetal influence on gestation length is exerted. Increased
cortisol output from the fetal adrenal gland is a common characteristic across animal species. In primates, there is, in addition,
increased output of estrogen precursor from the adrenal in late
gestation. The end result, however, in primates and in sheep is
similar: an increase in estrogen production from the placenta and
intrauterine tissues. We have revised the pathway by which endocrine events associated with parturition in the sheep come about
and suggest that fetal cortisol directly affects placental PGHS
expression. In human pregnancy we suggest that cortisol increases
PGHS expression, activity, and PG output in human fetal membranes in a similar manner. Simultaneously, cortisol contributes to
decreases in PG metabolism and to a feed-forward loop involving
elevation of CRH production from intrauterine tissues. In human
pregnancy, there is no systemic withdrawal of progesterone in late
gestation. We have argued that high circulating progesterone concentrations are required to effect regionalization of uterine activity, with predominantly relaxation in the lower uterine segment,
allowing contractions in the fundal region to precipitate delivery.
This new information, arising from basic and clinical studies,
should further the development of new methods of diagnosing the
patient at risk of preterm labor, and the use of scientifically based
strategies specifically for the management of this condition, which
will improve the health of the newborn. (Endocrine Reviews 21:
514 –550, 2000)
I. Introduction
I. Introduction
P
II. Regulation of Myometrial Contractions
ARTURITION is the process by which the fetus is expelled from the uterus to the extrauterine environment.
Parturition results from a complex interplay of maternal and
fetal factors. It requires that the uterus, which has been maintained in a relative state of quiescence during pregnancy,
develops coordinated contractility and that the cervix dilates
in a manner that allows passage of the fetus through the birth
canal. To be successful, parturition requires also that maturation of those fetal organ systems necessary for extrauterine
survival has occurred, and that the maternal organism has
undergone the changes necessary for lactation in the postpartum period. It is not surprising, therefore, that synchronous maturation of the fetus and stimulus to increased uterine activity should be desirable, and much evidence suggests
that it is the fetus itself that triggers both these series of
events.
Preterm birth, where there is asynchrony between the
labor process and fetal maturation, occurs in 8 –10% of all
pregnancies, and its incidence has changed little in the past
40 yr (1). Indeed, factors such as low socioeconomic status of
some inner-city populations, the tendency for women to
choose to start a family at an older age, and the impact of
fertility treatment are contributing to an increase in the incidence of preterm delivery (2, 3). Improved neonatal care,
however, continues to reduce the mortality rate due to pre-
III. Pregnancy: Phase 0 of Parturition
IV. Myometrial Activation: Phase 1 of Parturition
A. Activation: role of fetal hypothalamic-pituitary-adrenal (HPA) maturation
B. Activation mechanism by which cortisol changes
placental steroid and PG synthesis
C. HPA function in the primate fetus and activation of
parturition
D. HPA maturation in the primate fetus
E. Placental progesterone and human pregnancy: the
enigma of the progesterone block
V. Myometrial Stimulation: Phase 2 of Parturition
A. Stimulation: role of oxytocin
B. Stimulation: role of PGs
C. Stimulation: role of CRH
VI. Application to Clinical Preterm Labor
Address reprint requests to: Dr. J. R. G. Challis, Department of
Physiology, Medical Sciences Building, 1 King’s College Circle, University of Toronto, Toronto, Ontario M5S 1A8 Canada. E-mail: j.challis@
utoronto.ca
* Work in the authors’ laboratories has been supported by Medical
Research Council (MRC) Group and operating grants from the MRC of
Canada.
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PARTURITION
maturity, although preterm birth remains the primary cause
of neonatal death. In North America, the cost of caring for
infants in the neonatal intensive care nursery during the first
months of life has been estimated at $5– 6 billion annually (3).
That figure does not take into account the extraordinary
emotional stress to the family of the prematurely delivered
infant. Nor does it take into account the long-term costs
required for chronic care of these infants, some of whom have
major motor and/or mental handicaps and/or long-term
neuro-developmental complications. To prevent preterm
birth effectively, we need to understand the fundamental
processes that switch the myometrium from its relative quiescence during pregnancy to the activated and contractile
state at the time of labor. We will develop the thesis that
regulation of myometrial function requires both endocrine
and mechanical controls. Furthermore, it is now evident that
the cause of preterm labor may vary at different times during
pregnancy and will not necessarily reflect acceleration of the
processes at term gestation. The ability to recognize these
various causes of premature delivery, in a clinical setting,
and then provide appropriate treatment remains a major
clinical challenge. Furthermore, it is evident that prevention
of preterm delivery may not always be desirable, particularly
if the fetus is allowed to develop in a hostile intrauterine
environment.
Causes of preterm birth in general fall into three categories: iatrogenic, where there is demonstrable complication of
pregnancy such as preeclampsia or fetal distress that requires
obstetrical intervention; premature rupture of the fetal membranes with or without infection; and, idiopathic preterm
labor. The relative importance of these causes varies. However, most sources consider that approximately 30 – 40% of
preterm birth is associated with an underlying infective process, and 40 –50% of preterm births are idiopathic.
In this review, we will focus attention on experimental
studies in the sheep, the species of choice for many investigators concerned with understanding the processes of birth
(4). We shall then extrapolate from the sheep to an understanding of parturition in primates, particularly in the human. Our central thesis is that the processes of birth are
remarkably similar, at a fundamental level, across species,
and in both sheep and human the fetus, through activation
of its hypothalamic-pituitary-adrenal (HPA) axis, plays a
central and crucial role. We shall examine how the fetal HPA
axis may be activated in response to a stress circumstance
during pregnancy, e.g., hypoxemia, such as that perhaps
associated with reduced uteroplacental perfusion in preeclampsia. It will be apparent that the fetal signal provokes
increased outputs of stimulatory PGs and other uterotonins
from intrauterine tissues. It is evident now that there is a
progression from fetal to maternal control of intrauterine PG
production. Furthermore, the regulation of PG synthesis and
metabolism in fetal trophoblasts and maternal uterus is effected by different mechanisms.
II. Regulation of Myometrial Contractions
During pregnancy, myometrial activity is characterized by
poorly coordinated contractures, or the Braxton-Hicks con-
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tractions of human gestation (5). In late pregnancy, the uterus
undergoes preparedness for the stimuli that lead to contractility and labor (6, 7). Those stimuli may be local, maternal,
mechanical, or fetal (8). The contracture pattern of uterine
activity has been observed in several species, including the
sheep, baboon, and rhesus monkey (9). The development of
coordinated uterine contractions at term results in a myometrium that is excitable, generating high-frequency, highamplitude contractions. It is spontaneously active and responds to exogenous uterotonins. The transition of the
myometrium from a quiescent to an active state has been
termed “activation.” When this has occurred the myometrium can then undergo “stimulation” in response to endogenous and/or exogenous agonists (8).
We have found it useful to divide the uterine phenotype
into different stages of the parturition process (10). The
uterus is relatively quiescent during 95% of pregnancy, corresponding to phase 0 of parturition. Activation corresponds
to phase 1 and is effected predominantly by mechanical
input, and through regulation by uterotrophins such as estrogen. Stimulation corresponds to phase 2, when endogenous uterotonins, including PGs and oxytocin (OT), act on
the activated myometrium. Postpartum involution corresponds to phase 3. In this sequence of events, the “initiation”
of parturition corresponds to the transition from phase 0 to
phase 1, although clearly one could argue that initiation
started much earlier in gestation (11).
Contraction of the myometrium at term or preterm depends upon conformational changes in the actin and myosin
molecules, which allow actin and myosin filaments to slide
over each other, ultimately leading to a shortening of the
myocyte (Fig. 1 and Refs. 12 and 13). The confirmational
changes (involving cross-bridge cycling of the myosin head)
require ATP, which is generated by myosin after phosphorylation of the 20-kDa light chains of myosin by the enzyme
myosin light chain kinase (MLCK). This enzyme is central to
signaling pathways that both stimulate and inhibit myometrial contractions (14, 15). MLCK is activated through interaction with the calcium binding protein calmodulin (CAM),
which in turn requires 4 Ca2⫹ ions for its own activation.
Binding of calcium-activated CAM to MLCK induces a conformational change in the enzyme, allowing MLCK to phosphorylate the 20-kDa light chains of myosin. MLCK can also
undergo phosphorylation by protein kinase A (PKA, cAMPactivated protein kinase), which reduces the affinity of the
enzyme for calcium calmodulin (Ca-CAM) and leads to its
inactivation (14, 16). Regulation of MLCK has been reviewed
extensively (17, 18). It is evident that activity of this enzyme
is altered by intracellular pathways that regulate levels of
calcium and of cAMP and is critical for the development of
uterine contractility. Uterotonins generally increase intracellular calcium levels ([Ca2⫹]i), by increased influx of Ca2⫹
through receptor-operated channels, or release of calcium
from intracellular stores including sarcoplasmic reticulum
(see Ref. 19). Agents that inhibit myometrial activity do so by
increasing intracellular levels of cyclic nucleotides cAMP or
cGMP, which in turn inhibit release of calcium from intracellular stores or reduce MLCK activity. Binding of agents
such as ␤-adrenergic agonists, relaxin and prostacyclin, to
myometrial receptors activates adenylate cyclase activity,
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Vol. 21, No. 5
FIG. 1. Cartoon of a myometrial cell indicating the intracellular biochemical
pathways involved in regulating contractions. MLCK is central to uterine
contractility. It is activated by Ca-CAM
after an increase in intracellular calcium levels. This increase is generated
by the action of various uterotonins:
PGF acting through PGF receptor (FP),
OT acting through OTR. Agents that
increase cAMP (␤-agonists) or cyclic
GMP, or NO donors decrease uterine
contractility. AA, Arachidonic acid;
Atosiban OTR antagonist.
leading to an increase in cAMP generation, while uterine
inhibitors such as nitric oxide (NO) activate guanyl cyclase,
increasing cGMP. In collaborative studies, Pato et al. (20)
characterized MLCK purified from pregnant sheep myometrium. The enzyme had an apparent molecular mass of 160
kDa and high substrate specificity for myosin light chains.
Sheep myometrial MLCK has an absolute requirement for
Ca2⫹ and CAM for activation; in the absence of Ca-CAM,
MLCK is inactive. On binding Ca-CAM, MLCK undergoes a
conformational change that exposes the catalytic site, which
can then phosphorylate the 20-kDa myosin light chains to
initiate contraction. Relaxation is achieved either by dephosphorylation of MLC-20 by the catalytic subunits of type 2A
phosphatase (21) or by reduction in MLCK activity. The latter
is achieved, as discussed, by reduction in [Ca2⫹]i, resulting
in dissociation of Ca-CAM from MLCK. Sheep myometrial
MLCK is also a substrate for PKA, which phosphorylates
serine residues on the sheep myometrial enzyme in the presence or absence of bound Ca-CAM. The ability of PKA to
inhibit myometrial MLCK activity, even in the presence of
agonists that increase [Ca2⫹]i, provides a biochemical rationale for the finding that agents that increase intracellular
cAMP inhibit uterine contractions even in the presence of
calcium-activating agents such as OT and stimulatory PG.
Ca-CAM can also activate phosphodiesterase to increase the
breakdown of cAMP.
Inhibition of myometrial activity by ␤-adrenergic agonists,
relaxin, and PGI2 is mediated by increases in intracellular
cAMP (see Ref. 12). Binding of the inhibitor to its specific cell
membrane receptor causes dissociation of the receptorlinked heterotrimeric GTP-binding protein Gs into ␤-, ␥-, and
␣-subunits. The ␣-subunit activates adenylate cyclase to initiate cAMP synthesis. cAMP, in turn, activates PKA, which
then phosphorylates a series of regulatory proteins. Activated PKA either phosphorylates MLCK to reduce its ability
to bind Ca-CAM or phosphorylates a membrane-binding site
for Ca2⫹ that increases calcium binding and reduces free
intracellular calcium concentrations.
Regulation of myometrial calcium levels has been reviewed extensively (see Refs. 12 and 22–24). Free resting
Ca2⫹ increases from 150 nm to about 500 nm during contraction through influx of extracellular Ca2⫹ or by the release
of Ca2⫹ from intracellular binding sites or intracellular organelles (25, 26). Extracellular Ca2⫹ enters cells through receptor-operated or voltage-gated channels. Release of intracellular Ca2⫹ from sarcoplasmic reticulum is activated
through the phosphoinositol (PI) pathway. Binding of a
uterotonin to its plasma membrane receptor activates a G
protein transducer, coupled to phospholipase C, which frees
inositol trisphosphate (IP3) and diacylglycerol (27, 28). Free
IPs, especially IP3, increase cellular calcium from intracellular storage sites. Interestingly, IP3 binding in myometrium
was inhibited by calcium, suggesting that this might provide
a mechanism for regulating the IP3 response by oscillating
[Ca2⫹]i. Diacylglycerol formed during IP3 turnover may
stimulate PKC to phosphorylate cellular proteins such as
MLCK or be rapidly phosphorylated by diacylglycerol kinase to phosphatidic acid, a naturally occurring Ca2⫹ ionophore, or lead to release of arachidonic acid by cellular
lipases, resulting in production of eicosanoids (see below).
Function of the myometrium during labor at term or preterm requires highly developed cell-to-cell coupling, effected
through formation of intercellular GAP junctions within adjacent cell membranes (14, 29). The proteins forming GAP
junctions are termed connexins and are classified according
to their apparent molecular weights (30). Connexins are arranged into hexameric hemichannels, which become aligned
across adjacent cells to form an interconnecting pore that
allows low-resistance electrical or ionic coupling between the
cells and provides a pathway for metabolite transfer (31).
Hundreds of individual channels arrange themselves into an
organized plaque to form a GAP junction. Regulation of
connexins occurs at the level of transcription and translation
(31, 32); mechanisms also operate to control transport of
connexin protein to the cell membrane and to direct assembly
into connexons, through apposition, clustering, and formation of functional channels (33, 34). This complex process is
poorly understood, although it is influenced by steroids and
by mechanical stretch (35). GAP junction formation requires
the presence of cell adhesion molecules, and in early studies,
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Meyer et al. (36) showed that appearance of GAP junctions
in transfected S180 cells was blocked by coincubation with
antisera to liver cell adhesion molecule.
Garfield (see Refs. 14 and 16) established clearly that an
absence of GAP junctions in the pregnant myometrium was
responsible for high-input resistance of these smooth muscle
cells and poor coordination of uterine contractions. There is
a massive increase in numbers of GAP junctions with the
onset of labor, which significantly enhances electrical coupling and allows the myometrium to develop synchronous
high amplitude contractions (37). An increase in GAP junctions with labor onset has been found in all species studied.
In the rat, levels of connexin-43 (CX-43) mRNA and protein
were low during pregnancy but increased some 48 h before
labor (38, 39). Highest levels of mRNA and protein were
found during delivery itself. This is critical because the halflife of GAP junctions may be as short as 1–2 h, and hence
continued synthesis would be required to maintain labor.
Increases in CX-43 mRNA have been reported in sheep and
human myometrium with the onset of labor and correlated
with increases in CX-43 protein (37, 38). Permeability of GAP
junctions may be facilitated through phosphorylation at consensus serine and tyrosine sites within the cytoplasmic domain of CX-43. Garfield (14) demonstrated that cell-to-cell
communication in the myometrium is reduced by elevated
[Ca2⫹]i and increased levels of cAMP. Importantly, more
recent studies have shown that the pattern of CX-43 in myometrium during pregnancy differs from that of CX-26. Connexin-26 expression is elevated in midgestation in the rat and
appears to be associated more with uterine quiescence (7, 8).
