Preterm extremely low birth weight infants (ELBWi) are known to be at greater risk of developing neuropsychiatric diseases. Identifying early predictors of outcome is essential to refer patients for early intervention. Few studies have investigated neurodevelopmental outcomes in Italian ELBWi. This study aims to describe neurodevelopmental outcome at 24 months of corrected age in an eleven-year single-center cohort of Italian ELBWi and to identify early risk factors for adverse neurodevelopmental outcomes.All infants born with birth weight < 1000 g and admitted to the Neonatal Intensive Care Unit of the “Fondazione IRCCS Policlinico San Matteo” hospital in Pavia, Italy, from Jan 1, 2005 to Dec 31, 2015 were eligible for inclusion. At 24 months, Griffiths’ Mental Developmental Scales Extended Revised (GMDS-ER) were administered. Neurodevelopmental outcome was classified as: normal, minor sequelae , major sequelae . Univariate and multivariate multinomial logistic regression models were performed to analyze the correlation between neonatal variables and neurodevelopmental outcome.176 ELBWi were enrolled . 67% showed a normal outcome at 24 months, 17% minor sequelae and 16% major sequelae . The most frequent major sequela was cognitive impairment (8.52%).In the entire sample the median score on the Hearing-Speech subscale was lower than the median scores recorded on the other subscales and showed a significantly weaker correlation to each of the other subscales of the GMDS-ER.Severely abnormal cUS findings (RRR 10.22 p 0.043) and bronchopulmonary dysplasia (RRR 4.36 p 0.008) were independent risk factors for major sequelae and bronchopulmonary dysplasia for minor sequelae (RRR 3.00 p 0.018) on multivariate multinomial logistic regression.This study showed an improvement in ELBWI survival rate without neurodevelopmental impairment at 24 months compared to previously reported international cohorts. Cognitive impairment was the most frequent major sequela. Median scores on GMDS-ER showed a peculiar developmental profile characterized by a selective deficit in the language domain. Severely abnormal cUS findings and bronchopulmonary dysplasia were confirmed as independent risk factors for major sequelae.The online version contains supplementary material available at 10.1186/s13052-022-01303-9. In the last decades, the global increase in preterm birth rate and the concurrent reduction in mortality rate has led to a growing number of children who survived neonatal complications of preterm birth , 2. NeveReports of the neurodevelopmental outcome of large prospective Italian ELBW infants’ cohorts still lack . TherefoAll consecutive infants born with birth weight < 1000 g and admitted within 6 h from birth to the neonatal intensive care unit (NICU) of the “Fondazione IRCCS Policlinico San Matteo” hospital in Pavia, Italy, from Jan 1, 2005, to Dec 31, 2015 were eligible for inclusion  database collection criteria . The cliPer institutional protocol, serial cerebral ultrasound (cUS), was performed in all preterm infants. All infants born before 35 weeks GA underwent cerebral cUS evaluation within the first 24 h of life. In infants born before 30 weeks GA, cUS was performed at 24, 48 and 72 h of life, at 7 days of life, then weekly up to 31 weeks post-menstrual age (PMA) and then at 36 and 40 weeks PMA. In infants born between 30 and 33 weeks GA, a first evaluation was performed within the first week of life, a second one at 3 weeks, and then at 40 weeks PMA . IntraveFollowing the Rademaker classification, cUS scans performed at 40 weeks of postmenstrual age (PMA) were classified as “normal”, “slightly abnormal” in the presence of grade I/II IVH, grade I PVL, germinal layer necrosis, or a combination of these features, or isolated VD; “severely abnormal” cUS scans was defined in the presence of grade III/IV IVH, cystic grade II/III PVL, thalamic lesions, focal infarction, or post-hemorrhagic VD needing surgical intervention .A child neuropsychiatrist, unaware of the cUS findings, examined each subject at 38–42 weeks of PMA. The neurological evaluation of the infant was based on the methods of Amiel-Tison: this assessment is divided into six sections and includes 34 items covering cranial assessment, neurosensory function and spontaneous motor activity, passive muscle tone, axial motor activity (active tone), primitive reflexes, palate and tongue, adaptedness to manipulations during assessment, feeding autonomy . NeonataEach child underwent neurodevelopmental assessment every three months during the first year and every six months in the second year . The GriThe continuous variables were reported as mean and standard deviation if normally distributed, as median and interquartile range (IQR: 25th–75th percentile) if not normally distributed. The categorical variables were expressed as counts and percentages.2 or with the Fisher’s exact test.Comparison between two groups for continuous variables were performed using Student’s t test if normally distributed or the analogous non parametrical test of Mann Whitney if not normally distributed; continuous variables were compared between three groups with the ANOVA test or with the analogous non parametrical Kruskall-Wallis test. Association between categorical variables were studied with the Pearson’s χCorrelation between the scores reported in the GMDS-ER subscales were tested by calculating the Spearman correlation coefficient rho.Each variable of potential clinical interest was tested in a univariate multinomial (polytomous) regression model in order to assess a potential statistical significance in the association with the outcome sequelae.The risk factors of clinical  interest for which a statistical significance in the association with sequelae at the univariate analysis and for which no collinearity was reported were included as regressors in a multivariate multinomial logistic model.p value < 0.05 was considered statistically significant. Data analysis was performed using the STATA statistical package .All tests were two-sided. The significance level was set at α = 0.05. A During the study period, two hundred and ninety ELBWi were admitted to the NICU of “Fondazione IRCCS Policlinico San Matteo” hospital. Mean GA at birth was 26.2  weeks, and mean birth weight was 750.8 (sd 154.6) g.p-value < 0.001;BW 668 g [sd 164.5 g] versus 782.68 g [sd 138.29 g], p-value < 0.001 respectively). None of the surviving infants died after NICU discharge home. Two hundred and eight surviving children met the inclusion criteria and were enrolled. One hundred seventy-six children (84.6%) completed the follow-up evaluation and constituted the study group. No differences in perinatal medical data, cUS findings, or neurological assessment at 40 weeks of PMA were found between the study group and the subjects lost to follow-up.Twenty-two infants had a birth weight ≤ 500 g. There were 262 inborn infants (90.3%) while outborn were 28 (9.7%). Two infants were excluded from the analysis due to genetic disorders. Eighty infants (27.6%) died during NICU stay. All infants with birth weight ≤ 500 g died in NICU. GA and BW were significantly lower among infants who died compared with survivors  infants showed a neurodevelopmental outcome within the normal range at 24 months of CA. Fifty-eight patients 32.95%) had an “non-optimal” outcome: 30 cases (17.04%) showed minor sequelae and 28 cases (15.91%) major sequelae. In the “major sequelae” group, eight infants (4.54% of the whole sample) had CP, 4 of them with a non-disabling form and could walk unassisted. Severe developmental delay without other associated deficits was observed in 15 infants (8.52% of the sample). Sensory deficits were present in 7 children . More details are shown in Table 2.95% hadp < 0.001), as reported in Additional Table p < 0.001), as reported in Additional Table GMDS-ER scores are available for 174/176 patients. The median GQ was 100 . The median scores obtained on the subscales are shown in Fig. p value = 0.048).Gestational age was significantly lower in the group with cerebral palsy with 25 (sd = 0.53) week of GA versus 26.59 (sd = 0.17) weeks reported in the group without cerebral palsy; p value for the comparison = 0.002). Results of comparison of GQ Scale and related subscales between the two groups with different gestational age  were reported in Additional Table Children who reported a GQ score > 75 had significantly higher gestational age (26.71 [sd 0.18] weeks) than children who reported a GQ score ≤ 75  grams versus a mean birth weight of 687.22 8sd 0 40.82) among children with a GQ score ≤ 75.p value for the comparison = 0.030).Gender was found to be associated with GQ score, with 19.23%  of males versus 8.16%  of females reporting GQ score ≤ 75 .Severely abnormal cUS findings and bronchopulmonary dysplasia (BPD) were the most important predictors of major sequelae . Pathological neurological examination at term age has been identified as a moderate risk factor for minor sequel too.Abnormal neurological examination at 40 weeks PMA . In contrast, female gender resulted as a moderate protective factor for major sequelae , as well as a greater gestational age  Post-natal steroid administration is a moderate risk factor only for minor sequelae (RRR 3.10 p = 0.008).Further weak association, even if with statistical significance of the association with major sequelae, was reported for CRIB, time needed for birthweight recovery and total parenteral nutrition  and only BPD as predictor for minor sequelae .Multivariate multinomial logistic regression analysis confirmed severely abnormal cUS findings and BPD as independent risk factors significantly associated to major sequelae  . MoreoveSeveral reasons could explain differences in neurodevelopmental impairment rates and severity. First, inclusion criteria are based on gestational age or birth weight; even though most ELBW infants are preterm, gestational age may vary depending on the number of small for gestational age infants enrolled. Indeed, fetal growth restriction is a well-known risk factor for neurodevelopmental impairment . TherefoAnother explanation of results discrepancies among studies may rely on the recruitment period. Indeed, our cohort includes infants born between 2005 and 2015, while others were based on cohorts enrolled between 1998- and 2007 , 7, 28,,Assuming that in the last decade improvement in antenatal, perinatal and postnatal care has led to neurodevelopmental outcome improvement, we may argue that more recent case studies might reflect clinical progression of the management.Differently, the CP prevalence reduction demonstrated for VLBWI , 30 is nTo our knowledge, a comparable CP rate (around 5%) was described by Wilson-Costello et al. only. Another point to underline is that our sample's most frequent major sequela was a developmental delay (8.52%). These data align with those recently reported by an Italian study in which 9.1% of ELBW infants showed a cognitive score below 2SD at 24 months of corrected age . Other rRegarding GQ values and developmental subscale scores, we found a peculiar profile maintained regardless of normal GQ and characterized by a significant selective deficit in the language domain . A left-Several studies suggested that language difficulties in preterm children are not a mere expression of co-occurring cognitive disabilities; instead, they seem to emerge as complications of prematurity that may selectively interfere with language acquisition .Recent studies also highlighted that prematurity is associated with an increased risk for autism spectrum disorders . At oldeAs previously reported , severelCerebral US is the standard bedside examination in NICU. Despite its limits in identifying specific types of brain damage, such as diffuse white matter abnormalities and micro-cerebellar hemorrhages, our results confirmed its importance in clinical practice in defining the neurological prognosis in the short term and referring infants to specialized early intervention programs.Our sample identified bronchopulmonary dysplasia as the other independent risk factor for both minor and major neurodevelopmental sequelae.Children who developed BPD are at increased risk of brain injury and abnormal brain development , 44. It Moreover, we found that the