Intrauterine infection is an important cause leading to preterm delivery, which occupies approximately 25% of preterm delivery cases [11]. Infection can occur between maternal decidua and fetal chorion (choriodecidual space) by bacteria ascending from vagina [12]. The most commonly identified bacteria are Ureaplasma urealyticum, Mycoplasma hominis, Gardnerella vaginalis, Peptostreptococcus, and Bacteroides species—all vaginal organisms of relatively low virulence [12]. Prevalence of a Lactobacillus-poor vaginal community is inversely correlated with gestational age at delivery [13]. Nearly one-third of women with Lactobacillus-poor vaginal community delivered very preterm infants. In contrast, at least three-quarters of women who carried their pregnancies to term had Lactobacillus-dominant vaginal microbiota. Risk for preterm birth was more prominent for subjects with elevated Gardnerella or Ureaplasma abundances [13]. Interestingly, hyaluronic acids play an important role in epithelial barrier protection of the lower reproductive tract from bacteria. Depletion of hyaluronic acids in the cervix and vagina resulted in increased epithelial and mucosal permeability of bacteria and increased preterm delivery rates in mice [14]. Thus, keeping healthy vaginal microbiota is a key for successful pregnancy.

The mechanisms by which intrauterine infections lead to preterm delivery are related to activation of the innate immune system (Fig. 15.2). Microorganisms are recognized by pattern recognition receptors—such as toll-like receptors (TLRs). TLRs are a family of transmembrane receptors that are involved in the regulation of the innate immune system [15]. TLR4 exists in both epithelial cells and mesenchymal cells of amnion [16]. TLR4-deficient mice are resistant to preterm delivery by intrauterine inoculation of heat-killed bacteria or LPS [17]. Activation of TLRs elicits the release of inflammatory chemokines and cytokines—such as IL-8, IL-1β, and tumor necrosis factor (TNF) α. Microbial endotoxins and proinflammatory cytokines stimulate the production of prostaglandins and matrix-degrading enzymes (matrix metalloproteinases, MMPs), which lead to preterm rupture of membrane. In the myometrium and cervix, proinflammatory cytokines activate inflammation-associated transcriptional factors such as NFκB and AP-1, inhibiting progesterone receptor (PR) function, which induces the expression of myometrial contractile genes [18]. In the cases of preterm delivery, concentrations of proinflammatory cytokines increase in amniotic fluid and migration of neutrophils and macrophages into the myometrium, cervix, and fetal membranes is observed [8, 19, 20]. Thus, inflammation plays an important role in both term and preterm delivery. In addition, intra-amniotic infection also attacks a fetus, causing a fetal systemic inflammatory response (FIRS). The concept of FIRS is determined by elevated fetal plasma IL-6 level [21]. FIRS is a risk factor for severe neonatal morbidity such as respiratory distress syndrome, neonatal sepsis, pneumonia, chronic lung disease, necrotizing enterocolitis, intraventriclular hemorrhage, and cerebral palsy [21]. Thus, although it is important to prevent intrauterine infection, immediate medical intervention to delivery is required once the sign of intra-amniotic infection appears to prevent a fetus from damage.

Throughout most of pregnancy, uterine quiescence is maintained by elevated progesterone acting through progesterone receptor (PR) [18]. In human, serum progesterone concentrations do not fall as labor approaches, so a decrease in local progesterone concentrations or number of receptors is a plausible mechanism of decline in PR function [22]. Progesterone antagonizes the inflammatory pathway such as NFκB and AP-1 by acting nuclear progesterone receptor (PR) and suppresses proinflammatory cytokines and chemokines. When pregnancy comes close to term, circulating estradiol-17β (E2) levels increases [23, 24], and enhanced estrogen receptor α (ERα) activity is enhanced [25, 26], which promote a proinflammatory cascade that contribute to the decline in PR function and initiate myometrial contraction (Fig. 15.2). Estrogens also induce an influx of macrophages and neutrophils into the uterus and further enhance proinflammatory event [27]. ERα activation facilitates myometrial contraction by enhancing transcription of the contraction-associated genes of the uterus, such as oxytocin receptor, connexin-43, and COX2 [25, 28–30]. The expressions of these contraction-associated genes are low throughout most of pregnancy but are highly upregulated at term.

A microRNA is a small noncoding RNA molecule (containing about 22 nucleotides) found in plants, animals, and some viruses, which functions in RNA silencing and posttranscriptional regulation of gene expression [31]. Recently, miR-200 family is found to be closely associated with labor. In both mouse and human uterus, miR-200 family (miR-200b and miR-429) is highly induced at term, whereas its target genes, ZEB1 and ZEB2, zinc finger E-box binding homeobox proteins, are downregulated [32]. ZEB1 and ZEB2 are transcriptional factors that are associated with epithelial mesenchymal transition. ZEB1 is directly upregulated by the action of P4/PR. ZEB1 and ZEB2 not only inhibit expression of the contraction-associated genes, oxytocin receptor and connexin-43, but also block oxytocin-induced contractility in human myometrial cells. The downregulation of ZEB1 and ZEB2 was observed in LPS- or RU486- induced mouse preterm delivery model. Thus, the miR-200 family and their targets, ZEB1 and ZEB2, are P4/PR-mediated regulators of uterine quiescence and contractility during pregnancy and labor.

