There are two general contemporaneous theorems concerning labor initiation. Viewed simplistically, the first is the functional loss of pregnancy maintenance factors, whereas the second focuses on synthesis of factors that induce parturition. Some investigators also speculate that the mature fetus is the source of the initial signal for parturition commencement. Others suggest that one or more uterotonins, produced in increased amounts, or an increased population of myometrial uterotonin receptors is the proximate cause. Indeed, an obligatory role for one or more uterotonins is included in most parturition theories, as either a primary or a secondary phenomenon in the final events of childbirth. Both rely on careful regulation of smooth muscle contraction.
Anatomical and Physiological Considerations
There are unique characteristics of smooth muscle, including myometrium, compared with those of skeletal muscle that may confer advantages for uterine contraction efficiency and fetal delivery. First, the degree of smooth-muscle cell shortening with contractions may be one order of magnitude greater than that attained in striated muscle cells. Second, forces can be exerted in smooth muscle cells in multiple directions. In contrast, the contraction force generated by skeletal muscle is always aligned with the axis of the muscle fibers. Third, smooth muscle is not organized in the same manner as skeletal muscle. In myometrium, the thick and thin filaments are found in long, random bundles throughout the cells. This plexiform arrangement aids greater shortening and force-generating capacity. Last, greater multidirectional force generation in the uterine fundus compared with that of the lower uterine segment permits versatility in expulsive force directionality. These forces thus can be brought to bear irrespective of the fetal lie or presentation.
Regulation of Myometrial Contraction and Relaxation
Myometrial contraction is controlled by transcription of key genes, which produce proteins that repress or enhance cellular contractility. These proteins function to: (1) enhance the interactions between the actin and myosin proteins that cause muscle contraction, (2) increase excitability of individual myometrial cells, and (3) promote intracellular cross talk that allows development of synchronous contractions.
The interaction of myosin and actin is essential to muscle contraction. This interaction requires that actin be converted from a globular to a filamentous form. Moreover, actin must be attached to the cytoskeleton at focal points in the cell membrane to allow development of tension (Fig. 21-11). Actin must partner with myosin, which is composed of multiple light and heavy chains. The interaction of myosin and actin activates adenosine triphosphatase (ATPase), hydrolyzes adenosine triphosphate, and generates force. This interaction is brought about by enzymatic phosphorylation of the 20-kDa light chain of myosin (Stull, 1988, 1998). This is catalyzed by the enzyme myosin light-chain kinase, which is activated by calcium. Calcium binds to calmodulin, a calcium-binding regulatory protein, which in turn binds to and activates myosin light-chain kinase.
Uterine myocyte relaxation and contraction. A. Uterine relaxation is maintained by factors that increase myocyte cyclic adenosine monophosphate (cAMP). This activates protein kinase A (PKA) to promote phosphodiesterase activity with dephosphorylation of myosin light-chain kinase (MLCK). There are also processes that serve to maintain actin in a globular form, and thus to prevent fibril formation necessary for contractions. B. Uterine contractions result from reversal of these sequences. Actin now assumes a fibrillar form, and calcium enters the cell to combine with calmodulin to form complexes. These complexes activate MLCK to bring about phosphorylation of the myosin light chains. This generates ATPase activity to cause sliding of myosin over the actin fibrils, which is a uterine contractor. AC = adenylyl cyclase; Ca++ = calcium; DAG = diacylglycerol; Gs and Gα = G-receptor proteins; IP3 = inositol triphosphate; LC20 = light chain 20; PIP3 = phosphatidylinositol 3,4,5–triphosphate; PLC = phospholipase C; R-PKA = inactive protein kinase. (Redrawn from Smith, 2007.)
Agents that promote contraction act on myometrial cells to increase intracellular cytosolic calcium concentration—[Ca2+]i. Or, they allow an influx of extracellular calcium through ligand- or voltage-regulated calcium channels (see Fig. 21-11). For example, prostaglandin F2α and oxytocin bind their respective receptors during labor to open ligand-activated calcium channels. Activation of these receptors also releases calcium from the sarcoplasmic reticulum to cause decreased electronegativity within the cell. Voltage-gated ion channels open, additional calcium ions move into the cell, and cellular depolarization follows. The increase in [Ca2+]i is often transient, but contractions can be prolonged through the inhibition of myosin phosphatase activity (Woodcock, 2004).
Conditions that decrease [Ca2+]i and increase intracellular concentrations of cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP) ordinarily promote uterine relaxation. Corticotropin-releasing hormone is one of several factors reported to regulate [Ca2+]i and subsequently modulate expression of the large-conductance potassium channels (BKCa) in the human myometrium (Xu, 2011; You, 2012). Genetic studies in humans and transgenic overexpression in mice reveal that small-conductance calcium-activated K+ isoform 3 (SK3) channels may also be important in maintenance of uterine relaxation (Day, 2011; Rada, 2012). SK3 channel expression declines at the end of term pregnancy as contractility is increased and overexpression of SK3 in transgenic mice dampens uterine contraction force to prevent delivery. Yet another potential mechanism for maintenance of relaxation shown in Figure 21-11 is the promotion of actin in a globular form rather than in fibrils required for contraction (Macphee, 2000; Yu, 1998).
In addition to myocyte contractility, myocyte excitability is also regulated by changes in the electrochemical potential gradient across the plasma membrane. Before labor, myocytes maintain a relatively high interior electronegativity. This state is maintained by the combined actions of the ATPase-driven sodium-potassium pump and the large conductance voltage- and Ca2+-sensitive K channel—maxi-K channel (Parkington, 2001). During uterine quiescence, the maxi-K channel is open and allows potassium to leave the cell to maintain interior electronegativity. At the time of labor, changes in electronegativity lead to depolarization and contraction (Brainard, 2005; Chanrachakul, 2003). And, as parturition progresses, there is increased synchronization of electrical uterine activity.
Cellular signals that control myometrial contraction and relaxation can be effectively transferred between cells through intercellular junctional channels. Communication is established between myocytes by gap junctions, which aid the passage of electrical or ionic coupling currents as well as metabolite coupling. The transmembrane channels that make up the gap junctions consist of two protein “hemi-channels” (Sáez, 2005). These connexons are each composed of six connexin subunit proteins (Fig. 21-12). These pairs of connexons establish a conduit between coupled cells for the exchange of small molecules that can be nutrients, waste, metabolites, second messengers, or ions.