time needed to recover birth weight and red blood cells transfusion were other risk factors associated with impaired neurodevelopment, although only at univariate analysis.One possible explanation for the relationship between delayed time to birth weight recovery and impaired neurodevelopment could rely on a possible inadequate amount of specific macro and micronutrients during the critical phases of neurogenesis, neuronal differentiation, myelination and synaptogenesis . HoweverIt is already reported that RBC transfusion negatively impacts the long-term neurodevelopment of preterm newborns . In the Our study is one of the few investigating the neurodevelopmental outcome at two years of corrected age of ELBWi born in the 2000s in Italy , 49. OveThe single-center design of the study meant that highly homogeneous standards of care were applied to all participants throughout the study period and ensured methodological consistency of data collection during the follow-up.In this retrospective study obstetrical data are complete and available only for a subgroup of infants. Among incomplete maternal data, there were maternal emotional well-being and stress perception, stressful life events, depressive symptoms and pregnancy-related anxiety, known to be associated with neurodevelopmental outcomes in extremely preterm infants .Additionally, the predictive value of neurological examination improves with the analysis of GMs’ trajectories; however, video-recordings with the strict methodology needed for research are difficult to obtain for every patient in routine clinical settings. Nevertheless, the quality of spontaneous motility has been evaluated during the neurological examination performed at 40 weeks PMA. Unfortunately, we do not have neonatal brain MRI data available for all patients: it would be interesting to evaluate them, in particular in the proportion of children with cognitive delay at 24 months of corrected age.Our study showed an improvement of ELBW infants' survival rate without neurodevelopmental impairment compared to previous studies and a tendency to change the clinical type of major sequelae, with a prevalence of cognitive impairment on cerebral palsy. Even though the follow-up period until 24 months corrected age can identify major sequelae, long-term follow-up is mandatory for ELBW infants because neuropsychological and behavioral deficits related to preterm birth are ultimately expressed only at older ages. Finally, standardized follow-up protocols to detect and quantify infants with emotional dysregulation and risk of developing autism spectrum disorder are required.Additional file 1: Table S1. Correlation between GMDS-ER subscales scores reported for overall sample (n=176). rho Spearman correlation coefficient is reported in the first line of each cell; in the second line of each cell p value for the statistical significance of the correlation is shown.Additional file 2: Table S2. Correlation between GMDS-ER subscales scores reported for children with normal GQ score (n=153). rho Spearman correlation coefficient is reported in the first line of each cell; in the second line of each cell p value for the statistical significance of the correlation is shown.Additional file 3: Table S3. Comparison of GMDS-ER GQ and related subscales between the two groups with different gestational age . Data are reported as mean (standard deviation).


Aspergillus fumigatus has become a significant threat in clinical settings. Cases of invasive infections with azole-resistant A. fumigatus isolates (ARAF) increased recently. Developing strategies for dealing with ARAF has become crucial. We here investigated the in-vitro and in-vivo activity of the imidazole luliconazole (LLCZ) against clinical ARAF. In total, the LLCZ minimum inhibitory concentrations (MICs) were tested for 101 A. fumigatus isolates (84 ARAF and 17 azole-susceptible A. fumigatus as wild-type controls) according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Additionally, antifungal activity was assessed in vitro, including an XTT planktonic growth kinetics assay and biofilm assays . Further, a single-dose LLCZ treatment (152 mg/L) was tested for seven days in vivo in a Galleria mellonella infection model. LLCZ showed an MIC50 of 0.002 mg/L and no significant difference was found between triazole-resistant and wild-type isolates. Growth inhibition took place between 6 and 12 h after the start of incubation. LLCZ inhibited biofilm formation when added in the pre-adhesion stages. In vivo, single-dose LLCZ-treated larvae show a significantly higher survival percentage than the control group (20%). In conclusion, LLCZ has activity against planktonic cells and early biofilms of ARAF. Aspergillus fumigatus is among the most frequently isolated fungi in invasive mold diseases. A. fumigatus is a saprotrophic filamentous fungus, often described as an airborne opportunistic pathogen [A. fumigatus (ARAF) has only made this problem worse, with some isolates being capable of mutations that help them evade the effects of triazoles [cyp51A gene), an important enzyme in the ergosterol synthesis pathway. Through point mutations and a tandem repeat promoter alteration in the cyp51A gene, ARAFs manage to evade this mechanism [tinea pedis (athlete’s foot) [Aspergillus spp. have been reported [pathogen . It is kpathogen . With inpathogen . Azole-rriazoles . Triazolechanism . Anotherechanism . Biofilmechanism . Strategechanism ,10. Toda’s foot) , as of 2’s foot) . No syst’s foot) . Clinicareported ,15,16,17A. fumigatus isolates, which were identified through fungal ITS sequencing [n = 84 with different mutations of the cyp51A gene) or non-resistant (n = 17 wild type controls). Ten of these isolates were further used for the growth kinetics assay, biofilm assay, and in vivo assay. Of these, seven isolates carried a TR34/L98H mutation, one carried a TR46 mutation and two were wild-type isolates. All isolates used in this study and their specific mutations are summarized in The study includes a total of 101 quencing . The isoA. fumigatus isolates were grown on Sabouraud agar  for 2 days at 35 °C. An H2O + 0.1% Tween solution was added to the plates, the fungal suspension was collected with a syringe and filtered . After two washing steps with 1 × phosphate-buffered saline (PBS), the inoculum was set to an appropriate concentration, via dilution in RPMI 1640, depending on the experiment. Colony-forming units (CFU) were determined as an inoculum control.LLCZ was purchased from Sigma-Aldrich . The drug was diluted in DMSO and a stock solution was stored until use at −20 °C. For use, the stock was diluted to working concentrations in RPMI 1640 medium. For further information on concentrations and dilutions, refer to the corresponding sections below.To determine the specific minimal inhibitory concentration (MIC) of LLCZ, the broth microdilution according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) was performed . LLCZ wa5 CFU/mL) was incubated together with the different concentrations of LLCZ in flat-bottomed 96-well plates at 35 °C. The growth was assessed through OD492 measurement after 0 h, 6 h, 12 h, 24 h, and 48 h. Two hours prior to the end of incubation time, 50 µL of 2.5 mg/mL XTT  plus 125 µM menadione (Sigma) solution were added to each well. A total of 150 µL of the suspensions were transferred to new 96-well plates with a U-shaped bottom. The absorbance for each well was read at 492 nm with a microplate reader .A colorimetric micro-broth kinetic growth assay was performed to assess the impact of LLCZ on the growth of the above-mentioned isolates . The iso5 CFU/mL was used for the experiments. To assess LLCZ anti-biofilm activity, the agent was used in doses equal to the previously determined isolate-specific MICs, as well as in sub-MIC concentrations: a half, a quarter, and an eighth of the specific MIC value. A negative and a non-treated control were also included in this experiment. The drugs were added at different time points after incubation start at 35 °C: 0 h, 4 h, 12 h, 24 h, and 48 h. The plates were incubated at 35 °C for biofilm development. After incubation, they were washed with 1 × PBS and 200 µL LLCZ was added. The biofilm was incubated with LLCZ for a further 24 h at 35 °C. Afterward, it was washed twice with 1 × PBS and analyzed via XTT assay. Therefore, 100 µL/well of an XTT (0.5 mg/mL) plus 25 µM menadione solution were pipetted onto the plates and incubated at 36 °C for 3 h. The optical density was measured in a plate reader at 492 nm.Biofilm formation has been assessed as described previously . A final®, Gräfelfing, Germany). The formed biofilms were fixed with 100% methanol for 1 min. Images were processed using the ZEN black software (Zeiss) [The anti-biofilm effect was visualized in a confocal laser scanning microscopy CLSM using the ELYRA LSM 710  with a laser at 405 nm and a 20-fold magnification objective. Fungal suspensions were incubated and treated as described above in 1 µ-Slide 8-well glass-bottom dishes (ibidi GmbH (Zeiss) . G. mellonella) was chosen for in vivo experiments. The protocols for handling the larvae were previously optimized and published [The larval stage of the greater wax moth . The statistical significance level was determined by Dunnett’s multiple comparison test, p < 0.05 was stated as significant and the confidence score was indicated by asterisks: *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.All experiments were performed in triplicates. The survival data were analyzed with the program GraphPad Prism 9 (GraphPad Software Inc.). Survival curves were plotted with the Kaplan-Meier estimator. To compile results from multiple repetitions, the raw data was calculated as a single experiment on the datasheet. The statistical significance of the survival curves was determined with a log-rank (Mantel-Cox) test. 50 value was 0.002 mg/L and the MIC90 value was 0.015 mg/L. No significant differences in the MIC values could be noticed between mutants and wild-type isolates  or partially (4 × and 16 × MIC) recover from the effect of the drug .Afterwards, the effect of LLCZ on ARAF biofilm formation was tested via XTT and CV assay and visualized via CLSM: Both, crystal violet and XTT assay indicated that the best biofilm growth inhibition occurred when LLCZ was added 4 h after incubation start, in the pre-adhesion phase. On the more mature biofilm , LLCZ treatment was less effective or had no effect at all . Upon vi5 cells/mL and treated with a therapeutic dose of 152 mg/L LLCZ, had a survival percentage of 41% over the course of 7 days. The larvae treated with a placebo (PBS) had a survival outcome of 21%, similar to the result of the infection assay  but is inefficient against mature biofilm (24 h and 48 h). In vivo experiments showed one injection of LLCZ could significantly improve G. mellonella survival after infection with ARAF. Considering the above-mentioned results and the evident in vitro and in vivo activity of LLCZ against ARAF growth and biofilm formation, this drug might be considered as an alternative for a future systemic treatment of ARAF.In conclusion, we were able to demonstrate LLCZ activity against


