Pathogenesis of Spontaneous Preterm Birth

Progesterone Withdrawal and Parturition

Progesterone (P4) is essential for the establishment and maintenance of pregnancy. P4 maintains pregnancy, in part, by blocking labor. In all species examined so far, disruption of P4 synthesis and/or action induces parturition. The labor-blocking actions of P4 are mediated by its interaction with specific nuclear P4 receptors (PRs) in uterine myometrial, decidual, and cervical cells. The PRs function as ligand-activated transcription factors and, as such, P4/PR-dimer signaling is thought to block labor by affecting the transcription of genes whose net effect promotes myometrial relaxation, cervical closure, and decidual quiescence. In all species examined so far, disruption of P4/PR signaling induces parturition, and therefore it is generally considered that withdrawal of P4/PR signaling is a common terminal event in the physiology of parturition. In most mammals this is caused by a systemic decrease in maternal P4 levels. For example, in mice, P4 required to sustain pregnancy is provided by maternal corpus luteum (CL), and parturition is triggered by CL regression leading to a systemic P4 withdrawal. In sheep, P4 is produced by the placenta and parturition is triggered by a prepartum surge of cortisol production accompanying maturation of the fetal hypothalamic-pituitary-adrenal axis. Rising cortisol increases activity of trophoblast 17-hydroxylase and 17,20-lyase that convert P4 to androstenedione, which is aromatized to estrogen, leading to systemic P4 withdrawal.

It is believed that human parturition is also induced by functional P4 withdrawal. As with other species, disruption of P4/PR signaling at all stages of pregnancy induces parturition. In women, the placenta produces P4 de novo from cholesterol beginning at around the end of the first trimester. At this time, referred to as the luteo-placental shift, the placenta becomes the principal source of P4. Placental P4 synthesis markedly increases with advancing gestation, leading to high P4 levels in the maternal circulation, declining only after delivery of the placenta. The human placenta lacks 17-hydroxylase/17,20-lyase. Thus human parturition occurs without systemic P4 withdrawal. One explanation for this paradox is that human parturition is probably triggered by a “functional” progesterone withdrawal whereby cells in the myometrium, cervix, and decidua become refractory to P4/PR signaling. Several potential mechanisms for PR-mediated functional P4 withdrawal have been proposed, including reductions in overall genital tract PR expression. Another hypothesis is that it occurs via changes in the net transcriptional activity of the PR isoforms, PR-A and PR-B. Although both PRs function as ligand-activated transcription factors, at some gene promoters PR-A represses the activity of PR-B. Thus P4 responsiveness can be decreased by increased transrepressive activity of PR-A. Interestingly, abundance and transrepressive activity of PR-A in myometrial cells are each increased by site-specific phosphorylation and by exposure to proinflammatory stimuli. This suggests a mechanism for inflammation-induced parturition whereby proinflammatory/prolabor stimuli induce PR-A-mediated functional P4 withdrawal by promoting phosphorylation of PR-A. Other potential mechanisms for functional P4 withdrawal include decreased abundance of critical PR coactivators, inhibition of PR-B transcriptional activity by the NF-κB transcription factor complex, and increased abundance of specific miRNAs that alter the expression of transcriptional coregulators required for PR action. P4 withdrawal may also be caused by the conversion of P4 to a less active form within uterine target cells and potentially lead to excess unliganded PR-A promoting expression of genes encoding contraction-associated proteins. Thus various upstream pathways to parturition may induce specific P4 withdrawal mechanisms to trigger human parturition.

As discussed earlier, it is now generally accepted that parturition is induced by inflammatory processes, with the prelude to labor associated with edema, neutrophil infiltration, and expression of chemical mediators of inflammation, especially proinflammatory cytokines, chemokines, and prostaglandins in the myometrium, cervix, and decidua. Implicit in the hypothesis that labor is induced by intrauterine and tissue-level inflammation is the concept that parturition is blocked by P4, at least in part, by repressing responsiveness to proinflammatory and prolabor stimuli. Indeed, in vitro studies show that P4, via its interaction with PR-B, inhibits the responsiveness of myometrial cells to proinflammatory stimuli and that this effect is inhibited by PR-A. ^, Thus it is possible that withdrawal of PR-B–mediated antiinflammatory activity by PR-A allows for the activation of proinflammatory pathways that induces tissue-level inflammation that transforms the myometrium, cervix, and decidua to the laboring state.

