Next Article in Journal
A Commentary on Electrographic Seizure Management and Clinical Outcomes in Critically Ill Children
Next Article in Special Issue
Evaluation of the Hypotensive Preterm Infant: Evidence-Based Practice at the Bedside?
Previous Article in Journal
Open Pyeloplasty in Infants under 1 Year—Proven or Meaningless?
Previous Article in Special Issue
Severe Hypercapnia Requiring 48-h Whole-Body Hypothermia in an Infant with Acute Bronchiolitis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Children
Volume 10
Issue 2
10.3390/children10020256
children-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Perspective

Placing Ureaplasma within the Context of Bronchopulmonary Dysplasia Endotypes and Phenotypes

by
Karen Van Mechelen
1,
Elke van Westering-Kroon
1,
Matthias Hütten
1,
Ludo Mahieu
2 and
Eduardo Villamor
1,*
1
Department of Pediatrics, Maastricht University Medical Center, School for Oncology and Reproduction (GROW), Maastricht University, 6229 HX Maastricht, The Netherlands
2
Department of Neonatology, Antwerp University Hospital, 2650 Edegem, Belgium
*
Author to whom correspondence should be addressed.
Children 2023, 10(2), 256; https://doi.org/10.3390/children10020256
Submission received: 22 December 2022 / Revised: 17 January 2023 / Accepted: 29 January 2023 / Published: 31 January 2023
(This article belongs to the Special Issue Acute and Chronic Lung Disease of the Newborn: Open Challenges and Novel Insights)
Review Reports Versions Notes

Abstract

:
Different pathophysiological pathways (endotypes), leading to very preterm birth may result in distinct clinical phenotypes of bronchopulmonary dysplasia (BPD). Ureaplasma is a unique player in the pathogenesis of BPD. The interaction between factors inherent to Ureaplasma (virulence, bacterial load, duration of exposure), and to the host (immune response, infection clearance, degree of prematurity, respiratory support, concomitant infections) may contribute to BPD development in a variable manner. The data reviewed herein support the hypothesis that Ureaplasma, as a representative of the infectious/inflammatory endotype, may produce pulmonary damage predominantly in parenchyma, interstitium, and small airways. In contrast, Ureaplasma may have a very limited role in the pathogenesis of the vascular phenotype of BPD. In addition, if Ureaplasma is a key factor in BPD pathogenesis, its eradication by macrolides should prevent BPD. However, various meta-analyses do not show consistent evidence that this is the case. The limitations of current definitions and classifications of BPD, based on respiratory support needs instead of pathophysiology and phenotypes, may explain this and other failures in strategies aimed to prevent BPD. The precise mechanisms through which Ureaplasma infection leads to altered lung development and how these pathways can result in different BPD phenotypes warrant further investigation.

1. Introduction

More than 50 years after its initial description [1], bronchopulmonary dysplasia (BPD) continues to be one of the most significant complications of prematurity and still poses clinical and public health challenges [2,3,4,5,6,7]. Northway et al. coined the term BPD to describe a chronic pulmonary condition observed in relatively late preterm infants (32–34 weeks) with respiratory distress syndrome and treated with mechanical ventilation and high oxygen concentrations [1]. However, the population of infants currently diagnosed with BPD differs significantly from the original description. What we now call BPD is the consequence of the exposure to extrauterine life of lungs in the late canalicular or early saccular stage of development (i.e., below 28 weeks of gestation) [3,4]. This results in alterations in both alveolar and pulmonary vascular development that can also occur without exposure to mechanical ventilation or high oxygen concentrations. Therefore, the contemporary view of BPD is that of a heterogeneous condition in which the combination of genetic background, antenatal exposures, extreme prematurity, and early postnatal exposures lead to different patterns of injury and repair [3,4]. Moreover, there is a growing awareness that distinct intrauterine stresses may result in different clinical phenotypes of BPD beyond the traditional categorization of mild/moderate/severe [8,9,10].
The term endotype refers to “a subtype of a condition, which is defined by a distinct functional or pathophysiological mechanism” [11]. The different pathophysiological pathways that trigger extremely preterm birth and concurrently induce alterations in fetal lung development can be grouped into two endotypes: infectious/inflammatory and placental dysfunction [8,9,10,12]. In addition, a growing body of evidence indicates that airways, alveoli, lung parenchyma, and pulmonary vasculature are variably affected within each infant, which leads to different clinical phenotypes of BPD. The following seven phenotypes of BPD have been proposed: parenchymal, peripheral airway, central airway, interstitial, congestive, vascular, and mixed phenotype [8,13,14,15,16,17,18]. These phenotypes may have a partial or complete origin in prematurity endotypes [8,9,10]. Therefore, endotyping and phenotyping BPD will increase our understanding of the condition and facilitate the design of more targeted preventive and therapeutic approaches [3,8,9,10,13].
Ureaplasma is recognized as a major pathogen in prenatal infection triggering preterm birth and is frequently claimed to be involved in the pathogenesis of BPD [19,20,21,22]. As Ureaplasma is the most frequently isolated bacteria in the female genital tract [23,24], fetal or neonatal colonization may occur prenatally due to ascending intrauterine or placental infection, during birth by passage through the birth canal, or postnatal [19,24,25,26,27,28]. The incidence of Ureaplasma colonization in preterm neonates ranges from 15 to 36% [19,29,30,31,32,33,34,35,36]. The lower the gestational age (GA) or birth weight (BW), the higher the colonization rate [19,27].
Numerous preclinical and clinical studies, reviews, and meta-analyses have addressed the association between Ureaplasma and BPD but the results and conclusions are often contradictory [22,27,37,38,39]. Some of these contradictions may stem from considering BPD as a homogeneous entity rather than accounting for the possibility that different endotypes result in different BPD phenotypes. Therefore, we still do not fully understand whether Ureaplasma plays a major role in BPD development, is just a cofactor, or is just an innocent bystander. Our aim in this narrative review is to summarize and update information on the role of Ureaplasma in the pathogenesis of BPD and to frame it within the context of endotyping and phenotyping BPD.

2. Virulence of and Immune Response to Ureaplasma

Ureaplasma are spherical or coccobacillus-shaped bacteria that belong to the Mycoplasmataceae family. Ureaplasma bacteria look much like Mycoplasma but they hydrolyze urea to generate adenosine triphosphate, rather than arginine or glutamine. Furthermore, Ureaplasma lack a cell wall, are not visible on Gram staining and are resistant to β-lactam antibiotics. There are two separate species, namely Ureaplasma parvum and Ureaplasma urealyticum, with 14 serovars that can infect humans [40]. Ureaplasma bind to mucosal surfaces and are typically found in the mouth, upper respiratory and urogenital tract [41]. In women, they are the most frequently isolated commensal in the lower genital tract [23,41].
Ureaplasma can be virulent through different mechanisms, including multiple banded antigen (MBA), production of ammonia by urease, immunoglobulin A1 protease, lack of cell wall, and phospholipases [42,43]. These virulence factors evade the host immune response and can cause tissue damage [42]. The immune system then reacts producing cytokines such as tumor necrosis factor α, interleukin (IL)-6, IL-8, or IL-10 [44], activating the nuclear factor kappa B (NFkB) pathway via toll-like receptors (TLR)-1, TLR-2, and TLR-6 [45], stimulating macrophage activity through TLR-2 and TLR-4 [46], and increasing an immunoglobulin response [47]. At least part of this immune response has a protective character [47]. Another protective mechanism shown in in vitro models is surfactant protein A, which clears Ureaplasma in mouse lungs [48]. However, since surfactant production is inversely proportional to GA, extremely preterm newborns partially lack this protective mechanism.

3. Ureaplasma and Pregnancy

Ureaplasma are the most frequent commensal in the female lower genital tract and are also capable of causing ascending asymptomatic infections of the upper genital tract [23,24,49]. Ascending Ureaplasma in pregnant women can induce an inflammatory response in choriodecidua and amnion, resulting in cytokines and prostaglandins production, and stimulation of uterine contractions [50]. These reactions are associated with chorioamnionitis, premature rupture of membranes (PROM), preterm birth, and abortion [22,24,49,51,52,53,54,55]. Interestingly, only a small group of women with Ureaplasma colonization/infection develop these complications [49,51,52,53,54]. As reviewed by Sweeny et al., among the factors claimed to explain these differences are the virulence of the infecting serovar, the bacterial load, genetic background/ethnicity, and variations in the host immune response [49,56,57].
One of the main arguments for questioning the role of Ureaplasma as a major pathogen in chorioamnionitis is the frequent polymicrobial nature of the condition [49,58,59,60]. This seems to be particularly true in the case of chorioamnionitis below 32 weeks of gestation in which the presence of two or more microorganisms has been demonstrated in more than 60% of cases [49]. Of note, Ureaplasma is almost always one of these microorganisms and as GA advances, the proportion of cases in which it is the only bacteria identified increases [49].

