Myometrial Contractility

The mechanisms involved in the regulation of uterine smooth muscle (myometrium) contractility during human pregnancy and labour are poorly understood. Such information is essential to both an understanding of the physiology of human parturition and the clinical management of human labour and disorders. The major clinical problem associated with human labour is that of preterm labour, which is the leading cause of perinatal mortality and morbidity in the developed world. It is clear that the myometrium is transformed from a state of relative quiescence during pregnancy, to one of maximal contractile activity at the time of labour. It is also established that the state of contractility of uterine smooth muscle is intrinsically linked to cell membrane ion channel activity (Egarter CH, Husslein P. , 1992) . Myometrial contractility is regulated by both intracellular calcium concentration Ca++ and by the calcium sensitivity of myofilaments. An increase in Ca++ results in phosphorylation of myosin light chain (MLC) catalysed by the calcium-calmodulin activated myosin light chain kinase (MLCK). The more recently described process of calcium sensitisation, refers to an increase in smooth muscle tension and/or phosphorylation of MLC at a constant Ca++ by inhibition of myosin light chain phosphatase (MLCP) (Lopez Bernal A, 2003) . Cell membrane ion channels play a major role in regulation of smooth muscle activity by controlling cell membrane potential and in the regulation of intracellular calcium concentration. Potassium (K+) channels are the largest category of ion channels in the cell and hence are of primary importance in the regulation of membrane potential and cell excitability in the myometrium, and thus are functionally important in the regulation of uterine smooth muscle tone (Sanbor et al, 2000) . Since the uterus is endowed with this potent muscular structure (whose development and trophicity depends on the estrogens), this organ is capable not only of modifying its basal tonus, but also of contracting and relaxing through systoles and diastoles that vary greatly in intensity and duration. The variations in the uterine contractile intensity are so wide that they include:

weak and moderate contractions that occur in the periods in which the organ is predominantly relaxed, as during the greatest part of the menstrual cycle and pregnancy; stronger contractions that characterize menstruation and the orgasmic response, as well as those resulting from pathological conditions; the extremely powerful contractions of parturition. The degree of contractility/relaxation of the uterus depends on the excitability level of the smooth muscle cells of the myometrium. The increase in the excitability of the myometrial cells stimulates the uterine contractility, while its reduction induces to a relaxation of the organ. These phenomena depend on an interaction of hormonal, biochemical and neurovegetative factors, the most important of which are the first two ones. By reducing the excitability of the myometrial cells, progesterone is the hormonal factor that promotes uterine relaxation, keeping the uterus quiescent (in "repose")

(Mesiano S, 2004) . Differently, the estrogens have the opposite effect - by increasing the myometrial excitability, they promote a moderate stimulation of the uterine contractility. Due to the estrogenic action, the uterus also becomes highly sensitive to the action of oxytocin - the most important hormone in the causation of uterine contractions. Oxytocin has a powerful contractile action upon the myometrium, being the hormonal factor responsible for the very strong uterine contractions of parturition. Oxytocin production takes place in the hypothalamus and its release is triggered by a reflex neurogenic pathway that starts in nerve endings especially sensitive to pressure located inside the cervical canal and in the nipples. During parturition, it is the strong mechanical stimulation of the endocervix that causes the hypothalamic liberation of a great quantity of oxytocin, giving rise to increasingly potent uterine contractions (Shmygal A et al, 2006) . OT has been demonstrated to be
synthetized and to act within peripheral reproductive organs in both females and males. OT also exerts neuroendocrine effects on the anterior pituitary and is a neuroactive substance within the central nervous system Just before the onset of labour, uterine myometrium becomes extremely sensitive to OT because of the large increase in the number of myometrial OT receptors (OTRs). The amount of myometrial OTRs returns to basal levels shortly after birth. Although OT has a strong pharmaceutical potential, its physiological role in normal labour has long been equivocal. Indeed, circulating OT is not essential for the initiation or maintenance of labour. There is no evidence that a rise of plasmatic OT actually precedes the onset of labour. Administration of OT antiserum does not affect parturition. Moreover, normal parturition can be observed in the absence of plasmatic OT in cases of experimental or clinical pituitary gland dysfunction. OT may act primarly as a local mediator in the uterus at labour time. Endogeneous OT may exert a paracrine control of myometrial contractions both directly by interacting with myometrial OTRs, and indirectly by stimulating the production of Prostaglandine in particular F2a (PGF2a ) by endometrial cells where OTRs have also been identified.

