Lung Function in a Cohort of Italian Children Born Preterm

Background and aim: in children born preterm lung development may be affected with impairment of lung function in later ages. The aim of the study was assessing lung function in a cohort of school-aged children born preterm and assess the inuence of perinatal variables. Methods: we measured lung function with impulse oscillometry system (IOS) and spirometry in 54 children [(male 48.1%, mean age 8.1 (0.8)] born preterm [mean birth weight (BW) 1462 (546.9) g, mean gestational age 31.5 (3) weeks] at Parma Children University Hospital (Italy). Maternal and perinatal data and respiratory medical history were also collected. Results: compared to predicted values, children born preterm showed higher mean values of airway impedance Z, airway resistance R5, R20, R5-20, area under the reactance curve AX and resonance frequency Fres, lower mean values of reactance X5 and FEF 25-75 . The mean difference between observed and predicted values of R5-R20 was higher in children born small for gestational age than in those born appropriate for gestational age. An inverse relationship was found between BW z-score and Z (r -0.40, r 2 0.16; p 0.012) and R5 (r -0.44, r 2 0.20; p 0.005) in children born with a BW <1500 g. Thirty-one per cent of children had a history of wheezing and 14.8% a history of lower respiratory infection requiring admission to the hospital. Conclusions: school-aged children born preterm had an impaired lung function, especially in the peripheral airways, as resulted by IOS and spirometry assessment. BW may have a role in lung development.


Introduction
Preterm birth (< 37 weeks of gestation) rate ranges from 5-10% of live births depending on the geographical area and a very low birth weight (VLBW) < 1500 g is reported in 1% of all infants 1,2 . Over the years, infant survival is improved but respiratory morbidity in the short and in the long-term remain an important complication.
Depending on gestational age (GA), fetal lung development is interrupted by preterm birth during the canalicular, saccular or alveolar phase of lung maturation. Perinatal exposures to in ammation, infection, mechanical ventilation and hyperoxia may lead to further insult to the immature lung with potentially long-term consequences on lung function. Bronchopulmonary dysplasia (BPD) is the most signi cant respiratory disease related to prematurity but the spectrum of respiratory impairment is wide involving also children without exposure to oxygen therapy or children born late preterm. Although the growth of the lung may catch-up during the rst years of life 3 , follow-up studies of subjects born preterm showed a higher risk of wheezing 4 and airway obstruction regardless a diagnosis of BPD 5,6 . These results suggest that preterm birth alone can negatively affect lung development 7,8 with potential consequences persisting into adulthood 9,10 .
The aim of the present study was to investigate lung function using the impulse oscillometry system (IOS) and spirometry in school-aged children born preterm who were followed-up in the outpatient clinic of Parma Children University Hospital (Italy) assessing the associations between lung function and perinatal variables. Maternal (age, smoking during pregnancy, pregnancy complications, gestational diabetes, prenatal steroid administration, cause and type of delivery) and neonatal (sex, weight, length and cranial circumference at birth, Apgar score, surfactant administration, duration of invasive and non-invasive ventilation, duration of oxygen therapy and BPD diagnosis) data were collected from medical records.