Diffusion tensor imaging (DTI) has demonstrated the potential to assess the pathophysiology of mild traumatic brain injury (mTBI) but correlations of DTI findings and pathological changes in mTBI are unclear. We evaluated the potential of ex vivo DTI to detect tissue damage in a mild mTBI rat model by exploiting multiscale imaging methods, histology and scanning micro-X-ray diffraction (SμXRD) 35 days after sham-operation (n = 2) or mTBI (n = 3). There were changes in DTI parameters rostral to the injury site. When examined by histology and SμXRD, there was evidence of axonal damage, reduced myelin density, gliosis, and ultrastructural alterations in myelin that were ongoing at the experimental time point of 35 days postinjury. We assessed the relationship between the 3 imaging modalities by multiple linear regression analysis. In this analysis, DTI and histological parameters were moderately related, whereas SμXRD parameters correlated weakly with DTI and histology. These findings suggest that while DTI appears to distinguish tissue changes at the microstructural level related to the loss of myelinated axons and gliosis, its ability to visualize alterations in myelin ultrastructure is limited. The use of several imaging techniques represents a novel approach to reveal tissue damage and provides new insights into mTBI detection. Mild traumatic brain injury (mTBI) is a major public health problem worldwide affecting approximately 200–700 out of 100 000 people per year , 4. HoweNoninvasive imaging methods, such as computed tomography (CT) or magnetic resonance imaging (MRI), are the gold standard techniques used in the diagnosis of TBI patients in the clinic , 15. WhiIn this study, we used a multiscale imaging approach to evaluate the potential for DTI to detect the tissue damage in the rat brain at 35 days after mTBI. We used ex vivo DTI to assess tissue microstructural changes, light microscopy to examine disturbances in the cellularity and morphology of myelinated axons and scanning micro-X-ray diffraction (SμXRD) to evaluate the ultrastructure of myelin at the nanometer scale. Subsequently, we devised a regression model to examine the relationships between these 3 techniques in terms of tissue structure. This study highlights the potential pros and cons of DTI for detecting tissue microstructural changes after mTBI by using a multiscale imaging approach.Adult male Sprague-Dawley rats were used in all the experiments . They were housed individually in cages in a climate-control room under a 12-hour/12-hour light/dark cycle with ad libitum diet. All animal procedures were approved by the Animal Ethics Committee of the Province Government of Southern Finland and carried out according to the guidelines set by the European Community Council Directives 2010/63/EEC.Detailed experimental procedures were performed as in . In brieAt 35 days post-mTBI, all of the rats were anesthetized with 5% isoflurane in 70% nitrogen/30% oxygen, after which they were transcardially perfused with 0.9% NaCl for 5 minutes (30 mL/minute) followed by 4% paraformaldehyde in 0.1 M PB pH 7.4 for 25 minutes (30 mL/minute). After perfusion, the brains were removed from the skull and postfixed in 4% PFA for 4 hours. Before ex vivo DTI evaluation, the brains were transferred to a solution containing 1 mM gadopentetate dimeglumine  in 0.1 M PBS for at least 72 hours. In order to achieve an effective suppressed background signal imaging acquisition, we placed the brains inside a polyethylene tube filled with perfluoro-polyether .3, number of b0 images = 4, 30 directions per b-value , resolution of 0.15 mm3 isotropic (zero-filling interpolation to 0.075 mm3 isotropic) scan time of ∼21 hours.The fixed rat brains were scanned in an 11.7 T NMR spectrometer  with a Micro2.5 gradient system (maximum gradient strength = 1000 mT/m) using a birdcage volume coil (diameter = 20 mm) for transmission and signal reception. A 3D diffusion-weighted gradient- and spin-echo (DW-GRASE) sequence  was usedFor the ex vivo DTI analyses, the k-space data using an in-house code in IDL  was first processed to reconstruct the DW data into Nifti format. The reconstructed DW data were then preprocessed using MRtrix3 software  by perfoAfter ex vivo DTI imaging, the brains were washed in 0.9% NaCl for at least 2 hours at 4°C and then, cryoprotected for 36 hours in a cryoprotective solution . After cryoprotection, the brains were frozen in dry ice and stored at −70°C until cutting. Brains were sectioned in the coronal plane with a sliding microtome . The first series of sections were stored in 10% formalin, and the 4 remaining series in cryoprotectant-tissue collecting solution  at −20°C until processing.2/pixel) were acquired using a ZeissAxioImager2 light microscope equipped with a digital camera (Zeiss Axiocam color 506). Each photomicrograph of Nissl-stained sections was analyzed using an in-house developed MATLAB code for automated cell counting analysis available at https://github.com/aAbdz/cell_counter.Nissl (thionin) staining of the first series of the sections were performed to assess the cytoarchitectonics, gliosis or increased cell density (CD), and the extent of neurodegeneration at 35 days post-mTBI. To assess cell density, high-resolution photomicrographs of the whole brain section . We calculated the anisotropy index (AI) as a histological derived parameter by applying the pixelwise ST-based method . Therefore, an exposure time of about 10 ms was used for each frame in each 2 µm2 × 2 µm2 spot, avoiding in this way the radiation damage in our SµXRD measurements and at the same time, ensuring sufficient photon counts statistics. At each point reached by the y–z translator, we collected a 2D diffraction frame in transmission geometry using a fast Eiger 4 M detector (Dectris) 135 mm distant from the sample. The beam center, detector tilt and sample-to detector distance were calibrated with a NIST SRM 676 Al2O3 standard using pyFAI.One section per animal at −2.00 mm from bregma was selected from the third series of brain sections, rostrally to the lesion site (−3.60 mm from bregma). SμXRD measurements were performed on the ID13 beamline of the European Synchrotron Radiation Facility . Each brain section was mounted on a 4-µm-thin ultralene film, which was glued to a ring holder. The X-ray beam was monochromatized with a liquid nitrogen cooled Si-111 double monochromator and then focused by compound refractive lenses on a 2 × 2 μm−1 (L(q) + IH(q) ascribed to a main multilamellar phase (L) and a minority hexagonal phase (H), respectively (L(q) contribution presents the quasi-Bragg peaks at:h = 2, 3, and λL is the mean interlamellar separation representing the myelin period or lamellar lattice unit  profiles were fitted by Gaussian line shapes to provide the lattice units λL and λH in the lamellar and hexagonal phases and the myelin content (C) in lamellar (CL) and hexagonal (CH) phases by (Phase is IL(q) or IH(q) for the lamellar and hexagonal phase, respectively. The period and the content of the lamellar and hexagonal phase in each selected region of interest (ROI) were mapped. We defined a commensurability parameter, η, given by the ratio between the unit cell of the lamellar and hexagonal phases (η = λL/λH). Image processing and data analyses were performed by using a customized in-house developed code written in MATLAB   by obtaining the 1D intensity profiles, I, versus momentum transfer12] nm−1 . In the ectively . As showice unit . Beyond hases by :CPhase=ick, MA) .An ROI-based approach on specific selected brain areas was used to extract imaging parameters with each technique. We manually outlined ROIs in the external capsule, layer VI of the somatosensory cortex, cingulum, corpus callosum and internal capsule at approximately −2.00 mm from bregma ipsilaterally to the sham-operation or injury in the brain. The location of the ROIs in the 3 methodologies was anatomically delineated in the same location by an expert (I.S.M.M.) , and enshttp://aedes.uef.fi/) in MATLAB  was used to derive the ROI values from the ex vivo DTI parameters. The ZEN software  was used in the evaluation of the high-resolution photomicrographs of Nissl and myelin staining. For SμXRD, the ROIs using an in-house MATLAB code in the intensity maps were outlined. GraphPad Prism , KaleidaGraph  and MATLAB  software were used to obtain the graphs.An in-house software  when the values from all the 3 mTBI animals were higher (lower) than the values from the values from the 2 sham-operated animals. Similarly, as in DTI, for the histological findings, we reported changes from a single mTBI animal when the histological metrics values were higher (lower) than the values from both sham-operated animal values. For SµXRD results, the measured values in each individual animal were compared to the standard values for the lamellar period and commensurability of myelin reported in the literature , 48.xkj is the vector of predictor parameters for the region j of the animal k. b and c are the regression parameters, and ekj represents normally distributed i.i.d. errors. The parameter to be explained and predictor parameters originate from different modalities. For DTI, the following were considered: fractional anisotropy index (FA), axial diffusivity (AD), radial diffusivity (RD), mean diffusivity (MD), linear anisotropy index (CL), planar anisotropy index (CP), and spherical anisotropy index (CS); for histology, anisotropy index (AI) and cell density (CD) were selected; for diffraction, lamellar phase content (CL), lamellar phase period (λL), hexagonal phase period (λH), and hexagonal phase content (CH) were selected. The regions evaluated were external capsule, layer VI of the somatosensory cortex, cingulum, corpus callosum and internal capsule  and expanded λL > λL* (mTBI2) lamellar periods in all the brain areas , whereas DTI explained over 25% in CD  . In this regard, DTI explained over 25% of the variation in CL  and CH   and λH   and CH   and λH   . We then–0.410]) . In the –0.120]) . Histolo–0.450]) . In the –0.120]) . Additio–0.120]) . Subsequ–0.120]) . With re–0.120]) . These rIn this study, we evaluated the potential of DTI to detect tissue damage after an experimental mTBI in combination with a multiscale tissue assessment by histology and SμXRD. We found that DTI may reflect changes in white and grey matter areas after the injury; our histological analyses of myelin and Nissl staining illustrated axonal damage, loss of myelinated axons and gliosis. In mTBI animals, SμXRD also demonstrated alterations in the local structure of myelin, such as periodicity and content. In summary, these findings demonstrate that DTI distinguished the microstructural changes associated with loss of myelinated axons and gliosis only to a limited extent. Furthermore, the axonal injury and the damage of myelin visible by light microscopy and by SµXRD were not detected by DTI.In the present study, DTI showed moderate alterations in the values in mTBI animals as compared to sham-operated ones in the areas closest to the primary injury, that is, the external capsule and somatosensory cortex. DTI parameters from the corpus callosum and cingulum in mTBI animals exhibited moderate deviations from the DTI values in sham-operated animals, while parameters from the internal capsule did not show any apparent changes in DTI values between these animal groups. The animals selected for these analyses were representative of larger groups included in our previous studies , 33. WheDTI changes have been associated with axonal damage in white matter , 32, 33 SμXRD distinguished alterations in the ultrastructure of myelin, which may be an indication of either ongoing degenerating or regenerating processes in the myelin sheath of axons in mTBI brains. SμXRD is a technique which can sensitively detect local fluctuations in the dynamics of the ultrastructure of myelin in the central and peripheral nervous system . Our datWhen comparing DTI and SμXRD outcomes, we found that DTI did not explain well changes in the ultrastructure of myelin detected by SμXRD. Previously, Georgiadis et al  evaluateWe conducted extensive assessment of the tissue damage at the micro- and nanoscale levels, but a few limitations should be taken into consideration. The main limitation of our study is the sample size , which revealed variability between animals in DTI, histology and SµXRD findings indicating uncertainty of the statistical parameters. Second, it is important to consider that chemical fixation can potentially affect the tissue microstructural properties and therefore, in vivo and ex vivo DTI results should be compared , 69. HowIn conclusion, the findings suggest that DTI was moderately sensitive at distinguishing changes at the microstructural level in the subacute phase of mTBI, such as gliosis and loss of myelinated axons, but was less successful at detecting features at the nanometer scale such as axonal damage and ultrastructural-level alterations in myelin. The adoption of a multimodal imaging approach can offer new ways to study tissue changes after mild brain injury and may allow a better interpretation of DTI findings in future mTBI studies.This work was supported by the Academy of Finland , and Vilho, Yrjö and Kalle Väisälä Foundation of the Finnish Academy of Science and Letters.nlac100_Supplementary_DataClick here for additional data file.


