Steroid hormone control of myometrial contractility and parturition

Seminars in Cell & Developmental Biology 18 (2007) 321–331

Invited article

Steroid hormone control of myometrial contractility and parturition Sam Mesiano a,b,∗ , Toni N. Welsh a,b a

Department of Reproductive Biology, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106-5034, United States b Department of Ob/Gyn, University Hospitals Case Medical Center, 11100 Euclid Avenue, Cleveland, OH 44106-5034, United States Available online 18 May 2007

Abstract The precise temporal control of uterine contractility is essential for the success of pregnancy. For most of pregnancy, progesterone acting through genomic and non-genomic mechanisms promotes myometrial relaxation. At parturition the relaxatory actions of progesterone are nullified and the combined stimulatory actions of estrogens and other factors such as myometrial distention and immune/inflammatory cytokines, transform the myometrium to a highly contractile and excitable state leading to labor and delivery. This review addresses current understanding of how progesterone and estrogens affect the contractility of the pregnancy myometrium and how their actions are coordinated and controlled as part of the parturition cascade. © 2007 Elsevier Ltd. All rights reserved. Keywords: Progesterone; Estrogen; Myometrial contractility; Parturition

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The relaxatory actions of progesterone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Genomic actions of progesterone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Non-genomic actions of progesterone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transformation to a contractile phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Progesterone withdrawal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Estrogen activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Genomics of myometrial transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Progesterone and estrogens are key regulators of myometrial growth and contractility. Progesterone is a “pro-gestational” agent; it sustains the pregnant state and promotes myometrial relaxation. In contrast, estrogens (mainly estradiol) oppose the relaxatory actions of progesterone and augment myometrial contractility and excitability. The balance between the relaxatory actions of progesterone and the stimulatory actions of estrogens

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Corresponding author. Tel.: +1 216 844 1553; fax: +1 216 844 7095. E-mail address: [email protected] (S. Mesiano).

1084-9521/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2007.05.003

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is pivotal in determining the contractile state of the pregnancy myometrium and the timing and process of parturition. In the 1950s Arpad Csapo proposed the “progesterone block” hypothesis, which posits that progesterone promotes myometrial relaxation by blocking the development of a contractile phenotype and that parturition is initiated by its withdrawal [1]. Indeed, in all viviparous species studied so far, treatments that inhibit progesterone synthesis or actions induce labor, and in most species natural parturition is preceded by a fall in circulating progesterone levels (i.e., a systemic progesterone withdrawal). Parturition is also associated with increased estrogenic activity (i.e., estrogen activation), which in most animals is mediated by increased circulating estrogen levels. The combined effects of

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progesterone withdrawal and estrogen activation initiate parturition by transforming the myometrium to a highly contractile and excitable state. This review focuses on recent progress in understanding of how progesterone maintains myometrial relaxation for most of pregnancy and how estrogens promote a contractile state. We also examine recent data regarding the mechanism for progesterone withdrawal and estrogen activation in human parturition and how these key events are mediated, coordinated and controlled. 2. The relaxatory actions of progesterone Progesterone affects myometrial contractility through genomic and non-genomic pathways. Genomic pathways function by altering the expression of specific contraction associated genes to modulate the long-term contractile phenotype. Nongenomic pathways in contrast are more rapid and directly affect the contractile machinery by modulating intracellular signal transduction pathways. 2.1. Genomic actions of progesterone Genomic actions of progesterone are mediated by the classic nuclear progesterone receptors (nPRs) that function as ligandactivated transcription factors. The human nPR gene encodes two major products, the full-length PR-B and the truncated (by 164 N-terminal amino acids) PR-A, under the control of two separate promoters [2–5]. Other proteins generated by exon deletions and intronic insertions have also been reported (see [6] for review); however, their physiological roles are uncertain. Multiple in vitro studies suggest that PR-B is the principal mediator of genomic progesterone actions. PR-A also modulates the transcription of some genes; however, it mainly acts to repress the transcriptional activity of PR-B [7–9]. The extent to which PR-A decreases PR-B activity depends on its amount relative to PRB. Thus, genomic progesterone responsiveness is determined by the dual and opposing actions of PR-A and PR-B, and is inversely related to the PR-A/PR-B ratio. The induction of labor and delivery by treatment with nPR antagonists such as RU486, reflects the importance of nPR-mediated progesterone actions for the maintenance of myometrial relaxation during pregnancy. The principal genomic mechanism by which progesterone represses myometrial contractility is by modulating the expression of genes encoding contraction-associated proteins (CAPs). Some important CAPs include the oxytocin receptor (OTXR) and the prostaglandin (PG)-F2␣ (PGF2␣ ) receptor (FP), the gap-junction protein connexin-43 (Cx43) and the PG-metabolizing enzyme 15hydroxy-PG-dehydrogenase (PGDH). Progesterone decreases myometrial OT and PGF2␣ responsiveness by inhibiting OTXR and FP expression, respectively [10–14]. In pregnant rats, removal of endogenous progesterone by ovariectomy or inhibition of progesterone action with RU486 treatment increases myometrial OTXR expression and OT responsiveness. Progesterone decreases myometrial OTXR levels indirectly by inhibiting estrogen-induced OTXR expression [15,16]. Interestingly, progesterone also inhibits stretch-induced myometrial