III. Pregnancy: Phase 0 of Parturition
Studies in different species have indicated that a variety of
different inhibitors may play upon the myometrium during
pregnancy. Withdrawal of one or more of these may predict
the onset of delivery; precocious withdrawal may predict the
onset of premature parturition. Such an inhibitor, PTHrelated peptide (PTHRP), is produced in myometrium, and
its rate of transcription is increased by progesterone and
transforming growth factor ␤ (TGF␤) (40). PTHRP receptor
mRNA has also been localized to rat myometrial tissue, suggesting that the protein may act in an autocrine/paracrine
fashion through specific receptors to activate the G␣s subunits of G proteins and increase intracellular levels of cAMP
(40 – 42).
Relaxin also elevates myometrial cAMP and inhibits OTinduced turnover of phosphoinositide (PI) by the action of
cAMP-dependent protein kinase. Relaxin exerts a dual role
in the inhibition of myometrial contractility and in the regulation of connective tissue changes in the cervix (43, 44).
Porter and colleagues (45, 46) were among the first to show
that relaxin suppressed spontaneous uterine contractility in
the rat and guinea pig, but sensitivity to OT was preserved.
Thus, the major action of relaxin is one of frequency modulation (47). Hansell et al. (48) and others have demonstrated
that relaxin is expressed in the human fetal membranes,
decidua, and placenta, consistent with its exerting paracrine/autocrine effects on intrauterine tissues (49 –51). Re-
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laxin gene expression is dramatically up-regulated in patients with preterm, premature rupture of membranes
(PPROM) (49). Relaxin receptors have been localized to decidua and chorionic trophoblast cells, and the protein acts
through these to up-regulate expression of matrix metalloproteinases (MMP), especially MMP1, MMP3, and MMP9.
Similarly, relaxin increases MMP expression in cervical tissue at term. Administration of exogenous relaxin stimulates
separation of the pubic symphysis in those species in which
it is a prerequisite for delivery (52). In addition, in pigs and
rats, relaxin appears necessary for maintaining evolution of
spontaneous uterine contractility in late pregnancy and for
maintaining a high frequency of live births (43). In vitro
studies have shown that relaxin blocks the action of stimulants such as OT, carbachol, and norepinephrine on the myometrium, through mechanisms involving PKA-mediated
phosphorylation of PLC-linked G proteins. This in turn inhibits IP3 turnover and the increase in [Ca2⫹]i (22). Although
the precise role that relaxin plays during pregnancy remains
to be determined, it may be particularly useful in maintaining uterine quiescence during the period when progesterone
concentrations are falling and estrogen levels are beginning
to increase before the onset of labor (see Ref. 12). In addition,
there are reports that relaxin may act centrally to increase
circulating plasma OT and vasopressin concentrations by an
opioid-independent mechanism (53). It is now known that
OT is produced within human intrauterine, choriodecidual
tissues. It remains to be established whether a similar relationship exists between relaxin and OT synthesized within
the intrauterine compartment in women.
Lye and Challis (54, 55) first showed, some 20 yr ago, that
prostacyclin infused into nonpregnant sheep inhibited uterine contractility in vivo. In parallel studies a similar inhibitory
effect of prostacyclin was observed on human myometrium
(56), and it is clear now that prostacyclin represents the major
eicosanoid present within the pregnant myometrium of
many species (57), including human. In human term pregnant myometrial strips maintained in vitro, the initial response to PGI2 was contraction, but this was followed by
relaxation (58, 59). It is now recognized that PGI2 acts
through specific IP receptor species to increase adenylate
cyclase activity and elevate intracellular cAMP (60). Other
agents such as CRH also stimulate output of cAMP from
myometrial cells and act synergistically with PGI2 in a paracrine/autocrine fashion (61). The role of CRH in pregnancy
maintenance and parturition will be discussed later in this
review.
More recently, interest has arisen over the potential role of
NO as an endogenous inhibitor of myometrial contractility
(62). Increases in endogenous synthesis of NO by administration of the NO precursor l-arginine, or the NO donor
sodium nitroprusside, inhibit myometrial contractions in the
rat and human (62). Nitroprusside has been shown to decrease force and 20-kDa myosin light chain phosphorylation
in human myometrial strips, although the tissue is not as
sensitive as vascular smooth muscle. Nitric oxide synthase
(NOS) isoforms have been detected using RT-PCR in human
fetal membranes and choriodecidua (62). Levels of mRNAencoding inducible NOS (iNOS) are highest in human myometrium at preterm, not in labor patients, and decrease with
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CHALLIS ET AL.
a corresponding fall in iNOS protein in myometrium collected at term (see Ref. 10). Several authors have suggested
that NO acts in a paracrine manner, potentially in conjunction with progesterone to effect myometrial quiescence during pregnancy, although this position has been disputed.
There is a decrease in NOS activity of decidua and myometrium in species such as rat before parturition in a manner
that would presumably diminish its inhibitory influence on
the uterus. Furthermore, studies by Chwalisz and Garfield
(62) have shown that, at term in the rat, there is a corresponding increase in NO production by inflammatory cells
of the cervix, indicating a role for NO in cervical effacement
and relaxation as its influence on the myometrium is diminished.
Other inhibitors of uterine activity include calcitonin generelated peptide (CGRP), vasoactive intestinal polypeptide
(VIP), and endogenous ␤-adrenergic agonists (63). These
compounds act through increasing intracellular cAMP
and/or decreasing intracellular calcium availability (64).
IV. Myometrial Activation: Phase 1 of Parturition
The switch from myometrial quiescence to myometrial
activation is essential to enable the muscle to respond to the
stimulation provided by the high levels of uterotonic agonists and to generate the synchronous, high-amplitude, highfrequency contractions of labor. We have proposed that myometrial activation results from coordinated expression of a
cassette of proteins, termed contraction-association proteins,
or CAPs (12). CAPs include ion channels [which determine
the resting membrane potential and hence excitability of
myocytes (65)], agonist receptors [e.g., to OT and PG (60)] and
GAP junctions [permitting cell-to-cell coupling (16)].
Overall regulation of myometrial activity is genetically
regulated (Fig. 2). Different species have gestations of
varying lengths, and studies involving embryo transfer
suggest that it is the genotype of the fetus that controls the
length of pregnancy. For example, when sheep embryos
from short gestation or long gestation breeds were implanted into random gestation-age recipients, parturition
occurred at the appropriate time for the fetal rather than
FIG. 2. The onset of labor is dictated by the fetal genome proceeding
through either a fetal growth pathway with increases in uterine
stretch or fetal endocrine pathway involving activation of the fetal
HPA axis. These two arms are not independent because changes in
progesterone and estrogen modulate the ability of uterine stretch to
increase expressions of genes associated with myometrial activation.
Vol. 21, No. 5
maternal genotype (66, 67). There is a variety of mechanisms by which the fetal genotype can influence pregnancy length, and we have proposed that it includes both
endocrine and mechanical signals. In initial studies, Ou
and Lye (68) found, using unilaterally pregnant rats, that
while expression of CAP genes, CX-43 and OT receptor
(OTR), increased in the gravid uterine horn in labor, there
was no parallel increase in the nongravid horn, even
though both horns were exposed to the same systemic
hormonal changes. Next, these workers showed that when
a small 3-mm diameter tube was placed into one uterine
horn of bilaterally ovariectomized nonpregnant animals,
there was a significant increase in mRNA levels encoding
CX-43 in that horn, compared with the contralateral horn.
Control experiments showed that this result was not due
to the presence of a foreign body within the uterus. Administration of progesterone to these animals blocked
stretch-induced increases in CX-43 expression.
Subsequent experiments examined whether the endocrine
environment of pregnancy influenced the ability of stretch to
up-regulate CAP gene expression (see Ref. 8). In unilaterally
pregnant rats, at day 15 of gestation, the nonpregnant horn
received either the 3-mm Silastic tube or was left as control.
Other animals were operated on at day 18. Five days after
implanting the tubes, levels of transcripts encoding CX-43,
PGF2␣ receptor (FP receptor), or OTR were measured. In
animals treated at day 15 and studied at day 20, there was
no effect of the Silastic tube in increasing CX-43 transcripts,
but in animals studied at the time of labor there was a
dramatic increase in the numbers of CX-43 transcripts to
values similar to those seen in the contralateral pregnant
horn. There was little change in CX-43 transcripts in the
nonpregnant control horn. These data suggest that stretch of
the myometrium appears capable of up-regulating contraction-associated proteins, but the ability to do so is highly
dependent on the endocrine environment. If the stretch stimulus is applied during pregnancy, it is inadequate to induce
CX-43, and presumably its activity is inhibited by circulating
concentrations of progesterone. However, at term, when maternal systemic progesterone levels have decreased, stretch
itself is adequate to produce the same level of CX-43 expression as in the pregnant horn containing the fetus.
The molecular mechanisms by which stretch increases
CX-43 and OTR expression remain to be determined (69).
In other systems, such as cardiac myocytes, stretch activates multiple intracellular signaling pathways through
shear stress response elements in the promoter of some
stretch-responsive genes (70). The CX-43 gene contains
such an element, suggesting that if wall tension contributes to the regulation of CAP genes in the myometrium,
regulation of uterine growth through pregnancy will be
important in determining the level of shear stress. Lye and
colleagues (8) have argued that, during pregnancy, uterine
growth follows three distinct phases: an initial phase during the first trimester where uterine growth is due to
hyperplasia and controlled by endocrine factors, a second
phase during the second and third trimester in which
growth is closely matched to increased fetal size, and a
final phase in which there is a decline in uterine growth
in comparison to fetal growth, and hence an increase in
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uterine wall stretch and tension. They speculate that progesterone is necessary to support stretch-induced hypertrophy of the uterus during midgestation in concert with
increasing fetal size. Near term, the fall in progesterone,
observed in most animal species (see below), leads to a
decline in uterine growth relative to fetal growth and
hence increased tension development, which in turn results in increased CAP gene expression and contributes to
myometrial activation. Since the decrease in circulating
progesterone appears critical for the altered influence of
stretch on myometrial CAP gene expression, we shall consider the endocrine pathways that result in progesterone
withdrawal.
A. Activation: role of fetal HPA maturation
More than 35 yr ago, Professors Sir Graham (Mont) Liggins
and Geoffrey Thorburn, working in the sheep and goat,
showed conclusively in those species that the fetus, in utero,
appeared to provide the trigger mechanism for the onset of
parturition and that it did so through activation of the fetal
HPA axis. An endpoint of activation of this axis is progesterone withdrawal. We shall suggest that the primate fetus
similarly affects gestation lengths through activation of the
HPA axis. However, in human gestation there is no systemic
progesterone withdrawal, and we shall argue that, in
women, sustained circulating concentrations of progesterone
are indeed required at term to effect regionalization of myometrial contractility and promote relaxation of the lower
uterine segment.
Early studies in the fetal sheep showed that ablation of the
fetal pituitary gland, the fetal adrenal gland, pituitary stalk
section, or lesioning of the fetal paraventricular nucleus
(PVN) resulted in prolongation of gestation (71–73), whereas
the infusion to the fetal lamb in utero of ACTH or of a glucocorticoid resulted in premature parturition within 3–5
days of beginning the infusion. These studies provided experimental verification of the concept developed from observations of naturally occurring prolonged gestation in
sheep attributable to ingestion of the teratogen Veratrum
californicum at a specific time of gestation. In those animals,
gestation length was prolonged by up to 60 or 70 days,
although fetal growth continued. Fetuses exhibited gross
malformations, including cyclopean characteristics. At autopsy, the pituitary and adrenal glands were remarkably
hypoplastic as a result of impaired pituitary development at
an early gestational age (see Ref. 81).
Several groups of workers provided clear evidence for
maturation of fetal HPA function in the sheep fetus during
late gestation (74 –76). There are progressive increases in fetal
plasma ACTH1–39 and cortisol in the plasma of the lategestation fetal sheep (77– 80); the initial increases in ACTH
precede the rise in cortisol (79), but fetal cortisol increases in
an exponential fashion over the last 10 days of gestation, with
highest concentrations being established immediately before
term (80). This is consistent with the fact that ACTH is important in the development of the adrenal cortex in late
gestation. Similar maturation of pituitary adrenocortical
function has been demonstrated in several other species,
including the guinea pig, which represents a species that
519
gives birth to mature young. The prepartum surge of cortisol
is important for the maturation of several organ systems,
particularly the lungs and kidneys (see Ref. 81). It is also
critical for normal development of programming of the brain.
However, the simultaneous increase in fetal plasma ACTH
and cortisol has remained somewhat of a paradox because,
under normal circumstances, one would expect elevations in
fetal plasma cortisol concentration to inhibit further ACTH
secretion. Mechanisms have developed to override the influence of negative feedback in the fetus in late gestation, a
relationship now described in the guinea pig as well as in the
sheep (see below).
Recent studies have explored the molecular mechanisms
underlying changes in fetal pituitary adrenocortical activation in late gestation sheep. Developmental changes in CRH
mRNA in the fetal hypothalamic PVN were examined by in
situ hybridization (82). By day 60 of gestation, CRH mRNA
was detectable in the fetal PVN. There was an increase in
CRH mRNA expression by day 120 of gestation and a further
substantial up-regulation of CRH gene expression in the last
20 days of pregnancy. This was followed by a decrease in
CRH expression in the PVN of the newborn lamb. Throughout development, expression of CRH mRNA appears to be
confined to parvocellular fields of the PVN, with no expression detected in magnocellular neurons (82). Recent studies
have confirmed that the changes in CRH mRNA are translated to CRH peptide in the fetal hypothalamus, indicating
a close association between transcription and translation of
the CRH gene during development.
In the fetal pituitary, expression of the ACTH precursor,
POMC, is detectable in the inferior region of the pars distalis
by day 60 of gestation. Levels of POMC mRNA in the superior and inferior regions of the pars distalis increased with
progression of gestation until around day 120, when there
was a further increase in expression, peaking at term (83, 84).
The increase in POMC expression is combined with a remarkable reorganization of the corticotrophs toward the inferior aspect of the fetal pituitary gland. This pattern of
expression was sustained in the newborn lamb. In the fetal
pars intermedia, the developmental profile of POMC mRNA
was quite different. Relatively high levels were present by
day 60 of gestation; these increased between days 60 and 100
and then remained relatively constant for the remainder of
gestation. Early controversy concerning changes in expression of POMC mRNA in fetal pituitary tissue appears to
result from differences in methodologies. The use of in situ
hybridization clearly allows separation of different zones of
the fetal pituitary gland, whereas erroneous results may have
been obtained through use of Northern blot analysis (85, 86).
In a recent carefully conducted study obtaining pituitary
tissue from fetuses at specific times in late gestation and
during the labor process itself, the lack of negative feedback
on POMC mRNA, and the sustained increase in POMC
mRNA levels, was clearly demonstrated (87). The change in
regional distribution of POMC mRNA in the pars distalis
may indicate the transition of fetal-like to adult-like corticotrophs that has been described at this time (see below).
Changes in POMC mRNA in the pars distalis are reflected by
increased levels of ir-ACTH and by increased immunostaining for ACTH in pituitary corticotrophs (83, 84); at term
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CHALLIS ET AL.
ir-ACTH-positive cells represent about 15% of the total cell
number in the pars distalis.
