ABSTRACT Introduction Despite extensive efforts to identify effective treatments for COVID‐19, preventive strategies remain essential. Bromhexine hydrochloride (BRH), an over‐the‐counter mucolytic with potential TMPRSS2 inhibition, may reduce viral entry and thus serve as a prophylactic agent. Methods A retrospective questionnaire‐based study included 125 individuals reporting prophylactic BRH use. Demographic and clinical data were collected. COVID‐19 infection rates before and after BRH use were compared. Subgroup analyses were performed by vaccination status, prior infection, and duration of prophylaxis. Results Participants had a mean age of 56.5 years and a mean BMI of 25.6; 20% were vaccinated. Prior to BRH use, 62.4% reported COVID‐19 infection, compared to 11.2% after prophylaxis (p < 0.0001). Significant reductions were observed in both vaccinated (48.1% vs. 3.7%, p = 0.002) and unvaccinated individuals (66.3% vs. 13.3%, p < 0.0001). Prior infection did not significantly influence outcomes (p = 0.9248). Longer BRH use was associated with lower reinfection rates, decreasing from 33.3% (≤ 10 days) to 2.6% (≥ 30 days) (p = 0.003). Conclusions BRH prophylaxis was associated with reduced reported COVID‐19 infection rates, independent of vaccination or prior infection, with greater benefit observed with longer use. Prospective controlled studies are required to confirm these findings.

Understanding how chromosome 21 gene dosage contributes to neurodevelopmental phenotypes in trisomy 21 (T21) remains a fundamental challenge. Here, we perform transcriptome-wide RNA-sequencing of fetal cortical and hippocampal tissues from T21 cases and euploid controls collected during mid-gestation, a critical window for human brain development. We identify widespread gene expression dysregulation with significant enrichment for chromosome 21 genes and perturbation of neurodevelopmental, synaptic, and immune-related pathways. Among the most strongly dysregulated genes is ADARB1, a chromosome 21-encoded RNA editing enzyme, whose overexpression associates with increased adenosine-to-inosine RNA editing, with consistent over-editing at functionally important recoding sites in glutamate and GABA receptor-related genes, including GRIK2, GRIA2, GRIA3, and GABRA3, across cortex and hippocampus. Meta-analyses across independent transcriptomic datasets validate robust chromosome 21 dosage effects, including ADARB1 overexpression and over-editing at 3'UTRs and GRIA3. These findings implicate dysregulated RNA editing as a post-transcriptional mechanism contributing to fetal neuropathology in T21.

OBJECTIVE:The pericoronal dental follicle is a connective tissue structure encased in epithelium present around the developing tooth germ before erupting and emerging in the oral cavity. Stem cells from pericoronal follicles can be obtained and cultured under suitable conditions in vitro. This study builds upon our previous research, with the primary objective of identifying stem cell subpopulations in the oral cavity based on marker expression and differentiation potential. MATERIALS AND METHODS:Dental follicle stem cells (DFSCs) were obtained and cultured under standard cell culturing conditions. Cellular phenotype and the expression of stem cell and tissue markers were analyzed using high-throughput fluorescent analysis. Additionally, the cells were cultured for 10 days in differentiation media to assess their osteogenic, adipogenic, and chondrogenic differentiation potential in vitro. Stem cell differentiation was evaluated using Alcian Blue, Oil Red O, and Alizarin Red staining. RESULTS:The stem cells isolated by the research team showed positive fluorescent expression for epithelial markers and mesenchymal markers (CD44H, CD71, CD90). High-content cellular analysis enabled the quantitative profiling of DFSC marker expression. Under appropriate conditions, DFSCs demonstrated the ability to differentiate into osteoblast-like, chondroblast-like, and adipocyte-like cells in vitro. CONCLUSION:The dental follicle contains a population of stem cells with a specific phenotype capable of multilineage differentiation. It may represent another promising source of human adult stem cells. The observed differentiation profile may limit the application of the isolated cells in the hard tissue regeneration and suggest a potentially more suitable role in soft tissue engineering or immunomodulatory therapies.

