Serum Periostin Levels at Birth as a Predictor for Bronchopulmonary Dysplasia in Premature Infants.

Background: Bronchopulmonary dysplasia (BPD) is the most common morbidity complicating preterm birth and affects long-term respiratory outcomes. Periostin plays an important role in the development of various disease such as allergic and pulmonary diseases. The objectives of this study were to evaluate the perinatal factors affecting serum periostin levels at birth and to establish whether serum periostin at birth, day of life (DOL) 28 and corrected 36 week’s gestational age could be potential biomarkers for BPD. Methods: A total of 139 preterm (n=98) and healthy (n=41) infants were included in this study. Among of them, 98 infants born < 32 weeks were divided into BPD (n=44) and non-BPD infants (n=54). Serum periostin levels were measured using an enzyme-linked immunosorbent assay. Results: The median serum periostin levels at birth in preterm infants born < 32 weeks were signicantly higher than those in healthy infants. Furthermore, there were signicant inverse correlations between gestational age, birth weight, and serum periostin levels at birth among all 139 preterm and healthy infants. Among preterm infants born < 32 weeks, with BPD and without BPD infants, the median serum periostin levels at birth were higher with BPD than without (345.0 ng/mL vs 278.0 ng/mL, P=0.002). Multivariate analysis revealed that serum periostin levels at birth was signicantly associated with BPD (P=0.032). Receiver operating characteristic analysis for serum periostin levels at birth in infants with and without BPD revealed that the area under the curve were 0.725 (95% CI 0.627- 0.822, P=0.0001). Serum periostin levels at birth with moderate/severe BPD were signicantly higher than those with non-BPD/mild BPD (338.5 ng/mL vs 283.5 ng/mL, P=0.0032). Conclusions: Serum periostin levels at birth were signicantly correlated with BW and GA. Furthermore, serum periostin


Background
Bronchopulmonary dysplasia (BPD) is the most common morbidity complicating preterm birth and affects neurodevelopmental impairment and long-term respiratory outcomes such as childhood wheezing and asthma (1,2). BPD results from various perinatal factors including maternal in ammation, surfactant de ciency, ventilation and oxygen toxicity (3,4). Premature infants are often exposed to positive pressure ventilation, and supplemental oxygen, contributing to the development of BPD. An important pathophysiological feature of infants affected with BPD is developmental arrest of alveolarization (4). Such structural alterations are accompanied by characteristic in ammatory changes and extensive remodeling of the extracellular matrix (ECM), together with increased smooth muscle mass in small pulmonary arteries and airways (5). Periostin is characterized as both a matricellular protein as well as ECM protein belonging to the fasciclin family (6,7).
Periostin plays an important role in the development of allergic, pulmonary, and the other diseases (7,8). Lung periostin is expressed in human lung broblasts and human bronchial epithelial cells (9). Since periostin is regulated by interleukin (IL)-4 and IL-13 and is involved in pathogenesis of brosis and allergy in various diseases, many studies reported that serum and plasma periostin levels were a potential biomarker for various disease such as idiopathic lung brosis in adults and asthma (7,10,11,12). Furthermore, various factors such as transforming growth factor-beta (TGF-β), IL-4, IL-13, mechanical stress, and connective tissue growth factor upregulate periostin (13). In the pathogenesis of BPD, TGF-β is involved in lung vascular development. Periostin are associated with TGF-β mediated brosis and lung development exposed to hyperoxia (8,14). TGF-β, in turn, is associated with the pathogenesis of BPD during lung vascular development. Although periostin expression is increased in autopsy lungs of preterm neonates with BPD (14), few reports have been suggesting the relationship between serum periostin levels at birth and BPD. Although Ahlfeld et al reported that elevated plasma periostin levels in BPD patients on day 28 of life (DOL28) compared with that's' of non-BPD patients, their study had several limitations, such as a low sample number and their choice of sampling times (DOL7 and DOL28) (15).
While some studies propose reference intervals for serum periostin in children (16,17), reports correlating serum periostin levels in term and preterm births with other perinatal factors are lacking.
In this study, we hypothesized that serum periostin at birth might increase in BPD patients, and could serve as biomarkers of BPD. The objectives of the present study were to evaluate the perinatal factors affecting serum periostin levels at birth in preterm and healthy infants and to validate whether serum periostin at birth, DOL28 and corrected 36 week's gestational age could be potential biomarkers for BPD.