As non-hormonal factors that also cause strong myometrial contractions, we must mention several prostaglandins (Norwitz et al , 1991) . The formation of these prostaglandins in the endometrium during the menstrual necrosis of this tissue generates the increase in the uterine contractility typical of this phase of the cycle, giving rise to the menstrual cramps. Together with several other factors, these substances also play a role in the triggering of parturition labour. Certain prostaglandins are involved with the induction of labour and other reproductive processes. PGE2 causes uterine contractions and has been used to induce labour (Olson DM, 2003) . PGs contribute to the transition from phase 1 to phase 2 rather than initiating
the labor process. Perhaps the best indicator for a role of PG in parturition is the measurement of increased PG output before the appearance of labor-like myometrial contractions and the effectiveness with which drugs that block PG synthesis suppress myometrial contractility and prolong gestation length. The general consensus is that in human pregnancy, expression of PLA2 increases gradually in fetal membranes during gestation but does not increase appreciably at the time of labor. There are some suggestions that these products can weakly stimulate contractility of smooth muscle. It has also been suggested that arachidonic acid metabolism in human fetal membranes during pregnancy is directed preferentially toward lipoxygenase products, but there is a progressive switch toward the more potent PGHS (also cyclooxygenase, COX) activity at term (Loudon JA et al, 2003) . In human pregnancy, the PG synthesizing and metabolizing enzymes are compartmentalized discretely between the amnion and chorion, decidua, and myometrium (Egarter CH, Husslein P, 1992 ) . PGHS activity predominates in amnion, PGE2 is the principal PG formed and there is an increase in PG synthesis and levels of PGHS-2, but not PGHS-1 mRNA at preterm and term labor. Immunohistochemical and in situ hybridization studies have localized the PGHS-2 enzyme and mRNA to the amnion epithelium

the subepithelial cells in the mesenchyme and in the chorion laeve trophoblasts with lower expression found in decidua (Bethin KE et al, 2003) . Decidua has been reported to produce increased amounts of PGs at the time of labor, but this is not a consistent observation . Human decidua is made up of decidualized stromal cells, bone marrow-derived macrophages, and other cell types including trophoblasts that interface with chorion.

In spite of the fact that the uterine contractility is predominantly commanded by the hormonal and biochemical factors mentioned here, there are indications that the sympathetic and parasympathetic innervation of the uterus may also have a considerable influence upon it. Independently of the majority of the uterine contractions being endocrinally and biochemically triggered, the myometrial contractile activity exhibits a very peculiar characteristic that seems to demonstrate the existence of a precise and subtle nervous coordination: it is the "triple descending gradient." This gradient gives the uterine contractility its typical expulsive pattern. The uterine contractile waves originate in "pacemakers" situated around the uterine insertion of the Fallopian tubes (Wray S et al, 2001) , one on the right and the other on the left. This author describes the "triple descending gradient" with the following characteristics: 1. the propagation of the contractile wave along the uterus has a descending direction. That happens because, after starting in one of the "pacemakers," the contractile wave spreads throughout the uterine fundus and propagates downwards; 2. the systolic phase of the contraction lasts more at the uterine fundus and less at the inferior parts of the organ; 3. the contractions are stronger in the upper parts of the uterus than in the lower ones. The lack of steroid changes in the maternal circulation has prompted other hypotheses to explain the transition from relative uterine quiescence during continuing pregnancy to the initiation of uterine contractions at the onset of labour. These include the possibility of electrophysiological changes in myometrial cells. Myometrial smooth muscle contractions are phasic in nature and are driven by action potentials. The electrophysiological basis for myometrial contractions is not well understood, but it is the subject of investigation by several groups. There is precise synchronisation between electrical activity, influx of calcium inside myometrial cells and the development of tension. Voltage-operated calcium channels and calciumactivated potassium channels have been shown to be involved in the regulation of contractility by allowing calcium entry, or by setting the threshold of activation of the cell membrane, respectively. Changes in the functional activity of ion channels are likely to be an important mechanism to increase contractility in labour.