OTXR expression in rats [17,18], suggesting that it represses multiple pathways that induce OTXR expression. Administration of RU486 [19] or epostane [20,21] (a progesterone synthesis inhibitor) to pregnant women at early and midgestation increases the effectiveness of PG treatment to induce labor, indicating that progesterone represses PG responsiveness. Importantly, progesterone also increases the inactivation of PGs by increasing expression of PGDH in the myometrium and chorion [22–25]. Progesterone also decreases the development of coordinated uterine contractions by inhibiting expression of Cx43, a major component of myometrial gap-junctions that serve to synchronize contractions over the entire uterus. Expression of Cx43 in the human pregnancy myometrium increases with the onset of labor [26], and in human myometrial cell cultures its expression is up-regulated by estrogen and inhibited by progesterone [27,28]. Studies in rats showed that progesterone not only decreases expression of Cx43 but also its translocation through the Golgi and its assembly into functional gap-junctions at the plasma membrane [29]. Thus, progesterone decreases contractile capacity by inhibiting Cx43 expression and gap-junction formation. Progesterone also augments activity of the cAMP/protein kinase-A (PK-A) signaling cascade in myometrial cells. The cAMP/PK-A pathway promotes smooth muscle relaxation in part by PK-A-mediated inhibition of the phospholipase C (PLC)/Ca2+ pathway (for review see [30]; see also Lopez Bernal, this issue; Sanborn, this issue). Activity of PK-A is modulated by its association with A-kinase anchoring proteins (AKAPs) that localize PK-A to specific intracellular compartments and facilitate its capacity to phosphorylate specific targets, especially PLC [31–33]. In rats, labor is preceded by a decrease in PK-A association with the AKAP complex [34], and progesterone treatment prevents the parturition-related PK-A/AKAP decline [35]. Thus, progesterone, through its effects on the PK-A/AKAP interaction, augments PK-A-mediated inactivation of PLC. This opposes the capacity for stimulatory uterotonins such as OT and PGF2␣ to increase intracellular Ca2+ levels, via the PLC/Ca2+ pathway. That this action of progesterone was inhibited by RU486 suggests that it is nPR-mediated [35]. Thus, progesterone via its interactions with nPRs, maintains myometrial relaxation by: (1) directly inhibiting CAP expression; (2) decreasing estrogen-induced CAP expression, and (3) increasing the effectiveness of PK-A to inhibit PLC activity. In vitro studies suggest that these actions are most likely mediated by PR-B in the human pregnancy myometrium [36–38]. However, in PR-B-knockout mice, PR-A alone is sufficient to mediate the pro-gestational actions of progesterone [39,40], suggesting that species diversity exists in the role of the two nPRs in pregnancy and parturition. 2.2. Non-genomic actions of progesterone Non-genomic actions of progesterone are characterized by: (1) a rapid time-course for response with a latency of minutes, rather than hours; (2) no requirement for nPR activity or occupancy; (3) no requirement for RNA or protein synthe-