The functional interaction between inflammation and P4/PR signaling suggests a model for the hormonal control of parturition. ^, It is plausible that the gravid uterus is exposed to multiple physiologic (e.g., uterine distention, fetal maturation, placental senescence, maternal metabolic stress) and pathologic (e.g., intrauterine infection, abruption, maternal environmental stress) proinflammatory stressors throughout pregnancy. An inflammatory load threshold may exist that, when surpassed, leads to functional P4/PR withdrawal. This paradigm predicts that the timing of parturition is determined by the trajectory of the inflammatory load increase and the threshold for inflammation-induced functional P4 withdrawal. It is also possible that P4 withdrawal in the decidua in response to infection and abruption may involve downregulation of both PR isoforms mediated by interleukin (IL)-1β and thrombin, respectively, ^, which would explain the higher incidence of PTB associated with intrauterine infection and decidual hemorrhage. Studies in myometrial cells also show that factors acting via the cAMP/PKA signaling cascade augment antiinflammatory activity of P4/PR-B, further supporting the hypothesis that a balance exists between pro- and antiinflammatory stimuli that determines the contractile phenotype of the uterus.

Prostaglandins as Key Activators of the Common Pathway of Parturition

Prostaglandins are viewed as crucial mediators for the onset of labor because they can induce myometrial contractility, ^{, , ,} promote proteolysis of cervical and fetal membrane extracellular matrices to cause cervical ripening and fetal membrane rupture, ^{, , , ,} and stimulate decidual/membrane activation. Evidence of a role for prostaglandins in the initiation of human parturition includes:

• 1.

  Administration of prostaglandins induces termination of pregnancy. ^,

• 2.

  Inhibition of prostaglandin synthesis by indomethacin delays the spontaneous onset of parturition in animals.

• 3.

  Concentrations of prostaglandins in plasma and amniotic fluid increase during labor.

• 4.

  Intraamniotic injection of the prostaglandin precursor arachidonic acid induces abortion.

• 5.

  Expression of myometrial prostaglandin receptors increases in labor. ^,

Labor is associated with increased expression of prostaglandin endoperoxide synthase 2 (PTGS-2) messenger RNA (mRNA). Human PTGS2, also known as cyclooxygenase-2 (COX-2), is encoded by the PTGS2 gene and is involved in conversion of arachidonic acid to prostaglandin H ₂ , an important precursor of prostacyclin, which is expressed in inflammation. Increased activity of PTGS2, but not PTGS1 (COX-1), in the amnion (a rate-limiting step in the production of prostaglandins) is observed during human parturition with significant upregulation prior to and in association with labor. This increase in fetal membranes PTGS-2 activity is accompanied by decreased expression of the prostaglandin-metabolizing enzyme 15-hydroxyprostaglandin dehydrogenase in the chorion, and this combined action allows prostaglandins produced in the amnion to traverse the chorion and decidua and then reach the myometrium and stimulate contractions. The increase in PTGS-2 activity is induced by an increase in NF-κB activity in both amnion and myometrim. The importance of NF-κB and cytokine-mediated inflammation in the induction of PTB is further underscored by the demonstration that an NF-κB inhibitor can reduce lipopolysaccharide (LPS)-induced PTB in a mouse model.

NF-κB and prostaglandins activate common pathways of parturition by the following biochemical mechanisms:

• 1.

  Prostaglandins directly promote uterine contractions by increasing sarcoplasmic and transmembrane calcium fluxes and through increased transcription of oxytocin receptors, connexin-43 (gap junctions), and the prostaglandin E ₂ (PGE ₂ ) receptors EP ₁ , EP ₂ , EP ₃ , and EP ₄ (although EP ₃ appears to be the predominant receptor subtype ) and the PGF _2α receptor FP.

• 2.

  Prostaglandins induce synthesis of matrix metalloproteinases (MMPs) by fetal membranes and cells in the uterine cervix to promote membrane rupture and cervical ripening. ^,

• 3.