The protein subunits of gap junction channels are called connexins. Six connexins form a hemichannel (connexon), and two connexons (one from each cell) form a gap junction channel. Connexons and gap junction channels can be formed from one or more connexin proteins. The composition of the gap junction channel is important for their selectivity with regard to passage of molecules and communication between cells.
Optimal numbers of gap junctions are believed to be important for electrical myometrial synchrony. Four described in the uterus are connexins 26, 40, 43, and 45. Connexin 43 junctions are scarce in the nonpregnant uterus, and they increase in size and abundance during human parturition (Chow, 1994). Also, mouse models deficient in connexin 43-enriched gap junctions exhibit delayed parturition, further supporting their role (Döring, 2006; Tong, 2009).
There are various cell surface receptors that can directly regulate myocyte contractile state. Three major classes are G-protein-linked, ion channel-linked, and enzyme-linked. Multiple examples of each have been identified in human myometrium. These further appear to be modified during the phases of parturition. Most G-protein-coupled receptors are associated with adenylyl cyclase activation. Examples are the CRHR1α and the LH receptors (Fig. 21-13). Other G-protein-coupled myometrial receptors, however, are associated with G-protein-mediated activation of phospholipase C. Ligands for the G-protein-coupled receptors include numerous neuropeptides, hormones, and autacoids. Many of these are available to the myometrium during pregnancy in high concentration via endocrine or autocrine mechanisms (Fig. 21-14).
G-protein-coupled receptor signal transduction pathways. A. Receptors coupled to heterotrimeric guanosine-triphosphate (GTP)-binding proteins (G proteins) are integral transmembrane proteins that transduce extracellular signals to the cell interior. G-protein-coupled receptors exhibit a common structural motif consisting of seven membrane-spanning regions. B. Receptor occupation promotes interaction between the receptor and the G protein on the interior surface of the membrane. This induces an exchange of guanosine diphosphate (GDP) for GTP on the G protein α subunit and dissociation of the α subunit from the βγ heterodimer. Depending on its isoform, the GTP-α subunit complex mediates intracellular signaling either indirectly by acting on effector molecules such as adenylyl cyclase (AC) or phospholipase C (PLC), or directly by regulating ion channel or kinase function. cAMP = cyclic adenosine monophosphate; DAG = diacylglycerol; IP3 = inositol triphosphate.
Theoretical fail-safe system involving endocrine, paracrine, and autocrine mechanisms for the maintenance of phase 1 of parturition, uterine quiescence. CRH = corticotropin-releasing hormone; hCG = human chorionic gonadotropin; PGE2 = prostaglandin E2; PGI2 = prostaglandin I2; PGDH = 15-hydroxyprostaglandin dehydrogenase.
Cervical Dilatation During Labor
There is a large influx of leukocytes into the cervical stroma with cervical dilatation (Sakamoto, 2004, 2005). Cervical tissue levels of leukocyte chemoattractants such as IL-8 are increased just after delivery, as are IL-8 receptor levels. Identification of genes upregulated just after vaginal delivery further suggests that dilatation and early stages of postpartum repair are aided by inflammatory responses, apoptosis, and activation of proteases that degrade extracellular matrix components (Hassan, 2006; Havelock, 2005). The composition of glycosaminoglycans, proteoglycans, and poorly formed collagen fibrils that were necessary during ripening and dilatation must be rapidly removed to allow reorganization and recovery of cervical structure. In the days that follow parturition, recovery of cervical structure involves processes that resolve inflammation, promote tissue repair, and regenerate dense cervical connective tissue with structural integrity and mechanical strength.
Phase 1: Uterine Quiescence and Cervical Competence
Myometrial quiescence is so remarkable and successful that it probably is induced by multiple independent and cooperative biomolecular processes. Individually, some of these processes may be redundant to ensure pregnancy continuance. It is likely that all manners of molecular systems—neural, endocrine, paracrine, and autocrine—are called on to implement and coordinate a state of relative uterine unresponsiveness. Moreover, a complementary “fail-safe” system that protects the uterus against agents that could perturb the tranquility of phase 1 also must be in place (see Fig. 21-14).
As shown in Figure 21-15, phase 1 of human parturition and its quiescence are likely the result of many factors that include: (1) actions of estrogen and progesterone via intracellular receptors, (2) myometrial cell plasma membrane receptor-mediated increases in cAMP, (3) generation of cGMP, and (4) other systems, including modification of myometrial-cell ion channels.
The key factors thought to regulate the phases of parturition. CRH = corticotropin-releasing hormone; hCG = human chorionic gonadotropin; SPA = surfactant protein A. (Adapted from Challis, 2002.)
Progesterone and Estrogen Contributions
In many species, the role of the sex steroid hormones is clear—progesterone inhibits and estrogen promotes the events leading to parturition. In humans, however, it seems most likely that both estrogen and progesterone are components of a broader-based molecular system that implements and maintains uterine quiescence. In many species, the removal of progesterone, that is, progesterone withdrawal, directly precedes progression of phase 1 into phase 2 of parturition. In addition, providing progesterone to some species will delay parturition via a decrease in myometrial activity and continued cervical competency (Challis, 1994). Studies in these species have led to a better understanding of why the progesterone-replete myometrium of phase 1 is relatively noncontractile.
Plasma levels of estrogen and progesterone in normal pregnancy are enormous and in great excess of the affinity constants for their receptors. For this reason, it is difficult to comprehend how relatively subtle changes in the ratio of their concentrations could modulate physiological processes during pregnancy. The teleological evidence, however, for an increased progesterone-to-estrogen ratio in the maintenance of pregnancy and a decline in the progesterone-to-estrogen ratio for parturition is overwhelming. In all species studied to date, including humans, administration of the progesterone-receptor antagonists mifepristone (RU-486) or onapristone will promote some or all key features of parturition. These include cervical ripening, increased cervical distensibility, and increased uterine sensitivity to uterotonins (Bygdeman, 1994; Chwalisz, 1994a; Wolf, 1993).
The exact role of estrogen in regulating human uterine activity and cervical competency is less well understood. That said, it appears that estrogen can act to promote progesterone responsiveness and, in doing so, promote uterine quiescence. The estrogen receptor, acting via the estrogen-response element of the progesterone-receptor gene, induces progesterone-receptor synthesis, which allows increased progesterone-mediated function.