We aims to determine the relationship of amniotic fluid sludge (AFS) and/or short cervical length  with a high rate of preterm birth in women after cervical cerclage.A retrospective cohort study was conducted among singleton pregnancies after cervical cerclage between January 2018 and December 2021. A total of 296 patients who underwent transvaginal ultrasound to evaluate CL and the presence of AFS within 2 weeks after cerclage were included. Pregnancy outcome after cerclage was analyzed in accordance with the presence of AFS and CL ≤25 mm.P = .000). The Cox regression analysis showed that these results remained significant after adjusting for confounding factors (P = .000). The negative linear relationships between AFS and CL  also explained the outcome.In patients with cerclage, AFS was an independent risk factor for preterm birth at <28 and <36 weeks but not for preterm birth at <32 weeks, and CL ≤25 mm was an independent risk factor for preterm birth at <28, <32, and <36 weeks. The Kaplan–Meier analysis showed that the association between the presence of AFS and short gestational age at delivery was statistically significant in women with CL ≤25 mm (log rank test, AFS and short cervix have a direct effect on pregnancies after cerclage. Mid‐trimester AFS can become a supplementary ultrasound index for detecting preterm birth after cerclage in pregnant women with a short cervix. Ureaplasma urealyticum, Streptococcus mutans, and Mycoplasma hominis, are previously isolated.As a serious pregnancy complication, cervical insufficiency contributes to adverse pregnancy outcomes, such as abortion or preterm delivery.In this study, we have deeply explored whether the interaction between AFS and short cervix (CL ≤25 mm) influences preterm birth after cerclage. On the basis of this original study, we hope to determine an objective and efficient indicator of preterm birth for women after cerclage.A retrospective cohort study was conducted between January 2018 and December 2021 in The Third Affiliated Hospital of Guangzhou Medical University. The subjects were singleton pregnancies who underwent McDonald cerclage for the following diagnosis of cervical insufficiency based on a history of one or more mid‐trimester miscarriages or preterm birth (history‐indicated cerclage), transvaginal ultrasound evidence of short cervix (ultrasound‐indicated cerclage), and cervical dilatation found on speculum examination with amniotic sac bulging (rescue‐indicated cerclage).Perioperative antibiotic treatment was administered to women with cerclage to reduce the rate of complications during the procedure. However, different regimens, such as antibiotic type and treatment duration, were provided throughout the observation period. Cerclage suture was removed between 36 and 37 weeks or at the time when the patient developed progressing preterm birth. Pregnancy outcomes were monitored and recorded.All patients underwent transvaginal ultrasound at 14 to 24 weeks of gestation within 2 weeks after cerclage placement to measure CL and evaluate the presence of AFS. Ultrasound images were reassessed retrospectively by three ultrasound specialists who were blinded to pregnancy outcomes. Transvaginal ultrasound screening was conducted in accordance with the following standard technique.This study was approved by the Institutional Review Board of The Third Affiliated Hospital of Guangzhou Medical University (IRB no. 2021013), and all participants provided informed consent before enrolling in the study.t‐test and Mann–Whitney test were used to assess continuous variables when appropriate. The interaction between AFS and CL was analyzed using the chi‐square test for trend and Pearson analyses. The multivariable logistic regression analysis was performed to adjust for confounding factors and predict preterm birth before 28, 32, and 36 weeks. The Kaplan–Meier survival analysis of gestational age at delivery was conducted in accordance with the presence of AFS in pregnant women with or without CL ≤25 mm. The Cox regression analysis was performed to assess the gestational age at delivery. Variables with P values <.05 in the univariate analysis were entered into multivariable logistic and Cox regression analyses. P < .05 was considered statistically significant.All data were analyzed using IBM SPSS Statistics 25. Categorical variables between the cases and control subjects were compared by Fisher's exact test or Pearson's chi‐square test. The Student's A total of 296 pregnant women were included in the analysis. The maternal characteristics of patients with and without AFS were compared as follows Table . About 1P < .001), <32 , and <36  weeks were higher than those without AFS , <32 , and <36  weeks were higher than those with CL >25 mm , and the proportions of patients after cerclage with AFS that had preterm birth at <28 . Specifically, patients with AFS(+) and CL ≤25 mm had shorter median gestational age at delivery than those with AFS(−) and CL ≤25 mm (33 vs 36 weeks). Patients with AFS(+) and CL ≤25 mm had a significantly shorter median gestational age at delivery than those with AFS(+) and CL >25 mm (33 vs 38 weeks). However, patients with AFS(+) and CL >25 mm had a same median gestational age at delivery with those with AFS(−) and CL >25 mm (38 vs 38 weeks). The Cox regression analysis showed that these results remained significant after adjusting for the presence of AFS, CL ≤25 mm, and prepregnancy body mass index (BMI) .The Kaplan–Meier analysis of gestational age at delivery (in weeks) was performed in accordance with AFS and/or CL results Figure . The assP < .001; Table R = −0.454, P < .001).AFS and CL had a negative relationship .In addition, other factors, such as BMI and cervical dilation, affect pregnancy outcomes after cerclage.This study has some limitations. First, this study is a retrospective study and cannot estimate the exact natural frequency and history of occurrence of AFS. Second, a selection bias may arise because the decision of whether a cervical cerclage needs to be performed depends on the clinical criteria of the attending physician. The exact composition of AFS in this population is not confirmed in this study. Because of the lack of dynamic videos, we did not act a double‐check on the AFS cases.In conclusion, the combination of AFS and a short cervix improves the prediction of preterm birth in women after cervical cerclage. The clinical characteristics of AFS are not fully validated in clinical practice. The ultrasound features of AFS among patients presenting preterm labor need to be determined in further studies. AFS and a short cervix, which have implications for the management of preterm birth in women after cervical cerclage, can be used as biomarkers to detect preterm birth and offer clues to identify their mechanism in preterm birth.


Ethnic minority status and maternal socio-economic deprivation are linked to delayed access to health care during pregnancy. The link between late antenatal care initiation and neonatal outcomes in settings with high ethnic diversity and social disadvantage is seldom explored. This study examined associations between late antenatal care initiation  and neonatal outcomes of preterm birth (<37 weeks gestation) and low birth weight (<2500 g) in an ethnically diverse socially disadvantaged maternal cohort.A retrospective cross sectional study using routinely collected anonymous data of singleton births between April 2007 - March 2016 from a large UK National Health Service maternity unit in an ethnically diverse, socially disadvantaged area. Univariate and multivariate logistic regression models were used to examine the associations between late antenatal care initiation and prevalence of preterm birth and low birth weight.Of the 46,307 singleton births recorded, more than one third (34.8%) were to mothers from Black African, Black Caribbean, Indian, Pakistani, and Bangladeshi mothers. Gestational week at first antenatal appointment was available for 99.31% births among which 79.2% had their first appointment at ≤ 12 weeks, 12% at 13-20 weeks, and 8.8% at > 20 weeks. Mothers who booked at 13+ weeks were significantly more likely to have a preterm and/or low birth weight baby. Compared to mothers who booked at ≤ 12 weeks, those booking at > 20 weeks were 4.08 times  as likely to have an extremely preterm baby (<28 weeks of gestation) and 3.12  times as likely to have a baby born with extremely low birthweight (<1500g).Mothers in ethnically diverse socially deprived areas who started antenatal care late were at increased risk of adverse neonatal outcomes. Targeted intervention programmes and services are needed to support these mothers.Mothers who start antenatal care late are more likely to have a preterm and/or low birth weight baby in ethnically diverse socially disadvantaged areas.There is a need for targeted programmes and services to support mothers in ethnically diverse socially disadvantaged areas to start antenatal care on time.