Ethics approval and compliance
This research was approved by the Institutional Review Board of Fukushima Medical University, which is guided by local policy, national law, and the World Medical Association Declaration of Helsinki. As our human subjects were neonates, informed consent was solicited from parents or other legal guardians, and documented in writing.

NICU Patients
Blood samples were obtained from mechanically ventilated or oxygenated patients with parental consent in the neonatal intensive care unit (NICU) of Fukushima Medical University from November 2014 to July 2020. We examined cord blood at birth and venous blood at 36 weeks postmenstrual age and DOL28. Newborns with congenital anomalies or those who died prior to postnatal day 28 were excluded. Data for analysis included gestational age, phenotypic sex, body weight at birth, invasive mechanical ventilation at DOL28, supplemental oxygen at DOL14, respiratory distress syndrome (RDS), being small for gestational age (SGA), patent ductus arteriosus (PDA), Apgar scores and maternal complications: chorioamnionitis (CAM), premature rupture of membrane (PROM), hypertensive disorders of pregnancy (HDP) were recorded. BPD was de ned in accordance with the National Institutes of Health consensus de nition for infants (18). At a postmenstrual age of 36 weeks, the infants were classi ed into the following groups: mild BPD was de ned as the need for supplemental oxygen at ≥ 28 days but not at 36 weeks postmenstrual age; moderate BPD was de ned as the need for supplemental oxygen at 28 d, in addition to supplemental oxygen at FiO 2 (fraction of inspired oxygen) ≤ 0.30 at 36 weeks postmenstrual age; and criteria for severe BPD included the need for supplemental oxygen at 28 days and, at 36 weeks postmenstrual age, the need for mechanical ventilation and/or FiO 2 (fraction of inspired oxygen) > 0.30 (18). SGA was de ned as a birth weight of <-1.5 standard deviations that was corrected for the gestational age and sex in accordance with the criteria from previous study (19).

Healthy neonatal subjects
Healthy neonates who were born from 36.6 weeks to term in our hospital were included if informed consent was obtained from parents and/or legal guardians and documented in writing.

Statistical analysis
All data are presented as the medians. The Mann-Whitney U-test was used to compare continuous variables, and χ2 test was used for nominal variables. To evaluate the correlation between two parameters, Pearson's correlation coe cient was calculated. We performed multivariate analyses to determine factors signi cantly associated with serum periostin levels at birth as BW, GA, RDS, BPD, oxygen supplementation at DOL 14, invasive mechanical ventilation at DOL 28, and Apgar score at 1 min < 3 in premature infants born at less than 32 weeks. Next, we also performed multivariate analyses to determine factors signi cantly associated with BPD as potential confounding factors such as BW, GA, invasive mechanical ventilation at DOL 28, Apgar Score at 1 min < 3, oxygen supplementation at DOL 14 and serum periostin levels at birth in premature infants born at less than 32 weeks. The accuracy of diagnosing of classifying BPD was evaluated by receiver operating characteristics (ROC) curves with area under the curve (AUC) use to quantify the sensitivity of independent risks for BPD. The levels of signi cance were set 0.05 (P < 0.05). Data analysis was performed with SPSS (version 21.0) and GraphPad Prism version 8 software.