Regulation of Uterine Contractility

The basis for contractility in the uterus, as in other types of smooth muscle, is the interaction between actin and myosin, which is regulated by the enzyme myosin light chain kinase (MLCK). Contraction of the myometrium at term or preterm depends upon conformational changes in the actin and myosin molecules, which allow actin and myosin filaments to slide over each other, ultimately leading to a shortening of the myocyte. The confirmational changes (involving cross-bridge cycling of the myosin head) require ATP, which is generated by myosin after phosphorylation of the 20-kDa light chains of myosin by the enzyme myosin light chain kinase (MLCK). This enzyme is central to signaling pathways that both stimulate and inhibit myometrial contractions.

MLCK is activated through interaction with the calcium binding protein calmodulin (CAM), which in turn requires four Ca2+ ions for its own activation. Binding of calcium-activated CAM to MLCK induces a conformational change in the enzyme, allowing MLCK to phosphorylate the 20-kDa light chains of myosin. MLCK can also undergo phosphorylation by protein kinase A (PKA, cAMP-activated protein kinase), which reduces the affinity of the enzyme for calcium calmodulin (Ca-CAM) and leads to its inactivation. Regulation of MLCK has been reviewed extensively. It is evident that activity of this enzyme is altered by intracellular pathways that regulate levels of calcium and of cAMP and is critical for the development of uterine contractility. Uterotonins generally increase intracellular calcium levels ([Ca2+]i), by increased influx of Ca2+ through receptor-operated channels, or release of calcium from intracellular stores including sarcoplasmic reticulum. Agents that inhibit myometrial activity do so by increasing intracellular levels of cyclic nucleotides cAMP or cGMP, which in turn inhibit release of calcium from intracellular stores or reduce MLCK activity. Binding of agents such as -adrenergic agonists, relaxin and prostacyclin, to myometrial receptors activates adenylate cyclase activity, leading to an increase in cAMP generation, while uterine inhibitors such as nitric oxide (NO) activate guanyl cyclase, increasing cGMP (Lopez Bernal A, 2003) . Inhibition of myometrial activity by -adrenergic agonists, relaxin, and PGI2 is mediated by increases in intracellular cAMP. Binding of the inhibitor to its specific cell membrane receptor causes dissociation of the receptor-linked heterotrimeric GTP-binding protein Gs into -subunits. The -subunit activates adenylate cyclase to initiate cAMP synthesis. cAMP, in turn, activates PKA, which then phosphorylates a series of regulatory proteins. Activated PKA either phosphorylates MLCK to reduce its ability to bind Ca-CAM or phosphorylates a membrane-binding site for Ca2+ that increases calcium binding and reduces free intracellular calcium concentrations ( Price SA, Bernal AL, 2001) . Regulation of myometrial calcium levels has been reviewed extensively. Free resting Ca2+ increases from 150 nM to about 500 nM during contraction through influx of extracellular Ca2+ or by the release of Ca2+ from intracellular binding sites or intracellular organelles . Extracellular Ca2+ enters cells through receptor-operated or voltage-gated channels (Luckas M. J. et al , 1999) . Release of intracellular Ca2+ from sarcoplasmic reticulum is activated through the phosphoinositol (PI) pathway. Binding of a uterotonin to its plasma membrane receptor activates a G protein transducer, coupled to phospholipase C, which frees inositol trisphosphate (IP3) and diacylglycerol . Free IPs, especially IP3, increase cellular calcium from intracellular storage sites. Interestingly, IP3 binding in myometrium was inhibited by calcium, suggesting that this might provide a mechanism for regulating the IP3 response by oscillating [Ca2+]. Diacylglycerol formed during IP3 turnover may stimulate PKC to phosphorylate cellular proteins such as MLCK or be rapidly phosphorylated by diacylglycerol kinase to phosphatidic acid, a naturally occurring Ca2+ ionophore, or lead to release of arachidonic acid by cellular lipases, resulting in production of eicosanoids . (Lopez Bernal A, 2003) Function of the myometrium during labor at term or preterm requires highly developed cell-to-cell coupling, effected through formation of intercellular GAP junctions within adjacent cell membranes . The proteins forming GAP junctions are termed connexins and are classified according to their apparent molecular weights . Connexins are arranged into hexameric hemichannels, which become aligned across adjacent cells to form an interconnecting pore that allows low-resistance electrical or ionic coupling between the cells and provides a pathway for metabolite transfer . Hundreds of individual channels arrange themselves into an organized plaque to form