In natural populations, quantitative traits seldom show short‐term evolution at the rate predicted by evolutionary models. Resolving this “paradox of stasis” is a key goal in evolutionary biology, as it directly challenges our capacity to predict evolutionary change. One particularly promising hypothesis to explain the lack of evolutionary responses in a key offspring trait, body weight, is that positive selection on juveniles is counterbalanced by selection against maternal investment in offspring growth, given that reproduction is costly for the mothers. Here, we used data from one of the longest individual‐based studies of a wild mammal population to test this hypothesis. We first showed that despite positive directional selection on birth weight, and heritable variation for this trait, no genetic change has been observed for birth weight over the past 47 years in the study population. Contrarily to our expectation, we found no evidence of selection against maternal investment in birth weight—if anything, selection favors mothers that produce large calves. Accordingly, we show that genetic change in birth weight over the study period is actually lower than that predicted from models including selection on maternal performance; ultimately our analysis here only deepens rather than resolves the paradox of stasis. One of the most puzzling and persistent problems in evolutionary ecology is the “paradox of stasis,” whereby heritable phenotypic traits under strong selection do not evolve , rather than from antagonistic genetic covariances , as defined by the single‐trait maternal performance model proposed by Cheverud   population on the Isle of Rum, Scotland. In previous analyses, we have shown that maternal effects explain 35% of the total phenotypic variance in birth weight, that maternal effects are mostly genetic in origin, and that the correlation between direct and indirect genetic effects for birth weight  does not decrease the evolutionary potential of this trait  between 1972 and 2019. Deer living in this study area are recognizable either from ear tags or by facial characteristics, and individuals are closely monitored throughout the year. Mortality searching provides accurate information on death date for most resident individuals. There is no culling in the study area, but individuals are occasionally shot if they range elsewhere on the island. Most calves are caught soon after birth (often within 24 h) to be weighed, measured, tagged, and sampled for genetic analysis. Based on census data, each individual can be categorized as living in one of six regions of the study area , annual survival was evaluated from the 1st of May to the same date the following year. For individuals who were shot, data were recorded and analyzed up to the year prior to death.Adult annual breeding success (ABS). From observational and pedigree data, we calculated the number of offspring calved/sired each year by an individual from the age of three (maturity) to death. Females can give birth to a single calf per year, but males can mate multiply each rutting season. These data only recorded the ABS of males seen during the rut the year before.Lifetime breeding success (LBS). Lifetime breeding success was estimated for both sexes as the total number of calves produced across an individual lifetime. This data set includes individuals that died as juveniles and never get to reproduce. LBS was estimated for all the individuals born within the study area in the cohorts 1972–2005 that either died of natural causes or were still alive in 2019 . Only the males seen in the study area during the rut most of their lifetime were included in this data.All models were fitted using a Bayesian framework, with the R‐package MCMCglmm v2.32 , maternal age , and maternal breeding status in the previous year, i.e. whether she (i) calved and the calf survived to at least May 1 the year after birth (milk), (ii) calved and the calf died during the winter after birth, between October 1 and May 1 (winter yeld), (iii) calved and the calf died during the summer, before October 1 (summer yeld), (iv) female did not calve (true yeld), and (v) had never calved before (naive) , maternal genetic effect variance (σMg2), maternal environmental effect variance (σMe2), and variance due to cohort effects (σC2). Because birth weight was measured once per individual, permanent environmental effects were not fitted separately from residual effects. We also modeled the covariance between direct additive and maternal genetic effects (σAMg), but we neglected the covariance with maternal environmental effects since these effects are negligible . We included year as a fixed covariate in our model to avoid confounding phenotypic changes due to the plastic response to some environmental covariate with genetic change due to a response to selection  for birth weight. Total genetic effects for birth weight, including maternal genetic effects, were estimated as: atot=a+12mg. For each sample of the posterior distribution, we fitted linear regressions of the breeding values  against offspring birth year. We thus generated a posterior distribution for the linear slopes of genetic change, which allowed us to test for change over time while accounting for the uncertainty in BLUP estimation . We transformed this estimate of the annual genetic change for birth weight into one for genetic change per generation, considering a generation time of 8 years  lifetime breeding success, and (2) annual fitness components, namely adult annual breeding success and annual survival. Analysis using annual components allows the inclusion of shot individuals, increasing our sample size, and also allow us to decompose selection via breeding success versus survival. In addition, annual fitness components can be modeled as binary traits , whereas modeling LBS is notoriously challenging  to investigate the different phenotypic associations required for full representation of selection pressures. These associations are represented in Figure Model (a): Effect of birth weight on juvenile survival. Model (a) was a univariate model with juvenile survival as a response variable and birth weight as a covariate. The model also considered the effect of offspring sex, maternal age (linear and quadratic effects), spatial region, population density, offspring birth date, and maternal reproductive status as fixed effects potentially affecting juvenile survival. Birth year (or cohort) was included as a random effect to account for temporal heterogeneity in survival rates. A binomial model with a probit function (‘threshold’ model) was used to analyze this binary trait.We also extended model (a) to allow the effect of birth weight on juvenile survival to vary between years, by defining an interaction term between birth weight and cohort (treated here as a fixed effect), thereby modeling the potential for fluctuating selection on birth weight.Model (b): Effect of birth weight on ABS and LBS. Based on previous evidence, we assumed that late‐life fitness effects of birth weight could only occur through its effect on breeding success : Association between maternal investment and mother's breeding success and survival. Model (c) was a bivariate model with response variables of the birth weight of the calf produced by a mother (in a given year t) and the mother's subsequent annual breeding success (ABS) or survival (SurvA) . The model for female annual breeding success/survival accounted for the effect of age (linear and quadratic effects), population density, and spatial location of the female during spring , as well as random effects of the year of measurement and maternal identity as random effects. As offspring birth weight and mother's annual breeding success are repeated measurements at the level of the mother, this model thus defines a covariance structure between birth weight and fitness at both the among‐mother and within‐mother levels . These two covariances respectively capture the association between mothers' average maternal effect for birth weight and their average for the annual fitness component, and the association between the deviation in birth weight from the average maternal effect and the mother's average prospect of surviving or reproducing the following year. Female ABS and annual survival were treated as a threshold response.Model (d): Association between maternal investment and mother's LBS. Finally, we fitted a bivariate model of birth weight of a mother's offspring and mother's (relative) LBS. The model used for birth weight was the same as model (c), and for female relative LBS it included the effect of cohort as a random effect. A female thus had one value of LBS and can have zero, one or several values for the birth weight of the offspring it calved. In model (d), the residual effects for LBS were allowed to covary with the maternal random effect for birth weight  using the “covu = TRUE” model structure in MCMCglmm. σBWM,LBSR estimates the among‐female covariance between LBS and offspring birth weights and was then used to estimate selection on maternal performance for birth weight (see below).A major assumption when measuring phenotypic selection is that the associations between traits and fitness are causal  for both sexes, using the output of model (b). We estimated the selection gradient on maternal performance for birth weight as: βm = σBWM,LBSR/σm2(BW), with σm2(BW) being the maternal effects variance for birth weight, using the output of model (d). Variance‐standardized selection gradients (βSD) were also estimated, with respect to the phenotypic standard deviation of birth weight, to provide a dimensionless measure of selection that allows comparison across traits and studies.Because the approaches using LBS and annualized fitness components provide qualitatively similar results (see Results section), we only used the outputs of the models using relative LBS as a response variable to predict the response to selection. We estimated directional selection on birth weight as the linear regression coefficient of relative LBS on birth weight ΔzσMg2 is the maternal genetic variance for birth weight and σAMg the covariance between direct and maternal genetic effects and βm is the selection gradient on maternal performance. All the (co)variance components used in eq.  and antagonistic selection (βo and βm of opposite signs) can impose constraints on the evolutionary response of birth weight. Maternal effects interfere with evolutionary responses if |Δz¯eq.2| <|Δz¯eq.1|. With σAMg∼ 0  and we found no fluctuation of the sign of selection on birth weight , we found a positive linear effect of birth weight on juvenile survival  showed no evidence of an effect of birth weight on female ABS  at the among‐mother level, with σM) = 0.067 [0.010 to 0.152], meaning that females who consistently produced large calves also had the largest number of calves. We also found a negative association between offspring birth weight and maternal ABS at the within‐mother level, with σR) = −0.079 [−0.118 to −0.010] ) = 0.111  and σR) = −0.071 [−0.215 to 0.025]. The larger uncertainty in parameter estimates is consistent with the fact that survival data is less variable than breeding success (Table From model (c), we investigated the costs/benefits of maternal investment in birth weight, decomposing its potential effects on mothers' breeding success and survival. There was a positive association between offspring birth weight and maternal ABSt+ at the aσBWM,LBSR = 0.15 [−0.02 to 0.27]. The resulting selection gradient on maternal performance was βm = 0.28 [−0.05 to 0.51]. The variance standardized selection gradient, βmSD = 0.28 [−0.04 to 0.58], is in the upper range of the values reported in the literature  LBS using model (d). The estimated covariance between maternal effects for birth weight and residual effects for relative LBS was positive, marginally overlapping zero, with βm.Finally, we compared the predicted and observed responses to selection for birth weight per generation. Using a model that neglected the evolutionary consequences of maternal effects eq. , we predDespite positive directional selection on birth weight, and heritable variation for this trait, we observed no genetic change for birth weight over the past 47 years in the Isle of Rum red deer population. One possible explanation for this evolutionary stasis is the hypothesis of “parent‐offspring conflict”  directly measuring the quality of individuals and accounting for it in models , they are unlikely to resolve the paradox of stasis on their own  to (d).Click here for additional data file.


