Zhen Li

Bénédicte Leynaert

Orianne Dumas

Oscar Diaz Gil

Fito Colomer

Nicole Le Moual

Christophe M Pison

Isabelle Romieu

Valérie Siroux

Judith Garcia- Aymerich

Montserrat Fito Colomer

Carlos A Camargo

Raphaëlle Varraso

Rachel Nadif

Role of Leptin in the Association Between Body Adiposity and Persistent Asthma: A Longitudinal Study

Keywords: adipokines, adiposity, epidemiology, respiratory leptin, adiposity, and persistent asthma

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Study Importance Questions

-What is already known about this subject?  Obesity, or high body adiposity, has been associated with poor asthma control.

 Leptin, an adipocyte-derived pro-inflammatory protein, has been associated with asthma in both basic research and cross-sectional epidemiological studies. However, prospective data are limited in the literature.

 The role of leptin in the association between body adiposity and persistent asthma remained unclear due to methodological challenges of traditional analytic methods.

-What does your study add?

 This study provided prospective data regarding the associations between body adiposity, leptin, and persistent asthma.

 Using a causal analytic approach and including multiple measures of adiposity, we reported for the first time an indirect effect mediated by leptin in the association between adiposity and persistent asthma over time.

MAIN TEXT Introduction

Obesity, or high body adiposity, has been associated with severity, poor control, and reduced response to standard therapy of asthma [START_REF] Novosad | Role of Obesity in Asthma Control, the Obesity-Asthma Phenotype[END_REF]. Potential underlying mechanisms by which obesity affects asthma include genetic and nutritional factors, alterations in the gut microbiome, systemic inflammation, metabolic abnormalities and changes in lung anatomy and function [START_REF] Peters | Obesity and asthma[END_REF]. Among them, the role of the non-T H 2 components and in particular leptin, an adipocyte-derived pro-inflammatory protein, has raised great interest in the obesity-asthma activity association [START_REF] Leiria | Obesity and asthma: beyond T(H)2 inflammation[END_REF]. Using cross-sectional data, a few studies have observed positive associations of serum leptin concentration with asthma severity, asthma control, or lung function in children, and to a lesser extent in adults [START_REF] Kattan | Asthma Control, Adiposity and Adipokines among Inner-City Adolescents[END_REF][START_REF] Tsaroucha | Leptin, adiponectin, and ghrelin levels in female patients with asthma during stable and exacerbation periods[END_REF][START_REF] Leivo-Korpela | Adipokine resistin predicts anti-inflammatory effect of glucocorticoids in asthma[END_REF]. Nevertheless, whether leptin mediates the adiposity-asthma activity association remains unclear, partly due to limited prospective data and methodological challenges raised by traditional regression analyses, such as insufficient power for the tests of direct and total effects [START_REF] Kenny | Power Anomalies in Testing Mediation[END_REF]. A causal approach to mediation analyses (8), using longitudinal data, can help to address these issues.

When studying the role of biological markers on disease, prospective data are warranted to avoid reverse causation. Indeed, using a cross-sectional design does not allow concluding whether the modification in the concentration of the biological marker is a cause or a consequence of the disease status. Mediation analysis in the counterfactual framework (8) using longitudinal data can minimize such reverse causation and offers an appropriate approach to study the effect of leptin in the association between body adiposity and asthma activity.

When investigating the obesity-asthma activity association, most of the studies in the literature used BMI to measure body composition. However, BMI is a mixed measure of lean and fat mass, which may lead to potential misclassification of body adiposity. It has been recently proposed that other measures rather than only BMI are needed to understand the obesity-asthma association [START_REF] Dixon | Metabolic dysfunction: mediator of the link between obesity and asthma?[END_REF], such as body fat percentage measured through BIA and waist circumference [START_REF] Duren | Body Composition Methods: Comparisons and Interpretation[END_REF]. Investigating the association between these different measures of body composition and asthma activity may provide more insight into the obesity-asthma activity association.

In this context, we investigated the role of leptin as a potential mediator, using a causal approach to mediation analysis, in the association between three measures of adiposity (BMI, WC, and BFP) and change in asthma activity over time.

Methods

We used data from the French Epidemiological study on the Genetics and Environment of Asthma (EGEA, https://egeanet.vjf.inserm.fr/index.php/fr/) [START_REF] Kauffmann | EGEA (Epidemiological study on the Genetics and Environment of Asthma, bronchial hyperresponsiveness and atopy)--design issues. EGEA Co-operative Group[END_REF], with data from the second survey (EGEA2, 2003-2007) as baseline, and the third survey (EGEA3, 2011-2013) as follow-up. We only included adults with current asthma at EGEA2 (n=558). After excluding missing data on body adiposity (n=53 for BMI, n=59 for WC, and n=232 for BFP respectively) and leptin (n=77), participants lost to follow-up (n=65), and those with missing data on current asthma assessed at EGEA3 (n=32), 331 participants were included in the analyses using BMI, 325 in the analyses using WC, and 152 in the analyses using BFP.

Ever asthma was defined by being recruited as an asthma case in chest clinics at EGEA1 or responding positively to at least one of the two questions at EGEA1 or EGEA2: 1) "Have you ever had attacks of breathlessness at rest with wheezing?" and 2) "Have you ever had asthma attacks?". Among participants with ever asthma (n=715), current asthma was defined by having had during the past 12 months 1) asthma attacks, 2) asthma symptoms (wheezing, nocturnal chest tightness, attack of shortness of breath), or 3) medication use because of breathing problems. Persistent asthma was defined by having current asthma at both surveys (EGEA2 and EGEA3); remitted asthma was defined by having current asthma at EGEA2 only.

Measurements of body adiposity were performed by trained health technicians at EGEA2 according to standardized protocols [START_REF] Callaway | Anthropometric standardization reference manual[END_REF]. Using data obtained from BIA (800 μA, 50 kHz), BFP was computed according to the prediction equation proposed by Sun et al. [START_REF] Sun | Development of bioelectrical impedance analysis prediction equations for body composition with the use of a multicomponent model for use in epidemiologic surveys[END_REF].

Serum leptin concentration (ng/ml) was measured at EGEA2 using the Luminex xMAG® technique in a BioPlex system (Bio-Rad Laboratories, Inc. Berkeley, California, USA). An aliquot of the same serum pool was run by duplicate in each plate. In order to minimize inter-plate variability, values of leptin concentration were divided by the pool result of the same plate and expressed as ratios [START_REF] Nadif | EGEA collection: a biobank devoted to asthma and asthma-related phenotypes[END_REF]. Age (continuous), sex, smoking status (never, ever), educational level (university or equivalent or not), physical activity (metabolic equivalents per week as previously defined ( 15)), and total energy intake (continuous), assessed at EGEA2, were considered as potential confounders because they have been reported to be associated with both body adiposity and asthma activity in the literature. In our study, as expected, participants with a greater body adiposity were more often classified as persistent asthma (Table 1), and they were older, more often men, never smokers, with a low educational level, not physically active, and with a high energy intake, as compared to participants with lower body adiposity [START_REF] Li | Cured meat intake is associated with worsening asthma symptoms[END_REF].

To disentangle the direct effect of adiposity on persistent asthma from the indirect effect mediated by leptin, we applied a mediation model in the counterfactual framework as previously described (8) (Figure 1). When relevant, the proportion of the adiposity effect explained by leptin in the relationship between adiposity and persistent asthma was calculated as (OR TE -OR DE ) / (OR TE -1), where OR TE and OR DE were respectively the odds ratio of total and direct effects [START_REF] Hafeman | Proportion Explained": A Causal Interpretation for Standard Measures of Indirect Effect?[END_REF].