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sis; and (4) occurrence in response to conjugated progesterone (e.g., progesterone-BSA) that cannot enter the target cell. These effects are potentially mediated by the interaction of progesterone with specific mPRs that are coupled to intracellular signaling pathways; activation of Src/MAPK cascade intracellular signaling pathways by ligand activated nPRs, and/or by progesterone interaction with neurotransmitter and peptide hormone receptors (e.g., GABAA and OTXR). Studies examining the rapid effects of progesterone on isolated myometrial strips from various species generally showed that it inhibits OT-induced contractions and that it uncouples the excitation–contraction process (for review see [41]). However, in vitro studies using human pregnancy myometrium yielded mixed results with some investigators reporting a rapid relaxatory effect of progesterone and progesterone metabolites [42–46] while others reporting that progesterone augments contraction frequency but decreases duration, amplitude and activity area in term myometrial strips [47–49]. The reason for this variability is not readily apparent but could be due to difference in the progestins used and how they were prepared, and the contractile state of the tissue before it was mounted on the myograph. Nonetheless, the studies clearly demonstrated that progesterone has a rapid non-genomic affect on myometrial contractility. Several in vivo studies support the hypothesis that progesterone non-genomically influences myometrial contractility. In one of the first clinical trials of progestin tocolysis, Hendricks et al. [50] found that administration of a large bolus of progesterone into the amniotic fluid of women at term decreased the frequency of spontaneous contractions and attenuated OT responsiveness. The tocolytic effect of intra-amniotic progesterone therapy was rapid and in some women persisted for several days. However, the number of subjects was small and the data were somewhat anecdotal. In the mid-1960s Pinto et al. [51] re-addressed the issue and reported that large amounts of progesterone (100–200 mg bolus iv; this is almost as much as the placenta produces in 24 h at term) administered to women in term labor inhibited the frequency and intensity of uterine contractions within minutes. In one woman, progesterone completely silenced the laboring uterus within 10 minutes of administration. In two other women, progesterone inhibited spontaneous contractions but failed to decrease the intensity of contractions elicited by OT. Those data demonstrated direct non-genomic relaxatory actions of progesterone at high doses; however, study cohorts were small. Importantly, Pinto and colleagues later found the same effects of progesterone on isolated myometrial strips [46]. Two recent clinical studies reported that administration of moderate doses of progesterone (100 mg daily by vaginal suppository) or 17-hydroxyprogesterone caproate (250 mg intramuscular injection in oil weekly) starting at 16 weeks’ gestation to women at high risk for preterm birth reduced the incidence of preterm birth and improved neonatal outcome [52,53]. The mechanism by which progestin supplementation decreased the rate of preterm birth is uncertain. Interestingly, da Fonseca et al. [52] reported that women receiving progesterone via vaginal suppositories and who presented with preterm labor responded more favorably than women in the placebo group

