We aimed to evaluate diffusion tensor imaging (DTI) in infants born extremely preterm, to determine the effect of erythropoietin (Epo) on DTI, and to correlate DTI with neurodevelopmental outcomes at 2 years of age for infants in the Preterm Erythropoietin Neuroprotection (PENUT) Trial. Infants who underwent MRI with DTI at 36 weeks postmenstrual age were included. Neurodevelopmental outcomes were evaluated by Bayley Scales of Infant and Toddler Development (BSID-III). Generalized linear models were used to assess the association between DTI parameters and treatment group, and then with neurodevelopmental outcomes. A total of 101 placebo- and 93 Epo-treated infants underwent MRI. DTI white matter mean diffusivity (MD) was lower in placebo- compared to Epo-treated infants in the cingulate and occipital regions, and occipital white matter fractional isotropy (FA) was lower in infants born at 24–25 weeks vs. 26–27 weeks. These values were not associated with lower BSID-III scores. Certain decreases in clustering coefficients tended to have lower BSID-III scores. Consistent with the PENUT Trial findings, there was no effect on long-term neurodevelopment in Epo-treated infants even in the presence of microstructural changes identified by DTI. Advances in neonatology have led to unprecedented improvements in neonatal survival such that infants born at 22-0/7 to 25-6/7 weeks’ gestation now have a 70% survival rate . UnfortuAt least 50% of infants born prematurely have qualitative evidence of cerebral white matter injury on magnetic resonance imaging (MRI) at term-equivalent age ,7. TheseMicrostructural descriptions provide a valuable insight into the anatomic effect of injury on premature brain development; however, it is not well understood how these changes affect subsequent brain function. To address this question, DTI imaging can be used to model the brain as a complex grid of interconnected regions (nodes) with visual and quantitative characterization of the organization between nodes, referred to as connectivity . One comThe PENUT Trial was a randomized placebo-controlled trial studying the effects of erythropoietin (Epo) on death or severe NDI in infants born extremely preterm . Using aThe PENUT Trial included 19 sites across the United States, and enrolled inborn infants 24-0/7 to 27-6/7 weeks’ gestation between December 2013 and September 2016. Patients were excluded based on known life-threatening disorders, chromosomal anomalies, disseminated intravascular coagulopathy, twin-twin transfusion, hematocrit > 65%, hydrops fetalis, or known congenital infection [The PENUT Trial enrolled infants after informed parental consent. This study was IRB approved at all participating sites, and registered with ClinicalTrials.gov (NCT01378273) and the FDA (IND#12656) .p value of 0.32 (no significant difference).Five study sites acquired data from Siemens 3T MR scanners and three acquired data from Philips 3T MR scanners. Acquisition protocols were similar, although not identical, between scanners, and the number of diffusion gradient angles obtained was limited by the duration of scan time tolerated by the primary study protocol. Phantoms were scanned using the same infant MRI protocol to test for scanner quality at all sites. At the beginning of the trial, an MRI scan of the same person at all sites (SEJ) was performed to improve comparison across centers. Whole brain DTI FA values were compared taking an average and a standard deviation and then calculating a percent of the standard deviation of the average across all sites . The FA means of the Siemens and Philips scanners were 0.198 and 0.194, respectively, with a two-tailed 3, 55 slices, interleaved acquisition of slices, field of view 224 mm, fat suppression on, 30 different diffusion gradient angles + one non-diffusion volume, b-value 1000 s/mm2, 2 averages.Siemens MR acquisition protocol: The diffusion-weighted MR sequence was collected in the axial plane with an echo-planar diffusion weighted spin-echo pulse sequence with the following parameters: echo spacing 0.71 ms, EPI factor 112, TR/TE 7000/73 ms, voxel size 2 × 2 × 2 mm3, 55 slices, field of view 180 mm, 32 different diffusion gradient angles + one non-diffusion volume, b-value 1000 s/mm2, reconstructed matrix 128 × 128 × 55.Philips MR acquisition protocol: The diffusion-weighted MR sequence was collected in the axial plane with an echo-planar diffusion weighted spin-echo pulse sequence with the following parameters: TR/TE 7733/73 ms, echo-train-length 45, voxel size 1.4 × 1.4 × 2 mmAs described in the FMRIB Software Library v6.0, motion and distortion of diffusion-weight images were corrected for using FSL eddy software ,26. For MD is a mean measure of the magnitude of water diffusion detected across three gradient directions  in mm2/s. Diffusivity along and parallel to the principal axis is measured by L1  and has been associated with the axon diameter, whereas diffusivity in directions perpendicular to the principal axis of diffusion are measured by the average of L2 and L3  and has been associated with the degree of myelination and number of branching exiting fibers ,30,31. Ep < 0.05 [http://stnava.github.io/ANTs/, accessed on 10 February 2019). Instead of relying on standard tract-based spatial statistic (TBSS) skeleton projection (which is dependent on adult white matter/DTI characteristics), this procedure builds a template from all of the subjects and also co-registers the individual FA maps to the same whole brain template. The ANTs procedure has been validated as a rigorous non-linear approach to co-register subject brains to a subject template [p-value map corrected for multiple-voxel comparisons using the threshold-free cluster enhancement (TFCE) option. Per Spisák et al., “TFCE integrates cluster information into voxel-wise statistical inference to enhance detectability of neuroimaging signals” [Following transformation of all subjects into the same space for direct comparison, voxelwise statistical analysis of the DTI data was carried out using a two-group design with statistical significance defined as p < 0.05 ,33. Co-rtemplate ,35. Indetemplate . ANTS sotemplate ,38. 