Exacerbation‐prone asthma is a feature of severe disease. However, the basis for its persistency remains unclear.To determine the clinical and transcriptomic features of frequent exacerbators (FEs) and persistent FEs (PFEs) in the U‐BIOPRED cohort.We compared features of FE (≥2 exacerbations in past year) to infrequent exacerbators  and of PFE with repeat ≥2 exacerbations during the following year to persistent IE (PIE). Transcriptomic data in blood, bronchial and nasal epithelial brushings, bronchial biopsies and sputum cells were analysed by gene set variation analysis for 103 gene signatures.CEACAM5) was the only differentially expressed transcript in bronchial biopsies between PE and IE. There were no differentially expressed genes in the other four compartments. There were higher expression scores for type 2, T‐helper type‐17 and type 1 pathway signatures together with those associated with viral infections in bronchial biopsies from FE compared to IE, while there were higher expression scores of type 2, type 1 and steroid insensitivity pathway signatures in bronchial biopsies of PFE compared to PIE.Of 317 patients, 62.4% had FE, of whom 63.6% had PFE, while 37.6% had IE, of whom 61.3% had PIE. Using multivariate analysis, FE was associated with short‐acting beta‐agonist use, sinusitis and daily oral corticosteroid use, while PFE was associated with eczema, short‐acting beta‐agonist use and asthma control index. CEA cell adhesion molecule 5  and persistent frequent exacerbator (PFE) are associated with poorer asthma control compared to infrequent exacerbator (IE) and persistent IE, respectively.CEACAM5 was the differentially‐expressed transcript in bronchial biopsies in FE compared to IE.In FE and persistent FE, there was an increase in the expression of type 1 and type 2 inflammatory pathways. They were asked the number of exacerbations that needed systemic corticosteroid therapy they had experienced in the previous year. Patients were classified as either FE (two or more exacerbations in the past year) or IE (a maximum of one exacerbation in the past year). At a longitudinal visit at 1 year, asthma patients who attended had the baseline investigations repeated, and clinical data were collected, including clinical symptoms and severe exacerbations during the past year. The persistence of the exacerbation phenotype was analysed by classifying those patients who were IE at both visits  and those who were FE at both visits .2.2RNA from baseline blood samples was isolated using the PAXgene Blood RNA kit  with on‐column DNase treatment . Total RNA was extracted from nasal and bronchial biopsies, epithelial brushings and sputum cells using the miRNeasy mini kit .® Human Genome U133 Plus 2.0 Array . Pathway analysis, enrichment analysis and functional clustering of differentially expressed genes were performed as described previously, and protein interaction analysis using annotated protein‐coding genes was performed by STRING version 10.0 .www.somalogic.com) and reported as relative fluorescence units, cross‐plate calibrated and median normalised.Expression profiling of the transcriptome was performed using GeneChip2.3http://software.broadinstitute.org/gsea/msigdb) or from published studies10 fold‐change of 2.0 and a p value <0.05.Gene set variation analysis (GSVA) was used to calculate sample‐wise enrichment scores (ESs)2.4p<0.05 were modelled in a joint multivariate logistic regression model. Three categories of smoking, namely, nonsmoker, ex‐smoker and current smoker, were coded using indicator variables to avoid any assumptions of a dose effect. Backward step‐wise regression was applied using the Akaike information criterion (AIC).Clinical variables were summarised as the mean ± standard deviation when continuously symmetrically distributed, as the median and interquartile range when skewed and as their frequencies (proportion) when categorical. Between‐group comparisons were performed with the Mann–Whitney U test or chi‐square tests, as appropriate. Variables with raw All clinical as well as transcriptomic and proteomic data sets were uploaded into tranSMART, an open‐source knowledge management platform.33.1n = 264; 62.9%) had a greater proportion of female participants  and earlier age of diagnosis  than IE , but there were more current smokers in IE than in FE (Table p = 0.018) and eczema  were more prevalent in the FE group. FE had higher daily short‐acting β‐agonist (SABA) usage  and greater symptom scores  at baseline, but there was no difference in lung function parameters. Serum IgE was higher in IE .In total, 420 participants with severe asthma were studied at baseline. FE  score, Asthma Quality of Life Questionnaire (AQLQ) score, Hospital Anxiety and Depression Scale (score) and sleep score (ESS) score, were removed from consideration. The final model resulted from applying backward step‐wise regression on 17 variables, for which 302 patients had complete data. Variables retained in the model included being an ex‐smoker (compared to nonsmokers and current smokers combined), SABA use, diagnosis of sinusitis and age of onset  (Table Those variables with 5) Table .3.2p<0.001) in the PFE group, and the age of onset was earlier  than in the PIE group (Table p = 0.002) and osteoporosis  was also greater in the PFE group, and they had a greater use of maintenance OCS, SABA and xanthines. PFE were more symptomatic with worse AQLQ and ACQ scores. Levels of serum IgE, exhaled NO and blood eosinophil counts were lower in PFE, but blood neutrophil counts were not significantly different . Current smokers had an estimated reduced risk of being a PFE, possibly due to reverse causality , FeNO and ACQ 5 score  were differentially‐enriched in FE compared to IE Figure . No sign(ii)Innate and adaptive immune system‐associated signaturesTwo gene signatures comprising genes whose expression was previously implicated in bronchial biopsies of chronic obstructive pulmonary disease (COPD) patients treated with fluticasone and salmeterolIn sputum, signatures of downregulated genes after stimulation of macrophages by Interferonγ (IFNγ), lipopolysaccharide and Tumor Necrosis Factor/Prostaglandin E2/Toll‐like receptor 2 (TNF/PGE2/TLR2) activation were least enriched in FE Inflammation‐ and fibrosis‐associated signaturesIn bronchial biopsies, there was a greater enrichment of a gene signature associated with the differentiation of the Innate Lymphoid Cell 3 (ILC3) subset of innate lymphoid cells  and sputum (downregulated) in FE , while 61.3% of IEs remained IEs (persistent IEs), indicating that the FE phenotype as well as the IE phenotype was unstable. Persistent FE was more likely to be female, with an earlier onset of asthma, more commonly reported eczema, greater SABA use and OCS use, more symptoms and worse quality of life parameters. Contrary to what might be expected, FeNO and blood eosinophil counts were lower in the PFE group, and this could be explained by the greater OCS use in this group. Multivariate logistic regression analysis confirmed the association with the presence of eczema, high SABA use and higher mean ACQ5. Interestingly, being a current smoker was associated with a lower risk of PFE.Haemophilus influenzae.To understand the underlying pathways that may be associated with FEs, we compared the transcriptomic data of patients with FE to those with IE in various compartments, including blood, sputum, bronchial brushings and nasal brushings. We only found one differentially‐expressed gene between the two groups, with CEACAM5 expression in bronchial biopsies being more highly expressed in FE compared to IE. CEACAM5 is a member of the carcinoembryonic gene family, involved in cell signalling, cell proliferation, cell repair processes and maintenance of the bronchial epithelium, with evidence that this receptor is exploited by airway pathogens such as CEACAM5 is also one of the most highlyranked hub genes in the bronchial epithelium of patients with asthma.Nasal brushings also show enrichment for signatures associated with activated neutrophils and monocytes as well as for macrophages activated by IFNγ, LPS or TNF/PGE2/TLR2 exposure. Interestingly, the macrophage upsignatures were enriched in nasal brushings, while the downsignatures were less expressed in sputum. Together with the significant enrichment of the Th1‐associated multiple sclerosis peripheral blood mononuclear cell (PBMC) signature suggests a skewing of the immune response in FE towards a viral‐induced Th1 response. In contrast to expectation, we found mixed data regarding the enrichment of IL‐17/Th17‐associated signatures, with one reflecting the effect of IL‐17α‐stimulated airway smooth muscle cells being enriched in nasal brushings but other IL‐17 signatures being reduced in other compartments. These findings are in line with reports showing a lack of evidence for a role for Th‐17/IL‐17 pathways in asthma.GSVA of the five U‐BIOPRED compartments was also used to examine signatures that differentiated PFE from PIE. We found 27 differentially enriched GSVA signatures but only across nasal brushings and bronchial biopsies. There was enrichment of fibrotic signatures and a reduced steroid response signature compared with that seen with FE subjects in bronchial biopsies. For example, the signature for the downregulated gene response to combination therapy in COPD biopsies was enriched in bronchial biopsies of PFE subjects, while the signature reflecting the ability of prednisolone to upregulate ASM gene expression was reduced in nasal brushings from PFE subjects. In addition, there was a mixed Th1 and Th2 activated T‐cell response in bronchial biopsies of PFE subjects. The persistence of the enrichment of the IL‐13‐stimulated type 2 signature from bronchial brushings and an element of steroid resistance may underlie the pathways that might drive the need for higher steroid doses. We previously reported that the presence of tissue oeosinophilia, lower steroid responsiveness and a high T2 signature was associated with genes such as CD44In contrast, most immune cell signatures were either not differentially‐expressed between PFE and PIE or had a significantly lower ES in nasal brushings of PFEs. There were also reduced expression scores for poly(I:C)/viral response signatures in both nasal brushings and bronchial biopsies. Thus, PFE patients may have a greater tendency for remodelling to occur in the absence of clear immune cell recruitment. However, we did not examine the expression of all immune cells and their activation states, which could confirm their important role in PFE.One interesting observation is the lower risk of persistent exacerbations in severe asthmatic patients who were current smokers compared to nonsmokers and ex‐smokers. We did not find evidence of an increase in the type 1 response that could suppress type 2 inflammation, which could be the basis for exacerbations. In our previous analysis, we reported that GSVA of the bronchial epithelial cell transcriptome from current smokers showed enrichment of xenobiotic metabolism, oxidative stress and endoplasmic reticulum stress compared to the other groups.One weakness of the study is that we only used the MARS questionnaire as a measure of adherence that indicated good adherence to medication, particularly inhaled and oral corticosteroid therapy. This should have been backed by a direct assessment of prescriptions provided by the participant's doctor.In summary, multivariate regression analysis identified being an ex‐smoker, high SABA use, presence of sinusitis and a lower age of onset of asthma as differentiating FE from IE in the U‐BIOPRED cohort. On the other hand, the subgroup of FE patients with PFE compared with PIE had more uncontrolled asthma needing SABA and OCS use, a greater presence of eczema, and higher ACQ5 scores. CEACAM5 was the only differentially expressed gene that differentiated FE from IE, but this was not the case for PFE compared with PIE. Persistence of frequent exacerbations in PFE was associated with increased T1‐cell activation, IL‐13 overexpression and fibrosis pathways, with a reduced response to viral infections, providing some insight into the driving mechanism for the persistence of frequent exacerbations.www.imi.europa.eu). KFC and IMA are funded by UK Research and Innovation (UKRI). KFC is an Emeritus Senior Investigator of the UK National Institute for Health Research (NIHR). CEW is funded by the Swedish Heart Lung Foundation . We acknowledge the contribution of the whole U‐BIOPRED team as listed in the Supporting Information repository file.Dr Singer reports honoraria for lectures and presentations from Novartis Pharma Switzerland and Vertex Pharmaceuticals Switzerland, outside the submitted work. Dr Shaw has received speaker fees from Chiesi and Astra Zeneca and advisory board fees from Adherium, Nuvoair, Astra Zeneca and Chiesi. Dr Loza is employed by and own stock in Johnson & Johnson. Dr. SE Dahlén reports personal fees from AstraZeneca, Cayman Chemicals, GSK, Novartis, Regeneron, Sanofi and Teva for consultancies outside the submitted work. Drs Auffray, Demulder and Lefaudeux report grants from the IMI (U‐BIOPRED n°115010 and eTRIKS n°115446). Dr Fowler has received grants from Boehringer Ingelheim and fees from Chiesi, outside of the current work. Dr Djukanovic reports receiving fees for lectures at symposia organised by Novartis, AstraZeneca and TEVA, consultation for TEVA and Novartis as members of advisory boards, and participation in a scientific discussion about asthma organised by GlaxoSmithKline. He is a co‐founder and current consultant and has shares in Synairgen, a University of Southampton spin out company. Dr Sterk reports grants from public–private funding by the Innovative Medicines Initiative (IMI), outside the submitted work. Dr Chung has received honoraria for participating in Advisory Board meetings of GSK, AZ, Roche, Novartis, Merck, BI, TEVA and Shionogi regarding treatments for asthma, chronic obstructive pulmonary disease and chronic cough and has also been remunerated for speaking engagements. The other authors have no disclosures in relation to this work. U‐BIOPRED was supported by an Innovative Medicines Initiative Joint Undertaking (No. 115010), resources from the European Union's Seventh Framework Programme (FP7/2007‐2013) and EFPIA companies’ in‐kind contribution (SUPPORTING INFORMATIONClick here for additional data file.


