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Endocrine and Paracrine Regulation of Birth at Termand Preterm*

JOHN R.G. CHALLIS, STEPHEN G. MATTHEWS, WILLIAM GIBB, AND

STEPHEN J. LYE

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 inDevelopment and Fetal Health (J.R.G.C., S.J.L.), Samuel Lunenfeld Research Institute, Mount SinaiHospital, Toronto, Ontario, Canada M5G 1X5; MRC Group in Fetal and Neonatal Health andDevelopment (J.R.G.C., S.J.L.); Department of Obstetrics and Gynaecology, and Cellular andMolecular Medicine (W.G.), University of Ottawa, Ottawa, Ontario, Canada K1H 8L6

ABSTRACTWe have examined factors concerned with the maintenance of

uterine quiescence during pregnancy and the onset of uterine ac-tivity at term in an animal model, the sheep, and in primatespecies. We suggest that in both species the fetus exerts a criticalrole in the processes leading to birth, and that activation of the fetalhypothalamic-pituitary-adrenal axis is a central mechanism bywhich the fetal influence on gestation length is exerted. Increasedcortisol output from the fetal adrenal gland is a common charac-teristic across animal species. In primates, there is, in addition,increased output of estrogen precursor from the adrenal in lategestation. The end result, however, in primates and in sheep issimilar: an increase in estrogen production from the placenta andintrauterine tissues. We have revised the pathway by which en-docrine events associated with parturition in the sheep come aboutand suggest that fetal cortisol directly affects placental PGHS

expression. In human pregnancy we suggest that cortisol increasesPGHS expression, activity, and PG output in human fetal mem-branes in a similar manner. Simultaneously, cortisol contributes todecreases in PG metabolism and to a feed-forward loop involvingelevation of CRH production from intrauterine tissues. In humanpregnancy, there is no systemic withdrawal of progesterone in lategestation. We have argued that high circulating progesterone con-centrations are required to effect regionalization of uterine activ-ity, 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 thepatient at risk of preterm labor, and the use of scientifically basedstrategies specifically for the management of this condition, whichwill improve the health of the newborn. (Endocrine Reviews 21:514 –550, 2000)

I. Introduction

II. Regulation of Myometrial Contractions

III. Pregnancy: Phase 0 of Parturition

IV. Myometrial Activation: Phase 1 of ParturitionA. Activation: role of fetal hypothalamic-pituitary-ad-

renal (HPA) maturationB. Activation mechanism by which cortisol changes

placental steroid and PG synthesisC. HPA function in the primate fetus and activation of

parturitionD. HPA maturation in the primate fetusE. Placental progesterone and human pregnancy: the

enigma of the progesterone blockV. Myometrial Stimulation: Phase 2 of Parturition

A. Stimulation: role of oxytocinB. Stimulation: role of PGsC. Stimulation: role of CRH

VI. Application to Clinical Preterm Labor

I. Introduction

PARTURITION is the process by which the fetus is ex-pelled from the uterus to the extrauterine environment.

Parturition results from a complex interplay of maternal andfetal factors. It requires that the uterus, which has been main-tained in a relative state of quiescence during pregnancy,develops coordinated contractility and that the cervix dilatesin a manner that allows passage of the fetus through the birthcanal. To be successful, parturition requires also that matu-ration of those fetal organ systems necessary for extrauterinesurvival has occurred, and that the maternal organism hasundergone the changes necessary for lactation in the post-partum period. It is not surprising, therefore, that synchro-nous maturation of the fetus and stimulus to increased uter-ine activity should be desirable, and much evidence suggeststhat it is the fetus itself that triggers both these series ofevents.

Preterm birth, where there is asynchrony between thelabor process and fetal maturation, occurs in 8–10% of allpregnancies, and its incidence has changed little in the past40 yr (1). Indeed, factors such as low socioeconomic status ofsome inner-city populations, the tendency for women tochoose to start a family at an older age, and the impact offertility treatment are contributing to an increase in the in-cidence of preterm delivery (2, 3). Improved neonatal care,however, continues to reduce the mortality rate due to pre-

Address reprint requests to: Dr. J. R. G. Challis, Department ofPhysiology, Medical Sciences Building, 1 King’s College Circle, Uni-versityofToronto,Toronto,OntarioM5S1A8Canada.E-mail: [email protected]

* Work in the authors’ laboratories has been supported by MedicalResearch Council (MRC) Group and operating grants from the MRC ofCanada.

0163-769X/00/$03.00/0Endocrine Reviews 21(5): 514–550Copyright © 2000 by The Endocrine SocietyPrinted in U.S.A.

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maturity, although preterm birth remains the primary causeof neonatal death. In North America, the cost of caring forinfants in the neonatal intensive care nursery during the firstmonths of life has been estimated at $5–6 billion annually (3).That figure does not take into account the extraordinaryemotional stress to the family of the prematurely deliveredinfant. Nor does it take into account the long-term costsrequired for chronic care of these infants, some of whom havemajor motor and/or mental handicaps and/or long-termneuro-developmental complications. To prevent pretermbirth effectively, we need to understand the fundamentalprocesses that switch the myometrium from its relative qui-escence during pregnancy to the activated and contractilestate at the time of labor. We will develop the thesis thatregulation of myometrial function requires both endocrineand mechanical controls. Furthermore, it is now evident thatthe cause of preterm labor may vary at different times duringpregnancy and will not necessarily reflect acceleration of theprocesses at term gestation. The ability to recognize thesevarious causes of premature delivery, in a clinical setting,and then provide appropriate treatment remains a majorclinical challenge. Furthermore, it is evident that preventionof preterm delivery may not always be desirable, particularlyif the fetus is allowed to develop in a hostile intrauterineenvironment.

Causes of preterm birth in general fall into three catego-ries: iatrogenic, where there is demonstrable complication ofpregnancy such as preeclampsia or fetal distress that requiresobstetrical intervention; premature rupture of the fetal mem-branes with or without infection; and, idiopathic pretermlabor. The relative importance of these causes varies. How-ever, most sources consider that approximately 30–40% ofpreterm birth is associated with an underlying infective pro-cess, and 40–50% of preterm births are idiopathic.

In this review, we will focus attention on experimentalstudies in the sheep, the species of choice for many inves-tigators concerned with understanding the processes of birth(4). We shall then extrapolate from the sheep to an under-standing of parturition in primates, particularly in the hu-man. Our central thesis is that the processes of birth areremarkably similar, at a fundamental level, across species,and in both sheep and human the fetus, through activationof its hypothalamic-pituitary-adrenal (HPA) axis, plays acentral and crucial role. We shall examine how the fetal HPAaxis may be activated in response to a stress circumstanceduring pregnancy, e.g., hypoxemia, such as that perhapsassociated with reduced uteroplacental perfusion in pre-eclampsia. It will be apparent that the fetal signal provokesincreased outputs of stimulatory PGs and other uterotoninsfrom intrauterine tissues. It is evident now that there is aprogression from fetal to maternal control of intrauterine PGproduction. Furthermore, the regulation of PG synthesis andmetabolism in fetal trophoblasts and maternal uterus is ef-fected by different mechanisms.

II. Regulation of Myometrial Contractions

During pregnancy, myometrial activity is characterized bypoorly coordinated contractures, or the Braxton-Hicks con-

tractions of human gestation (5). In late pregnancy, the uterusundergoes preparedness for the stimuli that lead to contrac-tility and labor (6, 7). Those stimuli may be local, maternal,mechanical, or fetal (8). The contracture pattern of uterineactivity has been observed in several species, including thesheep, baboon, and rhesus monkey (9). The development ofcoordinated uterine contractions at term results in a myo-metrium that is excitable, generating high-frequency, high-amplitude contractions. It is spontaneously active and re-sponds to exogenous uterotonins. The transition of themyometrium from a quiescent to an active state has beentermed “activation.” When this has occurred the myome-trium can then undergo “stimulation” in response to endog-enous and/or exogenous agonists (8).

We have found it useful to divide the uterine phenotypeinto different stages of the parturition process (10). Theuterus is relatively quiescent during 95% of pregnancy, cor-responding to phase 0 of parturition. Activation correspondsto phase 1 and is effected predominantly by mechanicalinput, and through regulation by uterotrophins such as es-trogen. Stimulation corresponds to phase 2, when endoge-nous uterotonins, including PGs and oxytocin (OT), act onthe activated myometrium. Postpartum involution corre-sponds to phase 3. In this sequence of events, the “initiation”of parturition corresponds to the transition from phase 0 tophase 1, although clearly one could argue that initiationstarted much earlier in gestation (11).

Contraction of the myometrium at term or preterm de-pends upon conformational changes in the actin and myosinmolecules, which allow actin and myosin filaments to slideover each other, ultimately leading to a shortening of themyocyte (Fig. 1 and Refs. 12 and 13). The confirmationalchanges (involving cross-bridge cycling of the myosin head)require ATP, which is generated by myosin after phosphor-ylation of the 20-kDa light chains of myosin by the enzymemyosin light chain kinase (MLCK). This enzyme is central tosignaling pathways that both stimulate and inhibit myome-trial contractions (14, 15). MLCK is activated through inter-action with the calcium binding protein calmodulin (CAM),which in turn requires 4 Ca21 ions for its own activation.Binding of calcium-activated CAM to MLCK induces a con-formational change in the enzyme, allowing MLCK to phos-phorylate the 20-kDa light chains of myosin. MLCK can alsoundergo phosphorylation by protein kinase A (PKA, cAMP-activated protein kinase), which reduces the affinity of theenzyme for calcium calmodulin (Ca-CAM) and leads to itsinactivation (14, 16). Regulation of MLCK has been reviewedextensively (17, 18). It is evident that activity of this enzymeis altered by intracellular pathways that regulate levels ofcalcium and of cAMP and is critical for the development ofuterine contractility. Uterotonins generally increase intracel-lular calcium levels ([Ca21]i), by increased influx of Ca21

through receptor-operated channels, or release of calciumfrom intracellular stores including sarcoplasmic reticulum(see Ref. 19). Agents that inhibit myometrial activity do so byincreasing intracellular levels of cyclic nucleotides cAMP orcGMP, which in turn inhibit release of calcium from intra-cellular stores or reduce MLCK activity. Binding of agentssuch as b-adrenergic agonists, relaxin and prostacyclin, tomyometrial receptors activates adenylate cyclase activity,

October, 2000 PARTURITION 515

leading to an increase in cAMP generation, while uterineinhibitors such as nitric oxide (NO) activate guanyl cyclase,increasing cGMP. In collaborative studies, Pato et al. (20)characterized MLCK purified from pregnant sheep myome-trium. The enzyme had an apparent molecular mass of 160kDa and high substrate specificity for myosin light chains.Sheep myometrial MLCK has an absolute requirement forCa21 and CAM for activation; in the absence of Ca-CAM,MLCK is inactive. On binding Ca-CAM, MLCK undergoes aconformational change that exposes the catalytic site, whichcan then phosphorylate the 20-kDa myosin light chains toinitiate contraction. Relaxation is achieved either by dephos-phorylation of MLC-20 by the catalytic subunits of type 2Aphosphatase (21) or by reduction in MLCK activity. The latteris achieved, as discussed, by reduction in [Ca21]i, resultingin dissociation of Ca-CAM from MLCK. Sheep myometrialMLCK is also a substrate for PKA, which phosphorylatesserine residues on the sheep myometrial enzyme in the pres-ence or absence of bound Ca-CAM. The ability of PKA toinhibit myometrial MLCK activity, even in the presence ofagonists that increase [Ca21]i, provides a biochemical ratio-nale for the finding that agents that increase intracellularcAMP inhibit uterine contractions even in the presence ofcalcium-activating agents such as OT and stimulatory PG.Ca-CAM can also activate phosphodiesterase to increase thebreakdown of cAMP.

Inhibition of myometrial activity by b-adrenergic agonists,relaxin, and PGI2 is mediated by increases in intracellularcAMP (see Ref. 12). Binding of the inhibitor to its specific cellmembrane receptor causes dissociation of the receptor-linked heterotrimeric GTP-binding protein Gs into b-, g-, anda-subunits. The a-subunit activates adenylate cyclase to ini-tiate cAMP synthesis. cAMP, in turn, activates PKA, whichthen phosphorylates a series of regulatory proteins. Acti-vated PKA either phosphorylates MLCK to reduce its abilityto bind Ca-CAM or phosphorylates a membrane-binding sitefor Ca21 that increases calcium binding and reduces freeintracellular calcium concentrations.

Regulation of myometrial calcium levels has been re-viewed extensively (see Refs. 12 and 22–24). Free resting

Ca21 increases from 150 nm to about 500 nm during con-traction through influx of extracellular Ca21 or by the releaseof Ca21 from intracellular binding sites or intracellular or-ganelles (25, 26). Extracellular Ca21 enters cells through re-ceptor-operated or voltage-gated channels. Release of intra-cellular Ca21 from sarcoplasmic reticulum is activatedthrough the phosphoinositol (PI) pathway. Binding of auterotonin to its plasma membrane receptor activates a Gprotein transducer, coupled to phospholipase C, which freesinositol trisphosphate (IP3) and diacylglycerol (27, 28). FreeIPs, especially IP3, increase cellular calcium from intracel-lular storage sites. Interestingly, IP3 binding in myometriumwas inhibited by calcium, suggesting that this might providea mechanism for regulating the IP3 response by oscillating[Ca21]i. Diacylglycerol formed during IP3 turnover maystimulate PKC to phosphorylate cellular proteins such asMLCK or be rapidly phosphorylated by diacylglycerol ki-nase to phosphatidic acid, a naturally occurring Ca21 iono-phore, or lead to release of arachidonic acid by cellularlipases, resulting in production of eicosanoids (see below).

Function of the myometrium during labor at term or pre-term requires highly developed cell-to-cell coupling, effectedthrough formation of intercellular GAP junctions within ad-jacent cell membranes (14, 29). The proteins forming GAPjunctions are termed connexins and are classified accordingto their apparent molecular weights (30). Connexins are ar-ranged into hexameric hemichannels, which become alignedacross adjacent cells to form an interconnecting pore thatallows low-resistance electrical or ionic coupling between thecells and provides a pathway for metabolite transfer (31).Hundreds of individual channels arrange themselves into anorganized plaque to form a GAP junction. Regulation ofconnexins occurs at the level of transcription and translation(31, 32); mechanisms also operate to control transport ofconnexin protein to the cell membrane and to direct assemblyinto connexons, through apposition, clustering, and forma-tion of functional channels (33, 34). This complex process ispoorly understood, although it is influenced by steroids andby mechanical stretch (35). GAP junction formation requiresthe presence of cell adhesion molecules, and in early studies,

FIG. 1. Cartoon of a myometrial cell in-dicating the intracellular biochemicalpathways involved in regulating con-tractions. MLCK is central to uterinecontractility. It is activated by Ca-CAMafter an increase in intracellular cal-cium levels. This increase is generatedby the action of various uterotonins:PGF acting through PGF receptor (FP),OT acting through OTR. Agents thatincrease cAMP (b-agonists) or cyclicGMP, or NO donors decrease uterinecontractility. AA, Arachidonic acid;Atosiban OTR antagonist.

516 CHALLIS ET AL. Vol. 21, No. 5

Meyer et al. (36) showed that appearance of GAP junctionsin transfected S180 cells was blocked by coincubation withantisera to liver cell adhesion molecule.