INTRODUCTION:Human pregnancy requires acceptance and support for the semi-allogeneic embryo and effective protection of both mother and fetus. A failure to adapt, from either side, may cause abortion. The placenta-derived extracellular vesicles (EVs) have a crucial role in human implantation and pregnancy. These are lipid bilayer membrane-delimited, nano-to-micro sized extracellular microvesicles of endosomal origin, containing diverse signaling molecules, and functioning as short and long-distance messengers. We have already shown that first-trimester placenta releases the soluble HLA-C and HLA-G KIR ligands to modulate maternal cytotoxicity via the KIR/HLA axis. This study is to find whether extravillous trophoblast (EVT) secretes these HLA class I molecules via small EVs. METHODS:sEVs were isolated by ultrafiltration or precipitation from serum-free conditioned media from primary trophoblast-derived EVT, and non-tumor EVT-like model Sw71 cell line, cultured as monolayer and spheroids. sEVs from cultured placental explants served as a positive control. Combined data from several methods was used for their characterization including BCA, DLS, TEM, IEM, Dot blot, and FACS. RESULTS:Primary trophoblast-derived EVT and Sw71 EVT-like cells produced intact and well-visible CD63+, HLA-G- and HLA-C-bearing sEVs, regardless of culture mode and type of isolation. Both methods yielded sEVs sized 30-100 nm. DISCUSSION:We show original data on the HLA-C secretion via sEVs by early pregnancy EVT and confirm the production of HLA-G-positive sEVs. A new asset to the usefulness of the Sw71 spheroid model as an implanting blastocyst surrogate is added as a tool to elucidate the sEV-based signalization in the implantation.

In a series of publications, we attempt to demonstrate the prophylactic role of bromhexine hydrochloride (BHH) and the curative role of high colchicine doses against COVID-19. Effects of long-term prophylaxis with BHH are present with two cases of elderly women with severe comorbidity. After long-term prophylactic use of BHH from 3 to 6 months, one did not fall sick from COVID-19 and the other passes it almost asymptomatically, even though all their family members fell ill. The dramatic healing effect of colchicine was demonstrated in a family in which both spouses became ill with COVID-19 almost simultaneously. They are treated in different hospitals, and where colchicine is administered in high doses, the patient recovered. The other patient developed complications and died on standard treatment without high doses of colchicine. An outpatient accidentally overdosed with colchicine (12.5mg). The therapy was stopped immediately. Clinically, on the third day, there are absolutely no symptoms. These cases re-affirm the prophylactic effect of BRH and the curative effect of high-dose colchicine.

Neonatal infection with herpes simplex virus (HSV) is associated with significant morbidity, high mortality, and long-term neurological sequelae. We report the clinical case of an infant with HSV encephalitis, where the initial diagnosis was established based on cranial ultrasound (CUS) findings. These findings revealed localized, asymmetrically distributed hyperechoic areas in the parenchyma and signs of brain swelling. CUS dynamics on days 7 and 14 after the onset of clinical symptoms demonstrated multiple subcortical and perivascular zones of encephalomalacia in the right hemisphere, accompanied by ex vacuo ventricular dilatation. The cerebellum, left basal ganglia, and left hemisphere appeared to be less affected by the pathological process. We report the clinical case of an infant with HSV encephalitis whose initial diagnosis was based on the results of cranial ultrasound with localized, asymmetrically distributed hyperechoic areas in the parenchyma and signs of brain swelling. CUS dynamics at 7th and 14th day revealed multiple subcortical and perivascular zones of encephalomalacia. image

Oral cavity stem cells (OCSCs) have been the focus of intense scientific efforts due to their accessibility and stem cell properties. The present work aims to compare the different characteristics of 6 types of dental stem cells derived from the oral cavity: dental pulp stem cells (DPSC), stem cells from human exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSC), stem cells from the apical papilla (SCAP), bone marrow mesenchymal stem cells (BMSC), and gingival mesenchymal stem cells (GMSC). Using immunofluorescence and real-time polymerase chain reaction techniques, we analysed the cells for stem cell, differentiation, adhesion, and extracellular matrix markers; the ability to proliferate in vitro; and multilineage differentiation potential. Markers such as vimentin, CD44, alkaline phosphatase, CD146, CD271, CD49f, Oct 3/4, Sox 9, FGF7, nestin, and BMP4 showed significant differences in expression levels, highlighting the heterogeneity and unique characteristics of each cell type. At the same time, we confirmed that all cell types successfully differentiated into osteogenic, chondrogenic, or adipose lineages, with different readiness. In conclusion, our study reveals the distinct properties and potential applications of various dental-derived stem cells. These findings contribute to a deeper understanding of OCSCs and their significance in future clinical applications.