Ethical approval was obtained from the relevant institutional review board committees (Cochin Port-Royal Hospital and Necker-Enfants Malades Hospital, Paris). All participants provided written informed consent.

Results

Main characteristics are presented in Table 1. The mean follow-up time was 7 years.

Participants with persistent asthma and those with remitted asthma were similar regarding age (P=0.12), sex (P=0.44), educational level (P=0.99), total energy intake (P=0.67), physical activity (P=0.83), and smoking status (P=0.18). However, compared with participants with remitted asthma, participants with persistent asthma (n=305; 92%) had higher BMI (24.3 vs. 22.6 kg/m 2 , P=0.05), greater waist circumference (83 vs. 77 cm, P=0.03), higher BFP (25.0% vs. 21.5%, P=0.06), and higher concentration of leptin (4.4 vs. 3.0 ng/ml, P=0.03). WC correlated strongly with BMI in men and in women (both r>0.8). BFP correlated more strongly with BMI in women (r=0.89) than in men (r=0.74).

For BMI, we observed a positive borderline significant total effect on persistent asthma over 7 years, with a significant positive indirect effect mediated by leptin (Figure 2) and a negative direct effect. The latter precludes the calculation of the proportion of the association mediated by leptin [START_REF] Hafeman | Proportion Explained": A Causal Interpretation for Standard Measures of Indirect Effect?[END_REF]. For WC and BFP, we observed similarly a significant positive total effect on persistent asthma, with a borderline significant positive indirect effect; the indirect effect mediated through leptin accounted for 64% and 63% of the total effect respectively for WC and for BFP.

Discussion

Applying a causal approach to mediation analysis, we reported for the first time an indirect effect mediated by leptin in the association between adiposity and persistent asthma over time. Nevertheless, due to the limited sample size and borderline significant findings, and to the many other inflammatory biomarkers that are potentially involved in asthma, the results need to be interpreted with caution.

A recent study using the traditional approach to mediation analysis concluded that leptin mediated the association between BFP and asthma control assessed by Asthma Control Questionnaire [START_REF] Zhang | Systemic inflammation mediates the detrimental effects of obesity on asthma control[END_REF]. However, this study was mainly limited by the lack of causality in study design (i.e. cross-sectional) and in analytic approach.

Animal studies support the biological plausibility of the hypothesis that adiposity may affect asthma activity through increased leptin level. Leptin administration to wildtype mice resulted in increased airway inflammation [START_REF] Sood | Adiponectin, Leptin, and Resistin in Asthma: Basic Mechanisms through Population Studies, Adiponectin, Leptin, and Resistin in Asthma: Basic Mechanisms through Population Studies[END_REF]. Indeed, leptin enhances phagocytosis, activation, proliferation, and alternation of macrophages, and induces production of pro-inflammatory cytokines, including tumour-necrosis factor (TNF) α, interleukin-6 (IL-6), and reactive oxygen species (ROS) [START_REF] Tilg | Adipocytokines: mediators linking adipose tissue, inflammation and immunity[END_REF], which are likely involved in pathophysiological processes of asthma. Taken together, we believe that leptin is a likely mediator in the association between body adiposity and persistent asthma activity.

Other non-T H 2 components may also be involved in the association between body adiposity and persistent asthma activity. Low concentration of serum adiponectin, which is an anti-inflammatory biological marker mainly secreted by adipocytes [START_REF] Bruun | Regulation of adiponectin by adipose tissue-derived cytokines: in vivo and in vitro investigations in humans[END_REF], is associated with higher asthma incidence in women according to a prospective study [START_REF] Sood | Low serum adiponectin predicts future risk for asthma in women[END_REF]. In addition, given the complex effect of insulin in the metabolism and inflammation process [START_REF] Dandona | Increase in the mediators of asthma in obesity and obesity with type 2 diabetes: Reduction with weight loss[END_REF][START_REF] Ghanim | Suppressive Effect of Insulin on the Gene Expression and Plasma Concentrations of Mediators of Asthmatic Inflammation[END_REF], the role of insulin in the association between obesity and asthma activity merits further investigation. Besides, as leptin and adiponectin can regulate the production of pro-inflammatory cytokines (e.g. IL-6 and TNF-α) by macrophages, such cytokines may also lie in the causal pathway of the association between body adiposity and persistent asthma activity [START_REF] Sood | Adiponectin, Leptin, and Resistin in Asthma: Basic Mechanisms through Population Studies, Adiponectin, Leptin, and Resistin in Asthma: Basic Mechanisms through Population Studies[END_REF]. Beyond adipocyte-derived pro-inflammatory protein, many other inflammatory biomarkers play a key role in asthma, including matrix metalloproteinase (MMP), chemokines and their receptors (CCR), more recently LIGHT and its receptor, and thymic stromal lymphopoeitin (TSLP) [START_REF] Ghanim | Suppressive Effect of Insulin on the Gene Expression and Plasma Concentrations of Mediators of Asthmatic Inflammation[END_REF]. Interestingly, in obese patients, the gene expression and/or concentrations of interleukin (IL)-4, ADAM-33, LIGHT, MMP-9, and CCR-2 are reduced following gastric bypass surgery and weight loss [START_REF] Dandona | Increase in the mediators of asthma in obesity and obesity with type 2 diabetes: Reduction with weight loss[END_REF]. Further studies, with a larger study sample, using a model including multiple pro-inflammatory factors as well as the interrelations between these factors would be helpful to understand the association between body adiposity and asthma activity. A better understanding of the underlying mechanisms by which obesity affects asthma is the first step toward the development of effective treatment to address this unmet medical need.

Compared with BMI, for which the sample size was the largest among the three measures of adiposity, we reported stronger total effects for WC and BFP on persistent asthma. This finding may be partly explained by the inaccurate measure of body adiposity using BMI (i.e. mixed measure of both lean and fat mass), particularly in men.

Using longitudinal data, this study adds further evidence on the role of leptin in the adiposity-asthma activity association and introduced an interesting analytic approach. However, because of the relatively small sample size, it was not possible to study the subgroup effects; the mechanisms underlying the adiposity-asthma activity association might be different according to sex, age of asthma-onset, and allergic sensitization.

Conclusion

In conclusion, leptin is likely to have partly mediated the association between adiposity and persistent asthma over time. Further larger prospective studies, using causal analytic approaches and including multiple measures of adiposity, are likely to provide new insight into the mechanisms by which obesity affects asthma activity.

Figure 1

 1 FIGURE LEGEND:

Figure 2

 2 Figure 2 Association between measures of body adiposity and persistent asthma,

ACKNOWLEDGMENTS

The authors thank the EGEA cooperative group, who participated in the setting of the study. Coordination: V Siroux (epidemiology, PI since 2013); F Demenais (genetics); I Pin (clinical aspects); R Nadif (biology); F Kauffmann (PI 1992-2012).

This work was supported in part by the French Agency of Health Safety, Environment and Work (AFSSET, EST-09-15), the National Research Agency (Health Work program ANR-CES-2009), the Region Nord Pas-de-Calais, Merck Sharp & Dohme (MSD), the GA2LEN project, Global Allergy and Asthma European Network, the National Hospital program of clinical research (PHRC-national EvAdA), and the Conseil scientifique AGIR pour les maladies chroniques. declared no conflict of interest.