Garfield (see Refs. 14 and 16) established clearly that anabsence of GAP junctions in the pregnant myometrium wasresponsible for high-input resistance of these smooth musclecells and poor coordination of uterine contractions. There isa massive increase in numbers of GAP junctions with theonset of labor, which significantly enhances electrical cou-pling and allows the myometrium to develop synchronoushigh amplitude contractions (37). An increase in GAP junc-tions with labor onset has been found in all species studied.In the rat, levels of connexin-43 (CX-43) mRNA and proteinwere low during pregnancy but increased some 48 h beforelabor (38, 39). Highest levels of mRNA and protein werefound during delivery itself. This is critical because the half-life of GAP junctions may be as short as 1–2 h, and hencecontinued synthesis would be required to maintain labor.Increases in CX-43 mRNA have been reported in sheep andhuman myometrium with the onset of labor and correlatedwith increases in CX-43 protein (37, 38). Permeability of GAPjunctions may be facilitated through phosphorylation at con-sensus serine and tyrosine sites within the cytoplasmic do-main of CX-43. Garfield (14) demonstrated that cell-to-cellcommunication in the myometrium is reduced by elevated[Ca21]i and increased levels of cAMP. Importantly, morerecent studies have shown that the pattern of CX-43 in myo-metrium during pregnancy differs from that of CX-26. Con-nexin-26 expression is elevated in midgestation in the rat andappears to be associated more with uterine quiescence (7, 8).

III. Pregnancy: Phase 0 of Parturition

Studies in different species have indicated that a variety ofdifferent inhibitors may play upon the myometrium duringpregnancy. Withdrawal of one or more of these may predictthe onset of delivery; precocious withdrawal may predict theonset of premature parturition. Such an inhibitor, PTH-related peptide (PTHRP), is produced in myometrium, andits rate of transcription is increased by progesterone andtransforming growth factor b (TGFb) (40). PTHRP receptormRNA has also been localized to rat myometrial tissue, sug-gesting that the protein may act in an autocrine/paracrinefashion through specific receptors to activate the Gas sub-units of G proteins and increase intracellular levels of cAMP(40–42).

Relaxin also elevates myometrial cAMP and inhibits OT-induced turnover of phosphoinositide (PI) by the action ofcAMP-dependent protein kinase. Relaxin exerts a dual rolein the inhibition of myometrial contractility and in the reg-ulation of connective tissue changes in the cervix (43, 44).Porter and colleagues (45, 46) were among the first to showthat relaxin suppressed spontaneous uterine contractility inthe rat and guinea pig, but sensitivity to OT was preserved.Thus, the major action of relaxin is one of frequency mod-ulation (47). Hansell et al. (48) and others have demonstratedthat relaxin is expressed in the human fetal membranes,decidua, and placenta, consistent with its exerting para-crine/autocrine effects on intrauterine tissues (49–51). Re-

laxin gene expression is dramatically up-regulated in pa-tients with preterm, premature rupture of membranes(PPROM) (49). Relaxin receptors have been localized to de-cidua and chorionic trophoblast cells, and the protein actsthrough these to up-regulate expression of matrix metallo-proteinases (MMP), especially MMP1, MMP3, and MMP9.Similarly, relaxin increases MMP expression in cervical tis-sue at term. Administration of exogenous relaxin stimulatesseparation of the pubic symphysis in those species in whichit is a prerequisite for delivery (52). In addition, in pigs andrats, relaxin appears necessary for maintaining evolution ofspontaneous uterine contractility in late pregnancy and formaintaining a high frequency of live births (43). In vitrostudies have shown that relaxin blocks the action of stimu-lants such as OT, carbachol, and norepinephrine on the myo-metrium, through mechanisms involving PKA-mediatedphosphorylation of PLC-linked G proteins. This in turn in-hibits IP3 turnover and the increase in [Ca21]i (22). Althoughthe precise role that relaxin plays during pregnancy remainsto be determined, it may be particularly useful in maintain-ing uterine quiescence during the period when progesteroneconcentrations are falling and estrogen levels are beginningto increase before the onset of labor (see Ref. 12). In addition,there are reports that relaxin may act centrally to increasecirculating plasma OT and vasopressin concentrations by anopioid-independent mechanism (53). It is now known thatOT is produced within human intrauterine, choriodecidualtissues. It remains to be established whether a similar rela-tionship exists between relaxin and OT synthesized withinthe intrauterine compartment in women.

Lye and Challis (54, 55) first showed, some 20 yr ago, thatprostacyclin infused into nonpregnant sheep inhibited uter-ine contractility in vivo. In parallel studies a similar inhibitoryeffect of prostacyclin was observed on human myometrium(56), and it is clear now that prostacyclin represents the majoreicosanoid present within the pregnant myometrium ofmany species (57), including human. In human term preg-nant myometrial strips maintained in vitro, the initial re-sponse to PGI2 was contraction, but this was followed byrelaxation (58, 59). It is now recognized that PGI2 actsthrough specific IP receptor species to increase adenylatecyclase activity and elevate intracellular cAMP (60). Otheragents such as CRH also stimulate output of cAMP frommyometrial cells and act synergistically with PGI2 in a para-crine/autocrine fashion (61). The role of CRH in pregnancymaintenance and parturition will be discussed later in thisreview.

More recently, interest has arisen over the potential role ofNO as an endogenous inhibitor of myometrial contractility(62). Increases in endogenous synthesis of NO by adminis-tration of the NO precursor l-arginine, or the NO donorsodium nitroprusside, inhibit myometrial contractions in therat and human (62). Nitroprusside has been shown to de-crease force and 20-kDa myosin light chain phosphorylationin human myometrial strips, although the tissue is not assensitive as vascular smooth muscle. Nitric oxide synthase(NOS) isoforms have been detected using RT-PCR in humanfetal membranes and choriodecidua (62). Levels of mRNA-encoding inducible NOS (iNOS) are highest in human myo-metrium at preterm, not in labor patients, and decrease with


a corresponding fall in iNOS protein in myometrium col-lected at term (see Ref. 10). Several authors have suggestedthat NO acts in a paracrine manner, potentially in conjunc-tion with progesterone to effect myometrial quiescence dur-ing pregnancy, although this position has been disputed.There is a decrease in NOS activity of decidua and myome-trium in species such as rat before parturition in a mannerthat would presumably diminish its inhibitory influence onthe uterus. Furthermore, studies by Chwalisz and Garfield(62) have shown that, at term in the rat, there is a corre-sponding increase in NO production by inflammatory cellsof the cervix, indicating a role for NO in cervical effacementand relaxation as its influence on the myometrium is dimin-ished.

Other inhibitors of uterine activity include calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide(VIP), and endogenous b-adrenergic agonists (63). Thesecompounds act through increasing intracellular cAMPand/or decreasing intracellular calcium availability (64).

IV. Myometrial Activation: Phase 1 of Parturition

The switch from myometrial quiescence to myometrialactivation is essential to enable the muscle to respond to thestimulation provided by the high levels of uterotonic ago-nists and to generate the synchronous, high-amplitude, high-frequency contractions of labor. We have proposed that myo-metrial activation results from coordinated expression of acassette of proteins, termed contraction-association proteins,or CAPs (12). CAPs include ion channels [which determinethe resting membrane potential and hence excitability ofmyocytes (65)], agonist receptors [e.g., to OT and PG (60)] andGAP junctions [permitting cell-to-cell coupling (16)].

Overall regulation of myometrial activity is geneticallyregulated (Fig. 2). Different species have gestations ofvarying lengths, and studies involving embryo transfersuggest that it is the genotype of the fetus that controls thelength of pregnancy. For example, when sheep embryosfrom short gestation or long gestation breeds were im-planted into random gestation-age recipients, parturitionoccurred at the appropriate time for the fetal rather than

maternal genotype (66, 67). There is a variety of mecha-nisms by which the fetal genotype can influence preg-nancy length, and we have proposed that it includes bothendocrine and mechanical signals. In initial studies, Ouand Lye (68) found, using unilaterally pregnant rats, thatwhile expression of CAP genes, CX-43 and OT receptor(OTR), increased in the gravid uterine horn in labor, therewas no parallel increase in the nongravid horn, eventhough both horns were exposed to the same systemichormonal changes. Next, these workers showed that whena small 3-mm diameter tube was placed into one uterinehorn of bilaterally ovariectomized nonpregnant animals,there was a significant increase in mRNA levels encodingCX-43 in that horn, compared with the contralateral horn.Control experiments showed that this result was not dueto the presence of a foreign body within the uterus. Ad-ministration of progesterone to these animals blockedstretch-induced increases in CX-43 expression.

Subsequent experiments examined whether the endocrineenvironment of pregnancy influenced the ability of stretch toup-regulate CAP gene expression (see Ref. 8). In unilaterallypregnant rats, at day 15 of gestation, the nonpregnant hornreceived either the 3-mm Silastic tube or was left as control.Other animals were operated on at day 18. Five days afterimplanting the tubes, levels of transcripts encoding CX-43,PGF2a receptor (FP receptor), or OTR were measured. Inanimals treated at day 15 and studied at day 20, there wasno effect of the Silastic tube in increasing CX-43 transcripts,but in animals studied at the time of labor there was adramatic increase in the numbers of CX-43 transcripts tovalues similar to those seen in the contralateral pregnanthorn. There was little change in CX-43 transcripts in thenonpregnant control horn. These data suggest that stretch ofthe myometrium appears capable of up-regulating contrac-tion-associated proteins, but the ability to do so is highlydependent on the endocrine environment. If the stretch stim-ulus is applied during pregnancy, it is inadequate to induceCX-43, and presumably its activity is inhibited by circulatingconcentrations of progesterone. However, at term, when ma-ternal systemic progesterone levels have decreased, stretchitself is adequate to produce the same level of CX-43 expres-sion as in the pregnant horn containing the fetus.

The molecular mechanisms by which stretch increasesCX-43 and OTR expression remain to be determined (69).In other systems, such as cardiac myocytes, stretch acti-vates multiple intracellular signaling pathways throughshear stress response elements in the promoter of somestretch-responsive genes (70). The CX-43 gene containssuch an element, suggesting that if wall tension contrib-utes to the regulation of CAP genes in the myometrium,regulation of uterine growth through pregnancy will beimportant in determining the level of shear stress. Lye andcolleagues (8) have argued that, during pregnancy, uterinegrowth follows three distinct phases: an initial phase dur-ing the first trimester where uterine growth is due tohyperplasia and controlled by endocrine factors, a secondphase during the second and third trimester in whichgrowth is closely matched to increased fetal size, and afinal phase in which there is a decline in uterine growthin comparison to fetal growth, and hence an increase in

FIG. 2. The onset of labor is dictated by the fetal genome proceedingthrough either a fetal growth pathway with increases in uterinestretch or fetal endocrine pathway involving activation of the fetalHPA axis. These two arms are not independent because changes inprogesterone and estrogen modulate the ability of uterine stretch toincrease expressions of genes associated with myometrial activation.


uterine wall stretch and tension. They speculate that pro-gesterone is necessary to support stretch-induced hyper-trophy of the uterus during midgestation in concert withincreasing fetal size. Near term, the fall in progesterone,observed in most animal species (see below), leads to adecline in uterine growth relative to fetal growth andhence increased tension development, which in turn re-sults in increased CAP gene expression and contributes tomyometrial activation. Since the decrease in circulatingprogesterone appears critical for the altered influence ofstretch on myometrial CAP gene expression, we shall con-sider the endocrine pathways that result in progesteronewithdrawal.

A. Activation: role of fetal HPA maturation

More than 35 yr ago, Professors Sir Graham (Mont) Ligginsand 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 ofparturition and that it did so through activation of the fetalHPA axis. An endpoint of activation of this axis is proges-terone withdrawal. We shall suggest that the primate fetussimilarly affects gestation lengths through activation of theHPA axis. However, in human gestation there is no systemicprogesterone withdrawal, and we shall argue that, inwomen, sustained circulating concentrations of progesteroneare indeed required at term to effect regionalization of myo-metrial contractility and promote relaxation of the loweruterine segment.

Early studies in the fetal sheep showed that ablation of thefetal pituitary gland, the fetal adrenal gland, pituitary stalksection, or lesioning of the fetal paraventricular nucleus(PVN) resulted in prolongation of gestation (71–73), whereasthe infusion to the fetal lamb in utero of ACTH or of a glu-cocorticoid resulted in premature parturition within 3–5days of beginning the infusion. These studies provided ex-perimental verification of the concept developed from ob-servations of naturally occurring prolonged gestation insheep attributable to ingestion of the teratogen Veratrumcalifornicum 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 grossmalformations, including cyclopean characteristics. At au-topsy, the pituitary and adrenal glands were remarkablyhypoplastic as a result of impaired pituitary development atan early gestational age (see Ref. 81).

Several groups of workers provided clear evidence formaturation of fetal HPA function in the sheep fetus duringlate gestation (74–76). There are progressive increases in fetalplasma ACTH1–39 and cortisol in the plasma of the late-gestation fetal sheep (77–80); the initial increases in ACTHprecede the rise in cortisol (79), but fetal cortisol increases inan exponential fashion over the last 10 days of gestation, withhighest concentrations being established immediately beforeterm (80). This is consistent with the fact that ACTH is im-portant in the development of the adrenal cortex in lategestation. Similar maturation of pituitary adrenocorticalfunction has been demonstrated in several other species,including the guinea pig, which represents a species that

gives birth to mature young. The prepartum surge of cortisolis important for the maturation of several organ systems,particularly the lungs and kidneys (see Ref. 81). It is alsocritical for normal development of programming of the brain.However, the simultaneous increase in fetal plasma ACTHand cortisol has remained somewhat of a paradox because,under normal circumstances, one would expect elevations infetal plasma cortisol concentration to inhibit further ACTHsecretion. Mechanisms have developed to override the in-fluence of negative feedback in the fetus in late gestation, arelationship now described in the guinea pig as well as in thesheep (see below).

Recent studies have explored the molecular mechanismsunderlying changes in fetal pituitary adrenocortical activa-tion in late gestation sheep. Developmental changes in CRHmRNA in the fetal hypothalamic PVN were examined by insitu hybridization (82). By day 60 of gestation, CRH mRNAwas detectable in the fetal PVN. There was an increase inCRH mRNA expression by day 120 of gestation and a furthersubstantial up-regulation of CRH gene expression in the last20 days of pregnancy. This was followed by a decrease inCRH expression in the PVN of the newborn lamb. Through-out development, expression of CRH mRNA appears to beconfined to parvocellular fields of the PVN, with no expres-sion detected in magnocellular neurons (82). Recent studieshave confirmed that the changes in CRH mRNA are trans-lated to CRH peptide in the fetal hypothalamus, indicatinga close association between transcription and translation ofthe CRH gene during development.