Arginine vasopressin (AVP) is also an important regulator
of fetal pituitary ACTH secretion and is expressed in the fetal
PVN relatively early in gestation (88). AVP mRNA is present
in the supraoptic nucleus, PVN, and the accessory magnocellular nuclei by day 60 of gestation (82). Differential expression of magnocellular and parvocellular AVP is evident
in the PVN by day 80. In magnocellular neurons, AVP mRNA
increases with gestational age, whereas parvocellular expression of AVP remains relatively unchanged. Levels of AVP
mRNA increase dramatically in both regions of the PVN in
the newborn lamb. It is suggested that magnocellular AVP is
involved primarily in fetal fluid homeostasis, while parvocellular AVP is important in stimulation of the pituitary
corticotroph (84). There is a close correlation between AVP
mRNA levels and ir-AVP in the anterior hypothalamus, as
there is for CRH. The increase in parvocellular AVP mRNA
in the newborn may be associated with the stress of the novel
extrauterine environment. Axons containing AVP and OT
have been identified in a zone of the pars distalis adjacent to
the pars intermedia in fetal sheep. These axons are probably
those of magnocellular neurons and may represent a mechanism by which magnocellular AVP and OT directly affect
ACTH release in vivo.
CRH and AVP induced dose-dependent increases in
ACTH output from ovine fetal pituitary cells in vitro (89); at
equimolar concentrations AVP was more potent than CRH.
Simultaneous administration of CRH and AVP showed an
additive interaction between the neuropeptides (90). Treatment with CRH significantly increases POMC mRNA levels
in sheep pituitary cells harvested at day 120 and day 138 of
gestation. However, CRH treatment of cells collected from
fetuses at term failed to affect POMC synthesis. AVP increased POMC mRNA levels in cells obtained at day 138 of
gestation; in pituitary cells from late-gestation fetuses, AVP
and CRH are equally potent in the induction of POMC synthesis. Cortisol has little negative feedback effect on basal
output of ACTH in these cells but inhibits CRH-stimulated
ACTH output and POMC gene expression.
Studies by Lu et al. (91) showed that ovine fetal pituitary
membranes expressed CRH receptor activity as early as day
100 of gestation. CRH binding increased to its highest levels
at around day 135 (term, 145–150 days) and then decreased
progressively through late gestation (92). Recent studies
have extended these measurements to show that levels of
mRNA encoding fetal pituitary CRH-receptor type I may
follow a similar profile (J. C. Rose, personal communication),
and this may account for the altered outputs of ACTH in
response to CRH stimulation in vivo (see below). Factors
regulating CRH receptor expression have been examined in
vivo and in vitro. In vitro studies indicated that CRH downregulated expression of its own receptor and cortisol produced a similar attenuation of binding activity (92).
In vivo studies demonstrated that CRH was more potent
than AVP in stimulating ACTH output by pituitary tissue
from chronically catheterized fetal sheep in late gestation (93,
94). The response profiles, however, are quite different. AVP
induced a transient rise in plasma ACTH while CRH stimulated a more sustained increase (95). Subsequently, it was
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demonstrated that AVP concentrations are about 5 times
those of CRH in the hypophyseal portal circulation of adult
sheep (96), and it remains possible that the relative importance of AVP in fetal corticotroph activation in utero may be
greater than that of CRH (97). Fetal pituitary responsiveness
to CRH increases between day 110 and 125 and then decreases toward term (79). This relative insensitivity of the
pars distalis to CRH may reflect the increase in negative
feedback influence of rising endogenous cortisol concentrations, or the decrease in CRH binding sites indicated above
(79). Simultaneous administration of CRH and AVP results
in an ACTH response that is greater than when either neuropeptide is administered independently, and the interaction
is synergistic in nature, at least at around day 115 of gestation
(95). CRH and AVP affect the corticotrophs through different
second messenger systems. CRH exerts this action through
up-regulating a G␣s-adenylate cyclase-linked membrane receptor and increasing intracellular levels of cAMP (89). AVP
acts through V1b receptors to stimulate PI turnover, stimulating phospholipase C and activating protein kinase C.
POMC is processed through different endoproteases, prohormone convertase 1 (PC-1) and prohormone convertase 2
(PC-2), to yield a spectrum of products. Recent studies have
demonstrated that both PC-1 and PC-2 are present in fetal
sheep pituitary tissue in late gestation. However, expression
of these enzymes does not change with labor, and it seems
unlikely that the increase in ACTH output is attributable to
altered prohormone convertase activity (87, 98). However,
the pattern of POMC-derived peptides from the fetal pituitary does change in the plasma of the fetal lamb in late
pregnancy (99). Several groups of investigators have reported that large molecular weight POMC-derived ACTH
precursor peptides are present in the circulation (100). The
concentrations of these larger molecular weight forms decrease prepartum, whereas those of ACTH1–39 increase. Because the larger molecular weight peptides may act to antagonize the action of ACTH1–39 on adrenocortical cells (101–
103), a decrease in their concentration prepartum would
presumably facilitate ACTH action and an increase in adrenal glucocorticoid secretion (104). The sources of these peptides may be different (105–107). Studies in hypothalmopituitary-disconnected fetuses have led to the suggestion
that the pars intermedia may be a potential source of large
molecular weight peptides, whereas the pars distalis is the
primary source of ACTH1–39. In addition, the ovine fetal lung
and placenta express POMC mRNA and contain ir-ACTH. It
is not clear whether these potential sources of ACTH contribute to circulating ACTH1–39 in a meaningful manner or
whether the peptides have paracrine/autocrine actions
within the tissues of origin.
Thus, the temporal relationship between hypothalamicCRH and pituitary POMC expression is consistent with the
simultaneous increase in plasma ACTH and cortisol observed in late gestation (84, 108 –110). Nevertheless, the
mechanism by which CRH mRNA and POMC mRNA increase in the presence of high plasma glucocorticoid concentrations is not clear. One possible mechanism is that, in
the fetus, glucocorticoid feedback thresholds within the brain
and pituitary become modified. This may occur at several
levels (Fig. 3). We have reported that glucocorticoids up-
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FIG. 3. Summary of events associated with maturation and development of the HPA axis in the fetal sheep. Increased expression of
CRH from the PVN of the hypothalamus drives increased expression
of POMC in the anterior pituitary. POMC is processed to ACTH,
which drives the adrenal gland. In the fetus normal negative effects
of cortisol on the hypothalamus and pituitary are diminished through
increases in systemic corticosteroid binding globulin (CBG), pituitary
11␤ HSD, and diminished expression of GR in the pituitary and
hypothalamus.
regulate expression of corticosteroid binding globulin (CBG)
mRNA in the fetal liver, and of circulating CBG, which is the
opposite of the response in adult sheep (111, 112). In the fetus,
the pattern of CBG glycosylation varies from that in adult
animals, but the glycoprotein increases in concentration in
the fetal circulation and maintains a relatively constant free
cortisol concentration for most of pregnancy (112, 113). Near
term, however, the increase in adrenal cortisol output exceeds the CBG binding capacity, resulting in a sudden increase in free cortisol concentration over the final hours before birth (114). It appears that this increase in free cortisol
before parturition is a consistent observation across different
animal species (115). More recently, we have demonstrated
expression of CBG mRNA and the presence of CBG immunoreactive protein in other fetal tissues including the kidney,
pancreas, and pituitary (115). CBG mRNA has been localized
to fetal pituitary cells by in situ hybridization, and its pattern
of distribution appears to differ from that of POMC, with
greater abundance in superior regions of the gland. As yet,
there are no studies demonstrating colocalization of CBG
with ACTH-producing cells in fetal pituitary tissue.
Levels of glucocorticoids may also be modified by interconversion of biologically active cortisol and biologically
inactive cortisone, through the activity of 11␤-hydroxysteroid dehydrogenase (11␤-HSD) enzymes (116). We will discuss these later in the context of the placenta as a barrier to
the transfer of maternal cortisol to the fetus. In the pituitary
of fetal sheep, 11␤-HSD-1 activity predominates and appears
to operate somewhat unusually in a dehydrogenase direction, i.e., inactivating cortisol to cortisone (116). Presumably,
this would effect a local mechanism to inactivate circulating
521
cortisol and diminish the potential for negative feedback.
This pattern of 11␤-HSD activity in the pituitary needs substantiating and differs from that in other fetal tissues, e.g., the
liver, where 11␤-HSD-1 operates predominantly as a reductase, converting cortisone to cortisol, and suggesting a potential intrahepatic source of cortisol generation.
A further mechanism by which glucocorticoid feedback
could be altered locally is through modification of corticosteroid receptor expression (117). The ovine fetal pituitary
expresses type II glucocorticoid receptor (GR) from relatively
early in gestation, and the levels of GR mRNA increase
toward term (118), consistent with glucocorticoid effects in
modulating the switch from fetal to adult corticotroph cell
types in the pituitary (106). During the course of labor, there
is a dramatic decrease in levels of GR mRNA in the fetal pars
distalis, suggesting that the potential for glucocorticoid negative feedback decreases in the pituitary during the course of
labor. More important, perhaps, is the demonstration that
there are decreases in immunoreactive GR in the hypothalamic PVN near term. These changes were specific to CRHand AVP-positive parvocellular neurons. More recently, we
showed that GR mRNA levels in the PVN of fetal sheep and
guinea pigs decrease in late gestation, and in fetal sheep
levels of GR mRNA in the hippocampus also fall prepartum.
The hippocampus represents a major site of glucocorticoid
feedback for HPA function, and there are a number of direct
and indirect connections between the limbic system and the
PVN. Together these data suggest that a reduction in the
potential for glucocorticoid feedback occurs in late gestation
in brain structures that are central to glucocorticoid negative
feedback action (119).
In addition to classic feedback processes, there are several
other mechanisms by which fetal HPA axis activation may
occur. Expression of pro-enkephalin mRNA rises to a maximum in the parvocellular PVN of fetal sheep at day 135 of
gestation and then decreases in older animals (120). A fall in
hypothalamic pro-enkephalin mRNA occurs with intrafetal
infusion of cortisol at day 135, suggesting that the prepartum
rise in endogenous cortisol may inhibit parvocellular proenkephalin synthesis. CRH and met-enkephalin are present
in the same secretory granules in rodents, and met-enkephalin inhibits CRH-stimulated ACTH secretion from fetal pituitary cells in vitro. Thus, a decrease in met-enkephalin
production may facilitate corticotroph function near term
(120). OT has been implicated in the control of ACTH secretion in adult sheep, and OT stimulates ACTH output from
the fetal pituitary cells in vitro. OT mRNA is present in both
magnocellular and parvocellular fields of the PVN and SON
and follows a similar developmental profile to AVP mRNA,
raising the possibility that it too may influence fetal pituitary
function.
In fetal sheep, the kinetically determined production of
cortisol from the adrenal gland increases during the last
20 –25 days of gestation (77, 121). In part, this results from the
increase in drive to the adrenal from rising levels of ACTH,
but, in part, it is attributable to maturation of fetal adrenal
function (122). Indeed, in hypophysectomized fetuses treated
with a continuous low-level infusion of ACTH, plasma cortisol concentrations increased and parturition occurred at
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CHALLIS ET AL.
around the normal time, consistent with fetal adrenal maturation as the overriding influence (123).
Ovine fetal adrenal responsiveness changes dramatically
during the course of pregnancy (124, 124 –126). Adrenal cells
collected from animals at days 50 –70 of gestation secrete
cortisol in response to ACTH stimulation in amounts similar
to or greater than adrenal tissue from term fetuses (127).
However, between approximately days 90 –110 of pregnancy
the adrenal is relatively insensitive to ACTH stimulation
(124). It is now clear that this pattern of response is due, in
large part, to decreased gene expression of P450C17 and
P450SCC steroidogenic enzymes in fetal adrenal cortical cells
at midgestation (128, 129). The abundance of mRNAs for
these enzymes is increased by ACTH administration to the
fetus (130, 131). Although 3␤-HSD may be rate limiting to
cortisol production in the first half of pregnancy (132), immunoreactive (ir)-3␤-HSD-positive cells are present
throughout the zona fasiculata of the fetal adrenal cortex
from day 50 until term (133). The midgestational decrease in
P450C17 may result by TGF␤ inhibiting ACTH-induced stimulation to P450C17, as demonstrated in vitro in ovine fetal and
adult adrenal cells (134). Recent studies have demonstrated
that ACTH receptor mRNA is detectable from around day 60
of gestation (135). There is a modest increase through pregnancy and then a substantial increase between days 126 –128
and days 140 –141 (135). Thus, the low level of basal adrenal
responsiveness to ACTH around day 100 of gestation is not
due to lack of ACTH receptor expression, but may be attributable, in part, to very low concentrations of ACTH in the
fetal circulation at that time (136). The increase in ACTH
receptor expression in late gestation would appear to contribute to increased adrenal responsiveness near term. The
factors responsible for up-regulating ACTH receptor mRNA
abundance are unclear (137). These may include ACTH itself,
cortisol, or local intraadrenal interaction with IGF-II and/or
decreased influence of TGF␤ (138 –140).
Both in vivo and in vitro studies have shown that fetal
adrenal maturation can be advanced by ACTH1–24 administration (110, 141–143). Exogenous ACTH in vivo enhances
coupling between ACTH receptor and adenylate cyclase and
enhanced capacity for cAMP generation (144 –146). ACTH
treatment in vivo also increased expression and activity of
P450C17, P450C11, P450C21, and 3␤-HSD (130, 147). The adrenal responds to ACTH early in gestation, although continued
trophic input is required to maintain increased levels of gene
expression. Interestingly, when ACTH was administered to
fetuses in vivo as pulses, rather than as a continuous infusion,
it led to a pattern of fetal adrenal steroidogenesis that favored
cortisol over corticosterone output (i.e., directed P450C17 activity). Thus, the pulse pattern of endogenous ACTH secretion in vivo may affect the pattern of adrenal activation (148,
149).
These studies suggest that ACTH-induced increases in
adrenal steroidogenic enzymes, particularly P450C17, is essential to allow C21 steroids to proceed through the 17hydroxy pathway leading to cortisol biosynthesis (130, 150).
An obligatory role for an increase in ACTH drive to the fetal
adrenal as a prerequisite for increased responsiveness, however, has been challenged recently. When hypophysectomized fetal sheep were infused at a constant, but low level
Vol. 21, No. 5
of ACTH, there was a normal rise in fetal cortisol concentration; later, maternal progesterone levels decreased and
birth occurred at about the expected time (123). The molecular mechanisms underlying this fascinating result clearly
require elucidation.
We have hypothesized that fetal stress, perhaps reflected
in diminished fetal arterial P02, constitutes a stimulus for
preterm birth. Experimental hypoxemia has been used extensively to investigate fetal HPA activation (151, 152). Many
studies have shown that even modest reductions in fetal
arterial P02 induce robust increases in fetal plasma ACTH
and cortisol concentrations (153, 154). Release of CRH and
AVP into the hypophysial portal system is abolished in the
hypothalamo-pituitary-disconnected (HPD) fetus (152), and
these animals are incapable of mounting an ACTH response
to stress, implying that increased ACTH output requires
hypothalamic input. Studies by Akagi and colleagues (155)
demonstrated that changes in fetal P02 of only 4 –5 mm Hg
were adequate to elicit increased ACTH concentrations in the
circulation of the fetal lamb. This level of oxygen change is
similar to that seen during spontaneous contractures in late
gestation sheep, raising the possibility that uterine activity
itself may contribute part of the stimulus to increased fetal
HPA maturation. Whether chronic stress is a stimulus to
birth at term (156) or contributes only to some cases of preterm labor is unclear at the present time.