  PGE ₂ and PGF _2α increase the ratio of myometrial expression of the PR isoforms PR-A and PR-B, inducing functional progesterone withdrawal.

• 4.

  PGF _2α but not PGE ₂ suppresses term decidual cell PR expression.

• 5.

  NF-κB activation induces activation of a cassette of inflammatory genes, which may also induce a functional progesterone withdrawal.

Fig. 7.3 describes the molecular mechanisms implicated in the common pathway of parturition.

Biochemical Pathways Linking Preterm Birth to Inflammation and Intraamniotic Infection

That intrauterine infection induces PTB is suggested by at least three lines of evidence. First and most compelling, either intrauterine infection or systemic administration of microbial products (e.g., bacterial endotoxin) to pregnant animals results in spontaneous preterm labor and PTB. Second, subclinical intrauterine infections are consistently associated with preterm labor and PTB in humans. ^, Third, pregnant women with either intraamniotic infection or intraamniotic inflammation (defined as an elevation of amniotic fluid concentrations of proinflammatory cytokines, ^, matrix-degrading enzymes, and/or the presence of a specific set of antimicrobial peptides [e.g., defensins, calgranulins ] in midtrimester) are at increased risk for spontaneous PTB later in that pregnancy ( Fig. 7.6 ).

Culture-based data suggest that a large number of intraamniotic infections are polymicrobial. ^, Based solely on microbial cultures, the most common microorganisms identified in the fetal membranes and amniotic fluid of patients with infection-associated PTB are Ureaplasma urealyticum, Mycoplasma hominis, Gardnerella vaginalis, group B Streptococcus (GBS), Bacteroides species, and Escherichia coli. ^, Listeria monocytogenes is a much rarer participant. However, culture techniques are limited as a diagnostic test for infection in the amniotic cavity and elsewhere. Metagenomics, which uses genomics techniques to study communities of microbial organisms without the need to isolate and culture them, has shown that many environmental and human microbial species cannot be cultured. ^, Reasons include the low number of these organisms, their slow growth or resistance to being cultured in conventional media, and their specialized growth requirements. The cornerstone of genomics-based detection methods involves sequencing of full-length or variable regions of the bacterial 16 S ribosomal RNA (16 S -rRNA) gene. This gene is characterized by a high degree of conservation and a clustering of the variable regions of the 16 S -rRNA gene into discrete taxonomic units; the latter allows an in-depth characterization of species richness and the diversity of complex microbial communities. Metagenomic studies show that the diversity of the amniotic fluid microbiome is rich and is characterized by the presence of “uncultivated” and difficult-to-cultivate species, such as Sneathia, Fusobacterium nucleatum, Bergeyella, Clostridiales, Peptostreptococcus, and Bacteroides. ^, Furthermore, genes specific to microbial strains that are present in the amniotic fluid are now identified through the use of molecular techniques. These genes code for surface proteins with potential roles in invasiveness and the ability to cross biological barriers, such as fetal membranes.

The Human Microbiome Project was conceived to provide resources to enable the study of the human microbial communities that live in symbiosis with humans or play a role in human disease (see https://commonfund.nih.gov/hmp ). Advances in next-generation sequencing and metagenomic computational analysis have revealed that maternal oral, vaginal, gut, cervical, and placental microbiomes affect pregnancy outcomes. The project demonstrated that traditional culture techniques no longer meet all clinical needs. Using 16S-rRNA techniques, Ravel and coworkers classified nonpregnant vaginal microbial communities into five community groups, dominated by (I) Lactobacillus iners, (II) L. crispatus, (III) L. gasseri, or (IV) L. jensenii. In the fifth community, Lactobacillus had lower preponderance with a dominance of strictly anaerobic organisms. Major differences characterized pregnancy status and ethnic groups. Interestingly, in one study, the vaginal microbiome diversity and richness were reduced in pregnancy, with predominance of Lactobacillus species. In pregnancy, the presence of various Lactobacillus species is a major determinant to the stability of this microflora. Longitudinal studies performed from the first to the third trimester showed that L. crispatus promotes stability of the normal vaginal microflora, whereas both L. gasseri and L. iners predispose to abnormal vaginal microbiomes. A longitudinal analyses of 16S ribosomal RNA, metagenomic, metatranscriptomic, and cytokine profiles from preterm and term birth controls found that women who delivered preterm exhibited a significant increase in diversity with significantly lower vaginal levels of Lactobacillus crispatus but increased levels of bacterial vaginosis–associated bacterium-1 (BVAB1), Prevotella cluster 2, and Sneathia amnii. That the vaginal microbiome is different in pregnant versus nonpregnant women was also confirmed by other investigators. Whereas some authors report an increased risk of PTB in African American women with vaginal microbial community type IV (L. jensenii), accompanied by elevated Gardnerella or Ureaplasma abundance, others report no association between vaginal microbiome diversity and abundance and PTB.