Objective: To investigate the influence of citric acid on the osteogenic and angiogenic potential of stem cells from apical papillae (SCAPs). Materials and methods: Stem cells from apical papillae were isolated from freshly extracted third permanent molars. These cells were treated with 20 and 100 μM citric acid. Alizarin red staining was used to evaluate mineral deposition. The secreted levels of vascular endothelial growth factor (VEGF) were assessed by ELISA on days 18, 24 and 28. Immunofluorescence analysis was performed to assess the expression of surface markers after exposure to 20 and 100 μM citric acid. Results: Different mineralisation patterns were observed. Supplemented with citric acid, media showed more diffuse and less dense crystals. On day 18, most VEGF was secreted from the cells with no added citric acid. On day 24, there was a significant increase (p < 0.05) in the levels of VEGF secreted from cells treated with 20 μM citric acid. On day 28, cells from the control group did not secrete VEGF. There was a reduction in the levels of VEGF secreted by cells treated with 20 μM citric acid and a significant increase in the cells exposed to 100 μM citric acid (p < 0.05). Conclusion: Citric acid can promote the differentiation of SCAPs and angiogenesis.

Background. KL-6 is a biomarker of interstitial lung injury and increases during repair. Aim. Our aim was to determine the predictive value of plasma KL-6 for the development of bronchopulmonary dysplasia (BPD) in preterm infants. Methods. Ninety-five extremely preterm infants (EPIs), born at <28 gestational age (GA), were divided into two main BPD groups as follows: the moderate/severe and the no/mild group. KL-6 was analyzed on days 7 and 14. Binary logistic regression analyses and ROC curve analyses were performed. Results. Infants <26 + 0 weeks’ GA have higher mean KL-6 than infants >25 + 6 weeks’ GA on 7 and 14 days (335 vs. 286 U/ml and 378 vs. 260 U/ml; p=0.005 and 0.018, respectively). In the binary regression model at KL-6 day 7, three of the prognostic factors remained significant—mechanical ventilation OR: 10.38 (95% CI: 3.57–30.14), PDA OR: 6.39 (95% CI: 0.87–46.74), and KL-6 OR: 4.98 (95% CI: 1.54–16.08). The AUC was 0.86 with a sensitivity and specificity of 79% at a cutoff value ≥0.34. In the binary regression model at KL-6 day 14, six of the prognostic factors were significant—PDA OR: 23.34 (95% CI: 2.14–254.24), KL-6 OR: 13.59 (95% CI: 3.19–57.96), GA OR: 4.58 (95% CI: 1.16–18.06), mechanical ventilation OR: 4.45 (95% CI: 1.23–16.16), antenatal steroids OR: 0.19 (95% CI: 0.04–0.95), and gender (female OR: 0.30 (95% CI 0.08–1.12)). The AUC was 0.91, and the sensitivity and accuracy for a cutoff ≥0.37 were 89% and 85%, respectively. Conclusion. KL-6 could be a useful screening biomarker for early detection of infants at increased risk for developing BPD.