Myometrial Cell-to-Cell Communication
Progesterone maintains uterine quiescence by various mechanisms that cause decreased expression of the contraction-associated proteins (CAPs)(Phase 2 of Parturition: Preparation for Labor). Progesterone can promote expression of the inhibitory transcription factor ZEB1—zinc finger E-box binding homeobox protein 1—which can inhibit expression of the CAP genes, connexin 43, and oxytocin receptor (Renthal, 2010). As another mechanism, progesterone bound to the progesterone receptor (PR) can recruit coregulatory factors. These include PSF—polypyrimidine tract binding protein-associated splicing factor—and Sin3A/HDACs—yeast switch-dependent3 homologue A/histone deacetylase corepressor complex—which inhibit expression of the gene encoding the gap junctional protein connexin 43 in rat and human myocytes (Xie, 2012).
At the end of pregnancy, increased stretch along with increased estrogen dominance results in a decline in PSF and Sin/HDAC levels, thus abrogating the suppression of connexin 43 expression by progesterone. Additionally, with loss of progesterone function at term, ZEB1 levels decline due to increased production of small regulatory RNAs termed microRNAs. This releases inhibition of connexin 43 and oxytocin receptor levels to promote increased uterine contractility (Renthal, 2010; Williams, 2012b).
A number of G-protein-coupled receptors that normally are associated with Gαs-mediated activation of adenylyl cyclase and increased levels of cAMP are present in myometrium. These receptors together with appropriate ligands may act in concert with sex steroid hormones as part of a fail-safe system to maintain uterine quiescence (Price, 2000; Sanborn, 1998).
These receptors are prototypical examples of cAMP signaling causing myometrium relaxation. Agents binding to these receptors have been used for tocolysis with preterm labor and include ritodrine and terbutaline (Chap. 42, β-Adrenergic Receptor Agonists). β-Adrenergic receptors mediate Gαs-stimulated increases in adenylyl cyclase, increased levels of cAMP, and myometrial cell relaxation. The rate-limiting factor is likely the number of receptors expressed and the level of adenylyl cyclase expression.
Luteinizing Hormone (LH) and Human Chorionic Gonadotropin (hCG) Receptors
These hormones share the same receptor, and this G-protein-coupled receptor has been demonstrated in myometrial smooth muscle and blood vessels (Lei, 1992; Ziecik, 1992). Levels of myometrial LH-hCG receptors during pregnancy are greater before than during labor (Zuo, 1994). Chorionic gonadotropin acts to activate adenylyl cyclase by way of a plasma membranereceptor–Gαs-linked system. This decreases contraction frequency and force and decreases the number of tissue-specific myometrial cell gap junctions (Ambrus, 1994; Eta, 1994). Thus, high circulating levels of hCG may be one mechanism causing uterine quiescence.
This peptide hormone consists of an A and B chain and is structurally similar to the insulin family of proteins (Bogic, 1995; Weiss, 1995). Relaxin mediates lengthening of the pubic ligament, cervical softening, vaginal relaxation, and inhibition of myometrial contractions. There are two separate human relaxin genes, designated H1 and H2. The H1 gene is primarily expressed in the decidua, trophoblast, and prostate, whereas the H2 gene is primarily expressed in the corpus luteum.
Relaxin in plasma of pregnant women is believed to originate exclusively from corpus luteum secretion. Plasma levels peak at approximately 1 ng/mL between 8 and 12 weeks’ gestation. Thereafter, they decline to lower levels that persist until term. Its plasma membrane receptor—relaxin family peptide receptor 1 (RXFP1)—mediates activation of adenylyl cyclase. Relaxin inhibits contractions of nonpregnant myometrial strips, but not those of uterine tissue taken from pregnant women. It also effects cervical remodeling through cell proliferation and modulation of extracellular matrix components such as collagen and hyaluronan (Park, 2005; Soh, 2012).
Corticotropin-Releasing Hormone (CRH)
This hormone is synthesized in the placenta and hypothalamus. As discussed later, CRH plasma levels increase dramatically during the final 6 to 8 weeks of normal pregnancy and have been implicated in the mechanisms controlling the timing of human parturition (Smith, 2007; Wadhwa, 1998). CRH appears to promote myometrial quiescence during most of pregnancy and aids myometrial contractions with onset of parturition. Recent studies suggest that these opposing actions are achieved by differential actions of CRH via its receptor CRHR1. In the term nonlaboring myometrium, the interaction of CRH with its CRHR1 receptor results in activation of the Gs-adenylate cyclase-cAMP signaling pathway. This results in inhibition of inositol triphosphate (IP3) production and a stabilization of [Ca2+]i (You, 2012). In term laboring myometrium, [Ca2+]i is increased by CRH activation of G proteins Gq and Gi and leads to stimulation of IP3 production and increased contractility. Another aspect of CRH regulation is union of CRH to its binding protein, which can limit bioavailability. CRH-binding protein levels are high during pregnancy and are reported to decline at the time of labor.
These interact with a family of eight different G-protein-coupled receptors, several of which are expressed in myometrium (Myatt, 2004). Prostaglandins usually are considered as uterotonins. However, their effects are diverse, and some act as smooth muscle relaxants.
The major synthetic pathways involved in prostaglandin biosynthesis are shown in Figure 21-16. Prostaglandins are produced using plasma membrane-derived arachidonic acid, which usually is released by the action of the phospholipases A2 or C. Arachidonic acid can then act as substrate for both type 1 and type 2 prostaglandin H synthase (PGHS-1 and -2), which are also called cyclooxygenase-1 and -2 (COX-1 and -2). Both PGHS isoforms convert arachidonic acid to the unstable endoperoxide prostaglandin G2 and then to prostaglandin H2. These enzymes are the target of many nonsteroidal antiinflammatory drugs (NSAIDs). Indeed, the tocolytic actions of specific NSAIDs, as discussed in Chapter 42 (β-Adrenergic Receptor Agonists), were considered promising until they were shown to have adverse fetal effects (Loudon, 2003; Olson, 2003, 2007).
Overview of the prostaglandin biosynthetic pathway.
Through prostaglandin isomerases, prostaglandin H2 is converted to active prostaglandins, including PGE2, PGF2α, and PGI2. Isomerase expression is tissue-specific and thereby controls the relative production of various prostaglandins. Another important control point for prostaglandin activity is its metabolism, which most often is through the action of 15-hydroxyprostaglandin dehydrogenase (PGDH). Expression of this enzyme can be regulated in the uterus, which is important because of its ability to rapidly inactivate prostaglandins.