Because the amniotic fluid is normally sterile and most pathological intraamniotic bacteria are genital tract microorganisms, the current paradigm of intrauterine infection implies that bacteria originate from the lower genital tract and invade the pregnant uterus via an ascending route. Once the mechanical and innate immune barriers of the cervix are bypassed ( Fig. 7.7 ), microorganisms infect the decidua and then penetrate the fetal membranes to invade the amniotic fluid. ^, Finally, these microorganisms infect the fetus. This biologically plausible conceptual framework is based on studies demonstrating the following:

• 1.

  As noted above, microorganisms frequently implicated in intraamniotic infection (e.g., GBS, Mycoplasma, E. coli ) are common constituents of the vaginal microbiome as determined by both culturing and molecular techniques ^, and are cohabitants of the amniochorionic space.

• 2.

  The presence of Ureaplasma and Mycoplasma in amniochorion incites polymorphonuclear tissue infiltration and a higher degree of histologic chorioamnionitis in pregnancies complicated by PTB.

• 3.

  In vitro, GBS and E. coli can attach to chorioamniotic membranes.

• 4.

  In animal models of infection-induced PTB, transcervical and choriodecidual inoculation of GBS is followed by transmigration of bacteria from the choriodecidual space to the amniotic fluid cavity, a graded amniotic fluid leukocyte infiltration response, and elevated levels of proinflammatory cytokines (TNF-α, IL-6, IL-1β) and prostaglandins (PGE2, PGF2α), as well as increased uterine activity. ^,

Using mass spectrometry (MALDI-TOF) to profile bacteria in amniotic fluid and vaginal secretions in parallel with 16S-rRNA gene sequencing, women with intrauterine inflammation or infection or both (“Triple I”) were found to have amniotic fluid containing commensal bacteria, which were identified in the vagina at the time of the procedure. A secondary route of intraamniotic infection likely involves hematogenous transplacental seeding of the placenta and fetus by infectious microorganisms, in particular Haemophilus influenzae or F. nucleatum, originating from other parts of the body, including the mouth. ^, Using 16 S -rRNA DNA-based methods, Aagaard and colleagues identified a placental microbiome composed of nonpathogenic commensal microbiota, including Firmicutes, Tenericutes, Proteobacteria, Bacteroidetes, and Fusobacteria phyla. The authors concluded that placental microbiome profiles were closest to the human oral microbiome. In a follow-up study the same laboratory used in situ hybridization probes designed against highly conserved regions of the bacterial 16S ribosome and determined that low-abundance microbes were localized to villous parenchyma and/or syncytiotrophoblast even in the absence of histological chorioamnionitis.

The notion that the placenta is not “sterile” has been disputed by several groups of investigators. Lauder and associates compared placental samples from healthy deliveries with a set of contamination controls and matched oral and vaginal samples. Using 16 S -rRNA gene and quantitative polymerase chain reaction (PCR) technologies, the authors found that placental samples and negative controls contained low and indistinguishable copy numbers. This contrasted with oral and vaginal environments, which showed higher copy numbers. Based on this evidence the authors concluded placental samples have low to nonexistent bacterial loads. A similar conclusion was reached by Theis and collaborators. Leiby and colleagues compared bacterial levels in women whose pregnancies were complicated by PTB with those who delivered at term and reported only low bacterial levels in the placenta and disagreed with the concept that the placenta harbors a specific microbiome that could be involved in triggering inflammation and PTB.