Maternal blood pressure levels reflect cardiovascular adaptation to pregnancy and proper maternal-fetal exchanges through the placenta and are very sensitive to numerous environmental stressors. Maternal hypertension during pregnancy has been associated with impaired placental functions and with an increased risk for children to suffer from cardiovascular and respiratory diseases later on. Investigating changes in placental DNA methylation levels and cell-type composition in association with maternal blood pressure could help elucidate its relationships with placental and fetal development.Taking advantage of a large cohort of 666 participants, we investigated the association between epigenome-wide DNA methylation patterns in the placenta, measured using the Infinium HumanMethylation450 BeadChip, placental cell-type composition, estimated in silico, and repeated measurements of maternal steady and pulsatile blood pressure indicators during pregnancy.p-value < 0.05). At the regional level, our analyses highlighted 64 differentially methylated regions significantly associated with at least one blood pressure component, including 35 regions associated with mean arterial pressure levels during late pregnancy. These regions were found enriched for genes implicated in lung development and diseases. Further mediation analyses show that a significant part of the association between steady blood pressure—but not pulsatile pressure—and placental methylation can be explained by alterations in placental cell-type composition. In particular, elevated blood pressure levels are associated with a decrease in the ratio between mesenchymal stromal cells and syncytiotrophoblasts, even in the absence of preeclampsia.At the site-specific level, no significant association was found between maternal blood pressure and DNA methylation levels after correction for multiple testing , but 5 out of 24 previously found CpG associations were replicated (This study provides the first evidence that the association between maternal steady blood pressure during pregnancy and placental DNA methylation is both direct and partly explained by changes in cell-type composition. These results could hint at molecular mechanisms linking maternal hypertension to lung development and early origins of childhood respiratory problems and at the importance of controlling maternal blood pressure during pregnancy.The online version contains supplementary material available at 10.1186/s12916-022-02610-y. During pregnancy, placental proper development and functions are crucial for both maternal and fetal health. The placenta is involved in supplying nutrients and oxygen to the fetus, removing gas and waste products, and regulating fetal growth through the production of growth hormones . These edirect modifications, which affect all cell types, but also from indirect variations mediated by changes in cell-type composition [Accumulating evidence suggests that changes in placental DNA methylation (DNAm) may provide a readout of placental state and pregnancy history and a means to decipher the underlying mechanisms of action by which maternal health conditions during pregnancy affect fetal and later-life health , 10. Furposition . The impposition , 13. Thiposition . A few Eposition . Only onposition . Howeverposition –19.We hypothesized that maternal BP could influence placental DNAm, and taking advantage of recent experimental advances to estimate the proportion of six reference cell types in human term placenta , we inveThe present study relies on 668 mother-child pairs from the French cohort EDEN, who were recruited between 2003 and 2006 . RecruitSBP and DBP measurements in millimeters of mercury (mmHg) were abstracted from normal follow-up measurements during pregnancy and specific study measurements scheduled at 24 to 28 weeks of gestation. Based on clinical expertise and previous studies, we used upper thresholds of > 260 mmHg for SBP and > 200 mmHg for DBP for determining the plausibility of BP measures , 23. The3 were collected a few centimeters from the insertion of the cord under the chorio-amniotic membrane, washed in a saline solution, and immediately frozen at −80°C. The protocol was similar for both centers and all modes of delivery. DNA was extracted using the QIAsymphony instrument  [p-value < 0.01). Potential sex mix-ups were investigated by comparing placental DNAm-based sex prediction to the annotated sex, which led to exclude one sample from the subsequent analyses. Beta-values were then normalized using the Beta Mixture Quantile (BMIQ) normalization [DMRcate [maxprobes (https://github.com/markgene/maxprobes) [Alu elements (Alu) and long interspersed nucleotide elements 1 (LINE-1) [At delivery, placental tissue from the fetal side was sampled at one site by the specifically trained midwives of the study using the following standardized procedure. Samples of around 5 mmGermany) . The DNAGermany) . All samlization . To redu[DMRcate . As rece[DMRcate . Cross-hxprobes) , 30. 367(LINE-1) .The workflow of our main analyses is presented in Fig. In addition to the two usual measures of BP, diastolic and systolic blood pressure , we explored two indicators: mean arterial blood pressure (MAP) and pulse pressure (PP) defined respectively as: MAP = 1/3*SBP + 2/3*DBP and PP = SBP − DBP. MAP is an indicator of peripheral resistance and cardiac output . PP is sn = 1904), we relied on functional principal component analysis (fPCA) [fdapace [whole pregnancy, early pregnancy (before the 27th week of gestation), and late pregnancy   , 37 and [fdapace . This meIn the EDEN study, most BP measurements were abstracted from medical records and were thus irregularly collected among participants  and adjusted for potential confounders and technical effects described below.Regressions of BP onto DNAm levels were adjusted for known potential confounders, selected a priori, including recruitment center , maternal age (continuous), child sex, maternal education level (< 2 years after high school/ high school + 2 years/ ≥ high school + 3 years), maternal body mass index (BMI) before pregnancy  –43, pariM-values, owing to their better statistical properties [βij is the methylation beta-value for CpG j in subject i, Mij is the corresponding M-value, Zi is the set of adjustment factors, and εij is the random error term. For the interpretability of associations, we used the intercept method to report effect sizes on beta values  p-value was below 0.05. Moreover, we checked for genomic inflation and bias using the Bayesian method implemented in the R/Bioconductor package BACON [To evaluate the relationship between maternal BP indicators and placental DNAm of individual CpGs, while accounting for the repeated BP measurements, we assumed a two-stage mixed model for each CpG at a time . Preciseoperties :\documenmentclass2pt{minimge BACON .p-value was below 0.05.We sought whether our study in the EDEN cohort would replicate results from previous similar EWAS. To our knowledge, there is only one published EWAS, by Workalemahu et al., in which the association between continuous maternal SBP and DBP levels and DNAm from the fetal side of the placenta was explicitly examined, in a cohort of 301 participants . This wocomb-p method which was implemented in the R/Bioconductor package ENmix [p-values computed in the EWAS accounting for probe spatial correlation across the genome [p-values are then adjusted for multiple testing using the Benjamini-Hochberg method [p-value below 0.05 and including at least three probes at a maximum distance of 500 bp were considered significant. A maximal p-value of 0.001 was required to initiate a region. When possible, DMRs were then annotated to the nearest gene according to Illumina's annotation for the hg19 reference genome from the IlluminaHumanMethylation450kanno.ilmn12.hg19 R/Bioconductor package, or to an overlapping gene from the more recent ENSEMBL annotation (Homo_sapiens.GRCh38.104.gtf). Otherwise, they were labeled as “intergenic” regions. Additionally, we searched for enriched specific genomic locations, associated traits, GO terms, and KEGG pathways among CpGs included in detected DMRs  in women suffering from gestational hypertension, led us to hypothesize that cell type composition may be on the path between maternal blood pressure and placental DNAm [There is currently no consensus as to whether DNAm analyses should always be adjusted for cell mixtures , 12. Holtal DNAm , 60. Wittal DNAm .Reference-based placental cell-type composition was estimated in silico using DNAm data, based on a recent work by Yuan et al. in which they measured the methylation profiles of six reference cell types , Stromal, Syncytiotrophoblast, and Trophoblasts) in term placental tissues  Additio: Methodsglobal cellular composition through a multilinear regression model, adjusted for the confounders selected a priori. Association was tested for using an ANOVA [We first examined the possible influence of each maternal BP indicator on the an ANOVA . In a sean ANOVA –68.ccmm , which is able to handle multivariate compositional mediators as a whole and provides estimates for the direct and indirect effects along with component-wise indirect effects [. In a second analysis, we performed univariate mediation analyses to investigate the mediating role of the ratio of stromal cells over syncytiotrophoblasts. The