ABSTRACT

Objective: Obesity is a likely risk factor for asthma. However, underlying mechanisms by which obesity affects asthma activity remain poorly understood. We aimed to investigate the role of leptin, an adipocyte-derived pro-inflammatory protein, as a mediator in the association between body adiposity (assessed using body mass index (BMI), waist circumference (WC), and body fat percentage (BFP)) and persistent asthma.

Methods: A causal approach to mediation analysis was used to disentangle total and direct effects, and the indirect effect mediated by leptin, using data from the French prospective EGEA study (baseline: 2003-2007; follow-up: 2011-2013; mean followup time: 7 years). 331 participants with current asthma at baseline were included.

Results: Per 1-SD increment in BMI, WC and BFP, the adjusted odds ratios (ORs) of the total effect were 1.59 (95% CI: 0.95, 2.97), 2.06 (1.06-4.00), and 3.25 (1.01-9.41), respectively; the ORs of the indirect effect mediated by leptin were 1.68 (1.09, 2.46), 1.55 (0.99-2.57), and 1.99 (0.94-4.83), respectively.

Conclusions: Leptin partly (>60%) mediated the association between high body adiposity and persistent asthma over time. Using a newly developed analytic approach, this longitudinal study brought new insight into one mechanism by which obesity may affect asthma activity.


Julien Delrieu

Bruno Vellas

Sophie Guyonnet

Christelle Cantet

Vitaliy Ovod

Yan Li

James Bollinger

Randall Bateman

Sandrine Andrieu

Sophie Breinig
email: s.breinig@orange.fr

Virginie Ehlinger

Jean-Christophe Rozé

Laurent Storme

Heloise Torchin

Xavier Durrmeyer

Gilles Cambonie

Christophe Delacourt

Pierre-Henri Jarreau

Lionel Berthomieu

Olivier Brissaud

Valérie Benhammou

Geraldine Gascoin

Catherine Arnaud

Pierre-Yves Ancel

Cognitive impact of multidomain intervention and omega 3 according to blood Aβ42/40 ratio: a subgroup analysis from the randomized MAPT trial

Keywords:

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Pulmonary hypertension (also named Persistent Pulmonary Hypertension of the Newborn, PPHN, in term babies) results from failure to transition to a normal circulation pattern at birth. The syndrome causes labile hypoxemia and hemodynamic shock, due to decreased pulmonary blood flow and right to left shunting across the ductus arteriosus and/or foramen ovale. Its prevalence among term or near-term infants has been reported to be between 0.4 and 6.8 per 1000 live-births, with the mortality rate ranging from 4 to 33 % and low morbidity [START_REF] Greenough | Pulmonary hypertension in the newborn[END_REF][START_REF] Cabral | Persistent pulmonary hypertension of the newborn: recent advances in pathophysiology and treatment[END_REF][START_REF] Sharma | Persistent pulmonary hypertension of the newborn[END_REF][START_REF] Steurer | Persistent pulmonary hypertension of the newborn in late preterm and term infants in California[END_REF]. In very preterm and extremely preterm newborns, the prevalence of PH has been much more rarely described [START_REF] Danhaive | Pulmonary hypertension and right ventricular dysfunction in growth-restricted, extremely low birth weight neonates[END_REF][START_REF] Nakanishi | Neonatal Research Network, Japan, Persistent pulmonary hypertension of the newborn in extremely preterm infants: a Japanese cohort study[END_REF] and seems to be higher, between 3.4 % and 8.1 % [START_REF] Danhaive | Pulmonary hypertension and right ventricular dysfunction in growth-restricted, extremely low birth weight neonates[END_REF][START_REF] Nakanishi | Neonatal Research Network, Japan, Persistent pulmonary hypertension of the newborn in extremely preterm infants: a Japanese cohort study[END_REF], in the few studies addressing this topic.

PH is a serious condition which is potentially completely reversible but which causes significant mortality in preterm newborns. Very preterm infants are a vulnerable population with a high risk of neonatal mortality and morbidity due to their immaturity [START_REF] Pierrat | Neurodevelopmental outcomes at age 5 among children born preterm: EPIPAGE-2 cohort study[END_REF]. In this context, the additional burden of PH itself and aggressive management of the condition is not known. Besides, in preterm and extremely preterm neonates PH is frequently associated with sepsis or pulmonary hypoplasia secondary to preterm premature rupture of membranes (PPROM) or oligohydramnios. Management of this condition remains controversial [START_REF] Baczynski | Shortterm and long-term outcomes of preterm neonates with acute severe pulmonary hypertension following rescue treatment with inhaled nitric oxide[END_REF][START_REF] Giesinger | Controversies in the identification and management of acute pulmonary hypertension in preterm neonates[END_REF]. Extracorporeal oxygenation is not possible and inhaled nitric oxide (iNO) is not recommended [START_REF] Barrington | Nitric oxide for respiratory failure in infants born at or near term[END_REF][START_REF] Sherlock | Inhaled nitric oxide use in neonates: balancing what is evidence-based and what is physiologically sound[END_REF]. The efficacy of iNO in very preterm infants with PH is uncertain and its use remains off-label. Nevertheless, even though approval of iNO by the US Food and Drug Administration (FDA) or by the European Medicine Agency was limited to infants born term and near-term at >34 weeks gestation, iNO has been used in many neonatal intensive care units, with large variations in practice [START_REF] Lakshminrusimha | Just say no to iNO in preterms-really?[END_REF]. Moreover, most published trials assessed the effectiveness of iNO in preventing bronchopulmonary dysplasia or severe brain injury in preterm infants [START_REF] Barrington | Nitric oxide for respiratory failure in infants born at or near term[END_REF][START_REF] Greenough | Inhaled nitric oxide (iNO) for preventing prematurity-related bronchopulmonary dysplasia (BPD): 7-year follow-up of the European Union Nitric Oxide (EUNO) trial[END_REF] but not its effects on PH [START_REF] Ancel | EPIPAGE-2 Writing Group, Survival and morbidity of preterm children born at 22 through 34 weeks' gestation in France in 2011: results of the EPIPAGE-2 cohort study[END_REF].

The main objectives of this study were to determine the prevalence of PH and the rate of survival without severe adverse outcome among infants with PH in a large national prospective population-based cohort of infants born preterm at 22 through 32 weeks gestation (EPIPAGE-2).

Secondary objectives were to describe treatments received by infants diagnosed with PH, to estimate the rates and causes of neonatal mortality and morbidity and to compare these with infants of the same gestational age (GA) without PH.

Methods

Study design

EPIPAGE-2 is a national prospective population-based cohort of children born preterm in France in 2011. Live births, stillbirths and terminations of pregnancy between 22 and 34 weeks gestation were eligible for inclusion in the study. A systematic stratified sampling design was implemented. Three strata were defined, with specific duration of recruitment period: births occurring at 22 through 26 weeks of gestational age (GA) with a duration of recruitment period of 8 months, births occurring at 27 through 31 weeks GA with a duration of recruitment period of 6 months, and births occurring at 32 through 34 weeks GA with a duration of recruitment period of 5 weeks. Maternal, obstetric and neonatal data were collected from medical records following a standardized protocol. In total, 6.7 % of the eligible livebirths at 22 through 26 w GA and 6.0 % of the eligible livebirths at 27 through 31 w GA did not participate in the study. Details of the protocol have been published elsewhere, as well as the comparison between participants and nonparticipants [START_REF] Ancel | EPIPAGE 2 Writing Group, EPIPAGE 2: a preterm birth cohort in France in 2011[END_REF].