Iatrogenic infections can also occur during invasive procedures such as chorionic villous sampling, amniocentesis, and cordocentesis. Retrograde microbial seeding of the amniotic fluid through the fallopian tubes or colonization of the uterine endometrium before implantation has also been proposed. However, compelling evidence in support of these pathways remains to be provided.

Role of the Innate Immune Response in Inflammation-Associated Preterm Birth

Emerging evidence suggests that microorganisms are “sensed” by the innate components of the immune system, leading to a cascade of events that culminate in PTB. These sensing components include soluble pattern recognition receptors (PRRs), lectin, and C-reactive protein. The transmembrane PRRs include scavenger receptors, C-type lectins, and Toll-like receptors (TLRs). Intracellular PRRs include nucleotide-binding oligomerization domain (NOD)1 and NOD2, retinoic acid–induced gene type 1, and melanoma differentiation–associated protein 5, which mediate recognition of intracellular pathogens (e.g., viruses). The TLRs are the best-studied PRRs. Because of their strategic positioning at the maternal-fetal interface (the decidua), fetal membranes, and myometrium, TLR4 and TLR2 are considered major mediators by which the maternal and fetal reproductive tissues can respond to infection. TLR4 is recognized as the membrane-bound receptor that triggers LPS signaling by gram-negative microbes. A strain of mice bearing a spontaneous disabling mutation for TLR4 is more resistant to PTB than wild-type mice after intrauterine inoculation of heat-killed bacteria or administration of LPS. ^, Involvement of TLR2 has been shown in recognition of lipoproteins, peptidoglycan, and glycolipids of gram-positive bacteria and Mycoplasmataceae. How TLRs distinguish between commensal and pathogenic microorganisms in vaginal or other sites remains unknown. Studies exploring host-microbial interactions demonstrated that beneficial bacteria secrete effector molecules that activate a cell survival pathway involving p38 mitogen-activated protein kinase (MAPK) and protein kinase B (Akt). Although the effectors of such pathways have been previously established in the human myometrium, such interaction has not been proven.

Although the full spectrum of TLR-mediated responses remains to be elucidated, it is known that, once engaged by pathogen-associated molecular patterns (PAMPs), these pathogen sensors trigger a downstream molecular chain of events that leads to synthesis and release of proinflammatory cytokines such as TNF-α; interferon-γ; cytokines IL-12, IL-6, IL-1β; and many others via an NF-κB–mediated mechanism. Key chemokines secreted after TLR activation include IL-8; monocyte chemoattractant proteins 1, 2, 3, and 4; macrophage inflammatory proteins 1α and 1β; and RANTES (regulated on activation, normal T cell expressed and secreted).

Traditionally, activation of TLRs has been implicated in inducing a T helper cell 1 cytokine-type response (i.e., IL-2, interferon-γ, lymphotoxins). However, using genetically engineered animal models and a variety of cell culture systems, Pulendran and colleagues showed that TLR4 engagement can also trigger a T helper cell 2 cytokine reaction consistent with the release of IL-4, IL-5, IL-6, and the antiinflammatory cytokine IL-10, depending on bacterial type. The significance of this observation during human pregnancy remains to be clarified, but these results underline the concept that the balance among proinflammatory and antiinflammatory cytokine responses can dictate the intensity and potential resolution of an infectious process.

The biological activity of the TLRs depends not only on gestational age and presence of PAMPs but also on a palette of intracellular signaling adaptors (e.g., myeloid differentiation primary response 88) and coreceptor molecules (e.g., CD14) that associate with TLRs in complex supramolecular arrangements. Equally important is the concept that TLR signaling can be elicited by endogenous damage-associated molecular patterns (DAMPs). Like cytokines, DAMPs (e.g., high-mobility group box-1 protein [HMGB1, or amphoterin], S100β proteins) are endogenous proinflammatory and prooxidative stress molecules. Acting through TLR2, TLR4, and the receptor for advanced glycation end products (RAGE), DAMPs recruit inflammatory cells that in turn amplify innate immune responses and enhance levels of cytokine activation. The RAGE-DAMP system is active in women with PTB and intraamniotic infection. Activation of the RAGE-DAMP system correlates with the degree of inflammation and oxidative stress damage in amnion epithelial, decidual, and extravillous trophoblast cells ( Fig. 7.8 ). PAMPs and DAMPs may continue to keep active the processes that lead to fetal cellular damage.