4. Ureaplasma and the Developing Lung: Evidence from Preclinical Studies

The effects of Ureaplasma infection in the fetal and neonatal lung have been studied mainly in sheep and non-human primates [21]. Intraamniotic injection of Ureaplasma parvum in early gestation sheep (55 of 150 days) resulted in chorioamnionitis and lung colonization accompanied by a modest increase in neutrophils, monocytes, IL-1β and IL-8 [61]. Similarly, exposure to Ureaplasma parvum on day 80 of pregnancy in sheep also results in chorioamnionitis and modest fetal lung inflammation after 6 weeks [62]. This lung inflammation persists for 9-10 weeks [62]. Moreover, exposure to Ureaplasma in sheep during late-gestation (110 to 121 days) induced a mild acute inflammatory response with temporary increased neutrophils in preterm lungs [63]. In mice, a self-limiting lung inflammatory response was demonstrated after exposure to Ureaplasma parvum [64]. In contrast, non-human primates shortly exposed to Ureaplasma parvum during mid-gestation have strong lung inflammation [65]. Fetal baboons infected with Ureaplasma were seen to have increased cytokine expression in their lung fluid, causing severe bronchiolitis, and interstitial pneumonitis [66]. In addition, antenatal exposure to Ureaplasma can influence the hosts’ reaction to postnatal stimuli. For example, antenatal Ureaplasma exposure in sheep alters the innate immune system making the immature lung more vulnerable to inflammatory stimuli after birth [63]. Furthermore, studies in mice and baboons demonstrated an increased response to postnatal stimuli such as oxygen and mechanical ventilation [64,66]. It seems that the host response differs between non-primates and non-human primates, and influences lung inflammation and therefore lung morphology changes [21].
There is some evidence for long-term alveolar or vascular morphology changes in non-primate models [61,64]. Studies demonstrated an increased lung maturation, with increased surfactant and lung volumes, in sheep that had long-term Ureaplasma exposure [61], and increased surfactant but no change in lung volumes in sheep that had mid-long-term Ureaplasma exposure [62]. In sheep with short-term Ureaplasma exposure, there were decreased elastic foci with increased smooth muscle around bronchioles, and pulmonary vasculature [63]. This decreased elastin results in lung simplification [67]. These lung changes in non-primates were independent of the dose of Ureaplasma exposure or serovar [61]. In contrast, non-human primates exposed to Ureaplasma had more severe lung alternations compared to sheep exposed to Ureaplasma [21]. In non-human primate models, it seems that the acute lung inflammatory response can partially resolve but the structural lung changes such as epithelial hyperplasia, thickened alveolar walls, fibrosis and bronchiolitis remain [65,66,68,69]. Nevertheless, baboons that could clear their Ureaplasma infection had lower respiratory requirements on mechanical ventilation with improved lung function, in contrast to those which could not clear the infection [66].
The results of these preclinical studies on Ureaplasma are in contrast with the results of studies on chorioamnionitis caused by the bacterial endotoxin lipopolysaccharide (LPS). Animals exposed to chorioamnionitis due to injection of LPS developed a stronger inflammatory response than animals exposed to Ureaplasma. Sheep exposed to LPS had higher concentrations of inflammatory cells such as neutrophils, monocytes, IL-1β [62,70,71], and macrophages [72] in their lungs in comparison with sheep exposed to Ureaplasma. Furthermore, the inflammatory response from TLR4 receptors through LPS is stronger than the inflammatory response from TLR1, 2, and 6 through Ureaplasma [45,73]. Nevertheless, common underlying mechanisms are suggested. For example, pulmonary leukocyte-mediated lung inflammation stimulates lung maturation in sheep exposed to LPS and in sheep exposed to Ureaplasma [74]. Despite the more severe inflammatory endotype caused by LPS in the lungs of sheep, there was no sustained change in lung morphology such as decreased alveolarization [75]. In contrast, in LPS-exposed sheep the inflammation inhibited vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), platelet endothelial cell adhesion molecule-1, and Tie-2 protein, which led to vascular remodeling in small pulmonary arteries causing pulmonary hypertension [75].
In summary, preclinical studies demonstrated that Ureaplasma induces a mild inflammatory response in fetal lungs. Despite the milder inflammatory endotype, in comparison with the stronger inflammatory response caused by acute chorioamnionitis due to other etiologies (such as LPS), Ureaplasma causes more structural lung changes. Interestingly, a longer fetal Ureaplasma exposure induces lung maturation without sustained structural changes, whereas a shorter fetal Ureaplasma exposure causes more sustained structural changes [61,63]. This underlines the unique character of Ureaplasma.

5. Ureaplasma: Causative Agent, Cofactor, or Innocent Bystander in BPD Pathogenesis?

The positive association between maternal colonization with Ureaplasma and BPD (OR 2.4; 95% CI 1.7–3.3) has been shown in a very recent meta-analysis [22]. To the best of our knowledge, four meta-analyses attempted to synthesize the evidence of the association between pulmonary colonization with Ureaplasma and BPD [27,37,38,39]. The results of these meta-analyses are summarized in Table 1. Overall, the data suggest an increased risk of developing BPD in infants colonized with Ureaplasma but there are some limitations of the meta-analyses that should be taken into account. First, they had moderate/high degrees of heterogeneity. Part of this heterogeneity may be explained because the effect size decreased as the included studies became more recent. Of note, Zhang’s meta-analysis [37] only included studies published since 1995 and is the only one not reporting a significant association (Table 1). In addition, many of the studies included in the meta-analyses had very small sample sizes so there is a risk of so-called "small study effects" [76]. That is, smaller studies sometimes show different, often larger, treatment effects than larger studies [76]. Although there is no intrinsic inaccuracy in small studies with large effect sizes, selective publication of the results of smaller studies is more common than that of larger studies and this can seriously affect meta-analysis results [76]. Finally, the meta-analyses included two definitions of BPD: based on oxygen requirement at 28 days postnatal (hereinafter referred to as BPD28) and at 36 weeks postmenstrual age (hereinafter referred to as BPD36). The association of Ureaplasma colonization with BPD36 is weaker than the association with BPD28 (Table 1). Interestingly, the same is observed for the association between chorioamnionitis and BPD [77]. Neonatologists have long argued about the inadequacy of the diagnostic criteria for BPD and particularly about the fact that they are based on components of care (oxygen or respiratory support needs) and not on pathophysiology [3,4]. In any case, oxygen requirement at postnatal day 28 is probably only a proxy for pulmonary immaturity, while BPD36 is more reflective, albeit still imperfectly, of pulmonary damage or aberrant development [3,4].
Irrespective of the quality of the evidence on the association between Ureaplasma colonization and BPD, association does not mean causation. The possible role of Ureaplasma as a causative agent of BPD may depend on the interaction of some factors, not yet fully understood, between the bacteria and the host. Thus, it has been suggested that the type of serovar is a determining pathogenic factor for BPD [78] but this has not been confirmed by other studies [55]. In addition, the duration of colonization/infection with Ureaplasma, bacterial load, and longitudinal changes in the airway microbiome may be determining factors in the development of BPD [61,63,79,80,81]. Paradoxically, the data from preclinical studies suggest that longer fetal Ureaplasma exposure may accelerate lung maturation without inducing structural changes, whereas a shorter fetal exposure may cause more sustained structural changes [61,63]. Ureaplasma and Staphylococcus are the predominant genera of the early airway microbiome of very preterm infants [80,81]. In the first days after birth, infants with more severe BPD had a greater initial relative abundance of Ureaplasma and acquired less Staphylococcus [80,81].
Finally, if Ureaplasma is an important causative agent in BPD development, eradication by macrolides should significantly reduce BPD. However, this reduction has not been consistently demonstrated by meta-analyses [82,83,84]. The failure of macrolide treatment to reduce BPD might be due to some proportion of preterm infants requiring oxygen at 36 weeks postmenstrual age but having a BPD phenotype that is not associated with the infectious/inflammatory endotype. This phenotype would be therefore indifferent to antibiotic therapy. The relationship between endotype and phenotype is discussed in the following paragraphs.