For the current study, livebirths occurring between 22 +0 and 31 +6 weeks gestation were considered. Neonates with major malformation(s) and neonatal deaths in the delivery room were excluded.

Ethics

The study was approved by the National Data 160 Protection Authority (CNIL no. 911009), by the consultative Committee on the Treatment of Data on Personal Health for Research Purposes (reference no. 10.626), and the Committee for the Protection of People Participating in Biomedical Research (reference CPP SC-2873). After being informed of the aim of the study, families who agreed to participate gave informed consent.

Data collection of maternal and obstetric characteristics

The maternal sociodemographic factors collected included maternal age at delivery, pre-conception body mass index (BMI), country of birth, level of education, socioeconomic status and social insurance coverage at delivery. The obstetric characteristics studied were number of previous deliveries, infertility treatment, multiple pregnancies, monthly examination during pregnancy (considered as adequate follow-up of pregnancy), hospitalization during pregnancy, antenatal corticosteroid use, preterm premature rupture of membranes (PPROM), chorioamnionitis (whether suspected or proven), and induced prematurity defined as induction of labor or cesarean section before onset of labor.

Neonatal characteristics

We considered the following neonatal characteristics: gestational age (GA) at delivery (defined as the best obstetric estimate combining last menstrual period and ultrasonogram assessment in completed weeks of gestation), mode of delivery, sex, Apgar score at 5 min, small for gestational age (SGA) at birth defined as birthweight <10th percentile according to EPOPé curves [START_REF] Ego | Customized and non-customized French intrauterine growth curves. IIcomparison with existing curves and benefits of customization[END_REF], presence of minor malformation(s), oxygenation in the delivery room, surfactant use, respiratory distress syndrome, and treatment for persistent ductus arteriosus (non-steroidal anti-inflammatory drugs, NSAIDs), surgery). Neonatal infections were classified as early-onset sepsis (before 72 h), distinguishing early-onset sepsis without blood culture or cerebrospinal fluid (CSF) from proven neonatal bacterial infection with positive cultures of CSF or blood before 72 h of life, and late-onset sepsis (after 72 h of life) among survivors after 3 days. Duration of mechanical and non-invasive ventilation (in days) and GA at hospital discharge were recorded for survivors at discharge.

Pulmonary hypertension (PH) and hemodynamic treatments

On prospectively recorded data, PH diagnosis was based on the binary item "proven persistent pulmonary hypertension" (yes/no); this item was assessed by clinician by the presence of a significant oxygen pre and post-ductal saturation gradient and/or echocardiographic criteria. Cases with a missing value on this item were (re)classified as having PH only if the items relating to inhaled nitric oxide (iNO) treatments indicated that iNO was administered and exclusively in case of refractory hypoxemia with pulmonary hypertension. Infants who clearly received iNO for an indication other than hypoxemia with pulmonary hypertension were excluded. Otherwise, the missing value for the item PH was maintained.

In the subsequent sections, PH+ and PH-denote neonates with a PH diagnosis and with no PH diagnosis, respectively. During the period of the study (2011) saturation targets were in Franc 88-96 %. Treatments of PH+ infants included the use of iNO due to hypoxemia with pulmonary hypertension. Age at start, duration of iNO and maximum administered dose of iNO were detailed. Administration of class III sedative or analgesic treatments, use of vasoactive and inotropic drugs in the first 72 h after birth and after 72 h (among day 3 survivors), with type of treatment (dobutamine, dopamine, norepinephrin) and volume expansion during the first 72 h after birth with volume of vascular filling were collected for all neonates, whatever the PH status.

Outcomes

The primary outcome was survival without severe neonatal morbidity at discharge. Severe neonatal morbidity was a composite endpoint defined as at least one adverse outcome among the following: severe bronchopulmonary dysplasia, defined as a need for oxygen for at least 28 days plus a need for oxygen concentration 30 % or greater, mechanical ventilator support, or continuous positive airway pressure at 36 weeks postmenstrual age [START_REF] Jobe | Bronchopulmonary dysplasia[END_REF]; severe cerebral lesions, defined as grade 3 or higher intraventricular hemorrhage [START_REF] Volpe | Brain injury in premature infants: a complex amalgam of destructive and developmental disturbances[END_REF]; grade 3 or higher retinopathy of prematurity according to the international classification (International Committee for the Classification of Retinopathy of Prematurity) [START_REF]International Committee for the Classification of Retinopathy of Prematurity, The International Classification of Retinopathy of Prematurity revisited[END_REF]; and grade II and III necrotizing enterocolitis according to Bell staging [START_REF] Bell | Neonatal necrotizing enterocolitis. Therapeutic decisions based upon clinical staging[END_REF].

Secondary outcomes were neonatal death before hospital discharge and severe neonatal morbidity among survivors at discharge.

Statistical analysis

Data were analyzed using STATA version 14.2 (StataCorp LLC, College Station, TX, USA). All statistical tests were two-sided and statistical significance was set at a 5 % level. In order to take the study design into account and ensure the representativeness of the sample, because of unequal probability of selection of children across GA groups due to varying recruitment durations for births occurring at 22 through 26 weeks GA and at 27 through 31 weeks GA, weighted estimations were computed. The observations weights were inversely proportional to the duration of the recruitment period in their GA group.

Descriptive statistics

Maternal, obstetrical and neonatal characteristics were described according to GA group (22-27, 28-31 weeks gestation) using bivariate analyses. Categorical variables were summarized using percentages with 95 % confidence intervals (CIs). They were compared between groups using the Pearson chi-square test with a Rao-Scott correction. Continuous variables were summarized using mean ± standard error if normally distributed, median, 1st and 3rd quartile if not normally distributed, and compared across groups using Wald tests. Variance estimates were adjusted for non-independence of observations due to multiple deliveries to the same mother, considering multiples as clustered within a pregnancy.

Multiple imputation of missing data

Maternal age, multiple pregnancy, gestational age at birth, infant's sex, birthweight and neonatal death were available for all the infants included in the study. Missing values were observed on the other variables, with around than 5 % of missing values for the majority of variables (Table 1 online). The missing-data pattern was explored (Table 1 online). Accordingly, missing data on exposure (PH), outcomes and confounders were multiply imputed by chained equations [START_REF] White | Multiple imputation using chained equations: issues and guidance for practice[END_REF] among the eligible sample, assuming the missing data were missing at random (MAR) as defined in Rubin's terminology.

The imputation model included the variable related to PH diagnosis (proven PH, iNO use and timing of iNO administration before or after 7 days after birth), the potential confounders for the planned analysis model and the primary outcome. Maternal data and neonatal characteristics found to be correlated with PH diagnosis and/or with the outcome in univariate analyses performed on observed data as well as factors predicting non-response to PH treatment, and/or with missing values on the primary outcome were entered in the imputation model as auxiliary variables. As recommended in the literature, these variables were selected to make the MAR assumption more plausible and to improve the prediction models. The list and coding of these variables are reported in Table 2 online. Binary variables were imputed using logistic regression models and categorical variables using multinomial logistic models, while predictive mean matching was used for continuous variables. The imputation model accounting for the sampling weights was applied to generate a total of M = 40 independent imputed datasets, with 10 iterations each. The imputed values were explored by descriptive statistics and compared to observed data, in order to verify that the imputed data were reasonable (Table 2 online). The convergence of the procedure was visually inspected.