The roles of soluble receptor modulators (soluble TLR2, soluble TNF receptor 1, soluble IL-6 receptor, soluble glycoprotein 130, and soluble RAGE) in fine-tuning human TLR-mediated signaling have just begun to be elucidated. Downstream of the TLR receptor, other molecules such as prokineticin amplify the inflammatory response. Accordingly, lentiviral knockout of the prokineticin receptor inhibits the ability of the myometrium to produce proinflammatory cytokines in response to LPS.

Transcriptomics: Gene Pathways and MicroRNAs in Inflammation-Associated Preterm Birth

Recent advances in transcriptomics enabled identification of gene pathways involved in parturition and the response of decidua and placenta to inflammatory stimuli. Stimulation of cultured decidual cells with IL-1β altered the expression of several microRNAs (miR-146a-5p, miR-525-5p, miR-143-3p, miR-145-5p, miR-924, miR-4454). The more significantly expressed genes induced by IL-1β were coding sequences for chemokines, cytokines, prostaglandin-synthesizing enzymes, microsomal PGE synthase, transcriptional regulators, and MMP-1. In vivo, in the placenta, unique long transcripts and mature microRNAs (miRNAs) differed significantly between pregnancies complicated by infection and idiopathic PTB. The upregulated transcripts included cathepsin S, lysozyme, hexokinase 3, miR-133a, and miR-223. In the setting of intraamniotic infection the top-ranking gene sets included TLR and IL-8 receptor signaling pathways, as well as integrin binding and cell cycle regulation genes (e.g., polo-like kinase signaling and the forkhead box protein M1 transcription factor networks). Interestingly, some of these gene profiles are associated with neurodevelopmental impairment at 18 to 24 months of age.

Similarly, Ackerman and associates reported that several miRNAs and long RNAs are differentially expressed in the myometrium of women who delivered preterm in the setting of histologic chorioamnionitis compared to that of women who had an idiopathic PTB (absent inflammation). Only a few altered miRNAs, exemplified by the primate-specific miR-888 cluster, were commonly expressed by both preterm and term laboring myometrium. More often PTB myometrium was characterized by alterations in genes associated with inflammatory and immune pathways, whereas the pathways associated with term labor were considerably more diverse. The variety of clinical phenotypes associated with PTB complicate our understanding of the complex landscape of prematurity. For example, a recent paper presented a two-hit hypothesis indicating that subclinical placental viral infections sensitize women to intrauterine bacterial infection or to placental microflora itself. Viral infection of the placenta downregulates type 1 interferon (IFN), an immunomodulator that suppresses the immune response. Loss of IFN changes the immune response to low concentrations of bacteria to a robust inflammatory response. Viral infection inhibits IFN-β expression and inhibits signal transducer and activator of transcription 3 activation and loss of Twist-related protein 1, leading to increased NF-κB and proinflammatory responses.

Role of Cytokines and Other Inflammatory Mediators in Inflammation-Associated Preterm Birth

Inflammation and its mediators (e.g., chemokines such as IL-8, proinflammatory cytokines such as IL-1β and TNF-α, and others, such as platelet activating factor and prostaglandins) are central to infection-induced PTB. IL-1β was the first cytokine implicated in the onset of infection-associated PTB. Evidence that IL-1β plays a crucial role in the pathogenesis of inflammation-associated PTB includes the following:

• 1.

  It is synthesized by human decidua in response to bacterial products.

• 2.

  It stimulates prostaglandin production by human amnion and decidua.

• 3.

  IL-1α and IL-1β concentrations and IL-1–like bioactivity are increased in the amniotic fluid of women with preterm labor and infection.

• 4.

  Intravenous administration of IL-1β stimulates uterine contractions.

• 5.

  Administration of IL-1 to pregnant animals induces preterm labor and birth, which can be blocked by the administration of its natural antagonist, the IL-1 receptor antagonist.

• 6.

  Chorioamnionitis is associated with increased decidual IL-1β expression, and the latter directly inhibits term decidual cell PR expression.

Evidence supporting the role of TNF-α in mediating infection-associated PTB includes the following:

• 1.