a GAP junction (Ciray HN et al, 1995) . Regulation of connexins occurs at the level of transcription and translation ; mechanisms also operate to control transport of connexin protein to the cell membrane and to direct assembly into connexons, through apposition, clustering, and formation of functional channels ( Sparey C et al, 1999 ) . This complex process is poorly understood, although it is influenced by steroids and by mechanical stretch. Stimulatory agonists can also enhance uterine contractility through a calcium independent pathway, which involves the activation of small GTPases of the Rho family. Their actions are mediated through Rho kinases, which can phosphorylate myosin phosphatase inhibiting its activity and potentiating the effect of MLCK. This is a mechanism of so called calcium sensitisation. Rho kinase may be able to phosphorylate myosin light chains directly,further stimulating the development of tension.

Initiation of Labour
Labor is the process by which the fetus is expelled from the uterus to the extrauterine environment. Parturition results from a complex interplay of maternal and fetal factors. It requires that the uterus, which has been maintained in a relative state of quiescence during pregnancy, develops coordinated contractility and that the cervix dilates in a manner that allows passage of the fetus through the canal birth. To be successful, parturition requires also that maturation of those fetal organ systems necessary for extrauterine survival has occurred, and that the maternal organism has undergone the changes necessary for lactation in the postpartum period. It is not surprising, therefore, that synchronous maturation of the fetus and stimulus to increased uterine activity should be desirable, and much evidence suggests that it is the fetus itself that triggers both these series of events (Lopez Bernal A, 2003) (Carbillon L et al, 2001) . During pregnancy, myometrial activity is characterized by poorly coordinated contractures, or the Braxton-Hicks contractions of human gestation . In late pregnancy, the uterus undergoes preparedness for the stimuli that lead to contractility and labor . Those stimuli may be local, maternal, mechanical or fetal. The development of coordinated uterine contractions at term results in a myometrium that is excitable, generating high-frequency, high-amplitude contractions. It is spontaneously active and responds to exogenous uterotonins. The transition of the myometrium from a quiescent to an active state has been termed "activation." When this has occurred the myometrium can then undergo "stimulation" in response to endogenous and/or exogenous agonists. The uterus is relatively quiescent during 95% of pregnancy, corresponding to phase 0 of parturition. Activation corresponds to phase 1 and is effected predominantly by mechanical input, and through regulation by uterotrophins such as estrogen. Stimulation corresponds to phase 2, when endogenous uterotonins, including PGs and oxytocin (OT), act on the activated myometrium. Postpartum involution corresponds to phase 3. In this sequence of events, the "initiation" of parturition corresponds to the transition from phase 0 to phase 1, although clearly one could argue that initiation started much earlier in gestation (Gary Cunningham et al, 2005 ) ( Roger W Harms, 2004) .

The physiological mechanism of labour initiation is not fully understood however here is the current text book theory: The 2 dominant hormones during pregnancy is Oestrogen and Progesterone. Progesterone is known to inhibit uterine contractions therefore under normal circumstances until the uses of progesterone have occured, labour cannot be initiated prematurely- however this does occur in premature labours. At the end of gestation there is a sharp rise in Oestrogen which subsequently overides the progesterone. This is due to the placental realease of CRH (corticotropin-releasing hormone). CRH provokes a release of ACTH (adrenocorticotrophic hormones) from the foetus's anterior pituitary gland. Which in turn initiates a release of Cortisol from the foetus's adrenal gland along with DHEA (dehydroepiandrosterone hormone) which is the major andrenal androgen. The placenta converts the DHEA into oestrogen - further increasing the oestrogen. Oestrogen has many functions throughout pregnancy and during parturition, one of thoses functions during the initiation of labour is to increase th number of oxytocin receptions within the uterine muscle fibres, along with increasing the gaps in between these fibres. Oestrogen additionally provokes a placental release of Prostaglandins which in turn releases enzymes to digest cervical collagen softening the cervix. Oxytocin is then released by the maternal posterior pituitary gland (however produced in the hypothalamus) causing uterine contractions. The placenta releases another hormone named Relaxin which increases the flexibility of the pubic symphysis and helps to dilate the uterine cervix. The progress of labour is aided by positive feedback. There are stress receptors throughout the cervix and as the baby is forced out of the vaginal canal, this further initiates a release of oxytocin, causing it to contact the myometrium more forcefully.