6. Linking Ureaplasma Endotype with BPD Phenotypes

There is overwhelming evidence of the relevance of Ureaplasma as a causative agent of chorioamnionitis [22] and chorioamnionitis is the main proxy of the infectious/inflammatory endotype of prematurity [8,9,10]. Previous meta-analyses have demonstrated a robust association between chorioamnionitis and risk of developing BPD28 and BPD36, but not severe BPD (defined as the need for more than 30% oxygen or mechanical ventilation at 36 weeks postmenstrual age) [77,85]. However, the effect of chorioamnionitis was strongly related to the fact that infants in the control group had significantly higher GAs than those in the chorioamnionitis group [77]. It is a well-known fact that the incidence of chorioamnionitis increases as GA decreases and therefore the majority of extremely preterm newborns belong to the infectious/inflammatory endotype [86,87]. In addition, the presence of funisitis did not add an additional risk of BPD when compared to chorioamnionitis in the absence of funisitis [77]. Funisitis is considered the histological equivalent of the fetal inflammatory response syndrome, whereas chorioamnionitis represents a maternal inflammatory response [86]. Taken together, the meta-analysis data suggest that a significant part of the association between chorioamnionitis and BPD is mediated by the role of chorioamnionitis as a trigger of prematurity more than by the alteration that it induces in lung development [77]. It is remarkable that infants colonized by Ureaplasma had a significantly lower GA than controls and Lowe et al. demonstrated by meta-regression that there was a correlation between this difference in GA and the effect size of the association with BPD [27]. In summary, the induction of a higher degree of prematurity seems to be an inherent feature of the inflammatory/infectious endotype when compared to the placental dysfunction endotype. This does not preclude an additional, endotype-specific, pulmonary damage mediated by the inflammatory cascade.
As mentioned in the introduction, up to seven different clinical phenotypes of BPD can be distinguished but there is still a great lack of consensus on how to diagnose these phenotypes [8,9,10]. The one that may be best defined is the vascular phenotype of BPD, which is characterized by the concomitant presence of pulmonary hypertension [15,16,17,18,88,89,90]. A great effort has been made in recent years to reach a consensus on how and when to assess BPD-associated pulmonary hypertension [91]. Nevertheless, the vascular phenotype of BPD is most frequently associated with the placental dysfunction endotype of prematurity [8,9,10]. This is because the imbalance of pro- and anti-angiogenic mediators that takes place in the dysfunctional placenta also affects pulmonary vascular development [8,9,10]. In contrast, the damage caused by the infectious/inflammatory endotype seems to affect more preferentially the development of alveoli and pulmonary interstitium [8,9,10]. In addition, there is growing evidence that pulmonary infection/inflammation may increase the sensitivity of the lung to postnatal stresses such as mechanical ventilation [92,93].
Autopsy studies in Ureaplasma-infected preterm lungs showed an increased concentration of alveolar macrophages, myofibroblast proliferation, abnormal septation, interstitial fibrosis, and disordered elastic fibers in the distal air spaces [94,95]. These findings are similar to those of the interstitial BPD phenotype, which is characterized by an increase in fibrotic tissue and a widening of the interstitial spaces [8,96]. Although it is difficult to derive specific information from chest radiographs, it is noteworthy that Ureaplasma infection/colonization in extremely preterm infants has been associated with cystic-emphysematous changes [97,98]. This may be suggestive of a parenchymal BPD phenotype [8]. Finally, Kitajima et al. reported that intrauterine Ureaplasma infection in extremely preterm infants was associated with signs of moderate to severe small airway obstruction in lung function tests at school age [97]. These findings are compatible with the peripheral airway BPD phenotype [8]. Taken together, all these data suggest that Ureaplasma may be associated with a mixed phenotype in which the pulmonary parenchyma and small airways would be more affected while the pulmonary vasculature would be less involved. It could be speculated that this particular form of BPD might be more susceptible to treatment with corticosteroids and/or β2-agonists. In contrast, screening for pulmonary hypertension and treatment with pulmonary vasodilators would be preferentially indicated in the vascular phenotype of BPD [8,9,10]. This phenotype is more linked to the placental dysfunction endotype than to the infectious/inflammatory endotype [8,9,10].

7. BPD and Eradication of Ureaplasma

7.1. Prenatal

Macrolides are the antibiotics of choice for eradicating Ureaplasma in neonates and pregnant women because they are not, unlike fluoroquinolones and tetracyclines, teratogenic. Maternal macrolides can reduce vertical Ureaplasma transmission to the fetus and prevent the development of the inflammatory cascade. The literature is contradictory as to whether or not maternal macrolides prevent adverse pregnancy outcomes [99]. It is difficult to compare studies, as there are large differences between particular macrolides, dosing regimens, and routes of administration. In primate models, intramuscular or intra-amniotic erythromycin seems insufficient to eradicate Ureaplasma [100] because erythromycin does not pass the placenta. In contrast, solithromycin and azithromycin seem sufficient either through intravenous or by combining intravenous and intra-amniotic, administration [101,102,103]. Others, however, state that a combination of maternal (intravenous or intramuscular) and intra-amniotic administration is necessary to achieve therapeutic concentrations in both maternal and amniotic compartments [104]. Research also noted that in women with preterm pre-labor rupture of membranes (PPROM), antibiotics prolong pregnancy, decrease chorioamnionitis, as well as neonatal infection, and oxygen therapy [101,105,106]. In contrast, other studies demonstrate no or partial benefits [107,108]. Therefore, the efficacy of maternal therapy for Ureaplasma remains uncertain.
Furthermore, it is important to notice the possible side effects of macrolides. A meta-analysis of 2020 identified a weak association between the use of macrolides in early pregnancy and congenital malformations of the musculoskeletal and digestive systems [109]. The developing immune and neurological systems can already be influenced by one course of antibiotics which alter the microbiome of the neonate and mother [110,111]. In addition, intrapartum antibiotics increase antibiotic resistance in neonates [112].

7.2. Postnatal

Three meta-analyses about macrolide treatment in Ureaplasma-positive neonates reported variable results [82,83,84]. A Cochrane review of 2003 included two small RCTs and found no significant reduction in BPD risk when erythromycin was used in intubated preterm neonates [84]. Similar findings were reported by Nair et al. that included six RCTs [83]. This meta-analysis found no significant reduction in BPD36 (RR 0.64; 95% CI 0.31–1.31) or the composite outcome BPD36 or death (RR 0.41; 95% CI 0.05–3.13) when clarithromycin, azithromycin or erythromycin was used in Ureaplasma-positive neonates [83]. They did, however, report a significant reduction in BPD36 and BPD36 or death with prophylactic azithromycin irrespective of Ureaplasma status [83]. A possible explanation for this reduction might be that azithromycin and clarithromycin achieve better drug concentrations in the lungs than erythromycin [113]. Furthermore, the used dose of the macrolide is important to determine Ureaplasma clearance [114]. A limitation of this meta-analysis is that they combined studies having important differences in population, the drug used, dosages, time of initiation, duration of treatment, and method of Ureaplasma detection. The third meta-analysis by Razak and Alshehri, published in 2021, only evaluated azithromycin therapy [82]. Five RCTs were included (four including Ureaplasma-positive neonates). The meta-analysis demonstrated that Ureaplasma-positive neonates treated with azitromycin had less BPD36 or death (RR 0.83; 95% CI 0.70–0.99), and a trend toward less BPD36 (RR 0.83; 95% CI 0.66–1.03) [82]. In addition, there was a reduction in supplemental oxygen days, in all neonates irrespective of Ureaplasma status [82]. These findings suggest that azithromycin can effectively attenuate the pathogenic effect of Ureaplasma on BPD. Azithromycin has high potency against Ureaplasma [32] and has anti-inflammatory capacities by reducing IL-2 and IL-8 levels [31] which is an important pathway in BPD development. Nevertheless, the quality of evidence was low due to the risk of bias among the RCTs and the imprecision of the effect estimate [82].
The number needed to treat with macrolides to prevent one case of BPD was 11 in the meta-analysis of Razak and Alsheri [82] and 10 in the meta-analysis of Nair et al. [83]. Therefore, it is very important to select the infants who will benefit the most from treatment with macrolides. Extremely preterm neonates and neonates who cannot be extubated, are considered at high risk for BPD. Poets et al. suggests testing tracheal aspirates of extremely preterm neonates requiring mechanical ventilation for Ureaplasma, and consider intravenous azithromycin in those who tested positive [115]. Nevertheless, today, an increasing number of infants with BPD were never intubated due to advances in non-invasive ventilation.
It is important to bear in mind that comparing individual studies is difficult because there is considerable heterogeneity due to the use of different macrolide regimes (drug, dosing, duration), inclusion criteria, baseline characteristics, ventilation techniques, BPD definitions, sample sizes, and statistical power. To conclude, the safety and effectiveness of macrolides to eradicate Ureaplasma remains uncertain. Although there is some evidence that the macrolide azithromycin has an overall anti-inflammatory effect [31] and reduces BPD in Ureaplasma-positive neonates [82], the use of macrolides is not recommended because the current level of evidence is weak. The ongoing AZTEC (azithromycin therapy for chronic lung disease of prematurity) aims to determine if a 10-day course of intravenous azithromycin improves rates of survival without BPD36 when compared with placebo in a population of infants with GA <30 weeks [116].

8. Conclusions and Suggestions for Future Research

Ureaplasma is a unique player in the pathogenesis of BPD. The interaction between factors inherent to Ureaplasma, (virulence, bacterial load, duration of exposure), and to the host (immune response, infection clearance, degree of prematurity, respiratory therapy needs, concomitant infections) contribute, in a manner yet to be fully determined, to BPD development. The data reviewed herein support the hypothesis that Ureaplasma, as a representative of the infectious/inflammatory endotype, produces pulmonary damage predominantly in parenchyma, interstitium, and small airways. However, the precise mechanisms through which Ureaplasma infection leads to altered lung development and how these pathways can result in different BPD phenotypes require ongoing exploration.
In addition to Ureaplasma, very preterm infants are exposed to multiple potent antenatal and postnatal inflammatory modulators that may modify pulmonary injury and repair pathways. This results in a marked variability in clinical manifestations and severity of BPD. Current definitions of BPD are based on components of care, such as the need for oxygen and/or respiratory support, and do not capture this variability [3,4]. Although the persistent requirement of oxygen or respiratory support at a given age may be a clinical predictor of respiratory outcome, it provides little information about BPD phenotypes or therapeutic interventions that may benefit individual patients. A classification of BPD based on endotypes and phenotypes requires the development of biomarkers targeting Ureaplasma infection as well as other pathogenic pathways [3,4].
A challenge in future research is diagnosing Ureaplasma infection. The gold standard for diagnosing Ureaplasma is bacterial culture in a specialized media. Ureaplasma bacteria lack a cell wall and are therefore highly sensitive to desiccation and heat. That means any test samples must be immediately transported to the laboratory, and culturing Ureaplasma remains difficult [117]. To overcome these difficulties, polymerase chain reaction (PCR) can be helpful. In this respect, some suggest that PCR and culture are almost equally effective [118,119], yet others suggest that PCR is superior [120]. PCR tests have the advantage of allowing the identification of Ureaplasma species, the quantification of bacterial load, and the evaluation of specimens overgrown by other bacteria using the culture method. As discussed above, all of these factors are relevant to the pathogenic effects of Ureaplasma [78,79,119]. Nevertheless, a disadvantage of PCR is that it is not possible to test antimicrobial susceptibility, in contrast to culturing. A disadvantage of both tests is that neither can distinguish between carrier status and infection.
Finally, the evidence for treating Ureaplasma, and therefore decreasing BPD, remains inconclusive. Future studies are necessary to find the optimal approach for Ureaplasma in pregnant women and their offspring. The ideal drug, dosage regimen, timing, and route of administration remains a matter of debate. Furthermore, it remains unclear whether treating the infection/colonization with Ureaplasma effectively decreases lung inflammation, reduces BPD rate, and reduces long-term respiratory and neurological morbidity.