Results
Clinical characteristics and Serum periostin levels at birth in preterm and term infants.
A total of 139 preterm (n = 98) and healthy (n = 41) infants were included in this study. The clinical characteristics of preterm infants born at less than 32 weeks and healthy control are summarized in Table 1. Figure 1 shows the serum periostin levels at birth in preterm and term infants. The median serum periostin levels at birth among preterm infants born at less than 32 weeks was signi cantly higher than those among healthy infants (292.0 ng/mL vs 142.0 ng/mL, P < 0.0001) (Fig. 1A). Furthermore, there were signi cant inverse correlations between BW (r=-0.672, P < 0.0001), GA (r=-0.640, P < 0.0001), and serum periostin levels at birth in 139 preterm and term infants ( Fig. 1B and C). Perinatal factors and serum periostin levels at birth in preterm infants born at less than 32 weeks.
Next, among preterm infants born less than 32 weeks, we correlated serum periostin levels at birth with perinatal factors (Table 2). GA and BW were negatively correlated with serum periostin levels at birth. Additionally, serum periostin levels at birth were signi cantly higher in RDS, BPD, invasive mechanical ventilation at DOL 28, and Apgar score at 1 min < 3. In particular, the median serum periostin levels at birth were higher with BPD than without (345.0 ng/mL vs 278.0 ng/mL, P = 0.002). Multivariate analysis revealed that serum periostin levels at birth was signi cantly associated with BPD (P = 0.032).

Serum periostin levels in BPD infants
To investigate whether serum periostin levels were associated with BPD, preterm infants born at less than 32 weeks were divided into BPD neonates (n = 44) and non-BPD neonates (n = 54) ( Table 3). The median GA in BPD infants was signi cantly lower than those in non-BPD neonates (24.4 weeks vs 27.2 weeks, P < 0.001). The median BW in BPD neonates was also signi cantly lower than those in non-BPD infants (638 g vs 952 g, P < 0.001). The occurrence of Apgar score at 1 min < 3 (40.9% vs 14.8%, P < 0.005) was signi cantly higher in BPD infants compared with those in non-BPD infants. Furthermore, the incidence of the invasive mechanical ventilation at DOL 28 and oxygen supplementation at DOL14 in BPD infants were signi cantly higher than those in non-BPD infants. There were no signi cant differences between BPD and non-BPD infants in terms of phenotypic sex, antenatal steroid usage, SGA, PDA, CAM, HDP and PROM (Table 3). In multivariate analysis of the correlation between serum periostin at birth and clinical parameters, serum periostin levels at birth signi cantly correlated with BPD (P = 0.013) and BW (P = 0.021) ( Table 3). Receiver operating characteristic analysis for serum periostin levels at birth in infants with and without BPD revealed that the area under the curve were 0.725 (95% CI 0.627-0.822, P = 0.0001) (Fig. 2). Using a threshold of serum perisostin > 305 ng/mL at birth identi ed BPD with 71.7% sensitivity and 63.4% speci city.  Figure 3 shows the serum periostin levels at birth, DOL 28 and corrected 36-week's postmenstrual age in BPD and non-BPD infants. The median periostin levels on DOL 28 in BPD infants were signi cantly lower compared with those at birth (345.0 ng.mL vs 281.5 ng/mL, P = 0.003). However, the median serum periostin levels on DOL 28 and corrected age of 36-week's gestational were not signi cantly differed in BPD infants compared with non-BPD infants (DOL 28: 281.0 ng/mL vs 238.5 ng/mL, corrected 36-week's postmenstrual age: 327.5 ng/mL vs 332.0 ng/mL). Next, we evaluated the relationships between serum periostin at birth and severity of BPD (Fig. 4). Serum periostin levels at birth with moderate/severe BPD were signi cantly higher than those with non-BPD/mild BPD (338.5 ng/mL vs 283.5 ng/mL, P = 0.0032).