ratio was first log-transformed and computations were carried out with the R package mediation [Postulating that maternal BP might have some effect on placental methylation through changes in cell composition, we performed mediation analyses in order to quantify the direct effect of BP on DNAm as well as its indirect effect mediated by cell-type composition. We considered two different approaches which both relied on two linear regression models: an exposure-mediator model and an outcome-mediator model , 70. Fir effects . In a seediation . Both men = 1334, Additional file A total of 666 mother-child pairs were included in this study. They had similar distributions on considered covariates than the rest of the EDEN cohort (Alu and 26.1 (± 1.9) for LINE-1. Analysis of Alu and LINE-1 methylation levels yielded no significant differences related to any of the BP indicators considered therein   for p-values for genomic inflation. In the EWAS, no individual CpG was found significantly associated with any BP indicator after correcting for multiple tests (FDR < 0.05) ; these included 12 CpGs (out of 43) measured in cord blood and found significant in a broad meta-analysis by Kazmi et al. [BIF coefficients were all close to 1 indicating no genomic inflation  with the levels of at least one maternal BP indicator. Average MAP during late pregnancy and over the whole pregnancy produced the highest number of associations (35 and 20 annotated regions respectively), followed by SBP over the same time windows (19 and 20 annotated regions respectively)  to 0.007 (meaning an increase of 0.07 in the DNAm level of TRH for an increase of 10 mmHg in the average PP during early pregnancy). The average absolute effect size was 0.001 , respiratory traits , and immune deficiencies and inflammation such as HIV frailty, systemic lupus erythematosus, psoriasis, B acute lymphoblastic leukemia, and various cancers . We found enrichment in genes linked to molecular functions leukotriene were still found significantly enriched  for all BP indicators over all time windows  mediated the effect of SBP, DBP, and MAP for at least one probe in more than half of the DMRs, with a stable magnitude of indirect effect sizes across CpGs  and steady BP indicators  over placental DNAm and to have evaluated and highlighted the role played by cell-type composition in these relationships.Only one EWAS of maternal SBP/DBP in the placenta has previously been reported and emphasized the association between maternal BP and the methylation of CpGs related to cardiometabolic traits . Our stuOur analyses did not detect strong and consistent global changes in placental DNAm associated with maternal BP and no significant CpG was found in the EWAS after correction for multiple testing (FDR < 0.05), but we found significant associations with regional changes. All in all, 64 DMRs were found associated to at least one BP indicator in our study. Steady blood pressure indicators SBP and MAP are tightly correlated. They thus yielded consistent results with a substantial overlap of the DMRs, mainly related to average BP levels observed during pregnancy and/or late pregnancy and moderate effect sizes. Interestingly, we observed more and stronger associations with MAP than with SBP or/and DBP. This could be explained by MAP being less dependent on measurement site or technique and least altered by measurement damping and would promote it as a valuable indicator for investigating the interplay between blood pressure and DNAm . FurtherTRH, a gene implicated in the reduction of blood pressure in rats [IGF1R a growth factor receptor which also participates in the regulation of blood pressure [RNF141 and C6orf138 (PTCHD4), a gene found to be sensitive to transmitted adaptation to stress and associated with chronic obstructive pulmonary disease (COPD) [CCNO has been implicated in reduced generation of multiple motile cilia (RGMC) [FOXF1 was involved in alveolar capillary dysplasia and pulmonary arterial hypertension [TBX2 is known to act on lung growth by maintaining cell proliferation in the embryonic lung mesenchyme [FOXP4 is a transcriptional repressor which regulates lung-specific gene expression. Consistent with these, DMR cognate genes were enriched in GO terms related to the leukotrienes, a family of molecules recognized to be mediators of pulmonary inflammation and thought to be critical in triggering acute asthma attacks and in causing longer-term hypersensitivity of the airways in chronic asthma [Pulse pressure was suggested to be a hallmark of maternal vascular adaptation to pregnancy and an early predictor of preeclampsia , 19. Con in rats , and IGFpressure . The twoe (COPD) , 78. Acca (RGMC) , 80, a crtension ; TBX2 issenchyme , 83; andc asthma . These rc asthma . In mostIn EWAS, cell-type heterogeneity is often treated as a mere technical factor to be controlled for . Yet, inThe mediation analysis framework relied on the assumption that maternal blood pressure would have unilaterally influenced observed placental cell-type composition and DNAm. Though placental DNAm was measured from tissue extracted at birth, we cannot rule out reverse causation where placental state would have partly influenced maternal blood pressure. Nonetheless, we note that our major results still hold when we excluded preeclamptic participants from the EWAS. Moreover, these analyses allow us to estimate the proportion of the association between BP and DNAm which is due to variations in the cell-type composition. Our mediation analysis then indicates that maternal BP during pregnancy is doubly tied to term placental DNAm. On the one side, our results show that BP is associated with alterations in DNAm which may be partly explained by changes in placental cell-type composition, especially in the ratio of stromal to syncytiotrophoblast cells. This compares with recent observations showing that preeclampsia, a pregnancy disorder characterized by gestational hypertension, is associated with a lower amount and an altered structure and activity of mesenchymal stromal cells in term placental tissues , 87. Int. acknowledged that reference cell-type definition is arguable and limited by experimental capacity to identify and isolate more subtypes. Cell types with clearly distinct histological profiles, such as trophoblasts and syncytiotrophoblasts, may also be difficult to discriminate from their methylation patterns, which could bias the estimation of their respective proportions [We acknowledge several strengths and limitations to the present study. First, variations in blood pressure measurements, due to irregular consultations across participants, technical and circadian variability, likely impaired our capacity to discover true associations , 89. Nonportions . That saportions . These aportions .We studied and compared the association of the four most common maternal hemodynamic parameters during pregnancy with placental DNA methylation at birth and pointed out that, in addition to SBP and DBP, maternal MAP and PP were worth being investigated and showed markedly distinct behaviors. Our study confirmed that steady blood pressure during pregnancy is associated with alterations in placental methylation and further highlights that this relationship is twofold: both direct, potentially affecting all cell types, and indirect through changes in cell-type composition. More specifically, an increase in maternal blood pressure was associated with a decrease in the ratio between stromal cells and syncytiotrophoblasts, even in absence of preeclampsia, suggesting that maternal blood pressure could be a primary determinant of alterations in the maturation of the placenta. Moreover, elevated blood pressure was associated with increased methylation of genomic regions involved in airway development and disease. These results could provide clues to enlighten the correlations hitherto noticed between maternal hypertension and airway diseases in children and inform recommendations for reducing maternal stress during pregnancy.Additional file 1: Supplementary methods and figures S1-S7. S1 - Workflow of DNA methylation data pre-processing; S2 - Overview of blood pressure data; S3 - Reference placental cell-type composition observed in the EDEN cohort; S4 - Pearson correlation between DMR effect sizes; S5.A - Sensitivity analyses for DMRs found in our main analysis; S5.B - Sensitivity analysis excluding preeclampsia cases for the DMRs found in our main analysis; S6.A - Magnitude and 95% confidence intervals of the association between each BP indicator and each reference cell-type; Figure S6.B - Compositional Principal Component Analysis performed on cell-type mixtures; S7.A - Diagnostic of the direct and indirect effect (mediated by cell-type composition) of BP on the methylation level of DMRs; S7.B - Total, direct and indirect effect of BP on the methylation level of DMRs.Additional file 2: Supplementary tables ST1-ST12. ST1 – Participants’ characteristics; ST2 - Global methylation analyses; ST3 - Inflation Factors; ST4 - Top 100 DMPs; ST5 - Replication Analysis; ST6 - DMRs; ST7 - Trait Enrichment; ST8 - Gene enrichment; ST9 - Sensitivity analyses of DMRs; ST10 - Association between blood pressure and cell-type composition; ST11 – Compositional mediation analysis; ST12 - Mediation analysis: role of the ratio Stromal/Syncytiotrophoblasts.