The exact role of both conventional αβT and unconventional γδT-cells in normal and pathological pregnancies is yet not fully clarified. However, T-cell mediated homeostasis may be a key feature for establishment of maternal immune tolerance and is crucial for the fetal survival. Unconventional γδT-cells have T-cell receptor built by gamma and delta chains, but also share common cytotoxic features with NK-cells, working on the innate/adaptive immunity boundary, maintaining the tissue homeostasis, and responding to damage, infection, inflammation, and malignancy in an MHC-unrestricted manner. In a previous study we found an enrichment of activated and terminally differentiated pro-inflammatory γδT-cells at maternal-fetal interface (MFI, decidua) during early pregnancy, while there were no significant changes in their counterparts in the blood. Here we reveal and compare cytotoxic potential of γδ and αβT-cell subsets in human pregnancy by expression of perforin, granzyme-A and granulysin. We used decidual and blood samples from healthy women in early (n=16) and term pregnancy (n=13) as well as blood from nonpregnant women (n=11). Decidual and peripheral blood mononuclear cells were isolated and processed to FACS to distinguish the αβT and γδT-cell subsets. Cytotoxic potential was evaluated as percentage of the perforin-, granzymeA- or granulysin-positive cells within αβT or γδT cells in comparative manner as follows: into decidua vs. blood (paired samples, early pregnancy); into decidua early vs. term pregnancy and in the blood of pregnant vs. nonpregnant women (early pregnancy). Overall we observed higher cytotoxic potential of γδT-cell subset as compared to αβT-cells at MFI and in the blood of pregnant and non-pregnant women. No significant changes were observed of perforin-positive cells in both subsets at MFI in early vs late pregnancy and in the blood of pregnant vs non-pregnant women. However, the perforin-positive cells within γδT-cells declined significantly at MFI compared to the paired blood. GranzymeA-positive cells, although low in number within αβT cells were doubled at MFI in early than in late pregnancy and comparable in the blood of pregnant and non-pregnant women. TCRγδ cells contained high and stable portion of granzymeA-positive cells regardless their localization and pregnancy status. Interestingly, we observed decidua-specific increase of granulysin-positive cells within both subsets early in pregnancy. During early human pregnancy we found decidua-specific 1) increase of granzymeA-positive cells within αβT cells; 2) decrease of perforin-positive cells within γδT cells and 3) high number of granulysin-positive cells within both subsets. We could conclude that early pregnancy is associated with higher cytotoxic potential of γδT-cells but "impaired" local spontaneous cytotoxicity, due to decreased perforin production. Term pregnancy is characterized by notable decrease of the T-cell cytotoxic potential combined with stable low perforin production. The higher cytotoxic potential of γδT cells is probably due to their intrinsic propensity to cytotoxicity which, however, needs to be precisely modulated at MFI.

Maternal immune cells must tolerate fetal allo- antigens to establish a healthy pregnancy and remain competent to respond to infections both systemically and in placental tissues. However, the semi-allogeneic embryo is normally accepted, indicating the presence of active mechanisms of immune tolerance and/or fetus strategy to avoid an attack by the maternal immune system. Such a strategy is the lack of HLA class I molecules expression by villous trophoblasts and their atypical expression on extravillous trophoblasts (EVTs) which invade uterine tissues contacting with maternal immune cells. EVTs are the most invasive cells of extra-embryonic origin and express the invariant non-classical HLA class I molecules HLA-G and HLA-E, and the only classical, polymorphic HLA class I molecule HLA-C. Recognition of paternally inherited HLA-C could initiate allo-responses from maternal T and NK cells and the HLA-C cell surface expression levels influence how this response is shaped. We and others have shown expression of HLA-C by EVTs in situ at maternal-fetal interface during early pregnancy. Here we showed the expression of HLA-C molecule by 3D native trophoblast models and by in vitro 3D Sw71 spheroids, resembling human blastocyst during the peri-implantation period. Generated Sw71 spheroids were functional proved by their ability to attach to the epithelial endometrial cells, to migrate and to invade into ECM. We used human first trimester trophoblast cells (Swan 71) and generated stable and functional trophoblast Sw71 spheroids. Early placenta samples obtained from healthy pregnant women directed to elective abortions (6–12 gw, n = 10) were used for preparation of trophoblasts explants. 3D Sw71 spheroids and 2D Sw71 monolayers were cultured in cell culture chambers for 48 hrs. In order to obtain EVTs the primary trophoblast-derived explants were cultured in the same way for 72 hrs. Some of the EVT cultures were propagate for 7 days for spontaneous aggregation and self-organization of spheroid-like structures. Sw71 spheroids and monolayers, as well as expanded EVTs and EVTs-derived spheroids were stained for HLA-C. Both Sw71 monolayers and functional 3D Sw71 spheroids were positive for HLA-C. In line with our findings about HLA-C-positive EVTs in situ at maternal-fetal interface we detected strong HLA-C expression by freshly expanded EVTs. Moreover, EVTs-derived self-organized spheroid-like structures as natural models of human placenta migrated as well and retain intense HLA-C expression. The strong HLA-C expression by 3D Sw71 spheroids which is detected in comparison with primary trophoblast 3D cultures confirm their applicability as models of human blastocyst surrogates to study trophoblasts-maternal immune cells interactions during human implantation.