In the fetal pituitary, expression of the ACTH precursor,POMC, is detectable in the inferior region of the pars distalisby day 60 of gestation. Levels of POMC mRNA in the su-perior and inferior regions of the pars distalis increased withprogression of gestation until around day 120, when therewas a further increase in expression, peaking at term (83, 84).The increase in POMC expression is combined with a re-markable reorganization of the corticotrophs toward the in-ferior aspect of the fetal pituitary gland. This pattern ofexpression was sustained in the newborn lamb. In the fetalpars intermedia, the developmental profile of POMC mRNAwas quite different. Relatively high levels were present byday 60 of gestation; these increased between days 60 and 100and then remained relatively constant for the remainder ofgestation. Early controversy concerning changes in expres-sion of POMC mRNA in fetal pituitary tissue appears toresult from differences in methodologies. The use of in situhybridization clearly allows separation of different zones ofthe fetal pituitary gland, whereas erroneous results may havebeen obtained through use of Northern blot analysis (85, 86).In a recent carefully conducted study obtaining pituitarytissue from fetuses at specific times in late gestation andduring the labor process itself, the lack of negative feedbackon POMC mRNA, and the sustained increase in POMCmRNA levels, was clearly demonstrated (87). The change inregional distribution of POMC mRNA in the pars distalismay indicate the transition of fetal-like to adult-like cortico-trophs that has been described at this time (see below).Changes in POMC mRNA in the pars distalis are reflected byincreased levels of ir-ACTH and by increased immunostain-ing for ACTH in pituitary corticotrophs (83, 84); at term


ir-ACTH-positive cells represent about 15% of the total cellnumber in the pars distalis.

Arginine vasopressin (AVP) is also an important regulatorof fetal pituitary ACTH secretion and is expressed in the fetalPVN relatively early in gestation (88). AVP mRNA is presentin the supraoptic nucleus, PVN, and the accessory magno-cellular nuclei by day 60 of gestation (82). Differential ex-pression of magnocellular and parvocellular AVP is evidentin the PVN by day 80. In magnocellular neurons, AVP mRNAincreases with gestational age, whereas parvocellular expres-sion of AVP remains relatively unchanged. Levels of AVPmRNA increase dramatically in both regions of the PVN inthe newborn lamb. It is suggested that magnocellular AVP isinvolved primarily in fetal fluid homeostasis, while parvo-cellular AVP is important in stimulation of the pituitarycorticotroph (84). There is a close correlation between AVPmRNA levels and ir-AVP in the anterior hypothalamus, asthere is for CRH. The increase in parvocellular AVP mRNAin the newborn may be associated with the stress of the novelextrauterine environment. Axons containing AVP and OThave been identified in a zone of the pars distalis adjacent tothe pars intermedia in fetal sheep. These axons are probablythose of magnocellular neurons and may represent a mech-anism by which magnocellular AVP and OT directly affectACTH release in vivo.

CRH and AVP induced dose-dependent increases inACTH output from ovine fetal pituitary cells in vitro (89); atequimolar concentrations AVP was more potent than CRH.Simultaneous administration of CRH and AVP showed anadditive interaction between the neuropeptides (90). Treat-ment with CRH significantly increases POMC mRNA levelsin sheep pituitary cells harvested at day 120 and day 138 ofgestation. However, CRH treatment of cells collected fromfetuses at term failed to affect POMC synthesis. AVP in-creased POMC mRNA levels in cells obtained at day 138 ofgestation; in pituitary cells from late-gestation fetuses, AVPand CRH are equally potent in the induction of POMC syn-thesis. Cortisol has little negative feedback effect on basaloutput of ACTH in these cells but inhibits CRH-stimulatedACTH output and POMC gene expression.

Studies by Lu et al. (91) showed that ovine fetal pituitarymembranes expressed CRH receptor activity as early as day100 of gestation. CRH binding increased to its highest levelsat around day 135 (term, 145–150 days) and then decreasedprogressively through late gestation (92). Recent studieshave extended these measurements to show that levels ofmRNA encoding fetal pituitary CRH-receptor type I mayfollow a similar profile (J. C. Rose, personal communication),and this may account for the altered outputs of ACTH inresponse to CRH stimulation in vivo (see below). Factorsregulating CRH receptor expression have been examined invivo and in vitro. In vitro studies indicated that CRH down-regulated expression of its own receptor and cortisol pro-duced a similar attenuation of binding activity (92).

In vivo studies demonstrated that CRH was more potentthan AVP in stimulating ACTH output by pituitary tissuefrom chronically catheterized fetal sheep in late gestation (93,94). The response profiles, however, are quite different. AVPinduced a transient rise in plasma ACTH while CRH stim-ulated a more sustained increase (95). Subsequently, it was

demonstrated that AVP concentrations are about 5 timesthose of CRH in the hypophyseal portal circulation of adultsheep (96), and it remains possible that the relative impor-tance of AVP in fetal corticotroph activation in utero may begreater than that of CRH (97). Fetal pituitary responsivenessto CRH increases between day 110 and 125 and then de-creases toward term (79). This relative insensitivity of thepars distalis to CRH may reflect the increase in negativefeedback influence of rising endogenous cortisol concentra-tions, or the decrease in CRH binding sites indicated above(79). Simultaneous administration of CRH and AVP resultsin an ACTH response that is greater than when either neu-ropeptide is administered independently, and the interactionis synergistic in nature, at least at around day 115 of gestation(95). CRH and AVP affect the corticotrophs through differentsecond messenger systems. CRH exerts this action throughup-regulating a Gas-adenylate cyclase-linked membrane re-ceptor and increasing intracellular levels of cAMP (89). AVPacts through V1b receptors to stimulate PI turnover, stimu-lating phospholipase C and activating protein kinase C.

POMC is processed through different endoproteases, pro-hormone convertase 1 (PC-1) and prohormone convertase 2(PC-2), to yield a spectrum of products. Recent studies havedemonstrated that both PC-1 and PC-2 are present in fetalsheep pituitary tissue in late gestation. However, expressionof these enzymes does not change with labor, and it seemsunlikely that the increase in ACTH output is attributable toaltered prohormone convertase activity (87, 98). However,the pattern of POMC-derived peptides from the fetal pitu-itary does change in the plasma of the fetal lamb in latepregnancy (99). Several groups of investigators have re-ported that large molecular weight POMC-derived ACTHprecursor peptides are present in the circulation (100). Theconcentrations of these larger molecular weight forms de-crease prepartum, whereas those of ACTH1–39 increase. Be-cause the larger molecular weight peptides may act to an-tagonize the action of ACTH1–39 on adrenocortical cells (101–103), a decrease in their concentration prepartum wouldpresumably facilitate ACTH action and an increase in adre-nal glucocorticoid secretion (104). The sources of these pep-tides may be different (105–107). Studies in hypothalmo-pituitary-disconnected fetuses have led to the suggestionthat the pars intermedia may be a potential source of largemolecular weight peptides, whereas the pars distalis is theprimary source of ACTH1–39. In addition, the ovine fetal lungand placenta express POMC mRNA and contain ir-ACTH. Itis not clear whether these potential sources of ACTH con-tribute to circulating ACTH1–39 in a meaningful manner orwhether the peptides have paracrine/autocrine actionswithin the tissues of origin.

Thus, the temporal relationship between hypothalamic-CRH and pituitary POMC expression is consistent with thesimultaneous increase in plasma ACTH and cortisol ob-served in late gestation (84, 108–110). Nevertheless, themechanism by which CRH mRNA and POMC mRNA in-crease in the presence of high plasma glucocorticoid con-centrations is not clear. One possible mechanism is that, inthe fetus, glucocorticoid feedback thresholds within the brainand pituitary become modified. This may occur at severallevels (Fig. 3). We have reported that glucocorticoids up-


regulate expression of corticosteroid binding globulin (CBG)mRNA in the fetal liver, and of circulating CBG, which is theopposite of the response in adult sheep (111, 112). In the fetus,the pattern of CBG glycosylation varies from that in adultanimals, but the glycoprotein increases in concentration inthe fetal circulation and maintains a relatively constant freecortisol concentration for most of pregnancy (112, 113). Nearterm, however, the increase in adrenal cortisol output ex-ceeds the CBG binding capacity, resulting in a sudden in-crease in free cortisol concentration over the final hours be-fore birth (114). It appears that this increase in free cortisolbefore parturition is a consistent observation across differentanimal species (115). More recently, we have demonstratedexpression of CBG mRNA and the presence of CBG immu-noreactive protein in other fetal tissues including the kidney,pancreas, and pituitary (115). CBG mRNA has been localizedto fetal pituitary cells by in situ hybridization, and its patternof distribution appears to differ from that of POMC, withgreater abundance in superior regions of the gland. As yet,there are no studies demonstrating colocalization of CBGwith ACTH-producing cells in fetal pituitary tissue.

Levels of glucocorticoids may also be modified by inter-conversion of biologically active cortisol and biologicallyinactive cortisone, through the activity of 11b-hydroxyste-roid dehydrogenase (11b-HSD) enzymes (116). We will dis-cuss these later in the context of the placenta as a barrier tothe transfer of maternal cortisol to the fetus. In the pituitaryof fetal sheep, 11b-HSD-1 activity predominates and appearsto operate somewhat unusually in a dehydrogenase direc-tion, i.e., inactivating cortisol to cortisone (116). Presumably,this would effect a local mechanism to inactivate circulating

cortisol and diminish the potential for negative feedback.This pattern of 11b-HSD activity in the pituitary needs sub-stantiating and differs from that in other fetal tissues, e.g., theliver, where 11b-HSD-1 operates predominantly as a reduc-tase, converting cortisone to cortisol, and suggesting a po-tential intrahepatic source of cortisol generation.

A further mechanism by which glucocorticoid feedbackcould be altered locally is through modification of cortico-steroid receptor expression (117). The ovine fetal pituitaryexpresses type II glucocorticoid receptor (GR) from relativelyearly in gestation, and the levels of GR mRNA increasetoward term (118), consistent with glucocorticoid effects inmodulating the switch from fetal to adult corticotroph celltypes in the pituitary (106). During the course of labor, thereis a dramatic decrease in levels of GR mRNA in the fetal parsdistalis, suggesting that the potential for glucocorticoid neg-ative feedback decreases in the pituitary during the course oflabor. More important, perhaps, is the demonstration thatthere are decreases in immunoreactive GR in the hypotha-lamic PVN near term. These changes were specific to CRH-and AVP-positive parvocellular neurons. More recently, weshowed that GR mRNA levels in the PVN of fetal sheep andguinea pigs decrease in late gestation, and in fetal sheeplevels of GR mRNA in the hippocampus also fall prepartum.The hippocampus represents a major site of glucocorticoidfeedback for HPA function, and there are a number of directand indirect connections between the limbic system and thePVN. Together these data suggest that a reduction in thepotential for glucocorticoid feedback occurs in late gestationin brain structures that are central to glucocorticoid negativefeedback action (119).

In addition to classic feedback processes, there are severalother mechanisms by which fetal HPA axis activation mayoccur. Expression of pro-enkephalin mRNA rises to a max-imum in the parvocellular PVN of fetal sheep at day 135 ofgestation and then decreases in older animals (120). A fall inhypothalamic pro-enkephalin mRNA occurs with intrafetalinfusion of cortisol at day 135, suggesting that the prepartumrise in endogenous cortisol may inhibit parvocellular pro-enkephalin synthesis. CRH and met-enkephalin are presentin the same secretory granules in rodents, and met-enkepha-lin inhibits CRH-stimulated ACTH secretion from fetal pi-tuitary cells in vitro. Thus, a decrease in met-enkephalinproduction may facilitate corticotroph function near term(120). OT has been implicated in the control of ACTH se-cretion in adult sheep, and OT stimulates ACTH output fromthe fetal pituitary cells in vitro. OT mRNA is present in bothmagnocellular and parvocellular fields of the PVN and SONand follows a similar developmental profile to AVP mRNA,raising the possibility that it too may influence fetal pituitaryfunction.

In fetal sheep, the kinetically determined production ofcortisol from the adrenal gland increases during the last20–25 days of gestation (77, 121). In part, this results from theincrease in drive to the adrenal from rising levels of ACTH,but, in part, it is attributable to maturation of fetal adrenalfunction (122). Indeed, in hypophysectomized fetuses treatedwith a continuous low-level infusion of ACTH, plasma cor-tisol concentrations increased and parturition occurred at

FIG. 3. Summary of events associated with maturation and devel-opment of the HPA axis in the fetal sheep. Increased expression ofCRH from the PVN of the hypothalamus drives increased expressionof POMC in the anterior pituitary. POMC is processed to ACTH,which drives the adrenal gland. In the fetus normal negative effectsof cortisol on the hypothalamus and pituitary are diminished throughincreases in systemic corticosteroid binding globulin (CBG), pituitary11b HSD, and diminished expression of GR in the pituitary andhypothalamus.


around the normal time, consistent with fetal adrenal mat-uration as the overriding influence (123).

Ovine fetal adrenal responsiveness changes dramaticallyduring the course of pregnancy (124, 124–126). Adrenal cellscollected from animals at days 50–70 of gestation secretecortisol in response to ACTH stimulation in amounts similarto or greater than adrenal tissue from term fetuses (127).However, between approximately days 90–110 of pregnancythe adrenal is relatively insensitive to ACTH stimulation(124). It is now clear that this pattern of response is due, inlarge part, to decreased gene expression of P450C17 andP450SCC steroidogenic enzymes in fetal adrenal cortical cellsat midgestation (128, 129). The abundance of mRNAs forthese enzymes is increased by ACTH administration to thefetus (130, 131). Although 3b-HSD may be rate limiting tocortisol production in the first half of pregnancy (132), im-munoreactive (ir)-3b-HSD-positive cells are presentthroughout the zona fasiculata of the fetal adrenal cortexfrom day 50 until term (133). The midgestational decrease inP450C17 may result by TGFb inhibiting ACTH-induced stim-ulation to P450C17, as demonstrated in vitro in ovine fetal andadult adrenal cells (134). Recent studies have demonstratedthat ACTH receptor mRNA is detectable from around day 60of gestation (135). There is a modest increase through preg-nancy and then a substantial increase between days 126–128and days 140–141 (135). Thus, the low level of basal adrenalresponsiveness to ACTH around day 100 of gestation is notdue to lack of ACTH receptor expression, but may be at-tributable, in part, to very low concentrations of ACTH in thefetal circulation at that time (136). The increase in ACTHreceptor expression in late gestation would appear to con-tribute to increased adrenal responsiveness near term. Thefactors responsible for up-regulating ACTH receptor mRNAabundance are unclear (137). These may include ACTH itself,cortisol, or local intraadrenal interaction with IGF-II and/ordecreased influence of TGFb (138–140).

Both in vivo and in vitro studies have shown that fetaladrenal maturation can be advanced by ACTH1–24 admin-istration (110, 141–143). Exogenous ACTH in vivo enhancescoupling between ACTH receptor and adenylate cyclase andenhanced capacity for cAMP generation (144–146). ACTHtreatment in vivo also increased expression and activity ofP450C17, P450C11, P450C21, and 3b-HSD (130, 147). The adre-nal responds to ACTH early in gestation, although continuedtrophic input is required to maintain increased levels of geneexpression. Interestingly, when ACTH was administered tofetuses in vivo as pulses, rather than as a continuous infusion,it led to a pattern of fetal adrenal steroidogenesis that favoredcortisol over corticosterone output (i.e., directed P450C17 ac-tivity). Thus, the pulse pattern of endogenous ACTH secre-tion in vivo may affect the pattern of adrenal activation (148,149).

These studies suggest that ACTH-induced increases inadrenal steroidogenic enzymes, particularly P450C17, is es-sential to allow C21 steroids to proceed through the 17-hydroxy pathway leading to cortisol biosynthesis (130, 150).An obligatory role for an increase in ACTH drive to the fetaladrenal as a prerequisite for increased responsiveness, how-ever, has been challenged recently. When hypophysecto-mized fetal sheep were infused at a constant, but low level

of ACTH, there was a normal rise in fetal cortisol concen-tration; later, maternal progesterone levels decreased andbirth occurred at about the expected time (123). The molec-ular mechanisms underlying this fascinating result clearlyrequire elucidation.