The effect of prostaglandins on tissue targets is complicated in that there are a number of G-protein-coupled prostaglandin receptors (Coleman, 1994). This family of receptors is classified according to the binding specificity of a given receptor to a particular prostaglandin. Both PGE2 and PGI2 could potentially act to maintain uterine quiescence by increasing cAMP signaling, yet PGE2 can promote uterine contractility through binding to prostaglandin E receptors 1 and 3 (EP1 and EP3). Also, PGE2, PGD2, and PGI2 have been shown to cause vascular smooth muscle relaxation and vasodilatation in many circumstances. Thus, either the generation of specific prostaglandins or the relative expression of the various prostaglandin receptors may determine myometrial responses to prostaglandins (Lyall, 2002; Olson, 2003, 2007; Smith, 2001; Smith, 1998).
In addition to gestational changes, other studies show that there may be regional changes in the upper and lower uterine segments. COX-2 expression is spatially regulated in the myometrium and cervix in pregnancy and labor, with an increasing concentration gradient from the fundus to the cervix (Havelock, 2005). Thus, it is entirely possible that prostanoids contribute to myometrial relaxation at one stage of pregnancy and to regional fundal myometrial contractions after parturition initiation (Myatt, 2004).
Atrial and Brain Natriuretic Peptides and Cyclic Guanosine Monophosphate (cGMP)
Activation of guanylyl cyclase increases intracellular cGMP levels, which promotes smooth muscle relaxation (Word, 1993). Intracellular cGMP levels are increased in the pregnant myometrium and can be stimulated by atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) receptors, and nitric oxide (Telfer, 2001). All of these factors and their receptors are expressed in the pregnant uterus. However, it remains unclear if these factors and intracellular cGMP play a role in uterine quiescence in normal pregnancy physiology (Itoh, 1994; Yallampalli, 1994a,b).
Accelerated Uterotonin Degradation
In addition to pregnancy-induced compounds that promote myometrial cell refractoriness, there are striking increases in the activity of enzymes that degrade or inactivate endogenously produced uterotonins. Some of these and their degradative enzymes include: PGDH and prostaglandins; enkephalinase and endothelins; oxytocinase and oxytocin; diamine oxidase and histamine; catechol O-methyltransferase and catecholamines; angiotensinases and angiotensin-II; and platelet-activating factor (PAF) acetylhydrolase and PAF. Activities of several of these enzymes are increased by progesterone, and many decrease late in gestation (Bates, 1979; Casey, 1980; Germain, 1994).
Phase 2: Uterine Activation and Cervical Ripening
Classic Progesterone Withdrawal and Parturition
Key factors in uterine activation are depicted in Figure 21-15. In species that exhibit progesterone withdrawal, parturition progression to labor can be blocked by administering progesterone to the mother. However, there are conflicting reports as to whether progesterone administration in pregnant women can delay the timely onset of parturition or prevent preterm labor. The possibility that progesterone-containing injections or vaginal suppositories may be used to prevent preterm labor has been studied in a number of randomized trials conducted during the past 15 years. These are discussed in Chapter 42 (Cervical Cerclage), but in general, their use has marginal clinical benefits in preventing recurrent preterm birth and its associated perinatal morbidity.
Progesterone Receptor Antagonists and Human Parturition
When the steroidal antiprogestin mifepristone (RU-486) is administered during the latter phase of the ovarian cycle, it induces menstruation prematurely. It is also an effective abortifacient during early pregnancy (Chap. 18, Menstrual Aspiration). Mifepristone is a classic steroid antagonist, acting at the level of the progesterone receptor. Although less effective in inducing abortion or labor in women later in pregnancy, mifepristone appears to have some effect on cervical ripening and on increasing myometrial sensitivity to uterotonins (Berkane, 2005; Chwalisz, 1994a,b). These data suggest that humans have a mechanism for progesterone inactivation, whereby the myometrium and cervix become refractory to the inhibitory actions of progesterone.
Functional Progesterone Withdrawal in Human Parturition
As an alternative to classic progesterone withdrawal resulting from decreased secretion, research has focused on mechanisms that inhibit progesterone action in human pregnancy. Functional progesterone withdrawal or antagonism is possibly mediated through several mechanisms: (1) changes in the relative expression of the nuclear progesterone-receptor isoforms, PR-A, PR-B, and PR-C; (2) changes in the relative expression of membrane-bound progesterone receptors; (3) posttranslational modifications of the progesterone receptor; (4) alterations in PR activity through changes in the expression of coactivators or corepressors that directly influence receptor function; (5) local inactivation of progesterone by steroid-metabolizing enzymes or synthesis of a natural antagonist; and (6) microRNA regulation of progesterone-metabolizing enzymes and transcription factors that modulate uterine quiescence.
There is evidence that progesterone-receptor activity is decreased late in gestation. A series of studies have shown that the relative ratio of PR-A to PR-B within the myometrium, decidua, and chorion shifts late in gestation (Madsen, 2004; Mesiano, 2002; Pieber, 2001). Specifically, increased PR-A levels during parturition depress the antiinflammatory actions of PR-B and thereby promote proinflammatory gene expression at term (Tan, 2012). Moreover, these activities have been shown to be specific for the upper and lower uterine segments (Condon, 2003, 2006). Similarly, studies of cervical stroma suggest changes in receptor isoform concentrations (Stjernholm-Vladic, 2004). In addition, membrane PR isoforms are also expressed in the myometrium and placenta. However, it remains to be determined if they play a role to promote the transition from myometrial quiescence to activation (Chapman, 2006; Karteris, 2006; Zachariades, 2012). There is evidence in rodent models that local action of enzymes such as steroid 5α-reductase type 1 or 20α-hydroxysteroid dehydrogenase (20α-HSD) catabolize progesterone to metabolites that have a weak affinity for the progesterone receptor (Mahendroo, 1999; Piekorz, 2005). In the human cervix, decreased activity of 17β-hydroxysteroid dehydrogenase type 2 at term results in a net increase in estrogen and decline in progesterone levels (Andersson, 2008). Recent studies provide new insights into the regulatory role of small noncoding RNAs (microRNAs) in regulating expression of the steroid metabolizing enzyme 20α-HSD (Williams, 2012a). Increased expression of microRNA200a in the term myometrium blunts the expression of STAT5b, an inhibitor of 20α-HSD. Reduced STAT5b function allows increased 20α-HSD levels that result in increased progesterone metabolism and reduced progesterone function.