Since the current commonly used birth growth curves are unsuitable for neonates in high-altitude areas; this study aimed to establish birth growth curves for full-term neonates residing at 2,000–3,000 m.This cross-sectional study retrospectively analyzed the physical measurement data of 1,546 full-term neonates delivered at the Red Cross Hospital of Qinghai province, China, from July 2021 to April 2022. The percentile curves of birth weight, length, and head circumference of neonates of different gestational ages and genders were developed using curve fitting. The newly developed birth-weight percentile reference was compared with the INTERGROWTH-21st Neonatal Growth Curve  and the Chinese Neonate Growth Curve (Chinese Standard).P < 0.05). We found differences between the newly developed birth-weight percentile curves in the high-altitude areas and the International and Chinese Standards.The median birth weight, length, and head circumference of the study population were 3,200 g, 52.0 cm, and 32.8 cm, respectively, except for the group with a gestational age of 37 weeks. The growth indicators of male infants in all groups were higher than those of the female infants (Establishing birth growth curves corresponding to altitude may be more suitable than the existing standards for local medical staff to conduct health assessments of neonates. Levels of physical development of newborns reflect their intrauterine growth and nutritional status, and they are also essential factors in the early survival and development after birth . TherefoSeveral widely used neonatal growth curves have emerged during the past decade. In 2013, Fenton et al. included data from developed countries, such as Italy, Germany, and the United States from 1991 to 2007 to develop the Fenton growth curve, which is widely used in several countries . DespiteSince these widely used growth curves are based on data from populations at sea level or low altitudes, they are unsuitable for newborns at high altitudes. Recently, two meta-analyses showed that the average birth weight decreases by 54.7–96.98 g for every 1,000 m increase in height. In contrast, altitude has little or no effect on gestational age at birth –12. TherGonzales et al.  and Al-SThis study aimed to establish percentile curves for the birth weight, length, and head circumference of local full-term neonates of different gestational ages and genders in the Qinghai-Tibet plateau of China at an altitude of 2,000–3,000 m, to identify neonates with abnormal physical development more accurately.The physical measurement data of neonates delivered at Qinghai Red Cross Hospital in Xining City, Qinghai province, China, on the Qinghai-Tibet plateau were retrospectively analyzed in this cross-sectional study. The hospital is the largest tertiary delivery center in Qinghai province, with an altitude of 2,261 m above sea level. It serves pregnant women from different areas of Qinghai province to give birth. The Medical Ethics Committee of Qinghai Red Cross Hospital approved the study protocol .+0–41+6 weeks. The exclusion criteria were: (1) incomplete information; (2) twin or multiple births; (3) artificial insemination of the mother; (4) birth with a congenital malformation, limb mutilation, fetal hydrops, or chromosomal abnormality; (5) mother age <18 years or >40 years; (6) the presence of any of the following diseases in the mother during pregnancy: severe anemia (hemoglobin ≤70 g/L), gestational diabetes mellitus, gestational hypertension, abnormal thyroid function, and heart or renal insufficiency, or (7) living in the stated residence <1 year. This information was obtained from the electronic medical record system of hospital.In June 2021, quality improvement and training on taking physical measurements of neonates were implemented by the nursing department of center. After training, the measurements were checked and found to be reliable. The inclusion criteria for this study were: (1) all live neonates delivered at the Qinghai Red Cross Hospital between July 2021 and April 2022, and the altitude of mother residence was 2,000–3,000 m; (2) the neonatal gestational age at birth was within the range of 37Regarding neonates, the collected data included: gender, mode of delivery, gestational age, birth weight, length, and head circumference. Concerning mothers, the collected data included: age, gravidity, and parity. The involved study variables were as follows: (1) gestational age: the method of gestational age assessment of the center was based on the comprehensive determination of the last menstrual period and ultrasound examination results of the first trimester (first three months) of the mother. When a gestational age determined using the two methods differed by <one week, the gestational age at the time of the last menstrual period prevailed. When a gestational age determined by the two methods varied by ≥1 week, the gestational age determined by ultrasound prevailed. (2) Birth weight: was measured and recorded within 12 h of birth, and the reading was rounded to 10 g. (3) Length and head circumference: were measured and recorded within 24 h of birth, and the reading was rounded to 0.1 cm. (4) Residence: where the pregnant woman lived during her admission to the hospital for delivery. An altitude ≥2,000 m is a high altitude .+0–37+6 weeks, and neonates in the 37–41 weeks of gestation group were divided into five groups with one group per week. Data collation, logistic checks, and basic statistical analysis were performed using SPSS v26.0 . Data with a normal distribution were expressed as mean ± standard deviation (SD), frequencies were expressed as numbers (%), and data with skewed distributions were expressed as interquartile ranges. Outliers (mean ± 5 SDs) were removed when a reference standard was established. The percentile curve fittings of birth weight, length, and head circumference were performed using the GAMLSS 4.3-1 software package in R v3.1.2 . The newly developed birth weight percentile reference values were compared with the reference values of the International and Chinese Standards, and a comparison chart was drawn using Origin version 2017 . All tests were two-tailed, and P < 0.05 was set as the study level of statistical significance.This study was divided into groups by weeks of gestational age, with one group per each gestational week, the gestational age of the 37-week of gestation group was 37+0–41+6 weeks were included. Herein, 1,546 neonates with a gestational age of 37+0–41+6 weeks. P < 0.05) ( < 0.05) .The newly developed percentile curves for the birth weight of newborns in high-altitude areas  were compared with the international standard . On the There are differences in the physical development of newborns between different populations and races, and it is not appropriate to use a one-size-fits-all approach, i.e., the same standard for all . The IntAltitude is an independent influencing factor significantly associated with low birth weight –12. UnfoThere are theoretical differences in neonatal physical development between 2,000 m and 3,000 m altitude. However, we considered 2,000 m–3,000 m as a whole for several reasons. First, creating separate growth curves for each altitude region is impractical. Second, the Chinese Qinghai province is located on the Tibetan plateau, and the population lives in a wide range of altitude distributions, and the same city may have multiple altitudes. Developing reference standards needs to consider their practicality and convenience fully. About 80% of the population of Qinghai province in China lives in the range of 2,000 m–3,000 m above sea level. Third, by comparison, the high-altitude standard we made is better than the current standard, although it still has a gap with the theory. Fourth, this practice is commonly used now. For example, Gonzales GF et al.  considerThis study treated the residence altitude of mother during her pregnancy as an involved variable. However, many previous studies on the effects of altitude on the physical development of neonates have examined the birthplace altitude as a study variable , 14, 20.Lubchenco et al.  found thThe level of physical development of neonates at birth is a significant public health indicator that reflects fetal health and predicts future health . Our resThis study has some limitations. First, the relatively small number of neonates in the 37- and 41-week gestational age groups, 123  and 48 , respectively. However, methods to calculate sample size for establishing growth standards have yet to be fully developed . There iIt is essential to base the selection of growth curves on altitude to assess the physical development of local neonates. This study was the first to develop birth growth curves for different genders of full-term neonates residing at an altitude of 2,000–3,000 m. Differences were observed between the newly developed birth-weight percentile curves in the high-altitude areas and the International and Chinese Standards. Meanwhile, it is necessary to further explore the birth growth curves of neonates at high altitudes, including preterm neonates.


With the increased availability of access to prenatal ultrasound in low/middle-income countries, there is opportunity to better characterize the association between fetal growth and birth weight across global settings. This is important, as fetal growth curves and birthweight charts are often used as proxy health indicators. As part of a randomized control trial, in which ultrasonography was utilized to establish accurate gestational age of pregnancies, we explored the association between gestational age and birthweight among a cohort in Western Kenya, then compared our results to data reported by the INTERGROWTH-21st study.th, 25th, median, 75th, and 90th BW percentiles for 36 to 42 weeks gestation were determined; resulting percentile points were plotted, and curves determined using a cubic spline technique. A signed rank test was used to quantify the comparison of the percentiles generated in the rural Kenyan sample with those of the INTERGROWTH-21st study.This study was conducted in 8 geographical clusters across 3 counties in Western Kenya. Eligible subjects were nulliparous women carrying singleton pregnancies. An early ultrasound was performed between 6 + 0/7 and 13 + 6/7 weeks gestational age. At birth, infants were weighed on platform scales provided either by the study team (community births), or the Government of Kenya . The 10n = 49) or stillbirth (n = 27). No significant differences were found between subjects who were lost to follow-up. Signed rank comparisons of the observed median of the Western Kenya data at 10th, 50th, and 90th birthweight percentiles, as compared to medians reported in the INTERGROWTH-21st distributions, revealed close alignment between the two datasets, with significant differences at 36 and 37 weeks. Limitations of the current study include small sample size, and detection of potential digit preference bias.A total of 1291 infants (of 1408 pregnant women randomized) were included. Ninety-three infants did not have a measured birth weight. The majority of these were due to miscarriage (st).A comparison of birthweight percentiles by gestational age estimation, among a sample of infants from rural Kenya, revealed slight differences as compared to those from the global population .This is a single site sub-study of data collected in conjunction with the Aspirin Supplementation for Pregnancy Indicated Risk Reduction In Nulliparas (ASPIRIN) Trial, which is listed at  The widespread use of obstetrical ultrasound early in gestation, particularly within high-income settings, has allowed very accurate and precise estimation of gestational age , 2. Coupst , 6.While early prenatal ultrasound is common in high resource countries, it is less common or even nonexistent in low resource settings. This has made the development of fetal growth curves difficult in these settings. Growing access to low-cost ultrasound (US) devices in these settings may begin to increase access to antenatal sonography for populations in low-to-middle income countries , 8. FetaThe Global Network for Women’s and Children’s Health Research recently completed a multi-site global clinical trial of low dose aspirin administered to women throughout pregnancy, beginning between 6–14 weeks gestation One of the largest, most comprehensive set of studies on multinational birth weight are those of the INTERGROWTH-21st trial , 17. TheThe data presented in this paper were acquired at the Kenya site as part of the Global Network for Women’s and Children’s Health Research ASPIRIN trial. Detailed study methods are described in Hoffman et al. , 18. Thep = 0.0167), but the difference of means was not clinically significant (~ 63 g). The analysis of variance for gestational age was not statistically or clinically significant.Eligible subjects were pregnant nulliparous women carrying singleton pregnancies. An early ultrasound was performed between 6 + 0/7 and 13 + 6/7 weeks