Introduction

Preterm birth (PTB) is defined as delivery before 37 completed weeks of gestation and its divided into two types: a) spontaneous PTB, which happens on 65-75% of the cases where pregnant women who develop preterm birth present with idiopathic onset of uterine contractions, cervical dilatation and/or premature preterm rupture of membranes (PPROM) [2]; and b) medically indicated PTB due to maternal or fetal indications on the other 25-35% of the cases [1, 3]. Its considered a major public health problem, as approximately 15 million preterm births occur annually worldwide, with an approximate incidence of 5-12% in developed countries [3], but it can be as high as 40% in the poorest ones. It’s the leading cause of mortality in children under 5 years old, as every year 1.1 million of neonates die from consequences of prematurity and its associated with 80% of all neonatal morbidity in developed countries [4]. PTB is highly dangerous for the neonates as it strongly associates with respiratory distress syndrome, intraventricular hemorrhage, periventricular leukomalacia, necrotizing enterocolitis, bronchopulmonary dysplasia [5], patent ductus arteriosus, increased risk for cognitive deficits, cerebral palsy and autism [6], among others. 

Spontaneous labor takes place following the activation of a pro-inflammatory pathway where increased levels of cytokines and prostaglandins lead to an enhanced uterine contractility, cervical dilatation and rupture of membranes before 37 weeks of gestation [7]. Even though its etiopathogenesis is still unknown, some risk factors have been suggested to trigger it, within which we can include a history of preterm birth, African-American (AA) race, smoking during pregnancy, multiple gestations, polyhydramnios, a history of mid-trimester loss, short cervical length which needed cervical cerclage [4], microbial infection or inflammation of the amniotic fluid, placenta and/or vaginal canal, periodontal disease, vascular disease, uterine over-distention [8], and the presence of comorbidities such as diabetes and hypertension. 

Nonetheless, despite the fact that maternal and fetal genetic factors play a determining role in the duration of gestation, ontogenetic factors, including the microbiome, are among the most important contributors [9]. Ascending intrauterine infection occurs on nearly 40% of preterm deliveries, as colonization of the lower genital tract affects a large percentage of preterm pregnancies with PPROM [10] and chorioamnionitis [11]. Bacterial vaginosis is a condition of imbalance of the commensal microbiota that involves reduction of lactobacilli and its replacement by anaerobic microorganisms such as Gardnerella vaginalis, Bacteroides spp., Mobiluncus spp. and Mycoplasma hominis, and this entity increases up 4 times the risk of spontaneous preterm birth [12, 13]. Moreover, a higher prevalence of bacterial vaginosis in AA women compared with non-AA women has been documented, so that bacterial vaginosis might be related to racial disparities [14]. 

Intrauterine infection and inflammatory response

The pathogenesis of premature birth is connected with cytokines, collagen-specific matrix metalloproteinases (MMPs) and prostaglandins, it has been found that the triggering of preterm labor in the presence of infection is the result of the interaction of bacterial products such as phospholipases A2 and C, proteinases and endotoxins [15] with the host monocyte-macrophage system present in the maternal, fetal and placental decidua, in such a way that the change to a contractile state of the myometrium is accompanied by an imbalance between anti-inflammatory and pro-inflammatory signaling. On first place, the pathogens are recognized by toll-like receptors (TLRs) activating the innate immune system and triggering the activation of the Nuclear Factor-Kappa-B (NF-κB) pathway for pro-inflammatory mediators production [16, 17], such as IL-1α, IL-1β, IL-6, IL-8, TNFα, which then stimulate the production and secretion of β-defensins, prostaglandin E2 (PGE2) and MMPs [18]. Finally the separation of the chorio-amniotic membranes from the decidua and its consequent rupture is related to the increased expression of TNFα and IL-1, mobilization of neutrophils that infiltrate the tissue and generate increased activity of proteases such as MMP-8 and MMP-9 that degrade the extracellular matrix of the fetal membranes and dissolution of fibronectin [19, 20].  Cervical maturation is mediated by changes in the proteins of the extracellular matrix such as the loss of collagen and the increase of glycosaminoglycans, which reduces the tension force of the cervix [21]. 

Neutrophil α and β-defensins in the vaginal immune response

Antimicrobial peptides (AMPs) provide the first line of defense against infectious pathogens in the mucosal epithelia, functionally, they can make microbial cells permeable by integrating into their membranes and by the formation of multimeric pores. The most studied are defensins and cathelicidins, the former having a dichotomous response that can suppress inflammation in some circumstances, acting as immunomodulatory agents, or that can enhance inflammation by exerting a microbicidal activity by exacerbating the response to danger in other leukocytes (Fig. 1). 

Figure 1. Functions of antimicrobial peptides.