Post-streptococcal glomerulonephritis (PSGN) has a good prognosis in children, but few studies have evaluated the long-term renal outcomes in adults with PSGN.Streptococcus zooepidemicus were reassessed 20 years after an outbreak in Nova Serrana, Brazil. We evaluated clinical characteristics, renal outcomes, and the trajectory of the estimated glomerular filtration rate (eGFR) by the creatinine-based chronic kidney disease-epidemiology collaboration equation from 5 follow-up assessments. Logistic regression and mixed-effects regression were used in the analysis.In a follow-up study, 47 predominantly adult patients with PSGN due to group C 2, 8 of 43 (18.6%) had urine protein-to-creatinine ratio >150 mg/g, and 25 (53%) had CKD (low eGFR and/or increased proteinuria). Increasing age was associated with CKD  in multivariate analysis. The mean eGFR decline in the last 11 years of follow-up was −3.2 ml/min per 1.73 m2 per year (95% CI: −3.7 to −2.7). Older age at baseline , and hypertension 5 years after the outbreak  were associated with lower eGFR during the whole study period.After 20 years, the participants’ mean age was 56.6±15.1 years. Thirty-four (72%) patients had hypertension, 21 (44.7%) had eGFR <60 ml/min per 1.73 mThere was a marked worsening of renal function and a high prevalence of CKD and hypertension after 20 years of PSGN outbreak. Long-term follow-up is warranted after PSGN, especially among older patients.  Streptococcus zooepidemicus in Minas Gerais, Brazil since 1998.6Outbreaks of PSGN are rare in adults, and few studies have evaluated patients’ long-term renal function after contracting the disease.S zooepidemicus.In 1998, 152 patients presented with fever, headache, and myalgia, followed by cervical adenopathy in a rural region near the city of Nova Serrana, in Minas Gerais, the second most populated Brazilian state. Then, 134 patients developed GN 7 to 10 days after the infection.,,,,,,,S zooepidemicus 20 years after the acute episode.Although PSGN has a good prognosis in children,This follow-up study consisted of 2 analysis subsets as follows: (i) a cross-sectional analysis of patients initially affected during the PSGN outbreak in 1998 and recontacted for a new clinical and laboratory evaluation in 2019; and (ii) a retrospective cohort analysis of the eGFR trajectory for all patients with serum creatinine values available in the last evaluation (2019) and taking into account their creatinine values in the 4 previous evaluations . The local nephrology team saved the medical record data of the affected patients with their contact addresses with 1 of the authors of this article (SWP). Therefore, 47 subjects were found for reevaluation 20 years after the outbreak, in the current study. The serum creatinine-based eGFR available from previous surveys of these 47 patients was used to assess their evolution.A total of 253 cases of GN were reported in the state of Minas Gerais, Brazil, from December 1997 to July 1998. Most (92%) patients resided in Nova Serrana and Quilombo da Gaia , both small towns in the center-west region of the state . The complete description of the outbreak has already been published.The medical team visited the participants in their homes, collected the clinical information, obtained early morning blood (drawn after fasting), and recently voided urine samples for analysis. An interim history was obtained, and a physical examination was performed.The study variables included gender, age, comorbidities , laboratory values , and the use of angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers. Blood pressure was measured with a mercury sphygmomanometer while the patient was sitting after 5 minutes of rest. The average of 3 measurements taken with 1-minute intervals was used in the analysis. Hypertension was defined as systolic blood pressure was higher than or equal to 140 mmHg, or diastolic blood pressure was higher than or equal to 90 mmHg (or use of antihypertensive medication). Diabetes was defined as glycated hemoglobin was higher than 7% or by hypoglycemic drug use. The creatinine-based chronic kidney disease-epidemiology collaboration equation, without the race correction, was used to estimate eGFR.Blood and urine laboratory analysis was conducted in the same laboratory using the same methodology as in all surveys performed since 1998. Blood and urine samples were appropriately stored and transported to the reference laboratory at 4°C. When examined within 1 day, urine samples were kept at 4°C; otherwise, they were frozen at −20°C. In all surveys performed since 1998, blood samples were analyzed for serum creatinine  using a spectrophotometer. Albuminuria values were assessed by radioimmunoassay using gamma counter equipment  in 2003, and the values were considered abnormal if greater than 20 mg per 24 hours. Urine protein was tested in 2019 using a colorimetric assay kit  and expressed as protein-to-creatinine ratio; the values were considered abnormal if greater than 150 mg/g.t-test or the Mann-Whitney test (whenever appropriate) were used to compare continuous variables. We evaluated the variables associated with CKD by logistic regression analysis; odds ratios and 95% CI were calculated. CKD was defined as eGFR less than 60 ml/min per 1.73 m2 and/or the presence of proteinuria equal to or greater than 150 mg/g. All clinically plausible variables with a P-value lower than 0.20 to the outcome in the univariate method were included in the multivariate logistic regression analysis. After using a manual backward elimination, a final multivariate model was proposed to adjust for all significant variables in the model. The average eGFR reduction was calculated by subtracting the eGFR in 2019 from the 2008 value, and then dividing by the number of years in the range for patients with measurements in both evaluations. We evaluated the eGFR trajectory over the 20-year follow-up in the available sample at the last survey (n = 47) by mixed-effect linear regression analysis, taking into account their measurements in the 1998, 2000, 2003, 2008, and 2019 surveys, with the eGFR being the outcome of this analysis. The random effect was the subject identification variable. The other covariates were fixed . Interaction terms between covariates and time were assessed in the models.The χ2 test was used for the comparison of categorical variables. P-value lower than 0.20 were included in the multivariate linear mixed-effect model analysis. A final multivariate model was proposed after using a manual backward elimination to adjust for all significant variables in the model. Tests were 2-sided and statistical significance was set at P < 0.05. All data were analyzed using STATA/IC 16.1 software program .We considered the presence of hypertension and albuminuria values from the 2003 assessment in the analysis because that was the visit with the most data available. Similarly, all variables associated with the outcome in the univariate method with a n = 65) or did not accept to undergo another medical evaluation (n = 10), thereby leaving 47 patients for the present study , 6 died while in a chronic dialysis program before 2 years of follow-up, and 3 with normal renal function died  between 2 and 5 years of follow-up. Some cases of the 122 surviving patients could not be located 20 years after the outbreak  patients with measured proteinuria had values greater than 150 mg/g. The whole group’s median (range) of proteinuria was 80 mg/g (30–1450 mg/g). The median (range) proteinuria was 215 mg/g (160–1450 mg/g) in the subgroup with proteinuria greater than 150 mg/g, and 70 mg/g (30–130 mg/g) in the subgroup with proteinuria less than 150 mg/g. Moreover, 25 (53%) patients had CKD (low eGFR and/or proteinuria). Eight patients had proteinuria greater than 150 mg/g; 4 (16%) of these had eGFR lower than 60 ml/min per 1.73 m2, and the other 4 (16%) had eGFR higher than 60 ml/min per 1.73 m2. The presence of CKD was higher among those older than 65 years than those younger than 65 years . Two of the 25 (8%) CKD patients were younger than 18 years during the outbreak . There was no significant association between diabetes, obesity, and degree of proteinuria (teinuria .Table 1CP = 0.003) and had higher median proteinuria  than those without CKD. In the logistic regression univariate analysis (P = 0.011). Diabetes mellitus was marginally associated with CKD only in the univariate analysis .analysis , increas2 per year (95% CI: −3.7 to −2.7) in the last 11 years of follow-up .2 per year; 95% CI: −1.28 to −0.81; P < 0.001) and the presence of hypertension 5 years after the outbreak  remained significantly associated with lower eGFR after adjusting for other confounders. Nevertheless, when the models included the interaction terms between these risk factors and follow-up time, these terms were not statistically significant (2 greater at the end of the study (P = 0.028). In a further analysis, including data of all 67 available individuals, the results on eGFR trajectories remained similar  in the acute phase of the current study outbreak were adults with a mean age of 37 years, and 65% were women.Long-term follow-up studies to properly evaluate the prognosis after acute PSGN have several obstacles. One such obstacle is the difficulty of asymptomatic individuals adhering to surveillance examinations years after the acute phase of the disease.et al.S zooepidemicus in Romania; about 90% of the patients were adults and one-third presented GN. As in the few previous reports of human disease due to S zooepidemicus,S zooepidemicus are primarily adults and not rarely need dialysis or intensive care unit admission in the early phase of the disease.,,Duca ,,,,,n = 60), persistent proteinuria, hypertension, or reduction of kidney function were found in 50% of the patients more than 7 years after the acute phase.,,,Streptococci could initiate and maintain glomerular injury have been postulated since the classical theoretical concept of glomerular trapping of immune complex and complement activation as other structural similarities between streptococcal fractions and kidney structural elements being responsible by a nephritogenic role.S zooepidemicus have not been identified.S zooepidemicus of this outbreak ruled out the belief that streptococcal pyrogenic exotoxin B or antibodies reacting with it singularly cause PSGN.Short-term recovery after acute PSGN has been a frequent outcome,In our study, 72% of the recently examined patients presented with SAH, a remarkably higher percentage than the general population. According to the Brazilian National Health Survey, the largest coverage survey based on self-reporting in Brazil, SAH prevalence is 21.4%.2.2 above the mean value observed during the outbreak, and half of the studied sample fulfilled CKD criteria. Our sample’s mean eGFR decline rate during the last 11 years of follow-up was notably greater than expected in the general population and among hypertensive patients (−0.85 to −1.50 ml/min/1.73 m2 per year).,2 in 4.8% and albumin-to-creatinine ratio >300mg/g in 0.6% of participants) than in our group.2) in the general adult population between 6.7%Decline of glomerular filtration rate was observed after the fourth decade of life in the general population,et al.Aging has been linked with a higher risk of CKD.The present study has some limitations. It is not possible to rule out a selection bias considering the patients who could not be included in this reassessment. It was difficult to find and contact the patients due to the very long follow-up since the outbreak and some individuals’ residence city changed. Most nonparticipants were not located, and just a few contacted individuals refused to participate during the whole follow-up. We did not observe significant differences between the participants and those not evaluated regarding sex and age. In addition, the limited sample size may have reduced the statistical power of some analyses. We could not obtain age and sex-matched community controls in this study. Kidney biopsies could have improved the comprehension of the CKD evolution. Nevertheless, because it was a disease of already recognized etiology and many patients were asymptomatic, ethical aspects hampered the procedure.S zooepidemicus, in the literature,. The participant’s cohort was followed by some of the same authors who described the outbreak more than 20 years ago. Univariate and multivariate analyses were performed using several clinical and laboratory parameters in the previous evaluation and eGFR measurements during the follow-up visits.Long-term follow-up studies after PSGN are rare. This report is the longest follow-up study of epidemic PSGN mainly due to S zooepidemicus had CKD, and 72% had hypertension. Mean eGFR declined markedly 2 decades after the outbreak. The present findings emphasize the importance of long-term continuous follow-up after PSGN in adults, especially older adults in the acute phase of the disease and those with hypertension.In conclusion, after a mean follow-up of 20 years, more than half of the patients with PSGN due to All the authors declared no competing interests.


