Preterm birth is defined by the onset of labor at a gestational age shorter than 37 weeks, and it can lead to premature birth and impose a threat to newborns’ health. The Puerto Rico PROTECT cohort is a well-characterized prospective birth cohort that was designed to investigate environmental and social contributors to preterm birth in Puerto Rico, where preterm birth rates have been elevated in recent decades. To elucidate possible relationships between metabolites and preterm birth in this cohort, we conducted a nested case-control study to conduct untargeted metabolomic characterization of maternal plasma of 31 women who experienced preterm birth and 69 controls who underwent full-term labor at 24–28 gestational weeks.P = 8e−04, FDR = 0.006) with preterm birth. After controlling for potential clinical confounders, a total of 38 metabolites demonstrated significant changes uniquely associated with preterm birth, where 17 of them were preterm biomarkers. Among 7 machine-learning classifiers, the application of random forest achieved a highly accurate and specific prediction (AUC = 0.92) for preterm birth in testing data, demonstrating their strong potential as biomarkers for preterm births. The 17 preterm biomarkers are involved in cell signaling, lipid metabolism, and lipid peroxidation functions. Additional modeling using only the 19 spontaneous preterm births (sPTB) and controls identifies 16 sPTB markers, with an AUC of 0.89 in testing data. Half of the sPTB overlap with those markers for preterm births. Further causality analysis infers that suberic acid upregulates several fatty acids to promote preterm birth.A total of 333 metabolites were identified and annotated with liquid chromatography/mass spectrometry. Subsequent weighted gene correlation network analysis shows that the fatty acid and carene-enriched module has a significant positive association (Altogether, this study demonstrates the involvement of lipids, particularly fatty acids, in the pathogenesis of preterm birth. Preterm birth is defined as deliveries that occur prior to 37 weeks of gestation, and it is one of the leading causes of newborn mortality and morbidity . We prevMetabolomics provides compositional and quantitative information about the state of an organism or cell at the macromolecular level . Blood mThe samples used in this study were maternal plasma collected in gestational weeks 24–28 from the women, who went on to experience preterm birth (N = 31) or full-term healthy deliveries (N = 69). Untargeted metabolomics liquid chromatography with tandem mass spectrometry (LC-MS/MS) assays were performed on these samples, followed by bioinformatics analysis. Our goals were the following: to (i) identify metabolites and metabolomic pathways that are associated with preterm birth; (ii) elucidate metabolomic processes that may have a causal relationship with preterm birth; and (iii) seek early gestational metabolomic biomarkers (weeks 24–28) that are predictive of preterm birth.in vitro fertilization, and lack of major health conditions or obstetrical complications in medical records. For preterm births, gestational ages <37 weeks were included. For controls, gestational ages between 39 weeks 0 days and 40 weeks 6 days were included.This study was conducted in an exploratory sample of the PROTECT cohort, which obtained its own institutional review board approval. This is a single-center study conducted in Puerto Rico. At the time of this study, the parent cohort consisted of 812 pregnant women, from which we randomly sampled 31 women who experienced preterm birth and 69 full-term controls for metabolomic analysis. Recruitment of the PROTECT cohort is ongoing and began in 2010. It is funded by the National Institute of Environmental Health Sciences Superfund Research Program. Participants were recruited in the first or second trimester of pregnancy (median 14 weeks gestation). Inclusion during early gestational age ranges allows for greater capacity to evaluate windows of vulnerability across pregnancy. Inclusion criteria for recruitment were as follows: age of 18–40 years, having residence in the Northern Karst aquifer region, disuse of oral contraceptives 3 months before pregnancy, disuse of Medical records were used to determine birth outcomes. Gestational age in complete pregnancies was estimated using the American College of Obstetricians and Gynecologists (ACOG) recommendations and previously described in greater detail ,15,16. D15N) anthranilic acid; and 20 µM of L-(15N)2 tryptophan, gibberellic acid, L-epibrassinolide. Plasma samples were subsequently vortexed and centrifuged for 10 minutes at 15,000g. The supernatant of the centrifuged samples was transferred to a clean vial and dried using nitrogen gas. The dried samples were reconstituted to 50 µL.Stored plasma samples, which were collected from the women between 24 and 28 weeks gestation and subsequently stored at −80°C, were thawed on ice in preparation for analysis. Deproteinization was then performed by taking 100 µL of plasma combined with 400 µL 1:1:1 ratio of methanol, acetone, and water. Internal standards were also incorporated for metabolite recovery assessment and included 5 µM of L-(D4) thymine, L-  with the Waters Acquity BEH C18 column . The temperature of the column heater was maintained at 55°C. The injection volume was 5 μL for all analyses. The lipid extract was injected onto a 1.7-μm particle diameter, 100 × 2 mm id Waters Acquity BEH C18 column  to separate the lipids. We used a linear gradient beginning with 98% Solvent A (water + 0.1% formic acid) and 2% Solvent B (methanol + 0.1% formic acid) to perform chromatographic elution. Solvent B was linearly increased to 98% over the first 22 minutes and was held at this level for 8 minutes. Thereafter, the composition was returned to the beginning and held for 3 minutes. The flow rate used for these experiments was 0.46 mL/min.m/z, mass accuracy was <1 ppm with in-line mass calibration, and scan rate was ∼118 scans per minute. Acquisition of MS/MS spectra was performed using the data-dependent acquisition (DDA) function of the Analyst TF software . The software was set to the following parameters: dynamic background subtraction, charge monitoring to exclude multiply charged ions and isotopes, and dynamic exclusion of former target ions for 9 s.Mass spectrometry data acquisition for each sample was performed in both positive and negative ionization modes using an Agilent 6445 Q-TOF . In positive ion mode runs, mobile phase A is 100% water that has 0.1% formic acid while mobile phase B is 100% methanol that has 0.1% formic acid. The formic acid is replaced with 0.1% (m/v) ammonium bicarbonate in negative ion mode runs. The column effluent was directed to the ESI source. For positive ionization mode, the voltage was set to 5,500 V. For negative ionization mode, the voltage was set to 4,500 V. For both modes, the declustering potential (DP) was set to 60 V and the source temperature was set to 450°C. The curtain gas flow was 30 (l/min). The nebulizer was 40 (l/min). The heater gas was 45 (l/min). The Q-TOF resolution according to specifications is >45,000 FWHM at 2,722 The collected DDA MS/MS spectra data were analyzed using the Masshunter Qualitative Analysis Kit . Using this kit, the “Find by Feature” algorithm is used to detect chromatographic peaks representative of metabolites. Between samples, feature alignment was performed using an in-house–written software package that matches features with identical mass and retention time between samples. Tgaps in chromatographic data, recursive feature identification was also performed by searching the data a second time with the list of aligned features using the “Find by Formula” algorithm in Agilent Masshunter Qualitative Analysis Software. Metabolites were putatively annotated using the mass spectral data annotation tool, Binner , to reduk-nearest neighbors method  = 0.02, odds ratio = 2.12), and FAs are generally lower in preterm samples. They have a striking pattern of negative associations with preterm birth. Moreover, FAs also have overall negative associations with age, income, and alcohol use, suggesting the biological, socioeconomic, and behavioral effects are intertwined at the metabolomic level. The other 2 clusters do not have enrichment in specific metabolite functional groups.A total of 333 lipid metabolites were identified by LC/MS in maternal plasma. A PLS-DA plot of the 100 samples using all identified lipid metabolites shows that preterm samples are well separated from healthy controls using the first 2 components Fig. . To examP = 8e−04, FDR = 0.006) with preterm birth  and carene (CAR) . This FA/CAR-enriched module also shows a significant negative association  with gestational age (GestAge)  . However, we did not find that the density of FA-enriched modules was higher in preterm cases compared to that in controls .To further elucidate the relationships between metabolomics and preterm birth, we next performed the WGCNA method on the 333 metabolites . WGCNA arth Fig. . This moge) Fig. . These rge) Fig. , demonstP < 0.05) between preterm and control samples exclusively, and are not associated with other confounders , F1 statistic, and balanced accuracy to evaluate the models. Among all classification methods, RF yields the highest balanced accuracy statistic (1.0) in the training dataset Fig. , so we sP < 0.01) between the 17 metabolites and the binary preterm outcome. As shown in Fig. We used the Granger causality test  to inferP < 0.05, 33 of which also appeared in the previous 38 metabolites significantly different in preterms vs controls . For the 33 metabolites, the differential patterns are consistent in both preterm birth and sPTB, with the fold changes being more extreme in sPTB .The 31 preterm samples include 19 sPTB cases and 12 samples from other conditions . To further investigate the association between metabolites and sPTB, we analyzed the cases with sPTB separately . We conducted differential analysis between sPTB and controls and identified 53 metabolites with Using the same procedures as in the previous metabolomics-based preterm biomarker model, we identified 16 of the 55 metabolites as markers for sPTB. Half of these 16 markers, including FA(24:6), FA(16:3), FA(17:1), FA(14:2), FA(19:1), FA(14:0(Ke)), FA(14:1), and heptadecanoic acid, are also among the previously identified 17 metabolite markers for preterm birth. We further investigated whether the identified markers can serve as good predictors of sPTB . The RF method again presents the best performance in the training data and achieves an AUC of 0.89 in the testing data. In the metabolite marker importance ranking, previously fifth-ranked hexanoylcarnitine appears again among the top metabolites . In summary, many preterm markers are also robust sPTB signatures.Preterm birth is one of the leading causes of newborn mortality and morbidity . To imprThe importance of FAs in preterm birth is highlighted by bioinformatics analysis in various aspects. First, correlation network analysis of