Human defensins are a family of cationic antimicrobial peptides that contain between 28-42 amino acids, they stimulate the production of cytokines, cell migration, proliferation, maturation, and synthesis of extracellular matrix. In humans there are 2 functional subfamilies on the basis of the spatial distribution of the cysteine residues and connectivity of disulfide-bonds [22]: 1) α-defensins or human neutrophil peptides (HNP), of which there are 4 types (1,2,3, and 4), all being produced by neutrophils and expressed constitutively, but are classically absent in the mucosa or vaginal fluid; 2) β-defensins are host-derived anti-microbial peptides located in the vaginal epithelium, placenta, amniotic epithelium and decidua [23]; type 1 β-defensins are produced by epithelial cells and have a constitutive expression, that is not transcriptionally regulated by inflammatory agents, while types 2 and 3 are induced by infection and inflammation [24] (Table 1). β-defensins 2 activate adaptive immunity through chemotaxis of T-lymphocytes and dendritic cells to the site of infection [25, 26] and β-defensins 3 are chemotactic for macrophages [27]. Defensins are commonly found expressed at basal levels along fetal and maternal tissues during pregnancy, but when an intra-uterine infection occurs, levels of HNP 1-3 [28] and β-defensins-2 become increased in amniotic fluid [29], and they can activate neutrophils and defensins production. 

Interestingly, whether these peptides could work as protective factors against infections or contribute to the risk of PTB is not clear, as conflicting results have been obtained by several research teams. For instance, Balu et al [30] found that bacterial vaginosis increases the expression of β-defensins, and that such response increases the risk of PTB [31], and these data were supported by Xu and colleagues [14], whom made a similar study. On the other hand, Mitchel et al [32] discovered that bacterial vaginosis was associated with lower levels of β-defensins, and according to Kotani et al [33] this may increase the risk of PTB. On frank contrast with these two tendencies, Elovitz and colleagues [2] found that higher β-defensin levels associate with lower potential for vaginosis, and thus a decreased risk for PTB. Finally, Manning and team [34] found no significant association between bacterial vaginosis, β-defensin expression and PTB. 

In this way it is not clear if a) low β-defensin levels increase the risk of developing vaginosis, b) if vaginosis develops and increases β-defensin levels as a consequence, c) if vaginosis is the direct responsible for PTB, and if d) an enhanced inflammatory response (expressed as a means of high β-defensin) is associated directly with an increased possibility of developing PTB. 

As a consequence of these opposing results, we developed four hypotheses: 1) a lower β-defensin expression leads to increased risk for vaginosis and thus PTB, 2) vaginosis itself enhances the inflammatory response and thus the risk for PTB, 3) a higher β-defensin response associates with increased PTB risk but lower vaginosis risk, and 4) none of these factors associate together. To prove whether of these hypotheses is true we performed a systematic review of the literature and a series of linear regression models to prove these hypotheses. Our results show a lack of data support, as none of these factors are strongly associated with each other.

Table 1. Genes, regulation and function of human defensins.

Materials and methods 

A comprehensive literature search in Pubmed, Google scholar, Cochrane library, Science direct and Medline was conducted regarding the relation between β-defensins and PTB using the following keywords: spontaneous preterm birth, beta defensins, alfa defensins, innate immune response, using the proper Boolean operator AND. We use as inclusion criteria: studies that included 1) longitudinal design for pregnant women aged between 16-35 years old, 2) beta defensins as immune biomarkers and determination of the presence or absence of cervico-vaginal infection. As exclusion criteria we used non pregnant women studies, chromosomal disorders identified by ultrasound, presence of immunocompromise (HIV infection, cancer, steroid therapy), antibiotic therapy, medically indicated PTB or no comparison between term and preterm birth. 

The number of patients with low (0-2.8 µg/mL), intermediate (2.9-8.2 µg/mL) or high (˃8.2 µg/mL) levels of either α or β-defensins, as well as the number of patients with negative, intermediate or positive levels of vaginosis were plotted into a table. The plotted data was then used to calculate the R² by a linear regression model to perform a correlational study. Low or intermediate vaginosis were plotted as negative, giving a value of 0, while positive vaginosis was given a value of 1. 

Results and discussion 

Article findings 

A total of 820 articles were found using the aforementioned inclusion criteria using Pubmed, Google Scholar, Cochrane Library, Science Direct and Medline databases, but 85 articles were duplicated. Of the remaining 735 articles only seven adjusted to the inclusion criteria, remaining only four articles to use for the study after the use of the exclusion criteria (Fig. 2).

Figure 2. Article findings. Pubmed, Google Scholar, Cochrane Library, Science Direct and Medline databases were screened for articles regarding spontaneous preterm birth, beta defensins, alfa defensins, and innate immune response. Articles including non-pregnant women, products with chromosomal disorders identified by ultrasound, patients with the presence of any immune compromise (HIV infection, cancer, steroid therapy), antibiotic therapy, medically indicated PTB or no comparison between term and preterm birth were discarded along with duplicates.