P = .0081). The risk of developing MMI in patients with proteinuria remained significant in model 2  after adjusting for estimated glomerular filtrate rate (eGFR) < 60ml/min/1.73 m2 in addition to variables in model 1. In model 3, proteinuria was still significantly associated with MMI after adjusting for age, sex, dyslipidemia, ASPECTS, hypertension, diabetes, and atrial fibrillation . In model 4, the risk of developing MMI in patients with proteinuria remained significant  after adjusting for eGFR < 60ml/min/1.73 m2 in addition to variables in model 3. Proteinuria is independently associated with MMI development. Proteinuria may be a clinically accessible predictor of MMI development.A disrupted blood-brain barrier (BBB) with extravasation of macromolecules plays a critical role in the development of malignant middle cerebral artery infarction (MMI). Proteinuria is considered a marker of generalized endothelial dysfunction, including BBB disruption. This study aimed to clarify whether proteinuria identified in the acute stage of stroke is associated with MMI development. Patients with infarctions involving the middle cerebral artery territory were reviewed. Urine samples collected within 8 hours after stroke were analyzed using urine dipsticks. Patients were divided into proteinuria (urine dipstick reading of 1 + to 4+) and nonproteinuria groups. MMI was present if either signs of uncal herniation or a progressive conscious disturbance were recorded along with a midline shift > 5 mm identified on follow-up computed tomography (CT). Among the 1261 patients identified between January 2010 and June 2019, 138 were eligible for final analyses. Patients in the MMI group had lower Alberta Stroke Program Early CT Scores (ASPECTS), higher National Institutes of Health Stroke Scale scores, and a greater proportion of proteinuria than those in the non-MMI group. Four multivariate logistic regression models were used to clarify the role of proteinuria in MMI development. In model 1, proteinuria was significantly associated with MMI after adjusting for age, sex, dyslipidemia and ASPECTS (OR = 2.987, 95% CI = 1.329–6.716,  MMI may not only lead to profound disability and/or a vegetative state but may also contribute to a high mortality rate (up to 80%) if left untreated, which causes a tremendous socioeconomic burden.,2 Early decompressive hemicraniectomy, within 48 hours after the onset of stroke, reduces mortality and severe disability.,4 Therefore, early identification of patients at risk of MMI development is essential for providing timely therapy.Approximately 10% of infarctions involving the middle cerebral artery (MCA) territory may develop into malignant MCA infarction (MMI) with progressive disturbances in consciousness or signs of herniation combined with a shift in the midline structure.–12 Analyses of these imaging findings are relatively technician-dependent and less clinically approachable. Additionally, few studies have investigated the predictors of MMI in terms of its pathophysiology. Pathologically, ischemia initiates cytotoxic and ionic edema. Subsequently, disruption of the blood-brain barrier (BBB) leads to extravasation of fluid, macromolecules, and blood cells, which contributes to vasogenic edema and hemorrhagic transformation.,14 Protection of the BBB may potentially reduce vasogenic edema and hemorrhagic transformation after ischemic stroke. Severe leukoaraiosis and micro- and macroalbuminuria, as surrogates of preexisting disrupted BBB, have been reported to predict hemorrhagic transformation.–18 Although a disrupted BBB also plays a critical role in MMI development,,14 no study till date, to the best of our knowledge, has been designed to assess whether patients with preexisting disrupted BBBs are more vulnerable to extravasation than those with intact BBBs.Studies on predictors of MMI have mainly focused on the analyses of demographic data, comorbidities, stroke severity, and imaging findings, including infarct volume, collateral circulation, or recanalization status.–21 It is hypothesized that the kidney and brain share similar hemodynamic properties and common pathophysiologic processes related to risks.,22,23 Therefore, proteinuria is considered a marker of generalized endothelial dysfunction, including glomerular dysfunction in impaired renal function and disrupted BBB integrity.,22,24 In comparison to the evaluation of infarct volume, collateral circulation, and recanalization status, the assessment of proteinuria is less technician-dependent and more clinically approachable in routine practice. Therefore, this study aimed to clarify whether the presence of proteinuria in the acute stage of stroke, as a potential marker of a disrupted BBB, is associated with further development of MMI.Proteinuria, a consequence of endothelial dysfunction in the kidney that allows hyperpermeability of macromolecules, is a predictor of stroke, coronary artery disease, and cardiovascular mortality.Patients with acute infarction involving the MCA territory who were admitted to the neuro-intensive care unit at Kaohsiung Medical University Hospital between January 2010 and June 2019 were retrospectively reviewed in this case-control study. The Institutional Review Board approved this study (KMUH-IRB-E(I)-20200147).On admission, all patients underwent National Institutes of Health Stroke Scale (NIHSS) evaluation, brain computed tomography (CT), and laboratory tests, including urine analysis. After admission, patients underwent brain magnetic resonance imaging (MRI), including diffusion-weighted imaging (DWI). A follow-up brain CT was performed if any neurological deterioration was identified. Demographic characteristics, medical histories, and baseline laboratory reports were collected from patients’ medical records.Patients were included in this study if they met the following criteria: (1) precisely established onset of symptoms; (2) DWI-confirmed infarction involving MCA territory with an Alberta Stroke Program Early CT Score (ASPECTS) of 7 or less; and (3) urine analysis using urine dipstick within 8 hours after stroke onset. One of the authors (M.N.W.) assessed the ASPECTS. intrarater reliability was represented by an intraclass correlation coefficient of 0.854.; (3) receiving mechanical thrombectomy or intravenous thrombolysis; (4) involvement of other vascular territories; (5) evidence of urinary tract infection, including an increased white blood cell count and detectable bacteria or nitrites in the urine sample; (6) premorbid kidney diseases which could have caused proteinuria before infarction, including past histories of nephritic or nephrotic syndrome; (7) other conditions potentially masking neurological deterioration, including shock, sepsis, seizure, severe hypernatremia (serum sodium > 155 mg/dl) or hyponatremia (serum sodium < 125 mg/dl), or severe hypoglycemia (serum glucose < 60 mg/dl) within 5 days after infarction.Patients who met the following criteria were excluded: (1) any preexisting intracranial mass or encephalomalacia affecting the development of brain edema; (2) hemorrhagic transformation with either hemorrhagic infarction (HI)-2 with confluent petechiae in the infarcted zone, parenchymal hematoma (PH)-1 with hemorrhage involving <30% of infarcted zone and some mass effect, or PH-2 with hemorrhage involving >30% of infarcted zone or beyond the border of infarcted zone and some mass effect Dyslipidemia was determined by a recorded history of dyslipidemia, use of cholesterol-lowering agents, total cholesterol > 160 mg/dL, or low-density lipoprotein cholesterol > 100 mg/dL.,28 Atrial fibrillation was determined by chart review or any atrial fibrillation identified on 12-lead electrocardiogram or 24-hour Holter electrocardiogram results.Hypertension and congestive heart failure were diagnosed based on the recorded history via medical chart review. Diabetes mellitus was determined based on the recorded history, use of oral antihyperglycemic agents or insulin, or a fasting sugar ≥ 126 mg/dL combined with a glycated hemoglobin > 6.5% identified after admission.:All patients underwent urine analysis and a survey of creatinine levels on admission. Using urine dipsticks, the urinary protein level was scored based on the dipstick reading as follows: negative (−), trace (+/−), 1+, 2+, 3+, and 4+. According to the urinary protein level, patients were divided into the proteinuria group (urine dipstick reading of 1 + to 4+) and the nonproteinuria group (urine dipstick reading that was negative or trace). The estimated glomerular filtration rate (eGFR) was calculated using the Modification of Diet in Renal Disease equation2.All patients were Asian. Patients were categorized into 2 groups: eGFR ≥ 60 and < 60 ml/min/1.73 m,30,31If any neurological deterioration was identified, a follow-up brain CT was performed. Patients were defined as having MMI if (1) either a progressive conscious disturbance  or signs of uncal herniation (a new onset of anisocoria with decreased light reflex or impaired ocular motility) were identified after admission; and (2) a midline shift > 5 mm at the level of the septum pellucidum was recorded in the follow-up brain CT images.t-test (NIHSS) or the Mann–Whitney U test , as indicated. Categorical variables are presented as numbers with proportions and were analyzed using the Chi-squared test. We used 4 multivariate logistic regression models to clarify the role of proteinuria in predicting MMI. In regression models 1 and 2, clinically meaningful variables (age and sex) and variables with a P-value < 0.1 in the univariate analysis (dyslipidemia and ASPECTS) were entered into the multivariate logistic regression analysis. In regression models 3 and 4, age, sex and variables with clinical significance , such as dyslipidemia, ASPECTS, hypertension, diabetes mellitus, and atrial fibrillation, were entered into multivariate logistic regression analysis. To clarify the impact of concurrent decreasing eGFR on the role of proteinuria in predicting MMI, models 2 and 4 were additionally adjusted for eGFR < 60 ml/min/1.73 m2 in combination with the variables already entered in models 1 and 3, respectively. There was no significant interaction between proteinuria and eGFR < 60 ml/min/1.73 m2 in both models 2 and 4 . Statistical significance was set at P < .05.Statistical analyses were conducted using the JMP software version 10 . Continuous variables are presented as mean ± standard deviation (SD) or median , as appropriate, and were analyzed using Student Among the 1261 patients with infarction involving the MCA territory, 1123 patients were excluded for the following reasons: 95 patients had HI-2; 60 had PH-1 or PH-2; 62 had involvement of the anterior cerebral artery territory; 137 had involvement of the brainstem, cerebellum or contralateral hemisphere; 208 had an infarction in the MCA territory with an ASPECTS of 8 or more; 84 were receiving intravenous thrombolytic therapy or mechanical thrombectomy; 32 had preexisting encephalomalacia; 129 did not have urine samples available within 8 hours after stroke onset; 197 had possible urinary tract infections; and, 119 had conditions potentially masking neurological deterioration . The remaining 138 patients were eligible for further analyses. Figure P = .1933). Patients in the MMI group had significantly higher NIHSS scores on admission , lower ASPECTSs , and a higher proportion of proteinuria  than those in the non-MMI group.Table 2. There were 28 (20.3%) patients who had both proteinuria and eGFR < 60 ml/min/1.73 m2. Table 2 groups. Patients in the proteinuria group and eGFR < 60 ml/min/1.73 m2 group had significantly higher NIHSS scores on admission, lower ASPECTSs, and higher proportions of hypertension, diabetes, and atrial fibrillation than those in the nonproteinuria group and the eGFR ≥ 60 ml/min/1.73 m2 group. A higher proportion of patients with proteinuria had eGFR < 60 ml/min/1.73 m2.Sixty (43.5%) patients had proteinuria, and 48 (34.8%) patients had an eGFR < 60 ml/min/1.73 mP = .0081) than