metabolomics reveals deregulated lipid modules that may contribute to preterm birth Fig. . The FA/Interestingly, the causality analysis shows the causal effect of decreased suberic acid for the excessive FAs. This is consistent with a previous finding that suberic acid is related to FA disorders . SubericChanges in these lipids collectively suggest that lipid metabolism may contribute to the pathogenesis of preterm birth Fig. . Indeed,A few caveats of this study should also be mentioned. First, participants in this study do not have specific dietary records; thus potential confounding from diet cannot be investigated. The metabolites are measured from maternal blood; therefore any biological mechanisms discussed here are inferred systematically rather than being directly measurable from relevant tissues . In addition, despite developing a potential biomarker panel from a classification model, these candidates are suggestive and not quantitatively validated yet. We plan to validate them in other independent cohorts in the future. Nonetheless, this study provides strong evidence of the involvement of a class of saturated and unsaturated FAs and PCs in preterm births, mediated by perturbation in biological functions including cell signaling and lipid peroxidation.Project name: Maternal lipids in the pathogenesis of preterm birthhttps://github.com/lanagarmire/pretermBirth_metabolomicsProject home page: Operating systems: Windows and LinuxProgramming language: RLicense: MIThttps://www.metabolomicsworkbench.org, where they have been assigned Project ID PR001155. The data can be accessed directly via their Project DOI: http://dx.doi.org/10.21228/M8DH5P. Other data further supporting this work are openly available in the GigaScience repository, GigaDB [These data are available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, , GigaDB .Supplementary Figure S1.  WGCNA network in preterm births (A) and healthy controls (B), respectively. Each node represents a metabolite, whose size is proportional to the node connectivity value in a WGCNA network. (C) The overlap between modules of networks in control and preterm samples. (D) Detailed information on overlapping module density was discovered in (C). (E) Bar plot of the connectivity scores of the 17 upregulated metabolites.Supplementary Figure S2. Metabolites show significantly different levels in sPTB and control samples. (A) Heat map of the 55 metabolites with a significant difference exclusively between sPTB and control samples (P < 0.05). (B) Bar plots on the averaged normalized intensities in cases vs controls.Supplementary Figure S3. Classification model for sPTB. (A) Comparison of 7 classification models using 16 metabolites on the hold-out testing. The dataset was randomly split into training data (80%) and testing data (20%) 10 times. The average value and standard error of the 10 repeats are shown for 3 performance metrics of the area under the ROC curve (AUC), F1 statistic, and balanced accuracy. The winning method RF in training data (left) was then applied to the testing data (right). (B) The heat map of correlation coefficients between the 16 metabolites and clinical variables. (C) The precision-recall curves of the RF model from (A) on classifying preterm, LGA , income, and maternal age (≥35 y or not), respectively, using the same set of testing data as in (A). (D) Normalized variable importance scores for the 16 lipid markers in the RF model. The normalization is done by making the sum of importance scores equal 100.Supplementary Figure S4. Principal component analysis plots for the QC of the metabolomics data in positive and negative modes.Supplementary Table S1. Fold change values of the 38 metabolites that are significantly different between preterm and control samples.ANOVA: analysis of variance; AUC: area under the receiver operating curve; CAR: carene; CYP4F/A: cytochrome P450 (CYP) 4 F/A; FA: fatty acid; FDR: false discovery rate; FWHM: full width at half-maximum; GBM: gradient boosting; HMDB: Human Metabolome Database; KEGG: Kyoto Encyclopedia of Genes and Genomes; LC-MS/MS: liquid chromatography with tandem mass spectrometry; LDA: linear discriminant analysis; LGA: large for gestational age; LOG: logistic regression with elastic net regularization; MI: mutual information; NIH: National Institutes of Health; PAM: partition around medoids; PC: phosphocholine; PE: diacylglycerophosphoethanolamines; PG: phosphatidylglycerol; PI: phosphatidyinositol; PS: acylglycerophosphoserines; QC: quality control; Q-TOF: quadrupole time-of-flight; RF: random forest; RPART: recursive partitioning and regression trees; SGA: small for gestational age; SOV: source of variation; SVM: support vector machine; WGCNA: weighted gene correlation network analysis.The authors declare that they have no competing interests.This study was supported by the Superfund Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health (grants P42ES017198). Additional support was provided from NIEHS grant numbers P50ES026049, R01ES032203, and P30ES017885 and the Environmental influences on Child Health Outcomes (ECHO) program grant NO. UH3OD023251, and the NIH grant U2C-DK119886. L.X.G. is supported by grants K01ES025434 awarded by NIEHS through funds provided by the trans-NIH Big Data to Knowledge (BD2K) initiative, R01 LM012373, and LM012907 awarded by NLM, and R01 HD084633 (L.X.G.) awarded by NICHD.Y.C. and B.H. conducted the bioinformatics analysis and modified code provided by Y.L. M.T.A. provided writing material. J.D.M. designed the study, obtained funding, supervised the metabolomics assays, and critically reviewed early drafts of the paper. L.X.G. supervised the analysis. Y.C., B.H., and L.X.G. wrote the manuscript. All authors have read and revised the manuscript.The authors declare that they have no competing interests.


P < 3.6 × 10−8) and 1767 differentially methylated regions. Functional analysis identified 14 enriched gene ontology terms pertaining to cell–cell contacts and cell–extracellular matrix contacts. Principal component analysis of probes with genome-wide significance revealed a first principal component that explained 23.5% of the variance in DNA methylation, and this was negatively associated with gestational age at birth. The first principal component was associated with peak width of skeletonized mean diffusivity  and peak width skeletonized neurite density index , but not with peak width skeletonized fraction anisotropy ; these relationships mirrored the imaging metrics’ associations with gestational age at birth. Low gestational age at birth has a profound and widely distributed effect on the neonatal saliva methylome that is apparent at term equivalent age. Enriched gene ontology terms related to cell–cell contacts reveal pathways that could mediate the effect of early life environmental exposures on development. Finally, associations between differential DNA methylation and image markers of white matter tract microstructure suggest that variation in DNA methylation may provide a link between preterm birth and the dysconnectivity of developing brain networks that characterizes atypical brain development in preterm infants.Preterm birth is associated with dysconnectivity of structural brain networks and is a leading cause of neurocognitive impairment in childhood. Variation in DNA methylation is associated with early exposure to extrauterine life but there has been little research exploring its relationship with brain development. Using genome-wide DNA methylation data from the saliva of 258 neonates, we investigated the impact of gestational age on the methylome and performed functional analysis to identify enriched gene sets from probes that contributed to differentially methylated probes or regions. We tested the hypothesis that variation in DNA methylation could underpin the association between low gestational age at birth and atypical brain development by linking differentially methylated probes with measures of white matter connectivity derived from diffusion MRI metrics: peak width skeletonized mean diffusivity, peak width skeletonized fractional anisotropy and peak width skeletonized neurite density index. Gestational age at birth was associated with widespread differential methylation at term equivalent age, with genome-wide significant associations observed for 8870 CpG probes ( et al. report genome-wide differential DNA methylation in association with gestational age at birth. Differential methylation was associated with white matter dysmaturation, suggesting that epigenetics could explain altered brain development in preterm infants following early exposure to extrauterine life.Wheater   These include autism spectrum disorder, social difficulties, language impairment, attention-deficit hyperactivity disorder, reduced intelligence quotient, educational underachievement and psychiatric diagnoses.2–9Preterm birth, defined as birth at <37 weeks of gestation, affects around 11% of births worldwide10 A consequence of the encephalopathy is generalized dysconnectivity of developing structural networks, which can be inferred from diffusion MRI (dMRI) and neurite orientation dispersion and density imaging (NODDI) during the neonatal period.11–15 Specifically, normal maturation is characterized by a reduction in mean diffusivity (MD) and increases in both fractional anisotropy (FA) and neurite density index (NDI) in white matter; but MD is increased, and FA and NDI are decreased in preterm infants at term equivalent age, compared with control infants born at term.16 These changes reflect an increase in water content and a decrease in white matter organization in preterm infants. The peak width of skeletonized mean diffusivity (PSMD) is a method for histogram-based calculation of MD distribution across the entire white matter skeleton; it provides a measure of generalized white matter microstructure, is robust to scanner variation and is predictive of cognition in later life.17–19 In previous work, we extended the histogram model to neonatal data and included other dMRI and NODDI metrics. We found that PSMD and peak width skeletonized neurite density index (PSNDI) are altered in preterm infants at term equivalent age and that histogram-based measures have utility for investigating upstream determinants of dysmaturation such as systemic inflammation.21The neural phenotypes that underlie long-term functional impairment include diffuse white matter injury and subsequent dysmaturation of white matter and grey matter neuroaxonal structures, collectively termed the ‘encephalopathy of prematurity’.22 Alterations in DNAm contribute to the pathogenesis of neurodevelopmental disorders .25 There is growing evidence that differential DNAm can mediate the effect of environmental pressures on brain structure and function across the life course.27 The neonatal methylome is sensitive to prenatal factors such as maternal smoking,28 maternal body mass index,29 as well as birth weight.30 It is altered in association with co-morbidities of preterm birth,32 and there is some evidence for legacy