Study population and data collection
A diagnosis of BPD was de ned as oxygen need for ≥ 28 days from birth until 36 weeks of postmenstrual age according to the de nition described elsewhere 11 .
The study was approved by the ethical committee of Parma University Hospital (protocol number 732/2018/OSS*7AOUPR).
Lung function tests IOS and spirometry were performed using the Spirometry-IOS system (CareFusion, Germany). The system was calibrated each day prior to the measurements using a 3-liter syringe.
To obtain a valid IOS maneuver the subject had to tidally breath into a mouthpiece for at least 30 seconds with the cheeks supported by the hands of parents or operator. At least 3 reproducible maneuvers without artifacts due to coughing, swallowing, vocalization or breath holding had to be obtained to consider valid the tests. The mean value of IOS indices [impedance at 5 Hz (Z5), reactance at 5 Hz (X5), resistance at 5 Hz (R5) and at 20 Hz (R20), area under the reactance curve (AX) and resonance frequency (Fres)] were calculated from the 3 maneuvers. IOS indices (X5, Z5, R5, R20, AX and Fres) were expressed as raw values and z-scores derived from published reference data from a Brasilian healthy population; a log base-10 transformation of the variables Z5, R5, R20, Fres and AX in females and of the variable AX for males was performed according to the reference equations 12 . The difference R5-R20 was calculated and the predicted value derived on the basis of the predicted values of R5 and R20.
IOS generates small pressure oscillations at 30 s intervals into the airways by a loud speaker and measures respiratory impedance Z that includes respiratory resistance (R5 and R20) and respiratory reactance X. The pulmonary resistance R is the in-phase component of the lung impedance Z and accounts for the energy required to propagate the pressure wave through the airways including the pulmonary parenchyma and the thorax. Because oscillation frequencies < 15 Hz can be transmitted more distally in the lungs compared to higher frequencies, R5 re ects obstruction in both small and large airways while R20 re ects large airways only. The difference between R5 and R20 (R5-R20) is a derived index that describes the small airways resistance 13 . Reactance X is the out-of-phase component related to the elastic recoil and inertive properties of the lung. AX is the total reactance (area under the reactance curve) at all frequencies between 5 Hz and Fres (the point where the total reactance is 0) and re ects the ability of the peripheral lung to store capacitative energy (i.e. compliance). As the peripheral lung becomes less compliant, it cannot store as much capacitative energy and requires a larger pressure to in ate: an increase in small airway wall tone will decrease the reactance and increase AX. Thus, X5, AX and Fres all re ect changes in the degree of obstruction in the peripheral airways 13 . IOS was performed prior to spirometry to avoid in uence of forced exhalation maneuvers on results.
Standard spirometry maneuvers were performed according to ATS/ERS standards 14  At the time of assessment children were clinically stable and none had shown respiratory infections in the previous 3 weeks. The respiratory medical history was recorded by means of the International Study of Asthma and Allergy in Childhood (ISAAC) questionnaire 16 .

Data analysis
Data are reported as number and proportions or mean and standard deviations (SD) or median and 10th and 90th centile when appropriate. Descriptive statistics was used for differences in proportions.
Associations between maternal or perinatal variables (preclampsia or eclampsia, gender, GA, BW z-score, surfactant administration, days of invasive ventilation and days of oxygen therapy) and lung function tests (Z, X5, R5, R20, R5-R20, AX, Fres, FEV 1 , FVC, FEF 25 − 75 ) were assessed by linear regression analysis and comparison between groups with Student's t-test, as appropriate. Stepwise multiple regression analysis was used to determine the best predictor variables (sex, GA, BW z-score and preclampsia) for IOS parameters (Z, X5, R5, R20, R5-R20, AX, Fres) as dependent variables. Percentage of total variance in the dependent variable is expressed as the adjusted square of the multiple correlation coe cient (r 2 ).
BW was converted to z-score according to the Italian speci c reference for fetal growth 17 . Children were then categorized as born with a BW small for gestational age (SGA) when BW was ≥ 2SDs below the mean (i.e. <10th centile) or appropriate for gestational age (AGA) (i.e. ≥10th centile).
A lung function below the lower limit of normal was de ned as a z-score less than − 1.64 (< 5th centile). The analyses were carried out with the SPSS Statistics software. A p value less than 0.05 was considered as signi cant.

Population
In 2014 and 2019 fty-ve children [mean age 8.1 (0.87) years] in respiratory follow-up for prematurity at the outpatient clinic of the Paediatric Respiratory Unit of Parma Children University Hospital (Parma, Italy) were recruited. One girl was not included in the study because of severe neurological impairment. All children but one (98.1%) performed IOS test while only 44 (83%) were able to perform spirometry. The perinatal characteristics of these 54 children are shown in Table 1. Table 1 Characteristics of the cohort of 54 preterm babies.