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to tocolytic treatment with ␤-mimetics. It appeared that prolonged exposure of the myometrium to exogenous progestin improved the effectiveness of tocolytic therapy. This was also observed by Chanrachakul et al. [42] who reported that in isolated term myometrial strips, progesterone, albeit at high doses, decreased contractility and augmented the capacity for ritodrine, a ␤-mimetic commonly used for tocolytic therapy, to block OT-induced contractions. They concluded that the mechanism for this action is through a non-genomic pathway, as it occurred soon after progesterone exposure. However, using a similar approach, Sexton et al. [54] reported that 17hydroxyprogesterone caproate had no effect on spontaneous or OT-induced contractions in myometrial strips. They suggested that 17-hydroxyprogesterone-caproate affects the rate of preterm birth via long-term genomic affects rather than by direct non-genomic mechanisms. Clearly, further studies are needed to determine the mechanism by which progestin treatment decreases the incidence of preterm birth and in particular whether this occurs through genomic or non-genomic pathways. Several groups have reported that progesterone interacts with the OTXR and that this interaction decreases contractility by inhibiting OT-induced inositol triphosphate production and Ca2+ mobilization [55–57]. However, this effect was species-specific; Grazzini et al. [57] reported that progesterone interacted with the rodent but not with the human OTXR. The human OTXR instead interacted with 5␤-dihydroprogesterone, a 5␤ reduced progesterone metabolite. This observation is consistent with myograph studies showing that 5␤-dihydroprogesterone, but not progesterone or 5␣-reduced progesterone metabolites, is a potent myometrial relaxant that decreases basal and OT-induced contractile activity [45,58–60]. Thus, progesterone could relax the myometrium in an intracrine manner through its conversion to 5␤-dihydroprogesterone. Sheehan et al. [61] found that in human pregnancy, circulating levels of 5␤-dihydroprogesterone and expression of the 5␤-reductase enzyme in the placenta and myometrium decrease in association with the onset of labor. Mitchell et al. [62] also found that the human pregnancy myometrium expresses the 5␤-reductase enzyme and has the capacity to generate 5␤-dihydroprogesterone. However, whether 5␤-dihydroprogesterone interacts with the human OTXR is controversial, as others [63,64] could not confirm the outcome reported by Grazzini et al. [57] and instead reported that the human OTXR does not bind 5␤-dihydroprogesterone. This suggests that 5␤-reduced metabolites of progesterone relax the myometrium by interacting with other receptors. One possibility is that the gamma butyric acid-A (GABAA ) receptor is involved. 5␤-Dihydroprogesterone binds to the GABAA receptor and this interaction is in part responsible for the anesthetic effect of these steroids [65]. Putnam et al. [66] found that in the rat myometrium, a GABAA -specific antagonist blocked inhibition of contractility by progesterone and its 5␤-reduced metabolites suggesting that the relaxatory actions of those steroids were mediated by the GABAA receptor. Importantly, GABAA receptors have been identified in the human uterus [67]. Thus, progesterone metabolites, especially ␤-reduced forms, may nongenomically relax the myometrium by interacting directly with the OTXR or with the GABAA receptor.

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Non-genomic actions of progesterone can also be mediated by nPRs. PR-A and PR-B have proline-rich motifs in their Ntermini that, upon ligand binding, interact with the Src tyrosine kinase at the plasma membrane to activate the Ras/Raf-1/MAPK pathway [68,69]. Src is a key intermediate that couples hormone signals at the plasma membrane with intracellular transduction pathways involved in regulating a variety of cellular processes including proliferation, differentiation, adhesion, migration, and apoptosis [68]. This may be an important mechanism by which progesterone exerts tropic action on the pregnancy myometrium. Whether this pathway affects contractility is uncertain. Putnam et al. [66] found that RU486 reversed the rapid inhibition of spontaneous contractility by progesterone in myometrial strips from non-pregnant rats, suggesting that non-genomic relaxatory actions of progesterone involve nPRs. A number of unique membrane associated PRs (mPRs) have recently been identified and characterized. Progesterone receptor membrane component-1 and -2 (PGRMC1 and PGRMC2) have single transmembrane spanning domains and interact with plasminogen activator inhibitor of RNA binding-1 (PAIRBP1), which recruits the mPRs to form a multimeric progesteronebinding complex on the plasma membrane [70–76]. In granulosa cells, binding of progesterone to the PGRMC–PAIRBP1 complex activates protein kinase G and decreases intracellular Ca2+ levels [77]. The role of these mPRs in the onset/process of labor and whether the human pregnancy myometrium expresses PAIRBP1 is not known at this stage. mPR␣, mPR␤ and mPR␥ are structurally related to G-protein coupled receptors, having a typical seven-transmembrane domain structure [78,79]. mPR␣ and mPR␤ are coupled to inhibitory G-proteins and therefore, upon ligand binding decrease intracellular cAMP levels [80]. Several studies have shown that mPR␣ and -␤ are expressed in the human pregnancy myometrium, whereas mPR␥ expression is barely detectable [80–82]. Karteris et al. [80] found that ligand activation of mPR␣ and mPR␤ in primary cultures of human term myometrial cells decreased cAMP levels and increased phosphorylation of myosin. They proposed that these effects (decreased cAMP and activation of myosin) augment contractility. However, they also found that mPR␣ and mPR␤ increased the transcriptional activity of ligand-activated PR-B, which would be expected to decrease contractility via the genomic pathway. To reconcile those opposing activities, they proposed that for most of pregnancy the genomic actions of PR-B dominate to relax the myometrium, and that mPR␣ and mPR␤ augment this pathway by augmenting PR-B activity. At parturition, functional progesterone withdrawal (see below) negates PR-B actions, allowing non-genomic actions mediated by mPR␣ and -␤ to prevail, and therefore increase contractility by decreasing cAMP and increasing myosin phosphorylation. This model proposes that for most of pregnancy non-genomic and genomic actions of progesterone conspire to relax the myometrium, whereas nongenomic pathways mediated by mPR␣ and mPR␤ prevail after functional progesterone withdrawal and promote contraction. This may explain reports of progesterone increasing contractility of isolated strips of term human myometrium [49,83]. This would occur if the tissue was procured after genomic proges-