At 135 days’ gestation, hypoxia (P02 reduction by 8 mm
Hg) significantly increased CRH mRNA in parvocellular
PVN and POMC mRNA in the pars distalis within 6 h. This
response, however, was attenuated by concurrent infusion of
cortisol, indicating effective glucocorticoid feedback mechanisms in vivo at this time (157). After 48 h of sustained
hypoxemia, levels of POMC in the pars distalis were elevated, but expression in the pars intermedia was decreased
(158). This suggests differential regulation of these two zones
of the fetal pituitary, consistent with observations that dopamine, likely from the fetal arcuate nucleus, tonically inhibits pituitary POMC synthesis, and this inhibition is exacerbated in the presence of hypoxemia. Infusion of
bromocriptine, a dopamine D2 receptor agonist at day 130 of
gestation, produced a 50% decrease in pars intermedia
POMC mRNA levels, without affecting POMC mRNA in the
pars distalis (159). Thus, the fetal D2 receptor system is functional in late pregnancy, but the fetal pars intermedia does
not appear to secrete ACTH1–39 in amounts that alter fetal
adrenal function.
Activation of fetal HPA function in response to hypoxemia, however, is a critical aspect of the story leading to
preterm birth (160, 161). A sustained pulsatile hypoxemic
stimulus is adequate to up-regulate HPA gene expression,
plasma ACTH, and cortisol concentrations. It is reasonable to
predict that sustained hypoxemia in conditions of fetal compromise predisposes to fetal HPA activation and would result in premature birth (162, 163).
B. Activation mechanism by which cortisol changes
placental steroid and PG synthesis (Fig. 4)
Fetal cortisol acts on the sheep placenta to alter the pattern
of steroidogenesis; as a result, progesterone output falls and
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523
FIG. 4. Endocrine pathways leading to the onset of parturition in sheep. A, current model; B, proposed sequence of hormone events. In the
current model, activation of the fetal HPA axis leads to increased cortisol thought to up-regulate expression of P450C17 in the placenta. In the
new proposed hypothesis, increased fetal adrenal output of cortisol results in up-regulation of prostaglandin synthase (PGHS)-2 gene expression
in the placenta with increased production of PGE2. PGE2 feeds back to further up-regulate fetal HPA function but is itself responsible for
up-regulation of P450C17 gene expression in the placenta. Increased placental estrogen is required for up-regulation of PGHS-2 in maternal
tissues but not in fetal tissues. Thus, with the onset of parturition there is progression from fetal trophoblast within the placenta to the maternal
uterine tissues.
estrogen concentrations increase (164 –167). These changes in
placental steroid output are associated with increased expression and activity of placental P450C17 (168, 169). This is
a critical difference between the sheep and the human, where
this enzyme is not induced in the placenta at term. Ovine
placental tissue contains P450arom activity, and up-regulation
of this gene also occurs in late gestation. For many years the
general thesis has been that placental estrogen production is
limited in ovine pregnancy and occurs in abundance only at
term with the induction of placental P450C17 as a result of
glucocorticoid action (170 –172). The fall in progesterone and
later increase in maternal and fetal estrogen concentrations
have been considered as providing the stimulus to increased
PG output by intrauterine tissues, with consequent increase
in myometrial contractility (173–180).
For several reasons we have questioned the appropriateness of this model. It has been clearly established that the
sheep, like the human, has a feto-placental unit of estrogen
production by which C19 precursors from the fetal adrenal
gland can be secreted and aromatized in the placenta to form
estrogen (181). Later studies demonstrated output of C19
steroids including dehydroepiandrosterone (DHEA) and androstenedione by the ovine fetal adrenal gland, stimulation
of C19 fetal-adrenal steroid output by ACTH infusion and in
response to hypoxemia, and conversion of [3H]androstenedione infused into the fetus to estrogen measured in maternal
and fetal compartments (182, 183). Although unconjugated
estrogens increase sharply at the time of parturition in sheep
(165, 166), there is a progressive increase of conjugated estrogens in maternal plasma and urine throughout the latter
part of gestation, well before the terminal increase in placental P450C17 activity (184). The ratio of conjugated to unconjugated estrogen in maternal sheep plasma is high because, in ovine pregnancy, placental sulfotransferase activity
predominates over placental sulfatase activity (184). Thus, it
is clear that while increased expression of placental P450C17
may contribute to the sharp rise in maternal estrogen concentrations prepartum, its induction in the placenta is not a
prerequisite for ovine placental estrogen output at earlier
stages of gestation.
There are other troubling features of the currently accepted model (185). Several groups of investigators have
used either immunohistochemical techniques for localization
of PGHS-1/-2, or PGHS-2, or in situ hybridization for
PGHS-2 mRNA, or measurements of PGHS and/or PGHS-2
activity in ovine placental cells and microsomal preparations
(186 –189), to show that PG production by the sheep placenta
increases progressively through the last 20 –25 days of gestation (190 –195). Placental output of PGs is not confined to
the immediate 24 – 48 h before spontaneous parturition (196,
197). The increase in PGHS expression and activity in the
placenta correlates closely with the progressive increase in
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CHALLIS ET AL.
plasma PGE2 concentrations in the circulation of the chronically catheterized fetal lamb (191, 198). The increase in circulating PGE2 in the fetus bears a striking temporal relationship to the increase in plasma cortisol concentration (198,
199). Louis et al. (200) first reported, more than 25 yr ago, that
infusion of PGE2 into the ovine fetus in late gestation stimulated an increase in the plasma cortisol concentration at a
time when the fetal adrenal gland was relatively unresponsive to ACTH stimulation. Later studies have shown that the
effect of PGE2 infused into the fetus on fetal HPA function
could be exerted at any one or all of the hypothalamic, pituitary, or adrenal levels (201, 202). Thus, the progressive
increase in output of PGE2 appears to contribute to the drive
to fetal HPA function and augments the stimulus supplied
by ACTH to the fetal adrenal (201, 203). Indeed, fetal PGE2
infusion will provoke premature delivery of the ovine fetus
(204). Placental PGE2 output would not be subjected to negative feedback regulation by cortisol and may contribute to
the apparent lack of negative feedback relationship between
ACTH and cortisol in the late gestation ovine fetus.
Recent studies have suggested that in addition to PGE2
stimulating output of cortisol by the fetal adrenal gland (205,
206), fetal cortisol, and not estrogen, may affect placental
PGHS-2 activity and contribute to the rise in fetal plasma
PGE2 concentrations. Evidence in support of this suggestion
included the observation that infusion of estrogen into fetal
lambs in late pregnancy was without stimulatory effect on
levels of placental PGHS-2 mRNA (207), although estrogen
infusion into nonpregnant adult sheep did increase PGHS-2
expression in the endometrium (see also below). Studies with
human amnion cell cultures and chorion trophoblast cells
have suggested that glucocorticoids may up-regulate
PGHS-2 gene expression in these tissues. Infusion of cortisol
to fetal sheep in late gestation also increased levels of PGHS-2
mRNA and immunoreactive PGHS-2 protein (by Western
blotting) in placental trophoblast cells. This effect was independent of changes in estrogen, since a similar stimulation
of placental PGHS-2 mRNA levels was observed when cortisol was infused in the absence or presence of the aromatase
inhibitor, 4-hydroxyandrostenedione.
Using immunohistochemistry we showed that the P450C17
enzyme and PGHS-2 both localized to trophoblast epithelial
cells, but not binucleate cells in ovine placentomes (208).
Moreover, the appearance of ir-PGHS-2 clearly preceded that
of P450C17. Collectively, therefore, these data offer strong
reasons to refute the current model of endocrine events occurring in the placenta of the sheep in late gestation and
suggest that a different sequence likely pertains. This is summarized in Fig. 4. We have argued elsewhere that during late
gestation in the fetal sheep, increased output of cortisol from
the fetal adrenal gland progressively up-regulates PGHS-2
gene expression in placental trophoblast cells (208). The
mechanism of this action remains unresolved. It may depend
on trophoblast-specific transcription factors generated in response to elevations of cortisol, or it could be a direct action
of cortisol since early studies reported a full GRE consensus
sequence at approximately 760 bp upstream from the
PGHS-2 transcription start site. We suggest that increased
PGHS-2 expression in the sheep placenta contributes to increased PGE2 output into the fetal circulation. Fetal PGE2
Vol. 21, No. 5
drives the fetal HPA axis in a positive feed-forward fashion
(Fig. 4). PGE2, and not cortisol, is responsible for up-regulation of P450C17 in placental trophoblast cells. This occurs in
a manner analogous to the effect of PGE2 on P450C17 induction in ovine and bovine adrenal tissue. Ovine placental
tissue expresses PGE receptor subtypes (EP1-EP4), but any
changes in their expression during the course of late gestation remain to be determined (see Ref. 208). We have suggested further that increased P450C17 in the placenta allows
the conversion of C21 ⌬5 steroids directly through to ⌬5 C19
steroids, precursors for estrogen biosynthesis, as demonstrated by Flint et al. (209) and Mason and colleagues (210)
some years ago. A crucial difference of the current hypothesis
is that this change is superimposed on an already substantial
basal output of estrogen by the sheep placenta (measured as
conjugated estrogens in maternal plasma and urine), and
contributes principally to the terminal increase in maternal
estradiol concentrations. This increase in estrogen is required
for expression of CAP genes in the ovine myometrium and
for expression of PGHS-2 in maternal endometrial tissue,
predominantly endometrial epithelium. We have found that
whereas the increase in placental (fetal trophoblast) expression of PGHS-2 after intrafetal cortisol administration was
unaffected by concurrent infusion of 4-hydroxyandrostenedione, maternal endometrial up-regulation of PGHS-2
and output of 13–14 dihydro-15-keto PGF2␣ (PGFM) into the
maternal circulation occurred with cortisol infusion but was
blocked by concurrent administration of the aromatase inhibitor (211). Thus, in sheep it appears that the fetal placenta
and maternal endometrium exist as two separate sites of PG
synthesis in late gestation and that these are differentially
regulated. In fetal placenta, PGHS-2 is increased by cortisol,
independent of changes in estrogen output, whereas in maternal uterine tissue, up-regulation of PGHS-2 and maternal
plasma PGFM is dependent upon increased estrogen production (Fig. 4).
Current studies are directed at examining this hypothesis
further. Using immunohistochemistry and Western blot
analysis, it is evident that GR is expressed in ovine placental
tissue, predominantly in uninucleate trophoblast cells. Estrogen receptor (ER) mRNA and activity have been demonstrated in maternal endometrium but is apparently lacking
in placental trophoblast (212). Hence, it is difficult to envisage how estrogen could provide a stimulus to placental PG
production as previously hypothesized. It remains to be
shown whether glucocorticoids affect placental PGHS activity directly or indirectly. However, in early studies we have
demonstrated that glucocorticoids increase output of PGE2
by ovine placental trophoblast cells maintained in culture,
and this effect is abolished by addition of meloxicam, a specific inhibitor of PGHS-2 activity.
C. HPA function in the primate fetus and activation
of parturition
The role of the human and subhuman primate fetus in
controlling gestation length has been, until recently, less
clearly defined than that of the sheep fetus. However, over
the past few years it has become apparent that mechanisms
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leading to activation of fetal HPA function in primates bear
considerable similarity to processes in sheep, and that fetal
cortisol and fetal adrenal C19 steroids appear to play an
important role. In 1933, Malpas (213) in a study of gestation
length in human pregnancies complicated with anencephaly
concluded that “. . . . the fetal pituitary and adrenal glands
was responsible for the trigger to the neuromuscular expulsive mechanism that led to the onset of labor. ” Early observations indicated that the mean length of gestation in
anencephaly, after exclusion of cases with polyhydramnios,
was similar to controls, but the proportions of preterm and
postmature births were both higher (see Ref. 12). Similar
results have been obtained after experimental anencephaly
in rhesus monkeys (214). In monkeys, fetal hypophysectomy
predisposed to prolongation of gestation (215), but fetal adrenalectomy was without effect on gestational length, although five of eight fetuses died in that study (see Ref. 12).
Initial studies indicated that removal of the fetus, but leaving
the placenta in utero (fetectomy) had little effect on gestation
length. However, more recent studies have indicated clearly
that placental retention after fetectomy was significantly
longer (195 days) compared with 164 days in controls (216).
Fetectomy in baboon pregnancy did not affect gestation
length, although maternal estradiol concentrations fell to
basal values and progesterone concentrations were reduced
by 20 – 45% (217–219). Overall, these experiments are difficult
to interpret. The numbers and observations are invariably
small, no attempt is generally made to sustain uterine volume and the stretch stimulus to the myometrium, and it is
technically very difficult to operate on the primate fetus
without stimulating uterine contractility.
In intact rhesus monkeys, as in the baboon and human,
there is an increase in maternal estrogen concentrations in
late gestation that parallels an increase in the concentrations
of fetal adrenal C19 steroids, particularly DHEA and DHEAsulfate (DHAS) (220, 221). Maternal estrogen concentrations
increase progressively and then more rapidly in the later
phases of human gestation; estriol, derived in substantial
part from precursors of fetal adrenal origin, rises rapidly in
maternal plasma and urine in late pregnancy at term, and in
preterm labor (221). When androstenedione was infused into
pregnant rhesus monkeys at about three-quarters of the way
through gestation, there was an increase in maternal plasma
estrogen concentrations and premature birth (222). This effect was blocked by the coinfusion of the aromatase inhibitor
4-hydroxyandrostenedione, which prevented maternal endocrine changes and changes in fibronectin in the fetal membranes and inhibited the nocturnal increases in uterine myometrial contractility (223). Elevations of maternal systemic
estrogen concentrations by infusion increased myometrial
activity, but did not produce premature delivery or fetal
membrane changes. It was suggested that in the primate, as
in the sheep, estrogen is important for the normal processes
of parturition. The failure of exogenous estrogen to stimulate
sustained uterine contractility, even though locally produced
estrogen formed after C19 steroid infusion was effective, led
the authors to suggest that the estrogen had to be generated
near to its site of paracrine/autocrine action (223).
525
D. HPA maturation in the primate fetus
There is emerging strong evidence that maturation of HPA
function occurs in the primate fetus in a manner generally
analogous to that discussed above in the sheep fetus. Excellent reviews by Pepe and Albrecht (221, 224) and by Mesiano
and Jaffe (225) have provided detailed analyses of pituitaryadrenal function in the primate fetus. In the human, baboon,
and monkey fetus the pituitary is necessary for adrenal maturation and steroidogenesis, at least during the second half
of gestation. Adrenal development is impaired in anencephalic human fetuses. In the baboon fetus treated in late
gestation with betamethasone, there was suppression of fetal
pituitary POMC mRNA and reductions in fetal adrenal
weight, and 3␤-HSD fetal adrenal ACTH receptor mRNA
levels (221). The authors concluded that increased expression
of fetal adrenal ACTH receptor and mRNA species encoding
steroidogenic enzymes depended upon fetal pituitary ACTH
stimulation.
In the human fetus, ACTH activity is present in the pituitary by 5 weeks’ gestational age, and CRH- and AVP-like
activity is present in the fetal hypothalamus by approximately 12 weeks gestation (226). CRH1– 41, in addition to a
large molecular weight form of CRH, are contained within
the human fetal hypothalamic tissue. CRH and AVP synergize in promoting ACTH release from the human fetal pituitary tissue in early gestation, and the stimulatory effect of
CRH and ACTH output was reproduced by 8-bromo-cAMP
(see Ref. 12).