Author Contributions

Conceptualization, K.V.M. and E.V.; writing—original draft preparation, K.V.M. and E.V.; writing—review and editing, E.v.W.-K., M.H. and L.M.; supervision, E.V. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Northway, W.H., Jr.; Rosan, R.C.; Porter, D.Y. Pulmonary disease following respirator therapy of hyaline-membrane disease. Bronchopulmonary dysplasia. N. Engl. J. Med. 1967, 276, 357–368. [Google Scholar] [CrossRef] [PubMed]
  2. Stoll, B.J.; Hansen, N.I.; Bell, E.F.; Walsh, M.C.; Carlo, W.A.; Shankaran, S.; Laptook, A.R.; Sánchez, P.J.; Van Meurs, K.P.; Wyckoff, M.; et al. Trends in Care Practices, Morbidity, and Mortality of Extremely Preterm Neonates, 1993-2012. JAMA 2015, 314, 1039–1051. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Thébaud, B.; Goss, K.N.; Laughon, M.; Whitsett, J.A.; Abman, S.H.; Steinhorn, R.H.; Aschner, J.L.; Davis, P.G.; McGrath-Morrow, S.A.; Soll, R.F.; et al. Bronchopulmonary dysplasia. Nat. Rev. Dis. Prim. 2019, 5, 78. [Google Scholar] [CrossRef] [PubMed]
  4. Abman, S.H.; Bancalari, E.; Jobe, A. The Evolution of Bronchopulmonary Dysplasia after 50 Years. Am. J. Respir. Crit. Care Med. 2017, 195, 421–424. [Google Scholar] [CrossRef]
  5. Maitre, N.L.; Ballard, R.A.; Ellenberg, J.H.; Davis, S.D.; Greenberg, J.M.; Hamvas, A.; Pryhuber, G.S. Respiratory consequences of prematurity: Evolution of a diagnosis and development of a comprehensive approach. J. Perinatol. 2015, 35, 313–321. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Sriram, S.; Schreiber, M.D.; Msall, M.E.; Kuban, K.C.K.; Joseph, R.M.; TM, O.S.; Allred, E.N.; Leviton, A. Cognitive Development and Quality of Life Associated With BPD in 10-Year-Olds Born Preterm. Pediatrics 2018, 141, e20172719. [Google Scholar] [CrossRef] [Green Version]
  7. Álvarez-Fuente, M.; Arruza, L.; Muro, M.; Zozaya, C.; Avila, A.; López-Ortego, P.; González-Armengod, C.; Torrent, A.; Gavilán, J.L.; Del Cerro, M.J. The economic impact of prematurity and bronchopulmonary dysplasia. Eur. J. Pediatr. 2017, 176, 1587–1593. [Google Scholar] [CrossRef]
  8. Pierro, M.; Van Mechelen, K.; van Westering-Kroon, E.; Villamor-Martínez, E.; Villamor, E. Endotypes of Prematurity and Phenotypes of Bronchopulmonary Dysplasia: Toward Personalized Neonatology. J. Pers. Med. 2022, 12, 687. [Google Scholar] [CrossRef]
  9. Pierro, M.; Villamor-Martinez, E.; van Westering-Kroon, E.; Alvarez-Fuente, M.; Abman, S.H.; Villamor, E. Association of the dysfunctional placentation endotype of prematurity with bronchopulmonary dysplasia: A systematic review, meta-analysis and meta-regression. Thorax 2022, 77, 268–275. [Google Scholar] [CrossRef]
  10. Parsons, A.; Netsanet, A.; Seedorf, G.J.; Abman, S.H.; Taglauer, E.S. Understanding the Role of Placental Pathophysiology in the Development of Bronchopulmonary Dysplasia (BPD). Am. J. Physiol. Lung Cell. Mol. Physiol. 2022, 323, L651–L658. [Google Scholar] [CrossRef]
  11. Lötvall, J.; Akdis, C.A.; Bacharier, L.B.; Bjermer, L.; Casale, T.B.; Custovic, A.; Lemanske Jr, R.F.; Wardlaw, A.J.; Wenzel, S.E.; Greenberger, P.A. Asthma endotypes: A new approach to classification of disease entities within the asthma syndrome. J. Allergy. Clin. Immunol. 2011, 127, 355–360. [Google Scholar] [CrossRef] [PubMed]
  12. Amelio, G.S.; Provitera, L.; Raffaeli, G.; Tripodi, M.; Amodeo, I.; Gulden, S.; Cortesi, V.; Manzoni, F.; Cervellini, G.; Tomaselli, A. Endothelial dysfunction in preterm infants: The hidden legacy of uteroplacental pathologies. Front. Pediatr. 2022, 10, 1041919. [Google Scholar] [CrossRef] [PubMed]
  13. Cassady, S.J.; Lasso-Pirot, A.; Deepak, J. Phenotypes of bronchopulmonary dysplasia in adults. Chest 2020, 158, 2074–2081. [Google Scholar] [CrossRef] [PubMed]
  14. Wang, S.-H.; Tsao, P.-N. Phenotypes of bronchopulmonary dysplasia. Int. J. Mol. Sci 2020, 21, 6112. [Google Scholar] [CrossRef] [PubMed]
  15. Wu, K.Y.; Jensen, E.A.; White, A.M.; Wang, Y.; Biko, D.M.; Nilan, K.; Fraga, M.V.; Mercer-Rosa, L.; Zhang, H.; Kirpalani, H. Characterization of Disease Phenotype in Very Preterm Infants with Severe Bronchopulmonary Dysplasia. Am. J. Respir. Crit. Care Med. 2020, 201, 1398–1406. [Google Scholar] [CrossRef] [PubMed]
  16. Collaco, J.M.; McGrath-Morrow, S.A. Respiratory phenotypes for preterm infants, children, and adults: Bronchopulmonary dysplasia and more. Ann. Am. Thorac. Soc. 2018, 15, 530–538. [Google Scholar] [CrossRef]
  17. Durlak, W.; Thébaud, B. The vascular phenotype of BPD: New basic science insights—New precision medicine approaches. Pediatr. Res. 2022, 1–10. [Google Scholar] [CrossRef]
  18. Mandell, E.; Hysinger, E.B.; McGrath-Morrow, S.A. Disease Phenotyping of Infants with Severe Bronchopulmonary Dysplasia. Am. J. Respir. Crit. Care Med. 2020, 201, 1327–1329. [Google Scholar] [CrossRef] [Green Version]
  19. Viscardi, R.M.; Kallapur, S.G. Role of Ureaplasma respiratory tract colonization in bronchopulmonary dysplasia pathogenesis: Current concepts and update. Clin. Perinatol. 2015, 42, 719–738. [Google Scholar] [CrossRef] [Green Version]
  20. Silwedel, C.; Speer, C.P.; Glaser, K. Ureaplasma-associated prenatal, perinatal, and neonatal morbidities. Expert. Rev. Clin. Immunol. 2017, 13, 1073–1087. [Google Scholar] [CrossRef]
  21. Kallapur, S.G.; Kramer, B.W.; Jobe, A.H. Ureaplasma and BPD. Semin. Perinatol. 2013, 37, 94–101. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Xu, Y.P.; Hu, J.M.; Huang, Y.Q.; Shi, L.P. Maternal Ureaplasma exposure during pregnancy and the risk of preterm birth and BPD: A meta-analysis. Arch. Gynecol. Obstet. 2022, 306, 1863–1872. [Google Scholar] [CrossRef] [PubMed]
  23. Agbakoba, N.R.; Adetosoye, A.I.; Adewole, I.F. Presence of mycoplasma and ureaplasma species in the vagina of women of reproductive age. West Afr. J. Med. 2007, 26, 28–31. [Google Scholar] [CrossRef] [PubMed]
  24. Waites, K.B.; Katz, B.; Schelonka, R.L. Mycoplasmas and ureaplasmas as neonatal pathogens. Clin. Microbiol. Rev. 2005, 18, 757–789. [Google Scholar] [CrossRef] [Green Version]
  25. Sprong, K.E.; Mabenge, M.; Wright, C.A.; Govender, S. Ureaplasma species and preterm birth: Current perspectives. Crit. Rev. Microbiol. 2020, 46, 169–181. [Google Scholar] [CrossRef]
  26. Chu, A.; de St Maurice, A.; Sim, M.S.; Kallapur, S.G. Neonatal Mycoplasma and Ureaplasma Infections. Pediatr. Ann. 2020, 49, e305–e312. [Google Scholar] [CrossRef]
  27. Lowe, J.; Watkins, W.J.; Edwards, M.O.; Spiller, O.B.; Jacqz-Aigrain, E.; Kotecha, S.J.; Kotecha, S. Association between pulmonary ureaplasma colonization and bronchopulmonary dysplasia in preterm infants: Updated systematic review and meta-analysis. Pediatr. Infect. Dis. J. 2014, 33, 697–702. [Google Scholar] [CrossRef]
  28. Peretz, A.; Tameri, O.; Azrad, M.; Barak, S.; Perlitz, Y.; Dahoud, W.A.; Ben-Ami, M.; Kushnir, A. Mycoplasma and Ureaplasma carriage in pregnant women: The prevalence of transmission from mother to newborn. BMC Pregnancy Childbirth 2020, 20, 456. [Google Scholar] [CrossRef]