gestational age for accurate pregnancy dating. From this ultrasound, the estimated day of delivery was determined using the ACOG algorithm , 22, whiInfants born to subjects were weighed on platform scales either at a delivery in a health facility, or if born outside of a facility, at the home of the local village elder . For infFor subjects experiencing either a stillbirth or an infant death before the 42-day follow-up period, the assumed cause of death was determined using a previously published algorithm , 25. Estth, 25th, median, 75th, and 90th BW percentiles for 36 and 43 completed weeks gestation were determined, the resulting percentile points were plotted, and curves determined using a cubic spline technique. Percentile curves for gestational ages less than 36 weeks (n = 57) or greater than 43 weeks (n = 9) were not plotted due to paucity of data for these groups.Statistical analyses were performed using JMP software and SAS version 9.4 . The 10A signed rank test was used to quantify the comparison of the percentiles generated in this study with those of the INTERGROWTH-21st study. This test was performed twice within each gestational week – once testing the null hypothesis that the Kenya Male median equals the reported median of the INTERGROWTH-21st Male data, and once testing the null hypothesis that the Kenya Female median equals the reported median of the INTERGROWTH-21st Female data. This analysis was non-directional and performed at the alpha = 0.05 significance level.n = 1189.The consort diagram is shown in Fig. Table n = 1081) delivered in a health facility, while 210 delivered at home. Deliveries were conducted primarily by nurse-midwives (1103); 106 deliveries were conducted by Traditional Birth Attendants, 33 by either self or a family member, and 49 by a physician. The vast majority of birth weights were obtained on day of life (DOL) 0 (91%); 98% were obtained within DOL 0–3.The majority of subjects , either stillbirth or early infant death , are plotted in red, whereas survivors are plotted in black. Causes of death for the live born infants (n = 19) were primarily asphyxia (n = 10) followed by prematurity (n = 3), infection (n = 4), congenital anomalies (n = 1) and unknown (n = 1). Stillbirths (n = 13) were primarily due to asphyxia (n = 10), followed by infection (n = 2) and unknown (n = 1).Figure th, 25th, 50th, 75th, and 90th birth weight percentiles for 36–42 completed weeks of gestation are shown in Table Excluding stillborn and early neonatal death infants, the 10th, 50th, and 90th percentiles for birth weight for the present study and for the INTERGROWTH-21st study; p-values for the signed rank test comparison of medians are also reported in this table. To ensure comparability with the INTERGROWTH-21st data, this comparison uses a subset (n = 1189) of the Kenya population, excluding stillbirths, early infant deaths, and gestational ages < 36 or > 42. Births at 33–35 weeks and 43 weeks gestations were excluded due to paucity in data. Furthermore, due to limited public data for INTERGROWTH-21st, the analysis was limited to single data-point (median) comparisons, which does not address the intricacies of the full fetal growth curve distribution. Although this analysis had limitations the observed significant differences warrant further research with more robust data and methods in the rural, Western Kenyan population.Table st.Our data complements that reported by INTERGROWTH-21st, in that our Kenyan subjects were recruited from a rural agricultural setting with a significant malaria burden, whereas the Kenyan subjects in the INTERGROWTH-21st trial were recruited from the Parklands area of suburban Nairobi. Parklands is considered a middle to upper socioeconomic urban area, while the area of our study is largely defined as a World Bank rural poverty area. Thus, our population demographics are considerably different than those of the Kenyan subjects included in the INTERGROWTH-21st study. Despite these in-country geographical variations, our results, from rural Western Kenya, are quite similar to those reported for INTERGROWTH-21The incidence of prematurity and low birth weight are important public health indicators for a population. In addition, the construction of birth weight percentiles for gestational age are crucial for establishing the intrauterine growth of an infant, which is then used to track postnatal growth. Abnormal postnatal growth, as evidenced by deviations from the expected weight for age percentile that are established by the assessment of birth weight for gestational age, is then used as a warning sign for the need for nutritional intervention. However, it is known that the birth weight distribution curve varies among populations. In addition, despite extensive study, it has been challenging for clinicians and public health practitioners to find accurate prenatal surrogate markers that correlate reliably with actual birthweight , 27, 28.Birth weight and gestational age are intertwined, and it is crucial in evaluating a population to be able to separate low birth weight due to prematurity and low birth weight due to fetal factors, either constitutional or nutritional. Therefore, fetal growth curves with accurate birth weights are important tools for evaluating public health interventions designed to reduce rates of stillbirth , 32, preThe INTERGROWTH-21st study found that for most gestational weeks, median birthweights differed for male and female infants. The signed rank test used for this analysis was performed separately for male and female data but was limited by sample size disparities. Although this analysis had limitations and was based on a single metric (median), the observed significant differences at lower gestational ages for females and higher gestational ages for males warrant further investigation in the rural, western Kenyan population. A major strength of our cohort is the data originated from a prospectively designed study. Subjects were enrolled early in pregnancy as part of the ASPIRIN trial, and tracked prospectively through delivery to 42 days postpartum, as part of our established, population-based maternal newborn health registry, which employs rigorous quality assurance procedures . For thiIn the present study, another strength was our very low loss-to-follow up rate; we lost only 22 subjects (1.56%) to follow-up before birth. Another key strength is the rigorous quality-assurance standards that were utilized to train, monitor, and evaluate the sonographers in this study , 44. TheThere are several limitations of our study. First, the sample size is relatively small, especially for infants born below 36 weeks and of less than 2500 g. Furthermore, we used data only from women who had qualified for and enrolled in the ASPIRIN study, as these women all had accurate ultrasound gestational dating. This limited our subjects to primagravidas, and to singleton pregnancies, consistent with the ASPIRIN eligibility requirements. It is well established that multiple gestation pregnancies are more likely to result in low birthweight infants. Birth spacing can also impact the birthweights of subsequently born sibling infants, as compared to first born infants .Similar to challenges faced by other groups   another As mentioned previously, we had a very low loss-to-follow-up rate, overall. However, an additional 93 subjects, while not lost to follow-up, did not have measured birth weights. Of these, 49 pregnancies ended before 20 weeks and were considered miscarriages. Of the remaining subjects, 27 were stillbirths, 5 were neonatal deaths, and 12 had unknown status. A final challenge faced in this study is the limited precision of the scales used to determine birth weights. In general, these scales were graduated at 50-g increments. Combined with documented “digit preference” bias from other settings, including within the East African region , this laPremature birth continues to be a major problem in sub-Saharan Africa, including Kenya. The comparison of our data, with INTERGROWTH-21st results, found preliminary signals that this rural-dwelling population may have birthweight by gestational age percentiles that differ from that currently reported in global data sources. These results further indicate that continued efforts by clinicians and public health practitioners are needed to develop timely, accurate, effective, acceptable, and feasible evidence-based methods to accurately detect and predict regionally-specific rates of fetal growth restriction and low birthweight , 51, 52.


The present review considers some controversial management practices during extremely premature perinatal transition. We focus on perinatal prevention and treatment of respiratory distress syndrome (RDS) in immature infants. New concerns regarding antenatal corticosteroid management have been raised. Many fetuses are only exposed to potential adverse effects of the drug. Hence, the formulation and the dosage may need to be modified. Another challenge is to increase the fraction of the high-risk fetuses that benefit from the drug and to minimize the harmful effects of the drug. On the other hand, boosting anti-inflammatory and anti-microbial properties of surfactant requires further attention. Techniques of prophylactic surfactant administration to extremely immature infants at birth may be further refined. Also, new findings suggest that prophylactic treatment of patent ductus arteriosus (PDA) of a high-risk population rather than later selective closure of PDA may be preferred. The TREOCAPA trial (Prophylactic treatment of the ductus arteriosus in preterm infants by acetaminophen) evaluates, whether early intravenous paracetamol decreases the serious cardiorespiratory consequences following extremely premature birth. Lastly, is inhaled nitric oxide (iNO) used in excess? According to current evidence, iNO treatment of uncomplicated RDS is not indicated. Considerably less than 10% of all very premature infants are affected by early persistence of pulmonary hypertension (PPHN). According to observational studies, effective ventilation combined with early iNO treatment are effective in management of this previously fatal disease. PPHN is associated with prolonged rupture of fetal membranes and birth asphyxia. The lipopolysaccharide (LPS)-induced immunotolerance and hypoxia-reperfusion-induced oxidant stress may inactivate NO-synthetases in pulmonary arterioles and terminal airways. Prospective trials on iNO in the management of PPHN are indicated. Other pulmonary vasodilators may be considered as comparison drugs or adjunctive drugs. The multidisciplinary challenge is to understand the regulation of pregnancy duration and the factors participating the onset of extremely premature preterm deliveries and respiratory adaptation. Basic research aims to identify deficiencies in maternal and fetal tissues that predispose to very preterm births and deteriorate the respiratory adaptation of immature infants. Better understanding on causes and prevention of extremely preterm births would eventually provide effective antenatal and neonatal management practices required for the intact survival. The preterm birth (PTB) before full 37 weeks duration of pregnancy is the main underlying cause of neonatal deaths, and the predominant risk factor of the respiratory distress syndrome (RDS). Although the introduction of antenatal steroid, surfactant supplementation and other management practices have decreased both the incidence and the fatality of RDS, major morbidities continue to threaten the intact survival . The couafter the birth. However, the importance of the management of early perinatal transition is essential.The duration of pregnancy is the main factor influencing the fetal maturity, including the differentiation of surfactant which has dominant impact on RDS. Spontaneous premature births (SPTB) associate with histologic or clinical chorioamnionitis (CA) and prolonged rupture of fetal membranes (PPROM) . IntrautDuring the 1990s, both antenatal corticosteroid (ACS) therapy for acceleration of the organ maturity and surfactant supplementation for treatment of RDS became common management policy of very premature perinatal transition . AccordiPreviously, nearly all VLGA infants used to receive surfactant shortly after birth. Recently, surfactant treatment has become more selective. The current aim to practice less invasive surfactant administration does not require intubation and improves survival without BPD –19. The Very premature birth often triggers an inflammatory and oxidant storm spreading to multiple organs. In many cases, it is a continuation of the fetal inflammatory syndrome , 21, 22.Research on organ maturation and on regulation of the length of pregnancy may lead to new drug discoveries for the prevention and therapy of RDS. The new candidates include stem cell, exosome, and growth factor therapies. Systematic research on selective treatments toward increasing the defense functions of premature children and very preterm pregnancies is crucial. Progress in the management practices of early perinatal transition will decrease the risk of severe RDS and improve the early recovery from cardiopulmonary distress. This is expected to decrease the risk of BPD, severe multiorgan consequences, and to contribute toward improving neurosensory outcome. In present review, we focus on a few key therapies that are available and require further development.The global risk of the premature birth ranges from 5 to 20% with a mean risk of approximately 11%, or about 15 million PTBs each year. The influence of premature birth to the loss of disability-adjusted life years amounts to 80 million each year . MultiplThe most common cause of prematurity is SPTB . The SPTAccording to the reported incidence figures, there are nearly three million cases of RDS annually , and theMost of the preterm infants, born at 32–36 week, do not develop RDS, apparently due to advanced structural maturity and genetic predisposition to surfactant maturity , whereasIn most cases of SPTB, the labor starts early, without identifiable cause and progresses regardless of the tocolytic therapies. This is by far the most common cause of births at weeks 22–25 . PPROM pChorioamnionitis often presents as a non-infectious inflammation in fetal membranes. Intrauterine infection/ inflammation spreads from the ascending route via maternal blood or through the Fallopian tube, and it may manifest as clinical CA with prominent maternal and fetal symptoms. CA is present in 70–95% extremely premature (gestation < 28 weeks) and in 5–15% of term births . AntibioHereditary factors play significant roles in predisposing to SPTB , and manWNT-4, HSPA1L, and others)  , 37–39.  