terone withdrawal. The idea of functional cross-talk between the genomic and non-genomic pathways and the notion that progesterone switches from promoting relaxation to enhancing contraction are novel concepts that warrant further investigation. However, Krietsch et al. [84] have recently suggested that mPR␣, -␤ and -␥ are not activated by progesterone and do not localize to the plasma membrane. Clearly, further studies are needed to confirm the model proposed by Karteris et al. and determine the roles of mPR␣ and -␤ in the pregnancy myometrium. 3. Transformation to a contractile phenotype Transformation of the myometrium from a relaxed to a highly contractile state is an early and key event in the parturition process. The biochemical and physical changes include: increased Cx43 expression leading to increased coupling between myocytes so that contractions are synchronized across the whole uterus; increased sensitivity and contractile responsiveness to stimulatory uterotonins such as OT and PGF2␣ due respectively to increased OTXR and FP expression; increased production of PGs by the gestational tissues and decreased inactivation of PGs in the myometrium; lowered threshold for myocyte excitability; and decreased capacity for the cAMP/PK-A signaling pathway to maintain relaxation. These events are controlled primarily by the combined effects of progesterone withdrawal and estrogen activation. 3.1. Progesterone withdrawal In most animals the onset of labor is preceded by a fall in circulating maternal progesterone levels (i.e., a systemic progesterone withdrawal). In some species this is due to decreased placental progesterone secretion (e.g., sheep), while in others (e.g., rabbit, mouse, rat) it is caused by regression of the corpus luteum (CL) [85–88]. The mechanism by which progesterone withdrawal increases myometrial contractility is not clearly understood. As mentioned above, studies with the progesterone antagonist RU486 demonstrate that inhibition of nPR-mediated progesterone action induces the full parturition cascade. This suggests that withdrawal of genomic progesterone actions is a key parturition-triggering event. Whether withdrawal of nongenomic progesterone actions, including those mediated by nPRs, is also required for myometrial transformation is uncertain. The mechanism for progesterone withdrawal in human parturition is uncertain. Unlike most other species, labor and delivery in humans occur without a decrease in maternal, fetal and amniotic fluid progesterone levels [89–91], suggesting that progesterone withdrawal is not necessary. However, as RU486 treatment induces labor at all stages of human pregnancy, it is generally considered that human parturition involves a form of progesterone withdrawal that does not depend on a decrease in circulating progesterone levels. Proposed mechanisms include: (1) sequestration of free active progesterone by a circulating progesterone binding protein; (2) intracrine inactivation of local progesterone bioactivity by myometrial cells; (3) production of an endogenous progesterone antagonist; and (4) decreased

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myometrial progesterone responsiveness (i.e., a functional progesterone withdrawal) mediated by changes in the levels of specific nPRs or nPR co-activator/co-repressors (for review see [92,93]). Indirect evidence for each of these mechanisms has been reported but their definitive roles in human parturition remain uncertain. Several research groups, including ours, have tested the hypothesis that functional progesterone withdrawal is mediated by specific changes in myometrial nPR expression. Studies of nPR protein [37,38,94] and mRNA [95] levels in myometrial biopsies indicated that human parturition involves an increase in the myometrial PR-A/PR-B ratio due to increased expression of PR-A. Functional studies in myometrial cells showed that an increase in the PR-A/PR-B ratio decreased genomic progesterone responsiveness mediated by PR-B [37,38]. We found that PR-A and PR-B were exclusively expressed in myocytes in the human pregnancy uterus and that the PR-A/PR-B protein ratio was 0.5 (a PR-B-dominant state) at around 30 weeks, increased to 1.0 (equal amounts of PR-A and PR-B) at term before labor onset and increased further to around 3 (a PR-A-dominant state) in laboring myometrium [37]. The pregnancy stage- and laborassociated increase in the PR-A/PR-B ratio was almost identical to data reported by Haluska et al. [94] in the rhesus monkey (Fig. 1), a species that also lacks a systemic progesterone withdrawal at parturition. Another truncated nPR, known as PR-C, that also represses PR-B activity, has been found to increase in fundal myometrium in association with labor at term [36]. Thus, studies so far suggest that functional progesterone withdrawal in human parturition is mediated by an increase in the myometrial PR-A (or PR-C)/PR-B ratio and that regionalization exists in the uterus such that progesterone responsiveness is differentially regulated in fundal (by PR-C) and lower segment (by PR-A) myometrium. Functional progesterone withdrawal could also be mediated by the inhibition of nPR interaction with target DNA. nPR binding to nuclear extracts of term human decidua is reduced in laboring compared with non-laboring tissue indicating that the onset of labor involves changes in the nPR