Levels of POMC mRNA in anterior pituitary tissue from
fetal baboons increased significantly from mid (day 100) and
late (day 165) gestation (term ⫽ day 184) in nontreated animals, and there was a corresponding increase in pituitary
cells expressing ACTH peptide (227, 228). In the baboon it
has been suggested that this increase in fetal pituitary POMC
mRNA levels might be associated with increased pituitary
CRH receptor activity, rather than increased expression of
CRH peptide in hypothalamic nuclei. However, administration of estrogen to midgestation baboons resulted in an increase in levels of POMC mRNA- and ACTH-positive corticotrophs in pituitary tissue to values that approached, but
remained significantly different from, those at term (228).
Pepe et al. (229) have argued that this increase in POMC is
secondary to an effect of estrogen on placental 11␤-HSD
activity, particularly 11␤-HSD-2. In previous studies, these
investigators have shown increased expression of placenta
11␤-HSD-2 in the baboon during pregnancy and have shown
that activity of this enzyme is increased by treatments that
increase estrogen and decreased with inhibition of estrogen
production or action (221, 229). In midgestation, the relatively lower levels of placenta 11␤-HSD-2 allow passage of
maternal cortisol into the fetal compartment and relative
suppression of fetal HPA activity (221). With increased 11␤HSD-2 activity at day 160, there would be diminished maternal cortisol reaching the fetus (230), allowing the fetal
HPA axis to escape from the presumed negative feedback of
maternal cortisol. This would allow increases in POMC gene
expression, ACTH output, and fetal adrenal maturation.
These results are compatible with observations that production of cortisol by the primate fetal adrenal gland is relatively
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526
CHALLIS ET AL.
low for much of gestation (231, 232). The bulk of the gland
is occupied by the fetal zone with relative deficiency of 3␤HSD, and predominant formation of C19 ⌬5 steroids, particularly DHAS (233–235). In late gestation, there is an increase in ACTH receptor mRNA and 3␤-HSD activity in the
definitive zone of the fetal adrenal, and a decrease in ACTH
receptor mRNA and formation of DHAS in the fetal zone
(236 –238). The expression of fetal adrenal enzymes P450C17
and P450SCC remained relatively unchanged during gestation. Thus, there are subtle differences between fetal adrenal
development in the primate and sheep. In the former, expression of 3␤-HSD appears rate limiting toward adrenal
cortisol output whereas in the ovine species, expression of
P450C17 appears to regulate fetal adrenal steroidogenesis.
In primate pregnancy, estrogen production in the placenta
depends extensively on the provision of C19 precursor steroids, predominantly from the fetal adrenal gland (239, 240).
Fetal adrenal DHAS can be converted to estrone and estradiol in the placenta, and approximately 50% of circulating
maternal estrone and estradiol are derived from placental
aromatization of fetal DHAS; the remainder is formed from
maternal adrenal C19 steroids (239, 241). Activation of the
pituitary-adrenal axis of the fetus occurs in late gestation.
There is a progressive increase in the concentration of DHAS
in the fetal circulation, which mirrors an increase in maternal
plasma estriol concentration (maternal estriol is formed in
the placenta from the precursor 16-hydroxy-DHAS that is
90% of fetal origin and formed in the fetal liver from adrenal
DHAS). This pattern of fetal adrenal activation, reflected in
plasma DHAS concentrations, resembles the time course of
increase for plasma cortisol in the fetal sheep. Recent studies
have shown that the fetal adrenal in primates is divided into
the outer adult zone that produces predominantly aldosterone, the fetal zone that produces DHAS, and the transitional
zone, interposed between the adult and fetal cortex, which
produces predominantly cortisol (225). Thus, the elegant
studies of Mesiano and Jaffe (225) and Coulter and colleagues
(242), have shown that P450scc is expressed throughout the
primate fetal adrenal gland. P450C17 is not expressed in the
definitive zone but is expressed in the transitional and fetal
zones. P450C21 is expressed throughout the gland. 3␤-HSD is
not expressed in the fetal adrenal at midgestation but is
expressed in the definitive and transitional zone in late gestation fetuses. P450C11 is expressed in the transitional zone in
midgestation and throughout the fetal adrenal cortex in late
gestation. ACTH stimulates steroidogenesis in the transitional and fetal zone; the major products in late pregnancy
are cortisol from the former and DHAS from the latter. Both
in vitro and in vivo studies show dependence on ACTH for
fetal adrenal steroidogenesis. More recent studies, however,
have indicated that CRH, potentially of placental origin (see
below), can also stimulate the fetal zone to produce DHAS
(243). In addition, this zone of the fetal adrenal appears to
respond to trophic inputs from the fetal pituitary other than
ACTH. ER-␣/␤ mRNA is also expressed in fetal and definitive-transitional zones of the baboon fetal adrenal cortex at
mid- and at late gestation (244). The presence of ER in the
adrenal cortical cells provides an additional mechanism by
which estrogen mediates ACTH-dependent functional mat-
Vol. 21, No. 5
uration of the primate fetal adrenal gland. In addition, previous studies had shown that estrogens increase availability
of LDL-cholesterol as precursor for adrenal steroidogenesis
(245, 246).
The difference in fetal adrenal architecture between the
sheep and primate fetus has been regarded by many as a clear
obstacle to extrapolating from the sheep model of parturition
to the primate. However, it is now apparent that similarities
between these species are greater than the perceived differences (247). In both the sheep and primate fetus the fetal
adrenal produces increased amounts of cortisol in late gestation (247). It is relatively unprofitable to make detailed
comparison of the minutiae of temporal changes in plasma
cortisol because of differences in binding to circulating CBG,
transplacental transfer from the mother, and tissue levels of
11␤-HSD isozymes in the fetus that could locally regulate
cortisone-to-cortisol interconversion. In both sheep and primate, the feto-placental unit also produces increased
amounts of estrogen. In the primate, that estrogen results
primarily from placental aromatization of precursors generated within the fetal (and to a certain extent maternal)
adrenal. There is no induction of placental P450C17 at term,
and the primate placenta does not metabolize C21 steroids
through to estrogen. In the sheep, a similar fetal-placental
unit of estrogen production exists in pregnancy. The major
fetal adrenal precursors are both ⌬5 and ⌬4 C19 steroids
produced from the developing zona fasiculata reticularis. At
term, the prepartum rise in fetal cortisol results directly or
indirectly in increased expression of P450C17 in the ovine
placenta, which at that time becomes capable of metabolizing
⌬5 C21 steroids to estrogen. Thus, the apparent difference in
the pattern of estrogen biosynthesis between sheep and primate at term, in its simplest term, reflects the source of C19
precursor steroid. The mechanisms of HPA activation may
vary. However, in the primate, the C19 precursor comes from
the fetal zone of the fetal adrenal gland. In the sheep, that
precursor comes in part from the fetal adrenal, but there are
additional estrogen precursors produced in the placenta under the influence of cortisol from the fetal adrenal gland. We
suggest that these differences are ones of degree rather than
of absolute distinction.
The role of estriol in the processes leading to the onset of
human parturition has remained unresolved over many
years. Maternal estriol concentrations reflect fetal hepatic
16-hydroxylation of DHAS produced from the fetal adrenal
gland. It might be anticipated that estriol concentrations in
the maternal circulation would increase in response to fetal
stress and might be predictive of impending preterm delivery. Maternal estriol levels increase exponentially toward
normal term. Lachelin and colleagues (248, 249) have shown
that maternal plasma and salivary estriol concentrations are
elevated further in a subset of patients with diagnosis of
preterm labor. Since estriol may affect uterine CAP gene
expression (249), it could contribute to the progressive increase in uterine responsiveness in primate pregnancy during the third trimester of gestation, and its measurements
may be of predictive value in delineating patients at risk of
premature delivery (249, 250).
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E. Placental progesterone and human pregnancy: the
enigma of the progesterone block
A fall in the plasma progesterone concentration is the
single most common endocrine event associated with parturition across species (12, 250, 251). Administration of exogenous progesterone at term not only blocks the expression
of CAP genes, but blocks the onset of labor (252). Even in the
human, where there is no evidence of a fall in maternal
plasma or uterine tissue progesterone, administration of the
progesterone receptor (PR) antagonist RU486 leads to increased uterine activity and induction of labor (253). In human pregnancy, the luteoplacental shift in progesterone production occurs by 5– 6 weeks’ gestation (254). Progesterone
is synthesized from pregnenolone by placental syncytiotrophoblast and by chorionic trophoblasts (Fig. 6 and Ref. 255).
However, the levels of 3␤-HSD mRNA, protein, and activity
do not change in these tissues with labor at term or preterm
(256), although regional changes in 3␤-HSD expression
might still occur (257). For example, expression of 15hydroxyprostaglandin dehydrogenase (PGDH; the major PG
metabolizing enzyme) in chorion is regulated by progesterone (see below) and levels correlated with 3␤-HSD in tissue
collected adjacent to the placenta, but not in the cervical
region. In this lower segment, it was suggested that the action
of progesterone in maintaining PGDH tonically was overcome near term by the inhibitory influence of proinflammatory cytokines (see below). There are reports of an increase
in the estrogen-progesterone (E:P) ratio in amniotic fluid of
women during labor; however, these changes are not impressive (250). We have referred to suggestions that maternal
estriol, which increases during term and preterm labor,
might promote myometrial activation and labor contractions, but this possibility requires stronger experimental verification (249). Alternatively, another progesterone-like steroid, possibly a progesterone metabolite that interacts with
the PR, might serve as the active progestagen in human
pregnancy and decline before labor, or progesterone could be
converted to an inactive metabolite that displaces progesterone from its receptor (258 –260). To date, there are no clear
data to support either of these possibilities. Erb et al. (261)
reported recently that levels of allopregnanolone, the 3␣,5␣reduced metabolite of progesterone that can bind to ␥-aminobutryic acid-A receptors and inhibits uterine smooth muscle, did not decrease with labor. The 5␤- metabolite blocks OT
binding to its receptor and inhibits OT-induced contractions
in the human myometrium. However, there is also no evidence that levels of this metabolite decrease at term.
Studies of gene expression in the human myometrium
have focused on the lower uterine segment. These studies
suggest that the PR system is functional in this region during
labor. Increased expression of progesterone-responsive
genes such as CX26 (which would promote relaxation) raise
the possibility that elevated levels of progesterone are required to support establishment of a functional (inhibitory)
lower uterine segment during labor. If this were so, it would
also require mechanisms within the fundus that would block
the actions of progesterone, allowing the expression of CAPS,
and promote contractility in that region.
Although recent exciting data have shown that proges-
527
terone can bind directly to the oxytocin receptor (OTR) and
inhibit its signaling (262), the majority of the actions of progesterone are mediated through a nuclear ligand-inducible
transcription factor, the PR. It has been suggested that a
functional withdrawal of progesterone may involve antagonism of its action at the level of the PR or PR interaction with
transcriptional machinery (8). This might include a decrease
in PR expression, a switch in PR isoforms, a change in expression of receptor accessory proteins (e.g., heat shock proteins and receptor coactivators/repressors), or increased expression of endogenous antagonists of progesterone or PR
(such as cortisol, TGF␤, or phospholipids). Three isoforms of
the PR have been described: the full-length PR-B and the
truncated isoforms, PR-A and PR-C. In mammals, PR-B functions predominantly as an activator of progesterone-responsive genes, while PR-A acts as a modulator or repressor of
PR-B function and of other nuclear receptors including the
GR, possibly because it lacks one of the three activation
function domains (AF3) contained within PR-B (263). Notably, progesterone repression of estrogen-induced gene expression was effected through PR-B and not through PR-A.
The expression of PR-A and PR-B isoforms is regulated differentially during development and by hormone treatment.
The PR-C isoform (⬃60 kDa), which has C-terminal transactivating domains and lacks the first zinc finger of the DNA
binding domain, can dimerize with and modify (possibly
inhibit) transcriptional activity of both PR-A and PR-B.
Analysis of PR expression is complicated by the multiple
mRNA and protein species of the receptor. A decrease in PR
immunostaining in myometrium at term has been reported
but, given the multiple isoforms of PR, these data are difficult
to interpret. There is no change in PR mRNA in myometrium
or membranes with labor, and no evidence of change in PR-B
or A ⫹ B mRNA nor in any immunoreactive PR isoforms in
samples of lower segment myometrium during labor that might
indicate a decrease in progesterone signaling (G. Erb, N.
McLusky, and S. J. Lye, unpublished results). There was
increased expression of heat shock proteins (HSP)-90 and
HSP-56 as well as the steroid receptor coactivators SRC-1 and
TIF-2 (G. Erb and S. J. Lye, unpublished results). These coactivators may interact with several steroid receptors, but
any interaction with PR should increase rather than decrease
its transcriptional capability. There are limited data on ER
expression in myometrium with labor. However, in the
lower uterine segment at term, ER mRNA, protein, and highaffinity binding all appear to be very low.
There are several candidates for potential endogenous antagonists of progesterone action. TGF␤ has been proposed as
an endogenous antiprogestin that reduces progesterone
stimulation of genes such as enkephalinase (264). Others
have reported that a phospholipid extract of human fetal
membranes was capable of inhibiting progesterone binding,
but not estrogen binding. Cortisol itself may compete with
progesterone in the placenta or membranes to regulate the
gene for CRH (263). We have found (see below) that while
progestagens such as medroxyprogesterone acetate (MPA)
increase PGDH activity in human placental and chorion trophoblasts, this effect is reversed by cortisol. At the present
time, it is not clear whether these are separate actions through
GR and PR, or whether cortisol and MPA compete for PR-GR
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CHALLIS ET AL.
binding. Although four upstream GREs have been identified
within the PGDH promoter, no putative PRE has been identified. Cytokines [interleukin-1␤ (IL-1␤), tumor necrosis factor-␣ (TNF␣)] also decrease PGDH activity, but their interaction with progesterone as putative antiprogestins remains
unexplored. In recent studies, Stevens et al. (265) reported
that CRH receptor type 1 (CRH-R1) was expressed preferentially in myometrium and fetal membranes of human gestation. Levels of CRH-R1 increased in myometrium collected
from patients in term and preterm labor but, importantly,
levels of CRH-R1 in lower segment myometrium were consistently much higher than levels of CRH-R1 in the fundal
region (265). CRH acts through CRH-R1 to increase levels of
cAMP and promote uterine relaxation (61). We therefore
proposed that the role of CRH-R1 in the lower uterine segment was to promote relaxation of this region during labor
and to facilitate descent of the fetus (61, 265). These data
indicated that there might be mechanisms by which CRH-R1
expression was regulated differentially in the fundus and the
lower segment during labor. In independent studies, Sparey
et al. (266) reported that levels of PGHS-1 and PGHS-2 proteins were also expressed at greater levels in the lower than
upper uterine segment. Connexin-43 protein, in contrast, was
expressed at much greater levels in the upper uterine segment. Myometrial GS␣ protein was uniformly expressed in
both lower and upper segments and down-regulated at the
time of parturition. These authors also concluded that differential expression of these genes might be important to
allow cervical ripening before and dilatation during labor,
with orderly propagation of uterine contractions (266).
Our own data suggest considerable differences in the expression of CAP genes in the human myometrium during
labor compared with other species. In contrast to observations in myometrium of rats, sheep, and cows, Teoh et al. (267,
268) did not observe any increase in the expression of CAP
genes, including CX-43, OTR, and the PG receptors that are
linked to stimulation of contractile pathways (FP, EP1, and
EP3 receptor subtypes, including four splice variants of the
EP3 receptor) in lower segment myometrium at labor. However, Teoh et al. (267) did observe increased expression of
connexin-26, the EP4 receptor and CRH-R1 receptor that
might be expected to promote myometrial relaxation after an
increased generation of cAMP. It is known that connexin-26
is positively regulated by progesterone.