Taken together, all of these observations support the concept that multiple pathways exist for a functional progesterone withdrawal that includes changes in PR isoform and receptor coactivator levels, microRNA regulation, and increased local hormone metabolism to less active products.
Because of its longstanding application for labor induction, it seemed logical that oxytocin must play a central role in spontaneous human labor. But this venerable hormone may have only a minor supporting role. Currently, it still is controversial whether oxytocin plays a role in the early phases of uterine activation or whether its sole function is in the expulsive phase of labor. Most studies of regulation of myometrial oxytocin-receptor synthesis have been performed in rodents. Disruption of the oxytocin receptor gene in the mouse does not affect parturition. This suggests that, at least in this species, multiple systems likely ensure that parturition occurs. There is little doubt, however, that there is an increase in myometrial oxytocin receptors during phase 2 of parturition. Moreover, their activation results in increased phospholipase C activity and subsequent increases in cytosolic calcium levels and uterine contractility.
Progesterone and estradiol appear to be the primary regulators of oxytocin receptor expression. Estradiol treatment in vivo or in myometrial explants increases myometrial oxytocin receptor concentrations. This action, however, is prevented by simultaneous treatment with progesterone (Fuchs, 1983). Progesterone also may act within the myometrial cell to increase oxytocin-receptor degradation and inhibit oxytocin activation of its receptor at the cell surface (Bogacki, 2002). These data indicate that one of the mechanisms whereby progesterone maintains uterine quiescence is through the inhibition of myometrial oxytocin response.
The increase in oxytocin receptor levels in nonhuman species appears to be mainly regulated either directly or indirectly by estradiol. Treatment of several species with estrogen leads to increased uterine oxytocin receptor levels (Blanks, 2003; Challis, 1994). Moreover, the level of oxytocin receptor mRNA in human myometrium at term is greater than that found in preterm myometrium (Wathes, 1999). Thus, increased receptors at term may be attributable to increased gene transcription. An estrogen response element, however, is not present in the oxytocin receptor gene, suggesting that the stimulatory effects of estrogen may be indirect.
Human studies suggest that inflammatory-related rapid-response genes may regulate oxytocin receptors (Bethin, 2003; Kimura, 1999; Massrieh, 2006). These receptors also are present in human endometrium and in decidua at term and stimulate prostaglandin production. In addition, these receptors are found in the myometrium and at lower levels in amniochorion-decidual tissues (Benedetto, 1990; Wathes, 1999).
Although relaxin may contribute to uterine quiescence, it also has roles in phase 2 of parturition. These include remodeling of the extracellular matrix of the uterus, cervix, vagina, breast, and pubic symphysis as well as promoting cell proliferation and inhibiting apoptosis. Its actions on cell proliferation and apoptosis are mediated through the G-protein-coupled receptor, RXFP1, whereas some but not all actions of relaxin on matrix remodeling are mediated through this receptor (Samuel, 2009; Yao, 2008). The precise mechanisms for modulation of matrix turnover have not been fully elucidated. However, relaxin appears to mediate glycosaminoglycan and proteoglycan synthesis and degrade matrix macromolecules such as collagen by induction of matrix metalloproteases. Relaxin promotes growth of the cervix, vagina, and pubic symphysis and is necessary for breast remodeling for lactation. Consistent with its proposed roles, mice deficient in relaxin or the RXFP1 receptor have protracted labor; show reduced growth of the cervix, vagina, and symphysis; and are unable to nurse because of incomplete nipple development (Feng, 2005; Park, 2005; Rosa, 2012; Soh, 2012; Yao, 2008).
Fetal Contributions to Initiation of Parturition
It is intellectually intriguing to envision that the mature human fetus provides the signal to initiate parturition. Teleologically, this seems most logical because such a signal could be transmitted in several ways to suspend uterine quiescence. The fetus may provide a signal through a blood-borne agent that acts on the placenta. Research is ongoing to better understand the fetal signals that contribute to parturition initiation (Mendelson, 2009). Although signals may arise from the fetus, the uterus and cervix likely first must be prepared for labor before a uterotonin produced by or one whose release is stimulated by the fetus can be optimally effective (Casey, 1994).
Uterine Stretch and Parturition
There is now considerable evidence that fetal growth is an important component in uterine activation in phase 1 of parturition. In association with fetal growth, significant increases in myometrial tensile stress and amnionic fluid pressure follow (Fisk, 1992). With uterine activation, stretch is required for induction of specific contraction-associated proteins (CAPs). Specifically, stretch increases expression of the gap junction protein—connexin 43 and of oxytocin receptors. Gastrin-releasing peptide, a stimulatory agonist for smooth muscle, is increased by stretch in the myometrium (Tattersall, 2012). Others have hypothesized that stretch plays an integrated role with fetal-maternal endocrine cascades of uterine activation (Lyall, 2002; Ou, 1997, 1998).
Clinical support for a role of stretch comes from the observation that multifetal pregnancies are at a much greater risk for preterm labor than singletons. And preterm labor is also significantly more common in pregnancies complicated by hydramnios. Although the mechanisms causing preterm birth in these two examples are debated, a role for uterine stretch must be considered.
Cell signaling systems used by stretch to regulate the myometrial cell continue to be defined. This process—mechanotransduction—may include activation of cell-surface receptors or ion channels, transmission of signals through extracellular matrix, or release of autocrine molecules that act directly on myometrium (Shynlova, 2009; Young, 2011). For example, the extracellular matrix protein fibronectin and its cell-surface receptor, alpha 5 integrin receptor, are induced in the rodent by stretch (Shynlova, 2007). This interaction may aid force transduction during labor contraction by anchoring hypertrophied myocytes to the uterine extracellular matrix.
Fetal Endocrine Cascades Leading to Parturition
The ability of the fetus to provide endocrine signals that initiate parturition has been demonstrated in several species. Liggins and associates (1967, 1973) demonstrated that the fetus provides the signal for the timely onset of parturition in sheep. This signal was shown to come from the fetal hypothalamic-pituitary-adrenal axis (Whittle, 2001).