PROBLEM:Human implantation is a limiting factor for the success of natural and IVF reproduction since about 60% of pregnancy losses occur in the peri-implantation period. The in vitro modeling of human implantation challenges the researchers in accurate recreation of the complex in vivo differentiation and function of human blastocyst in the peri-implantation period. In previous studies, we constructed Sw71-spheroid models, which like human blastocyst undergo compactization, attaches to the endometrial epithelium, invade, and migrate. The aim of this study was to validate the trophoblast Sw71-spheroid model with primary trophoblast cells, derived from healthy women in early pregnancy.METHOD OF STUDY:We performed a direct comparison of Sw71-spheroid model with placenta-derived primary trophoblasts regarding their hybrid phenotype and HLA status, as well as the ability to generate spheroids able to migrate and invade. From the primary trophoblast cells, isolated by mild enzymatic treatment and Percoll gradient separation, were generated long-lived clones, which phenotype was assessed by FACS and immunocytochemistry.RESULTS:Our results showed that cultured primary trophoblasts have the EVT phenotype (Vim+/CK7+/HLA-C+/HLA-G+), like Sw71 cells. In both 3D culture settings, we obtained stable, round-shaped, multilayered spheroids. Although constructed from the same number of cells, the primary trophoblast spheroids were smaller. The primary trophoblast spheroids migrate successfully, and in term of invasion are equally potent but less stable as compared to Sw71 spheroids.CONCLUSIONS:The Sw71 cell line and cultured native trophoblast cells are interchangeable regarding their EVT phenotype (HLA-C+/HLA-G+/Vim+/CK7+). The blastocyst-like spheroids sourced by both types of cells differentiate in the same time frame and function similarly. We strongly advise the use of Sw71 spheroids as blastocyst surrogate for observation on trophectoderm differentiation and function during early human implantation.

Maternal immune cells must tolerate fetal allo- antigens to establish a healthy pregnancy and remain competent to respond to infections both systemically and in placental tissues. However, the semi-allogeneic embryo is normally accepted, indicating the presence of active mechanisms of immune tolerance and/or fetus strategy to avoid an attack by the maternal immune system. Such a strategy is the lack of HLA class I molecules expression by villous trophoblasts and their atypical expression on extravillous trophoblasts (EVTs) which invade uterine tissues contacting with maternal immune cells. EVTs are the most invasive cells of extra-embryonic origin and express the invariant non-classical HLA class I molecules HLA-G and HLA-E, and the only classical, polymorphic HLA class I molecule HLA-C. Recognition of paternally inherited HLA-C could initiate allo-responses from maternal T and NK cells and the HLA-C cell surface expression levels influence how this response is shaped. We and others have shown expression of HLA-C by EVTs in situ at maternal-fetal interface during early pregnancy. Here we showed the expression of HLA-C molecule by 3D native trophoblast models and by in vitro 3D Sw71 spheroids, resembling human blastocyst during the peri-implantation period. Generated Sw71 spheroids were functional proved by their ability to attach to the epithelial endometrial cells, to migrate and to invade into ECM. We used human first trimester trophoblast cells (Swan 71) and generated stable and functional trophoblast Sw71 spheroids. Early placenta samples obtained from healthy pregnant women directed to elective abortions (6–12 gw, n = 10) were used for preparation of trophoblasts explants. 3D Sw71 spheroids and 2D Sw71 monolayers were cultured in cell culture chambers for 48 hrs. In order to obtain EVTs the primary trophoblast-derived explants were cultured in the same way for 72 hrs. Some of the EVT cultures were propagate for 7 days for spontaneous aggregation and self-organization of spheroid-like structures. Sw71 spheroids and monolayers, as well as expanded EVTs and EVTs-derived spheroids were stained for HLA-C. Both Sw71 monolayers and functional 3D Sw71 spheroids were positive for HLA-C. In line with our findings about HLA-C-positive EVTs in situ at maternal-fetal interface we detected strong HLA-C expression by freshly expanded EVTs. Moreover, EVTs-derived self-organized spheroid-like structures as natural models of human placenta migrated as well and retain intense HLA-C expression. The strong HLA-C expression by 3D Sw71 spheroids which is detected in comparison with primary trophoblast 3D cultures confirm their applicability as models of human blastocyst surrogates to study trophoblasts-maternal immune cells interactions during human implantation.