What is the relevance of these observations to the effect of
progesterone on the myometrium and the apparent lack of
withdrawal of the progesterone block to the myometrium in
human pregnancy? We propose that the biological basis for
the onset of labor in animals and in humans is essentially
similar. Both require activation of the myometrium and the
generation of uterotonins to generate labor contractions. In
human fetal membranes and myometrium, however, regional differences in gene expression allow functional autonomy during labor. We suggest that this functional autonomy may be critical for the efficient and effective delivery
of the fetus and speculate that this is a mechanism associated
with evolution to bipedal life. We have suggested that this
regionalization is established through the action of progesterone. Early studies, e.g., those of Wiqvist and colleagues
(269), support this hypothesis. These authors found that
Vol. 21, No. 5
PGF2␣ had little effect on the fundal myometrium, but was
stimulatory in lower segment specimens taken before labor.
PGE2 induced a biphasic dose-dependent response. However, PGF2␣ and PGE2 always stimulated fundal myometrium collected during spontaneous labor. PGE2 induced
inhibition in lower segment samples collected at that time
while PGF2␣ had no effect.
We speculate that during pregnancy, progesterone limits
the generation of stimulatory PG in chorion by inducing high
expression of PGDH (see below), and it also inhibits the
expression of CAP genes within the myometrium, thereby
maintaining the muscle in a quiescent state (8). Functional
regionalization of both chorion and myometrium at term is
engineered by progesterone. In the cervical, but not fundal,
region of chorion, there is a local decrease in PGDH (10),
increased production of PGE, and later matrix remodeling.
In the myometrium, functional withdrawal of progesterone
in the fundus induces CAP gene expression and myometrial
activation. Enhanced progesterone signaling in the lower
uterine segment, however, promotes the expression of genes
that induce relaxation, facilitating descent of the fetus (8). The
mechanisms inducing functional withdrawal of progesterone in fundal myometrium and cervical chorion need not
necessarily be the same (270). Cortisol and/or cytokines may
antagonize progesterone- induced PGDH activity in chorion
(see below). In myometrium, potential mechanisms include
changes in PR isoforms, steroid receptor co-activator/repressors, or other putative antagonists of progesterone action. We
speculate that this concept of human labor provides an explanation as to why progesterone levels remain high in this
species. Rather than being an impediment to labor onset, we
suggest that progesterone is required to induce lower segment relaxation and the safe and efficient delivery of the
primate fetus.
Recent exciting studies have pointed to a role for progesterone in maintaining cervical function during pregnancy,
and to metabolism of progesterone within the cervix as being
a critical step in cervical dilatation and parturition. Mahendroo and colleagues (271, 272) showed that parturition was
delayed in mice lacking steroid 5␣-reductase type 1 enzyme.
They showed subsequently that basal and stimulated levels
of uterine contractility were similar in these animals and in
wild-type controls. However, cervical distention did not occur in 5␣-reductase-deficient animals, and cervical compliance was less on day 20 of gestation than earlier in pregnancy. As expected, relaxin, which is known to promote
cervical ripening, induced delivery in both wild-type and
5␣-reductase knockout animals. Subsequent studies demonstrated that while serum progesterone concentrations declined in knockout animals in a manner generally similar to
that of controls, the concentration of progesterone in cervical
tissue and in whole uterus remained elevated. As expected,
cervical ripening and parturition occurred after ovariectomy.
Thus, these studies point to the role of progesterone metabolism in facilitating normal cervical dilatation that must accompany uterine contractility to allow birth (273). In the
uterus of pregnant mice, progesterone can be metabolized at
term through either 5␣-reductase or 20␣ -HSD pathways. In
the cervix, however, there is limited 20␣-HSD activity, and
normally 5␣-reductase provides the pathway for progester-
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PARTURITION
one metabolism, progesterone withdrawal, and cervical ripening and dilatation (272). Further studies of other genes
associated with cervical ripening are clearly warranted in
this fascinating model, as are measurements of 5␣-reductase
activity in human cervix from patients at term and preterm
labor.
V. Myometrial Stimulation: Phase 2 of Parturition
Activation prepares the myometrium to respond optimally to the production of those myometrial stimulants that
provoke myometrial contractility during labor. Although
many agonists have been described to stimulate myometrial
contractions, convincing information is available only for OT
and stimulatory PGs (274). The physiological role of other
putative agonists such as CRH is uncertain and equivocal.
The actions of these three groups of compounds are discussed below.
A. Stimulation: role of OT
OT is a nonapeptide synthesized by hypothalamic magnocellular neurons located in the supraoptic and paraventricular nuclei (275–277). Hypothalamic OT is released into
the circulation from the posterior pituitary. Its classical effects include promoting myometrial contractility during late
pregnancy and parturition and stimulating milk release from
the mammary gland in lactation (275, 278, 279). The dilemma
surrounding the role of OT in the process of labor arose when
it was unclear whether levels of OT in the maternal circulation actually increased before the onset of labor (279, 280).
The recent report that mice bearing a null mutation in the OT
gene have normal pregnancies and labors may reflect a compensatory effect of AVP (281, 282). Studies showing the relative ineffectiveness of OTR antagonists in preventing preterm labor, however, suggest that while this hormone
contributes to labor, it may not be an essential element (283).
One aspect of the solution to the apparent discrepancy
between circulating OT levels and parturition was the dramatic increase in myometrial sensitivity to OT before and
during labor, associated with a several-fold increase in myometrial OTR gene expression, which coincides with peak
uterine responsiveness (276, 284 –286). Thus, changes in circulating OT levels would not be necessary for the peptide to
have a physiological role in labor (280). A parallel conclusion
is drawn from the 24-h pattern of OT secretion, and myometrial sensitivity (287). Recent studies also suggest that OT
may act as a local mediator of parturition. OT gene expression has been demonstrated in the human and rat uterus and
fetal membranes (288 –290). In the rat, fetal membranes, placenta, and uterus synthesize OT mRNA transcripts with
extended poly-A tails (289). Levels of OT mRNA in rat fetal
membranes declined from gestational day 14 to term, but
uterine OT transcripts increased during gestation 150-fold
and exceeded levels of OT mRNA in the hypothalamus at
term (289). Human fetal membranes, amnion, chorion, and
decidua synthesize OT mRNA, and levels of OT mRNA
transcripts increased in these tissues at the time of parturition
(290). In vitro studies with rat and human chorio-decidual
tissue have indicated that estrogen, generated locally, could
529
up-regulate OT gene expression (291–293), consistent also
with the presence of an ERE in the OT promoter region (293).
Other, fascinating studies have indicated that OT may promote uterine activity by antagonizing the relaxant effect of
CRH through receptors coupled to adenylate cyclase (see
below). The general consensus is that OT appears to have a
role to play in the stimulus to uterine contractility at term and
in uterine involution (294). Whether that role is indispensable
remains in dispute.
B. Stimulation: role of PGs
There is a substantial body of evidence to support a role
for PGs in the labor process, at term and preterm (207, 295).
PGs contribute to the transition from phase 1 to phase 2
rather than initiating the labor process. Mice carrying null
mutations for genes encoding the PGF2␣ receptor (296), cytosolic phospholipase A2, and prostaglandin synthase type
1 (PGHS-1) (297) have delayed labor onset although neonatal
viability is diminished. Mice lacking the PGHS-2 gene (298)
have not been studied in relation to gestation length and
pregnancy outcome because fertility is impaired, and ovulation and implantation are blocked. Lack of PGF2␣ (FP)
receptor prevents effective luteolysis at the end of gestation,
so plasma progesterone concentrations are maintained. In
these animals OTR expression in the uterus is suppressed,
presumably in response to the elevation in progesterone,
since ovariectomy allowed OTR up-regulation and delivery.
The extent to which information from these murine models
is applicable to human gestation may be questioned, since
the primary site of PG action is at the level of the corpus
luteum, which is not required for pregnancy maintenance in
women after the first 5– 6 weeks of pregnancy. Perhaps the
best indicator for a role of PG in parturition in primates as
well as sheep and other species is the measurement of increased PG output before the appearance of labor-like myometrial contractions (299 –301) and the effectiveness with
which drugs that block PG synthesis suppress myometrial
contractility and prolong gestation length.
PGs are formed from membrane phospholipids through
the initial activity of phospholipase A2 or C isozymes forming unesterified arachidonic acid (302–304). PLA2 isozymes,
localized by immunostaining to fetal membranes and myometrium (305), may include the larger molecular mass (85–
110 kDa) cytosolic form (cPLA2), as well as secretory types
I, II, and III, extracellular 14-kDa forms. Activation of secretory PLA2 (sPLA2) requires millimolar concentrations of calcium, whereas cPLA2 is activated at micromolar calcium
concentrations (see Ref. 8).
Cytosolic PLA2 translocates to the cell membrane in response to agonist stimulation and liberates arachidonic acid
from the sn-2 position of phospholipid (306). Activity of
cPLA2 is reportedly greater in amnion from patients not in
labor at term or preterm than from patients in labor, explained as depletion of cPLA2 at this time (304). Previous
studies had shown that cPLA2 expression was up-regulated
in WISH cells, a transformed amnion epithelial cell line, in
response to cytokine stimulation, and that this occurs in
parallel with increased expression of PGHS-2 by these cells
(307, 308). The general consensus, however, is that in human
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CHALLIS ET AL.
pregnancy, expression of PLA2 increases gradually in fetal
membranes during gestation but does not increase appreciably at the time of labor (309).
Arachidonic acid is further metabolized to the intermediate PGH2 by PGHS enzymes, which have both cyclooxygenase and peroxidase activities (310, 311). There are two forms
of PGHS; both are heme proteins composed of two approximately 70-kDa subunits. The constitutive form (PGHS-1)
and the inducible form (PGHS-2) are distinct gene products
although they have considerable sequence homology, and
their cDNAs are 60 – 65% homologous (312). PGHS-1 has
similar properties to other housekeeping genes. PGHS-2 is
characteristically up-regulated by growth factors and cytokines. The activity of PGHS-1 and PGHS-2 is inhibited by a
wide spectrum of nonsteroidal antiinflammatory drugs.
These differ in their Ki values for the two PGHS isoforms,
suggesting the potential to develop specific inhibitors of either isoform for therapeutic management (313–315).
Arachidonic acid may also be metabolized through different lipoxygenase pathways including 5-lipoxygenase,
platelet-type-12-lipoxygenase, leukocyte-type-12-lipoxygenase, and 15-lipoxygenase (316). Arachidonic acid metabolism
through 5-lipoxygenase forms 5 H(P)ETE, which can be converted to leukotriene A4 (LTA4), which is subsequently hydrolyzed to LTB4 or LTC4. 12-Lipoxygenase or 15-lipoxygenase activity results in the formation of 12-H(P)ETE and
15H(P)ETE. There are some suggestions that these products
can weakly stimulate contractility of smooth muscle. It has
also been suggested that arachidonic acid metabolism in
human fetal membranes during pregnancy is directed preferentially toward lipoxygenase products, but there is a progressive switch toward the more potent PGHS (also cyclooxygenase, COX) activity at term (317). Primary PGs are
formed from PGH2 through the activity of specific isomerases and synthases. There is unfortunately very little information concerning the expression, localization, and change
in activity of these enzymes in intrauterine tissues at term or
preterm labor, and this will be an obvious area of further
investigation.
The major pathway in the metabolism of PGE2 and PGF2␣
involves the action of a type 1 NAD⫹- dependent PGDH that
catalyzes oxidation of 15-hydroxy groups resulting in formation of 15-keto and 13,14 dihydro-15-keto metabolites
with reduced biological activity (318, 319). We have reported
that PGDH expression and activity are decreased in choriodecidual tissue of women at spontaneous and preterm labor
(see below), raising the possibility that failure to inactivate
PGs produced within intrauterine tissues during pregnancy
may be one cause of preterm labor (320).
The action of PGs is exerted through specific receptors
including the four main subtypes for PGE2, EP1, EP2, EP3,
and EP4, and FP for PGF2␣ (60, 321). EP1 and EP3 receptors
mediate contractions of smooth muscle through intracellular
signaling pathways that elevate free calcium and decrease
intracellular cAMP (27). EP2 and EP4 receptors are coupled
through adenylate cyclase and increase cAMP formation,
leading to relaxation of smooth muscle. Consistent with this,
various groups have reported that EP2 expression in myometrium is higher preterm than at term. In the rat, parturition
is associated with down-regulation of EP receptor subtypes
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and with up-regulation of myometrial FP receptors, effecting
a switch from inhibition to stimulation.
1. PG synthesis. Regulation of PGHS-2 and PGHS-1 genes are
clearly multifactorial (322–324). There are two nuclear factor
(NF)-␬B binding elements within the proximal promoter region of PGHS-2 (325, 326). p50 And p65, key members of the
NF-␬B Rel family of proteins are present in trophoblasts and
likely serve as mediators of cytokine-induced up-regulation
of PGHS-2 expression (327). The PGHS-2 promoter also includes response elements resembling NF-IL6, GRE, CRE, and
AP2 sites (323, 325). Levels of PGHS-2 are increased up to
80-fold in response to various cytokines and growth factors,
whereas levels of PGHS-1 are usually increased only 2- to
3-fold in response to these stimulators (328, 329). Studies in
several species, including the human, have indicated that the
PGHS-2 isoform is the principal form of the enzyme involved
in the increased PG production seen at the time of parturition. Effects of CRH in up-regulating PG output, at least
within fetal membranes (see below), is likely mediated
through proximal CRE sequences (326). Although glucocorticoids inhibit PGHS-2 expression in WISH cells and in most
other cell types, apparently by interference with the NF-␬B
signaling system (330), they stimulate PGHS expression and
activity in trophoblast-derived cells including amnion, and
chorionic trophoblast (58, 331–334). Kniss (327) reported a
similar effect of dexamethasone in stimulating PGHS-2
mRNA expression in human breast adenocarcinoma cells.
The stimulatory effect of glucocorticoids on PGHS gene expression in human fetal membranes is central to our current
hypotheses of human parturition and will be discussed in
more detail below.
In human pregnancy, the PG synthesizing and metabolizing enzymes are compartmentalized discretely between
the amnion and chorion, decidua, and myometrium (Fig. 5;
Refs. 335 and 336). PGHS activity predominates in amnion,
PGE2 is the principal PG formed (337), and there is an increase in PG synthesis and levels of PGHS-2, but not PGHS-1
mRNA at preterm and term labor (338 –343). Immunohistochemical and in situ hybridization studies have localized the
PGHS-2 enzyme and mRNA to the amnion epithelium (344 –
346), the subepithelial cells in the mesenchyme and in the
chorion laeve trophoblasts with lower expression found in
decidua (347–349). Decidua has been reported to produce
increased amounts of PGs at the time of labor, but this is not
a consistent observation (348). Human decidua is made up
of decidualized stromal cells, bone marrow-derived macrophages, and other cell types including trophoblasts that interface with chorion (350). Variability in cell populations
used for in vitro studies may contribute to the variability of
responses that have been obtained. In chorion, interposed
between amnion and decidua, PGDH activity predominates,
although PGHS is also expressed (347, 351). Output of PGs
and PGHS activity is greater in chorion from patients at
spontaneous labor than at elective term cesarean section; in
preterm labor chorion both PGHS-1 and PGHS-2 mRNA
levels are increased (352, 353).