Fig. 1. Comparison of the PR-A/PR-B protein ratio in human and rhesus monkey pregnancy myometrium. *P < 0.001 (Adapted from Merlino et al. [37] and Haluska et al. [94]).

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transcriptional complex [96]. In the myometrium, labor is associated with a decline in specific nPR co-activators, particularly cAMP-response element-binding protein-binding protein and steroid receptor coactivators-2 and -3 [97]. The reduction in co-activators may decrease histone acetylation that effectively closes chromatin around the progesterone response element, making it inaccessible to the nPR transcriptional complex. Such a scenario would explain the decrease in nPR binding to nuclear response elements in decidual cells [96]. As a variation on this theme, Dong et al. [98] identified a protein known as polypirimidine tract-binding protein-associated splicing factor (PTB) that specifically inhibits nPR transactivation and whose expression in rat myometrium increases at term. They proposed that this factor contributes to functional progesterone withdrawal by acting as an additional nPR co-repressor. Interestingly, PTB also controls the splicing of myosin phosphatase targeting protein mRNAs, and therefore the functional activity of myosin phosphatase, a key determinant of smooth muscle contractility [99]. These data demonstrate the complexity that underlies the genomic actions of progesterone on myometrial contractility and the multiple levels at which functional progesterone withdrawal could occur. 3.2. Estrogen activation In 1967, Pinto et al. [46] examined the role of estrogens in human parturition by administrating a large amount of 17␤-estradiol (200 mg intravenously in 1 h) to non-laboring pregnant women at term. They found that estradiol treatment increased uterine contractility and OT responsiveness within 4–6 h and accelerated the time to delivery. Those findings were consistent with the stimulatory actions of estrogens on myometrial contractility and showed that the progesterone block is not absolute and can be overcome by estrogenic drive. Studies in rats and sheep also demonstrated that treatment with estradiol at mid-gestation induces preterm labor [100–102]. Interestingly, Pinto et al. [46] also found that administration of progesterone rapidly (within 10 minutes) blocked the stimulatory actions induced by 17␤-estradiol, supporting the hypothesis that progesterone acts non-genomically to promote relaxation. In the rhesus monkey, Nathanielsz et al. found that augmenting placental estrogen production (by the administration of androstenedione, which is readily converted to estrogens by the placenta) increases myometrial contractility and OT responsiveness and induces preterm birth [103,104]. Inhibition of aromatase activity eliminated the induction of parturition by androstenedione, confirming that its conversion to estrogens was essential for the initiation of parturition [105]. However, others found that estradiol treatment alone had no effect on parturition in the rhesus monkey even though circulating estradiol levels were markedly elevated [106]. Those inconsistent outcomes suggest that local production of estrogens from androgen precursor is more important than circulating estrogen levels. Further studies are needed to resolve this controversy. Nevertheless, data so far demonstrate the critical role of estrogenic drive (i.e., estrogen activation) for myometrial transformation to a contractile state. In most species estrogen activation is mediated by an increase in circulating estrogen levels and is coordinated with systemic