Preterm premature rupture of membrane (pPROM) is associated with about one-third of preterm delivery cases and occurs in 1–3% of all pregnancies. The primary load-bearing structure of the fetal membranes is the amnion, which comprises a single layer of epithelial cells and an underlying layer of mesenchymal cells [33]. Mesenchymal cells are the primary source of collagen and matrix support in the amnion. Interstitial collagens (types I, III, and V) maintain the mechanical integrity of the amnion. Fetal membrane rupture is preceded by the degradation of collagen that is mediated primarily by matrix metalloproteinase (MMPs) in the amnion. Interstitial collagenase, MMP1, cleaves the triple helix of fibrillar collagen, which is then further degraded by the gelatinases, MMP2 and MMP9. pPROM and interauterine MMPs activity is closely correlated. MMP1 in amniotic fluid and MMP9 in amniotic membranes are elevated in women with pPROM [34–37]. Ehlers-Danlos syndrome, inheritable connective tissue disorder, is a risk factor of PROM by a defect in the structure, production, or processing of collagen or proteins that interact with collagen [38, 39].

Fibronectin (FN) is a large extracellular glycoprotein that helps cells attach to the matrix. Fetal FN (fFN) is one of the FN proteins produced by fetal cells. It is diffusely distributed in the fetal membrane, from the amnion to decidua, providing structural support and adhesion of the fetal membranes to the uterine lining, and fFN in cervical and vaginal secretions has been used as a clinical marker of preterm delivery [40]. In vitro, fFN treatment results in increased expression of MMP1 and MMP9, mRNA, and enzymatic activity, as well as COX2 mRNA and PGE₂ synthesis in amnion mesenchymal cells, activating both NFκB and MAPK pathway [41] (Fig. 15.3). fFN has a unique alternatively spliced exon encoding extra domain-A (EDA) [42]. The treatment of amnion mesenchymal cells with recombinant EDA also resulted in increases in MMP1 and MMP9 mRNA levels and enzymatic activity, as well as in the COX2 mRNA level and PGE₂ synthesis, indicating that EDA is a functional domain of fFN, and function of EDA was mediated via TLR4 [41]. Thus, neutralization of fFN-EDA domain or antagonism of TLR4 may have therapeutic potential for preterm delivery and pPROM.

A question is how fFN is increased and released in preterm delivery. Fibronectin-1 (FN1) protein and mRNA were dose-dependently increased by lipopolysaccharide (LPS) or TNF-α treatment in epithelial cells. This data show that epithelial cells of the amnion function as a sensor to harmful inflammatory stimuli and sends a “danger signal” by releasing fFN in the extracellular matrix. Then, mesenchymal cells receive the fFN danger signal from epithelial cells and begin producing MMPs and PGE₂. In other words, fFN “amplifies” the dangerous signal produced by endotoxins and proinflammatory cytokines in order to cause the rupture of the membrane or a preterm delivery via activation of MMPs and PGE2. This amplification of inflammation by fFN function would be evolutionally important because once infection has occurred, a fetus should be immediately released from harmful intrauterine inflammation by delivery. Moreover, the delivery of an already infected fetus is the only way to protect a mother from fatal inflammation such as maternal sepsis.

Intrauterine bleeding or hematoma during early pregnancy is correlated with an increased risk for adverse maternal and neonatal complications. Nagy et al. reported a 2-fold increase in preterm delivery in the hematoma group [43]. Moreover, pregnancy-induced hypertension, preeclampsia, placental abruption, and fetal growth restriction were also frequent in this group. Similarly, Tuuli et al. reported that subchorionic hematoma was associated with a 1.5-fold increase in preterm delivery and pPROM, a 2-fold increase in spontaneous abortion and stillbirth, and a 5-fold increase in placental abruption [44]. These reports indicate that intrauterine bleeding during pregnancy is a strong risk factor of perinatal complications, especially of preterm delivery.

Thrombin is a trypsin-like serine proteinase, the most abundant enzyme associated with blood coagulation. In addition to its role in hemostasis, thrombin also influences normal and pathological processes, such as inflammation, tissue repair, embryogenesis, angiogenesis, and tumor invasion [45]. There is considerable clinical evidence pointing to a role of thrombin in preterm delivery. Thrombin-antithrombin complexes, markers of in vivo generation of thrombin, are increased in the plasma [46, 47] and amniotic fluid [46] of women in preterm labor or pPROM. Placental abruption-induced thrombin generation has been associated with fetal membrane weakening and pPROM [47, 48], and treatment of amnion explants with thrombin results in increased levels of MMP9 and mechanical weakening [49]. In an animal model, intrauterine administration of whole blood to pregnant rats stimulates myometrial contractility, whereas blood containing heparin or a thrombin inhibitor does not [50].

Thrombin activity was significantly increased in amniotic membranes from women who delivered preterm [51]. Considering that the decidua is the primary source of thrombin [52], increased thrombin activity is probably due to the bleeding from the decidua in the early stage of pregnancy, and thrombin activity would remain in the amnion for several months until the preterm delivery finally occurs. In primary amnion cells, thrombin treatment resulted in an increase of MMP1 and MMP9 mRNA and enzymatic activity, conversion of MMP2 to its active form, and COX2 mRNA and PGE₂ synthesis in amnion mesenchymal cells (Fig. 15.3). These activations were mediated by G protein-coupled thrombin receptor, protease-activated receptor-1 (PAR-1), and TLR4 [51]. When thrombin or PBS was locally injected into the uterus of pregnant mice, all thrombin-injected mice delivered preterm, whereas PBS did not [51]. In these mice, collagenase-2 (MMP8) and collagenase-3 (MMP13), gelatinase MMP9 mRNA as well as PGE₂ synthesis were all increased in fetal membranes. Thus, thrombin weakens the membrane by degrading collagen through upregulation of MMPs and stimulates cervical ripening and myometrial contraction through the production of PGE₂.