It is generally considered that activity of PGDH predominates in chorion (354), forming a relative metabolic barrier
that prevents passage of PGs generated within amnion or
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531
FIG. 5. Diagrammatic representation of sites of PG synthesis and metabolism at term labor (panel A) and preterm labor (panel B). PGHS-2,
prostaglandin H synthase 2; PGDH, 15-OH prostaglandin dehydrogenase.
chorion from reaching underlying decidua or myometrium
through most of pregnancy (320, 355). The presence of high
PGDH activity in chorion trophoblasts (356) implies that at
full term those PGs acting on the myometrium would more
likely be derived from decidua, or from the myometrium
itself (41, 357). There are variable reports, however, of
changes in PGHS activity in human myometrium at the time
of labor (59). Some workers have reported increased PGHS-2
expression and activity, while others have reported no
change, or even decreased activity. In myometrium through
pregnancy, PGHS-1 or PGHS-2 must be present to generate
the predominant PGI2 which, as discussed above, contributes
to maintenance of uterine quiescence (59). It has been suggested that PGI2 formation in myometrium may be decreased
by glucocorticoids. Unfortunately, it is difficult, experimentally, to obtain consistent specimens of human myometrium
for biochemical analysis. Generally, tissue is obtained from
lower segment uterus, but at term with ensuing cervical
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CHALLIS ET AL.
dilatation, the proportion of myocytes in the tissue is likely
to have changed. Further, recent studies indicating that there
are regional differences in CAP genes between the fundus
and lower segment of the human uterus in late pregnancy
(see above) may suggest the need for further reexamination
of these issues, ideally combined with experimental manipulation in subhuman primates.
It remains crucial to understand regulation of PGHS-1 and
PGHS-2 expression in human fetal membranes and to delineate the major site of PG production at term and preterm
labor (Fig. 5). These may not necessarily be the same. For
example, instances of preterm labor may be associated with
elevated PG production in amnion or chorion, whereas term
labor may require increased PGHS-2 expression in decidua
and myometrium (344). Given that PGs act generally as paracrine or autocrine regulators, it will be exceedingly difficult
to obtain in vivo evidence for altered PG production specifically at these sites. Amniotic fluid concentrations of PGs
increase at labor, and the initial changes precede the onset of
myometrial contractility. Levels of PGF2␣ in amniotic fluid
presumably reflect, in part, production from decidua, since
PGE2 and not PGF2␣ is the major eicosanoid formed from
amnion and chorion (Figs. 5 and 6). However, these measurements probably provide no more than a crude estimate
of the pattern of PG change at a local cellular level and give
no information concerning receptor subtypes and distribution (358).
Primary cultures of mixed and purified cells from human
amnion or chorion have been used extensively as models to
Vol. 21, No. 5
study the regulation of PG formation in response to cytokines, growth factors, CRH, and lipopolysaccharides. In addition, the amnion-derived epithelial cell line (WISH cells)
has also been used extensively (359 –361). A crucial reservation with all of these in vitro studies is that, in general, single
compounds have been studied in isolation of the in vivo
environment; the extent to which results can be extrapolated
from in vitro to in vivo will remain, unfortunately, a matter
of conjecture.
Many cytokines have been shown to act on amnion, chorion leave, and decidua to increase PG output (360, 362–364).
IL-1␤ stimulates PG output by cultured amnion, chorion
leave, and decidua (195, 317, 365) while IL-6 stimulates PG
output by decidua and amnion (366, 367). IL-8 did not alter
PG production by chorion or decidua, but augmented the
stimulatory action of other cytokines (368). The effect of IL-1␤
is certainly associated with increased expression of PLA-2
and PGHS-2 (329). The action of IL-1␤ can be reduced by the
naturally occurring receptor antagonist, which has been
shown to prevent IL-1␤-induced labor in mice (369). IL-1␤
stimulation of PGHS in amnion and chorion may be mediated through the NF-␬B system (370 –372). In WISH cells
stimulated with interleukin-1␤, I-␬B␣ was degraded by more
than 90% within 15 min of stimulation, and this was associated temporally with nuclear translocation and binding of
NF-␬B (373). PGHS-2 mRNA was increased within 30 min
and reached steady state by 4 h. PGHS-2 protein then increased more than 80-fold, and this was associated with a
corresponding time-dependent increase in PG production.
FIG. 6. Summary to indicate factors leading to up-regulation (⫹) or down-regulation (⫺) of prostaglandin H2 synthase in intrauterine tissues.
The role of progesterone remains equivocal.
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Inhibition of I-␬B␣ degradation by calpain-I inhibition
blocked NF-␬B translocation, and increases in PGHS-2
mRNA and protein, and PG synthesis (373). Wang and Tai
(374) provided similar information and showed that in WISH
cells, dexamethasone blocked IL-1␤-mediated stimulation of
PGE2 output consistent with the general model of mutual
transcriptional antagonism from GR/NF-␬B interaction
(330).
Human amnion cells can be maintained as mixed populations in culture or can be separated into primary epithelial
cells and cells of the subepithelial mesenchymal layer (375).
We have reported recently that the output of PG by mesenchymal cells exceeds that of epithelial cells in the basal state.
Epithelial cell production of PGs was stimulated by glucocorticoids, whereas there was no significant change in the
already elevated output of PGs from mesenchymal cells
(375). Previously, in mixed cultures, glucocorticoids and
IL-1␤ were shown to increase PGHS-2 mRNA, protein, and
PGE2 output predominantly from the subepithelial mesenchymal cells (331, 376). It remains possible that this apparent
difference can be explained by epithelial-mesenchymal cell
interaction, and current studies are directed at resolving this
issue.
The effect of glucocorticoids on primary cultures of amnion cells and on chorion trophoblast cells is surprising (377)
and striking (323, 378, 379). Although dexamethasone inhibited PGE2 output by freshly dispersed amnion cells, it stimulated PGE2 output by amnion cells after 4 –5 days in culture
(376). The effect was dose dependent and associated with
increased expression of PGHS-2 mRNA and protein. The
activity of glucocorticoids is also receptor mediated and can
be inhibited by addition of GR antagonist (380). In previous
studies, we had localized GR to amnion epithelial cells, subepithelial fibroblasts, and chorion laeve trophoblasts in human pregnancy (381). GR exists as both ␣-form and ␤-form
(330). GR␣ is retained in the cytoplasm in an inactive state by
its association with the regulatory heat shock proteins such
as HSP-56 and HSP-90. GR␤, formed from alternate splicing
of the same mRNA transcript as GR␣, is localized in the cell
nucleus independent of binding to ligand. It appears that
GR␤ functions as a dominant negative regulator of GR␣
transactivation. Thus, earlier studies of GR localization to cell
types within human fetal membranes require repeating with
specific identification of GR␣ and GR␤ forms.
Peptides such as CRH could be released from amnion
epithelial cells to act in a local paracrine manner and upregulate PGHS-2 expression in mesenchymal cells (see Ref.
207). Full thickness fetal membranes treated in culture with
CRH were stimulated to increase output of PGE2 and increased levels of PGHS-2 mRNA within 4 h in culture. Thus,
the stimulatory effect of glucocorticoids on PG production by
amnion, known to involve an intermediary protein synthetic
step, could be the result of synergistic epithelial-mesenchymal interaction, in addition to, or instead of, any direct effect
on amnion cell types. Similar interactions may contribute to
the response to cytokines such as IL-1␤ in vitro (382). Interestingly, recent studies have shown that in amnion explants,
in contrast to chorion and decidua, the antiinflammatory
cytokine IL-10 stimulates rather than inhibits PG production,
and the normally antiinflammatory cytokine IL-4 stimulates
533
PGE2 output in amnion cultures (329). The authors have
suggested that amnion may therefore be refractory to inhibitory cytokines as part of an evolutionary mechanism designed to expedite the parturition processes.
Over the past 10 yr, in vitro studies have generated an
impressive list of substances capable of increasing PG output
by human fetal membranes in culture (383–386). Clearly,
availability of free calcium is a critical requirement. Epidermal growth factor (EGF), platelet activating factor (PAF), and
agents that activate protein kinase C stimulate PG output
(387, 388). Importantly, ␤-sympathomimetic drugs and
agents that increase intracellular cAMP levels also increased
PG output by cultured chorion and decidual cells (389). Catecholamines are present in increasing concentrations in human amniotic fluid in late gestation (390), and both amnion
and decidua express components of the adenylate cyclase
system, which undergoes stimulation with ␤-agonists such
as isoproteronol (391). Effects of these activators of adenylate
cyclase can be mimicked by (Bu)2cAMP or phosphodiesterase inhibitors such as methylxanthine (389). Studies such as
these may help explain the disappointing lack of efficacy of
␤2-sympathomimetic drugs in sustaining uterine quiescence
when used in the treatment of preterm labor (392). Although
these compounds are effective in the short term by elevating
cAMP and decreasing activity of MLCK, in the longer term
elevations of cAMP may up-regulate PGHS-2 through a
proximal CRE, resulting in increased output of stimulatory
PGs, uterotonins whose action the administration of ␤2mimetic was intended to antagonize.
2. PG metabolism. The major metabolizing enzyme for PGs
(393), PGDH, is exquisitely localized in fetal membranes to
trophoblast cells of chorion (Fig. 5). Thus, it could act as a
metabolic barrier to the passage of unmetabolized PGs, generated in amnion or chorion, and prevent their reaching the
underlying decidua or myometrium in a biologically active
form (354, 394, 395). Some years ago, we identified a group
of patients presenting in idiopathic preterm labor with deficiency of PGDH in chorion trophoblast cells (396). There
was a further reduction of ir-PGDH, PGDH mRNA, and
PGDH activity in chorion trophoblast cells, but not placental
trophoblast, in patients in preterm labor with an underlying
infective process (397). Thus, with preterm labor in the presence of an inflammatory response, loss of chorion trophoblast cells leads to loss of PGDH activity. PGs generated, for
example in response to elevations of cytokines, will not be
metabolized and will be available to stimulate underlying
myometrium.
In idiopathic preterm delivery, in the absence of infection,
it is clear that PGDH activity is specifically regulated in
chorion trophoblast (Fig. 7). During in vitro studies with
chorion trophoblast cells maintained in culture, we found
that the glucocorticoids, cortisol and dexamethasone, inhibited PGDH activity and decreased levels of PGDH mRNA
(398). Cortisone was as effective as cortisol, since chorion
trophoblasts contain 11␤-HSD Type 1 (11␤-HSD-1) capable
of reducing cortisone to biologically active cortisol (399). This
activity could be inhibited by carbenoxolone, an active ingredient of licorice. Chorion trophoblast cells also expressed
3␤-HSD and converted pregnenolone to progesterone (400,
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CHALLIS ET AL.
Vol. 21, No. 5
FIG. 7. Diagrammatic summary of factors regulating expression of the acitivity of PGDH in human chorion.
401). Inhibition of 3␤-HSD activity with trilostane led to
decreased PGDH activity and reduced levels of PGDH
mRNA in the cells. These could be restored by concurrent
addition of progesterone, or of the synthetic progestagens,
MPA or R5020 (398). Effects of these compounds, in turn,
were antagonized by onapristone and RU486, inhibitors of
progesterone action (398, 402). Furthermore, inhibition of
PGDH mRNA and activity by cortisol could be reversed by
addition of progesterone (320).
These data could be explained by glucocorticoids and progesterone acting through independent receptors, or by their
interaction at the same binding sites on GR␣ (403). Previously, Karalis and Majzoub (404) provided evidence that
similar interaction between progesterone and cortisol for
binding to GR explains the interactive effect of these compounds on the output of CRH by placenta trophoblast cells.
In recent studies we found that CRH also decreased PGDH
activity in chorion trophoblast cells in a dose-dependent
fashion (F. Patel and J. R. G. Challis, unpublished observations). We believe this activity is mediated through cAMP
generation, since CRH binds to CRH-R1 species in fetal membranes where it may increase cAMP, and cAMP decreases
PGDH activity (405), presumably acting through a consensus
CRE in its promoter region. Thus a pattern is emerging that
several agents which up-regulate PGHS-2 in human fetal
membranes (CRH, cortisol, IL-1␤, TNF) down-regulate
PGDH in chorion (Fig. 8). Effects of cortisol in the membranes
may be enhanced by local conversion of cortisone to cortisol,
through the reductase activity of chorionic 11␤-HSD-1 (406).
The activity of this enzyme is increased by PGE2 and PGF2␣
in a dose-dependent fashion that is associated with, and
dependent upon, a transient increase in intracellular Ca2⫹ (N.
Alfaidy and J. R. G. Challis, unpublished results). Therefore,
a further feed-forward paracrine/autocrine loop exists in
which increased output of PG should stimulate 11␤-HSD-1,
resulting in increased production of cortisol, which leads to
further increases in PGHS-2 and decreases in PGDH (Fig. 8).
We have referred previously to the finding of regional
variation in PGDH activity. We suggest that this might reflect
progesterone stimulation of the enzyme (407, 408) in a regional pattern. Chorion collected from patients at elective
cesarean section at term in the absence of labor had higher
PGDH activity in the region of the membranes overlying the
internal os than chorion collected from a region adjacent to
the placenta or between the placenta and cervix (41). However, at cesarean section in labor, there was a dramatic reduction in PGDH activity in chorion from the lower uterine
segment. We suggested that this altered response could reflect an antagonism of the effect of progesterone on the enzyme by elevations of cytokines derived from vaginal
and/or cervical fluids. We and others have shown that
whereas IL-1␤ and TNF␣ increase PG synthesis, these cytokines decrease PGDH activity and PGDH gene expression
(409, 410). Importantly, IL-10, the antiinflammatory cytokine
that attenuates IL-1␤-induced up-regulation of PGHS, also
reverses IL-1␤ down-regulation of PGDH (409). The importance of this observation is that PGs generated within amnion
and chorion in the lower segment may escape metabolism in
chorion specifically in that region at the time of labor to reach
the cervix and effect effacement and dilatation.
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535
FIG. 8. Interrelationship between cortisol, PGHS-2, PGDH, and CRH. In chorion, cortisone can be converted to cortisol through the activity
of 11␤ HSD-1, and the activity of this enzyme is increased locally by PGs.
3. PGs and infection. Approximately 30 – 40% of preterm labors are associated with an underlying infective process.
Romero, Mitchell, and collaborators (411– 413) have demonstrated elegantly the role for infection in preterm labor. Bacterial organisms themselves secrete phospholipases, resulting in increased release of arachidonic acid from intrauterine
tissues and increased PG production. Alternatively, bacterial
endotoxin, such as lipopolysaccharide, acts on amniotic or
membrane macrophages, causing either PG release or further
release of cytokines (414 – 417). Cytokines in turn elevate PG
production within amnion, chorion, and decidua as discussed previously (418). Administration of cytokines or bacterial endotoxins to pregnant mice provokes premature delivery and allows examination of the precise temporal
sequence of events in infection-driven preterm labor (419,
420). A number of cytokines including IL-1␤, TNF␣, IL-6, and
IL-8 (neutrophil-activating protein-1) are increased in amniotic fluid of patients undergoing preterm labor associated
with infection (421– 424). Cytokines are produced not only by
macrophages, but are synthesized and secreted by human
fetal membranes in decidua, and these tissues may be the
sources of the cytokines found in amniotic fluid. IL-1␤, IL-6,
and IL-8 mRNA were expressed in amnion, chorion leave,
and decidua, particularly in tissues obtained after labor. In
addition, cultured decidual and chorion cells produce IL-6
and IL-8 when stimulated with IL-1␤ and TNF␣, and amnion
produces IL-8 in response to IL-1␤ (425). Thus, these studies
have led to the suggestion that there is a complex cytokine
network at the chorio-decidual interface, as has been proposed to exist in other tissues (269). It is also possible that
cytokines cause release of other uterotonins, including OT
and CRH in decidua (426, 427), myometrium, and/or placenta. These compounds may affect the myometrium directly or indirectly. Lipopolysaccharide also inhibits replication of amnion cells, and it has been suggested that this
might be a mechanism by which lipopolysaccharide contributes to premature ruptured membranes.