We have hypothesized that fetal stress, perhaps reflectedin diminished fetal arterial P02, constitutes a stimulus forpreterm birth. Experimental hypoxemia has been used ex-tensively to investigate fetal HPA activation (151, 152). Manystudies have shown that even modest reductions in fetalarterial P02 induce robust increases in fetal plasma ACTHand cortisol concentrations (153, 154). Release of CRH andAVP into the hypophysial portal system is abolished in thehypothalamo-pituitary-disconnected (HPD) fetus (152), andthese animals are incapable of mounting an ACTH responseto stress, implying that increased ACTH output requireshypothalamic input. Studies by Akagi and colleagues (155)demonstrated that changes in fetal P02 of only 4–5 mm Hgwere adequate to elicit increased ACTH concentrations in thecirculation of the fetal lamb. This level of oxygen change issimilar to that seen during spontaneous contractures in lategestation sheep, raising the possibility that uterine activityitself may contribute part of the stimulus to increased fetalHPA maturation. Whether chronic stress is a stimulus tobirth at term (156) or contributes only to some cases of pre-term labor is unclear at the present time.

At 135 days’ gestation, hypoxia (P02 reduction by 8 mmHg) significantly increased CRH mRNA in parvocellularPVN and POMC mRNA in the pars distalis within 6 h. Thisresponse, however, was attenuated by concurrent infusion ofcortisol, indicating effective glucocorticoid feedback mech-anisms in vivo at this time (157). After 48 h of sustainedhypoxemia, levels of POMC in the pars distalis were ele-vated, but expression in the pars intermedia was decreased(158). This suggests differential regulation of these two zonesof the fetal pituitary, consistent with observations that do-pamine, likely from the fetal arcuate nucleus, tonically in-hibits pituitary POMC synthesis, and this inhibition is ex-acerbated in the presence of hypoxemia. Infusion ofbromocriptine, a dopamine D2 receptor agonist at day 130 ofgestation, produced a 50% decrease in pars intermediaPOMC mRNA levels, without affecting POMC mRNA in thepars distalis (159). Thus, the fetal D2 receptor system is func-tional in late pregnancy, but the fetal pars intermedia doesnot appear to secrete ACTH1–39 in amounts that alter fetaladrenal function.

Activation of fetal HPA function in response to hypox-emia, however, is a critical aspect of the story leading topreterm birth (160, 161). A sustained pulsatile hypoxemicstimulus is adequate to up-regulate HPA gene expression,plasma ACTH, and cortisol concentrations. It is reasonable topredict that sustained hypoxemia in conditions of fetal com-promise predisposes to fetal HPA activation and would re-sult in premature birth (162, 163).

B. Activation mechanism by which cortisol changesplacental steroid and PG synthesis (Fig. 4)

Fetal cortisol acts on the sheep placenta to alter the patternof steroidogenesis; as a result, progesterone output falls and


estrogen concentrations increase (164–167). These changes inplacental steroid output are associated with increased ex-pression and activity of placental P450C17 (168, 169). This isa critical difference between the sheep and the human, wherethis enzyme is not induced in the placenta at term. Ovineplacental tissue contains P450arom activity, and up-regulationof this gene also occurs in late gestation. For many years thegeneral thesis has been that placental estrogen production islimited in ovine pregnancy and occurs in abundance only atterm with the induction of placental P450C17 as a result ofglucocorticoid action (170–172). The fall in progesterone andlater increase in maternal and fetal estrogen concentrationshave been considered as providing the stimulus to increasedPG output by intrauterine tissues, with consequent increasein myometrial contractility (173–180).

For several reasons we have questioned the appropriate-ness of this model. It has been clearly established that thesheep, like the human, has a feto-placental unit of estrogenproduction by which C19 precursors from the fetal adrenalgland can be secreted and aromatized in the placenta to formestrogen (181). Later studies demonstrated output of C19steroids including dehydroepiandrosterone (DHEA) and an-drostenedione by the ovine fetal adrenal gland, stimulationof C19 fetal-adrenal steroid output by ACTH infusion and inresponse to hypoxemia, and conversion of [3H]androstenedi-one infused into the fetus to estrogen measured in maternal

and fetal compartments (182, 183). Although unconjugatedestrogens increase sharply at the time of parturition in sheep(165, 166), there is a progressive increase of conjugated es-trogens in maternal plasma and urine throughout the latterpart of gestation, well before the terminal increase in pla-cental P450C17 activity (184). The ratio of conjugated to un-conjugated estrogen in maternal sheep plasma is high be-cause, in ovine pregnancy, placental sulfotransferase activitypredominates over placental sulfatase activity (184). Thus, itis clear that while increased expression of placental P450C17may contribute to the sharp rise in maternal estrogen con-centrations prepartum, its induction in the placenta is not aprerequisite for ovine placental estrogen output at earlierstages of gestation.

There are other troubling features of the currently ac-cepted model (185). Several groups of investigators haveused either immunohistochemical techniques for localizationof PGHS-1/-2, or PGHS-2, or in situ hybridization forPGHS-2 mRNA, or measurements of PGHS and/or PGHS-2activity in ovine placental cells and microsomal preparations(186–189), to show that PG production by the sheep placentaincreases progressively through the last 20–25 days of ges-tation (190–195). Placental output of PGs is not confined tothe immediate 24–48 h before spontaneous parturition (196,197). The increase in PGHS expression and activity in theplacenta correlates closely with the progressive increase in

FIG. 4. Endocrine pathways leading to the onset of parturition in sheep. A, current model; B, proposed sequence of hormone events. In thecurrent model, activation of the fetal HPA axis leads to increased cortisol thought to up-regulate expression of P450C17 in the placenta. In thenew proposed hypothesis, increased fetal adrenal output of cortisol results in up-regulation of prostaglandin synthase (PGHS)-2 gene expressionin the placenta with increased production of PGE2. PGE2 feeds back to further up-regulate fetal HPA function but is itself responsible forup-regulation of P450C17 gene expression in the placenta. Increased placental estrogen is required for up-regulation of PGHS-2 in maternaltissues but not in fetal tissues. Thus, with the onset of parturition there is progression from fetal trophoblast within the placenta to the maternaluterine tissues.


plasma PGE2 concentrations in the circulation of the chron-ically catheterized fetal lamb (191, 198). The increase in cir-culating PGE2 in the fetus bears a striking temporal relation-ship to the increase in plasma cortisol concentration (198,199). Louis et al. (200) first reported, more than 25 yr ago, thatinfusion of PGE2 into the ovine fetus in late gestation stim-ulated an increase in the plasma cortisol concentration at atime when the fetal adrenal gland was relatively unrespon-sive to ACTH stimulation. Later studies have shown that theeffect of PGE2 infused into the fetus on fetal HPA functioncould be exerted at any one or all of the hypothalamic, pi-tuitary, or adrenal levels (201, 202). Thus, the progressiveincrease in output of PGE2 appears to contribute to the driveto fetal HPA function and augments the stimulus suppliedby ACTH to the fetal adrenal (201, 203). Indeed, fetal PGE2infusion will provoke premature delivery of the ovine fetus(204). Placental PGE2 output would not be subjected to neg-ative feedback regulation by cortisol and may contribute tothe apparent lack of negative feedback relationship betweenACTH and cortisol in the late gestation ovine fetus.

Recent studies have suggested that in addition to PGE2stimulating output of cortisol by the fetal adrenal gland (205,206), fetal cortisol, and not estrogen, may affect placentalPGHS-2 activity and contribute to the rise in fetal plasmaPGE2 concentrations. Evidence in support of this suggestionincluded the observation that infusion of estrogen into fetallambs in late pregnancy was without stimulatory effect onlevels of placental PGHS-2 mRNA (207), although estrogeninfusion into nonpregnant adult sheep did increase PGHS-2expression in the endometrium (see also below). Studies withhuman amnion cell cultures and chorion trophoblast cellshave suggested that glucocorticoids may up-regulatePGHS-2 gene expression in these tissues. Infusion of cortisolto fetal sheep in late gestation also increased levels of PGHS-2mRNA and immunoreactive PGHS-2 protein (by Westernblotting) in placental trophoblast cells. This effect was inde-pendent of changes in estrogen, since a similar stimulationof placental PGHS-2 mRNA levels was observed when cor-tisol was infused in the absence or presence of the aromataseinhibitor, 4-hydroxyandrostenedione.

Using immunohistochemistry we showed that the P450C17enzyme and PGHS-2 both localized to trophoblast epithelialcells, but not binucleate cells in ovine placentomes (208).Moreover, the appearance of ir-PGHS-2 clearly preceded thatof P450C17. Collectively, therefore, these data offer strongreasons to refute the current model of endocrine events oc-curring in the placenta of the sheep in late gestation andsuggest that a different sequence likely pertains. This is sum-marized in Fig. 4. We have argued elsewhere that during lategestation in the fetal sheep, increased output of cortisol fromthe fetal adrenal gland progressively up-regulates PGHS-2gene expression in placental trophoblast cells (208). Themechanism of this action remains unresolved. It may dependon trophoblast-specific transcription factors generated in re-sponse to elevations of cortisol, or it could be a direct actionof cortisol since early studies reported a full GRE consensussequence at approximately 760 bp upstream from thePGHS-2 transcription start site. We suggest that increasedPGHS-2 expression in the sheep placenta contributes to in-creased PGE2 output into the fetal circulation. Fetal PGE2

drives the fetal HPA axis in a positive feed-forward fashion(Fig. 4). PGE2, and not cortisol, is responsible for up-regu-lation of P450C17 in placental trophoblast cells. This occurs ina manner analogous to the effect of PGE2 on P450C17 induc-tion in ovine and bovine adrenal tissue. Ovine placentaltissue expresses PGE receptor subtypes (EP1-EP4), but anychanges in their expression during the course of late gesta-tion remain to be determined (see Ref. 208). We have sug-gested further that increased P450C17 in the placenta allowsthe conversion of C21 D5 steroids directly through to D5 C19steroids, precursors for estrogen biosynthesis, as demon-strated by Flint et al. (209) and Mason and colleagues (210)some years ago. A crucial difference of the current hypothesisis that this change is superimposed on an already substantialbasal output of estrogen by the sheep placenta (measured asconjugated estrogens in maternal plasma and urine), andcontributes principally to the terminal increase in maternalestradiol concentrations. This increase in estrogen is requiredfor expression of CAP genes in the ovine myometrium andfor expression of PGHS-2 in maternal endometrial tissue,predominantly endometrial epithelium. We have found thatwhereas the increase in placental (fetal trophoblast) expres-sion of PGHS-2 after intrafetal cortisol administration wasunaffected by concurrent infusion of 4-hydroxyandro-stenedione, maternal endometrial up-regulation of PGHS-2and output of 13–14 dihydro-15-keto PGF2a (PGFM) into thematernal circulation occurred with cortisol infusion but wasblocked by concurrent administration of the aromatase in-hibitor (211). Thus, in sheep it appears that the fetal placentaand maternal endometrium exist as two separate sites of PGsynthesis in late gestation and that these are differentiallyregulated. In fetal placenta, PGHS-2 is increased by cortisol,independent of changes in estrogen output, whereas in ma-ternal uterine tissue, up-regulation of PGHS-2 and maternalplasma PGFM is dependent upon increased estrogen pro-duction (Fig. 4).

Current studies are directed at examining this hypothesisfurther. Using immunohistochemistry and Western blotanalysis, it is evident that GR is expressed in ovine placentaltissue, predominantly in uninucleate trophoblast cells. Es-trogen receptor (ER) mRNA and activity have been demon-strated in maternal endometrium but is apparently lackingin placental trophoblast (212). Hence, it is difficult to envis-age how estrogen could provide a stimulus to placental PGproduction as previously hypothesized. It remains to beshown whether glucocorticoids affect placental PGHS activ-ity directly or indirectly. However, in early studies we havedemonstrated that glucocorticoids increase output of PGE2by ovine placental trophoblast cells maintained in culture,and this effect is abolished by addition of meloxicam, a spe-cific inhibitor of PGHS-2 activity.

C. HPA function in the primate fetus and activationof parturition

The role of the human and subhuman primate fetus incontrolling gestation length has been, until recently, lessclearly defined than that of the sheep fetus. However, overthe past few years it has become apparent that mechanisms


leading to activation of fetal HPA function in primates bearconsiderable similarity to processes in sheep, and that fetalcortisol and fetal adrenal C19 steroids appear to play animportant role. In 1933, Malpas (213) in a study of gestationlength in human pregnancies complicated with anencephalyconcluded that “. . . . the fetal pituitary and adrenal glandswas responsible for the trigger to the neuromuscular expul-sive mechanism that led to the onset of labor. ” Early ob-servations indicated that the mean length of gestation inanencephaly, after exclusion of cases with polyhydramnios,was similar to controls, but the proportions of preterm andpostmature births were both higher (see Ref. 12). Similarresults have been obtained after experimental anencephalyin rhesus monkeys (214). In monkeys, fetal hypophysectomypredisposed to prolongation of gestation (215), but fetal ad-renalectomy was without effect on gestational length, al-though five of eight fetuses died in that study (see Ref. 12).Initial studies indicated that removal of the fetus, but leavingthe placenta in utero (fetectomy) had little effect on gestationlength. However, more recent studies have indicated clearlythat placental retention after fetectomy was significantlylonger (195 days) compared with 164 days in controls (216).Fetectomy in baboon pregnancy did not affect gestationlength, although maternal estradiol concentrations fell tobasal values and progesterone concentrations were reducedby 20–45% (217–219). Overall, these experiments are difficultto interpret. The numbers and observations are invariablysmall, no attempt is generally made to sustain uterine vol-ume and the stretch stimulus to the myometrium, and it istechnically very difficult to operate on the primate fetuswithout stimulating uterine contractility.

In intact rhesus monkeys, as in the baboon and human,there is an increase in maternal estrogen concentrations inlate gestation that parallels an increase in the concentrationsof fetal adrenal C19 steroids, particularly DHEA and DHEA-sulfate (DHAS) (220, 221). Maternal estrogen concentrationsincrease progressively and then more rapidly in the laterphases of human gestation; estriol, derived in substantialpart from precursors of fetal adrenal origin, rises rapidly inmaternal plasma and urine in late pregnancy at term, and inpreterm labor (221). When androstenedione was infused intopregnant rhesus monkeys at about three-quarters of the waythrough gestation, there was an increase in maternal plasmaestrogen concentrations and premature birth (222). This ef-fect was blocked by the coinfusion of the aromatase inhibitor4-hydroxyandrostenedione, which prevented maternal en-docrine changes and changes in fibronectin in the fetal mem-branes and inhibited the nocturnal increases in uterine myo-metrial contractility (223). Elevations of maternal systemicestrogen concentrations by infusion increased myometrialactivity, but did not produce premature delivery or fetalmembrane changes. It was suggested that in the primate, asin the sheep, estrogen is important for the normal processesof parturition. The failure of exogenous estrogen to stimulatesustained uterine contractility, even though locally producedestrogen formed after C19 steroid infusion was effective, ledthe authors to suggest that the estrogen had to be generatednear to its site of paracrine/autocrine action (223).