Defining the exact mechanisms regulating human parturition has proven more difficult, and all evidence suggests that it is not regulated in the exact manner seen in the sheep. Even so, activation of the human fetal hypothalamic-pituitary-adrenal-placental axis is considered a critical component of normal parturition. Moreover, premature activation of this axis is considered to prompt many cases of preterm labor (Challis, 2000, 2001). As in the sheep, steroid products of the human fetal adrenal gland are believed to have effects on the placenta and membranes that eventually transform myometrium from a quiescent to contractile state. A key component in the human may be the unique ability of the placenta to produce large amounts of CRH, as shown in Figure 21-17.
The placental–fetal adrenal endocrine cascade. In late gestation, placental corticotropin-releasing hormone (CRH) stimulates fetal adrenal production of dehydroepiandrosterone sulfate (DHEA-S) and cortisol. The latter stimulates production of placental CRH, which leads to a feed-forward cascade that enhances adrenal steroid hormone production. ACTH = adrenocorticotropic hormone.
Placental Corticotropin-Releasing Hormone Production
A CRH hormone identical to maternal and fetal hypothalamic CRH is synthesized by the placenta in relatively large amounts (Grino, 1987; Saijonmaa, 1988). One important difference is that, unlike hypothalamic CRH, which is under glucocorticoid negative feedback, cortisol has been shown to stimulate placental CRH production. This is by activation of the transcription factor, nuclear factor kappa B (NF-κB) (Jones, 1989; Marinoni, 1998; Thomson, 2013). This ability makes it possible to create a feed-forward endocrine cascade that does not end until delivery.
Maternal plasma CRH levels are low in the first trimester and rise from midgestation to term. In the last 12 weeks, CRH plasma levels rise exponentially, peaking during labor and then falling precipitously after delivery (Frim, 1988; Sasaki, 1987). Amnionic fluid CRH levels similarly increase in late gestation. CRH is the only trophic hormone-releasing factor to have a specific serum binding protein. During most of pregnancy, it appears that CRH-binding protein (CRH-BP) binds most maternal circulating CRH, and this inactivates it (Lowry, 1993). During later pregnancy, however, CRH-BP levels in both maternal plasma and amnionic fluid decline, leading to markedly increased levels of bioavailable CRH (Perkins, 1995; Petraglia, 1997).
In pregnancies in which the fetus can be considered to be “stressed” from various complications, concentrations of CRH in fetal plasma, amnionic fluid, and maternal plasma are increased compared with those seen in normal gestation (Berkowitz, 1996; Goland, 1993; McGrath, 2002; Perkins, 1995). The placenta is likely the source for this increased CRH concentration (Torricelli, 2011). For example, placental CRH content was fourfold higher in placentas from women with preeclampsia than in those from normal pregnancies (Perkins, 1995). Such increases in placental CRH production during normal gestation and the excessive secretion of placental CRH in complicated pregnancies may play a role in fetal adrenal cortisol synthesis (Murphy, 1982). They also may result in the supranormal levels of umbilical cord blood cortisol noted in stressed neonates (Falkenberg, 1999; Goland, 1994).
Corticotropin-Releasing Hormones and Parturition Timing
Placental CRH has been proposed to play several roles in parturition regulation. Placental CRH may enhance fetal cortisol production to provide positive feedback so that the placenta produces more CRH. Late in pregnancy—phase 2 or 3 of parturition—modification in the CRH receptor favors a switch from cAMP formation to increased myometrial cell calcium levels via protein kinase C activation (You, 2012). Oxytocin acts to attenuate CRH-stimulated accumulation of cAMP in myometrial tissue. And, CRH augments the contraction-inducing potency of a given dose of oxytocin in human myometrial strips (Quartero, 1991, 1992). CRH acts to increase myometrial contractile force in response to PGF2α (Benedetto, 1994). Finally, CRH has been shown to stimulate fetal adrenal C19-steroid synthesis, thereby increasing substrate for placental aromatization. Increased production of estrogens would shift the estrogen-to-progesterone ratio and promote the expression of a series of myometrial contractile proteins.
Some have proposed that the rising level of CRH at the end of gestation reflects a fetal-placental clock (McLean, 1995). CRH levels vary greatly among women, and it appears that the rate of increase in maternal CRH levels is a more accurate predictor of pregnancy outcome than is a single measurement (Leung, 2001; McGrath, 2002). In this regard, the placenta and fetus, through endocrinological events, influence the timing of parturition at the end of normal gestation.
Fetal Lung Surfactant and Parturition
Surfactant protein A (SP-A) produced by the fetal lung is required for lung maturation. Its levels are increased in amnionic fluid at term in mice. Studies in the mouse suggest that the increasing SP-A concentrations in amnionic fluid activate fluid macrophages to migrate into the myometrium and induce NF-κB (Condon, 2004). This factor activates inflammatory response genes in the myometrium, which in turn promote uterine contractility. This model supports the supposition that fetal signals play a role in parturition initiation. SP-A is expressed by the human amnion and decidua, is present in the amnionic fluid, and prompts signaling pathways in human myometrial cells (Garcia-Verdugo, 2008; Lee, 2010; Snegovskikh, 2011). The exact mechanisms by which SP-A activates myometrial contractility in women, however, remains to be clarified (Leong, 2008). SP-A selectively inhibits prostaglandin F2α in the term decidua, and amnionic fluid concentration of SP-A declines at term (Chaiworapongsa, 2008).
Fetal Anomalies and Delayed Parturition
There is fragmentary evidence that pregnancies with markedly diminished estrogen production may be associated with prolonged gestation. These “natural experiments” include fetal anencephaly with adrenal hypoplasia and those with inherited placental sulfatase deficiency. The broad range of gestational length seen with these disorders calls into question the exact role of estrogen in human parturition initiation.
Other fetal abnormalities that prevent or severely reduce the entry of fetal urine into amnionic fluid—renal agenesis, or into lung secretions—pulmonary hypoplasia, do not prolong human pregnancy. Thus, a fetal signal through the paracrine arm of the fetal-maternal communication system does not appear to be mandated for parturition initiation.
Some brain anomalies of the fetal calf, fetal lamb, and sometimes the human fetus delay the normal timing of parturition. More than a century ago, Rea (1898) observed an association between fetal anencephaly and prolonged human gestation. Malpas (1933) extended these observations and described a pregnancy with an anencephalic fetus that was prolonged to 374 days—53 weeks. He concluded that the association between anencephaly and prolonged gestation was attributable to anomalous fetal brain-pituitary-adrenal function. The adrenal glands of the anencephalic fetus are very small and, at term, may be only 5 to 10 percent as large as those of a normal fetus. This is caused by developmental failure of the fetal zone that normally accounts for most of fetal adrenal mass and production of C19-steroid hormones (Chap. 5, Directional Secretion of Steroids from Syncytiotrophoblast). Such pregnancies are associated with delayed labor and suggest that the fetal adrenal glands are important for the timely onset of parturition (Anderson, 1973).