An efficient immune defense against pathogens requires sufficient basal sensing mechanisms that can deliver prompt responses. Type I IFNs are protective against acute viral infections and respond to viral and bacterial infections, but their efficacy depends on constitutive basal activity that promotes the expression of downstream genes known as IFN-stimulated genes (ISGs). Type I IFNs and ISGs are constitutively produced at low quantities and yet exert profound effects essential for numerous physiological processes beyond antiviral and antimicrobial defense, including immunomodulation, cell cycle regulation, cell survival, and cell differentiation. Although the canonical response pathway for type I IFNs has been extensively characterized, less is known regarding the transcriptional regulation of constitutive ISG expression. Zika virus (ZIKV) infection is a major risk for human pregnancy complications and fetal development and depends on an appropriate IFN-β response. However, it is poorly understood how ZIKV, despite an IFN-β response, causes miscarriages. We have uncovered a mechanism for this function specifically in the context of the early antiviral response. Our results demonstrate that IFN regulatory factor (IRF9) is critical in the early response to ZIKV infection in human trophoblast. This function is contingent on IRF9 binding to Twist1. In this signaling cascade, Twist1 was not only a required partner that promotes IRF9 binding to the IFN-stimulated response element but also an upstream regulator that controls basal levels of IRF9. The absence of Twist1 renders human trophoblast cells susceptible to ZIKV infection.

Human γδ T cells are enriched at the maternal-fetal interface (MFI, decidua basalis) showing a highly differentiated phenotype. However, their functional potential is not well-known and it is not clear whether this decidua-enrichment is associated with specific γδ T cell receptors (TCR) as is observed in mice. Here we addressed these open questions by investigating decidual γδ T cells during early and late gestation, in comparison with paired blood samples, with flow cytometry (cytotoxic mediators, cytokines) and TCR high-throughput sequencing. While decidual γδ T cells expressed less perforin than their counterparts in the blood, they expressed significant more granulysin during early pregnancy. Strikingly, this high granulysin expression was limited to early pregnancy, as it was reduced at term pregnancy. In contrast to this granulysin expression pattern, decidual γδ T cells produced reduced levels of IFNγ and TNFα (compared to paired blood) in early pregnancy that then increased by term pregnancy. TCR repertoire analysis indicated that human decidual γδ T cells are not generated early in life as in the mouse. Despite this, a specific enrichment of the Vγ2 chain in the decidua in early pregnancy was observed that disappeared later onwards, reflecting dynamic changes in the decidual γδ TCR repertoire during human gestation. In conclusion, our data indicate that decidual γδ T cells express a specific and dynamic pattern of cytotoxic mediators, Th1 cytokines and TCR repertoire suggesting an important role for these unconventional T cells in assuring a healthy pregnancy in human.