D. HPA maturation in the primate fetus

There is emerging strong evidence that maturation of HPAfunction occurs in the primate fetus in a manner generallyanalogous to that discussed above in the sheep fetus. Excel-lent reviews by Pepe and Albrecht (221, 224) and by Mesianoand Jaffe (225) have provided detailed analyses of pituitary-adrenal function in the primate fetus. In the human, baboon,and monkey fetus the pituitary is necessary for adrenal mat-uration and steroidogenesis, at least during the second halfof gestation. Adrenal development is impaired in anence-phalic human fetuses. In the baboon fetus treated in lategestation with betamethasone, there was suppression of fetalpituitary POMC mRNA and reductions in fetal adrenalweight, and 3b-HSD fetal adrenal ACTH receptor mRNAlevels (221). The authors concluded that increased expressionof fetal adrenal ACTH receptor and mRNA species encodingsteroidogenic enzymes depended upon fetal pituitary ACTHstimulation.

In the human fetus, ACTH activity is present in the pitu-itary by 5 weeks’ gestational age, and CRH- and AVP-likeactivity is present in the fetal hypothalamus by approxi-mately 12 weeks gestation (226). CRH1–41, in addition to alarge molecular weight form of CRH, are contained withinthe human fetal hypothalamic tissue. CRH and AVP syner-gize in promoting ACTH release from the human fetal pi-tuitary tissue in early gestation, and the stimulatory effect ofCRH and ACTH output was reproduced by 8-bromo-cAMP(see Ref. 12).

Levels of POMC mRNA in anterior pituitary tissue fromfetal baboons increased significantly from mid (day 100) andlate (day 165) gestation (term 5 day 184) in nontreated an-imals, and there was a corresponding increase in pituitarycells expressing ACTH peptide (227, 228). In the baboon ithas been suggested that this increase in fetal pituitary POMCmRNA levels might be associated with increased pituitaryCRH receptor activity, rather than increased expression ofCRH peptide in hypothalamic nuclei. However, administra-tion of estrogen to midgestation baboons resulted in an in-crease in levels of POMC mRNA- and ACTH-positive cor-ticotrophs in pituitary tissue to values that approached, butremained significantly different from, those at term (228).Pepe et al. (229) have argued that this increase in POMC issecondary to an effect of estrogen on placental 11b-HSDactivity, particularly 11b-HSD-2. In previous studies, theseinvestigators have shown increased expression of placenta11b-HSD-2 in the baboon during pregnancy and have shownthat activity of this enzyme is increased by treatments thatincrease estrogen and decreased with inhibition of estrogenproduction or action (221, 229). In midgestation, the rela-tively lower levels of placenta 11b-HSD-2 allow passage ofmaternal cortisol into the fetal compartment and relativesuppression of fetal HPA activity (221). With increased 11b-HSD-2 activity at day 160, there would be diminished ma-ternal cortisol reaching the fetus (230), allowing the fetalHPA axis to escape from the presumed negative feedback ofmaternal cortisol. This would allow increases in POMC geneexpression, ACTH output, and fetal adrenal maturation.These results are compatible with observations that produc-tion of cortisol by the primate fetal adrenal gland is relatively


low for much of gestation (231, 232). The bulk of the glandis occupied by the fetal zone with relative deficiency of 3b-HSD, and predominant formation of C19 D5 steroids, par-ticularly DHAS (233–235). In late gestation, there is an in-crease in ACTH receptor mRNA and 3b-HSD activity in thedefinitive zone of the fetal adrenal, and a decrease in ACTHreceptor mRNA and formation of DHAS in the fetal zone(236–238). The expression of fetal adrenal enzymes P450C17

and P450SCC remained relatively unchanged during gesta-tion. Thus, there are subtle differences between fetal adrenaldevelopment in the primate and sheep. In the former, ex-pression of 3b-HSD appears rate limiting toward adrenalcortisol output whereas in the ovine species, expression ofP450C17 appears to regulate fetal adrenal steroidogenesis.

In primate pregnancy, estrogen production in the placentadepends extensively on the provision of C19 precursor ste-roids, predominantly from the fetal adrenal gland (239, 240).Fetal adrenal DHAS can be converted to estrone and estra-diol in the placenta, and approximately 50% of circulatingmaternal estrone and estradiol are derived from placentalaromatization of fetal DHAS; the remainder is formed frommaternal adrenal C19 steroids (239, 241). Activation of thepituitary-adrenal axis of the fetus occurs in late gestation.There is a progressive increase in the concentration of DHASin the fetal circulation, which mirrors an increase in maternalplasma estriol concentration (maternal estriol is formed inthe placenta from the precursor 16-hydroxy-DHAS that is90% of fetal origin and formed in the fetal liver from adrenalDHAS). This pattern of fetal adrenal activation, reflected inplasma DHAS concentrations, resembles the time course ofincrease for plasma cortisol in the fetal sheep. Recent studieshave shown that the fetal adrenal in primates is divided intothe outer adult zone that produces predominantly aldoste-rone, the fetal zone that produces DHAS, and the transitionalzone, interposed between the adult and fetal cortex, whichproduces predominantly cortisol (225). Thus, the elegantstudies of Mesiano and Jaffe (225) and Coulter and colleagues(242), have shown that P450scc is expressed throughout theprimate fetal adrenal gland. P450C17 is not expressed in thedefinitive zone but is expressed in the transitional and fetalzones. P450C21 is expressed throughout the gland. 3b-HSD isnot expressed in the fetal adrenal at midgestation but isexpressed in the definitive and transitional zone in late ges-tation fetuses. P450C11 is expressed in the transitional zone inmidgestation and throughout the fetal adrenal cortex in lategestation. ACTH stimulates steroidogenesis in the transi-tional and fetal zone; the major products in late pregnancyare cortisol from the former and DHAS from the latter. Bothin vitro and in vivo studies show dependence on ACTH forfetal adrenal steroidogenesis. More recent studies, however,have indicated that CRH, potentially of placental origin (seebelow), can also stimulate the fetal zone to produce DHAS(243). In addition, this zone of the fetal adrenal appears torespond to trophic inputs from the fetal pituitary other thanACTH. ER-a/b mRNA is also expressed in fetal and defin-itive-transitional zones of the baboon fetal adrenal cortex atmid- and at late gestation (244). The presence of ER in theadrenal cortical cells provides an additional mechanism bywhich estrogen mediates ACTH-dependent functional mat-

uration of the primate fetal adrenal gland. In addition, pre-vious studies had shown that estrogens increase availabilityof LDL-cholesterol as precursor for adrenal steroidogenesis(245, 246).

The difference in fetal adrenal architecture between thesheep and primate fetus has been regarded by many as a clearobstacle to extrapolating from the sheep model of parturitionto the primate. However, it is now apparent that similaritiesbetween these species are greater than the perceived differ-ences (247). In both the sheep and primate fetus the fetaladrenal produces increased amounts of cortisol in late ges-tation (247). It is relatively unprofitable to make detailedcomparison of the minutiae of temporal changes in plasmacortisol because of differences in binding to circulating CBG,transplacental transfer from the mother, and tissue levels of11b-HSD isozymes in the fetus that could locally regulatecortisone-to-cortisol interconversion. In both sheep and pri-mate, the feto-placental unit also produces increasedamounts of estrogen. In the primate, that estrogen resultsprimarily from placental aromatization of precursors gen-erated 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 steroidsthrough to estrogen. In the sheep, a similar fetal-placentalunit of estrogen production exists in pregnancy. The majorfetal adrenal precursors are both D5 and D4 C19 steroidsproduced from the developing zona fasiculata reticularis. Atterm, the prepartum rise in fetal cortisol results directly orindirectly in increased expression of P450C17 in the ovineplacenta, which at that time becomes capable of metabolizingD5 C21 steroids to estrogen. Thus, the apparent difference inthe pattern of estrogen biosynthesis between sheep and pri-mate at term, in its simplest term, reflects the source of C19precursor steroid. The mechanisms of HPA activation mayvary. However, in the primate, the C19 precursor comes fromthe fetal zone of the fetal adrenal gland. In the sheep, thatprecursor comes in part from the fetal adrenal, but there areadditional estrogen precursors produced in the placenta un-der the influence of cortisol from the fetal adrenal gland. Wesuggest that these differences are ones of degree rather thanof absolute distinction.

The role of estriol in the processes leading to the onset ofhuman parturition has remained unresolved over manyyears. Maternal estriol concentrations reflect fetal hepatic16-hydroxylation of DHAS produced from the fetal adrenalgland. It might be anticipated that estriol concentrations inthe maternal circulation would increase in response to fetalstress and might be predictive of impending preterm deliv-ery. Maternal estriol levels increase exponentially towardnormal term. Lachelin and colleagues (248, 249) have shownthat maternal plasma and salivary estriol concentrations areelevated further in a subset of patients with diagnosis ofpreterm labor. Since estriol may affect uterine CAP geneexpression (249), it could contribute to the progressive in-crease in uterine responsiveness in primate pregnancy dur-ing the third trimester of gestation, and its measurementsmay be of predictive value in delineating patients at risk ofpremature delivery (249, 250).


E. Placental progesterone and human pregnancy: theenigma of the progesterone block

A fall in the plasma progesterone concentration is thesingle most common endocrine event associated with par-turition across species (12, 250, 251). Administration of ex-ogenous progesterone at term not only blocks the expressionof CAP genes, but blocks the onset of labor (252). Even in thehuman, where there is no evidence of a fall in maternalplasma or uterine tissue progesterone, administration of theprogesterone receptor (PR) antagonist RU486 leads to in-creased uterine activity and induction of labor (253). In hu-man pregnancy, the luteoplacental shift in progesterone pro-duction occurs by 5–6 weeks’ gestation (254). Progesteroneis synthesized from pregnenolone by placental syncytiotro-phoblast and by chorionic trophoblasts (Fig. 6 and Ref. 255).However, the levels of 3b-HSD mRNA, protein, and activitydo not change in these tissues with labor at term or preterm(256), although regional changes in 3b-HSD expressionmight still occur (257). For example, expression of 15-hydroxyprostaglandin dehydrogenase (PGDH; the major PGmetabolizing enzyme) in chorion is regulated by progester-one (see below) and levels correlated with 3b-HSD in tissuecollected adjacent to the placenta, but not in the cervicalregion. In this lower segment, it was suggested that the actionof progesterone in maintaining PGDH tonically was over-come near term by the inhibitory influence of proinflamma-tory cytokines (see below). There are reports of an increasein the estrogen-progesterone (E:P) ratio in amniotic fluid ofwomen during labor; however, these changes are not im-pressive (250). We have referred to suggestions that maternalestriol, which increases during term and preterm labor,might promote myometrial activation and labor contrac-tions, but this possibility requires stronger experimental ver-ification (249). Alternatively, another progesterone-like ste-roid, possibly a progesterone metabolite that interacts withthe PR, might serve as the active progestagen in humanpregnancy and decline before labor, or progesterone could beconverted to an inactive metabolite that displaces proges-terone from its receptor (258–260). To date, there are no cleardata to support either of these possibilities. Erb et al. (261)reported recently that levels of allopregnanolone, the 3a,5a-reduced metabolite of progesterone that can bind to g-ami-nobutryic acid-A receptors and inhibits uterine smooth mus-cle, did not decrease with labor. The 5b- metabolite blocks OTbinding to its receptor and inhibits OT-induced contractionsin the human myometrium. However, there is also no evi-dence that levels of this metabolite decrease at term.

Studies of gene expression in the human myometriumhave focused on the lower uterine segment. These studiessuggest that the PR system is functional in this region duringlabor. Increased expression of progesterone-responsivegenes such as CX26 (which would promote relaxation) raisethe possibility that elevated levels of progesterone are re-quired to support establishment of a functional (inhibitory)lower uterine segment during labor. If this were so, it wouldalso require mechanisms within the fundus that would blockthe actions of progesterone, allowing the expression of CAPS,and promote contractility in that region.

Although recent exciting data have shown that proges-

terone can bind directly to the oxytocin receptor (OTR) andinhibit its signaling (262), the majority of the actions of pro-gesterone are mediated through a nuclear ligand-inducibletranscription factor, the PR. It has been suggested that afunctional withdrawal of progesterone may involve antag-onism of its action at the level of the PR or PR interaction withtranscriptional machinery (8). This might include a decreasein PR expression, a switch in PR isoforms, a change in ex-pression of receptor accessory proteins (e.g., heat shock pro-teins and receptor coactivators/repressors), or increased ex-pression of endogenous antagonists of progesterone or PR(such as cortisol, TGFb, or phospholipids). Three isoforms ofthe PR have been described: the full-length PR-B and thetruncated isoforms, PR-A and PR-C. In mammals, PR-B func-tions predominantly as an activator of progesterone-respon-sive genes, while PR-A acts as a modulator or repressor ofPR-B function and of other nuclear receptors including theGR, possibly because it lacks one of the three activationfunction domains (AF3) contained within PR-B (263). Nota-bly, progesterone repression of estrogen-induced gene ex-pression was effected through PR-B and not through PR-A.The expression of PR-A and PR-B isoforms is regulated dif-ferentially during development and by hormone treatment.The PR-C isoform (;60 kDa), which has C-terminal trans-activating domains and lacks the first zinc finger of the DNAbinding domain, can dimerize with and modify (possiblyinhibit) transcriptional activity of both PR-A and PR-B.

Analysis of PR expression is complicated by the multiplemRNA and protein species of the receptor. A decrease in PRimmunostaining in myometrium at term has been reportedbut, given the multiple isoforms of PR, these data are difficultto interpret. There is no change in PR mRNA in myometriumor membranes with labor, and no evidence of change in PR-Bor A 1 B mRNA nor in any immunoreactive PR isoforms insamples of lower segment myometrium during labor that mightindicate a decrease in progesterone signaling (G. Erb, N.McLusky, and S. J. Lye, unpublished results). There wasincreased expression of heat shock proteins (HSP)-90 andHSP-56 as well as the steroid receptor coactivators SRC-1 andTIF-2 (G. Erb and S. J. Lye, unpublished results). These co-activators may interact with several steroid receptors, butany interaction with PR should increase rather than decreaseits transcriptional capability. There are limited data on ERexpression in myometrium with labor. However, in thelower uterine segment at term, ER mRNA, protein, and high-affinity binding all appear to be very low.

There are several candidates for potential endogenous an-tagonists of progesterone action. TGFb has been proposed asan endogenous antiprogestin that reduces progesteronestimulation of genes such as enkephalinase (264). Othershave reported that a phospholipid extract of human fetalmembranes was capable of inhibiting progesterone binding,but not estrogen binding. Cortisol itself may compete withprogesterone in the placenta or membranes to regulate thegene for CRH (263). We have found (see below) that whileprogestagens such as medroxyprogesterone acetate (MPA)increase PGDH activity in human placental and chorion tro-phoblasts, this effect is reversed by cortisol. At the presenttime, it is not clear whether these are separate actions throughGR and PR, or whether cortisol and MPA compete for PR-GR


binding. Although four upstream GREs have been identifiedwithin the PGDH promoter, no putative PRE has been iden-tified. Cytokines [interleukin-1b (IL-1b), tumor necrosis fac-tor-a (TNFa)] also decrease PGDH activity, but their inter-action with progesterone as putative antiprogestins remainsunexplored. In recent studies, Stevens et al. (265) reportedthat CRH receptor type 1 (CRH-R1) was expressed prefer-entially in myometrium and fetal membranes of human ges-tation. Levels of CRH-R1 increased in myometrium collectedfrom patients in term and preterm labor but, importantly,levels of CRH-R1 in lower segment myometrium were con-sistently much higher than levels of CRH-R1 in the fundalregion (265). CRH acts through CRH-R1 to increase levels ofcAMP and promote uterine relaxation (61). We thereforeproposed that the role of CRH-R1 in the lower uterine seg-ment was to promote relaxation of this region during laborand to facilitate descent of the fetus (61, 265). These dataindicated that there might be mechanisms by which CRH-R1expression was regulated differentially in the fundus and thelower segment during labor. In independent studies, Spareyet al. (266) reported that levels of PGHS-1 and PGHS-2 pro-teins were also expressed at greater levels in the lower thanupper uterine segment. Connexin-43 protein, in contrast, wasexpressed at much greater levels in the upper uterine seg-ment. Myometrial GSa protein was uniformly expressed inboth lower and upper segments and down-regulated at thetime of parturition. These authors also concluded that dif-ferential expression of these genes might be important toallow cervical ripening before and dilatation during labor,with orderly propagation of uterine contractions (266).