  TNF-α stimulates prostaglandin production by amnion, decidua, and myometrium.

• 2.

  Human decidua produces TNF-α in response to bacterial products. ^,

• 3.

  Amniotic fluid TNF-α bioactivity and immunoreactive concentrations are elevated in women with preterm labor and intraamniotic infection.

• 4.

  In women with preterm PROM and intraamniotic infection, TNF-α concentrations are higher during labor.

• 5.

  In amniochorion explant systems, TNF-α stimulates production of MMPs, ^, which have been implicated in membrane rupture.

• 6.

  Application of TNF-α to the cervix induces changes that resemble cervical ripening.

• 7.

  TNF-α can induce preterm parturition when administered systemically to pregnant animals. ^,

TNF-α and IL-1β each enhance IL-8 expression by decidual cells, a chemokine strongly expressed by term decidual cells in the presence of chorioamnionitis. Other cytokines and chemokines (IL-6, IL-10, ^{, ,} IL-16, IL-18, colony-stimulating factors [CSFs], macrophage migration inhibitory factor, IL-8, ^, monocyte chemoattractant protein-1, epithelial cell–derived neutrophil-activating peptide 78, neutrophil gelatinase-associated lipocalin, ^, and RANTES ) have also been implicated in infection-induced PTB. The large number and redundancy of molecules involved in the cytokine network implicated in parturition indicate that blockade of a single cytokine is unlikely to be sufficient to prevent PTB in the setting of infection. For example, preterm labor after exposure to infection can occur in knockout mice for the IL-1 type 1 receptor, suggesting that IL-1β is sufficient, but not necessary, for the onset of parturition in the context of intraamniotic infection or inflammation. Of note, blockade of multiple signaling pathways (e.g., IL-1β and TNF-α) in a double-knockout mouse model decreased the rate of PTBs after the administration of microorganisms.

In the setting of intraamniotic infection, a large array of cytokines (e.g., IL-6, IL-8, IL-1β, and GM-CSF) are found in the amniotic fluid. The sources of cytokines in amniotic fluid include the decidua, fetal membranes, and the fetus. However, independent of source, amniotic fluid IL-6 and many other cytokines induce recruitment of fetal neutrophils. Cytokines also induce degranulation of granulocytes, which then release MMP-1. Except for TLR3, human leukocytes express mRNAs for TLR1 through TLR10. Expression of TLR2 is higher in circulating leukocytes obtained from women in labor than from pregnant women not in labor. Thus neutrophil secretion of cytokines and chemokines likely follows their recognition of a large repertoire of bacterial PAMPs and cellular DAMPs. Taken together, these observations highlight the maternal and fetal involvement in the process of intraamniotic inflammation and the role of mother and fetus in amplification of the inflammatory status of the amniotic fluid and tissue damage in a forward loop fashion.

The complement system favors placental development and function and is important in sustaining both host defense and fetal development. Increasing evidence points to a role for complement in inflammation-induced PTB. Increased cervical deposition of the split complement product C3 was noted in mouse models of preterm labor induced both by LPS and by progesterone withdrawal. Though complement activation was believed to be restricted to preterm labor and absent from physiologic parturition at term, ^, recent studies dispute these findings. Soluble terminal complement complex SC5b-9 levels are increased in the plasma of women during term labor, preterm labor, and preterm PROM compared to women not in labor.

Many antiinflammatory mediators are known to operate in the pregnant uterus. The most widely studied of these is the antiinflammatory cytokine IL-10, which is thought to be important for the maintenance of pregnancy. Its concentrations are increased in intraamniotic inflammation, suggesting that IL-10 may play a role in damping the inflammatory response and may have therapeutic value. IL-10 knockout mice are more sensitive to LPS-induced preterm labor than wild-type mice, a defect that is ameliorated by external IL-10 administration. In wild-type animals, exogenous IL-10 also attenuates the preterm labor phenotype. For example, pregnant rhesus monkeys were allocated to one of three interventional groups: (1) intraamniotic IL-1β infusion with maternal dexamethasone intravenously, (2) intraamniotic IL-1β and IL-10, or (3) intraamniotic IL-1β administered alone. Dexamethasone and IL-10 treatment significantly reduced IL-1β–induced uterine contractility. The amniotic fluid concentrations of TNF-α and leukocyte counts were also decreased by IL-10 treatment. In addition to these beneficial effects on inhibition of contractility and inflammation, administration of IL-10 in animal models of infection has been associated with improved pregnancy outcome. ^,