The paradigm of infection-driven preterm labor has been
proposed as a means of understanding regulation of PG
production in labor at term (348). However, preterm labor in
the absence of infection can occur without demonstrable
changes in amniotic fluid PGE concentrations and apparently without enhanced PG biosynthetic activity in fetal
membranes. It has been argued that changes in PG and
cytokine concentrations in the amniotic fluid of women in
preterm labor with infection are not reproducible, and that
these compounds accumulate there as a result of preterm
labor, rather than as a cause (428). It has also been argued that
invasion of the amniotic sac by microorganisms occurs when
labor has been initiated, when tissues of the forebag are
exposed. Furthermore, since parturition is an inflammatory
process, the presence of mediators of inflammation in amniotic fluid could be a natural event of parturition without
arguing for a role of infection as a cause of preterm labor. The
body of evidence currently available has tended to counter
this latter view. However, as in all human studies of this type,
it is extremely difficult to delineate precisely the cause-andeffect sequence of relationships. Furthermore, a low-grade
inflammatory response, where accumulation of cytokines
occurs without an infective process, may be present normally
at term and contribute to the stimulus of labor or remain as
a parallel, but unrelated, event.
C. Stimulation: role of CRH
Over the past 10 yr there has been considerable interest in
the possible role that CRH, produced from intrauterine tissues, plays in the regulation of human pregnancy and parturition (429, 430). Pro-CRH mRNA is present in placental
tissue (431) and decidua in increasing amounts during pregnancy. These levels correlate with increased concentrations
of ir-CRH peptides in the placenta and with the exponential
increase in CRH1– 41 concentrations in maternal peripheral
plasma (432– 435). CRH also increases in cord plasma, although the concentrations are generally lower than those in
the maternal compartment (432, 436, 437). Several groups of
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CHALLIS ET AL.
investigators have reported that maternal plasma CRH concentrations are elevated significantly in the plasma of patients presenting in preterm labor (433, 438 – 440) and may be
used to discriminate patients presenting in preterm labor
who will deliver within 24 – 48 h from those patients with a
similar diagnosis, but in whom labor is not imminent (441).
The biological activity of CRH in maternal plasma is attenuated by the presence of a circulating CRH binding protein (CRH-BP), produced in the liver and placenta (429, 442).
CRH-BP blocks the ability of circulating CRH to promote
ACTH release from pituitary corticotrophs, and it inhibits the
stimulatory effect of CRH on uterine PG production. Concentrations of CRH-BP decrease during the last 5– 6 weeks of
normal pregnancy and before preterm labor, coincident with
the increase in maternal CRH concentrations (443), and apparently in response to increased CRH secretion. In the placenta, CRH is produced by syncytiotrophoblast and intermediate trophoblasts (444), and immunoreactive CRH
localizes to these cell layers (429, 445). In culture, CRH output
from placental and chorion trophoblast cells is inhibited by
nitric oxide and progesterone and increased by catecholamines, OT, cytokines, and glucocorticoids (Fig. 9; Refs.
427 and 444). Majzoub and colleagues (446, 447) demonstrated that dexamethasone increases levels of CRH mRNA
in placental trophoblast cells maintained in culture in a timeand dose-dependent fashion, although later suggested that
this “apparent” stimulation resulted in fact from reversal of
progesterone-induced inhibition of CRH expression (263,
404). Glucocorticoids compete with and displace progesterone from GR␣ binding, and diminished inhibition is measured as an apparent increase in secretion of CRH.
Vol. 21, No. 5
We demonstrated in vivo that patients receiving prenatal
glucocorticoids to promote pulmonary maturation in
amounts that decreased maternal ACTH and cortisol concentrations by more than 80% provoked stimulation of maternal CRH concentration by almost 50% over pretreatment
values (448). Administration of glucocorticoids to pregnant
women with singleton or multiple fetuses at risk of preterm
labor actually stimulates uterine contractility, although the
effect may be transient (449, 450). From the foregoing discussion it is evident that this could be the result of upregulation of PGHS, down-regulation of PGDH, and/or
stimulation of placental CRH which, in turn, provokes a
further increase in PGHS-2 expression (451). Administration
of glucocorticoids eventually suppresses fetal HPA function,
decreases estrogen output from the placenta, and might be
expected to diminish uterotrophic activation of the myometrium, perhaps accounting in part for the (fortunately) transient nature of this response.
Based upon these results, and the demonstration of activation of fetal HPA function in response to hypoxemia in
animal fetuses, we proposed that the human fetus would also
respond to an adverse intrauterine environment such as
acute hypoxemia with activation of the fetal HPA axis (10).
With time, increased pituitary drive to the adrenal increases
steroidogenic enzyme potential and cortisol output. Fetal
cortisol, then acting through placental and/or membrane
GR␣, up-regulates placental CRH gene expression, leading to
the increased CRH concentrations in the plasma of patients
presenting in preterm labor. Accordingly, cord CRH concentrations are elevated in the presence of intrauterine
growth restriction (IUGR), or decreased values of cord PO2
FIG. 9. Summary of regulation of expression and output of CRH in human intrauterine tissues and placenta.
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PARTURITION
(440, 452). CRH is a vasodilator in the placental vascular bed
and reverses the vasoconstrictor influence of PGF2␣ (453). In
the placenta, the vasodilator action of CRH is associated with
up-regulation of the NO-cyclic GMP pathway. Hence, elevations of CRH within the placenta should signal increased
blood flow and correction of a hypoxemic insult to the fetus.
However, if the hypoxemia persists, placental CRH output
presumably remains elevated (Fig. 10). CRH, secreted into
the fetal circulation, drives further pituitary ACTH secretion
and also drives DHAS output from the fetal zone of the fetal
adrenal gland (243); hence, maternal estrogen output should
rise as a secondary response to fetal distress. Increased estrogen leads to uterine activation. CRH contributes to increased expression of PGHS (451) by up-regulating adenylate cyclase activity in placental and membrane cells (61). It
will be recalled that the PGHS promoter contains a CRE.
Thus, we speculate that activation of a feed-forward loop in
response to a hostile intrauterine environment is a mechanism by which a compromised fetus may signal preterm
labor and induce premature delivery (Fig. 10). In addition,
maternal stress with elevations of maternal glucocorticoid
concentrations may also contribute to elevations of placental
CRH output and preterm birth. Hobel and colleagues (454)
reported increases in maternal CRH concentrations in
women with elevated scores for perceived stress and anxiety.
These values predicted preterm labor, even as early as 20 –24
weeks of gestation.
It is extremely difficult to prove or disprove this hypothesis with in vivo studies in normal human pregnancy. Studies
cannot be performed in nonprimates, since these species do
not appear to produce placental CRH. The pattern of placental CRH output during pregnancy in the baboon and
rhesus monkey has been described but differs from the exponential increase of plasma CRH concentration observed in
human gestation (429). Women receiving betamethasone deliver at variable times after treatment. Current obstetric practice in North America, in fact, makes it difficult to obtain
“control” placental tissue from patients in preterm labor who
537
have not received exogenous corticosteroid; such patients
may have increased endogenous corticosteroids before tissue
collection in any case.
A further reservation is related to CRH receptor specificity.
CRH exerts its effects through activating specific G proteincoupled receptors, which exist in two subtypes: CRH-R1 and
CRH-R2. These arise from different genes with multiple
splice variants (455). The two receptors share approximately
70% homology at the amino acid level. CRH-R1 exists in at
least three variant forms (R1␣, R1␤, and R1C). Recently, an
additional form, CRH-R1D, has been isolated, which is identical to CRH-R1␣ except that it contains an exon deletion
resulting in loss of 14 amino acids in the seventh transmembrane domain (456). CRH-R2 exists in at least three splice
variant forms (R2␣, R2␤, and R2␥). CRH-R1 predominates in
human myometrium (455, 457). CRH-R2 is expressed in fetal
membranes, but at lower levels than CRH-R1. Parenthetically, this pattern is reversed in rats in which CRH-R2 predominates in myometrium (Y. Stevens and J. R. G. Challis,
unpublished observations). CRH-R2 has higher specificity
for urocortin than CRH, raising the possibility that in rodent
gestation, placental output of urocortin rather than CRH,
may determine activity of this pathway.
Because CRH-R1 is linked to the adenylate cyclase system
through GS␣ regulatory proteins, it is not surprising that CRH
stimulates cAMP output by human myometrial cells maintained in vitro (61). Herein lies the paradox. CRH-induced
increases in cAMP should inhibit myometrial activity,
through mechanisms described above, yet elevations in maternal peripheral plasma CRH concentration are suggested to
predict women at risk of increased uterine activity and preterm labor (61). This may be resolved if CRH action on
myometrium is independent of effects on PG synthesis in
other tissues (458). Affinity of CRH binding in myometrium
increases with pregnancy, and then decreases in late gestation (459). Hence, we (8) and others (61) have speculated that
during gestation CRH acts as a myometrial relaxant, rather
than as a uterotonin. At term, OT up-regulates protein kinase
FIG. 10. Diagram to indicate interrelationships between mother, placenta,
and fetus concerned with up-regulation
of placental CRH output in human gestation in response to stress. It is proposed that cortisol from either maternal
or fetal adrenal can up-regulate placental CRH expression. Placental CRH, in
turn, affects fetal adrenal function indirectly through stimulation of fetal pituitary ACTH release, and directly by
stimulating secretion of DHAS from the
fetal zone of the fetal adrenal gland.
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538
CHALLIS ET AL.
C, which phosphorylates CRH receptor protein resulting in
its desensitization and loss of inhibitory influence (61, 460).
Stevens et al. (265) showed that levels of mRNA for CRH-R1
heptohelical glycoprotein increase in lower segment myometrium from patients in labor whether at term or preterm.
Hence, CRH may contribute to regionalization of uterine
activity responses at this time, producing inhibition of activity, or relaxation in the lower segment, but stimulation of
activity through up-regulation of PG synthesis in the fundal
region of the uterus.
In our view, the putative role of CRH in pregnancy maintenance and parturition remains unclear. The concept of placental CRH as “a placental clock controlling the length of
human pregnancy” implied a stimulatory effect on the myometrium (461, 462), which is difficult to reconcile with the
known biochemical effects of CRH (61). Certainly, CRH augments OT- and PGF2␣-induced contractility of myometrial
strips in vitro (289, 463). However, it decreases output of PGI2
by myometrial cells and has no direct stimulatory action on
its own. Perhaps increased levels of CRH are required to
sustain relaxation, rather than stimulation, of the uterus
through late gestation. However, lowered concentrations of
CRH in maternal plasma are associated with postterm delivery in which, presumably, relative myometrial quiescence
has been maintained. Resolution of this interesting dilemma
in which a single ligand may have different actions depending upon differential expression of its receptor subtypes and
coupling through second messenger systems is required as
a scientific basis to understanding CRH action in pregnancy
(61).
VI. Application to Clinical Preterm Labor
Rates of preterm labor in North America have remained
relatively unchanged over the last 30 – 40 yr, despite substantial advances in our understanding of this process (1–3).
It is apparent, however, that new knowledge has not yet been
extrapolated to clinical diagnosis and management (464,
465), and that there may be reluctance to develop new drugs
for administration to women in pregnancy without guarantees of safety for mother and fetus. There is a clear need to
recognize first those preterm labors in which prevention is
undesirable because it constitutes a greater compromise to
fetal health. There is a need to develop diagnostic indicators,
likely specific for particular windows of gestation, to determine the patient in whom the diagnosis of preterm labor is
correct. Ideally, only these patients should be subjected to
tocolytics and to prenatal glucocorticoids. There is a need to
develop effective methods of tocolysis ideally related on a
patient-specific basis to the cause of preterm labor in that
individual. Hence, diagnosis of preterm labor should encompass a multiple-test approach. The new generation of
specific PGHS-2 inhibitors offers great promise, since increased expression of PGHS-2 appears to represent a common final pathway of birth and preterm labor mechanisms
among species (466). The ability to regulate CRH or PG
effects through specific and appropriate agonists and/or antagonists is a potential alternative approach.
Both these approaches, however, act on agents of phase 2
Vol. 21, No. 5
parturition, in which uterine activation has already taken
place. Inhibition of uterotonin action or secretion does not
necessarily affect myometrial activation, although recent
studies in sheep treated with nimesulide, a PGHS-2 inhibitor,
have shown reversal of some CAP gene expression. Ideally,
a future strategy for preterm labor diagnosis and management should address uterine activation. Those studies will
require careful animal studies before the introduction of new
drugs into clinical practice. A satisfactory outcome may be to
delay rather than actually to prevent preterm birth, providing that there is improvement in mortality and morbidity of
the newborn.
We remain concerned about the capricious use of glucocorticoids in preterm labor patients (467). There is no question of the beneficial effect of these compounds in promoting
pulmonary maturation in infants of women who give birth
prematurely within an appropriate time for treatment. However, a central thesis of this review is that glucocorticoids
provide a stimulus to the labor process and that evidence is
accumulating to suggest that the model derived from animal
experiments may have substantial applicability to the human. We recognize from animal studies that repeated administration of glucocorticoids to pregnant animals produces, in a dose-dependent fashion, inhibition of fetal
growth (468). Prenatal corticosteroids alter postnatal HPA
function and the setting of negative feedback. Prenatal corticosteroids, in animals, may result in the development of
hypertension postnatally, and in a pattern of pancreatic response to a glucose load that resembles insulin resistance
(469). Prenatal and postnatal administration of corticosteroids affect levels of type 1 and type 2 GRs in critical brain
regions, particularly the hippocampus, associated with
memory and, in later life, with memory loss and neurodegenerative disease. Future research into the control of preterm labor, and to the tocolytic management of the patient at
risk of preterm labor, will need to define the relative risks and
benefits of different management paradigms that may be
proposed (469).
Acknowledgments
We are indebted to Jenny Katsoulakos, Linda Vranic, and Fal Patel for
their help in the preparation of this manuscript and to Maggie Haworth
for her patience with us.
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Erratum
Figure 1 in the June 2000 Endocrine Reviews article by P. C. White and P. W. Speiser, “Congenital adrenal
hyperplasia due to 21-hydroxylase deficiency” (Endocrine Reviews 2000, 21:245–291) contained errors that
have been corrected in the following figure:
FIG. 1. Pathways of steroid biosynthesis. The pathways for synthesis of progesterone and mineralocorticoids (aldosterone), glucocorticoids
(cortisol), androgens (testosterone and dihydrotestosterone), and estrogens (estradiol) are arranged from left to right. The enzymatic activities
catalyzing each bioconversion are written in boxes. For those activities mediated by specific cytochromes P450, the systematic name of the
enzyme (“CYP” followed by a number) is listed in parentheses. CYP11B2 and CYP17 have multiple activities. The planar structures of cholesterol,
aldosterone, cortisol, dihydrotestosterone, and estradiol are placed near the corresponding labels.
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