Characteristics
No of subjects (% or median) Mean raw and z-score values for IOS lung function and spirometry are shown in Table 2.  Table 3. The regression equation generated by stepwise multiple regression analysis for Z5, R5, R20 and AX changes, as dependent variables, in children with BW < 1500 g included male sex and BW z-score as independent variable (Table 4). In this model BW z-score accounted for 29%, 20-43% of the total variance for Z5, R5 and AX respectively.   Table 4 Stepwise multivariable linear regression models using lung function indices z-scores as the dependent variable and perinatal factors as independent factors in the cohort of children born < 1500 g. Data are presented as coe cients (95%CI). Reported respiratory symptoms Thirty-one per cent of children (n 17) had a history of wheezing and 14.8% (n 8) a history of lower respiratory infection requiring admission to the hospital. Eleven children (20.3%) had experienced at least one episode of wheezing in the previous 12 months; there were no signi cant differences in lung function between these children and those without wheezing. Eight children (14.8%) were on regular antiasthmatic inhalers; 6 children were atopic (11.1%).

Discussion
In our cohort of school-aged children born preterm lung function measured by IOS and spirometry was signi cantly impaired as compared to predicted values. Notably, we found an increase in airway impedance with higher values of resistance and lower values of reactance, larger area under the reactance curve AX and higher resonant frequency Fres. Spirometry revealed normal forced expiratory volume and forced vital capacity but reduced values of FEF 25 − 75 compared to predicted values. Children with a diagnosis of BPD showed a signi cant lower FEV 1 than children without BPD. Having BPD did not in uence IOS parameters while being born SGA was associated to higher increase in the value of peripheral airway resistance R5-R20 compared to children born AGA. In children born with a BW < 1500 g airway impedance Z and airway resistance at 5 Hz were inversely related to BW z-score. Respiratory symptoms were reported by almost one third of our population.
The impairment of lung function in children born preterm is a well-known nding. Over the years, longitudinal studies demonstrated signi cant reduction of FEV 1 in cohorts of children born extremely preterm, especially in those with BPD 5,7,8,18,19 , and also in children born late preterm 20  One study conducted in a group of 6-8 years old Swedish children born preterm with a BW < 1500 g showed that, even if asymptomatic, those with BPD had worse IOS parameters with higher airway resistance and more negative reactance compared to children without BPD 28 . Same results were obtained in a similar cohort of 49 Finnish children studied at the age of 5-10 years 29 . Findings in adolescents born preterm between 24 and 31 weeks of gestation con rmed that those with a diagnosis of BPD had higher R5-R20 compared to peers without BPD 26 . In all the three studies FEV 1 was lower and often impaired in subjects with BPD. Thunqvist et al. found in 151 extremely preterm children aged 6 years a higher airway resistance R5-R20 compared to controls born at term irrespective of a diagnosis of BPD 20 . Compared to controls born at term, greater airway resistance was described in young children born late preterm (34-36 weeks of gestation) and higher values of R5, R5-R20 and AX were reported in male adolescents born moderate to late preterm (32-36 weeks of gestation) 30 suggesting that also slight prematurity can have a negative effect on pulmonary function.
Our results are in line with these previous ndings with a certain degree of respiratory impairment found in 7-9-year-old children born preterm. The IOS ndings of increased resistance of central and peripheral airways, lower reactance and increased AX and Fres suggest that children born preterm may have an increase in airway tone particularly in peripheral airways. Imaging studies performed in school children born very preterm revealed structural lung abnormalities of the peripheral airways such as airway wall thickening, increased subpleural opacities (that usually stand for alveolar septal brosis), mosaic perfusion and air trapping both in children with BPD 31 and in children without BPD 8 suggesting a xed peripheral airway narrowing 32 . These changes might be present especially in children who might have suffered from anatomical damages during fetal life like those with intrauterine growth restriction (IUGR).
IUGR is commonly suspected when BW is classi ed as SGA. IUGR is often the result of nutritional fetal de ciency and impaired oxygenation due to placental insu ciency 33 that usually manifests in the last phases of gestation when pulmonary parenchyma encounters acinar and alveolar phases. As a result, the growth of distal airways is the portion of the lung mainly affected as con rmed in animal studies 33 34 . Furthermore, in children with a VLBW (< 1500 g) we found that airway impedance Z and airway resistance R5 negatively correlated with BW z-score and that in the multivariable linear regression analysis BW z-score was an independent predictor for Z5, R5 and AX values. The relationship between low BW and impaired lung function has been extensively studied in infants 34,35 , childhood 5,8,36,37 and adults 38 . Our data may further con rm the association between fetal and airway growth, particularly in children with VLBW.
We speculate that the nding of lower FEF 25 − 75 in our cohort may re ect the impairment of small airways demonstrated with the increased values of the IOS indices. Previous studies showed concordant information between the oscillometric method and spirometry 26,28,29 but IOS is considered to be more sensitive in evaluating peripheral airways 13 . However, while we found a signi cant lower FEV 1 in subjects with BPD compared to those without BPD (-0.93 z-score), we did not nd a signi cant difference with IOS. However, all IOS indices were slightly but not signi cantly higher in children with BPD. In our population children born extremely preterm were only 9.2% of total while more than half (51.9%) was born moderate to late preterm; 10 children were diagnosed with BPD because exposed to long-term oxygen therapy.
These data point out that the number of subjects at risk for severe respiratory complications was small and that the not signi cant data obtained from IOS parameters when comparting children with and without BPD may be due to the small size of the sample. However, a recent prospective cohort study based in Italy on a large population of very preterm children (n 194) also reported no signi cant difference in airway resistance in subjects with and without BPD 22 . Regardless a diagnosis of BPD, preterm birth alone, interrupting the last phases of lung development, may have contributed to the abnormalities observed in our study in lung function tests.
Almost one third of children in our cohort reported at least one episode of wheezing con rming the higher prevalence of respiratory symptoms in preterms with and without BPD 4,25,36 . However, in contrast with other authors 27 , we did not nd higher airway resistance in children who had experienced wheezing in the previous year.
Our study has certain limitations. The cohort did not include a control population and we compared our IOS data with the published reference values of a Brasilian pediatric population. Our population cannot be considered representative of the total population of preterm children born in our area because we studied only those in regular follow-up in our respiratory clinic. Furthermore, we excluded from the study children with severe neurological impairment likely excluding the most severe cases. Our limited number of participants might have contributed to the non-signi cant associations between all the range of BW and the various IOS parameters because of power issues. The effect of bronchodilators on airway resistance was not evaluated, making it di cult to exclude that the higher resistance found in our study can be due to reversible airway obstruction. In addition, we did not assess lung function serially over time.
Further studies are needed to con rm our results.