Discussion
To our knowledge, this is the rst study to describe an association between serum periostin levels at birth and perinatal factors in preterm and term infants and the correlation between serum periostin levels at birth and BPD. The present study revealed that higher serum periostin levels at birth in preterm infants born at less than 32 week's gestational age are independent risk factors for BPD and re ects the severity for BPD. Although there are many studies trying to demonstrate an association between serum biomarkers and the risk of BPD, few suggest a correlation between blood periostin levels and BPD. In this study, we also demonstrated that serum periostin levels on DOL28 and corrected 36 week's postmenstrual age could not serve as potential biomarkers for BPD. Ahlfeld et al previously suggested that early elevation of plasma periostin on DOL28 is signi cantly associated with chronic ventilator-dependent bronchopulmonary dysplasia (15). This may be due to the differences in the type of sample, sample size, and method of analysis. Ahlfeld's study used plasma samples and did not include multivariate or measure periostin levels at birth. In terms of the relationship between periostin and lung disease, previous studies demonstrated that elevated serum periostin levels were associated with various lung disease such as asthma, idiopathic pulmonary brosis, and COPD in children and adults (5,8,23,24). Furthermore, the expression of lung periostin was upregulated in patients with idiopathic lung brosis (8,11). Bozyk et al. also reported that periostin expression increased in autopsy lungs of preterm neonates with BPD (14). In a murine model of BPD exposed to hyperoxia, hyperoxia upregulated periostin expression in neonatal mice lung (14). Furthermore, lung periostin levels were also increased during the saccular stage, as previously shown (25). Although the mechanism by which periostin is associated with the pathogenesis of BPD remains poorly understood, we speculate that the linkage of periostin and TGFβ might be associated with the pathogenesis of BPD. Periostin and TGF-β are known to play a critical role in the proliferation of lung broblasts (9). Furthermore, many studies in different animal models of BPD con rm elevated TGF-β expression levels and activation of its associated pathways as an important part of lung disease pathophysiology (22,26,27). Also, we previously reported that serum TGF-β levels were upregulated in BPD patients (28).
Another new nding in this study was signi cant correlation of serum periostin levels at birth with BW and GA. Fujitani et al. reported that periostin levels in non-allergic children from 0 years to 15 years were almost 91.9-124.8 ng/mL (29). They also suggested that serum periostin levels gradually increased after age 10 years. Anderson et al also reported that serum periostin levels at ages 2-6 years ranged from 120-150 ng/mL (16). In this study, serum periostin levels of healthy neonates were around 140 ng/mL. Furthermore, serum periostin levels at birth in neonates born at less than 32 week's gestational age was almost 340 ng/mL. On the other hand, a previous study proposed a periostin threshold of 95 ng/mL based on values from healthy adult controls (30). Thus, serum periostin levels in infants were the highest when comparing infants, children, and adult. These developmental changes of serum periostin levels may be related to metabolic turnover and growth as periostin is a component of the extracellular matrix and regulates serum type I collagen formation, which is essential component of skin, tendon, and bone development (16,31). Compared with term infants, the cord blood serum procollagen type I C-terminal propeptide (PICP) as bone information in preterm infants was signi cantly higher and in uenced by fetal age (32).
Our study has several limitations. First, it was performed at a single center and the sample size of BPD patients was small. To validate our observations, a larger sample size with multiple centers and different ethnic cohorts would be invaluable. Second, we could not evaluate lung periostin. A previous study demonstrated that lung periostin in BPD infants was higher than in healthy lungs at term (9). Third, we could not detect the cellular sources of periostin. Thus, our next goal is to determine the cell types secreting periostin as well as the mechanism(s) of upregulation of periostin in BPD neonates; this will advance understanding of the pathogenesis of BPD. Lastly, in this study, we did not investigate the correlation between periostin levels and Th2 cytokines such as IL-4 and IL-13. It is noteworthy that upon stimulation by IL-4 and IL-13, periostin could be detected in lung broblasts (2). One of the main consequences of BPD is lung brosis. Although a previous study suggested that IL-4 and IL-13 levels of tracheal aspirates from premature infants were very low and did not correlate with BPD (29), premature infants born at less than 32 week's gestational age have increased nasal airway IL-4 and IL-13 secretion during rhinovirus infections (30).
In summary, we conclude that serum periostin levels were signi cantly correlated with birth weight and gestational age. Furthermore, serum periostin levels at birth could serve as a biomarker for predicting BPD and severity of BPD. The mechanism by which serum periostin is upregulated in BPD infants and inversely correlated with gestational age and birth weight remains to be further elucidated.