Among the studies that were found for the present systematic review, we used a prospective study performed by Elovitz et al. [2] that includes 539 pregnant women from which 107 delivered preterm, they were on 74.5% African American (AA) women with a mean age of 28 yearls old. Samples for determine cervicovaginal community state types (CST) were collected during 3 visits done between 16-20, 20-24 and 24-28 gestacional weeks, respectively, and those used for the determination of β-defensin were measured at visit 1. They found that higher vaginal levels of β-defensin-2 lowered the risk of spontenous preterm birth (sPTB) associated with cervicovaginal microbiota  (whether the microbiota was dominated by Lactobacillus spp. or by Atopobium, M.curtsii/mulieris, S.sanguinegens, M.indolicus) but in an ethnicity-dependent manner, the association remains in AA, but not in non-AA women. 

A prospective study performed by Xu et al. [14] enrolled 1,031 pregnant women from which 244 delivered preterm, they measured α-defensins 1-3 levels and determine presence or abscense of bacterial vaginosis by samples collected between 15-27 weeks of gestation. They found that α-defensins 1-3 levels ≥median were associated with sPTB <32 weeks in both non-AA and AA women, but on this last group the association persisted for the 33-36 week sPTB as well.

A case-cohort study was made by Balu et al. [31] with a total of 749 pregnant women whom were studied by samples taken between the 24-29 gestational weeks, 242 cases develop preterm birth. Elevated levels of HNP-2 defensins compared to those women with no detectable defensins increased the risk of delivering before specifically 32 gestational weeks. 

A prospectively study done by Manning et al. [34] that includes 135 pregnant women from which 42 delivered preterm, the samples available were only for 132 of the 135 women and they were taken between 22-24 weeks gestation, the results show that β-defensins-1 levels did not differ between women with a term delivery compared to those with sPTB, but they also studied levels of cervical fluid IL-8 and IL-1β and found that their levels were reduced in women who delivered preterm compared to those who delivered at term, also were lower in women who subsequently developed chorioamnionitis. 

Preterm birth does not correlate with defensin levels or vaginosis

In the aforementioned studies some authors were able to find a corelation between vaginosis and high levels of defensins production [30], and a further relationship of high levels of such antimicrobial peptides with preterm birth [31], enhancing the notion that vaginosis may be corelated with preterm birth [34]. Nonetheless, controversy arises when other authors found that high levels of defensins are better predictors of PTB than vaginosis [14], that vaginosis does not strongly correlate with high defensin levels [36], or even that lower defensin levels do correlate with stronger vaginosis and thus with preterm birth [2, 37]. In such panorama, we think that the relationship between PTB, vaginosis and the innate immune response must be clarified, as means to get a better understanding about the factors that contribute to PTB.

Following such line of thought, we performed a linear correlation model using the data from these articles, but our model showed no relationship between PTB and high defensin levels (Fig. 3), or vaginosis (Fig. 4). Moreover, according to the same model, a positive relation between vaginosis and high or low levels of defensins does not exist (Fig. 5), and even a three-way correlation model was not able to find a relationship between these three factors when put together (Fig. 6).

Figure 3. Negative correlation between PTB and defensins.

Figure 4. Negative corelation between PTB and vaginosis.

Figure 5. Negative correlation between vaginosis and defensins.

Figure 6. Three-way analysis of the corelation beteween PTB, vaginosis and defensins.

The disparity within the data itself may be deemed as the culprit of such negative relationships, but the model is also parting from insufficient data to normalize the trend. Moreover, patients with different genetic backgrounds were included in each study, and finally, data regarding the expression of defensins and the bacterial colonization of the vagina was taken in different timepoints in each study. In this way, we think that the available data to date does not lend itself to make accurate predictions about preterm birth. 

Obtaining clarity in the mechanisms of PTB induction may impact in the choice of preventive pharmacotherapy, but whether antibiotics, immune modulating agents or both may be the drugs of choice remains undiscovered, as there is no data clearely supporting either desicion. In this way, we think that more research is necessary to achieve an understanding about the role of these factors in PTB induction.

Other inflammatory biomarkers for preterm birth

On the other hand, reduced levels of cytokines in the lower genital tract could indicate immune hypo responsiveness that can lead to an environment more conductive to ascending infections affecting the choriodecidual membranes. Kalinka et al. [15] performed a study with 114 pregnant women were they investigated the relationship between some proinflammatory cytokines (IL-1α, IL-1β, IL-6, IL-8) in cervicovaginal fluid and the risk of subsequent preterm birth, they found that women with lower genital tract pathological flora and more than one cytokine low concentration presented an increased risk of sPTB, so there is convincing information from some cytokines such as IL-6, IL-8, and IL-10 in amniotic fluid and its relation with preterm delivery and/or PPROM.

Conclusions 

Definitive conclusions from the studies that have been done about the relation between α and β-defensins with preterm birth are difficult to establish as the phenotype of spontaneous preterm birth is heterogenous, the number of cases is limited and their results are variable with poor agreement between studies. Thus, more studies should be done with the aim of preventing sPTB and improving neonatal outcomes. 

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