Phase 0 of labor Studies in different species have indicated that a variety of different inhibitors may play upon the myometrium during pregnancy. Withdrawal of one or more of these may predict the onset of delivery; precocious withdrawal may predict the onset of premature parturition. Such an inhibitor, PTH-related peptide (PTHRP), is produced in myometrium, and its rate of transcription is increased by progesterone and transforming growth factor (TGF). PTHRP receptor mRNA has also been localized to rat myometrial tissue, suggesting that the protein may act in an autocrine/paracrine fashion through specific receptors to activate the G subunits of G proteins and increase intracellular levels of cAMP . Relaxin also elevates myometrial cAMP and inhibits OT-induced turnover of phosphoinositide (PI) by the action of cAMP-dependent protein kinase. Relaxin exerts a dual role in the inhibition of myometrial contractility and in the regulation of connective tissue changes in the cervix. Thus, the major action of relaxin is one of frequency modulation. Relaxin is expressed in the human fetal membranes, decidua, and placenta, consistent with its exerting paracrine/autocrine effects on intrauterine tissues. Relaxin gene expression is dramatically up-regulated in patients with preterm, premature rupture of membranes (PPROM). Relaxin receptors have been localized to decidua and chorionic trophoblast cells, and the protein acts through these to up-regulate expression of matrix metallo-proteinases (MMP), especially MMP1, MMP3, and MMP9. Similarly, relaxin increases MMP expression in cervical tissue at term. Administration of exogenous relaxin stimulates separation of the pubic

symphysis in those species in which it is a prerequisite for delivery . In vitro studies have shown that relaxin blocks the action of stimulants such as OT, carbachol, and norepinephrine on the myometrium, through mechanisms involving PKA-mediated phosphorylation of PLC-linked G proteins. This in turn inhibits IP3 turnover and the increase in [Ca2+]i . Although the precise role that relaxin plays during pregnancy remains to be determined, it may be particularly useful in maintaining uterine quiescence during the period when progesterone concentrations are falling and estrogen levels are beginning to increase before the onset of labor. In addition, there are reports that relaxin may act centrally to increase circulating plasma OT and vasopressin concentrations by an opioid-independent mechanism. It is now known that OT is produced within human intrauterine, choriodecidual tissues. It remains to be established whether a similar relationship exists between relaxin and OT synthesized within the intrauterine compartment in women. In human term pregnant myometrial strips maintained in vitro, the initial response to PGI2 was contraction, but this was followed by relaxation . It is now recognized that PGI2 acts through specific IP receptor species to increase adenylate cyclase activity and elevate intracellular cAMP. Other agents such as CRH also stimulate output of cAMP from myometrial cells and act synergistically with PGI2 in a paracrine/autocrine fashion. More recently, interest has arisen over the potential role of NO as an endogenous inhibitor of myometrial contractility . Nitroprusside has been shown to decrease force and 20-kDa myosin light chain phosphorylation in human myometrial strips, although the tissue is not as sensitive as vascular smooth muscle. Nitric oxide synthase (NOS) isoforms have been detected using RT-PCR in human fetal membranes and choriodecidua . Levels of mRNAencoding inducible NOS (iNOS) are highest in human myometrium at preterm, not in labor patients, and decrease with a corresponding fall in NOS protein in myometrium collected at term. Several authors have suggested that NO acts in a paracrine manner, potentially in conjunction with progesterone to effect myometrial quiescence during pregnancy, although this position has been disputed. There is a decrease in NOS activity of decidua and myometrium in species such as rat before parturition in a manner that would presumably diminish its inhibitory influence on the uterus. Other inhibitors of uterine activity include calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), and endogenous -adrenergic agonists . These compounds act through increasing intracellular cAMP and/or decreasing intracellular calcium availability. Phase 1 of labor: myometrial activation The switch from myometrial quiescence to myometrial activation is essential to enable the muscle to respond to the stimulation provided by the high levels of uterotonic agonists and to generate the synchronous, high-amplitude, high-frequency contractions of labor. Myometrial activation results from coordinated expression of a cassette of proteins, termed contraction-association proteins, or CAPs . CAPs include ion channels (which determine the resting membrane potential and hence excitability of myocytes), agonist receptors and GAP junctions (permitting cell-to-cell coupling). Overall regulation of myometrial activity is genetically regulated . The onset of labor is dictated by the fetal genome proceeding through either a fetal growth pathway with increases in uterine stretch or fetal endocrine pathway involving activation of the fetal HPA axis. These two arms are not independent because changes in progesterone and estrogen modulate the ability of uterine stretch to increase expressions of genes associated with myometrial activation.