Our own data suggest considerable differences in the ex-pression of CAP genes in the human myometrium duringlabor compared with other species. In contrast to observa-tions in myometrium of rats, sheep, and cows, Teoh et al. (267,268) did not observe any increase in the expression of CAPgenes, including CX-43, OTR, and the PG receptors that arelinked to stimulation of contractile pathways (FP, EP1, andEP3 receptor subtypes, including four splice variants of theEP3 receptor) in lower segment myometrium at labor. How-ever, Teoh et al. (267) did observe increased expression ofconnexin-26, the EP4 receptor and CRH-R1 receptor thatmight be expected to promote myometrial relaxation after anincreased generation of cAMP. It is known that connexin-26is positively regulated by progesterone.

What is the relevance of these observations to the effect ofprogesterone on the myometrium and the apparent lack ofwithdrawal of the progesterone block to the myometrium inhuman pregnancy? We propose that the biological basis forthe onset of labor in animals and in humans is essentiallysimilar. Both require activation of the myometrium and thegeneration of uterotonins to generate labor contractions. Inhuman fetal membranes and myometrium, however, re-gional differences in gene expression allow functional au-tonomy during labor. We suggest that this functional au-tonomy may be critical for the efficient and effective deliveryof the fetus and speculate that this is a mechanism associatedwith evolution to bipedal life. We have suggested that thisregionalization is established through the action of proges-terone. Early studies, e.g., those of Wiqvist and colleagues(269), support this hypothesis. These authors found that

PGF2a had little effect on the fundal myometrium, but wasstimulatory in lower segment specimens taken before labor.PGE2 induced a biphasic dose-dependent response. How-ever, PGF2a and PGE2 always stimulated fundal myome-trium collected during spontaneous labor. PGE2 inducedinhibition in lower segment samples collected at that timewhile PGF2a had no effect.

We speculate that during pregnancy, progesterone limitsthe generation of stimulatory PG in chorion by inducing highexpression of PGDH (see below), and it also inhibits theexpression of CAP genes within the myometrium, therebymaintaining the muscle in a quiescent state (8). Functionalregionalization of both chorion and myometrium at term isengineered 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 progesteronein the fundus induces CAP gene expression and myometrialactivation. Enhanced progesterone signaling in the loweruterine segment, however, promotes the expression of genesthat induce relaxation, facilitating descent of the fetus (8). Themechanisms inducing functional withdrawal of progester-one in fundal myometrium and cervical chorion need notnecessarily be the same (270). Cortisol and/or cytokines mayantagonize progesterone- induced PGDH activity in chorion(see below). In myometrium, potential mechanisms includechanges in PR isoforms, steroid receptor co-activator/repres-sors, or other putative antagonists of progesterone action. Wespeculate that this concept of human labor provides an ex-planation as to why progesterone levels remain high in thisspecies. Rather than being an impediment to labor onset, wesuggest that progesterone is required to induce lower seg-ment relaxation and the safe and efficient delivery of theprimate fetus.

Recent exciting studies have pointed to a role for proges-terone in maintaining cervical function during pregnancy,and to metabolism of progesterone within the cervix as beinga critical step in cervical dilatation and parturition. Mahen-droo and colleagues (271, 272) showed that parturition wasdelayed in mice lacking steroid 5a-reductase type 1 enzyme.They showed subsequently that basal and stimulated levelsof uterine contractility were similar in these animals and inwild-type controls. However, cervical distention did not oc-cur in 5a-reductase-deficient animals, and cervical compli-ance was less on day 20 of gestation than earlier in preg-nancy. As expected, relaxin, which is known to promotecervical ripening, induced delivery in both wild-type and5a-reductase knockout animals. Subsequent studies demon-strated that while serum progesterone concentrations de-clined in knockout animals in a manner generally similar tothat of controls, the concentration of progesterone in cervicaltissue and in whole uterus remained elevated. As expected,cervical ripening and parturition occurred after ovariectomy.Thus, these studies point to the role of progesterone metab-olism in facilitating normal cervical dilatation that must ac-company uterine contractility to allow birth (273). In theuterus of pregnant mice, progesterone can be metabolized atterm through either 5a-reductase or 20a -HSD pathways. Inthe cervix, however, there is limited 20a-HSD activity, andnormally 5a-reductase provides the pathway for progester-


one metabolism, progesterone withdrawal, and cervical rip-ening and dilatation (272). Further studies of other genesassociated with cervical ripening are clearly warranted inthis fascinating model, as are measurements of 5a-reductaseactivity in human cervix from patients at term and pretermlabor.

V. Myometrial Stimulation: Phase 2 of Parturition

Activation prepares the myometrium to respond opti-mally to the production of those myometrial stimulants thatprovoke myometrial contractility during labor. Althoughmany agonists have been described to stimulate myometrialcontractions, convincing information is available only for OTand stimulatory PGs (274). The physiological role of otherputative agonists such as CRH is uncertain and equivocal.The actions of these three groups of compounds are dis-cussed below.

A. Stimulation: role of OT

OT is a nonapeptide synthesized by hypothalamic mag-nocellular neurons located in the supraoptic and paraven-tricular nuclei (275–277). Hypothalamic OT is released intothe circulation from the posterior pituitary. Its classical ef-fects include promoting myometrial contractility during latepregnancy and parturition and stimulating milk release fromthe mammary gland in lactation (275, 278, 279). The dilemmasurrounding the role of OT in the process of labor arose whenit was unclear whether levels of OT in the maternal circu-lation actually increased before the onset of labor (279, 280).The recent report that mice bearing a null mutation in the OTgene have normal pregnancies and labors may reflect a com-pensatory effect of AVP (281, 282). Studies showing the rel-ative ineffectiveness of OTR antagonists in preventing pre-term labor, however, suggest that while this hormonecontributes to labor, it may not be an essential element (283).

One aspect of the solution to the apparent discrepancybetween circulating OT levels and parturition was the dra-matic increase in myometrial sensitivity to OT before andduring labor, associated with a several-fold increase in myo-metrial OTR gene expression, which coincides with peakuterine responsiveness (276, 284–286). Thus, changes in cir-culating OT levels would not be necessary for the peptide tohave a physiological role in labor (280). A parallel conclusionis drawn from the 24-h pattern of OT secretion, and myo-metrial sensitivity (287). Recent studies also suggest that OTmay act as a local mediator of parturition. OT gene expres-sion has been demonstrated in the human and rat uterus andfetal membranes (288–290). In the rat, fetal membranes, pla-centa, and uterus synthesize OT mRNA transcripts withextended poly-A tails (289). Levels of OT mRNA in rat fetalmembranes declined from gestational day 14 to term, bututerine OT transcripts increased during gestation 150-foldand exceeded levels of OT mRNA in the hypothalamus atterm (289). Human fetal membranes, amnion, chorion, anddecidua synthesize OT mRNA, and levels of OT mRNAtranscripts increased in these tissues at the time of parturition(290). In vitro studies with rat and human chorio-decidualtissue have indicated that estrogen, generated locally, could

up-regulate OT gene expression (291–293), consistent alsowith the presence of an ERE in the OT promoter region (293).Other, fascinating studies have indicated that OT may pro-mote uterine activity by antagonizing the relaxant effect ofCRH through receptors coupled to adenylate cyclase (seebelow). The general consensus is that OT appears to have arole to play in the stimulus to uterine contractility at term andin uterine involution (294). Whether that role is indispensableremains in dispute.

B. Stimulation: role of PGs

There is a substantial body of evidence to support a rolefor PGs in the labor process, at term and preterm (207, 295).PGs contribute to the transition from phase 1 to phase 2rather than initiating the labor process. Mice carrying nullmutations for genes encoding the PGF2a receptor (296), cy-tosolic phospholipase A2, and prostaglandin synthase type1 (PGHS-1) (297) have delayed labor onset although neonatalviability is diminished. Mice lacking the PGHS-2 gene (298)have not been studied in relation to gestation length andpregnancy outcome because fertility is impaired, and ovu-lation and implantation are blocked. Lack of PGF2a (FP)receptor prevents effective luteolysis at the end of gestation,so plasma progesterone concentrations are maintained. Inthese 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 modelsis applicable to human gestation may be questioned, sincethe primary site of PG action is at the level of the corpusluteum, which is not required for pregnancy maintenance inwomen after the first 5–6 weeks of pregnancy. Perhaps thebest indicator for a role of PG in parturition in primates aswell as sheep and other species is the measurement of in-creased PG output before the appearance of labor-like myo-metrial contractions (299–301) and the effectiveness withwhich drugs that block PG synthesis suppress myometrialcontractility and prolong gestation length.

PGs are formed from membrane phospholipids throughthe initial activity of phospholipase A2 or C isozymes form-ing unesterified arachidonic acid (302–304). PLA2 isozymes,localized by immunostaining to fetal membranes and myo-metrium (305), may include the larger molecular mass (85–110 kDa) cytosolic form (cPLA2), as well as secretory typesI, II, and III, extracellular 14-kDa forms. Activation of secre-tory PLA2 (sPLA2) requires millimolar concentrations of cal-cium, whereas cPLA2 is activated at micromolar calciumconcentrations (see Ref. 8).

Cytosolic PLA2 translocates to the cell membrane in re-sponse to agonist stimulation and liberates arachidonic acidfrom the sn-2 position of phospholipid (306). Activity ofcPLA2 is reportedly greater in amnion from patients not inlabor at term or preterm than from patients in labor, ex-plained as depletion of cPLA2 at this time (304). Previousstudies had shown that cPLA2 expression was up-regulatedin WISH cells, a transformed amnion epithelial cell line, inresponse to cytokine stimulation, and that this occurs inparallel with increased expression of PGHS-2 by these cells(307, 308). The general consensus, however, is that in human


pregnancy, expression of PLA2 increases gradually in fetalmembranes during gestation but does not increase appre-ciably at the time of labor (309).

Arachidonic acid is further metabolized to the intermedi-ate PGH2 by PGHS enzymes, which have both cyclooxygen-ase and peroxidase activities (310, 311). There are two formsof PGHS; both are heme proteins composed of two approx-imately 70-kDa subunits. The constitutive form (PGHS-1)and the inducible form (PGHS-2) are distinct gene productsalthough they have considerable sequence homology, andtheir cDNAs are 60–65% homologous (312). PGHS-1 hassimilar properties to other housekeeping genes. PGHS-2 ischaracteristically up-regulated by growth factors and cyto-kines. The activity of PGHS-1 and PGHS-2 is inhibited by awide spectrum of nonsteroidal antiinflammatory drugs.These differ in their Ki values for the two PGHS isoforms,suggesting the potential to develop specific inhibitors of ei-ther isoform for therapeutic management (313–315).

Arachidonic acid may also be metabolized through dif-ferent lipoxygenase pathways including 5-lipoxygenase,platelet-type-12-lipoxygenase, leukocyte-type-12-lipoxygen-ase, and 15-lipoxygenase (316). Arachidonic acid metabolismthrough 5-lipoxygenase forms 5 H(P)ETE, which can be con-verted to leukotriene A4 (LTA4), which is subsequently hy-drolyzed to LTB4 or LTC4. 12-Lipoxygenase or 15-lipoxy-genase activity results in the formation of 12-H(P)ETE and15H(P)ETE. There are some suggestions that these productscan weakly stimulate contractility of smooth muscle. It hasalso been suggested that arachidonic acid metabolism inhuman fetal membranes during pregnancy is directed pref-erentially toward lipoxygenase products, but there is a pro-gressive switch toward the more potent PGHS (also cyclo-oxygenase, COX) activity at term (317). Primary PGs areformed from PGH2 through the activity of specific isomer-ases and synthases. There is unfortunately very little infor-mation concerning the expression, localization, and changein activity of these enzymes in intrauterine tissues at term orpreterm labor, and this will be an obvious area of furtherinvestigation.

The major pathway in the metabolism of PGE2 and PGF2a

involves the action of a type 1 NAD1- dependent PGDH thatcatalyzes oxidation of 15-hydroxy groups resulting in for-mation of 15-keto and 13,14 dihydro-15-keto metaboliteswith reduced biological activity (318, 319). We have reportedthat PGDH expression and activity are decreased in chorio-decidual tissue of women at spontaneous and preterm labor(see below), raising the possibility that failure to inactivatePGs produced within intrauterine tissues during pregnancymay be one cause of preterm labor (320).

The action of PGs is exerted through specific receptorsincluding the four main subtypes for PGE2, EP1, EP2, EP3,and EP4, and FP for PGF2a (60, 321). EP1 and EP3 receptorsmediate contractions of smooth muscle through intracellularsignaling pathways that elevate free calcium and decreaseintracellular cAMP (27). EP2 and EP4 receptors are coupledthrough adenylate cyclase and increase cAMP formation,leading to relaxation of smooth muscle. Consistent with this,various groups have reported that EP2 expression in myo-metrium is higher preterm than at term. In the rat, parturitionis associated with down-regulation of EP receptor subtypes

and with up-regulation of myometrial FP receptors, effectinga switch from inhibition to stimulation.

1. PG synthesis. Regulation of PGHS-2 and PGHS-1 genes areclearly multifactorial (322–324). There are two nuclear factor(NF)-kB binding elements within the proximal promoter re-gion of PGHS-2 (325, 326). p50 And p65, key members of theNF-kB Rel family of proteins are present in trophoblasts andlikely serve as mediators of cytokine-induced up-regulationof PGHS-2 expression (327). The PGHS-2 promoter also in-cludes response elements resembling NF-IL6, GRE, CRE, andAP2 sites (323, 325). Levels of PGHS-2 are increased up to80-fold in response to various cytokines and growth factors,whereas levels of PGHS-1 are usually increased only 2- to3-fold in response to these stimulators (328, 329). Studies inseveral species, including the human, have indicated that thePGHS-2 isoform is the principal form of the enzyme involvedin the increased PG production seen at the time of parturi-tion. Effects of CRH in up-regulating PG output, at leastwithin fetal membranes (see below), is likely mediatedthrough proximal CRE sequences (326). Although glucocor-ticoids inhibit PGHS-2 expression in WISH cells and in mostother cell types, apparently by interference with the NF-kBsignaling system (330), they stimulate PGHS expression andactivity in trophoblast-derived cells including amnion, andchorionic trophoblast (58, 331–334). Kniss (327) reported asimilar effect of dexamethasone in stimulating PGHS-2mRNA expression in human breast adenocarcinoma cells.The stimulatory effect of glucocorticoids on PGHS gene ex-pression in human fetal membranes is central to our currenthypotheses of human parturition and will be discussed inmore detail below.