others) . An actiSERPINA1 gene, which encodes alpha-1 antitrypsin (AAT), contained one of the three rare variants in some individuals affected by SPTB. AAT was localized and expressed in trophoblasts. It localizes within cytoplasmic granules of syncytiotrophoblasts and is secreted to placental intervillous space, potentially contributing to the known increase in serum AAT concentration during pregnancy  . Among sregnancy ,. AAT ising SPTB . These flacentas . AccordiSERPINA1. AAT deficiency is also evident in the airways of RDS infants developing BPD  as it increases the expression of PGR-A and decreases that of PGR-B  secretion does not induce labor. Instead, P4 secretion from villous syncytiotrophoblasts continues to increase during the pregnancy and labor. P4 supplementation has a capacity to prevent SPTB, but only in rare cases of the short cervix syndrome . The actof PGR-B . Certain species .Several anti-inflammatory agents, as inhibitors of proinflammatory mediators, have a potential in prevention of SPTB. These drugs have multiple effects, and the inhibitors of inflammation may perturb the balance of perinatal transition and cause serious adverse effects to the fetus and newborn. A desirable drug both prolongs the duration of a very short pregnancy and promotes the respiratory adaptation without adversely influencing growth or critical differentiation of the brain and other organs. Investigation on causes, consequences, and prevention of very premature birth is becoming a lucrative area of neonatal-perinatal research.Potential adverse long term consequences of ACS in imminent preterm birth must be weighed against the benefits of advancing the fetal maturity and prevention of serious diseases: survival without RDS, intraventricular hemorrhage (IVH), necrotizing enterocolitis (NEC), and possibly BPD. Unfortunately, the follow-up results of ACS therapy in young adults are based upon limited availability of data from randomized trials as they mostly originate from retrospective population-based registers. Increased risk of CP is reported after multiple repeat doses of ACS . Some coAntenatal fluorinated steroids, betamethasone (BM), or dexamethasone (DX) pass the uterine barrier and dose-dependently decrease the growth of the preterm fetus . Growth The debate whether to repeat the ACS dosage continues as more data accumulates . AccordiAnother evidence suggests that even the single ACS dosage used may be excessive. Liggins and Howie used a single dosage of BM  , and in According to Jobe et al., the slow release of i.m. BM-acetate leads to persistence of the drug in circulation . On the The beneficial effect of ACS after threatened premature birth was not evident in a cluster-randomized trial within six moderate-to-low income countries  . ACS treAt present, the optimal ACS drug formulation, dosage, and time of administration remain to be defined. Pharmacodynamic reevaluation, including pharmacokinetic studies, model studies of efficacy, and safety, are needed. An ACS drug formulation with rather fast adsorption rate and a dosage allowing moderate and continuous glucocorticoid exposure of the fetus for not longer than 3–4 days appears desirable. Another challenge is to define biomarkers that accurately predict the duration of the remaining preterm pregnancy and minimize the unnecessary ACS treatments. New randomized trials using a modified ACS-formulation with long-term follow-up appears to be indicated.Surfactant supplementation, together with a combination of new perinatal management practices, has virtually eliminated RDS deaths in infants born around 26 weeks or later and allowed the survival of preemies who are multiple weeks below the survival limit of 28 weeks gestation during 1980s. Nowadays, a large majority of ELGA infants survive in advanced perinatal centers. However, many of them still die of generalized cardiorespiratory failure during the first week despite maximal cardiopulmonary treatment, including surfactant therapy, advanced ventilation practice, and hemodynamic drugs. Insufficient gas exchange, caused by the small surface of the respiratory bronchioles, deficient capillary network, and the long distance between terminal airspaces and capillaries, are factors limiting the survival of extremely immature infants.vs. INSURE or LISA vs. non-invasive continuous airway pressure revealed that LISA was associated with the lowered likelihood of composite death or BPD at 36 weeks’ postmenstrual age   rather trual age . Accordirual age . These rAccording to meta-analysis of randomized trials, prophylactic surfactant treatment shortly after birth had no beneficial effects on survival without BPD compared to early selective treatment . Trials Direct prophylactic surfactant administration to the fetal lung fluid after delivery and before the onset of air breathing offered a striking benefit in experimental prophylactic surfactant treatment of very premature sheep compared to early surfactant administration within less than 1 h after birth in maximally ventilated animals . This obThe delayed cord clamping has been recently re-introduced in management of preterm infants at birth . The oxyIn a study reported in 1948, a detergent that lowered the surface tension was introduced to airways at birth by Gruenwald . After t4-peptide-containing synthetic surfactant aims to capture the surface-adsorption-promoting properties of SP-B , indomethacin and ibuprofen, are prominent soon after birth. They cause dose-dependent and non-specific inhibition of prostaglandin, thromboxane, and prostacyclin synthesis , 99. BesAfter the first evidence of paracetamol (acetaminophen)-induced closure of PDA , multiplParacetamol has a narrow therapeutic window, and the serious hepatic toxicity is a rather common self-inflicted event . The polThe studies on paracetamol for the prophylactic closure of PDA justify further multicenter randomized trials with long term follow-up. The multicenter TREOCAPA trial (Prophylactic treatment of the ductus arteriosus in preterm infants by acetaminophen) is currently recruiting infants, born between weeks 23 to 28 of pregnancy . The i.vLeft-to-right shunt across PDA and its complications are suggested to increase the risk of BPD. Whether the early paracetamol treatment increases survival without serious adverse effects, such as Grade 2-3 BPD, grade 3–4 IVH, or stage 2–3 NEC, remains to be shown. In addition, long term follow-up of the participants of the TREOCAPA trial is required to demonstrate the lack of adverse effects before prophylactic paracetamol may become an accepted therapy.Onset of pulmonary oxygen uptake associates with an acute decrease of high fetal pulmonary arterial resistance shortly after birth. Several regulators, including bradykinin, prostacyclin, and NO, decrease the fetal contractile state of the pulmonary resistance vessels. The NO synthesis is catalyzed by endothelial NO synthetase (eNOS). It activates membrane-bound guanylate cyclase in smooth muscle cells of the resistance vessels, forming the potent vasodilator cyclic GMP (cGMP) . The hal2) despite effective ventilation. Many affected infants respond to surfactant very shortly after birth. Once they develop PPHN, they fail to respond to conventional treatments, often after a grace period for few hours or more. In previous studies, in reporting the iNO-treatment of early PPHN of VLGA infants, nearly all were born after PPROM  generation and oxidative depletion of cofactor tetrahydropteridin (BH4)  by supplementation of N-acetylcysteine (NAC) potentially increases the NO synthesis  . The oxiynthesis , 128.A study of nitrate/nitrite levels in airway specimens from VLGA infants of mothers with severe preeclampsia revealed a consistent deficiency, indicating lack of NO generation . These VDuring the iNO treatment, the small lung volume in PPHN expands as the contractile state decreases. The size of the lung often remarkably expands during the treatment and appears normal in size during the recovery. In most cases of early PPHN, the duration of PPROM and oligohydramnios lasted more than a week, and the fetal compression of the chest likely restricted the lung growth. Severe early neonatal respiratory insult, such as severe RDS or lung hypoplasia, are well-known risk factors of BPD. In the previous study of the 17 iNO-treated VLGA-infants, two died and nine had grade 2 BPD . We antiIn some cases, the patient fails to respond to iNO . The difIn the placebo-controlled iNO-trials involving the VLGA-infants, the indications have ranged from mild to moderate-severe respiratory distress, and only a small minority have been identified as PPHN-like disease . AccordiFurther clinical trials comparing iNO and alternative vasodilator treatments in early PPHN of VLGA infants are indicated. Randomized phase 2 trials involving iNO treatment of the newborn with pulmonary hypertension and another comparison drug include endothelin receptor antagonists (Bosentan) , 133. InNew supplementation treatments or prophylactic treatments may eventually prove to be safe and effective for improving the less satisfactory outcome of the most immature infants. The best scenario would be a drug that may be given to the mother for prevention of extremely preterm labor and delivery and to an extremely premature newborn for early treatment of respiratory distress. Glucocorticoid supplementation is extensively used before and after birth. Critical pharmacodynamic studies are required for defining the effective and least damaging dosage. Respiratory distress in extremely preterm infants presents as a multiorgan failure that requires integrated cardiopulmonary management strategy. Less invasive surfactant treatment and non-invasive ventilation with caffeine supplementation is a preferred treatment strategy in most cases. However, it may not be successful for the most immature infants and for the rare cases of complicated and severe respiratory distress. For the most immature infants, surfactant may be given as soon as possible at birth, followed by fast and thoughtful weaning from mechanical to non-invasive ventilation. Despite this, how to further protect against injuries and promote healing and growth remains a challenge. Failure of less invasive strategy requires rapid diagnostics and modification of treatment. Establishing the preferred ventilation mode and settings, management of perfusion, avoiding excess of fluid intake, limiting excessive left-to-right shunts, and, if necessary, establishing effective pulmonary vasodilator treatment via the airways need to be included in the strategy. Rapid weaning from effective and at the same time damaging therapies is a priority. Lastly, new medications that strengthen the immature or dysfunctional defenses throughout the very premature perinatal transition, prolonging very premature pregnancies, and further decreasing respiratory distress and serious multi-organ consequences may provide the next leap in neonatal—perinatal medicine.MH conceptualized, discussed with collaborators and wrote the manuscript. ER collected and analyzed the data and contributed to conceptualization and writing. TS proposed and discussed the topics contributed to the writing. RM contributed to the conceptualization, discussion and writing of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.