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progesterone withdrawal [85]. However, in humans (and higher primates) circulating estrogens increase at around mid-gestation and continue to rise gradually until birth [89–91]. This has led to the concept that estrogen activation in human parturition, as with progesterone withdrawal, is mediated at the functional level, by an increase in myometrial estrogen responsiveness. Thus, for most of pregnancy the human myometrium appears to be refractory to estrogens at least in terms of CAP gene expression, and parturition involves functional estrogen activation whereby the myometrium becomes estrogen-responsive. Refractoriness of the myometrium to estrogens for most of pregnancy is likely due to very low levels of ER␣ and ER␤. Importantly, we found that ER␣ expression is low in nonlaboring term myometrium and increases in association with labor onset, suggesting that functional estrogen activation is mediated by increased ER␣ expression [95]. We also found that ER␣ mRNA levels correlate with the PR-A/PR-B mRNA ratio [95] indicating a functional link between the nPR and ER␣ systems. This association is consistent with studies in a variety of species showing that progesterone decreases uterine estrogen responsiveness by decreasing ER␣ expression [107–110]. In the pregnant rhesus monkey, treatment with RU486 at mid-gestation increased myometrial ER␣ expression indicating that progesterone decreases ER␣ expression through an nPR-mediated process [111]. Taken together the current data suggest that progesterone via its interaction with PR-B inhibits myometrial ER␣

expression and causes the myometrium to be refractory to circulating estrogens. The increase in myometrial PR-A expression with advancing gestation decreases PR-B transcriptional activity and eventually eliminates the PR-B-mediated inhibition of ER␣ expression. According to this model (Fig. 2) functional progesterone withdrawal, mediated by increased PR-A, induces functional estrogen activation mediated by increased expression of ER␣ and therefore coordinates these critical parturitiontriggering events. Circulating estrogens can then transform the myometrium to a contractile state. This paradigm implies that a fundamental mechanism by which progesterone maintains myometrial relaxation in human pregnancy is by blocking the stimulatory actions of estrogens and that functional progesterone withdrawal induces functional estrogen activation. This physiologic interaction explains why inhibition of nPR-mediated progesterone actions triggers the full parturition cascade, especially as estrogens are readily available to act on the myometrium for most of human pregnancy. It also implies that human parturition is triggered by any event (e.g., local PGs, myometrial stretch, inflammation) that increases myometrial PR-A expression and induces functional progesterone withdrawal. Thus, multiple parturition trigger pathways may converge on myometrial PR-A expression. A more detailed understanding at the molecular level of how the myometrial progesterone-nPR and estrogen-ER␣ signaling pathways interact and are regulated in human pregnancy may reveal novel targets to therapeuti-

Fig. 2. Schematic model of the genomic and non-genomic pathways by which steroid hormones affect contractility of the human pregnancy myometrium. For most of pregnancy progesterone, via its interaction with PR-B in myometrial cells, inhibits expression of CAP genes and decreases responsiveness to estrogens by inhibiting ER␣ expression. Progesterone and/or its metabolite 5␤-dihydroprogesterone also exert non-genomic effects on the pregnancy myometrium by interacting with a variety of mPRs. The effects of non-genomic progesterone actions on myometrial contractility are not well understood but it is generally considered that progesterone decreases contractility via this pathway. At parturition PR-A expression increases until the PR-A/PR-B ratio reaches a point whereby the relaxatory actions mediated through PR-B are repressed, i.e., functional progesterone withdrawal. As a consequence ER␣ expression increases and the myometrium becomes more responsive to circulating estrogens which increase CAP expression and transform the myometrium to a highly contractile and excitable phenotype leading to the onset of labor.