Conclusions
Children born preterm showed some evidence of impaired lung function (higher large and small airway resistance, more negative reactance, higher AX and Fres, lower FEF 25 − 75 ) irrespective of BPD and respiratory symptoms. Preterm birth alone interrupting the last phases of lung development may contribute to structural alterations with possible long-term consequences. In children born < 1500 g airway resistance may be related to BW z-score and being born SGA may affect the development of small airways. Our results suggest that IOS is a feasible and non-invasive method to assess lung function and add additional information to standard spirometry about the respiratory function of children born preterm. To follow-up this high risk population taking account of their neonatal history and identifying pulmonary function impairments is important to early recognise lung de cit and to predict long-term respiratory outcome in childhood and adulthood. Determining which are the clinical implications of the functional abnormalities seen in these subjects and nding new practises to promote their health remain an important subject for future studies. Availability of data and materials:

List Of Abbreviations
the dataset analysed in the current study is available from the corresponding author upon request.
Competing interests: The authors declare no competing interests. Funding: none.
Authors' contribution: VF developed the design of the work, analysed the data and wrote the manuscript; IB, ES and MC collected the data and contributed to the analysis; CM, AC and GP gave a substantial scienti c contribution in interpretation of data and critically reviewed the manuscript. All authors approved the nal version of the manuscript.