  29. Ballard, H.O.; Anstead, M.I.; Shook, L.A. Azithromycin in the extremely low birth weight infant for the prevention of bronchopulmonary dysplasia: A pilot study. Respir. Res. 2007, 8, 41. [Google Scholar] [CrossRef] [Green Version]
  30. Ballard, H.O.; Shook, L.A.; Bernard, P.; Anstead, M.I.; Kuhn, R.; Whitehead, V.; Grider, D.; Crawford, T.N.; Hayes, D., Jr. Use of azithromycin for the prevention of bronchopulmonary dysplasia in preterm infants: A randomized, double-blind, placebo controlled trial. Pediatr. Pulmonol. 2011, 46, 111–118. [Google Scholar] [CrossRef]
  31. Nunes, C.R.; Procianoy, R.S.; Corso, A.L.; Silveira, R.C. Use of Azithromycin for the Prevention of Lung Injury in Mechanically Ventilated Preterm Neonates: A Randomized Controlled Trial. Neonatology 2020, 117, 522–528. [Google Scholar] [CrossRef] [PubMed]
  32. Viscardi, R.M.; Terrin, M.L.; Magder, L.S.; Davis, N.L.; Dulkerian, S.J.; Waites, K.B.; Ambalavanan, N.; Kaufman, D.A.; Donohue, P.; Tuttle, D.J.; et al. Randomised trial of azithromycin to eradicate Ureaplasma in preterm infants. Arch. Dis. Child. Fetal Neonatal Ed. 2020, 105, 615–622. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Jónsson, B.; Rylander, M.; Faxelius, G. Ureaplasma urealyticum, erythromycin and respiratory morbidity in high-risk preterm neonates. Acta Paediatr. 1998, 87, 1079–1084. [Google Scholar] [CrossRef] [PubMed]
  34. Lyon, A.J.; McColm, J.; Middlemist, L.; Fergusson, S.; McIntosh, N.; Ross, P.W. Randomised trial of erythromycin on the development of chronic lung disease in preterm infants. Arch. Dis. Child. Fetal Neonatal Ed. 1998, 78, F10–F14. [Google Scholar] [CrossRef]
  35. Ozdemir, R.; Erdeve, O.; Dizdar, E.A.; Oguz, S.S.; Uras, N.; Saygan, S.; Karabulut, E.; Dilmen, U. Clarithromycin in preventing bronchopulmonary dysplasia in Ureaplasma urealyticum-positive preterm infants. Pediatrics 2011, 128, e1496–e1501. [Google Scholar] [CrossRef] [PubMed]
  36. Gharehbaghi MM, P.A.; Ghojazadeh, M.; Mahallei, M. Efficacy of azithromycin for prevention of bronchopulmonary dysplasia (BPD). Turk. J. Med. Sci. 2012, 42, 1070–1075. [Google Scholar] [CrossRef]
  37. Zheng, X.D.; Li, D.; Yang, D.H.; Xiang, X.; Mei, H.; Pu, J.R.; Tong, Q.S.; Zheng, L.D. Association of Ureaplasma urealyticum colonization with development of bronchopulmonary dysplasia: A systemic review and meta-analysis. J. Huazhong Univ. Sci. Technolog. Med. Sci. 2014, 34, 265–269. [Google Scholar] [CrossRef]
  38. Schelonka, R.L.; Katz, B.; Waites, K.B.; Benjamin, D.K., Jr. Critical appraisal of the role of Ureaplasma in the development of bronchopulmonary dysplasia with metaanalytic techniques. Pediatr. Infect. Dis. J. 2005, 24, 1033–1039. [Google Scholar] [CrossRef]
  39. Wang, E.E.; Ohlsson, A.; Kellner, J.D. Association of Ureaplasma urealyticum colonization with chronic lung disease of prematurity: Results of a metaanalysis. J. Pediatr. 1995, 127, 640–644. [Google Scholar] [CrossRef]
  40. Waites, K.B.; Schelonka, R.L.; Xiao, L.; Grigsby, P.L.; Novy, M.J. Congenital and opportunistic infections: Ureaplasma species and Mycoplasma hominis. Semin. Fetal Neonatal Med. 2009, 14, 190–199. [Google Scholar] [CrossRef]
  41. Cassell, G.H.; Waites, K.B.; Watson, H.L.; Crouse, D.T.; Harasawa, R. Ureaplasma urealyticum intrauterine infection: Role in prematurity and disease in newborns. Clin. Microbiol. Rev. 1993, 6, 69–87. [Google Scholar] [CrossRef] [PubMed]
  42. Venturelli, N.; Zeis, A.; De Beritto, T.; Hageman, J.R. Ureasplasma and Its Role in Adverse Perinatal Outcomes: A Review. Neoreviews 2021, 22, e574–e584. [Google Scholar] [CrossRef]
  43. Zheng, X.; Teng, L.J.; Watson, H.L.; Glass, J.I.; Blanchard, A.; Cassell, G.H. Small repeating units within the Ureaplasma urealyticum MB antigen gene encode serovar specificity and are associated with antigen size variation. Infect. Immun. 1995, 63, 891–898. [Google Scholar] [CrossRef] [Green Version]
  44. Li, Y.H.; Brauner, A.; Jonsson, B.; van der Ploeg, I.; Söder, O.; Holst, M.; Jensen, J.S.; Lagercrantz, H.; Tullus, K. Ureaplasma urealyticum-induced production of proinflammatory cytokines by macrophages. Pediatr. Res. 2000, 48, 114–119. [Google Scholar] [CrossRef] [Green Version]
  45. Shimizu, T.; Kida, Y.; Kuwano, K. Ureaplasma parvum lipoproteins, including MB antigen, activate NF-{kappa}B through TLR1, TLR2 and TLR6. Microbiology 2008, 154, 1318–1325. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Peltier, M.R.; Freeman, A.J.; Mu, H.H.; Cole, B.C. Characterization of the macrophage-stimulating activity from Ureaplasma urealyticum. Am. J. Reprod. Immunol. 2007, 57, 186–192. [Google Scholar] [CrossRef] [PubMed]
  47. Ireland, D.J.; Keelan, J.A. The Maternal Serological Response to Intrauterine Ureaplasma sp. Infection and Prediction of Risk of Pre-Term Birth. Front. Immunol. 2014, 5, 624. [Google Scholar] [CrossRef] [Green Version]
  48. Okogbule-Wonodi, A.C.; Chesko, K.L.; Famuyide, M.E.; Viscardi, R.M. Surfactant protein-A enhances ureaplasmacidal activity in vitro. Innate. Immun. 2011, 17, 145–151. [Google Scholar] [CrossRef]
  49. Sweeney, E.L.; Dando, S.J.; Kallapur, S.G.; Knox, C.L. The human Ureaplasma species as causative agents of chorioamnionitis. Clin. Microbiol. Rev. 2016, 30, 349–379. [Google Scholar] [CrossRef] [Green Version]
  50. Olomu, I.N.; Hecht, J.L.; Onderdonk, A.O.; Allred, E.N.; Leviton, A. Perinatal correlates of Ureaplasma urealyticum in placenta parenchyma of singleton pregnancies that end before 28 weeks of gestation. Pediatrics 2009, 123, 1329–1336. [Google Scholar] [CrossRef]
  51. Abele-Horn, M.; Peters, J.; Genzel-Boroviczény, O.; Wolff, C.; Zimmermann, A.; Gottschling, W. Vaginal Ureaplasma urealyticum colonization: Influence on pregnancy outcome and neonatal morbidity. Infection 1997, 25, 286–291. [Google Scholar] [CrossRef]
  52. Motomura, K.; Romero, R.; Xu, Y.; Theis, K.R.; Galaz, J.; Winters, A.D.; Slutsky, R.; Garcia-Flores, V.; Zou, C.; Levenson, D.; et al. Intra-Amniotic Infection with Ureaplasma parvum Causes Preterm Birth and Neonatal Mortality That Are Prevented by Treatment with Clarithromycin. mBio 2020, 11, e00797-20. [Google Scholar] [CrossRef]
  53. Kusanovic, J.P.; Vargas, P.; Ferrer, F.; Díaz, F.; Córdova, V.; Martinovic, C.; Valdés, R.; Rosas, A.; Luna, D.; Silva, P.; et al. Comparison of two identification and susceptibility test kits for Ureaplasma spp and Mycoplasma hominis in amniotic fluid of patients at high risk for intra-amniotic infection. J. Matern. Fetal Neonatal Med. 2020, 33, 3409–3417. [Google Scholar] [CrossRef]
  54. Bartkeviciene, D.; Opolskiene, G.; Bartkeviciute, A.; Arlauskiene, A.; Lauzikiene, D.; Zakareviciene, J.; Ramasauskaite, D. The impact of Ureaplasma infections on pregnancy complications. Libyan J. Med. 2020, 15, 1812821. [Google Scholar] [CrossRef]