Phase 3: Uterine Stimulation
This parturition phase is synonymous with uterine contractions that bring about progressive cervical dilatation and delivery. Current data favor the uterotonin theory of labor initiation. Increased uterotonin production would follow once phase 1 is suspended and uterine phase 2 processes are implemented. A number of uterotonins may be important to the success of phase 3, that is, active labor (see Fig. 21-15). Just as multiple processes likely contribute to myometrial unresponsiveness of phase 1 of parturition, other processes may contribute jointly to a system that ensures labor success.
Uterotonins that are candidates for labor induction include oxytocin, prostaglandins, serotonin, histamine, PAF, angiotensin II, and many others. All have been shown to stimulate smooth muscle contraction through G-protein coupling.
Oxytocin and Phase 3 of Parturition
Late in pregnancy, during phase 2 of parturition, there is a 50-fold or more increase in the number of myometrial oxytocin receptors (Fuchs, 1982; Kimura, 1996). This increase coincides with an increase in uterine contractile responsiveness to oxytocin. Moreover, prolonged gestation is associated with a delay in the increase of these receptors (Fuchs, 1984).
Oxytocin—literally, quick birth—was the first uterotonin to be implicated in parturition initiation. This nanopeptide is synthesized in the magnocellular neurons of the supraoptic and paraventricular neurons. The prohormone is transported with its carrier protein, neurophysin, along the axons to the neural lobe of the posterior pituitary gland in membrane-bound vesicles for storage and later release. The prohormone is converted enzymatically to oxytocin during transport (Gainer, 1988; Leake, 1990).
Role of Oxytocin in Phases 3 and 4 of Parturition
Because of successful labor induction with oxytocin, it was logically suspected in parturition initiation. First, in addition to its effectiveness in inducing labor at term, oxytocin is a potent uterotonin and occurs naturally in humans. Subsequent observations provide additional support for this theory: (1) the number of oxytocin receptors strikingly increases in myometrial and decidual tissues near the end of gestation; (2) oxytocin acts on decidual tissue to promote prostaglandin release; and (3) oxytocin is synthesized directly in decidual and extraembryonic fetal tissues and in the placenta (Chibbar, 1993; Zingg, 1995).
Although little evidence suggests a role for oxytocin in phase 2 of parturition, abundant data support its important role during second-stage labor and in the puerperium—phase 4 of parturition. Specifically, there are increased maternal serum oxytocin levels: (1) during second-stage labor, which is the end of phase 3 of parturition; (2) in the early puerperium; and (3) during breast feeding (Nissen, 1995). Immediately after delivery of the fetus, placenta, and membranes, which completes parturition phase 3, firm and persistent uterine contractions and myometrial retraction are essential to prevent postpartum hemorrhage. Oxytocin likely causes persistent contractions.
Oxytocin infusion in women promotes increased levels of mRNAs from myometrial genes that encode proteins essential for uterine involution. These include interstitial collagenase, monocyte chemoattractant protein-1, interleukin-8, and urokinase plasminogen activator receptor. Therefore, oxytocin action at the end of labor may be involved in uterine involution.
Prostaglandins and Phase 3 of Parturition
Although their role in parturition phase 2 of noncomplicated pregnancies is less well defined, a critical role for prostaglandins in phase 3 of parturition is clear (MacDonald, 1993). First, levels of prostaglandins—or their metabolites—in amnionic fluid, maternal plasma, and maternal urine are increased during labor (Fig. 21-18. Second, treatment of pregnant women with prostaglandins, by any of several administration routes, causes abortion or labor at all gestational stages. Moreover, administration of prostaglandin H synthase type 2 (PGHS-2) inhibitors to pregnant women will delay spontaneous labor onset and sometimes arrest preterm labor (Loudon, 2003). Last, prostaglandin treatment of myometrial tissue in vitro sometimes causes contraction, dependent on the prostanoid tested and the physiological status of the tissue treated.
Mean (±SD) concentrations of prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2) in amnionic fluid at term before labor and in the upper and forebag compartments during labor at all stages of cervical dilatation. (Data from MacDonald, 1993.)
Uterine Events Regulating Prostaglandin Production
During labor, prostaglandin production within the myometrium and decidua is an efficient mechanism of activating contractions. For example, prostaglandin synthesis is high and unchanging in the decidua during phase 2 and 3 of parturition. Moreover, the receptor level for PGF2α is increased in the decidua at term, and this increase most likely is the regulatory step in prostaglandin action in the uterus. The myometrium synthesizes PGHS-2 with labor onset, but most prostaglandins likely come from the decidua.
The fetal membranes and placenta also produce prostaglandins. Primarily PGE2, but also PGF2α, are detected in amnionic fluid at all gestational stages. As the fetus grows, prostaglandins levels in the amnionic fluid increase gradually. Their major increases in concentration within amnionic fluid, however, are demonstrable after labor begins (see Fig. 21-18). These higher levels likely result as the cervix dilates and exposes decidual tissue (Fig. 21-19). These increased levels in the forebag compared with those in the upper compartment are believed to follow an inflammatory response that signals the events leading to active labor. Together, the increases in cytokines and prostaglandins further degrade the extracellular matrix, thus weakening fetal membranes.
Sagittal view of the exposed forebag and attached decidual fragments after cervical dilatation during labor. (Redrawn from MacDonald, 1996.)
Findings of Kemp and coworkers (2002) and Kelly (2002) support a possibility that inflammatory mediators aid cervical dilatation and alterations to the lower uterine segment. It can be envisioned that they, along with the increased prostaglandin levels measured in vaginal fluid during labor, add to the relatively rapid cervical changes that are characteristic of parturition.