In human pregnancy, the PG synthesizing and metabo-lizing enzymes are compartmentalized discretely betweenthe 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 in-crease in PG synthesis and levels of PGHS-2, but not PGHS-1mRNA at preterm and term labor (338–343). Immunohisto-chemical and in situ hybridization studies have localized thePGHS-2 enzyme and mRNA to the amnion epithelium (344–346), the subepithelial cells in the mesenchyme and in thechorion laeve trophoblasts with lower expression found indecidua (347–349). Decidua has been reported to produceincreased amounts of PGs at the time of labor, but this is nota consistent observation (348). Human decidua is made upof decidualized stromal cells, bone marrow-derived macro-phages, and other cell types including trophoblasts that in-terface with chorion (350). Variability in cell populationsused for in vitro studies may contribute to the variability ofresponses that have been obtained. In chorion, interposedbetween amnion and decidua, PGDH activity predominates,although PGHS is also expressed (347, 351). Output of PGsand PGHS activity is greater in chorion from patients atspontaneous labor than at elective term cesarean section; inpreterm labor chorion both PGHS-1 and PGHS-2 mRNAlevels are increased (352, 353).

It is generally considered that activity of PGDH predom-inates in chorion (354), forming a relative metabolic barrierthat prevents passage of PGs generated within amnion or


chorion from reaching underlying decidua or myometriumthrough most of pregnancy (320, 355). The presence of highPGDH activity in chorion trophoblasts (356) implies that atfull term those PGs acting on the myometrium would morelikely be derived from decidua, or from the myometriumitself (41, 357). There are variable reports, however, ofchanges in PGHS activity in human myometrium at the timeof labor (59). Some workers have reported increased PGHS-2expression and activity, while others have reported no

change, or even decreased activity. In myometrium throughpregnancy, PGHS-1 or PGHS-2 must be present to generatethe predominant PGI2 which, as discussed above, contributesto maintenance of uterine quiescence (59). It has been sug-gested that PGI2 formation in myometrium may be decreasedby glucocorticoids. Unfortunately, it is difficult, experimen-tally, to obtain consistent specimens of human myometriumfor biochemical analysis. Generally, tissue is obtained fromlower segment uterus, but at term with ensuing cervical

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.


dilatation, the proportion of myocytes in the tissue is likelyto have changed. Further, recent studies indicating that thereare regional differences in CAP genes between the fundusand lower segment of the human uterus in late pregnancy(see above) may suggest the need for further reexaminationof these issues, ideally combined with experimental manip-ulation in subhuman primates.

It remains crucial to understand regulation of PGHS-1 andPGHS-2 expression in human fetal membranes and to de-lineate the major site of PG production at term and pretermlabor (Fig. 5). These may not necessarily be the same. Forexample, instances of preterm labor may be associated withelevated PG production in amnion or chorion, whereas termlabor may require increased PGHS-2 expression in deciduaand myometrium (344). Given that PGs act generally as para-crine or autocrine regulators, it will be exceedingly difficultto obtain in vivo evidence for altered PG production specif-ically at these sites. Amniotic fluid concentrations of PGsincrease at labor, and the initial changes precede the onset ofmyometrial contractility. Levels of PGF2a in amniotic fluidpresumably reflect, in part, production from decidua, sincePGE2 and not PGF2a is the major eicosanoid formed fromamnion and chorion (Figs. 5 and 6). However, these mea-surements probably provide no more than a crude estimateof the pattern of PG change at a local cellular level and giveno information concerning receptor subtypes and distribu-tion (358).

Primary cultures of mixed and purified cells from humanamnion or chorion have been used extensively as models to

study the regulation of PG formation in response to cyto-kines, growth factors, CRH, and lipopolysaccharides. In ad-dition, the amnion-derived epithelial cell line (WISH cells)has also been used extensively (359–361). A crucial reserva-tion with all of these in vitro studies is that, in general, singlecompounds have been studied in isolation of the in vivoenvironment; the extent to which results can be extrapolatedfrom in vitro to in vivo will remain, unfortunately, a matterof conjecture.

Many cytokines have been shown to act on amnion, cho-rion leave, and decidua to increase PG output (360, 362–364).IL-1b stimulates PG output by cultured amnion, chorionleave, and decidua (195, 317, 365) while IL-6 stimulates PGoutput by decidua and amnion (366, 367). IL-8 did not alterPG production by chorion or decidua, but augmented thestimulatory action of other cytokines (368). The effect of IL-1bis certainly associated with increased expression of PLA-2and PGHS-2 (329). The action of IL-1b can be reduced by thenaturally occurring receptor antagonist, which has beenshown to prevent IL-1b-induced labor in mice (369). IL-1bstimulation of PGHS in amnion and chorion may be medi-ated through the NF-kB system (370–372). In WISH cellsstimulated with interleukin-1b, I-kBa was degraded by morethan 90% within 15 min of stimulation, and this was asso-ciated temporally with nuclear translocation and binding ofNF-kB (373). PGHS-2 mRNA was increased within 30 minand reached steady state by 4 h. PGHS-2 protein then in-creased more than 80-fold, and this was associated with acorresponding time-dependent increase in PG production.

FIG. 6. Summary to indicate factors leading to up-regulation (1) or down-regulation (2) of prostaglandin H2 synthase in intrauterine tissues.The role of progesterone remains equivocal.


Inhibition of I-kBa degradation by calpain-I inhibitionblocked NF-kB translocation, and increases in PGHS-2mRNA and protein, and PG synthesis (373). Wang and Tai(374) provided similar information and showed that in WISHcells, dexamethasone blocked IL-1b-mediated stimulation ofPGE2 output consistent with the general model of mutualtranscriptional antagonism from GR/NF-kB interaction(330).

Human amnion cells can be maintained as mixed popu-lations in culture or can be separated into primary epithelialcells and cells of the subepithelial mesenchymal layer (375).We have reported recently that the output of PG by mesen-chymal cells exceeds that of epithelial cells in the basal state.Epithelial cell production of PGs was stimulated by glu-cocorticoids, whereas there was no significant change in thealready elevated output of PGs from mesenchymal cells(375). Previously, in mixed cultures, glucocorticoids andIL-1b were shown to increase PGHS-2 mRNA, protein, andPGE2 output predominantly from the subepithelial mesen-chymal cells (331, 376). It remains possible that this apparentdifference can be explained by epithelial-mesenchymal cellinteraction, and current studies are directed at resolving thisissue.

The effect of glucocorticoids on primary cultures of am-nion cells and on chorion trophoblast cells is surprising (377)and striking (323, 378, 379). Although dexamethasone inhib-ited PGE2 output by freshly dispersed amnion cells, it stim-ulated PGE2 output by amnion cells after 4–5 days in culture(376). The effect was dose dependent and associated withincreased expression of PGHS-2 mRNA and protein. Theactivity of glucocorticoids is also receptor mediated and canbe inhibited by addition of GR antagonist (380). In previousstudies, we had localized GR to amnion epithelial cells, sub-epithelial fibroblasts, and chorion laeve trophoblasts in hu-man pregnancy (381). GR exists as both a-form and b-form(330). GRa is retained in the cytoplasm in an inactive state byits association with the regulatory heat shock proteins suchas HSP-56 and HSP-90. GRb, formed from alternate splicingof the same mRNA transcript as GRa, is localized in the cellnucleus independent of binding to ligand. It appears thatGRb functions as a dominant negative regulator of GRatransactivation. Thus, earlier studies of GR localization to celltypes within human fetal membranes require repeating withspecific identification of GRa and GRb forms.

Peptides such as CRH could be released from amnionepithelial cells to act in a local paracrine manner and up-regulate PGHS-2 expression in mesenchymal cells (see Ref.207). Full thickness fetal membranes treated in culture withCRH were stimulated to increase output of PGE2 and in-creased levels of PGHS-2 mRNA within 4 h in culture. Thus,the stimulatory effect of glucocorticoids on PG production byamnion, known to involve an intermediary protein syntheticstep, could be the result of synergistic epithelial-mesenchy-mal interaction, in addition to, or instead of, any direct effecton amnion cell types. Similar interactions may contribute tothe response to cytokines such as IL-1b in vitro (382). Inter-estingly, recent studies have shown that in amnion explants,in contrast to chorion and decidua, the antiinflammatorycytokine IL-10 stimulates rather than inhibits PG production,and the normally antiinflammatory cytokine IL-4 stimulates

PGE2 output in amnion cultures (329). The authors havesuggested that amnion may therefore be refractory to inhib-itory cytokines as part of an evolutionary mechanism de-signed to expedite the parturition processes.

Over the past 10 yr, in vitro studies have generated animpressive list of substances capable of increasing PG outputby human fetal membranes in culture (383–386). Clearly,availability of free calcium is a critical requirement. Epider-mal growth factor (EGF), platelet activating factor (PAF), andagents that activate protein kinase C stimulate PG output(387, 388). Importantly, b-sympathomimetic drugs andagents that increase intracellular cAMP levels also increasedPG output by cultured chorion and decidual cells (389). Cat-echolamines are present in increasing concentrations in hu-man amniotic fluid in late gestation (390), and both amnionand decidua express components of the adenylate cyclasesystem, which undergoes stimulation with b-agonists suchas isoproteronol (391). Effects of these activators of adenylatecyclase can be mimicked by (Bu)2cAMP or phosphodiester-ase inhibitors such as methylxanthine (389). Studies such asthese may help explain the disappointing lack of efficacy ofb2-sympathomimetic drugs in sustaining uterine quiescencewhen used in the treatment of preterm labor (392). Althoughthese compounds are effective in the short term by elevatingcAMP and decreasing activity of MLCK, in the longer termelevations of cAMP may up-regulate PGHS-2 through aproximal CRE, resulting in increased output of stimulatoryPGs, uterotonins whose action the administration of b2-mimetic was intended to antagonize.

2. PG metabolism. The major metabolizing enzyme for PGs(393), PGDH, is exquisitely localized in fetal membranes totrophoblast cells of chorion (Fig. 5). Thus, it could act as ametabolic barrier to the passage of unmetabolized PGs, gen-erated in amnion or chorion, and prevent their reaching theunderlying decidua or myometrium in a biologically activeform (354, 394, 395). Some years ago, we identified a groupof patients presenting in idiopathic preterm labor with de-ficiency of PGDH in chorion trophoblast cells (396). Therewas a further reduction of ir-PGDH, PGDH mRNA, andPGDH activity in chorion trophoblast cells, but not placentaltrophoblast, in patients in preterm labor with an underlyinginfective process (397). Thus, with preterm labor in the pres-ence of an inflammatory response, loss of chorion tropho-blast cells leads to loss of PGDH activity. PGs generated, forexample in response to elevations of cytokines, will not bemetabolized and will be available to stimulate underlyingmyometrium.

In idiopathic preterm delivery, in the absence of infection,it is clear that PGDH activity is specifically regulated inchorion trophoblast (Fig. 7). During in vitro studies withchorion trophoblast cells maintained in culture, we foundthat the glucocorticoids, cortisol and dexamethasone, inhib-ited PGDH activity and decreased levels of PGDH mRNA(398). Cortisone was as effective as cortisol, since choriontrophoblasts contain 11b-HSD Type 1 (11b-HSD-1) capableof reducing cortisone to biologically active cortisol (399). Thisactivity could be inhibited by carbenoxolone, an active in-gredient of licorice. Chorion trophoblast cells also expressed3b-HSD and converted pregnenolone to progesterone (400,


401). Inhibition of 3b-HSD activity with trilostane led todecreased PGDH activity and reduced levels of PGDHmRNA in the cells. These could be restored by concurrentaddition of progesterone, or of the synthetic progestagens,MPA or R5020 (398). Effects of these compounds, in turn,were antagonized by onapristone and RU486, inhibitors ofprogesterone action (398, 402). Furthermore, inhibition ofPGDH mRNA and activity by cortisol could be reversed byaddition of progesterone (320).

These data could be explained by glucocorticoids and pro-gesterone acting through independent receptors, or by theirinteraction at the same binding sites on GRa (403). Previ-ously, Karalis and Majzoub (404) provided evidence thatsimilar interaction between progesterone and cortisol forbinding to GR explains the interactive effect of these com-pounds on the output of CRH by placenta trophoblast cells.

In recent studies we found that CRH also decreased PGDHactivity in chorion trophoblast cells in a dose-dependentfashion (F. Patel and J. R. G. Challis, unpublished observa-tions). We believe this activity is mediated through cAMPgeneration, since CRH binds to CRH-R1 species in fetal mem-branes where it may increase cAMP, and cAMP decreasesPGDH activity (405), presumably acting through a consensusCRE in its promoter region. Thus a pattern is emerging thatseveral agents which up-regulate PGHS-2 in human fetalmembranes (CRH, cortisol, IL-1b, TNF) down-regulatePGDH in chorion (Fig. 8). Effects of cortisol in the membranesmay be enhanced by local conversion of cortisone to cortisol,through the reductase activity of chorionic 11b-HSD-1 (406).The activity of this enzyme is increased by PGE2 and PGF2a

in a dose-dependent fashion that is associated with, anddependent upon, a transient increase in intracellular Ca21 (N.Alfaidy and J. R. G. Challis, unpublished results). Therefore,a further feed-forward paracrine/autocrine loop exists inwhich increased output of PG should stimulate 11b-HSD-1,resulting in increased production of cortisol, which leads tofurther increases in PGHS-2 and decreases in PGDH (Fig. 8).

We have referred previously to the finding of regionalvariation in PGDH activity. We suggest that this might reflectprogesterone stimulation of the enzyme (407, 408) in a re-gional pattern. Chorion collected from patients at electivecesarean section at term in the absence of labor had higherPGDH activity in the region of the membranes overlying theinternal os than chorion collected from a region adjacent tothe placenta or between the placenta and cervix (41). How-ever, at cesarean section in labor, there was a dramatic re-duction in PGDH activity in chorion from the lower uterinesegment. We suggested that this altered response could re-flect an antagonism of the effect of progesterone on the en-zyme by elevations of cytokines derived from vaginaland/or cervical fluids. We and others have shown thatwhereas IL-1b and TNFa increase PG synthesis, these cyto-kines decrease PGDH activity and PGDH gene expression(409, 410). Importantly, IL-10, the antiinflammatory cytokinethat attenuates IL-1b-induced up-regulation of PGHS, alsoreverses IL-1b down-regulation of PGDH (409). The impor-tance of this observation is that PGs generated within amnionand chorion in the lower segment may escape metabolism inchorion specifically in that region at the time of labor to reachthe cervix and effect effacement and dilatation.

FIG. 7. Diagrammatic summary of factors regulating expression of the acitivity of PGDH in human chorion.