  55. Sweeney, E.L.; Kallapur, S.G.; Gisslen, T.; Lambers, D.S.; Chougnet, C.A.; Stephenson, S.A.; Jobe, A.H.; Knox, C.L. Placental Infection With Ureaplasma species Is Associated With Histologic Chorioamnionitis and Adverse Outcomes in Moderately Preterm and Late-Preterm Infants. J. Infect. Dis. 2016, 213, 1340–1347. [Google Scholar] [CrossRef] [Green Version]
  56. Abele-Horn, M.; Scholz, M.; Wolff, C.; Kolben, M. High-density vaginal Ureaplasma urealyticum colonization as a risk factor for chorioamnionitis and preterm delivery. Acta Obstet. Gynecol. Scand. 2000, 79, 973–978. [Google Scholar] [CrossRef]
  57. Knox, C.L.; Dando, S.J.; Nitsos, I.; Kallapur, S.G.; Jobe, A.H.; Payton, D.; Moss, T.J.; Newnham, J.P. The severity of chorioamnionitis in pregnant sheep is associated with in vivo variation of the surface-exposed multiple-banded antigen/gene of Ureaplasma parvum. Biol. Reprod. 2010, 83, 415–426. [Google Scholar] [CrossRef] [Green Version]
  58. Taylor-Robinson, D. The role of mycoplasmas in pregnancy outcome. Best. Pr. Res. Clin. Obstet. Gynaecol. 2007, 21, 425–438. [Google Scholar] [CrossRef]
  59. Redline, R.W. Inflammatory responses in the placenta and umbilical cord. Semin. Fetal Neonatal Med. 2006, 11, 296–301. [Google Scholar] [CrossRef]
  60. Vogel, I.; Thorsen, P.; Hogan, V.K.; Schieve, L.A.; Jacobsson, B.; Ferre, C.D. The joint effect of vaginal Ureaplasma urealyticum and bacterial vaginosis on adverse pregnancy outcomes. Acta Obstet. Gynecol. Scand. 2006, 85, 778–785. [Google Scholar] [CrossRef]
  61. Polglase, G.R.; Dalton, R.G.; Nitsos, I.; Knox, C.L.; Pillow, J.J.; Jobe, A.H.; Moss, T.J.; Newnham, J.P.; Kallapur, S.G. Pulmonary vascular and alveolar development in preterm lambs chronically colonized with Ureaplasma parvum. Am. J. Physiol. Lung Cell. Mol. Physiol. 2010, 299, L232–L241. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  62. Moss, T.J.; Knox, C.L.; Kallapur, S.G.; Nitsos, I.; Theodoropoulos, C.; Newnham, J.P.; Ikegami, M.; Jobe, A.H. Experimental amniotic fluid infection in sheep: Effects of Ureaplasma parvum serovars 3 and 6 on preterm or term fetal sheep. Am. J. Obstet. Gynecol. 2008, 198, 122.e1–122.e8. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  63. Collins, J.J.; Kallapur, S.G.; Knox, C.L.; Nitsos, I.; Polglase, G.R.; Pillow, J.J.; Kuypers, E.; Newnham, J.P.; Jobe, A.H.; Kramer, B.W. Inflammation in fetal sheep from intra-amniotic injection of Ureaplasma parvum. Am. J. Physiol. Lung Cell. Mol. Physiol. 2010, 299, L852–L860. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  64. Normann, E.; Lacaze-Masmonteil, T.; Eaton, F.; Schwendimann, L.; Gressens, P.; Thébaud, B. A novel mouse model of Ureaplasma-induced perinatal inflammation: Effects on lung and brain injury. Pediatr. Res. 2009, 65, 430–436. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  65. Novy, M.J.; Duffy, L.; Axthelm, M.K.; Sadowsky, D.W.; Witkin, S.S.; Gravett, M.G.; Cassell, G.H.; Waites, K.B. Ureaplasma parvum or Mycoplasma hominis as sole pathogens cause chorioamnionitis, preterm delivery, and fetal pneumonia in rhesus macaques. Reprod. Sci. 2009, 16, 56–70. [Google Scholar] [CrossRef]
  66. Yoder, B.A.; Coalson, J.J.; Winter, V.T.; Siler-Khodr, T.; Duffy, L.B.; Cassell, G.H. Effects of antenatal colonization with ureaplasma urealyticum on pulmonary disease in the immature baboon. Pediatr. Res. 2003, 54, 797–807. [Google Scholar] [CrossRef] [Green Version]
  67. Kramer, B.W.; Albertine, K.H.; Moss, T.J.; Nitsos, I.; Ladenburger, A.; Speer, C.P.; Newnham, J.P.; Jobe, A.H. All-trans retinoic acid and intra-amniotic endotoxin-mediated effects on fetal sheep lung. Anat. Rec. 2008, 291, 1271–1277. [Google Scholar] [CrossRef]
  68. Viscardi, R.M.; Atamas, S.P.; Luzina, I.G.; Hasday, J.D.; He, J.R.; Sime, P.J.; Coalson, J.J.; Yoder, B.A. Antenatal Ureaplasma urealyticum respiratory tract infection stimulates proinflammatory, profibrotic responses in the preterm baboon lung. Pediatr. Res. 2006, 60, 141–146. [Google Scholar] [CrossRef] [Green Version]
  69. Walsh, W.F.; Butler, J.; Coalson, J.; Hensley, D.; Cassell, G.H.; deLemos, R.A. A primate model of Ureaplasma urealyticum infection in the premature infant with hyaline membrane disease. Clin. Infect. Dis. 1993, 17 (Suppl. 1), S158–S162. [Google Scholar] [CrossRef]
  70. Kallapur, S.G.; Willet, K.E.; Jobe, A.H.; Ikegami, M.; Bachurski, C.J. Intra-amniotic endotoxin: Chorioamnionitis precedes lung maturation in preterm lambs. Am. J. Physiol. Lung Cell. Mol. Physiol. 2001, 280, L527–L536. [Google Scholar] [CrossRef]
  71. Moss, T.J.; Nitsos, I.; Knox, C.L.; Polglase, G.R.; Kallapur, S.G.; Ikegami, M.; Jobe, A.H.; Newnham, J.P. Ureaplasma colonization of amniotic fluid and efficacy of antenatal corticosteroids for preterm lung maturation in sheep. Am. J. Obstet. Gynecol. 2009, 200, 96.e1–96.e6. [Google Scholar] [CrossRef] [Green Version]
  72. Kramer, B.W.; Joshi, S.N.; Moss, T.J.; Newnham, J.P.; Sindelar, R.; Jobe, A.H.; Kallapur, S.G. Endotoxin-induced maturation of monocytes in preterm fetal sheep lung. Am. J. Physiol. Lung Cell. Mol. Physiol. 2007, 293, L345–L353. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  73. Hillman, N.H.; Moss, T.J.; Nitsos, I.; Kramer, B.W.; Bachurski, C.J.; Ikegami, M.; Jobe, A.H.; Kallapur, S.G. Toll-like receptors and agonist responses in the developing fetal sheep lung. Pediatr. Res. 2008, 63, 388–393. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  74. Kallapur, S.G.; Moss, T.J.; Ikegami, M.; Jasman, R.L.; Newnham, J.P.; Jobe, A.H. Recruited inflammatory cells mediate endotoxin-induced lung maturation in preterm fetal lambs. Am. J. Respir. Crit. Care Med. 2005, 172, 1315–1321. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  75. Kallapur, S.G.; Nitsos, I.; Moss, T.J.; Kramer, B.W.; Newnham, J.P.; Ikegami, M.; Jobe, A.H. Chronic endotoxin exposure does not cause sustained structural abnormalities in the fetal sheep lungs. Am. J. Physiol. Lung Cell. Mol. Physiol. 2005, 288, L966–L974. [Google Scholar] [CrossRef] [Green Version]
  76. Hong, C.; Salanti, G.; Morton, S.C.; Riley, R.D.; Chu, H.; Kimmel, S.E.; Chen, Y. Testing small study effects in multivariate meta-analysis. Biometrics 2020, 76, 1240–1250. [Google Scholar] [CrossRef]
  77. Villamor-Martinez, E.; Álvarez-Fuente, M.; Ghazi, A.M.T.; Degraeuwe, P.; Zimmermann, L.J.I.; Kramer, B.W.; Villamor, E. Association of Chorioamnionitis With Bronchopulmonary Dysplasia Among Preterm Infants: A Systematic Review, Meta-analysis, and Metaregression. JAMA Netw. Open 2019, 2, e1914611. [Google Scholar] [CrossRef]
  78. Rittenschober-Böhm, J.; Waldhoer, T.; Schulz, S.M.; Pimpel, B.; Goeral, K.; Kasper, D.C.; Witt, A.; Berger, A. Vaginal Ureaplasma parvum serovars and spontaneous preterm birth. Am. J. Obstet. Gynecol. 2019, 220, 594.e1–594.e9. [Google Scholar] [CrossRef]