differences in the methylome two decades after preterm birth.33 A meta-analysis investigating DNAm from umbilical cord blood identified widespread differential methylation associated with GA at birth (across the range 27−42 weeks) affecting 2% of sites and involving both hypo- and hypermethylation, as measured on the Illumina 450k array.34 However, due to differences in the cellular composition of samples, epigenetic signatures observed in different tissues are likely to be distinct.35 The main cellular component of saliva, buccal epithelium, may be more representative of the brain than umbilical cord blood because of ectodermal origin.36–38The mechanisms that link the environmental stress of preterm birth with atypical brain development are uncertain. Variation in DNA methylation (DNAm) is a possible mechanism; DNAm is involved in the regulation of gene expression and cell fate during foetal brain development.Here, our first aim was to determine whether low gestational age at birth was associated with variation in the salivary methylome at term equivalent age and to characterize the biological pathways implicated in the DNAm response to preterm birth. Our second aim was to investigate whether the DNAm signal of gestational age explains variance in measures of white matter microstructure at term equivalent age. We tested the hypotheses that low gestational age at birth is associated with widespread differential methylation apparent at the end of neonatal intensive care and that DNAm contributes to variance in peak width skeletonized metrics of white matter connectivity.39 Exclusion criteria were major congenital malformations, chromosomal abnormalities, congenital infection, overt parenchymal lesions  or posthaemorrhagic ventricular dilatation. Ethical approval has been obtained from the National Research Ethics Service, South East Scotland Research Ethics Committee . Informed consent was obtained from a person with parental responsibility for each participant. The study was conducted according to the principles of the Declaration of Helsinki. DNAm data were available from 258 neonates, 214 of whom also had successful dMRI acquisition.All participants were born at the Royal Infirmary of Edinburgh, UK. Preterm infants, defined as GA of <33 weeks of gestation and term infants, defined as GA > 37 weeks based on first-trimester ultrasound scan dating, were recruited to the Theirworld Edinburgh Birth Cohort. This is a longitudinal study designed to investigate the effect of preterm birth on brain development.Saliva obtained at term equivalent age, on the same day as MRI acquisition, was collected in Oragene OG-575 Assisted Collection kits, by DNA Genotek, and DNA extracted using prepIT.L2P reagent . DNA was bisulphite converted and methylation levels were measured using Illumina HumanMethylationEPIC BeadChip  at the Edinburgh Clinical Research Facility . The arrays were imaged on the Illumina iScan or HiScan platform, and genotypes were called automatically using GenomeStudio Analysis software version 2011.1 (Illumina). DNAm was processed in two batches.41 The pfilter function in wateRmelon was used to exclude samples with 1% of sites with a detection P-value > 0.05; sites with beadcount <3 in 5% of samples and sites with 1% of samples with detection P-value > 0.05. Cross hybridizing probes and probes targeting single nucleotide polymorphisms with overall minor allele frequency ≥0.05 were also removed.42 Control probes were also removed. Samples were removed if there was a mismatch between predicted sex (minfi) and recorded sex (n = 3). Data were danet normalized which includes background correction and dye bias correction.41 Saliva contains different cells types, including buccal epithelial cells and leucocytes. Epithelial cell proportions were estimated with epigenetic dissection of intra-sample heterogeneity with the reduced partial correlation method implemented in the R package EpiDISH.43 Probes located on sex chromosomes were removed before analysis. Data from one of each twin pair were removed randomly (n = 20).Raw intensity (.idat) files were read into the R environment (version 3.4.4) using minfi. wateRmelon and minfi were used for preprocessing, quality control and normalization.2- and 64 diffusion-weighted (b = 750 s/mm2) single-shot spin-echo echo-planar imaging (EPI) volumes acquired with 2 mm isotropic voxels (echo time (TE) = 106 ms and repetition time (TR) = 7300 ms).MRI was obtained at the same appointment as saliva sample collection for DNAm analysis. Structural and dMRI were performed on 93 infants using a MAGNETOM Verio 3T clinical MRI scanner  and 12-channel phased-array head coil, which were used to acquire dMRI using a protocol consisting of 11 Tb = 0 s/mm2 (b0)] and 64 volumes with b = 750 s/mm2; the second consisted of 8 b0, 3 volumes with b = 200 s/mm2, 6 volumes with b = 500 s/mm2 and 64 volumes with b = 2500 s/mm2. An optimal angular coverage for the sampling scheme was applied.44 In addition, an acquisition of three b0 volumes with an inverse phase encoding direction was performed. All dMRI volumes were acquired using single-shot spin-echo EPI with 2-fold simultaneous multislice and 2-fold in-plane parallel imaging acceleration and 2 mm isotropic voxels; all three diffusion acquisitions had the same parameters (TR/TE 3500/78.0 ms). Images affected by motion artefact were re-acquired multiple times as required; dMRI acquisitions were repeated if the signal loss was seen in three or more volumes.One hundred and twenty-one infants were scanned using a MAGNETOM Prisma 3T clinical MRI scanner  and 16-channel phased-array paediatric head and neck coil. dMRI was acquired in two separate acquisitions: the first consisted of eight baseline volumes .53 FA and MD were calculated using single-shell data to match the Verio scanner.The two dMRI acquisitions from the MAGNETOM Prisma scanner were first concatenated and then denoised using a Marchenko–Pastur-PCA-based algorithm20 The diffusion tensor derived maps of each subject (FA and MD) were calculated after registration; NDI was then propagated to the template space using the previously calculated transformations. The data were skeletonized using the ENA50 skeleton and then multiplied by a custom mask. Finally, the peak width of the histogram of values computed within the skeletonized maps was calculated as the difference between the 95th and 5th percentiles.20All the subjects were registered to the Edinburgh Neonatal Atlas (ENA50) using DTI-TK.54 An overview of the analysis pipeline is shown in sva package in R.56 A fully adjusted model was specified before SVA to retain signal explained by biological variables of interest: CpG ∼ gestational age at birth + age at scan + birthweight Z-score + maternal smoking + sex + epithelial cells. SVA identified 17 significant surrogate variables (SVs) which were subsequently included in the analysis.Unless otherwise stated, analysis was completed in R version 3.4.4.limma package in R.57 Beta values of each of 776 025 CpG sites were regressed (as dependent variables) on gestational age (GA) at birth using linear regression. Covariates were added to adjust for sex, birthweight Z-score, age at sample collection, maternal smoking, estimated epithelial cell proportions and 17 surrogate variables. A significance threshold of 3.6 × 10−8 was selected, which represents genome-wide significance.58An epigenome-wide association study (EWAS) was performed using the dmrff package in R.59 Here, a differentially methylated region is a region containing two or more sites separated by ≤500 bp with EWAS analysis P ≤ 0.05 and methylation changes in a consistent direction. Following dmrff’s subregion selection step, DMRs with Bonferroni-adjusted P ≤ 0.05 were significant.Differentially methylated regions (DMRs) were assessed using the dmrff function in the missMethyl package, which controls for multiple probe bias.60 We performed an analysis that included those sites that reached genome-wide significance in EWAS and a second analysis that also incorporated those sites that contributed to differentially methylated regions.Gene set enrichment analysis was carried out using the gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) databases, and using the gometh function in prcomp function in R. CpGs were precorrected for the effects of biological covariates and surrogate variables via linear regression. The scree plot was visually inspected to select a principal component  to be carried forward for subsequent analysis.Principal component analysis (PCA) was conducted on CpG probes that reached genome-wide significance, using the 54 In models testing PSMD and PSFA, MRI scanner was included as a binary covariate as MRI data from two scanners were included. PSNDI was only available from data acquired on one scanner, so it was not necessary to include the scanner as a covariate. We report standardized regression coefficients and P-values.Pearson’s correlation coefficient was used to assess the relationship between the first PC identified from PCA and gestational age at birth. This PC was used in linear regression models, as an independent variable, to test the associations between peak width of skeletonised mean diffusivity (PSMD), peak width of skeletonised neurite density index (PSNDI) and peak width of skeletonised fractional anisotropy (PSFA) with DNAm, conducted in R version 4.0.1.https://git.ecdf.ed.ac.uk/jbrl/ena and the code necessary to calculate histogram-based metrics is at https://git.ecdf.ed.ac.uk/jbrl/psmd. Requests for original image data will be considered through the BRAINS governance process: www.brainsimagebank.ac.uk.61 DNAm data available upon request from Theirworld Edinburgh Birth Cohort, University of Edinburgh (james.boardman@ed.ac.uk or https://www.tebc.ed.ac.uk/2019/12/data-access-and-collaboration/). The DNAm and metadata are not publicly available due to them containing information that could compromise participant consent.The atlas with templates can be found at t = −0.157, P = 0.88) or for sex .DNAm data were collected from 311 neonates. Twenty-nine did not meet DNAm preprocessing QC criteria and were excluded. One participant with a congenital abnormality was removed, as were three participants whose sex predicted from DNAm data did not match their recorded sex. This group of 278 neonates included 20 sets of twins. After random removal of one twin from each set, there was no evidence of imbalance for birthweight between the twins that were removed and the twins that remained in the sample  and 103 were infants born at full term , see Z-score, infant sex, age at sample collection, maternal smoking, estimated epithelial cell proportion and surrogate variables. The genomic inflation factor was 1.72 (P < 3.6 × 10−8), P < 0.05. Of these, 11 had 10 or more CpG sites contributing to the DMR. The largest DMR mapped to a genomic region that encodes two genes: NNAT and BLCAP. The 29 probes mapped to this region were all located within islands and positively associated with gestational age at birth, indicating that longer