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cally control human labor and address the problem of preterm birth. 3.3. Genomics of myometrial transformation Determination of the human genome sequence has led to the development of novel and cutting-edge technologies to characterize the relationship between global gene transcriptional status (i.e., the transcriptome) and phenotype in cells and tissues specimens. Techniques such as suppression subtractive hybridization [112] and multidimensional cDNA and oligonucleotide array platforms [113–117] have been used to determine changes in the myometrial transcriptome, encompassing thousands of genes, that may account for its contractile transformation at parturition (for review see [118]). The fundamental hypothesis tested by these powerful techniques is that transformation of the pregnancy myometrium from quiescence to contraction involves changes in specific gene expression in myometrial cells. Although many of these genes have been identified using more conventional assay techniques, the global approach incorporates the entire transcriptome and therefore provides the opportunity to reveal the complete spectrum of genes involved. This approach generates novel hypotheses, which can then be tested using regular approaches. Studies so far have used a variety of platforms to compare the transcriptome in myometrial biopsy specimens obtained before and after the onset of labor at term [113–116] and from the fundal and lower uterine segments [117]. Most analyses identified multiple genes whose expression changed (increased or decreased by at least twofold) in association with the onset of labor, however, there was little overlap between the cohorts of genes identified in different studies. There was also marked variation between some studies. Notably, Havelock et al. [117] found little difference in gene expression profiles between fundal and lower uterine segment and in association with labor onset, whereas Charpigny et al. [115] found that labor was associated with a down-regulation of a large number of developmental-, cell adhesion- and proliferation-related genes and a concomitant increase in inflammatory- and contraction-associated genes. The differences between study outcomes likely stem from variability due to methodological factors related to the array platform, the number of samples studied in each of the experimental groups and the purity of the biopsy samples with respect to myometrial cell content (biopsy specimens contain multiple and varied cell types that contribute to total RNA content of the extract). These problems will likely be overcome as the array technology improves and price of microarrays decreases, allowing more replicates of laboring and non-laboring specimens to be performed. Thus, at this stage the body of data regarding the relationship between the myometrial transcriptome and contractile phenotype is inconclusive and no specific labor-associated genomic pathways have been identified. Interpretation of outcomes from array-based studies largely depends on the existing knowledge-base of specific gene function and the power of the computational and statistical techniques to analyze and align gene expression data, which often comprises large numbers of genes with unknown function, into

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functional networks. Advances in clustering and hierarchical techniques have allowed for the organization of gene expression data into functional networks; however, many of the genes identified as altered by labor onset have unknown functions and their relationship to phenotype is ambiguous at best. Clearly, this will be less of a problem as understanding of individual genes and gene networks advances. The use of multidimensional statistical analyses, such as directed graphs and principal component analysis, have been applied to determine how theoretical causal pathways generated by microarray data sets interact to impact on phenotype [119,120]. This approach utilizes probabilistic and statistical analyses of the expression data to develop hierarchical causal pathways for a particular phenotype or condition. For example, our analysis of qRT-PCR-based data-sets using directed graphs suggested a causal relationship between the activation of inflammatory pathways and functional progesterone withdrawal [119] whereby inflammation precedes and possibly causes functional progesterone withdrawal. That conclusion is consistent with our earlier study using the PHM1-31 immortalized human pregnancy myometrial cell line in which we found that PGF2␣ preferentially increases PR-A expression [121]. Thus, locally produced PGs may initiate myometrial transformation and the onset of labor by first inducing functional progesterone withdrawal via increased myometrial PR-A expression. This approach exemplifies the advantage of integrating outcomes from specific cause-effect studies (e.g., from animal models, cell lines, tissue specimens and clinical studies), multidimensional cDNA/oligonucleotide array studies and computational analyses of theoretical causal pathways in an iterative and informative paradigm to unravel the physiology of human parturition. 4. Conclusions In an evolutionary context, physiological triggers for parturition would have been subjected to strong selective pressures so that the timing for birth favors species survival by optimizing neonatal outcome and minimizing risks to the mother (and therefore future pregnancies) imposed by the pregnant and parturient states. This perspective helps explain the remarkable diversity in gestation length/birth timing and parturition control across viviparous species. In contrast, the physiologic systems that establish and maintain pregnancy exhibit relatively little diversity. In this case a single hormone, progesterone (and possibly its metabolites) maintains pregnancy and promotes myometrial relaxation through a combination of genomic and non-genomic mechanisms. In fact, the progesterone block appears to be a common trait among viviparous species. As pregnancy advances, stimulatory influences build up to progressively challenge the progesterone block. Although the principal stimulatory drive to myometrial contractility is imparted by estrogens (a trait that also appears to be conserved), other important factors such as uterine stretch, myometrial response to the intrauterine cytokine milieu and the activity of a fetal and/or placenta-based physiologic clock mechanism, are also involved. The combination of estrogenic and other physiologic stimulators for parturition and the level of co-operativity, functional overlap and redun-

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