Analysis of the primary outcome on the imputed datasets

The prevalence of PH+ diagnosis was estimated in the total sample and by gestational age group.

The rate of survival without severe neonatal morbidity was modelled by a modified Poisson regression model with robust estimation of variances [START_REF] Zou | Extension of the modified Poisson regression model to prospective studies with correlated binary data[END_REF] and estimated by generalized estimating equations (GEE) models that accounted for the clustering effect due to multiple pregnancies [START_REF] Ananth | Regression models for clustered binary 558 responses: implications of ignoring the intracluster correlation in an analysis of perinatal 559 mortality in twin gestations[END_REF]. The models were developed on the multiply imputed data, with estimates from each imputed dataset being pooled using Rubin's combination rules [START_REF] Rubin | Frontmatter[END_REF]. Results were expressed as adjusted incidence rate ratios (aIRR), which can be interpreted as relative risks, and their associated 95 % CIs. Significance of the regression coefficients was based on F statistics.

The primary outcome was assessed by comparing the crude rates of survival without severe morbidity, globally (Model 1) and within each GA group. Model 1 was further adjusted for GA (22-27, 28-31 weeks gestation) (Model 2). The interaction term between GA group and PH was tested at this stage. Baseline variables considered as potential confounding factors were then added to Model 2. Confounders included in the initial model were maternal age at delivery (divided into 3 categories: <25 years, 25-34 years, ≥35 years), mother's country of birth (France versus other country), maternal social insurance coverage (National Health Insurance versus state health aid, universal health insurance or none), maternal education level (higher than high school versus high school or less), antenatal corticosteroid use (no corticosteroids, one incomplete course, one complete course or more), PPROM (yes/no), suspected or proved chorioamnionitis (yes/no), sex of the neonate, Apgar score at 5 min (<7 versus ≥7), growth status at birth (small for gestational age (SGA) yes/no, <10th percentile, EPOPé curves according to Ego et al. [START_REF] Ego | Customized and non-customized French intrauterine growth curves. IIcomparison with existing curves and benefits of customization[END_REF], surfactant use and respiratory distress syndrome (RDS), categorized as no surfactant use or RDS, surfactant use but no RDS, and RDS), administration of catecholamines before 72 h of life (yes/no), early-onset sepsis (no versus probable or certain). These variables were selected on their clinical relevance, as they were expected to be related both to PH diagnosis and neonatal outcome, or because they were known as predicting the primary outcome [START_REF] Steurer | Persistent pulmonary hypertension of the newborn in late preterm and term infants in California[END_REF][START_REF] Ancel | EPIPAGE-2 Writing Group, Survival and morbidity of preterm children born at 22 through 34 weeks' gestation in France in 2011: results of the EPIPAGE-2 cohort study[END_REF]. Non-significant variables were sequentially eliminated from this initial model: the covariate with the largest P-value greater than the significance level was removed, unless its removal led to a relative change in the estimated regression coefficient for PH of 20 % or more.

Interactions between each adjustment variable in turn and GA, and between each maternal or pregnancy covariate in turn and PH diagnosis, were explored in the multivariate regression model. The aim was to detect subgroups of infants where the hypothesized reduction in the rate of survival without severe morbidity linked to PH was more marked. The interaction between PH status and the variable "Surfactant use and RDS" was judged as not relevant and was not tested, because we expected that there would be very few PH+ infants without surfactant administered and PH+ infants without RDS. Interactions found to be significant were removed in a sequential fashion if they did not reach the significance level in the multivariable model. The final adjusted model was named Model 3.

Finally, as we could not exclude the possibility that catecholamines were administered after PH diagnosis among PH+ infants and they could be an intermediary in the causal association between PH and the primary outcome, Model 3 was replicated excluding the covariate "use of catecholamines before 72 hours" [START_REF] Ananth | Confounding, causality, and confusion: the role of intermediate variables in interpreting observational studies in obstetrics[END_REF].

Analysis of secondary outcomes after on imputed datasets

Neonatal death rates were estimated and compared according to PH diagnosis and gestational age group. The incidence of neonatal death in each PH group, by gestational age, was presented using survivor functions estimated by the Kaplan-Meier product-limit estimator. Finally, the proportion of severe neonatal morbidity among survivors at discharge was described by PH diagnosis and by gestational age group. Statistical comparison tests between PH groups were performed by using a modified Poisson regression model applied on the multiply imputed datasets. We aimed to distinguish between PH profiles. The PH+ group was separated into two groups: PH+ without iNO treatment, PH+ with iNO. The regression models (Model 1, Model 2 and Model 3) were repeated using this detailed three-category PH variable as the exposition variable.

Sensitivity analyses

Two sensitivity analyses were performed on the multiply imputed datasets. Because we expected that most PH+ infants would have respiratory distress syndrome, the first sensitivity analysis consisted in applying Model 1, Model 2 and Model 3 after excluding PH+ infants without RDS in order to clarify the role of PH.

Results

Description of the population

Neonates were born in 276 hospitals and the PH+ neonates were hospitalized in 61 level 3 hospitals.

The eligible population for the study consisted of 3383 live-born infants, born between 22 and 31 +6 weeks gestation, without major malformation(s) and alive at discharge from the maternity unit. Of these infants, 3103 had available data on both the exposition to PH and the survival without severe morbidity (Fig. 1).

The maternal, obstetrical and neonatal characteristics for the total population and according to gestational age groups are presented in Table 3 online. Missing values ranged between 0 % and 18 % (Table 1 online). Among eligible, 1802 observations (53 % of the eligible population) had data available for all variables planned in the analysis model, and 1026 observations had one missing value among the variables planned in the analysis model (including PH and survival without severe morbidity). Missing data were more frequent among deceased infants, and even more frequent among infants deceased before 2 days of life (data not shown). Otherwise, no specific pattern was observed. As a consequence, we assumed that the missing data were MAR. The main results are based on the imputed data.

Main objectives

The prevalence of PH was estimated at 6.0 % (95 % CI, 5.2-6.9 1). The IRR did not significantly differ between the two GA groups. (Table 1).

Apart from the presence of chorioamnionitis and the absence of administration of antenatal corticosteroid therapy, no maternal characteristic appeared to be significantly associated with PH at the 5 % threshold in bivariate analyses (performed on the available data). Neonatal factors associated with PH were gestational age, Apgar score at 5 min, oxygenation in the delivery room, administration of surfactant, RDS, treatment of ductus arteriosus with NSAIDs and by surgery, and early onset infection within the first 72 h of life (Table 2).

After adjustment for confounding factors (Model 3), the rate of survival without severe morbidity in PH+ newborns was half that of PHnewborns (aIRR = 0.53, 95 % CI, 0.43-0.65, P < .001). This risk ratio did not differ significantly between the two gestational age groups. The Model 3 included three interaction terms with gestational age at birth, which reflected that the incidence rate ratios for the variables "small for gestational age", "antenatal steroids" and "surfactant use and RDS" differed between the gestational age groups. The sensitivity analyses produced similar results, with slightly lower IRRs. The sensitivity analyses produced similar results, with slightly lower IRRs.

As we could not exclude the possibility that catecholamines were administered to PH+ infants after the diagnosis of PH and that this could intervene in the causal association between PH and the primary outcome, Model 3 was replicated excluding the covariate "use of catecholamines before 72 hours". In this model, IRR = 0.50, 95 % CI, 0.41-0.61, P < .001. The regression model including Model 3 and the distinction between PH treated with iNO and not treated by iNO found an aIRR = 0.37, 95 % CI, 0.27-0.52, P < .001, and aIRR = 0.69, 95 % CI, 0.54-0.87, P < .002, respectively.

Secondary objectives

Treatments of PH

The most frequent administered treatment was sedative treatment: 92.2 % of infants (95 % CI, 87.7-95.2), with no significant difference between gestational age groups.

Inhaled NO was administered in 63.5 % (95 % CI, 56.6-69.9) of cases, 60.8 % (95 % CI, 52.5-68.5) for 22-27 +6 WG infants and 68.9 % (95 % CI, 56.2-79.2) for 28-31 +6 WG infants (P = .276). For all the 22-31 +6 WG infants, the median age at start of iNO treatment was 1 day of life (Q1-Q3: 0-8). Half of the infants received iNO on day 0 or day 1. Median duration of iNO administration was 2 days (Q1-Q3: 1-6). The median administered dose was 15 ppm (parts per million) (Q1-Q3: 10-15). None of these values differed significantly between the two gestational age groups. Among the PH+ neonates, 48.6 % (95 % CI, 41.9-55.3) received volume expansion within their first 72 h of life, with a median volume of 20 ml/kg (milliliter per kilogram of bodyweight). Volume expansion was more frequent (58.7 % vs 43.5 %, P = .043) and the amount received was greater (28 vs 20 ml/kg, P = .043) in infants born at 28-31 +6 weeks gestation compared with those born at 22-27 +6 weeks.

Lastly, 36.4 % (95 % CI, 30.3-43.1) of PH+ neonates received at least one vasoactive or inotropic drug (dopamine, dobutamine, norepinephrin) before 72 h of life, and 46.0 % (95 % CI, 39.1-53.0) after 72 h.

Death or severe morbidities at discharge

Neonatal death and severe comorbidities according to PH status are summarized in Table 3. Deaths before discharge were >5-fold higher in the PH+ group than in the PH-group (40.5 %, 95 % CI, 33.7-47.2 versus 7.4 %, 95 % CI, 6.5-8.3), respectively, P < .001), with differential estimates according to gestational age.

In the 28-31 +6 WG group, the neonatal death rate was almost 10-fold higher in PH+ than in PH-infants, whereas in the most immature group (22-27 +6 WG) it was only twice as high, even though the basic death rate was higher in the latter. Overall time between birth and death did not differ according to PH status or gestational age (Table 4 online). Fig. 1 online presents survival functions according to PH status and gestational age.

Respiratory causes accounted for the majority of deaths in the PH+ group. More than half the deceased neonates had severe neurologic impairment (Table 4 online) but this was not the cause of death. There was no difference between PH+ and PH-infants in relation to this factor and no difference regarding discontinuation or limitation of treatment.

Among survivors at discharge, the proportion of neonates with severe morbidity was >3-fold greater among PH+ neonates compared with PH-neonates (49.3 %, 95 % CI, 40.3-58.4 versus 13.4 %, 95 % CI, 12.1-14.7 respectively, P < .001), and also in the two gestational age groups (Table 3). In both gestational age groups the main morbidity was bronchopulmonary dysplasia. Intraventricular grade III or IV hemorrhage or cystic periventricular leukomalacia were significantly higher only in the 28-31 +6 WG group (and was almost 5-fold higher in the PPHN+ group). Conversely, severe retinopathy was higher in the 22-27 +6 WG group. No difference was observed in the incidence of necrotizing enterocolitis.

Discussion

In this study, the prevalence of PH among 22-31 +6 preterm infants in a large national prospective population-based cohort (EPIPAGE-2) was estimated at 6.0 % (95 % CI, 5.2-6.9) and survival without severe morbidity was 60 % lower in the PH+ group compared with the PHgroup. This is concordant with the rare studies on this subject, with a reported prevalence ranging from 3.4 % [START_REF] Seth | Risk factors and outcomes of early pulmonary hypertension in preterm infants[END_REF] to 8.1 % [START_REF] Nakanishi | Neonatal Research Network, Japan, Persistent pulmonary hypertension of the newborn in extremely preterm infants: a Japanese cohort study[END_REF]. This overall proportion is much higher than that reported in late preterm and term neonates (0.4 to 6.8 per 1000 live births [START_REF] Greenough | Pulmonary hypertension in the newborn[END_REF][START_REF] Cabral | Persistent pulmonary hypertension of the newborn: recent advances in pathophysiology and treatment[END_REF][START_REF] Sharma | Persistent pulmonary hypertension of the newborn[END_REF][START_REF] Steurer | Persistent pulmonary hypertension of the newborn in late preterm and term infants in California[END_REF]). By gestational age, the prevalence of PH was markedly higher in the 22-27 +6 WG group (14.5 %) than in the 28-31 +6 WG group (2.7 %), in agreement with the findings of Nakanishi et al. [START_REF] Nakanishi | Neonatal Research Network, Japan, Persistent pulmonary hypertension of the newborn in extremely preterm infants: a Japanese cohort study[END_REF] In this large multicenter cohort study of 12,954 extremely preterm infants (28 WG or less), these authors found that the prevalence of PH increased with decreasing gestational age: 4.4 % for infants born at 27 WG and 18.5 % for infants born at 22 WG.

To our knowledge, our study, which highlights a significantly increased rate of neonatal death and morbidity among PH survivors, is the first to address mortality and morbidity in a large cohort of both extremely preterm and very preterm neonates. In the literature, we found no results on the short-, medium-and long-term outcome of these severely affected infants. Although the prevalence of PH was higher in the group of more immature neonates, the absolute number of very

Table 1

Main outcome (survival without severe morbidity) by pulmonary hypertension, globally and by gestational age. Results from the observed data and from the pooled analysis of the imputed datasets. Data were inversely weighted according to duration of recruitment by GA groups. The variance estimation accounted for the clustering effect due to multiple pregnancies. preterm neonate births is known to be generally higher than that of extremely preterm neonates [START_REF] Ancel | EPIPAGE-2 Writing Group, Survival and morbidity of preterm children born at 22 through 34 weeks' gestation in France in 2011: results of the EPIPAGE-2 cohort study[END_REF]. This represents a larger number of infants requiring treatment for challenging and life-threatening PH, and there is no consensus on the limits of aggressive management of a severe condition among very vulnerable and immature neonates. As found in a few studies [START_REF] Nakanishi | Neonatal Research Network, Japan, Persistent pulmonary hypertension of the newborn in extremely preterm infants: a Japanese cohort study[END_REF][START_REF] Seth | Risk factors and outcomes of early pulmonary hypertension in preterm infants[END_REF], PH was associated with chorioamnionitis and with the administration of antenatal corticosteroids, but not with PROM. This result was unexpected because PROM can be responsible for infections and pulmonary hypoplasia, which are both major factors suspected of increasing pulmonary vascular resistances. Study of extremely preterm neonates with PROM at 22-25 WG (neonates also included in the EPIPAGE-2 study [START_REF] Lorthe | Preterm premature rupture of membranes at 22-25 weeks' gestation: perinatal and 2-year outcomes within a national population-based study (EPIPAGE-2)[END_REF] and born between 23 and 27 WG) found a mortality rate similar to our population (48.3 % in PROM neonates versus 48.1 % in PH neonates). Survival without severe morbidity was lower in our PH population (22.5 %) than in the PROM population (38.8 %), but PROM is only one of the numerous causal factors of PH. Besides, neonatal factors associated with PH were gestational age, Apgar score at 5 min, treatment of ductus arteriosus, RDS and the onset of early infection within the first 72 h of life. Only the first three of these factors were also found to be associated with PH in a Japanese cohort consisting exclusively of preterm infants aged <28 WG [START_REF] Nakanishi | Neonatal Research Network, Japan, Persistent pulmonary hypertension of the newborn in extremely preterm infants: a Japanese cohort study[END_REF].

Our analyses showed that survival without severe morbidity was significantly lower in the PH+ group (30.2 %) than in the PH-group (80.2 %). This is the first study to propose composite criteria for estimating the global impact of PH in preterm infants. Nakanishi et al. [START_REF] Nakanishi | Neonatal Research Network, Japan, Persistent pulmonary hypertension of the newborn in extremely preterm infants: a Japanese cohort study[END_REF] found a significantly higher prevalence of bronchopulmonary dysplasia and severe intraventricular hemorrhage (20 % in PH+ patients versus 7 % in PH-). Unlike us, these authors also observed an increased prevalence of necrotizing enterocolitis and severe retinopathy. In our study, the occurrence of grade III or IV intraventricular hemorrhage or cystic periventricular leukomalacia was increased only in the 28-31 +6 PH+ group. Overall, our study suggests that the impact of PH differs according to GA group. Although neonatal deaths were high in both groups, the difference tended to be more marked in the second group (28-31 +6 WG), probably because, as could be expected, the more immature infants had a higher death rate. This was also true of overall severe morbidity, which was proportionately higher in the 28-31 +6 WG group (4-fold higher) than in the 22-27 +6 WG group, where it was twice as high.

Treatment of PH is quite aggressive, generally including sedative drugs, optimization of alveolar recruitment (with high frequency oscillation ventilation if required), iNO, hemodynamic support by volume expansion (if required) and vasoactive drugs. These interventions are now well established for the management of PPHN of term neonates [START_REF] Storme | French Congenital Diaphragmatic Hernia Study Group, Pathophysiology of persistent pulmonary of the newborn: impact of the perinatal environment[END_REF][START_REF] Abman | American Heart Association Council on Cardiopulmonary, Critical Care, Perioperative and Resuscitation; Council on Clinical Cardiology; Council on Cardiovascular Disease in the Young; Council on Cardiovascular Radiology and Intervention; Council on Cardiovascular Surgery and Anesthesia; and the American Thoracic Society. Pediatric pulmonary hypertension: guidelines from the American Heart Association and American Thoracic Society[END_REF][START_REF] Lakshminrusimha | Considerations in the management of hypoxemic respiratory failure and persistent pulmonary hypertension in term and late preterm neonates[END_REF][START_REF] Jain | Persistent pulmonary hypertension of the newborn: advances in diagnosis and treatment[END_REF] but they are not so common in preterm infants, particularly in extremely preterm infants [START_REF] Giesinger | Controversies in the identification and management of acute pulmonary hypertension in preterm neonates[END_REF]. The use of iNO has been widely studied in preterm infants, but not in this indication: Mercier et al. [START_REF] Mercier | Inhaled nitric oxide for prevention of bronchopulmonary dysplasia in premature babies (EUNO): a randomised controlled trial[END_REF], Hascoet et al. [START_REF] Hascoet | The safety and efficacy of nitric oxide therapy in premature infants[END_REF] and more recently Greenough et al. [START_REF] Greenough | Inhaled nitric oxide (iNO) for preventing prematurity-related bronchopulmonary dysplasia (BPD): 7-year follow-up of the European Union Nitric Oxide (EUNO) trial[END_REF] concluded that iNO for the prevention of bronchopulmonary dysplasia in very premature infants with respiratory distress did not result in long-term benefits or adverse long-term sequelae. However, the neonates studied were very different from ours, as they did not have PH. Besides, Barrington et al. [START_REF] Barrington | Inhaled nitric oxide for respiratory failure in preterm infants[END_REF] in 2017 in a Cochrane database review examined the effects of iNO on death, severe morbidity and neurodevelopmental outcome when this treatment was prescribed for hypoxic respiratory failure (however, hemodynamic status and the presence or absence of pulmonary hypertension were not detailed). The authors concluded that iNO does not appear to be effective as rescue therapy for very ill preterm infants. Nevertheless, some case reports and a study by Baczynski et al. [START_REF] Baczynski | Shortterm and long-term outcomes of preterm neonates with acute severe pulmonary hypertension following rescue treatment with inhaled nitric oxide[END_REF] found that iNO was effective in 46 % of cases of PH among extremely preterm neonates, with improved oxygenation and a better survival rate without severe morbidity if immediate response to treatment was good. In the latter study [START_REF] Baczynski | Shortterm and long-term outcomes of preterm neonates with acute severe pulmonary hypertension following rescue treatment with inhaled nitric oxide[END_REF], all neonates were treated with iNO and the prognosis of non-responders was poor. However, comparison with our data is difficult. In the EPIPAGE-2 cohort study, iNO was not administered to all neonates with a diagnosis of pulmonary hypertension, but only to 63.5 % of them, with no difference between GA groups.

One of the limitations of our study is the mode of selection of PH neonates. The diagnosis was established from database entries but the time of onset of PH after birth was not stated for neonates who were not treated with iNO (for these ones on the contrary, we could date the beginning of the disease with the beginning of administration of iNO). We cannot exclude the possibility that in a few neonates (PH not treated with iNO or with delayed treatment) the condition arose from a secondary cause of pulmonary hypertension (late sepsis or bronchopulmonary dysplasia). This is another limitation of the study. Besides, in 2011 systematic screening of PH wasn't the routine and the diagnosis of PH is hence likely to be an underestimated. Nevertheless, all the neonates included had refractory hypoxemia and we believe that the pathophysiological mechanism of PH is increased pulmonary vascular resistance. This criterion was present in all our study population.

Conclusion

In this nationwide population-based cohort of extremely preterm and very preterm infants, the prevalence of PH was estimated at 6 %, with higher prevalence in the more immature infants. PH was associated with significantly decreased survival without severe morbidity at discharge. Supplementary data to this article can be found online at https://doi. org/10.1016/j.earlhumdev.2023.105837. Underweight is defined as body mass index (BMI) of <18.5 kg/m 2 ; normal weight is defined as BMI of 18.5 to 24.9 kg/m 2 ; overweight is defined as BMI of 25.0 to 29.9 kg/m 2 ; obesity is defined as BMI higher than 30.0 kg/m 2 . Small for gestational age is defined as a birthweight <10th percentile, EPOPé curves according to Ego et al.

Fig. 1 .

 1 Fig. 1. Flow chart. WG: weeks gestation.

  ). It differed significantly between the two gestational age groups: 14.5 % (95 % CI, 12.4-16.7) in the 22-27 weeks gestation (WG) group versus 2.7 % (95 % CI, 2.0-3.4) in the 28-31 WG group (P < .001). The survival rate without severe morbidity was 30.2 % (95 % CI, 23.7-36.6) in the PH+ group. It was 60 % lower (IRR = 0.39, 95 % CI, 0.32-0.48 in Model 1, P < .001) in the PH+ group compared with the PH-group (80.2 %, 95 % CI, 78.7-81.7, Table

Table 2

 2 Maternal, obstetric and neonatal characteristics according to the newborn's PPHN status among n = 3222 eligible newborns with known PH status. The analyses are based on observed data before multiple imputation. Weighted estimations are presented; data were inversely weighted according to duration of recruitment by gestational age groups. Taylor-linearized variance estimation is used to account for the clustering effect due to multiple pregnancies.

		22-27 +6 weeks gestation	28-31 +6 weeks gestation	22-31 +6 weeks gestation
		PH-	PH+	PH-	PH+	PH-	PH+	Total
	Complete cases analysis (n = 3103)
	% (95 % CI)	56.4	21.7	89.4	45.9	81.1	29.7	77.9
		(52.8-59.9)	(15.9-29.1)	(88.0-90.7)	(33.9-58.4)	(79.6-82.6)	(23.8-36.3)	(76.3-79.4)
	Pooled analysis of the MI dataset (M =
	40)
	% (95 % CI)	54.8	22.5	89.0	46.5	80.2	30.2	77.2
		(51.4-58.2)	(15.7-29.2)	(87.6-90.3)	(33.5-59.5)	(78.7-81.7)	(23.7-36.6)	(75.7-78.7)
	P-value	<0.001		<0.001		<0.001
	IRR PH+ vs PH-(95 % CI)	0.42		0.52		0.39
		(0.32-0.57)		(0.39-0.69)		(0.32-0.48)
	PH, pulmonary hypertension; IRR, incidence rate ratio; CI, confidence interval; MI, multiple imputations.

Table 2

 2

	(continued )
		PH-		PH+	P-value
		Percent	95 % CI	Percent	95 % CI
		(%)		(%)
	Mother				0.460
	hospitalized
	during pregnancy
	No	69.3	67.3-71.2	66.8	59.8-73.1
	Yes	30.7	28.8-32.7	33.2	26.9-40.2
	Follow-up of				0.495
	pregnancy
	Adequate	92.6	91.5-93.5	91.2	86.5-94.4
	Not adequate	7.4	6.5-8.5	8.8	5.6-13.5
	Antenatal steroids				0.006
	No steroids	16.1	14.7-17.7	24.5	19.1-30.7
	One incomplete	16.7	15.3-18.3	16.5	12.1-22.2
	course
	At least one	67.1	65.2-69.0	59.0	52.2-65.5
	complete course
	Premature				0.235
	rupture of
	membranes
	No	63.9	61.9-65.8	59.8	53.0-66.2
	Yes	36.1	34.2-38.1	40.2	33.8-47.0
	Chorioamnionitis				0.001
	No	77.6	75.7-79.4	66.1	58.5-72.9
	Yes	22.4	20.6-24.3	33.9	27.1-41.5
	Infant's delivery and
	neonatal
	characteristics
	Cesarean section				0.573
	No	34.4	32.5-36.3	36.2	30.1-42.9
	Yes	65.6	63.7-67.5	63.8	57.1-69.9
	Indicated				0.164
	pretermaturity
	No	51.8	49.7-53.9	46.7	39.8-53.7
	Yes	48.2	46.1-50.3	53.3	46.3-60.2
	Gestational age at				<0.001
	delivery
	22 to 27 weeks	25.3	23.6-27.0	66.8	60.0-72.9
	28 to 31 weeks	74.7	73.0-76.4	33.2	27.1-40.0
	Sex of the infant				0.514
	Female	47.9	46.0-49.7	45.6	39.1-52.3
	Male	52.1	50.3-54.0	54.4	47.7-60.9
	Small for				0.163
	gestational age
	No	65.4	63.6-67.1	70.1	63.6-75.9
	Yes	34.6	32.9-36.4	29.9	24.1-36.4
	Apgar score at 5				<0.001
	min
	Score of 7 to10	84.2	82.7-85.5	59.0	52.1-65.6
	Lower than 7	15.8	14.5-17.3	41.0	34.4-47.9
	Presence of minor				0.302
	malformation
	No	96.9	96.2-97.4	95.6	92.0-97.6
	Yes	3.1	2.6-3.8	4.4	2.4-8.0
	Oxygenation of				<0.001
	the infant in the
	delivery room
	No	26.7	25.0-28.5	11.9	8.1-17.1
	Yes	73.3	71.5-75.0	88.1	82.9-91.9
	Surfactant use				<0.001
	No	38.0	36.1-39.9	1.4	0.5-4.4
	Yes	62.0	60.1-63.9	98.6	95.6-99.5
	Respiratory				<0.001
	distress syndrome
	No	45.2	43.3-47.2	9.5	6.2-14.5
	Yes	54.8	52.8-56.7	90.5	85.5-93.8
	Early onset				0.001
	infection within
	the first 72 h of
	life
	No	80.6	79.0-82.1	70.5	63.7-76.4
	Probable or	19.4	17.9-21.0	29.5	23.6-36.3
	sure
					(continued on next page)

Table 2

 2 (continued )

		PH-		PH+	P-value
		Percent	95 % CI	Percent	95 % CI
		(%)		(%)
	Late onset sepsis				<0.001
	among survivors
	after 2 days of life
	No	80.5	79.0-81.9	65.3	58.6-71.5
	Yes	19.5	18.1-21.0	34.7	28.5-41.4
	Administration of				<0.001
	catecholamines
	0 -no	83.1	81.7-84.5	42.4	36.0-49.1
	1 -yes	16.9	15.5-18.3	57.6	50.9-64.0
	Administration of				<0.001
	catecholamines
	before 72 h of life
	No	93.8	92.8-94.6	63.6	56.9-69.7
	Yes	6.2	5.4-7.2	36.4	30.3-43.1
	Administration of				<0.001
	catecholamines
	after 72 h of life
	No	91.8	90.8-92.8	54.0	47.0-60.9
	Yes	8.2	7.2-9.2	46.0	39.1-53.0
	NSAIDs for				<0.001
	persistent ductus
	arteriosus
	No	77.8	76.2-79.4	51.7	44.9-58.4
	Yes	22.2	20.6-23.8	48.3	41.6-55.1
	Surgery for				<0.001
	persistent ductus
	arteriosus
	No	96.3	95.6-96.9	83.6	78.0-88.0
	Yes	3.7	3.1-4.4	16.4	12.0-22.0

P-values from Pearson Chi-square test with Rao-Scott correction. PH, persistent pulmonary hypertension of the newborn; CI, confidence interval; NSAIDs: non-steroidal anti-inflammatory drugs.

Funding

This project was funded with support from the following organizations: the French Institute of Public Health Research/Institute of Public Health and its partners: the French Health Ministry, the National Institute of Health and Medical Research (INSERM), the National Institute of Cancer, and the National Solidarity Fund for Autonomy (CNSA); the National Research Agency through the French EQUIPEX program of investments in the future (reference ANR-11-EQPX-0038); the PREMUP Foundation; the Fondation de France (reference 00050329); and the Fondation pour la Recherche Médicale (reference SPF20160936356). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Declaration of competing interest

None of the authors have any conflict of interest.

Table 3

Neonatal death among the eligible sample (n = 3383 observations) and severe neonatal morbidity among survivors at hospital discharge, by pulmonary hypertension, globally and by GA. Results of complete cases analysis and pooled analysis of the multiply imputed dataset. Weighted estimations are presented; data were inversely weighted according to duration of recruitment by gestational age groups. Variance estimation accounted for the clustering effect due to multiple pregnancies.