gestation corresponds to hypermethylation. Of the 10 most significant differentially methylated probes (DMPs), three probes were localized to the IRX4 gene, one probe to the GAL3ST4 gene, and one to LOXL4 , no KEGG terms remained significant following false discovery rate (FDR) correction for multiple comparisons. Two GO terms were enriched following FDR correction: one for anchoring  and one for adherens junction . In an analysis that incorporated all 11 752 distinct CpGs from both EWAS and DMR analysis, 14 GO terms were enriched  and PSNDI  but not with PSFA (β = −0.005 P = 0.933)  ; Table 5r = −0.622; P < 2.2 × 10−16) , and in models adjusted for scanner it was positively associated with PSMD  but not PSFA  (The first unrotated PC (PC1) derived from the 8870 genome-wide significant CpGs accounted for 23.5% of the variance, and the second PC accounted for 2.5% . There w× 10−16) . PC1 was= 0.510) . All modBy studying a unique database of DNA linked to brain imaging in a population of preterm and term infants, we have identified extensive differential methylation in association with gestational age at birth and revealed an association between the principal axis of methylation and brain dysmaturation within the same sample. Differentially methylated regions and probes were distributed widely across the genome, indicating that gestation duration has a global effect on DNAm. Gene enrichment analysis of changes associated with gestational age identified gene sets pertaining to cell contacts and cytoskeleton. A single principal component that explained 23.5% of the variance in differential DNAm linked to preterm birth was closely associated with markers of generalized dysconnectivity across the white matter skeleton.22 and umbilical cord blood;62 and widely distributed variation is reported in association with postmenstrual age at the sampling of preterm infants .31 The signature we identified in saliva sampled at term equivalent age included 233 probes that were previously shown to be differentially methylated in association with gestational age in a meta-analysis of umbilical cord blood samples that reported 8899 gestation-dependent CpGs.34 The limited overlap could be explained by differences in the cellular composition of assessed tissues, different array types used to measure DNAm, or due to the time of sampling. Cord blood is collected at birth and so methylation changes at this time reflect foetal maturity and/or prenatal experience, whereas the methylation signature at term equivalent age reflects the allostatic load of early postnatal experiences, as well as the prenatal environment. We chose to sample at term equivalent age because postnatal co-morbidities of preterm birth and NICU care practices such as painful stress exposures alter DNAm profiles, and because cumulative DNAm variations over this time period may link exposure to behavioural outcome in preterm infants.64The data are consistent with studies that have reported associations between length of gestation and genome-wide variation in DNAm within foetal brain34 The most significant DMR mapped to a site encoding two genes: NNAT and BLCAP. NNAT encodes the neural fate initiator neuronatin, the expression of which decreases throughout development;65 there was a positive association with increasing gestational age at birth. Hypomethylation of NNAT is associated with a corresponding increase in the expression of neuronatin.66BLCAP encodes the bladder cancer-associated protein. This is a tumour suppressor that induces apoptosis, with high expression in brain and B lymphocyte. The candidacy of this locus, encoding NNAT and BLCAP, as a region of interest whose expression may be modified by perinatal exposures is supported by previous EWAS of gestational age, maternal body mass index, maternal smoking and schizophrenia.67–70 In addition, BLCAP was found to have reduced methylation in placental samples from women who had preeclampsia.71Functional analyses of DMPs identified two enriched GO terms, for adherens and anchoring junctions. When distinct probes that contributed to both DMPs and DMRs were combined, GO analysis identified an additional 14 terms related to cell–cell adhesion, cell adhesions with the extracellular matrix and signalling from the extracellular membrane; 12 of these were also identified in the meta-analysis of gestational age effects on DNAm obtained at birth from umbilical cord blood.70 Hypermethylated probes were found in genes including ZBP1, which was identified in EWAS investigating gestational age and hypertensive disorders of pregnancy;72INTS1, which has been identified in EWAS of gestational age, hypertensive disorders of pregnancy, maternal body mass index, birthweight and breastfeeding duration.73 Hypomethylated probes were found in genes including UBXN11, which was identified in studies of gestational age;62 and IRX4. Three of the 10 most significant DMPs mapped to the IRX4 gene, all of which displayed a negative association with gestational age at birth. IRX4 is associated with cardiac development in vertebrates, including humans.74 Its homologues have been implicated in retinal axon guidance in zebrafish, and neural patterning in Xenopus,76 and it has been identified in previous EWAS of hypertensive disorders of pregnancy72 and prenatal maternal stress.77Probes that demonstrated the largest magnitude of effect in association with gestational age mapped to genes previously associated with gestational age or maternal risk factors in EWAS.27 For example, one of the genes identified in our EWAS has been implicated in neurodevelopmental disorders; biallelic mutations in INTS1 have been associated with a rare neurodevelopmental syndrome characterized by intellectual disability.78–80The novel pathways and genes implicated by EWAS studies of gestational age could provide a strong empirical basis for the selection of genes in targeted analyses in association with neuroimaging.81 PSNDI and PSMD were strongly associated with the first principal component of gestational age-dependent variation in DNAm but PSFA was not. This suggests that variations in DNAm could contribute to the higher variability in water content and intra-axonal volume that characterize preterm brain dysmaturation.20 We have previously reported that differential DNAm is associated with FA of the genu of the corpus callosum and tract shape of the right corticospinal tract;26 it is most likely that we did not observe an association between gestational age or DNAm with PSFA because this metric is subject to histogram shift,20 meaning that although there are groupwise differences in FA values across the skeleton, the spread of values is the same. Associations between gestational age at birth and both DNAm and image markers of dysconnectivity, and between DNAm and image features, suggest that differential DNAm contributes, in part, to the relationship between gestational age and brain network dysconnectivity in preterm infants. However, some of the DNAm signatures may be related to underlying causes of prematurity that operate in foetal life, such as infection or preeclampsia. We cannot rule out that such changes are involved in the aetiology of preterm birth, which might preclude them from mediating specific associations with brain development.We used metrics of generalized white matter connectivity to assess relationships between DNAm and brain development because generalized dysconnectivity of structural networks is a hallmark of preterm brain dysmaturation.17 There are some limitations. First, control for cell composition was based on estimation of cell proportions rather than measurement, so we cannot rule out the possibility that some of the signal identified was related to variation in cell composition. Second, mediation analysis to assess causation was not possible because the association between the DNAm PC1 and peak width skeletonized metrics might result from the DNAm PC being derived from CpG sites that are associated with gestational age, so variance attributable to the mediating variable cannot be assumed. This could be addressed by out-of-sample validation, which will require other neonatal cohorts with both saliva methylome and dMRI data. Cohorts, with such data, that recruit neonates from across the gestational age continuum would also provide valuable replication of analyses described here. There was also some evidence of inflation of test statistics based on the genomic inflation factor. However, the genomic inflation factor is thought to provide an overestimate of inflation and corrections based on it may be overly conservative.82 The value of the genomic inflation factor was also similar to those previously reported in neonatal cohorts.34 In addition, visual inspection of our results via Manhattan plots suggests that our finding, of widespread differences in DNAm in relation to gestational age at birth, is in line with previous studies that have investigated this in cord blood and foetal brain tissue.34 Finally, preterm birth itself can reflect maternal health or circumstance; in this cohort, mothers of preterm infants were more likely to have pregnancy-induced hypertension and the control mothers were more likely to have college or university qualifications. It is possible that these, or other maternal factors associated with preterm birth such as infection, chronic disease, poor nutrition and alcohol, tobacco or drug use, could influence the neonatal methylome at term equivalent age. A large prospective study with longitudinal sampling from birth would be required to test the hypothesis that maternal factors contribute to changes in the methylome apparent after neonatal intensive care.The strengths of this study are that we studied a population of preterm and term infants across the gestational age range of 23–42 weeks, who were uniquely phenotyped with DNAm and dMRI. We sampled after the period of NICU care to capture the allostatic load of preterm birth. We measured DNAm from neonatal saliva samples, which has consistency with brain DNAm patterns and is non-invasive. The Illumina EPIC platform provided extensive coverage of the methylome (850 000 sites) and we controlled for cell composition. Finally, we used an image phenotype that is robust to scanner variation.In conclusion, gestational age at birth has a profound and widely distributed impact on the neonatal saliva methylome at term equivalent age, which reflects the allostatic load of preterm birth itself and postnatal exposures during neonatal intensive care. GO terms related to cell–cell contacts were enriched, indicating that cell contacts and organization are implicated in the phenotype. Associations between DNAm and PSMD and PSNDI suggest that variations in DNAm could contribute to white matter dysconnectivity commonly seen in preterm infants, and this analysis identified several genes and gene regions that could provide further insight into the molecular mechanisms by which early exposure to extrauterine life influences neurodevelopment.fcac056_Supplementary_DataClick here for additional data file.