In healthy couples over half of the conceptions result in failed pregnancy and around 30% of them occur during implantation defining it as a rate-limiting step for the success of native and in vitro fertilization. The understanding of the factors regulating each step of implantation and immune recognition is critical for the pregnancy outcome. Creation of 3D-cell culture models, such as spheroids and organoids, is in the focus of placental tissue engineering in attempt to resemble the in vivo complexity of the maternal-fetal interface and to overcome the need of laboratory animals and human embryos. We constructed stable, reliable, and reproducible trophoblast Sw71 spheroids which are functional independently of the serum level in the culture media. These models resemble the hatched human blastocyst in size, shape and function and are useful for in vitro studies of the in vivo concealed human implantation. Since Sw71 spheroids produce HLA-C, the only classical MHC molecule indispensable for establishment of the immune tolerance and proper human implantation, they are applicable for the evaluation not only of implantation itself but also of maternal-trophoblasts immune interactions. In addition, Sw71-blastocyst-like spheroids are manipulable in low-volume platform, easy to monitor and analyze automatically under treatment with favorable/detrimental factors.

PURPOSE:Lung ultrasound (LUS) is a widely used technique in neonates to diagnose a lot of acute diseases. However, its role in the diagnosis of chronic lung conditions is still to be evaluated. We aimed to describe the changes in ultrasound images as well as the appearance of lung consolidations (LC) in very low birth weight (VLBW) infants with and without bronchopulmonary dysplasia (BPD). METHODS:For a period of 2 years we prospectively examined 124 VLBW infants, divided into two main groups: below 28 gestational weeks with two subgroups-BPD (moderate and severe) and non-BPD, and 28-32 gestational weeks with two subgroups-BPD (moderate and severe) and non-BPD group. We performed weekly LUS until 36 weeks' postmenstrual age (PMA). On the seventh postnatal day, we calculated the LUS score as a semiquantitative score that represents the aeration (0-3) in three different areas (upper anterior, lower anterior, lateral) of each lung. RESULTS:We performed around 750 LUS exams. The BPD groups had LUS scores above 8 on the seventh day, p < 0.001. The number of LC was higher in the BPD groups 14 (3-45) than in the mild or non-BPD groups 2.5 (0-6), p < 0.001, and the difference was significant for the period between 1 week and 1 month after birth, p = 0.001. In the BPD group <28 weeks' PMA, LC appeared equally in the anterior, lateral, and posterior fields, while in the BPD group 28-32 weeks' PMA, LC are more typical for the anterior-lateral fields. CONCLUSION:Serial LUS exams and identification of LC could be early predictors of moderate and severe forms of BPD. LUS score >8 on the seventh postnatal day is an early predictor of severe BPD. LC in more lung fields are typical for the severe forms of the disease.

BACKGROUND: Based on the last update of European Consensus Guidelines on Management of Respiratory Distress Syndrome, the following study is new for our clinic approach for minimally invasive respiratory support in preterm infants. AIM: The aim is to find out if the implementation of several minimally invasive procedures leads to a reduction of the frequency and severity of chronic complications and improved outcomes in extremely premature infants. MATERIALS AND METHODS: Infants are below 30 gestational weeks, divided into two groups – therapeutic – 37 infants on standardized early respiratory management protocol which includes: High-flow continuous positive airway pressure (20l/min ≥ 15 PEEP) in the delivery room, support of spontaneous breathing and non-invasive mechanical ventilation, avoidance of hypothermia and LISA shortly after birth, and control group – 46 infants that received standard respiratory support (positive pressure ventilation – invasive and non-invasive in the delivery room, most infants ≤ 27 weeks gestational age were intubated and received early surfactant, extubation “when being ready” – usually after few days). RESULTS: The duration of mechanical ventilation, oxygen therapy, and hospital stay is shorter in the therapeutic group (p < 0.05). Severe bronchopulmonary dysplasia is not found in the interventional group, 26% of the patients in the control group have severe form of the disease (p = 0.001). Severe intraventricular hemorrhages are found in 11% of the infants in the therapeutic group and 28% in the control group (p = 0.06). CONCLUSION: Due to the changed protocol, we report increased survival of ELBW infants without severe chronic complications. The acute pulmonary injury, acquired in the perinatal period, is directly connected with the development of BPD. For this reason, all the changes that we introduced in our clinic (heat management, support of spontaneous breathing, “open up” lungs – high flow PEEP/CPAP, and LISA during spontaneous breathing) contribute to the lower frequency of severe chronic complications and high percentage of ELBW infants, who do not develop severe BPD.