3. PGs and infection. Approximately 30–40% of preterm la-bors are associated with an underlying infective process.Romero, Mitchell, and collaborators (411–413) have demon-strated elegantly the role for infection in preterm labor. Bac-terial organisms themselves secrete phospholipases, result-ing in increased release of arachidonic acid from intrauterinetissues and increased PG production. Alternatively, bacterialendotoxin, such as lipopolysaccharide, acts on amniotic ormembrane macrophages, causing either PG release or furtherrelease of cytokines (414–417). Cytokines in turn elevate PGproduction within amnion, chorion, and decidua as dis-cussed previously (418). Administration of cytokines or bac-terial endotoxins to pregnant mice provokes premature de-livery and allows examination of the precise temporalsequence of events in infection-driven preterm labor (419,420). A number of cytokines including IL-1b, TNFa, IL-6, andIL-8 (neutrophil-activating protein-1) are increased in amni-otic fluid of patients undergoing preterm labor associatedwith infection (421–424). Cytokines are produced not only bymacrophages, but are synthesized and secreted by humanfetal membranes in decidua, and these tissues may be thesources of the cytokines found in amniotic fluid. IL-1b, IL-6,and IL-8 mRNA were expressed in amnion, chorion leave,and decidua, particularly in tissues obtained after labor. Inaddition, cultured decidual and chorion cells produce IL-6and IL-8 when stimulated with IL-1b and TNFa, and amnionproduces IL-8 in response to IL-1b (425). Thus, these studieshave led to the suggestion that there is a complex cytokinenetwork at the chorio-decidual interface, as has been pro-posed to exist in other tissues (269). It is also possible thatcytokines cause release of other uterotonins, including OTand CRH in decidua (426, 427), myometrium, and/or pla-centa. These compounds may affect the myometrium di-rectly or indirectly. Lipopolysaccharide also inhibits repli-cation of amnion cells, and it has been suggested that thismight be a mechanism by which lipopolysaccharide contrib-utes to premature ruptured membranes.

The paradigm of infection-driven preterm labor has been

proposed as a means of understanding regulation of PGproduction in labor at term (348). However, preterm labor inthe absence of infection can occur without demonstrablechanges in amniotic fluid PGE concentrations and appar-ently without enhanced PG biosynthetic activity in fetalmembranes. It has been argued that changes in PG andcytokine concentrations in the amniotic fluid of women inpreterm labor with infection are not reproducible, and thatthese compounds accumulate there as a result of pretermlabor, rather than as a cause (428). It has also been argued thatinvasion of the amniotic sac by microorganisms occurs whenlabor has been initiated, when tissues of the forebag areexposed. Furthermore, since parturition is an inflammatoryprocess, the presence of mediators of inflammation in am-niotic fluid could be a natural event of parturition withoutarguing for a role of infection as a cause of preterm labor. Thebody of evidence currently available has tended to counterthis latter view. However, as in all human studies of this type,it is extremely difficult to delineate precisely the cause-and-effect sequence of relationships. Furthermore, a low-gradeinflammatory response, where accumulation of cytokinesoccurs without an infective process, may be present normallyat term and contribute to the stimulus of labor or remain asa parallel, but unrelated, event.

C. Stimulation: role of CRH

Over the past 10 yr there has been considerable interest inthe possible role that CRH, produced from intrauterine tis-sues, plays in the regulation of human pregnancy and par-turition (429, 430). Pro-CRH mRNA is present in placentaltissue (431) and decidua in increasing amounts during preg-nancy. These levels correlate with increased concentrationsof ir-CRH peptides in the placenta and with the exponentialincrease in CRH1–41 concentrations in maternal peripheralplasma (432–435). CRH also increases in cord plasma, al-though the concentrations are generally lower than those inthe maternal compartment (432, 436, 437). Several groups of

FIG. 8. Interrelationship between cortisol, PGHS-2, PGDH, and CRH. In chorion, cortisone can be converted to cortisol through the activityof 11b HSD-1, and the activity of this enzyme is increased locally by PGs.


investigators have reported that maternal plasma CRH con-centrations are elevated significantly in the plasma of pa-tients presenting in preterm labor (433, 438–440) and may beused to discriminate patients presenting in preterm laborwho will deliver within 24–48 h from those patients with asimilar diagnosis, but in whom labor is not imminent (441).

The biological activity of CRH in maternal plasma is at-tenuated by the presence of a circulating CRH binding pro-tein (CRH-BP), produced in the liver and placenta (429, 442).CRH-BP blocks the ability of circulating CRH to promoteACTH release from pituitary corticotrophs, and it inhibits thestimulatory effect of CRH on uterine PG production. Con-centrations of CRH-BP decrease during the last 5–6 weeks ofnormal pregnancy and before preterm labor, coincident withthe increase in maternal CRH concentrations (443), and ap-parently in response to increased CRH secretion. In the pla-centa, CRH is produced by syncytiotrophoblast and inter-mediate trophoblasts (444), and immunoreactive CRHlocalizes to these cell layers (429, 445). In culture, CRH outputfrom placental and chorion trophoblast cells is inhibited bynitric oxide and progesterone and increased by cat-echolamines, OT, cytokines, and glucocorticoids (Fig. 9; Refs.427 and 444). Majzoub and colleagues (446, 447) demon-strated that dexamethasone increases levels of CRH mRNAin placental trophoblast cells maintained in culture in a time-and dose-dependent fashion, although later suggested thatthis “apparent” stimulation resulted in fact from reversal ofprogesterone-induced inhibition of CRH expression (263,404). Glucocorticoids compete with and displace progester-one from GRa binding, and diminished inhibition is mea-sured as an apparent increase in secretion of CRH.

We demonstrated in vivo that patients receiving prenatalglucocorticoids to promote pulmonary maturation inamounts that decreased maternal ACTH and cortisol con-centrations by more than 80% provoked stimulation of ma-ternal CRH concentration by almost 50% over pretreatmentvalues (448). Administration of glucocorticoids to pregnantwomen with singleton or multiple fetuses at risk of pretermlabor actually stimulates uterine contractility, although theeffect may be transient (449, 450). From the foregoing dis-cussion it is evident that this could be the result of up-regulation of PGHS, down-regulation of PGDH, and/orstimulation of placental CRH which, in turn, provokes afurther increase in PGHS-2 expression (451). Administrationof glucocorticoids eventually suppresses fetal HPA function,decreases estrogen output from the placenta, and might beexpected to diminish uterotrophic activation of the myome-trium, perhaps accounting in part for the (fortunately) tran-sient nature of this response.

Based upon these results, and the demonstration of acti-vation of fetal HPA function in response to hypoxemia inanimal fetuses, we proposed that the human fetus would alsorespond to an adverse intrauterine environment such asacute hypoxemia with activation of the fetal HPA axis (10).With time, increased pituitary drive to the adrenal increasessteroidogenic enzyme potential and cortisol output. Fetalcortisol, then acting through placental and/or membraneGRa, up-regulates placental CRH gene expression, leading tothe increased CRH concentrations in the plasma of patientspresenting in preterm labor. Accordingly, cord CRH con-centrations are elevated in the presence of intrauterinegrowth 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.


(440, 452). CRH is a vasodilator in the placental vascular bedand reverses the vasoconstrictor influence of PGF2a (453). Inthe placenta, the vasodilator action of CRH is associated withup-regulation of the NO-cyclic GMP pathway. Hence, ele-vations of CRH within the placenta should signal increasedblood flow and correction of a hypoxemic insult to the fetus.However, if the hypoxemia persists, placental CRH outputpresumably remains elevated (Fig. 10). CRH, secreted intothe fetal circulation, drives further pituitary ACTH secretionand also drives DHAS output from the fetal zone of the fetaladrenal gland (243); hence, maternal estrogen output shouldrise as a secondary response to fetal distress. Increased es-trogen leads to uterine activation. CRH contributes to in-creased expression of PGHS (451) by up-regulating adenyl-ate cyclase activity in placental and membrane cells (61). Itwill be recalled that the PGHS promoter contains a CRE.Thus, we speculate that activation of a feed-forward loop inresponse to a hostile intrauterine environment is a mecha-nism by which a compromised fetus may signal pretermlabor and induce premature delivery (Fig. 10). In addition,maternal stress with elevations of maternal glucocorticoidconcentrations may also contribute to elevations of placentalCRH output and preterm birth. Hobel and colleagues (454)reported increases in maternal CRH concentrations inwomen with elevated scores for perceived stress and anxiety.These values predicted preterm labor, even as early as 20–24weeks of gestation.

It is extremely difficult to prove or disprove this hypoth-esis with in vivo studies in normal human pregnancy. Studiescannot be performed in nonprimates, since these species donot appear to produce placental CRH. The pattern of pla-cental CRH output during pregnancy in the baboon andrhesus monkey has been described but differs from the ex-ponential increase of plasma CRH concentration observed inhuman gestation (429). Women receiving betamethasone de-liver at variable times after treatment. Current obstetric prac-tice in North America, in fact, makes it difficult to obtain“control” placental tissue from patients in preterm labor who

have not received exogenous corticosteroid; such patientsmay have increased endogenous corticosteroids before tissuecollection in any case.

A further reservation is related to CRH receptor specificity.CRH exerts its effects through activating specific G protein-coupled receptors, which exist in two subtypes: CRH-R1 andCRH-R2. These arise from different genes with multiplesplice variants (455). The two receptors share approximately70% homology at the amino acid level. CRH-R1 exists in atleast three variant forms (R1a, R1b, and R1C). Recently, anadditional form, CRH-R1D, has been isolated, which is iden-tical to CRH-R1a except that it contains an exon deletionresulting in loss of 14 amino acids in the seventh transmem-brane domain (456). CRH-R2 exists in at least three splicevariant forms (R2a, R2b, and R2g). CRH-R1 predominates inhuman myometrium (455, 457). CRH-R2 is expressed in fetalmembranes, but at lower levels than CRH-R1. Parentheti-cally, this pattern is reversed in rats in which CRH-R2 pre-dominates in myometrium (Y. Stevens and J. R. G. Challis,unpublished observations). CRH-R2 has higher specificityfor urocortin than CRH, raising the possibility that in rodentgestation, placental output of urocortin rather than CRH,may determine activity of this pathway.

Because CRH-R1 is linked to the adenylate cyclase systemthrough GSa regulatory proteins, it is not surprising that CRHstimulates cAMP output by human myometrial cells main-tained in vitro (61). Herein lies the paradox. CRH-inducedincreases in cAMP should inhibit myometrial activity,through mechanisms described above, yet elevations in ma-ternal peripheral plasma CRH concentration are suggested topredict women at risk of increased uterine activity and pre-term labor (61). This may be resolved if CRH action onmyometrium is independent of effects on PG synthesis inother tissues (458). Affinity of CRH binding in myometriumincreases with pregnancy, and then decreases in late gesta-tion (459). Hence, we (8) and others (61) have speculated thatduring gestation CRH acts as a myometrial relaxant, ratherthan as a uterotonin. At term, OT up-regulates protein kinase

FIG. 10. Diagram to indicate interrela-tionships between mother, placenta,and fetus concerned with up-regulationof placental CRH output in human ges-tation in response to stress. It is pro-posed that cortisol from either maternalor fetal adrenal can up-regulate placen-tal CRH expression. Placental CRH, inturn, affects fetal adrenal function in-directly through stimulation of fetal pi-tuitary ACTH release, and directly bystimulating secretion of DHAS from thefetal zone of the fetal adrenal gland.


C, which phosphorylates CRH receptor protein resulting inits desensitization and loss of inhibitory influence (61, 460).Stevens et al. (265) showed that levels of mRNA for CRH-R1heptohelical glycoprotein increase in lower segment myo-metrium from patients in labor whether at term or preterm.Hence, CRH may contribute to regionalization of uterineactivity responses at this time, producing inhibition of ac-tivity, or relaxation in the lower segment, but stimulation ofactivity through up-regulation of PG synthesis in the fundalregion of the uterus.

In our view, the putative role of CRH in pregnancy main-tenance and parturition remains unclear. The concept of pla-cental CRH as “a placental clock controlling the length ofhuman pregnancy” implied a stimulatory effect on the myo-metrium (461, 462), which is difficult to reconcile with theknown biochemical effects of CRH (61). Certainly, CRH aug-ments OT- and PGF2a-induced contractility of myometrialstrips in vitro (289, 463). However, it decreases output of PGI2by myometrial cells and has no direct stimulatory action onits own. Perhaps increased levels of CRH are required tosustain relaxation, rather than stimulation, of the uterusthrough late gestation. However, lowered concentrations ofCRH in maternal plasma are associated with postterm de-livery in which, presumably, relative myometrial quiescencehas been maintained. Resolution of this interesting dilemmain which a single ligand may have different actions depend-ing upon differential expression of its receptor subtypes andcoupling through second messenger systems is required asa scientific basis to understanding CRH action in pregnancy(61).

VI. Application to Clinical Preterm Labor

Rates of preterm labor in North America have remainedrelatively unchanged over the last 30–40 yr, despite sub-stantial advances in our understanding of this process (1–3).It is apparent, however, that new knowledge has not yet beenextrapolated to clinical diagnosis and management (464,465), and that there may be reluctance to develop new drugsfor administration to women in pregnancy without guaran-tees of safety for mother and fetus. There is a clear need torecognize first those preterm labors in which prevention isundesirable because it constitutes a greater compromise tofetal health. There is a need to develop diagnostic indicators,likely specific for particular windows of gestation, to deter-mine the patient in whom the diagnosis of preterm labor iscorrect. Ideally, only these patients should be subjected totocolytics and to prenatal glucocorticoids. There is a need todevelop effective methods of tocolysis ideally related on apatient-specific basis to the cause of preterm labor in thatindividual. Hence, diagnosis of preterm labor should en-compass a multiple-test approach. The new generation ofspecific PGHS-2 inhibitors offers great promise, since in-creased expression of PGHS-2 appears to represent a com-mon final pathway of birth and preterm labor mechanismsamong species (466). The ability to regulate CRH or PGeffects through specific and appropriate agonists and/or an-tagonists is a potential alternative approach.

Both these approaches, however, act on agents of phase 2

parturition, in which uterine activation has already takenplace. Inhibition of uterotonin action or secretion does notnecessarily affect myometrial activation, although recentstudies 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 manage-ment should address uterine activation. Those studies willrequire careful animal studies before the introduction of newdrugs into clinical practice. A satisfactory outcome may be todelay rather than actually to prevent preterm birth, provid-ing that there is improvement in mortality and morbidity ofthe newborn.

We remain concerned about the capricious use of glu-cocorticoids in preterm labor patients (467). There is no ques-tion of the beneficial effect of these compounds in promotingpulmonary maturation in infants of women who give birthprematurely within an appropriate time for treatment. How-ever, a central thesis of this review is that glucocorticoidsprovide a stimulus to the labor process and that evidence isaccumulating to suggest that the model derived from animalexperiments may have substantial applicability to the hu-man. We recognize from animal studies that repeated ad-ministration of glucocorticoids to pregnant animals pro-duces, in a dose-dependent fashion, inhibition of fetalgrowth (468). Prenatal corticosteroids alter postnatal HPAfunction and the setting of negative feedback. Prenatal cor-ticosteroids, in animals, may result in the development ofhypertension postnatally, and in a pattern of pancreatic re-sponse to a glucose load that resembles insulin resistance(469). Prenatal and postnatal administration of corticoste-roids affect levels of type 1 and type 2 GRs in critical brainregions, particularly the hippocampus, associated withmemory and, in later life, with memory loss and neurode-generative disease. Future research into the control of pre-term labor, and to the tocolytic management of the patient atrisk of preterm labor, will need to define the relative risks andbenefits of different management paradigms that may beproposed (469).

Acknowledgments

We are indebted to Jenny Katsoulakos, Linda Vranic, and Fal Patel fortheir help in the preparation of this manuscript and to Maggie Haworthfor 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 adrenalhyperplasia due to 21-hydroxylase deficiency” (Endocrine Reviews 2000, 21:245–291) contained errors thathave 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 activitiescatalyzing each bioconversion are written in boxes. For those activities mediated by specific cytochromes P450, the systematic name of theenzyme (“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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