The endothelins are a family of 21-amino acid peptides that powerfully induce myometrial contraction (Word, 1990). The endothelin A receptor is preferentially expressed in smooth muscle and effects an increase in intracellular calcium. Endothelin-1 is produced in myometrium of term gestations and is able to induce synthesis of other contractile mediators such as prostaglandins and inflammatory mediators (Momohara, 2004; Sutcliffe, 2009). The requirement of endothelin-1 in normal parturition physiology remains to be established. However, there is evidence of pathologies associated with aberrant endothelin-1 expression, such as premature birth and uterine leiomyomas (Tanfin, 2011, 2012).
There are two G-protein-linked angiotensin II receptors expressed in the uterus—AT1 and AT2. In nonpregnant women, the AT2 receptor is predominant, but the AT1 receptor is preferentially expressed in pregnant women (Cox, 1993). Angiotensin II binding to the plasma-membrane receptor evokes contraction. During pregnancy, the vascular smooth muscle that expresses the AT2 receptor is refractory to the pressor effects of infused angiotensin II (Chap. 4, Supine Hypotension). In myometrium near term, however, angiotensin II may be another component of the uterotonin system of parturition phase 3 (Anton, 2009).
Contribution of Intrauterine Tissues to Parturition
Although they have a potential role in parturition initiation, the amnion, chorion laeve, and decidua parietalis more likely have an alternative role. The membranes and decidua make up an important tissue shell around the fetus that serves as a physical, immunological, and metabolic shield to protect against untimely initiation of parturition. Late in gestation, however, the fetal membranes may indeed act to prepare for labor.
Virtually all of the tensile strength—resistance to tearing and rupture—of the fetal membranes is provided by the amnion (Chap. 5, Immunogenicity of the Trophoblasts). This avascular tissue is highly resistant to penetration by leukocytes, microorganisms, and neoplastic cells. It also constitutes a selective filter to prevent fetal particulate-bound lung and skin secretions from reaching the maternal compartment. In this manner, maternal tissues are protected from amnionic fluid constituents that could worsen decidual or myometrial function or could promote adverse events such as amnionic-fluid embolism (Chap. 41, Amnionic-Fluid Embolism).
Several bioactive peptides and prostaglandins that cause myometrial relaxation or contraction are synthesized in amnion (Fig. 21-20). Late in pregnancy, amnionic prostaglandin biosynthesis is increased, and phospholipase A2 and PGHS-2 show increased activity (Johnson, 2002). Accordingly, many hypothesize that prostaglandins regulate events leading to parturition. It is likely that amnion is the major source for amnionic fluid prostaglandins, and their role in activation of cascades that promote membrane rupture is clear. The influence of amnion-derived prostaglandins on uterine quiescence and activation, however, is less clear. This is because prostaglandin transport from the amnion through the chorion to access maternal tissues is limited by expression of the inactivating enzyme, prostaglandin dehydrogenase.
The amnion synthesizes prostaglandins, and late in pregnancy, synthesis is increased by increased phospholipase A2 and prostaglandin H synthase, type 2 (PGHS-2) activity. During pregnancy, the transport of prostaglandins from the amnion to maternal tissues is limited by expression of the inactivating enzymes, prostaglandin dehydrogenase (PGDH), in the chorion. During labor, PGDH levels decline, and amnion-derived prostaglandins can influence membrane rupture and uterine contractility. The role of decidual activation in parturition is unclear but may involve local progesterone metabolism and increased prostaglandin receptor concentrations, thus enhancing uterine prostaglandin actions and cytokine production. (Adapted from Smith, 2007.)
This tissue layer also is primarily protective and provides immunological acceptance. The chorion laeve is also enriched with enzymes that inactivate uterotonins. Enzymes include prostaglandin dehydrogenase (PGDH), oxytocinase, and enkephalinase (Cheung, 1990; Germain, 1994). As noted, PGDH inactivates amnion-derived prostaglandins. With chorionic rupture, this barrier would be lost, and prostaglandins could readily influence adjacent decidua and myometrium.
There is also evidence that PGDH levels found in the chorion decline during labor. This would allow increased prostaglandin-stimulated matrix metalloproteinase activity associated with membrane rupture. It would further allow prostaglandin entry into the maternal compartment to promote myometrial contractility (Patel, 1999; Van Meir, 1996; Wu, 2000). It is likely that progesterone maintains chorion PGDH expression, whereas cortisol decreases its expression. Thus, PGDH levels would decrease late in gestation as fetal cortisol production increases and as part of progesterone withdrawal.
A metabolic contribution of decidual activation to parturition initiation is an appealing possibility for both anatomical and functional reasons. The generation of decidual uterotonins that act in a paracrine manner on contiguous myometrium is intuitive. In addition, decidua expresses steroid metabolizing enzymes such as 20α-HSD and steroid 5αR1 that may regulate local progesterone withdrawal. Decidual activation is characterized by increased proinflammatory cells and increased expression of proinflammatory cytokines, prostaglandins, and uterotonins such as oxytocin receptors and connexin 43.
Cytokines produced in the decidua can either increase uterotonin production—principally prostaglandins. Or they can act directly on myometrium to cause contraction. Examples are tumor necrosis factor-α (TNF-α) and interleukins 1, 6, 8, and 12. These molecules also can act as chemokines that recruit to the myometrium neutrophils and eosinophils, which further increase contractions and labor (Keelan, 2003).
There is uncertainty whether prostaglandin concentration or output from the decidua increases with term labor onset. Olson and Ammann (2007) suggest that the major regulation of decidual prostaglandin action is not their synthesis but rather increased PGF2α receptor expression.
Summary: Regulation of Phase 3 and 4 of Parturition
It is likely that multiple and possibly redundant processes contribute to the success of the three active labor phases once phase 1 of parturition is suspended and phase 2 is initiated. Phase 3 is highlighted by increased activation of G-protein-coupled receptors that inhibit cAMP formation, increase intracellular calcium stores, and promote interaction of actin and myosin and subsequent force generation. Simultaneously, cervical proteoglycan composition and collagen structure are altered to a form that promotes tissue distensibility and increased compliance. The net result is initiation of coordinated myometrial contractions of sufficient amplitude and frequency to dilate the prepared cervix and push the fetus through the birth canal. Multiple regulatory ligands orchestrate these processes and vary from endocrine hormones such as oxytocin to locally produced prostaglandins.
In phase 4 of parturition, a complicated series of repair processes are initiated to resolve inflammatory responses and remove glycosaminoglycans, proteoglycans, and structurally compromised collagen. Simultaneously, matrix and cellular components required for complete uterine involution are synthesized, and the dense connective tissue and structural integrity of the cervix is reformed.