  79. Kasper, D.C.; Mechtler, T.P.; Reischer, G.H.; Witt, A.; Langgartner, M.; Pollak, A.; Herkner, K.R.; Berger, A. The bacterial load of Ureaplasma parvum in amniotic fluid is correlated with an increased intrauterine inflammatory response. Diagn. Microbiol. Infect. Dis. 2010, 67, 117–121. [Google Scholar] [CrossRef]
  80. Pammi, M.; Lal, C.V.; Wagner, B.D.; Mourani, P.M.; Lohmann, P.; Luna, R.A.; Sisson, A.; Shivanna, B.; Hollister, E.B.; Abman, S.H.; et al. Airway Microbiome and Development of Bronchopulmonary Dysplasia in Preterm Infants: A Systematic Review. J. Pediatr. 2019, 204, 126–133.e122. [Google Scholar] [CrossRef]
  81. Wagner, B.D.; Sontag, M.K.; Harris, J.K.; Miller, J.I.; Morrow, L.; Robertson, C.E.; Stephens, M.; Poindexter, B.B.; Abman, S.H.; Mourani, P.M. Airway Microbial Community Turnover Differs by BPD Severity in Ventilated Preterm Infants. PLoS ONE 2017, 12, e0170120. [Google Scholar] [CrossRef] [Green Version]
  82. Razak, A.; Alshehri, N. Azithromycin for preventing bronchopulmonary dysplasia in preterm infants: A systematic review and meta-analysis. Pediatr. Pulmonol. 2021, 56, 957–966. [Google Scholar] [CrossRef] [PubMed]
  83. Nair, V.; Loganathan, P.; Soraisham, A.S. Azithromycin and other macrolides for prevention of bronchopulmonary dysplasia: A systematic review and meta-analysis. Neonatology 2014, 106, 337–347. [Google Scholar] [CrossRef] [PubMed]
  84. Mabanta, C.G.; Pryhuber, G.S.; Weinberg, G.A.; Phelps, D.L. Erythromycin for the prevention of chronic lung disease in intubated preterm infants at risk for, or colonized or infected with Ureaplasma urealyticum. Cochrane Database Syst. Rev. 2003, 4, Cd003744. [Google Scholar] [CrossRef] [PubMed]
  85. Hartling, L.; Liang, Y.; Lacaze-Masmonteil, T. Chorioamnionitis as a risk factor for bronchopulmonary dysplasia: A systematic review and meta-analysis. Arch. Dis. Child. Fetal Neonatal Ed. 2012, 97, F8–F17. [Google Scholar] [CrossRef] [PubMed]
  86. Kim, C.J.; Romero, R.; Chaemsaithong, P.; Chaiyasit, N.; Yoon, B.H.; Kim, Y.M. Acute chorioamnionitis and funisitis: Definition, pathologic features, and clinical significance. Am. J. Obstet. Gynecol. 2015, 213, S29–S52. [Google Scholar] [CrossRef] [Green Version]
  87. Russel, P. Inflammatory lesion of the human placenta. 1. Clinical significance of acute chorioamnionitis. Am. J. Diagnost. Gynecol. Obstet. 1979, 1, 127–137. [Google Scholar]
  88. Mourani, P.M.; Abman, S.H. Pulmonary hypertension and vascular abnormalities in bronchopulmonary dysplasia. Clin. Perinatol. 2015, 42, 839–855. [Google Scholar] [CrossRef] [Green Version]
  89. Al-Ghanem, G.; Shah, P.; Thomas, S.; Banfield, L.; El Helou, S.; Fusch, C.; Mukerji, A. Bronchopulmonary dysplasia and pulmonary hypertension: A meta-analysis. J. Perinatol 2017, 37, 414–419. [Google Scholar] [CrossRef]
  90. Arjaans, S.; Wagner, B.D.; Mourani, P.M.; Mandell, E.W.; Poindexter, B.B.; Berger, R.M.; Abman, S.H. Early angiogenic proteins associated with high risk for bronchopulmonary dysplasia and pulmonary hypertension in preterm infants. Am. J. Physiol. Lung Cell. Mol. Physiol 2020, 318, L644–L654. [Google Scholar] [CrossRef]
  91. Levin, J.C.; Annesi, C.A.; Williams, D.N.; Abman, S.H.; McGrath-Morrow, S.A.; Nelin, L.D.; Sheils, C.A.; Hayden, L.P. Discharge practices for infants with bronchopulmonary dysplasia: A survey of national experts. J. Pediatr 2022, S0022-3476(22)00822-8. [Google Scholar] [CrossRef] [PubMed]
  92. Inatomi, T.; Oue, S.; Ogihara, T.; Hira, S.; Hasegawa, M.; Yamaoka, S.; Yasui, M.; Tamai, H. Antenatal exposure to Ureaplasma species exacerbates bronchopulmonary dysplasia synergistically with subsequent prolonged mechanical ventilation in preterm infants. Pediatr. Res. 2012, 71, 267–273. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  93. Van Marter, L.J.; Dammann, O.; Allred, E.N.; Leviton, A.; Pagano, M.; Moore, M.; Martin, C. Chorioamnionitis, mechanical ventilation, and postnatal sepsis as modulators of chronic lung disease in preterm infants. J. Pediatr. 2002, 140, 171–176. [Google Scholar] [CrossRef] [PubMed]
  94. Viscardi, R.M.; Manimtim, W.M.; Sun, C.C.; Duffy, L.; Cassell, G.H. Lung pathology in premature infants with Ureaplasma urealyticum infection. Pediatr. Dev. Pathol. 2002, 5, 141–150. [Google Scholar] [CrossRef]
  95. Viscardi, R.; Manimtim, W.; He, J.R.; Hasday, J.D.; Sun, C.C.; Joyce, B.; Pierce, R.A. Disordered pulmonary myofibroblast distribution and elastin expression in preterm infants with Ureaplasma urealyticum pneumonitis. Pediatr. Dev. Pathol. 2006, 9, 143–151. [Google Scholar] [CrossRef]
  96. Deutsch, G.H. Histologic Phenotypes of Bronchopulmonary Dysplasia and Childhood Interstitial and Diffuse Lung Disease. In Updates on Neonatal Chronic Lung Disease; Kallapur, S.G., Pryhuber, G.S., Eds.; Elsevier: Amsterdam, The Netherlands, 2020; pp. 13–31. [Google Scholar]
  97. Kitajima, H.; Fujimura, M.; Takeuchi, M.; Kawamoto, Y.; Sumi, K.; Matsunami, K.; Shiraishi, J.; Hirano, S.; Nakura, Y.; Yanagihara, I. Intrauterine Ureaplasma is associated with small airway obstruction in extremely preterm infants. Pediatr Pulmonol. 2022, 57, 2763–2773. [Google Scholar] [CrossRef]
  98. Theilen, U.; Lyon, A.J.; Fitzgerald, T.; Hendry, G.M.; Keeling, J.W. Infection with Ureaplasma urealyticum: Is there a specific clinical and radiological course in the preterm infant? Arch. Dis. Child. Fetal Neonatal Ed. 2004, 89, F163–F167. [Google Scholar] [CrossRef]
  99. Raynes-Greenow, C.H.; Roberts, C.L.; Bell, J.C.; Peat, B.; Gilbert, G.L.; Parker, S. Antibiotics for ureaplasma in the vagina in pregnancy. Cochrane Database Syst Rev 2011, 9, CD003767. [Google Scholar] [CrossRef]
  100. Kemp, M.W.; Miura, Y.; Payne, M.S.; Watts, R.; Megharaj, S.; Jobe, A.H.; Kallapur, S.G.; Saito, M.; Spiller, O.B.; Keelan, J.A.; et al. Repeated maternal intramuscular or intraamniotic erythromycin incompletely resolves intrauterine Ureaplasma parvum infection in a sheep model of pregnancy. Am. J. Obstet. Gynecol. 2014, 211, 134.e1–134.e9. [Google Scholar] [CrossRef]
  101. Grigsby, P.L.; Novy, M.J.; Sadowsky, D.W.; Morgan, T.K.; Long, M.; Acosta, E.; Duffy, L.B.; Waites, K.B. Maternal azithromycin therapy for Ureaplasma intraamniotic infection delays preterm delivery and reduces fetal lung injury in a primate model. Am. J. Obstet. Gynecol. 2012, 207, 475.e1–475.e14. [Google Scholar] [CrossRef] [Green Version]
  102. Miura, Y.; Payne, M.S.; Keelan, J.A.; Noe, A.; Carter, S.; Watts, R.; Spiller, O.B.; Jobe, A.H.; Kallapur, S.G.; Saito, M.; et al. Maternal intravenous treatment with either azithromycin or solithromycin clears Ureaplasma parvum from the amniotic fluid in an ovine model of intrauterine infection. Antimicrob. Agents Chemother. 2014, 58, 5413–5420. [Google Scholar] [CrossRef] [PubMed]
  103. Acosta, E.P.; Grigsby, P.L.; Larson, K.B.; James, A.M.; Long, M.C.; Duffy, L.B.; Waites, K.B.; Novy, M.J. Transplacental transfer of Azithromycin and its use for eradicating intra-amniotic ureaplasma infection in a primate model. J. Infect. Dis. 2014, 209, 898–904. [Google Scholar] [CrossRef] [PubMed]