those without proteinuria after adjusting for age, sex, dyslipidemia and ASPECTS. In the regression model 2, the risk of developing MMI in patients with proteinuria remained significant  after adjusting for eGFR < 60ml/min/1.73 m2 in addition to age, sex, dyslipidemia, and ASPECTS. In model 3, after adjusting for age, sex, dyslipidemia, ASPECTS, hypertension, diabetes mellitus, and atrial fibrillation, patients with proteinuria still had a significantly higher risk of developing MMI  than those without proteinuria. In patients with proteinuria, the risk of developing MMI remained significant in model 4  after adjusting for eGFR < 60 ml/min/1.73 m2 in addition to age, sex, dyslipidemia, ASPECTS, hypertension, diabetes mellitus, and atrial fibrillation. The details of regression model 1–4 are illustrated in .Tables This study revealed that proteinuria identified within 8 hours after stroke onset is associated with an approximately 2- to 3-fold increased risk of developing MMI, independent of age, sex, dyslipidemia, hypertension, diabetes mellitus, or atrial fibrillation. Conversely, no significant relationship was found between the eGFR and the development of MMI. Furthermore, the impact of proteinuria on MMI development was independent of the eGFR.,32 The Micro-vasculature in the brain and kidney may share similar hemodynamic and anatomical properties as well as common pathogenetic processes with similar risk factors, including diabetes, hypertension or hypercholesterolemia.,22 The juxta-medullary afferent arterioles, the earliest and most severely injured anatomical site in patients with proteinuria, are directly exposed to high and fluctuating pulsatile pressure from large vessels, similar to the perforating arterioles of the brain.,34 In a spontaneously hypertensive stroke-prone rat model, similar pathological vascular injuries were concurrently recorded in both perforating arterioles of the brain and juxtamedullary afferent arterioles. Therefore, proteinuria is considered a marker of generalized endothelial dysfunction, including disrupted BBB integrity.,22 We postulated that patients with proteinuria in the acute stage of stroke may have concomitant disrupted BBBs which may contribute toward vulnerability to extravasation during vasogenic edema and a tendency of developing MMI.Endothelial dysfunction with hyperpermeability to macromolecules underlies both glomerular protein leakage and disrupted BBB integrity.,14 Proteinuria, considered a marker of a disrupted BBB, has been reported to be a predictor of hemorrhagic transformation after ischemic stroke in patients with and without reperfusion therapy.,18 Despite the shared pathophysiology of MMI development and hemorrhagic transformation, to the best of our knowledge, no study, has assessed the association between proteinuria and MMI development.Pathologically, a disrupted BBB with extravasation of intravascular content not only leads to MMI development, but also contributes to hemorrhagic transformation when blood cells, in addition to fluid and macromolecules, penetrate through this impaired mechanical and electrical barrier into the extravascular space.–38 these methods are relatively time-consuming and facility-dependent. Conversely, proteinuria assessment is relatively convenient, inexpensive and clinically accessible.Although the evaluation of BBB permeability by means of perfusion CT or Tc-99 m diethylenetriamine-pentaacetic-acid single-photon emission CT has been reported as a predictor of MMI development, However, a urine dipstick was used to measure macroalbuminuria, rather than microalbuminuria, to reduce the impact of stress-related microalbuminuria. A prospective cohort study should be conducted using parameters measured before stroke to confirm whether a premorbid BBB disruption can predict MMI development. Third, only patients at risk of developing MMI (ASPECTS ≤ 7) were included in this study, which may affect generalizability. Fourth, we could not precisely clarify recanalization status because of the retrospective design. Therefore, we excluded patients receiving intravenous thrombolysis or mechanical thrombectomy to diminish the impact of recanalization; this exclusion criterion may also affect generalizability.This study has some limitations. First, owing to the retrospective design, the impact of the severity and duration of diabetes mellitus, hypertension, and atrial fibrillation on MMI could not be clarified, and a causal relationship between proteinuria and the development of MMI could not be inferred. Second, acute stress-related microalbuminuria may have confounded the interpretation.In conclusion, this study revealed that proteinuria identified in the acute stage of stroke is associated with the further development of MMI. We speculate that proteinuria may be considered a clinically accessible and relevant predictor of MMI development. Further prospective cohort studies should be conducted to confirm whether a premorbid BBB disruption is vulnerable to extravasation during vasogenic edema and indicates a tendency to develop MMI.The authors thank the help from Dr Fu-Wen Liang, a biostatistician in the Division of Medical Statistics and Bioinformatics, Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung Medical University.Conceptualization: Wu, M.N; Fang, P.T; Yang, I.H; Hsu, C.Y; Lai, C.L; Liou, L.MData curation: Wu, M.N; Fang, P.T; Yang, I.H; Liou, L.MFormal analysis: Wu, M.N; Fang, P.T; Yang, I.H; Liou, L.MFunding acquisition: Wu, M.NInvestigation: Wu, M.N; Fang, P.T; Yang, I.H; Liou, L.MMethodology: Wu, M.N; Fang, P.T; Yang, I.H; Hsu, C.Y; Lai, C.L; Liou, L.MProject administration: Wu, M.N; Liou, L.MResources: Wu, M.N; Fang, P.T; Yang, I.H; Liou, L.MSoftware: Yang, I.HSupervision: Hsu, C.Y; Lai, C.L; Liou, L.MValidation: Wu, M.N; Liou, L.MVisualization: Wu, M.N, Liou, L.MWriting-original draft: Wu, M.NWriting-review & editing: Wu, M.N; Hsu, C.Y; Lai, C.L; Liou, L.M


Arterial wave reflection is an important component of the left ventricular afterload, affecting both pressure and flow to the aorta. The aim of the present study was to evaluate the impact of wave reflection on transvalvular pressure gradients (TPG), a key parameter for the evaluation of aortic valve stenosis (AS), as well as its prognostic significance in patients with AS undergoing a transcatheter aortic valve replacement (TAVR).The study population consisted of 351 patients with AS  who underwent a complete hemodynamic evaluation before the TAVR. The baseline assessment included right and left heart catheterization, transthoracic echocardiography, and a thorough evaluation of the left ventricular afterload by means of wave separation analysis. The cohort was divided into quartiles according to the transit time of the backward pressure wave (BWTT). Primary endpoint was all-cause mortality at 1 year.p < 0.001) and higher aortic systolic blood pressure . TPG was significantly related to the BWTT, patients in the arrival group exhibiting the lowest TPG  for the same aortic valve area (AVA) . In multivariate analysis, BWTT remained an independent determinant of mean TPG . Moreover, the prevalence of low-flow, low-gradient AS with preserved ejection fraction was higher in patients with early arterial reflection arrival . Finally, patients with early arrival of the reflected wave (Q1) exhibited higher all-cause mortality at 1 year after the TAVR .Early arrival of the backward pressure wave was related to lower cardiac output (Q1: 3.7 ± 0.9 lt/min vs Q4: 4.4 ± 1.0 lt/min, Early reflected wave arrival to the aortic root is associated with poor prognosis and significant aortic hemodynamic alterations in patients undergoing a TAVR for AS. This is related to a significant decrease in TPG for a given AVA, leading to a possible underestimation of the AS severity. Afterload is the mechanical load imposed on the left ventricle by both the aortic valve and the systemic circulation and is determined by complex time-varying phenomena. The arterial part has different components and it is best described by the propagative model of the human circulation, which consists of a distensible tube terminating at the peripheral resistance . The comFrom a physiological standpoint, the best fitting propagative model also predicts the presence of retrograde pressure waves moving throughout the arterial tree in the opposite direction (from the periphery to the aortic root) . This moThe physiological implications of this phenomenon have been extensively studied, especially as a key mechanism for the development of arterial hypertension with advancing age . One of Aortic valve stenosis (AS) is the most common valvular heart disease in the Western world impacting significantly morbidity and mortality especially in the elderly population . In the Since TPG depends on the transvalvular blood flow –12, and n = 480). The study population comprised patients referred for symptomatic AS of a native valve while presenting a high or intermediate risk for a conventional surgical approach. Sixty-seven patients were excluded due to unavailable, low quality, or missing data from baseline heart catheterization. Twenty-nine patients were excluded due to missing or low-quality baseline echocardiographic Pulsed Wave Doppler measurements and/or invasive aortic pressure measurements during the TAVR. Finally, thirteen patients were excluded because of a delay exceeding 1 year between the baseline heart catheterization and the TAVR. The final cohort consisted of 351 patients, and all data were anonymized prior to analysis. Informed written consent was obtained from each patient for inclusion in the local TAVR database as part of the Swiss prospective registry (NCT1368250) approved by the local Ethics Committee. A detailed study flowchart is depicted in This is a retrospective study based on data collected from the medical records of all patients who underwent a successful TAVR in our department from June 2008 to December 2019 , n = 88, BWTT from 11.9 to 15.5% of the ED; Quartile 3 (Q3), n = 88, BWTT from 15.6 to 22.3% of the ED and; Quartile 4 (Q4), n = 88, BWTT ≥ 22.4% of the ED.The study population was divided into four groups corresponding to the quartiles (Q) of the transit time of the backward pressure wave (BWTT). For the purpose of the current study, meaningful comparisons in BWTT between subjects can only be performed after taking into account for the relative timing of the arrival of the reflected wave to the systolic period. For this reason, values are expressed as percentage of the ejection duration (ED): Quartile 1 (Q1), All patients underwent a baseline heart catheterization as part of the standard evaluation of the AS. During this examination, cardiac output (CO) was measured for all patients either by the thermodilution or the modified Fick method with estimated oxygen consumption. CO was also indexed to body surface area (BSA) and cardiac index (CI) was calculated. Stroke volume (SV) was calculated as the ratio of the CO to the heart rate (HR) and was indexed to BSA (SV index [SVi]). Pulmonary artery and wedge pressures were also obtained, while a diagnostic coronary angiography was performed on all patients.On the day of the TAVR, invasive recordings of the baseline pressure waveform in the aortic root were acquired. For all but five patients, simultaneous left ventricular pressure measurements were available. The heart catheterization protocol included a first 6F “pigtail” catheter (Cordis), which was advanced through the stenotic aortic valve into the left ventricle from the vascular access for the transcatheter prosthetic valve and a second 6F “pigtail” catheter which was advanced to the aortic root using a second vascular access. Both catheters were connected to a pressure line and a calibrated transducer. In some patients, a double lumen catheter (Langston) was used. The pressure curves were simultaneously recorded over several heartbeats and were subsequently analyzed offline.A complete transthoracic echocardiography (TTE) in supine position was performed prior to the TAVR in all study participants. All measurements were conducted by an experienced cardiologist according to standard recommendations for TTE . Acquire2 or AVAi < 0.6 cm2/m2 and mean TPG ≥ 40 mmHg; (ii) Low-gradient AS with reduced EF: AVA < 1 cm2 or AVAi < 0.6 cm2/m2, mean TPG < 40 mmHg and EF < 50%; (iii) Low-flow, low-gradient AS with preserved EF: AVA < 1 cm2 or AVAi < 0.6 cm2/m2, mean TPG < 40 mmHg, EF ≥ 50% and SVi ≤ 35 ml/m2; (iv) Normal-flow, low-gradient AS with preserved EF: AVA < 1 cm2 or AVAi < 0.6 cm2/m2, mean TPG < 40 mmHg, EF ≥ 50% and SVi > 35 ml/m2.AS classification was performed by the application of the diagnostic criteria proposed by the European Society of Cardiology Guidelines for the management of valvular disease ; (i) HigThe left ventricle and aortic root pressure curves recorded before the TAVR were digitized for each patient. A custom, in-house Matlab code was developed to identify the beginning and end of each heartbeat automatically, and the average pressure curves over several (4–8) heartbeats were computed. Subsequently, pressure waveform analysis was performed, and key features were determined, including (i) left ventricle systolic and end-diastolic pressures, (ii) the invasive TPG, calculated as the difference between the left ventricle and aortic pressures , (iii) the aortic systolic, diastolic, mean and pulse pressures and (iv) the valvulo-arterial impedance, defined as the ratio of systolic left ventricular pressure over SVi. According to the Gorlin formula , the AVAThe invasive aortic pressures were subsequently combined with the TTE flow curves for wave separation analysis. The two curves were synchronized for each patient by adopting the second derivative approach, whereby the time lag between the two signals was corrected by calculating the maxima of the second time derivatives . Note thKey features of the forward and backward pressure waves were identified, including the magnitude and timing of the peak pressure, the wave amplitude, and the BWTT, identified by the foot of the curve. Finally, the reflection coefficient was evaluated as the ratio of the backward wave to the forward wave amplitudes. The synchronized pressure and flow signals were additionally used for the calculation of the equivalent total vascular resistance (TVR) and total arterial compliance (TAC) via parameter-fitting on a 2-element Windkessel model as described by Stergiopulos et al. .n = 341, 97.1%), the Edwards Sapien S3  or the Boston neo Accurate . Device implantation success was systematically evaluated for all interventions according to the Valve Academic Research Consortium-2 consensus Document criteria . The primary study endpoint was all-cause mortality at 1 year. Events were adjudicated by an external clinical committee.A post-TAVR follow-up was performed for all patients at 1-, 6-, and 12-months intervals through a clinical visit. All baseline clinical characteristics and procedural and follow-up data were stored in a dedicated database using a secured online platformP-value level of 0.05. Statistical analysis was performed in IBM SPSS statistics .Categorical variables are reported as counts with percentages. Continuous variables are expressed as mean and standard deviation or as the median and interquartile range for variables with non-normal distribution . Categorical variables are compared among groups by the use of Pearson Chi-Square or the Fischer exact test as appropriate. Continuous variables were compared among the groups using analysis of variance (ANOVA) or the Kruskal-Wallis test for the non-normally distributed data. Levene’s test was used to assess the homogeneity of variance among the compared groups, and in case of violation, Welch’s ANOVA test was used. In order to assess the independent effect of the BWTT on TPG multiple linear regression model analysis was performed treating BWTT as a continuous variable and after adjusting for the following parameters: Aortic systolic blood pressure, AVA (estimated by the Gorlin formula), Zc, TVR, TAC, gender and height. One-year all-cause mortality rates was calculated from Kaplan-Meier analysis for patients presenting the earliest return of the reflected wave (Q1) as compared to the rest of the population (Q2–Q4). Cox-regression analysis for the same groups was performed to compute hazard ratios and the 95% confidence intervals after verification of the proportional hazard assumption. A multivariate Cox-regression model was used in order to adjust comparisons between the two groups for potential confounding mortality factors . Statistical significance was assumed at a 2-sided p = 0.009 and 0.002, respectively).The baseline characteristics of the study population are presented in p = 0.027), peak to peak (p = 0.012), and mean (p = 0.011) gradients, while AVA remained constant among groups. Two characteristic cases from the Q1 and Q4 groups are depicted in No significant differences were noted among groups in terms of HR, ED, left ventricular systolic, end-diastolic, mean pulmonary artery, and wedge pressures . Early ap = 0.005), maximum TPG (p = 0.003), and maximum transvalvular velocity (p = 0.005) were all associated with shorter BWTT (p = 0.031), as well as the prevalence of the low-flow, low-gradient AS with preserved EF , lower TAC (p = 0.041) and lower Zc .Early arrival of the backward wave was associated with higher TVR .In multivariate analysis, BWTT remained a strong, independent predictor of all TPG measures after adjusting for gender and height, Zc, TVR, and TAC (n = 341), followed by sub-clavian (n = 4) and trans-apical access (n = 2). 44 patients (12.5%) underwent a concomitant procedure (coronary angioplasty). Device success was achieved in 325 interventions (92.5%), which was comparable among the groups (Data on the TAVR procedure are presented in e groups .p = 0.016), as compared to the rest of the study population. This remained significant even after adjustment for baseline differences including gender and STS score , for device success rate , but not after adjustment for tricuspid regurgitation which was different between the groups .Clinical follow-up was completed for the totality of the study population. The main findings of the present study may be summarized as follows: In patients with AS, the early arrival of the wave reflection to the aorta is associated with (a) lower aortic TPG (as assessed either by Doppler echocardiography or heart catheterization) for the same AVA; (b) lower CO, lower SV, and lower mean flow rate during systole, (c) higher aortic systolic and mean pressures and finally (d) poor prognosis as assessed by all-cause 1 year mortality.Although a causal relationship cannot be established, the present study provides evidence that wave reflections may influence TPG measurements possibly through a flow-dependent mechanism affecting the accuracy of AS evaluation. The shift towards earlier arrival of the reflected waves increases pressure during systole and decelerates substantially the aortic transvalvular flow. According to the Gorlin formula, for a given AVA, TPG depends exclusively on the transvalvular flow, which explains the observed associations .2) corresponding to a severe AS, but with a mean TPG below 40 mmHg, classifying the stenosis as less severe. The low TPG in these patients is explained by a low-flow state, which is defined by a SVi ≤ 35 ml/m2. The “paradox,” though, lies in the fact that the SV is low while at the same time the EF is preserved (≥50%)  . The find (≥50%) .In our study, enhanced reflections were associated with stiffer arterial trees (low TAC), which, however, had lower Zc, i.e., lower proximal aortic stiffness. In young adults, the proximal aorta is highly compliant, whereas the peripheral arteries are relatively stiff. In terms of wave propagation, this suggests an important impedance mismatch between the compliant aorta and the branch vessels, which generates reflections . DispropIt is interesting to note that the decrease in SV observed with enhanced wave reflection was not associated with a concomitant decrease in EF. This in accordance with previous observations where enhanced arterial wave reflection was associated with preserved EF but reduced left ventricular function as reflected by ventricular longitudinal strain and tissue imaging . AnotherOur study highlights the importance of a detailed analysis of the left ventricular afterload for the accurate evaluation of the severity of the AS. Brachial systolic and diastolic pressures are not sufficient since they do not represent the whole spectrum of the mechanical load imposed on the left ventricle. On the other hand, it would be unrealistic to suggest invasive recordings of the aortic pressure for every patient with AS. The use of the handheld, high fidelity tonometers developed in the last years may be an excellent option since they provide accurate, non-invasive measurements of the pressure waveform of an artery close to the skin , 27. TheTo attenuate the impact of high after load on TPG, it has been suggested that the assessment of the AS should be repeated after the intravenous or sublingual administration of nitrates –34. At cAnother important finding of the present study was the association between early reflection wave arrival (Q1) and all cause 1 year mortality. This is in accordance with observations in other populations such as patients with arterial hypertension, end-stage renal disease and coronary artery disease where arterial reflections present a prognostic significance independently of the traditional risk factors –7. This The study is subjected to the limitations of the retrospective, cohort study design. Wave separation analysis was performed by combining flow and pressure data not simultaneously recorded. In case of difference in HR between the pressure and the flow waveforms, synchronization was achieved by truncating or extending the diastolic portion of the flow wave, which may influence accuracy. However, this can only increase the probability of a type II error . Moreover, CO was acquired invasively by two different techniques (thermodilution or the modified Fick method with estimated oxygen consumption) that may not be used interchangeably. Finally, AS classification was performed by the use of invasive SVi estimation, which is not readily available in clinical routine. Thus, the associations with the incidence of low-flow, low gradient severe AS with preserved EF may not apply when SVi is measured by other techniques with higher variability .Early reflected wave arrival at the aortic root, generated by arterial trees with pronounced impedance mismatch between peripheral and central arteries, is associated with poor prognosis and profound hemodynamic changes at the aortic level including a significant decrease in transvalvular aortic flow and concomitant increase in aortic pressures. This is related to a significant decrease in TPG for a given AVA, leading to the underestimation of the AS. Our study highlights the importance of a detailed analysis of the left ventricular afterload for the accurate evaluation of the AS severity for both diagnostic and prognostic purposes.dionysios.adamopoulos@hcuge.ch and SN, stephane.noble@hcuge.ch.The datasets presented in this article are not readily available because of institutional restrictions applying to data involving human subjects. Requests to access the datasets should be directed to DA, The studies involving human participants were reviewed and approved by Geneva Ethics Committee . The patients/participants provided their written informed consent to participate in this study.SP and DA conceived and designed the research, analyzed the data, prepared the figures, and drafted the manuscript. SN, GG, and HM performed the measurements. SP, NS, and SN interpreted the results. SP, DA, GR, VB, HM, GG, SM-W, M-JL, NS, and SN edited and revised the manuscript and approved the final version of the manuscript.Open access funding was provided by the University of Geneva.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.


What is missed?Standard cardiac rehabilitation (CR) programs do not typically consider respiratory symptoms.What are the main findings?Breathing exercises (BE) have a positive physiological effect on chronic heart failure (CHF).What are the implications of the main findings?Future CR programs for CHF have to manage respiratory symptoms.CR programs for CHF have to address patient-centered outcomes.n = 20); group (A): standard CR with BE and group (B): standard CR alone. Primary outcomes were respiratory parameters and secondary outcomes included cardiovascular and cardiopulmonary outcomes. All the participants attended a program of aerobic exercise  for 12 weeks, plus educational, nutritional, and psychological counseling. Group (A) patients attended the same program together with BE using inspiratory muscle training (IMT) and breathing calisthenics (BC)  for the same duration. Results: There was a significant improvement in the respiratory outcomes, and most of the cardiovascular and cardiopulmonary outcomes in both groups with a greater change percentage in group A (p < 0.05). Conclusions: These results indicate that the addition of BE to the CR programs in CHF is effective and is a “patient-centered” approach.Background: Although solid evidence has indicated that respiratory symptoms are common amongst patients with chronic heart failure (CHF), state-of-the-art cardiac rehabilitation (CR) programs do not typically include management strategies to address respiratory symptoms. This study investigated the effect of the addition of breathing exercises (BE) to the CR programs in CHF. Methods: In a two parallel-arm randomized controlled study (RCT), 40 middle-aged patients with CHF and respiratory symptoms were recruited and randomized into two equal groups ( Chronic heart failure (CHF) is a common and debilitating condition that has cardiovascular , cardioIn clinical practice, cardiac rehabilitation (CR) is considered standard care for CHF patients to mainly improve cardiovascular and/or cardiopulmonary manifestations . SkeletaSystematic reviews have confirmed that addressing respiratory symptoms in CHF is crucial and affects the outcome of the treatment, and they recommended the blending of exercise training with BE to optimize outcomes. Typically standard CR programs do not include strategies to control respiratory symptoms ,8.The most successful state-of-the-art CR programs globally, such as the EuroAction , Our heaAddressing these symptoms is important to CHF patients as it improves dyspnea. Improving ventilation in CHF would reduce ventricular preload and afterload, decrease extra-vascular lung water, and the work of breathing in CHF, improving many of the pathophysiologic mechanisms of the CHF ,15. HencBreathing training (BE) techniques that aim to improve the function of the respiratory muscles through specific exercises incorporating different methods like inspiratory Muscle Training (IMT) and breathing calisthenics (BC) . IMT hasThe objective of this study was to assess the effect of the addition of the BE techniques to the standard CR on the respiratory, cardiovascular, and cardiopulmonary symptoms in comparison with the standard CR alone, which is usually prescribed for CHF patients. ClinicalTrials.gov, with the registration number NCT04905433, registered 27 May 2021.This study was a two-parallel arm RCT study. The recruitment period was 9 months, from June 2019 to March 2020. All patients signed a written consent before enrollment in the study. This study conforms to the principles of the Declaration of Helsinki and relevant ethical guidelines : high frequency (HF), low frequency (LF), and low frequency to a high-frequency ratio (LF/HF). Resting heart rate (HR), resting systolic blood pressure (SBP), and resting diastolic blood pressure (DBP)].2max), anaerobic threshold (AT), maximum heart rate (MHR), and maximum minute ventilation (VE).Cardiopulmonary: maximal oxygen capacity  was used for data analysis. The sample size was calculated using the G*Power software (version 3.0.10). F-test MANOVA within and between the interaction effects was selected. Considering a power of 0.80, an α level of 0.05 (two-tailed), and an effect size of 0.46; two groups, a generated sample size of at least 20 participants per group was required and a total sample size of 40 subjects .Data were screened for normality assumption, the homogeneity of variance, and the presence of extreme scores. Shapiro–Wilk and Kolmogorov–Smirnov tests for normality showed that all measured variables are normally distributed. p = 0.77), weight (p = 0.09), height (p = 0.92), BMI (p = 0.75), or EF (p = 0.88) (No significant difference between the groups was found in age ( = 0.88) .p = 0.001 and 0.039), respectively, in favor of group A. There was a significant increase in MIP within group A and a significant decrease in the mean values of RPE within both groups A and B (p = 0.001), as shown in There was a significant difference in the mean values of MIP and RPE post-treatment between the two groups (p < 0.05) in favor of group A. There was a significant difference between HF, LF/HF, RHR, and RSBP within both groups; HF increased, whereas LF/HF, RHR, and RSBP decreased. Regarding RDBP, there was a significant difference within group A only (p = 0.001). No significant changes between groups were noticed in LF post-treatment (p = 0.460) and within both groups A and B (p = 0.074 and 0.140), respectively as shown in There was a significant difference between the two groups in the mean values of HF, RHR, RSBP, and RDBP post-treatment (2max and AT) and the VE post-treatment in favor of group A (p < 0.05). Additionally, there was a significant difference in these variables within group A; VO2max, AT, and MHR increased, and VE decreased significantly (p < 0.05). There was a significant increase in AT within group B (p = 0.007). There was a non-significant difference in MHR between both groups (p = 0.730), as shown in The results of the present study showed a significant increase in the parameters of the functional capacity indices  by 20.6% and 3%, and dyspnea scores (RPE) by 20.9% and 13.6% in groups A and B, respectively. The results of the current study coincided with the recommendations of a recent systematic review by Sadek et al. , which dHowever, the same authors mentioned above  performAfter careful investigation, it was found that the main difference between this RCT and the current one is the exercise frequency per week. In the current study, we followed the exercise recommendations mentioned in the systematic review mentioned previously . ConsequThere were increases in the post-treatment parameters of HRV , RHR by 9.6, 5.1%, RSBP by 7.6, 4.2%, and RDBP by 4.5, 1% in groups A and B, respectively.That came in agreement with a study carried out by Catella et al.  who examHowever, the results of the present study showed a non-significant reduction in the low-frequency component of HRV (LF) in group A. Previous investigators reported that the interpretation of the LF component is more controversial, as it represents a mixture of the sympathetic and parasympathetic modulation of the heart rate, yet the exact contribution of each component to LF is unknown .2max by 10.5, 3%, AT by 18.3, 6.6%, VE by 12.9, 0.4%, and MHR by 4, 2% in groups A and B, respectively.The results of the current study showed a post-treatment increase in the parameters of VOThis came in agreement with Lopes et al.  who eval2max among group A. This shows that the effect of inspiratory muscle training in patients with CHF is largely attributed to central [as the VO2 max is limited to cardiac output (VO2 max = arterio-venous O2 deference multiplied by cardiac output)] rather than peripheral (the ability of a muscle to move against a larger workload). This finding may not be surprising as it could be related to the absence of the effect of whole-body training and the conditioning effect of aerobic exercise [On the other hand, the percentage of change in the MHR is less than that reported with the VOexercise .One of the possible explanations for the above-mentioned results is that the IMT can delay the development of diaphragmatic fatigue in patients with CHF ,3, leadiIn addition, in CHF, there is enhanced ventilatory sensitivity to both central and peripheral chemoreceptor stimulation, which can reflexively induce sympathetic vasoconstriction and reduce blood flow to skeletal muscles during exercise . Therefore, diaphragm fatigue may elicit sympathetically mediated vasoconstriction in limb muscles, a reflex that might be attenuated by inspiratory muscle training which delayed the development of fatigue of the diaphragm . This coFurthermore, the significant improvements reported in anaerobic threshold during cardiopulmonary exercise may be attributed to the significant reduction in ventilation (VE) in group A. This came in parallel with the study of Audrey et al.  in whichIt is believed that the significant reduction in VE seen in the present study is partially attributed to the shifting of the respiratory muscle metabolism from a catabolic to an anabolic state secondary to training. This may result in the attenuation of ergoreceptors originating from this muscle, explaining the hyperventilation response in CHF. Therefore, improvement in ventilatory muscle strength and endurance may also attenuate the exaggerated ergoreceptors. The techniques improve not only ventilatory manifestations but also cardiovascular and cardiopulmonary outcomes that may be affected by the ergoreflex activation and sympathetic overactivity usually experienced in CHF . Limitations: This study has mainly a limitation about the accessibility to the full medical history of the participants  due to the nature of the clinical practice in Egypt. Further studies with the consideration of this confounder will be conducted in the future in a better-structured RCT.Breathing training using IMT and BC for 12 weeks in combination with CR may have a positive impact on the respiratory, cardiovascular, and cardiopulmonary outcomes in patients with CHF more than standard CR.CR programs have to be individually tailored as mentioned in the European and American guidelines ,39. As t