The CX-43 gene contains such an element, suggesting that if wall tension contributes to the regulation of CAP genes in the myometrium, regulation of uterine growth through pregnancy will be important in determining the level of shear stress. During pregnancy uterine growth follows three distinct phases: an initial phase during the first trimester where uterine growth is due to hyperplasia and controlled by endocrine factors, a second phase during the second and third trimester in which growth is closely matched to increased fetal size, and a final phase in which there is a decline in uterine growth in comparison to fetal growth, and hence an increase in uterine wall stretch and tension. They speculate that progesterone is necessary to support stretchinduced hypertrophy of the uterus during midgestation in concert with increasing fetal size. Near term, the fall in progesterone, observed in most animal species (see below), leads to a decline in uterine growth relative to fetal growth and hence increased tension development, which in turn results in increased CAP gene expression and contributes to myometrial activation. Since the decrease in circulating progesterone appears critical for the altered influence of stretch on myometrial CAP gene expression, we shall consider the endocrine pathways that result in progesterone withdrawal. Phase 2 of labor Activation prepares the myometrium to respond optimally to the production of those myometrial stimulants that provoke myometrial contractility during labor. Although many agonists have been described to stimulate myometrial contractions, convincing information is available only for OT and stimulatory PGs ( Roger W Harms, 2004) . Certain prostaglandins are involved with the induction of labour and other reproductive processes. PGE2 causes uterine contractions and has been used to induce labour (Olson DM, 2003) . PGs contribute to the transition from phase 1 to phase 2 rather than initiating the labor process. Perhaps the best indicator for a role of PG in parturition is the measurement of increased PG output before the appearance of labor-like myometrial contractions and the effectiveness with which drugs that block PG synthesis suppress myometrial contractility and prolong gestation length. The general consensus is that in human pregnancy, expression of PLA2 increases gradually in fetal membranes during gestation but does not increase appreciably at the time of labor. There are some suggestions that these products can weakly stimulate contractility of smooth muscle. It has also been suggested that arachidonic acid metabolism in human fetal membranes during pregnancy is directed preferentially toward lipoxygenase products, but there is a progressive switch toward the more potent PGHS (also cyclooxygenase, COX) activity at term (Loudon JA et al, 2003) . In human pregnancy, the PG synthesizing and metabolizing enzymes are compartmentalized discretely between the amnion and chorion, decidua, and myometrium (Egarter CH, Husslein P, 1992 ) . PGHS activity predominates in amnion, PGE2 is the principal PG formed and there is an increase in PG synthesis and levels of PGHS-2, but not PGHS-1 mRNA at preterm and term labor. Immunohistochemical and in situ hybridization studies have localized the PGHS-2 enzyme and mRNA to the amnion epithelium the subepithelial cells in the mesenchyme and in the chorion laeve trophoblasts with lower expression found in decidua (Bethin KE et al, 2003) . Decidua has been reported to produce increased amounts of PGs at the time of labor, but this is not a consistent observation . Human decidua is made up of decidualized stromal

cells, bone marrow-derived macrophages, and other cell types including trophoblasts that interface with chorion.

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