Another major group of antiinflammatory molecules, the lipoxins, is also expressed in the reproductive tract. Lipoxins are members of a group of proresolution molecules that appear to actively terminate the inflammatory process by promoting neutrophil engulfment and inhibiting proinflammatory cytokine expression. Lipoxins circulate in increasing concentrations as pregnancy advances, their receptor is present in the myometrium of pregnant women, and they attenuate the myometrium’s proinflammatory cytokine response to LPS. ^, In animal models of endotoxin-induced PTB, lipoxins did not delay PTB but reduced the mortality among premature pups, potentially via a prostaglandin-induced pathway. Therefore lipoxins are promising therapeutic agents to prevent prematurity-related fetal damage.

Bacterial Species and the Intensity of Intraamniotic Inflammatory Responses

Microorganisms can stimulate the production of proinflammatory cytokines through activation of fetal membrane TLR receptors. Several microorganisms (e.g., Ureaplasma, Mycoplasma ) are traditionally considered to have low virulence. ^, Studies describing the presence of Ureaplasma parvam and M. hominis in the amniotic fluid of second-trimester asymptomatic women are in support of this concept. Menon and coworkers demonstrated in vitro that in comparison with gram-positive and other gram-negative bacteria, Ureaplasma has a lower inflammatory effect on fetal membranes. Paradoxically, isolation of Ureaplasma and Mycoplasma from amniotic fluid is consistently associated with a wide range of adverse pregnancy outcomes, such as early abortion, stillbirth, prematurity, and neonatal morbidity and mortality. Although an intense intraamniotic inflammatory response is often encountered at the time of clinical onset of PTB, these studies prove association, not causation. Evidence that these so-called silent microorganisms can trigger an inflammatory response in vivo that can induce PTB was provided by Novy and colleagues. They inoculated U. urealyticum and M. hominis into the amniotic fluid of rhesus monkeys, which resulted in an increase in a myometrial contractile activity that was preceded by an intense inflammatory cytokine response and increased prostaglandin synthesis.

Invasion of the amniotic fluid by gram-positive anaerobes, E. coli, and GBS results in intraamniotic inflammation and fetal sepsis. However, intraamniotic inflammation can also occur in the absence of positive amniotic-fluid culture results. In this regard, as previously mentioned, “uncultivated” or difficult-to-cultivate bacteria may play an important role. The extent of an intraamniotic inflammatory response triggered by such bacteria, separately or as a group, remains to be determined.

Intraamniotic Inflammatory Responses in the Absence of Infection

The term sterile inflammation is used to describe the presence of intraamniotic inflammation in the absence of a demonstrable infection. Although this concept is not new, it can be related to inability of the traditional microbial cultures to detect bacteria, or to release of cellular mediators of inflammation, including HMGB1 and RAGE, or to bleeding. ^{, , ,} The relevance of DAMPS in non–microbial-induced inflammation was demonstrated in vivo in an animal model of HMGB1-induced PTB. Calciprotein particles have been proposed as a novel trigger of preterm PROM in the absence of infection. According to this mechanism, aberrant aggregation of hydroxyapatite into calciprotein particles and the hydroxyapatite-induced differentiation of mesenchymal cells into osteoblasts (ectopic osteogenesis) promote inflammation and prostaglandin synthesis. Fetal membranes from women with idiopathic preterm PROM frequently show evidence of ectopic calcification and expression of osteoblastic differentiation markers. Concentrations of fetuin-A, an endogenous inhibitor of ectopic calcification, were decreased in amniotic fluid of idiopathic preterm PROM cases, which reflected their reduced functional capacity to inhibit calcification. When applied to amniochorion explants, amniotic fluid–derived calciprotein particles induced structural and functional pathologic effects recapitulating those observed after preterm PROM. Recent data suggest that sterile inflammation is present in 5% of women who are displaying signs and symptoms of clinical chorioamnionitis at term, implying that uncultivable bacteria in this clinical scenario may play a role.