Association of morning glory disc anomaly (MGDA) with persistent hyperplastic primary vitreous (PHPV) has been reported earlier. Retinopathy of prematurity (ROP) like retinopathy in preterm babies with optic disc anomalies has also been published. Our case is unique in terms of presence MGDA, PHPV, unilateral ROP like retinopathy in a term infant with normal birth weight.A 5-month-old girl, born at term with a birth weight of 3750 g, presented with anterior PHPV, MGDA and ROP like retinopathy. In order to prevent retinal detachment, she received 360 degree barrage laser photocoagulation at the edge of the optic disc excavation of the left eye. In the follow-up a month later, laser scars were found in her left fundus without other complications.PHPV and MGDA with ROP like retinopathy in term and normal weight baby is rare. The peripheral avascular retinal area, caused by the dragging of the defected optic disc, might have been more vulnerable to the oxygen change after birth which resulted in ROP like retinopathy. High sensitivity to oxygen results in a series of changes such as upregulation of VEGF and IGF-1 may cause ROP-like retinopathy. Morning glory disc anomaly (MGDA) is a rare congenital optic disc malformation characterized by funnel shaped excavation of the dysplastic optic disc, covered with glial tissue, increasing number of straight radiating vessels from the disc margin, peripapillary pigmentation and excavation with or without macular capture . MagnetiOther congenital anomalies associated with MGDA are cataract, posterior lenticonus, lens coloboma, persistent hyperplastic primary vitreous (PHPV), retinal detachment and secondary glaucoma –5. AmongRetinopathy of prematurity is a potentially blinding condition associated with abnormal vasculature in response to abnormal angiogenesis in preterm and low birth weight babies. The pathogenesis of ROP has two phases: the first phase involves delayed physiologic retinal vascular development, and the second phase involves vasoproliferation , 8. VascA 5-month-old girl, born at term with a birth weight of 3750 g, was presented to our outpatient department with the chief complaint of deviation of both eyes to the left side by the parents. The baby had history of oxygen therapy in neonatal intensive care unit (NICU) for neonatal pneumonia for three days after birth, there was no other systemic abnormalities. The exact duration and mode of oxygen inhalation was not known. Except for the deviation of both eyes to the left, there was no other abnormal facial features of the baby. A preliminary torch light examination was done at the time of admission later we examined the child under anesthesia under an operating ophthalmic microscope. The anterior segment of the right eye and fundus were within normal limits Fig. . The lefROP-like retinopathy has been reported in full-term infants with low birth weight, multiple births, respiratory distress syndrome (RDS), phototherapy, and oxygen administration . But allMGDA associated with peripheral retinal nonperfusion has been reported and well recognized. The peripheral avascular retina attributed by the anomaly of defected optic disc is the character of MGDA , 12, 13,


Echinococcus species. Iran is endemic for Echinococcus granulosus. Here, we present a case of hydatidosis in an 85-year-old man, presented with acute, dull, constant, and generalized abdominal pain. A computed tomography scan (CT scan) showed an intact hydatid cyst on the bladder dome and several hydatid cysts in the liver. Open surgery revealed a cyst with hepatic origin, confirmed with histopathological studies.  Echinococcosis is a zoonotic infection caused by  Although a primary abdominal hydatid cyst is very rare, it can be transferred to the abdominal cavity without any rupture as secondary ones. In this case, rupture of the liver wall was the reason for this transfer. As a result, there should be a suspicion of hydatidosis in a patient with a similar presentation. Echinococcus from the Taeniidae family. Echinococcosis, based on its transmission cycle, has specific geographical distribution around the world. The Middle East, especially Iran, is among the endemic places for Echinococcus granulosus [Echinococcosis is a zoonotic infection caused by a parasitic tapeworm called anulosus . Accordianulosus .Here, we discuss a rare presentation of hydatid cyst and the purpose of this report is to draw the attention of physicians to include hydatid cyst in the differential diagnosis of patients with similar manifestations.An 85-year-old healthy man presented to the surgery clinic of Ghaem Hospital, complaining of abdominal pain, which was generalized, dull, and acute and started two days before he came to the clinic. The pain was constant, and he declared its intensity 4 out of 10, with no radiation. Moreover, he did not reveal any correlation between pain intensity and eating or activity. He did not mention any recent fever, nausea, or vomiting. He did not report any past medical history of surgery or hospitalization.On his medical examination, vital signs were as follows: heart rate = 98/min, respiratory rate = 14/min, blood pressure = 115/70 mmHg, and axial temperature = 37.3°C. His abdomen was soft, and there was just a mild generalized tenderness without rebound or guarding. There was no sign of organomegaly, scar, or herniation. The rest of his examination was normal.Complete blood count was normal, and blood chemistry did not show any abnormal findings other than high ESR (53 mm/hr) and high CRP (187 mg/l) with the normal range of ESR (0-22 mm/hr for men) and CRP < 10 mg/l.Coronal sections of the abdomen-pelvic CT scan with IV contrast showed a hepatic hydatid cyst with water lily sign in the right lobe of the liver with a compressive effect on the adjacent diaphragm and the left lobe of the liver and hydatid cyst of the bladder dome . In axiaOpen abdominal surgery was performed with the final diagnosis of hydatidosis. There was an intact cyst without any invasion right above the bladder with 10∗13 cm size and hepatic origin. There was no adhesion, but just a little fibrous tissue was seen between the cyst and bladder.Further examination of the liver revealed a perforation, and the source of intra-abdominal bloody discharge was from there. Isolated hepatic cysts were aspirated and washed with hypertonic serum. The cysts were not related to the bile ducts, and bile secretions were not seen at the site of the cyst cavity. Pathology samples were sent for further examination. On histopathological examination, hydatid cyst diagnosis was confirmed for the bladder dome cyst .The postoperative status was good and stable. The patient was discharged four days after the surgery with an albendazole prescription  and said to visit the surgical clinic next week.Echinococcus spp. Echinococcosis is endemic in Iran with a prevalence of 5% and an estimated expense of US$93.39 million. The disease is more frequent in southwest and south of Iran mostly in rural regions and is accountable for 1% of the admissions to surgical wards [Hydatid cyst occurs due to the cystic form of CE is considered to be endemic in all eastern Mediterranean region, and this region is one the major hotspots of CE. Despite this fact, CE is not among the health topic list of diseases in this region which requires more attention . In PakiMoreover, in India which is not far from Iran, the incidence varies from 1 to 200/100,000 in a year and most of the cases were from Kashmir, Andhra Pradesh, Tamil Nadu, and Central India .Most cases are asymptomatic for decades and are found accidentally or in the autopsies. However, there are several clinical signs and symptoms for cystic echinococcosis, which is different based on the size and involved organ. The cyst size is wavering, but often it is between 1 and 15 centimeters , 8.Different organs are involved in this disease, and the liver and lungs are the most common sites, respectively.Most symptoms of cystic echinococcosis (CE) are due to the gradual growth of the cyst, which puts pressure on the adjacent structures, which could cause abdominal discomfort, pain, nausea, and vomiting. The rupture of CE that can be caused by blunt or spontaneous trauma is an acute complication. The ruptures are of three different types, and based on the type, there could be no leakage or leakage to the biliary system or the abdominal cavity , 10.In this case, the CE was intact, mobile in the peritoneal cavity right above the bladder with hepatic origin, and the rupture happened in the liver wall. There were multiple foci of fibrin deposition on CE and no adhesion to the bladder. Just Kahraman et al. have reported a similar wandering cyst in the peritoneal cavity. However, it was a daughter cyst caused by a ruptured primary cyst in the liver . TherefoIn conclusion, a hydatid cyst should be considered as one of the differential diagnoses of acute abdominal pain. Although the primary abdominal hydatid cyst is very rare, it can transfer to the abdominal space without any rupture as a secondary abdominal hydatid cyst. Therefore, it is crucial to look for other hydatid cysts in other organs, such as the liver.


A previous study showed that the lungs are involved in the biogenesis of platelets (PLTs). Thus, the present study aimed to investigate the association between bronchopulmonary dysplasia (BPD), a chronic lung disease, and PLT parameters in very premature infants.The study subjects were premature infants with a gestational age of ≤ 30 weeks and birth weight of ≤ 1500 g in a preterm birth cohort study recruited between January 1, 2015, and August 31, 2019. BPD was defined as the need for oxygen supplementation more than 28 days after birth. The PLT count, mean platelet volume (MPV), platelet distribution width (PDW), and plateletcrit (PCT) level were compared between BPD and non-BPD infants. A generalized estimating equation model was used to adjust for confounding factors. A forward stepwise logistic regression model was used to calculate the adjusted odds ratio (OR) for thrombocytopenia in the BPD group. Receiver operating characteristic curve analysis was performed to assess the predictive value of PLT count combined with gestational age (GA) and birth weight (BW) for BPD.F = 4.44, P = 0.03) and PCT levels  than the non-BPD infants. However, the MPV  and PDW  were higher in the BPD group. After adjusting for potential confounding factors, the BPD infants had a higher risk of thrombocytopenia than the non-BPD infants , and the risk of BPD was increased in very premature infants with a PLT count ≤ 177*109/L  at the end of the second week. In the multivariate predictive model, it was showed that the AUC area (0.85), sensitivity (0.88), specificity (0.70) and Youden index (0.58) are improved using PLT counts ≤ 177*109/L combined with GA and BW.The final study subjects were 134 very premature infants, namely, 64 infants with BPD and 70 infants without BPD. The BPD infants had lower PLT counts (9/L was a potential risk factor for the development of BPD in very premature infants.Abnormal PLT parameters were observed in BPD infants, and a PLT count ≤ 177*10The online version contains supplementary material available at 10.1186/s12890-022-01895-2. Bronchopulmonary dysplasia (BPD) is the most common respiratory complication of very-low-birth-weight (VLBW) and very premature infants . Over thNeonatal thrombocytopenia is a common hematological abnormality in newborn infants, particularly in premature infants . GeneralA nested case–control study was conducted in very premature infants recruited in a preterm birth cohort from January 1, 2015, to August 31, 2019. Written informed consent was accepted from the guardian while the infants were enrolled in the cohort. The present study is the secondary analysis of the cohort study and the protocol was reviewed and approved by the institutional research ethics committee of the Affiliated Hospital of Southwest Medical University.(1) Gestational age (GA) at birth ≤ 30 weeks, (2) birth weight ≤ 1500 g, (3) admission to the hospital within 24 h after birth, and (4) hospital stay more than 28 days.(1) Severe congenital constructional and/or chromosome malformations, such as congenital heart or lung disease, central nervous system malformation, diaphragmatic hernia, congenital deformity of the head, face or thorax, genetic metabolic disease, or chromosomal disease; (2) hematological disease or pulmonary disease; or (3) death in the first 7 days of life.A total of 3,242 preterm infants were recruited in the preterm birth cohort, 185 of whom had a GA ≤ 30 weeks and a birth weight ≤ 1500 g, and all were admitted to the neonatal intensive care unit. Infants with congenital malformations (n = 6) or admission exceeding 24 h after birth (n = 19) were excluded. Infants with a hospital stay less than 28 days, including 11 infants who died and 15 infants discharged after recovery, were also excluded. The study subsequently enrolled 134 infants, including 64 infants in the BPD group and 70 infants in the non-BPD group , BPD was defined as a need for supplemental oxygen for 28 days and was categorized at 36 weeks postmenstrual age (PMA) or 56 days of age for infants > 32 weeks GA as “mild, moderate, or severe” based on oxygen use and/or respiratory support . The pat9/L confirmed by peripheral blood smears [9/L [Thrombocytopenia was defined as a PLT count < 150 × 10ars [9/L . As throA uniform questionnaire was used to extract the medical data of the eligible premature infants from the hospital information system. Epidata was used to record data by two researchers independently to ensure the accuracy of the data. Inconsistent data were assessed by a third researcher.Maternal factors: premature rupture of membranes > 18 h, gestational diabetes mellitus, gestational hypertension, antenatal use of glucocorticoids and magnesium sulfate, chorioamnionitis, systemic antibiotics 48 h before delivery, infection during pregnancy, and thrombocytopenia during pregnancy.Infantile factors: GA, sex, birth weight, Apgar score at one minute and five minutes after birth, mode of delivery, presence of infection at admission, presence of intrauterine growth restriction, pulmonary surfactant replacement therapy, mechanical ventilation support and lasting time, nasal continuous positive airway pressure (nCPAP), noninvasive ventilator support and lasting time, reuse of noninvasive ventilator or invasive ventilator, duration of oxygen supplementation, and maximum oxygen fraction use.Blood cell counts: Based on the clinical guidelines of our department, the first blood cell counts were measured at patient admission. During hospitalization, blood cell counts were performed weekly. Tests for infection were performed immediately if infection was suspected. The tests were performed with an XS-800i automatic blood cell analyzer. The PLT count, mean platelet volume (MPV), plateletcrit (PCT) level, and platelet distribution width (PDW) data were extracted and used as outcomes in the analysis.The potential risk factors and confounders were as follows.Mean ± SD), and abnormally distributed data are presented as the median and quartile range. Comparisons of the continuous data were made using an independent sample t-test or rank sum test. Categorical data are presented as the frequency and percentage and were compared by means of a chi-square test. Multivariate forward stepwise logistic regression was used to calculate the odds ratio (OR) of thrombocytopenia in the BPD group and adjust for potential confounders . The PLT parameters were subsequently compared between the BPD and non-BPD groups at each time point (two-way ANOVA). A generalized estimating equation (GEE) model for repeated measurement data was established to examine the effects of the interaction between BPD and age on the PLT parameters. The GEE model was also used to adjust for infection status or other potential confounding factors. Finally, receiver operating characteristic (ROC) curve analysis was performed to assess the predictive value of PLT count at each time point for BPD. After getting the cut-off value of PLT count, a multivariate ROC curve was used to test whether combining PLT count can improve the predictive value of GA and BW for BPD.Statistical analysis was performed with SPSS 21.0 software. The normality of the continuous data was tested. Normally distributed variables are presented as the mean ± standard deviation (P < 0.05). The incidence of infection during pregnancy, duration of nCPAP use, reuse of invasive and noninvasive ventilation, duration of invasive ventilation, duration of oxygen supplementation, highest fractional inspired oxygen, and intrauterine growth restriction were higher in the BPD group (P < 0.05).As shown in Table P < 0.05) and severe thrombocytopenia  in the BPD group was higher than that in the non-BPD group. BPD was associated with an increased risk of thrombocytopenia using multiple forward stepwise Logistic regression to adjust for confounders   met the definition of thrombocytopenia at least once during hospitalization, and 46 (34.3%) were diagnosed after the first week of life. From the 7th to 28th day of life, the incidence of thrombocytopenia , but there were no significant variations in the BPD group. With the exception of the first day, the PLT counts in the BPD group were lower than those in the non-BPD group . Using the GEE model to adjust for confounding factors, the PLT counts in the BPD group were lower than those in the non-BPD group . The differences in PCT between the two groups were similar to the differences in PLT counts.As shown in Table P < 0.05 for all). Using the GEE model to adjust for confounding factors, the MPV  and PDW  in the BPD group were higher than those in the non-BPD group . Compared to those with a PLT count > 177*109/L, infants with a PLT count ≤ 177*109/L in the second week had a significantly higher risk of BPD . Subsequently, in the multivariate predictive model, it was showed that the predictive value for BPD was improved using PLT counts ≤ 177*109/L combined with gestational age and birth weight  and its corresponding PLT count at each time point are shown (Additional file 9/L in the second week of life is a potential risk factor for the development of BPD in very premature infants.In this nested case–control study, BPD was found to be associated with a higher risk of thrombocytopenia and abnormal PLT parameters. The abnormal development of the lungs seems to influence the biogenesis of PLTs, and a PLT count ≤ 177*109/L in newborn infants [Thrombocytopenia is a common complication in NICUs, with 18% to 35% of patients developing this problem before hospital discharge. Neonatal thrombocytopenia is defined as a PLT count < 150 × 10 infants , and it  infants . The pre infants . The frePLTs are usually considered to be small pieces of cytoplasm that disintegrate from the cytoplasm of mature megakaryocytes in bone marrow and are involved in a variety of physiological activities. Megakaryocytes, the parent cells that spawn PLTs, are found in the mammalian lung and in the pulmonary vascular beds. The PLT counts in postpulmonary vessels are significantly higher than those in pulmonary arteries . This inBPD is a common chronic inflammatory lung disease of VLBW infants and very preterm infants and is associated with arrested lung development and microvascular dysplasia . ReducinMPV, PDW, and PCT level are three common parameters related to PLT function. MPV is a measurement of the average PLT size, and PDW is an indicator of the variation in PLT size. Normally, MPV and PDW are inversely associated with the PLT count . In someThere are several limitations to the present study. First, although the GEE model was used to adjust for potential confounders, the findings in the present analysis could not totally avoid bias from the differences in GA and birth weight between the two groups. However, in the same conditions, there were no differences in the baseline platelet parameters between the two groups in the first day of life. Thus, we have some confidence that the changes of the platelet parameters in the BPD group after the first week were partly associated with the lung impairment. Second, as an observational study, a causal relationship between BPD and changes in PLT parameters was not confirmed. Third, due to the relatively small size of the study population, it was not possible to perform stratification analysis according to the severity of BPD.9/L as a potential risk factor for the development of BPD.Abnormal PLT parameters were observed in infants with developing and developed BPD, and a reduce PLT count ≤ 177*10Additional file 1. Table S1 Calculation of cut-off discriminating bronchopulmonary dysplasia status.


The simulation results show the driving forces during the charging and discharging process, where there is a variation for the angles of the domain walls from 180° to 90° (and then an increase to 135°), which are the external electric field and domain wall evolution, respectively. As for the phase states, there is a transformation between the orthorhombic and rhombohedral phases which can’t be explained by the traditional polarization switching theory. This work provides a reasonable understanding of the alternating current field effect, which is essential in information and energy storage.In this work, atomically K Aly fields .The discovery of KNN began in 1962, when Jona  first reThe mechanism of domain switching under an alternating current field of ferroelectrics has been widely investigated , e.g., tIn addition, reducing dimension has become a common and effective way to modify the electric property of materials because of their high sensibility to electric fields . Thus, wIn this work, we describe and explain the domain pattern, phase state, and energy evolution in KNN thin films that are a few atoms thick. In particular, an interesting mechanism is proposed, i.e., a z-axis external electric field that can promote the formation of in-plane vortexes, and, further, to realize ultrahigh data processing and enhance the storage abilities of electric energy .The contributions of the domains and domain walls drift are expressed as polarization 1,P2,P3) . The dom1,P2,P3) :(1)∂PifLandgiven as :(5)fgradessed as :(6)     ribed as :(8)felecIn this paper, we use quasi-2D discrete grids of The initial state of the simulation is the same preset domain structure , i.e., tThe phase states and average polarization are shown in For the A state in 3 while there is a contrast tendency for both K0.75Na0.25NbO3 and K0.5Na0.5NbO3, but the average polarization of all KNN thin films remains lower in the C state than those in the A state. It should be noticed that the tendency of average polarization shows the same with the R-phase. Thus, the preliminary conclusion can be speculated, i.e., maybe the average polarization of the R phase is higher than the others.There is a reduction for the average polarization, which then increases in KNbOThe above results indicate that although there are some differences in the changes of the KNN thin films with different Na ratios after loading an alternating current field, these changes are mostly reversible, with the unloading of the electric field, and a few irreversible changes also exist.Since the electric field applied in this paper is along the z-axis, only the distribution of polarization in the XOY plane needs to be considered. For reasons of this paper’s length, the situations of Subsequently, the external electric field begins to unload, as shown in However, the new In order to further reveal the relative contribution of the electric field to the domain evolution and phase transition, the different energies for loading and unloading electric fields are systematically studied, as shown in An amazing result shows that an external electric field can alter the direction of polarization in the XOY plane, e.g., the transformation between the In particular, several interesting phase transitions between in-plane phases and the R phase or O phase are shown during the loading and unloading process, which are caused by the gradient energy.Thus, a formal calculation method is constructed to understand the phase transitions during the physical process, and the vortex domains are demonstrated to drive the above transitions. This is what has never been reported in previous work and can be used to modify the energy or information storage properties via a domain project.


Midwives experiencing traumatic births are emotionally affected by this process, lose their self-confidence, and may intend to leave the profession. This study aims to carry out a meta-synthesis of current qualitative research exploring the experiences of midwives witnessing traumatic births.The meta-synthesis consisted of 18 full-text studies in English, obtained from PubMed, Scopus, Web of Sciences, Cumulative Index of Nursing and Allied Health Literature (CINAHL), EMBASE, and PsycINFO databases. The results of the studies were analyzed using the thematic analysis technique. The study includes qualitative, mixedmethod, and full-text studies published between 2000 and 2020 that explored the experiences of midwives and obstetric nurses witnessing birth trauma.The thematic analysis identified seven themes: post-traumatic feelings, posttraumatic stress symptoms, the impact of trauma on professional values, social support, learning from experience, legal process, and reflection of emotions of women experiencing traumatic birth on the midwife.Midwives who witnessed traumatic birth were mostly emotionally affected. They lost their self-confidence and intended to leave their profession. They emphasized the importance of peer support through which they could share their experiences after trauma. Psychological education should be provided to midwives who witness the trauma by specialists, and midwives should be strengthened against the effects of trauma in terms of both the institutional policies where the birth takes place and midwifery-specific legal policies. It does not have to occur due to a major disaster, but it carries an elevated risk of the stress response2. It suddenly occurs without any symptoms and weakens the individual’s defence mechanisms. Thus, it causes individuals to experience loss of control and fear3. Although childbirth is considered to be a positive life experience, adverse events occurring during the intra-natal period may increase the risk for maternal, fetal, and neonatal mortality and morbidity4. Therefore, complications occurring during childbirth can create a situation that fits the post-traumatic stress criteria of the American Psychiatric Association Diagnostic and Statistical Manual5. While it is mothers and/or fetus/neonate who experience the trauma at birth, midwives who care for mothers and communicate with them emotionally and empathically are also witnessing this trauma. The two most significant components of professional midwifery care at birth are compassion and empathy. There is a strong bond between midwives, birthing women, and the birth process, in which intense emotions are experienced1. Such strong feelings increase the risk and severity of the emotional stress felt by the midwife in case of an undesirable or traumatic circumstance. This may even result in the midwife experiencing secondary traumatic stress disorder6.A traumatic event is defined as an unpleasant event that may result from an injury, violence, or emotional shock5. Secondary traumatic stress disorder (STSD), on the other hand, occurs as a result of witnessing the trauma and develops symptoms similar to those of PTSD such as sad dreams, constant anxiety, anger, depression, hopelessness, irritability, discomfort stemming from remembering the event, difficulty in concentrating and insomnia7. Studies examining the experiences of midwives following the traumatic event have focused on STSD and PTSD. A study conducted in the Netherlands reported that 13% of midwives feel traumatized following birth trauma, and 2.2% of midwives experience STSD8. Another study in the United States, Beck and Gable1 found that 35% of midwives had PTSD. Wallbank et al.9 also indicated that midwives often felt symptoms of PTSD after a neonatal death, miscarriage, and difficult birth, and consequently avoided similar events as they constantly thought about the trauma. Although exposure to traumatic events may not always fit PTSD criteria, it could cause symptoms that adversely affect the psychological health of the individual10.According to DSM-V, Post-Traumatic Stress Disorder (PTSD) is triggered by an important traumatic event and develops as a state of excessive arousal in a person, an avoidance of stimuli that reminds the person of the trauma, and re-experiencing the traumatic event through dreams, nightmares, and flashbacks. PTSD is a mental issue in which a person experiences symptoms for at least one month following a traumatic event11. Rice and Warland6 describe the experiences of midwives witnessing traumatic births with ‘guilt, weakness, and responsibility’. Another study noted that midwives had difficulty in providing professional care and maintaining professional values for the mother experiencing trauma and suffered from witnessing the traumatic birth1. Calvert and Benn3 also reported that midwives experienced such intense feelings as loss, grief, loss of self-confidence, and violence after a traumatic birth that they needed support. Evidence shows that midwives experience guilt, failure, frustration, shame, intend to leave the profession, and PTSD13. Hence, it seems critical to explore how midwives’ experiences of traumatic births could adversely affect their mental health and the quality of midwifery care during childbirth and the early postpartum period employing a meta-synthesis of qualitative studies focusing on the experiences of midwives witnessing traumatic births. The study aimed to conduct a meta-synthesis of current qualitative research exploring the experiences of midwives witnessing traumatic births. The research question was: ‘What are the experiences of midwives witnessing a traumatic birth?’.Traumas experienced by midwives during the birth process may lead to burnout, unmet care, emotional fatigue, deterioration of interpersonal relations, increase in conflict, dejection, and other similar problemsA meta-synthesis was carried out to synthesize and interpret qualitative research about the experiences of midwives witnessing traumatic births.This review was limited to articles published in English between 2000 and 2020. First, we scanned the relevant literature in PubMed, Scopus, Web of Sciences, CINAHL, EMBASE, and PsycINFO databases using the following keywords:Population: Midwife OR midwives OR nurse-midwives OR nurse-midwife OR nurse-midwives OR midwifery OR obstetric nurse OR nurse in the delivery room.Outcomes: Trauma OR secondary trauma OR posttraumatic stress OR post-traumatic stress OR secondary traumatic stress OR Second victim.Study design: Not quantitative, not review. Finally, we performed a general search on Google Scholar with keywords and obtained related studies.The study covers the qualitative, mixed-method, and fulltext studies published in English between 2000 and 2020 that explored the experiences of midwives and obstetric nurses on witnessing birth trauma. Case studies, book chapters, abstracts, reviews, theses, editorials, and studies investigating the traumatic experience of other health professionals such as obstetricians and women who give birth were excluded from the review.As a result of the literature scanning, 550 studies were obtained, of which 292 were duplicates and 4 could not be reached in full text. The remaining 214 studies did not meet the research criteria according to their titles and abstracts. The detailed review of other 40 studies showed that 4 studies were related to midwifery students, 4 were related to other health professionals, 3 articles were related to intervention experiences, 2 studies were a qualitative survey study, 1 was conducted over the phone, and 1 was related to post-education experiences. After excluding all the irrelevant articles, 18 studies were left meeting the research criteria .Two researchers  scanned the articles by reading their titles and abstracts. An opinion exchange was made by the researchers when a disagreement arose regarding the inclusion criteria. The number of studies included in the meta-synthesis is shown in 14. The checklist consists of 10 questions evaluating the purpose, the research question, data collection and analysis method, supporting the results with the findings, the context, validity, and reliability of the research. The scoring of the checklist is evaluated as Yes (2), Partial (1), and No (0), with a range in points in the checklist 0–20. The total score for each article is calculated by summing the total score from 10 items and dividing it by 20. In this study, the total score of 10 items was divided by 20 and the quality of the articles was given as a percentage. The quality of the articles varied between 80% and 100% (Supplementary file).The researchers assessed each of the qualitative studies to determine their quality using the checklist developed by Kmet et al.15 was used for the analysis of the data. We first read the data to become familiar with them and identify remarkable statements about the participants’ experiences. In the second step, initial codes were generated by putting all the common expressions together, and then listed and organized into meaningful groups. In the third step, themes were generated by bringing together similar initial codes. The generated themes were subsequently reexamined, defined and named in the fourth step. In the fifth step, the themes were identified, and which aspects of the data are effective in identifying the themes were clarified, followed by reporting the analysis. The results of the analysis were checked by an external person.The thematic analysis approach including six steps by Braun and Clarke16. All the steps mentioned above were carefully followed in this review.As in all scientific studies, qualitative research also deals with producing valid and reliable information within the ethical rules. To ensure the validity and reliability of the meta-synthesis studies, the research problem, data scanning databases, keywords, the inclusion criteria for the meta-synthesis, the synthesis of the data, the duration of synthesis, and by whom it was made, must be clearly statedAccording to systematic search analysis, 18 studies meeting the research criteria were included in the review. The studies were mostly published between 2013 and 2020, six had the mixed-method, and 12 had the qualitative research design. A semi-structured interview form was applied in all the studies. Generally, thematic and content analyses were used in the studies. The number of samples determined using the purposive sampling method varied between six and twenty-two. The studies have been published in journals of midwifery, birth, nursing, women’s health, and healthcare. Details of the articles are presented in Seven themes were identified in the thematic analysis: post-traumatic feelings, post-traumatic stress symptoms, the impact of trauma on professional values, social support, learning from experience, legal process, and reflection of emotions of women experiencing traumatic birth on the midwife .Post-traumatic feelings of midwives after trauma were identified as ten sub-themes: being shocked, crying, feeling the chaos, feeling helpless, being disrespected as a midwife, feeling powerless, sad, guilty, shame, disappointed, and feeling like a failure .After a traumatic birth, midwives had post-traumatic symptoms such as flashbacks, nightmares, inability to forget, and avoidance. Sub-themes and expressions of midwives are given in The impact of trauma on midwifery professional values was investigated under three sub-themes including loss of confidence in professional practice, intention to leave the profession, and maintenance of professional values. The subthemes and expressions of midwives are given in Midwives emphasized that they need post-traumatic support, which is very important. Two sub-themes were identified.Post-traumatic midwives expressed their need for social support as follows:1‘We need a safe forum to share with our colleagues. If there is an adverse outcome, we are told to keep silent. There is no place to talk to unburden our souls.’ 19‘Maternity ward manager is absent. Support after a critical incident depends on which colleagues you work with that day.’ A midwife said that after being traumatized, people were disrespectful towards her and her inability to get support from her colleagues made her feel like a neglected animal.17‘I talked to the doctor, but I needed my colleagues to support me. People treated me with disrespect. I came to work the next day, and one of the midwives asked me when I would stop causing tears for all the women that I am taking care of because she heard that I was constantly doing this ...’ Some midwives felt lucky for receiving support from their colleagues and expressed their feelings as follows:18‘I feel very fortunate to be working in an environment where I am always supported as a novice nurse, and I mean I'm working with these people … I think that it is a tremendously gifted position to be in, to be respected as a novice nurse.’ One midwife explained her relief after talking to someone about her post-traumatic experiences as follows:21‘Once you've talked to somebody about it properly, it feels as if a weight is just lifted off your shoulders, and you can speak about it, and you feel like you, you know you've just got it off your chest and you can sort of move on in a way.’ Midwives explained that past traumatic experiences were a learning process for them:7‘I think back to that first situation that I had, and I think of how better I responded in that emergency. How much I had grown and how … much more comfortable I felt just… I know that those experiences are important to help us learn ...’ 11‘I've used it as a learning tool, I've kind of tried to turn it the other way round and think what I can use from this, and I've used it to regain my confidence, I've used it to cope with similar scenarios, how I deal with those kinds of stressful scenarios …’ The legal process was examined under two sub-themes: sadness due to the lawsuit and collecting evidence for it.After the traumatic birth, the midwives expressed their sadness due to the lawsuit filed against them with the following:24‘I was involved in the resuscitation of a baby who did poorly at birth. I had not delivered the baby but saw the family at the birth center for office hours and offered my assistance when resuscitation was needed. I visited the mother and baby in the neonatal intensive care unit, and she thanked me for saving her baby's life. I was shocked when several months later I was named in a lawsuit … I could not eat and sleep. The case was eventually settled. It was all about money.’ 20‘The obstetrician and the hospital attempted to apportion blame me during the three years and five separate investigative processes. I was very upset.’ 22‘Battling all the time against the system at traumatic childbirth.’ It is stated that gathering evidence is very important for the midwifery profession with the possibility of a lawsuit that will be filed after the traumatic birth:24‘I had a patient who experienced a placental abruption in labor several years ago. Although I knew I had not done anything clinically wrong, I feared that there would be a lawsuit, so I took copious notes to help me remember the clinical facts. Sure enough, the case came to trial several years later; the jury found in my favor. A student studying midwifery should accept today, that one day they could be sued for a poor outcome in practice.’ Some of the midwives stated that women suffered a lot and felt hopeless in traumatic births:26‘Some birthing women had longer first stage of labor, and they felt discouraged and hopeless. The long labor time and the pressure from other birthing women may make things worse.’ Some of the midwives expressed that attending the funeral is important for them and the family when the baby is lost as a result of traumatic birth:19‘It was just the parents, the priest, and me. I spent the evening after the funeral with the parents. I think it was as much my needs as for theirs … A good dialogue with the couple in retrospect helps to process the event. It is difficult when you can't communicate with the parent.’ The study aimed to systematically examine the studies exploring the experiences of midwives who witnessed traumatic births. Although all studies have different objectives, they all contributed to the experiences of midwives in traumatic births, and their themes mainly focus on post-traumatic stress disorder symptoms. As a result of the secondary qualitative analysis of the primary qualitative studies, seven themes emerged. These themes were: post-traumatic feelings, post-traumatic stress symptoms, the impact of trauma on professional values, social support, learning from experience, legal process, and reflection of emotions of women experiencing traumatic birth on the midwife.23. A study conducted with 18 midwives in Israel reported that midwives experienced sadness, crying, disappointment, and shock due to the loss of the baby17.Midwives in this study experienced negative feelings during and after the trauma. As stated in the sub-themes, midwives felt shocked, guilt, chaos, and helpless, and they cried during the trauma and felt helpless, powerless, sad, guilt, shame, and failure after the trauma27. In a mixed-method study by Toohill et al.20 it was reported that approximately 94% of midwives experienced a traumatic birth, and one in ten midwives experienced fear at birth. The findings of this study are consistent with the literature. The traumatic birth experience of the midwife may increase her/his fear of birth. The midwife’s fear of birth is a risk for decreased quality of midwifery care in the philosophy of maintaining the normality of birth12. Female midwives can also be psychologically more sensitive in stressful and traumatic births. The fact that the midwife has experienced the birth itself and the experience of trauma in the birth of her baby are the factors that increase the risk of being negatively affected by the birth trauma10.In a study in Australia, more than half of midwives felt fear and guilt during or immediately after trauma5. This study revealed that midwives experienced some of the symptoms of post-traumatic stress . Negative emotions such as shock, crying, guilt, and helplessness experienced by midwives during and after birth trauma may have been predisposing factors for post-traumatic stress disorder24. As a result of the analysis, it was seen that midwives having PTS symptoms experience birth traumas due to maternal and infant death, shoulder dystocia, and deep perineal tears. Like this study, in the study of Leinweber and Rowe27, midwives who cared for women with birth trauma reported that they experienced secondary traumatic stress.Birth is defined as a condition with a high potential to fit post-traumatic stress criteria in DSM-V24. Besides, the literature emphasizes that the midwives may experience post-traumatic stress disorder in cases in which the empathic approach cannot be controlled in midwife–woman relationships, and occupational burnout that may damage the nature of midwifery care29. Trauma witnessed by midwives in this study caused loss of confidence in professional practices, intention to leave the profession, and difficulty in sustaining professional values17. Halperin et al.17 reported that some midwives quit their jobs after being involved in a birth with a negative outcome and rested for the rest of their lives.Negative feelings and post-traumatic stress symptoms experienced by the midwife during and after trauma adversely affect the current and future mental health of the midwife, the quality of midwifery care, and their private and professional lives23. In a qualitative study, non-empathic behaviors, and some applications  of midwives30 and the negative attitude and ability of midwifery in the previous birth of multigravidas in another study31 were found to have an impact on negative birth experience and traumatic birth perception. However, the midwife’s empathic and emotional relationship with women can reduce the risk of trauma. Rice and Warland6 emphasized that midwives’ interest in the feelings and emotions of women who give birth may affect the successful management of traumatic births. In this study, some midwives needed social support from their colleagues in the post-traumatic period, which was valuable for them in coping with trauma. Literature also cites that the midwife needs more social support, especially in challenging deliveries requiring medical intervention23. In traumatic births, the midwife, the birthing woman, the other healthcare workers, and the hospital management should have an empathic relationship. The midwife, who cannot get enough social support from her colleagues, institution, and manager, feels lonely and helpless23. This situation leads to the midwife’s inability to use his/her professional capacity sufficiently, professional burnout, a decrease in job satisfaction7, and the desire to quit the profession18. It should be remembered that adequate social support to the midwives will contribute to their self-confidence in their knowledge and skills to promote normal birth21.If the midwives exposed to trauma are not treated, the mental health of the women that these midwives care for is at risk33. Following traumatic birth, some midwives experience growth in personal strength, appreciation of life, relating to others, existential and spiritual change, and new possibilities33. Post-traumatic peer support and personal characteristics affect traumatic growth. Growth in midwives after a traumatic birth experience provides the empathic approach of the midwife to women who experience a traumatic birth, increases the quality of midwifery care, ensures that their experiences are shared with their colleagues, and strengthens professional cooperation10. After the trauma, some midwives in this study considered their trauma as something contributing to their learning and awareness30. Elmir et al.32 stated that midwives in difficult births experienced weaknesses, failures, and pain, but later these experiences turn into positive contributions, which helped them to develop strategies and problemsolving skills to cope with difficult situations and to guide their colleagues. Moreover, post-traumatic growth can reduce fear and increase trust34. Research has demonstrated that midwives can professionally be stronger, establish a more positive midwife–woman relationship after trauma, and this experience will increase the power to cope with future traumatic events34. One of the midwives in the study by Fontein-Kuipers et al.10 expressed the posttraumatic growth after a traumatic birth as follows:Traumatic experiences do not always result in negative consequences. They may contribute to post-traumatic growth such as becoming more confident, and growing personally, spiritually, or professionally‘At the end of the day, it allowed me to grow … I wouldn't be where I am right now if it [event] wouldn't have happened … maybe it even made me a better person … a better midwife.’As seen in the expression of the midwife, posttraumatic growth can provide learning from experiences and psychological growth.31. This study revealed that the midwife’s sadness due to the lawsuit filed against her or her collecting evidence against the possibility of a lawsuit was a further trauma for her. In countries where midwifery education, relevant associations, and laws, are not strong enough, midwives are at greater risk of birth trauma35. Besides, the state does not pay compensation insurance for midwives in some countries, so midwives are supposed to pay it36.The legal process after birth trauma can further deteriorate the birth trauma of the midwife31. Haung et al.26 determined in their study that prolonged labor, high severity of birth pain, and baby loss as the reasons for the traumatic perceptions of women from the midwives’ perspective.Factors such as prolonged labor, severe perception of birth pain, interventions applied at birth , and negative communication of midwives during birth could lead women to perceive birth as traumatic25. In some traditionally structured communities, having a male newborn baby is seen as the continuation and status gain of the lineage. In addition, the misconception that ‘the female chromosomes determine’ the gender of the baby is dominant28. When a woman gives birth to a baby girl, her maternity status is ignored, and she is subjected to psychological violence by the family and the environment21. In a study conducted in China, midwives explained that women who gave birth to a baby girl are not accepted by their families, and they feel collapsed and experience traumatic birth22.Midwives experience secondary trauma after birth due to being exposed to the negative attitudes and behaviors of the mother, spouse, and family, reacting to the gender of the newborn baby35. The midwives in the study by Christoffersen et al.19 emphasized that it felt good to attend the funeral of the baby lost at the end of the traumatic birth with the family.To reframe and make sense of the situation after a traumatic situation, healthcare professionals use emotional  and task-oriented  coping methodsOur results showed that midwives who experience traumatic births have similar experiences even in different countries. Midwives who witness the trauma are mostly emotionally affected by this process. They lose their self-confidence and feel a desire to leave their profession. They emphasize the importance of peer support, through which they can share their traumatic experiences.Psychological education should be primarily provided to midwives who witness the trauma by health professionals. Besides, since all health professionals working in the delivery unit  are also at risk of trauma, psychological education should be given to them at regular intervals for their mental health, even if they have not experienced trauma so far. It is seen that birth traumas are generally experienced after a difficult birth. Therefore, in-service training with the most realistic training methods (such as simulation) should be implemented to promote the ability to cope with problems at difficult births, to benefit from the experiences of other midwives, to prevent malpractice, and to reduce occupational burnout. The mother and her family, who have undergone trauma at birth, can experience trauma by committing psychological and physical violence against the midwife. Midwives, therefore, need to be strengthened for the effects of trauma  both in terms of institutional and midwifery-specific legal policies. In the post-traumatic period, associations supporting all reproductive health, especially midwifery associations, should take a supportive approach to protect the rights of midwives and to protect/ increase their commitment and their sense of belonging to the midwifery profession. To increase midwives’ ability to cope with trauma, to avoid its long-term effects, and to maintain their professional values and autonomy, they should be mentally and socially supported by their colleagues, maternity unit, and institution managers.