  104. Keelan, J.A.; Nitsos, I.; Saito, M.; Musk, G.C.; Kemp, M.W.; Timmins, M.; Li, S.; Yaegashi, N.; Newnham, J.P. Maternal-amniotic-fetal distribution of macrolide antibiotics following intravenous, intramuscular, and intraamniotic administration in late pregnant sheep. Am. J. Obstet. Gynecol. 2011, 204, 546.e10–546.e17. [Google Scholar] [CrossRef] [PubMed]
  105. Kenyon, S.; Boulvain, M.; Neilson, J.P. Antibiotics for preterm rupture of membranes. Cochrane. Database Syst. Rev. 2013, 12, Cd001058. [Google Scholar] [CrossRef]
  106. Kenyon, S.L.; Taylor, D.J.; Tarnow-Mordi, W. Broad-spectrum antibiotics for preterm, prelabour rupture of fetal membranes: The ORACLE I randomised trial. ORACLE Collaborative Group. Lancet 2001, 357, 979–988. [Google Scholar] [CrossRef]
  107. Kirchner, L.; Helmer, H.; Heinze, G.; Wald, M.; Brunbauer, M.; Weninger, M.; Zaknun, D. Amnionitis with Ureaplasma urealyticum or other microbes leads to increased morbidity and prolonged hospitalization in very low birth weight infants. Eur. J. Obstet. Gynecol. Reprod. Biol. 2007, 134, 44–50. [Google Scholar] [CrossRef]
  108. Kenyon, S.L.; Taylor, D.J.; Tarnow-Mordi, W. Broad-spectrum antibiotics for spontaneous preterm labour: The ORACLE II randomised trial. ORACLE Collaborative Group. Lancet 2001, 357, 989–994. [Google Scholar] [CrossRef]
  109. Mallah, N.; Tohidinik, H.R.; Etminan, M.; Figueiras, A.; Takkouche, B. Prenatal Exposure to Macrolides and Risk of Congenital Malformations: A Meta-Analysis. Drug Saf. 2020, 43, 211–221. [Google Scholar] [CrossRef]
  110. Pronovost, G.N.; Hsiao, E.Y. Perinatal Interactions between the Microbiome, Immunity, and Neurodevelopment. Immunity 2019, 50, 18–36. [Google Scholar] [CrossRef]
  111. Grech, A.; Collins, C.E.; Holmes, A.; Lal, R.; Duncanson, K.; Taylor, R.; Gordon, A. Maternal exposures and the infant gut microbiome: A systematic review with meta-analysis. Gut Microbes 2021, 13, 1897210. [Google Scholar] [CrossRef]
  112. Nogacka, A.; Salazar, N.; Suárez, M.; Milani, C.; Arboleya, S.; Solís, G.; Fernández, N.; Alaez, L.; Hernández-Barranco, A.M.; de Los Reyes-Gavilán, C.G.; et al. Impact of intrapartum antimicrobial prophylaxis upon the intestinal microbiota and the prevalence of antibiotic resistance genes in vaginally delivered full-term neonates. Microbiome 2017, 5, 93. [Google Scholar] [CrossRef] [PubMed]
  113. Togami, K.; Chono, S.; Morimoto, K. Distribution characteristics of clarithromycin and azithromycin, macrolide antimicrobial agents used for treatment of respiratory infections, in lung epithelial lining fluid and alveolar macrophages. Biopharm. Drug Dispos. 2011, 32, 389–397. [Google Scholar] [CrossRef] [PubMed]
  114. Hassan, H.E.; Othman, A.A.; Eddington, N.D.; Duffy, L.; Xiao, L.; Waites, K.B.; Kaufman, D.A.; Fairchild, K.D.; Terrin, M.L.; Viscardi, R.M. Pharmacokinetics, safety, and biologic effects of azithromycin in extremely preterm infants at risk for ureaplasma colonization and bronchopulmonary dysplasia. J. Clin. Pharmacol. 2011, 51, 1264–1275. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  115. Poets, C.F.; Lorenz, L. Prevention of bronchopulmonary dysplasia in extremely low gestational age neonates: Current evidence. Arch. Dis. Child. Fetal Neonatal. Ed. 2018, 103, F285–F291. [Google Scholar] [CrossRef] [PubMed]
  116. Lowe, J.; Gillespie, D.; Hubbard, M.; Zhang, L.; Kirby, N.; Pickles, T.; Thomas-Jones, E.; Turner, M.A.; Klein, N.; Marchesi, J.R.; et al. Study protocol: Azithromycin therapy for chronic lung disease of prematurity (AZTEC)-a randomised, placebo-controlled trial of azithromycin for the prevention of chronic lung disease of prematurity in preterm infants. BMJ Open 2020, 10, e041528. [Google Scholar] [CrossRef] [PubMed]
  117. Horner, P.; Donders, G.; Cusini, M.; Gomberg, M.; Jensen, J.S.; Unemo, M. Should we be testing for urogenital Mycoplasma hominis, Ureaplasma parvum and Ureaplasma urealyticum in men and women?-A position statement from the European STI Guidelines Editorial Board. J. Eur. Acad. Dermatol. Venereol. 2018, 32, 1845–1851. [Google Scholar] [CrossRef] [Green Version]
  118. Aaltone, R.; Jalava, J.; Laurikainen, E.; Kärkkäinen, U.; Alanen, A. Cervical ureaplasma urealyticum colonization: Comparison of PCR and culture for its detection and association with preterm birth. Scand. J. Infect. Dis. 2002, 34, 35–40. [Google Scholar] [CrossRef]
  119. Frølund, M.; Björnelius, E.; Lidbrink, P.; Ahrens, P.; Jensen, J.S. Comparison between culture and a multiplex quantitative real-time polymerase chain reaction assay detecting Ureaplasma urealyticum and U. parvum. PLoS ONE 2014, 9, e102743. [Google Scholar] [CrossRef]
  120. Colaizy, T.T.; Morris, C.D.; Lapidus, J.; Sklar, R.S.; Pillers, D.A. Detection of ureaplasma DNA in endotracheal samples is associated with bronchopulmonary dysplasia after adjustment for multiple risk factors. Pediatr. Res. 2007, 61, 578–583. [Google Scholar] [CrossRef]
Table
Table 1. Summary of meta-analyses on the association between neonatal pulmonary colonization with Ureaplasma and bronchopulmonary dysplasia.
Table 1. Summary of meta-analyses on the association between neonatal pulmonary colonization with Ureaplasma and bronchopulmonary dysplasia.
Meta-AnalysisBPD28BPD36
Effect SizeKp-Value
Heterogeneity 		Effect SizeKp-Value
Heterogeneity
Wang et al. 1995 [39]RR 1.8
(95% CI 1.5–2.0)		170.09
Schelonka et al. 2005 [38]OR 2.8
(95% CI 2.3–3.5)		23<0.01OR 1.6
(95% CI 1.1–2.3)		8<0.01
Lowe et al. 2014
[27]		OR 3.0
(95% CI 2.4–3.8)		310.32OR 2.22
(95% CI 1.4–3.5)		17<0.001
Zhang et al. 2014 [37] OR 1.1
(95% CI 0.7–1.7)		11<0.001
BPD28: Bronchopulmonary dysplasia defined by oxygen requirement at 28 days postnatal; BPD36: Bronchopulmonary dysplasia defined by oxygen requirement at 36 weeks postmenstrual age; CI: confidence interval; K: number of studies included in the meta-analysis; OR: odds ratio; RR: risk ratio.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Van Mechelen, K.; van Westering-Kroon, E.; Hütten, M.; Mahieu, L.; Villamor, E. Placing Ureaplasma within the Context of Bronchopulmonary Dysplasia Endotypes and Phenotypes. Children 2023, 10, 256. https://doi.org/10.3390/children10020256

AMA Style

Van Mechelen K, van Westering-Kroon E, Hütten M, Mahieu L, Villamor E. Placing Ureaplasma within the Context of Bronchopulmonary Dysplasia Endotypes and Phenotypes. Children. 2023; 10(2):256. https://doi.org/10.3390/children10020256

Chicago/Turabian Style

Van Mechelen, Karen, Elke van Westering-Kroon, Matthias Hütten, Ludo Mahieu, and Eduardo Villamor. 2023. "Placing Ureaplasma within the Context of Bronchopulmonary Dysplasia Endotypes and Phenotypes" Children 10, no. 2: 256. https://doi.org/10.3390/children10020256

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop