Chrono-proteomics of human saliva: variations of the salivary proteome during human development.

An important contribution to the variability of any proteome is given by the time dimension that should be carefully considered to define physiological modifications. To this purpose, whole saliva proteome was investigated in a wide age range. Whole saliva was collected from 17 preterm newborns with a postconceptional age at birth of 178-217 days. In these subjects sample collection was performed serially starting immediately after birth and within about 1 year follow-up, gathering a total of 111 specimens. Furthermore, whole saliva was collected from 182 subjects aged between 0 and 17 years and from 23 adults aged between 27 and 57 years. The naturally occurring intact salivary proteome of the 316 samples was analyzed by low- and high-resolution HPLC-ESI-MS platforms. Proteins peculiar of the adults appeared in saliva with different time courses during human development. Acidic proline-rich proteins encoded by PRH2 locus and glycosylated basic proline-rich proteins encoded by PRB3 locus appeared following 180 days of postconceptional age, followed at 7 months (±2 weeks) by histatin 1, statherin, and P-B peptide. The other histatins and acidic proline-rich proteins encoded by PRH1 locus appeared in whole saliva of babies from 1 to 3 weeks after the normal term of delivery, S-type cystatins appeared at 1 year (±3 months), and basic proline-rich proteins appeared at 4 years (±1 year) of age. All of the proteinases involved in the maturation of salivary proteins were more active in preterm than in at-term newborns, on the basis of the truncated forms detected. The activity of the Fam20C kinase, involved in the phosphorylation of various proteins, started around 180 days of postconceptional age, slowly increased reaching values comparable to adults at about 2 years (±6 months) of age. Instead, MAPK14 involved in the phosphorylation of S100A9 was fully active since birth also in preterm newborns.

Determination of Lung Volume and Hemodynamic Changes During High-Frequency Ventilation Recruitment in Preterm Neonates With Respiratory Distress Syndrome.

OBJECTIVES: To evaluate the changes in end-expiratory lung volume during an oxygenation-guided stepwise recruitment procedure in elective high-frequency ventilation. We hypothesized that high continuous distending pressure impedes pulmonary blood flow as evidenced by reduced lung volume measurements using respiratory inductive plethysmography. Changes in oxygenation, ventilation, and peripheral perfusion were evaluated as secondary outcomes. DESIGN: A prospective, single center, observational, nonrandomized study. SETTING: The study was conducted in a neonatal ICU in Italy. PATIENTS: High-frequency ventilated preterm infants with respiratory distress syndrome. INTERVENTIONS: During the recruitment procedure, end-expiratory lung volume measured by respiratory inductive plethysmography, oxygen saturation, perfusion index, regional cerebral and perirenal tissue oxygenation, heart rate, transcutaneous PCO2, and tidal volume were simultaneously recorded at each airway pressure step. MEASUREMENTS AND MAIN RESULTS: In 12 preterm newborns (gestational age, 27.4 ± 0.2 wk; birth weight, 979 ± 198 g), high-frequency ventilation was initiated at a continuous distending pressure of 10 cm H2O and incrementally increased by 1-2 cm H2O every 2-5 minutes until FIO2 was less than or equal to 0.25. End-expiratory lung volume progressively increased during the initial recruitment, but decreased at the maximum airway pressure in nine patients, indicative of a reduction in pulmonary perfusion. At the end of recruitment, tidal volume was significantly higher (p = 0.002) and oxygenation was significantly improved (p = 0.002); however, mean perfusion index, postductal saturation, and mean renal tissue oxygenation values were significantly reduced (p < 0.05) compared with baseline. Mean cerebral tissue oxygenation and mean transcutaneous PCO2 values were reduced but failed to reach significance. CONCLUSIONS: High distending lung pressures increased oxygenation but decreased peripheral perfusion with no adverse cerebral side effects. Coupled with the reduction in respiratory inductive plethysmography-derived lung volume, high continuous distending pressure had adverse cardiopulmonary effects. Incorporation of lung volume and hemodynamic and oxygenation variables may guide optimum lung volume determination during high-frequency ventilation recruitment procedure while preventing adverse effects on the pulmonary circulation.

Thymosin ß4 and ß10 Expression in Human Organs during Development: A Review.

This review summarizes the results of a series of studies performed by our group with the aim to define the expression levels of thymosin ß4 and thymosin ß10 over time, starting from fetal development to different ages after birth, in different human organs and tissues. The first section describes the proteomics investigations performed on whole saliva from preterm newborns and gingival crevicular fluid, which revealed to us the importance of these acidic peptides and their multiple functions. These findings inspired us to start an in-depth investigation mainly based on immunochemistry to establish the distribution of thymosin ß4 and thymosin ß10 in different organs from adults and fetuses at different ages (after autopsy), and therefore to obtain suggestions on the functions of ß-thymosins in health and disease. The functions of ß-thymosins emerging from these studies, for instance, those performed during carcinogenesis, add significant details that could help to resolve the nowadays so-called "ß-thymosin enigma", i.e., the potential molecular role played by these two pleiotropic peptides during human development.

Top-down HPLC-ESI-MS detection of S-glutathionylated and S-cysteinylated derivatives of cystatin B and its 1-53 and 54-98 fragments in whole saliva of human preterm newborns.

Analysis by a HPLC-ESI-MS top-down proteomic platform of specimens of human preterm newborn whole saliva evidenced high relative amounts of cystatin B and its S-glutathionylated,S-cysteinylated, and S-S 2-mer (on Cys(3)) derivatives, decreasing as a function of postconceptional age (PCA). The percentage of S-unmodified cystatin B was higher than the S-modified isoforms in the early PCA period, differently from adults where cystatin B was detectable only as S-modified derivatives. The percentage of S-modified derivatives increased as a function of PCA, reaching at the normal term of delivery values similar to those determined in at-term newborns, babies, and adults. Moreover, in the early PCA period, high relative amounts of the 1-53 and 54-98 cystatin B fragments were detected, decreasing as a function of PCA and disappearing at the normal term of delivery. In agreement with intact cystatin B, fragment 1-53 was detectable as S-unmodified and S-modified derivatives, and their percentages changed accordingly with the percentages of intact proteins, suggesting that the fragmentation process could be subsequent to and independent from the S-modification of the protein. This study highlights specific enzymatic activity in the oral cavity of preterm newborns not present in at-term newborns and adults, which can be a clue to specialized pathways occurring during fetal oral development.

Association of high levels of α-defensins and S100A proteins with Candida mannan detection in bronchoalveolar lavage fluid of preterm neonates.

BACKGROUND: Candida mannan (Mn) detection in bronchoalveolar lavage fluid (BALF) was shown to be useful for earlier identification and preemptive therapy targeting in preterm infants at high risk of invasive Candida infection. We investigated whether early detection of Candida Mn in BALF is associated with the presence of some neutrophilic products, as markers of prenatal infection/inflammation. METHODS: BALF specimens were collected during the first 48 h of life from mechanically ventilated preterm newborns. Samples were analyzed by high-performance liquid chromatography-electrospray ionization-mass spectrometry. The relative amounts of α-defensins 1-4 and S100A proteins were measured by extracted ion current peak area. Absolute and differential white cell counts in BALF were obtained. Mn antigen concentrations were determined by the Platelia Candida antigen kit. RESULTS: Twenty-five studied neonates were divided into two groups: Mn-positive group and Mn-negative group. Levels of α-defensins 1-4 and S100A12 were significantly higher in the Mn-positive group than in the Mn-negative group. Moreover, positive significant correlations between the absolute number of neutrophils and the levels of α-defensins 1-4 and S100A8 were observed. CONCLUSION: The detection of Mn antigen in BALF of preterm infants is consistent with evidence of an innate immune response in their lungs as demonstrated by higher levels of α-defensins and S100A proteins.

The surprising composition of the salivary proteome of preterm human newborn.

Saliva is a body fluid of a unique composition devoted to protect the mouth cavity and the digestive tract. Our high performance liquid chromatography (HPLC)-electrospray ionization-MS analysis of the acidic soluble fraction of saliva from preterm human newborn surprisingly revealed more than 40 protein masses often undetected in adult saliva. We were able to identify the following proteins: stefin A and stefin B, S100A7 (two isoforms), S100A8, S100A9 (four isoforms), S100A11, S100A12, small proline-rich protein 3 (two isoforms), lysozyme C, thymosins ß(4) and ß(10), antileukoproteinase, histone H1c, and α and γ globins. The average mass value reported in international data banks was often incongruent with our experimental results mostly because of post-translational modifications of the proteins, e.g. acetylation of the N-terminal residue. A quantitative label-free MS analysis showed protein levels altered in relation to the postconceptional age and suggested coordinate and hierarchical functions for these proteins during development. In summary, this study shows for the first time that analysis of these proteins in saliva of preterm newborns might represent a noninvasive way to obtain precious information of the molecular mechanisms of development of human fetal oral structures.

Efficacy of exogenous surfactant during conventional and high-frequency oscillatory ventilation in preterm infants with RDS.

INTRODUCTION: The immediate effects of exogenous surfactant on lung volume and hemodynamics in preterm infants have been poorly studied. MATERIALS AND METHODS: Lung volume, SpO2, perfusion index, regional SO2, transcutaneous partial pressure of CO2 were simultaneously monitored and recorded ten minutes after surfactant tracheal instillation in elective HFOV ventilated preterm infants with RDS. OUTCOMES: Despite a reduction in gas exchange during the first 5 minutes post surfactant instillation, most likely dependent on airway obstruction, lung volume increases rapidly in HFOV preterm infants with RDS. After 5 minutes from administration of surfactant, lung volume, gas exchange and PI reach stable values and CDP can be safely reduced.

Endobronchial ultrasound: a pictorial essay.

BACKGROUND AND AIM: endobronchial ultrasound has gained widespread popularity in the last decade, becoming the primary technique for minimally invasive evaluation of the mediastinum and staging of lung cancer. Several tertiary and quaternary care institutes use this method, performed by trained and accredited specialists. Its main indications are (I) diagnosis and staging of lung cancer, (II) mediastinal lymphadenopathy diagnosis (III) sampling peripheral pulmonary lesions. CONCLUSIONS: this manuscript aims to describe the operational potential of both convex endobronchial ultrasound probe and radial endobronchial ultrasound probe technology, focusing on lung cancer. This narrative review is complemented with by the description of peculiar clinical cases in which endobronchial ultrasound played a pivotal role in reaching the diagnosis.

Respiratory Management of the Preterm Infant: Supporting Evidence-Based Practice at the Bedside.

Extremely preterm infants frequently require some form of respiratory assistance to facilitate the cardiopulmonary transition that occurs in the first hours of life. Current resuscitation guidelines identify as a primary determinant of overall newborn survival the establishment, immediately after birth, of adequate lung inflation and ventilation to ensure an adequate functional residual capacity. Any respiratory support provided, however, is an important contributing factor to the development of bronchopulmonary dysplasia. The risks correlated to invasive ventilatory techniques increase inversely with gestational age. Preterm infants are born at an early stage of lung development and are more susceptible to lung injury deriving from mechanical ventilation. Any approach aiming to reduce the global burden of preterm lung disease must implement lung-protective ventilation strategies that begin from the newborn's first breaths in the delivery room. Neonatologists today must be able to manage both invasive and noninvasive forms of respiratory assistance to treat a spectrum of lung diseases ranging from acute to chronic conditions. We searched PubMed for articles on preterm infant respiratory assistance. Our narrative review provides an evidence-based overview on the respiratory management of preterm infants, especially in the acute phase of neonatal respiratory distress syndrome, starting from the delivery room and continuing in the neonatal intensive care unit, including a section regarding exogenous surfactant therapy.

The future of neonatal lung ultrasound: Validation of an artificial intelligence model for interpreting lung scans. A multicentre prospective diagnostic study.

BACKGROUND: Artificial intelligence (AI) is a promising field in the neonatal field. We focused on lung ultrasound (LU), a useful tool for the neonatologist. Our aim was to train a neural network to create a model able to interpret LU. METHODS: Our multicentric, prospective study included newborns with gestational age (GA) ≥ 33 + 0 weeks with early tachypnea/dyspnea/oxygen requirements. For each baby, three LU were performed: within 3 h of life (T0), at 4-6 h of life (T1), and in the absence of respiratory support (T2). Each scan was processed to extract the region of interest used to train a neural network to classify it according to the LU score (LUS). We assessed sensitivity, specificity, positive and negative predictive value of the AI model's scores in predicting the need for respiratory assistance with nasal continuous positive airway pressure and for surfactant, compared to an already studied and established LUS. RESULTS: We enrolled 62 newborns (GA = 36 ± 2 weeks). In the prediction of the need for CPAP, we found a cutoff of 6 (at T0) and 5 (at T1) for both the neonatal lung ultrasound score (nLUS) and AI score (AUROC 0.88 for T0 AI model, 0.80 for T1 AI model). For the outcome "need for surfactant therapy", results in terms of area under receiver operator characteristic (AUROC) are 0.84 for T0 AI model and 0.89 for T1 AI model. In the prediction of surfactant therapy, we found a cutoff of 9 for both scores at T0, at T1 the nLUS cutoff was 6, while the AI's one was 5. Classification accuracy was good both at the image and class levels. CONCLUSIONS: This is, to our knowledge, the first attempt to use an AI model to interpret early neonatal LUS and can be extremely useful for neonatologists in the clinical setting.

Early Respiratory Physiotherapy versus an Individualized Postural Care Program for Reducing Mechanical Ventilation in Preterm Infants: A Randomised Controlled Trial.

BACKGROUND: Tactile stimulation manoeuvres stimulate spontaneous breathing in preterm newborns. The aim of this study is to evaluate the effect of early respiratory physiotherapy on the need for mechanical ventilation during the first week of life in preterm infants with respiratory failure. METHODS: This is a monocentric, randomised controlled trial. Preterm infants (gestational age ≤ 30 weeks) not intubated in the delivery room and requiring non-invasive respiratory support at birth were eligible for the study. The intervention group received early respiratory physiotherapy, while the control group received only a daily physiotherapy program (i.e., modifying the infant's posture in accordance with the patient's needs). RESULTS: between October 2019 and March 2021, 133 preterm infants were studied, 68 infants in the study group and 65 in routine care. The study group showed a reduction in the need for mechanical ventilation (not statistically significant) and a statistically significant reduction in hemodynamically significant patent ductus arteriosus with respect to the control group (19/68 (28%) vs. 35/65 (54%), respectively, p = 0.03). CONCLUSIONS: early respiratory physiotherapy in preterm infants requiring non-invasive respiratory support at birth is safe and has proven to be protective against haemodynamically significant PDA.

Early lung ultrasound score to predict noninvasive ventilation needing in neonates from 33 weeks of gestational age: A multicentric study.

OBJECTIVE: To propose an early lung ultrasound (LUS) score for the prediction of the need for respiratory assistance in newborns of gestational age (GA) ≥ 33 weeks presenting respiratory distress. STUDY DESIGN AND SETTING: Multicenter, prospective observational study in third-level neonatal intensive care units. PATIENT SELECTION: Infants with GA ≥ 33 + 0 weeks with respiratory distress within 3 h of life. METHODS: Three LUS for each patient were collected: within 3 h of life (T0), at 4-6 h of life (T1), and at the resolution of symptoms (T2). The primary aim was to assess the validity of the early LUS score in predicting the need for continuous positive airway pressure (CPAP). We also evaluated the validity of the score in predicting the need for surfactant, the scores' trend in our population, and any correlation with the duration of ventilation and oxygen therapy. RESULTS: Sixty-two patients were enrolled in the study. The mean GA was 36 weeks. The receiver operating characteristic analysis for the LUS T0 and T1 yielded area under the curves of 0.91 and 0.82 in predicting the need for CPAP, respectively. LUS score cut off of 6 (sensitivity 84.8%, specificity 86.2%) and 5 (sensitivity 66.7%, specificity 100%) were calculated at T0 and T1, respectively. We found significant correlations between LUS score and respiratory assistance, surfactant administration, and SpO2 /FiO2 ratio. CONCLUSION: An early LUS score is a good noninvasive predictor of the need for respiratory assistance with CPAP and surfactant administration in newborns with GA ≥ 33 weeks.

Lowering of the Neonatal Lung Ultrasonography Score after nCPAP Positioning in Neonates over 32 Weeks of Gestational Age with Neonatal Respiratory Distress.

Respiratory distress (RD) is one of the most common causes of admission to the neonatal intensive care unit. Correct diagnosis and timely intervention are crucial. Lung ultrasonography (LU) is a useful diagnostic tool for the neonatologist in the diagnosis of RD; the neonatal lung ultrasonography score (nLUS) can be used in the diagnostic process, but some authors hypothesise that it is also useful for the management of some neonatal RD. The aim of this study is to analyse the changes in nLUS score before (T0) and after (T1) the start of respiratory support with nasal CPAP in neonates over 32 weeks of age with RD. Thirty-three newborns were enrolled in this retrospective study. LU was performed before and after the start of CPAP. The median nLUS scores at T0 and T1 were 9 (IQR 7−12) and 7 (IQR 4−10), respectively, and showed a significant difference (p < 0.001). The magnitude of reduction in nLUS score, expressed as a percentage, was inversely related to the need for subsequent administration of exogenous surfactant. The study suggests the usefulness of the nLUS score in assessing the response to CPAP in neonates over 32 weeks gestational age.

The Relationship Between Maternal and Neonatal Microbiota in Spontaneous Preterm Birth: A Pilot Study.

The newborn's microbiota composition at birth seems to be influenced by maternal microbiota. Maternal vaginal microbiota can be a determining factor of spontaneous Preterm Birth (SPPTB), the leading cause of perinatal mortality. The aim of the study is to investigate the likelihood of a causal relationship between the maternal vaginal microbiota composition and neonatal lung and intestinal microbiota profile at birth, in cases of SPPTB. The association between the lung and/or meconium microbiota with the subsequent development of bronchopulmonary dysplasia (BPD) was also investigated. Maternal vaginal swabs, newborns' bronchoalveolar lavage fluid (BALF) (1st, 3rd, 7th day of life) and first meconium samples were collected from 20 women and 23 preterm newborns with gestational age ≤ 30 weeks (12 = SPPTB; 11 = Medically Indicated Preterm Birth-MIPTB). All the samples were analyzed for culture examination and for microbiota profiling using metagenomic analysis based on the Next Generation Sequencing (NGS) technique of the bacterial 16S rRNA gene amplicons. No significant differences in alpha e beta diversity were found between the neonatal BALF samples of SPPTB group and the MIPTB group. The vaginal microbiota of mothers with SPPTB showed a significant difference in alpha diversity with a decrease in Lactobacillus and an increase in Proteobacteria abundance. No association was found between BALF and meconium microbiota with the development of BPD. Vaginal colonization by Ureaplasma bacteria was associated with increased risk of both SPPTB and newborns' BPD occurrence. In conclusion, an increase in α-diversity values and a consequent fall in Lactobacillus in vaginal environment could be associated to a higher risk of SPPTB. We could identify neither a specific neonatal lung or meconium microbiota profiles in preterm infants born by SPPTB nor a microbiota at birth suggestive of subsequent BPD development. Although a strict match has not been revealed between microbiota of SPPTB mother-infant couples, a relationship cannot be excluded. To figure out the reciprocal influence of the maternal-neonatal microbiota and its potential role in the pathogenesis of SPPTB and BPD further research is needed.

Weaning from the Ventilator in Bronchopulmonary Dysplasia.

For the newborns needing respiratory support at 36 weeks postmenstrual age, regardless of the type of ventilation used, it is critical to take into account the mechanics properties of both airways and lungs affected by severe bronchopulmonary dysplasia (sBPD). Ventilator strategies, settings, and weaning must change dramatically after sBPD is established, but to date there is almost no high-quality evidence base supporting a specific approach to guide the optimal ventilator management and weaning in patients with sBPD. Weaning from invasive mechanical ventilation, management of the immediately postextubation period, and weaning from noninvasive ventilation in patients with sBPD are the topics covered in this chapter.

Effects of High-Frequency Oscillatory Ventilation With Volume Guarantee During Surfactant Treatment in Extremely Low Gestational Age Newborns With Respiratory Distress Syndrome: An Observational Study.

Objective: To evaluate the effect of volume guarantee (VG) combined with high-frequency oscillatory ventilation (HFOV) on respiratory and other physiological parameters immediately after lung recruitment and surfactant administration in HFOV elective ventilated extremely low gestational age newborns (ELGAN) with respiratory distress syndrome (RDS). Design: Observational study. Setting: Tertiary neonatal intensive care unit. Patients: Twenty-two ELGANs of 25.5 ± 1.1 weeks of gestational age requiring invasive mechanical ventilation and surfactant administration for RDS during the first 6 h of life. Interventions: All infants intubated in delivery room, were managed with elective HFOV and received surfactant after a lung recruitment manoeuver. Eleven infants received HFOV + VG and were compared with a control group of 11 infants receiving HFOV alone. HFOV was delivered in both groups by Dräger Babylog VN500 ventilator (Dräger, Lubeck, Germany). Main Outcome Measures: Variations and fluctuations of delivered high-frequency tidal volume (VThf), fluctuation of pressure amplitude (ΔP) and partial pressure of CO2 (pCO2) levels after recruitment manoeuver and immediately after surfactant administration, in HFOV + VG vs. HFOV ventilated infants. Results: There were no significant differences in the two groups at starting ventilation with or without VG. The mean applied VThf per kg was 1.7 ± 0.3 ml/kg in the HFOV group and 1.7 ± 0.1 ml/kg in the HFOV + VG group. Thirty minutes after surfactant administration, HFOV group had a significant higher VThf/Kg than HFOV + VG (2.1 ± 0.3 vs. 1.6 ± 0.1 ml/kg, p < 0.0001) with significantly lower pCO2 levels (43.1 ± 3.8 vs. 46.8 ± 1.5 mmHg, p = 0.01), 54.4% of patients having pCO2 below 45 mmHg. Measured post-surfactant ΔP values were higher in HFOV group (17 ± 3 cmH2O) than in HFOV + VG group (13 ± 3 cmH2O, p = 0.01). Conclusion: HFOV + VG maintains pCO2 levels within target range and reduces VThf delivered variations more consistently than HFOV alone after surfactant administration.

Oxidative and Proteolytic Inactivation of Alpha-1 Antitrypsin in Bronchopulmonary Dysplasia Pathogenesis: A Top-Down Proteomic Bronchoalveolar Lavage Fluid Analysis.

The study investigates the role of the oxidative and proteolytic inactivation of alpha-1 antitrypsin (AAT) in the pathogenesis of bronchopulmonary dysplasia (BPD) in premature infants. Bronchoalveolar lavage fluid (BALF) samples were collected on the 3rd day of life from mechanically ventilated neonates with gestational age ≤ 30 weeks and analyzed without previous treatment (top-down proteomics) by reverse-phase high-performance liquid chromatography-electrospray ionization mass spectrometry. AAT fragments were identified by high-resolution LTQ Orbitrap XL experiments and the relative abundances determined by considering the extracted ion current (XIC) peak area. Forty preterm neonates were studied: 20 (50%) did not develop BPD (no-BPD group), 17 (42.5%) developed mild or moderate new-BPD (mild + moderate BPD group), and 3 (7.5%) developed severe new-BPD (severe BPD group). Eighteen fragments of AAT and a fragment of AAT oxidized at a methionine residue were identified: significantly higher values of AAT fragments 25-57, 375-418, 397-418, 144-171, and 397-418 with oxidized methionine were found in the severe BPD group. The significantly higher levels of several AAT fragments and of the fragment 397-418, oxidized in BALF of preterm infants developing BPD, underlie the central role of an imbalance between proteases and protease inhibitors in exacerbating lung injury and inducing most severe forms of BPD. The study has some limitations, and between them, the small sample size implies the need for further confirmation by larger studies.

HPLC-ESI-MS top-down analysis of salivary peptides of preterm newborns evidenced high activity of some exopeptidases and convertases during late fetal development.

To have information on the proteolytic activity of convertases and exo-peptidases on human salivary proteins, this study investigated the relative amounts of the truncated proteoforms in the saliva of preterm newborns and compared them with the relative amounts measured in saliva of at-term newborns, of babies (0-10 years old) and of adults. Results indicated that convertase(s), acting on acidic proline-rich proteins and histatin 3, and carboxypeptidase(s) acting on acidic proline-rich proteins, P-C peptide, histatin 6 and statherin were many folds more active in preterm newborns than in the other groups. Conversely, the aminopeptidase responsible for the removal of the N-terminal Asp residue of statherin was not active in preterm newborns, becoming active only several months after the normal term of delivery. The high activity of convertases determined in preterm newborns suggests that it is required for the molecular events connected to the fetus development, and encourages further studies devoted to the characterization of their specific substrates.

Effects of early respiratory physiotherapy on spontaneous respiratory activity of preterm infants: study protocol for a randomized controlled trial.

BACKGROUND: Tactile maneuvers stimulating spontaneous respiratory activity in preterm infants are recommended since birth, but data on how and how often these maneuvers are applied in clinical practice are unknown. In the last years, most preterm newborns with respiratory failure are preferentially managed with non-invasive respiratory support and by stimulating spontaneous respiratory activity from the delivery room and in neonatal intensive care unit (NICU), in order to avoid the risks of intubation and prolonged mechanical ventilation. METHODS: Preterm infants with gestational age < 31 weeks not intubated in the delivery room and requiring non-invasive respiratory support at birth will be eligible for the study. They will be randomized and allocated to one of two treatment groups: (1) the study group infants will be subject to the technique of respiratory facilitation within the first 24 h of life, according to the reflex stimulations, by the physiotherapist. The newborn is placed in supine decubitus and a slight digital pressure is exerted on a hemithorax. The respiratory facilitation technique will be performed for about three minutes and repeated for a total of 4/6 times in sequence, three times a day until spontaneous respiratory activity is achieved; thus, no respiratory support is required; (2) the control group infants will take part exclusively in the individualized postural care program. They will perform the technique of respiratory facilitation and autogenous drainage. OBJECTIVE: To evaluate the efficacy of early respiratory physiotherapy in reducing the incidence of intubation and mechanical ventilation in the first week of life (primary outcome). DISCUSSION: The technique of respiratory facilitation is based on reflex stimulations, applied early to preterm infant. Slight digital pressure is exerted on a "trigger point" of each hemithorax, to stimulate the respiratory activity with subsequent increase of the ipsilateral pulmonary minute ventilation and to facilitate the contralateral pulmonary expansion. This mechanism will determine the concatenation of input to all anatomical structures in relation to the area being treated, to promote spontaneous respiratory activity and reducing work of breathing, avoiding or minimizing the use of invasive respiratory support. TRIAL REGISTRATION: UMIN-CTR Clinical Trial UMIN000036066. Registered on March 1, 2019. Protocol 1. https://www.umin.ac.jp/ctr.

Lung recruitment before surfactant administration in extremely preterm neonates with respiratory distress syndrome (IN-REC-SUR-E): a randomised, unblinded, controlled trial.

BACKGROUND: The importance of lung recruitment before surfactant administration has been shown in animal studies. Well designed trials in preterm infants are absent. We aimed to examine whether the application of a recruitment manoeuvre just before surfactant administration, followed by rapid extubation (intubate-recruit-surfactant-extubate [IN-REC-SUR-E]), decreased the need for mechanical ventilation during the first 72 h of life compared with no recruitment manoeuvre (ie, intubate-surfactant-extubate [IN-SUR-E]). METHODS: We did a randomised, unblinded, controlled trial in 35 tertiary neonatal intensive care units in Italy. Spontaneously breathing extremely preterm neonates (24 + 0 to 27 + 6 weeks' gestation) reaching failure criteria for continuous positive airway pressure within the first 24 h of life were randomly assigned (1:1) with a minimisation algorithm to IN-REC-SUR-E or IN-SUR-E using an interactive web-based electronic system, stratified by clinical site and gestational age. The primary outcome was the need for mechanical ventilation in the first 72 h of life. Analyses were done in intention-to-treat and per-protocol populations, with a log-binomial regression model correcting for stratification factors to estimate adjusted relative risk (RR). This study is registered with ClinicalTrials.gov, NCT02482766. FINDINGS: Of 556 infants assessed for eligibility, 218 infants were recruited from Nov 12, 2015, to Sept 23, 2018, and included in the intention-to-treat analysis. The requirement for mechanical ventilation during the first 72 h of life was reduced in the IN-REC-SUR-E group (43 [40%] of 107) compared with the IN-SUR-E group (60 [54%] of 111; adjusted RR 0·75, 95% CI 0·57-0·98; p=0·037), with a number needed to treat of 7·2 (95% CI 3·7-135·0). The addition of the recruitment manoeuvre did not adversely affect the safety outcomes of in-hospital mortality (19 [19%] of 101 in the IN-REC-SUR-E group vs 37 [33%] of 111 in the IN-SUR-E group), pneumothorax (four [4%] of 101 vs seven [6%] of 111), or grade 3 or worse intraventricular haemorrhage (12 [12%] of 101 vs 17 [15%] of 111). INTERPRETATION: A lung recruitment manoeuvre just before surfactant administration improved the efficacy of surfactant treatment in extremely preterm neonates compared with the standard IN-SUR-E technique, without increasing the risk of adverse neonatal outcomes. The reduced need for mechanical ventilation during the first 72 h of life might facilitate implementation of a non-invasive respiratory support strategy. FUNDING: None.