1000signals” .Region of interest (ROI) analysis and identification of brain region anatomy of the infant MRI brains was based on atlases and templates created in the laboratory of Dr. John E. Richards . While tp values < 0.02) found on the axial diffusivity TFCE randomise statistical map created by comparing the 2 main groups. These ROIs/clusters were near AAL regions described as follows: AAL region 50 superior occipital gyrus, AAL region 52 middle occipital gyrus, AAL region 34 middle cingulate cortex, AAL region 20 supplemental motor area. The ROIs chosen in the basal ganglia were based on prior reports of brain injury found in the basal ganglia, and these regions were close to AAL region 73 Putamen Left and AAL region 74 Putamen Right [Two ROIs in the white matter  and three in the grey matter  as described in the Automated Anatomical Labelling atlas were evaluated . The ROIhttps://sites.google.com/site/bctnet/construction, accessed on 15 February 2019) uses clustering coefficients to perform the complex network/graph theory analysis, as described in Rubinov and Sporns [As an indicator of network segregation, clustering coefficient measures functionally distinct networks that have been linked to separate and measurable cognitive processes ,43. We cd Sporns . DTI quad Sporns . The clud Sporns ,51. Braid Sporns .Neurodevelopmental outcomes at 22–26 months CA were evaluated using the Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID-III). Individuals performing the BSID-III assessments were centrally certified and blinded to the child’s medical history, treatment arm, and brain imaging studies.A modified intent-to-treat (ITT) approach was used in all analyses, with all randomized infants who received the first dose of study treatment included in the analyses. For all statistical comparisons between groups, Generalized Estimating Equations (GEE) with robust standard errors and a working independence correlation structure were used to account for inclusion of infants from a multiple gestation . ComplicThe primary analysis was a comparison of DTI values (extracted by the FSL software) between randomized treatment groups. Specifically, a GEE-based Wald test was used to examine differences in diffusion values and clustering coefficients between treatment groups, with adjustment for gestational age (GA) at birth used to stratify treatment randomization (24 + 0 to 25 + 6 vs. 26 + 0 to 27 + 6 in weeks + days of GA), sex, and scanner type used at the enrolling hospital. In secondary analyses, for any DTI measurements found to significantly differ in the main comparison, an interaction term between treatment and GA at birth was used to explore treatment effect moderation by GA.p-values generated from the m = 15 tests of DTI diffusion values by treatment group were sorted from smallest to largest and compared against nominal levels of 0.05/m, 0.05/(m−1), 0.05/1, where statistical significance was declared for all p-values smaller than the smallest indexed p-value such that Pk > 0.05/(m + 1−k). Clustering coefficients (N = 66) were assessed overall and by region using a Manhattan plot of the p-values, with a similar Bonferroni-Holm correction procedure for declaration of statistical significance. Tests of treatment interactions with GA were considered exploratory analyses.Adjustment for multiple testing among the 15 DTI measurements  was handled with the Bonferroni-Holm procedure, with the aim of controlling the overall type I error rate at 0.05. L1, L2, and L3 values were excluded from analysis as the correlation coefficient between these gradients and MD in all ROIs was close to 1 (data not shown). GEE analyses were used to estimate the association between DTI measures and BSID-III cognitive outcomes at 2 years CA with adjustment for treatment assignment, GA, sex, and study recruitment site. All statistical analyses were performed using the R statistical software package .Funders did not have any role in study design, data collection, data analyses, interpretation, or writing of report.Of the 741 infants enrolled in the PENUT Trial, 469 infants were enrolled across eight designated MRI sites (229 placebo-treated and 240 Epo-treated), with approximately equal stratification within each group for 24–25 week and 26–27 week GAs. One hundred and one infants in the placebo group and 93 infants in the Epo group underwent an MRI  and were included. Of these, 81 and 73 underwent 2 year neurodevelopmental assessments, respectively .As entry into the MRI cohort was conditional upon surviving until 36 weeks PMA, demographic and clinical characteristics were compared between treatment groups (Epo vs. Placebo) within the imaging cohort as well as between the MRI cohort and infants who were enrolled at MRI recruitment sites, survived to 36 weeks PMA, but were not in the MRI cohort. p < 0.05) and were more likely to demonstrate attributes associated with survival including delayed cord clamping , higher mean birth weight , and higher Apgar score at 5 min .While there were no substantial differences between the treatment groups within the MRI cohort, those in the MRI group more commonly had a Hispanic mother  or grade III/IV intraventricular hemorrhage (IVH) .There were no statistically significant differences between the Epo and placebo groups or between the MRI and non-MRI cohorts in necrotizing enterocolitis (NEC), spontaneous intestinal perforation (SIP), or retinopathy of prematurity (ROP). When compared to the non-MRI cohort, the MRI cohort had significantly fewer infants with culture proven sepsis  and motor : −6.6 (−10.1 to −3.1); p < 0.001) scores at 2 years of age were significantly higher in the MRI cohort compared to the non-MRI cohort, again reflecting a healthier cohort overall. There was no statistically significant difference in BSID-III language scores between groups (adjusted difference (95%CI): −0.6 (−6.1 to −4.9); p = 0.73).Iron deficiency evaluated by serum ferritin was also queried as significant iron deficiency can result in delayed myelination ,56. In c2/s, mean ×10,000: 13.25 vs. 13.70; adjusted difference: 0.49; 95% CI: 0.24 to 0.73; p < 0.0001) and occipital  ROIs  ROIs c,d. No oThere were no statistically significant differences in clustering coefficients between placebo- and Epo-treated infants b. An exap = 0.0027) despite obtaining the MRIs at the same PMA   relative to those born at 26–27 weeks’ GA  b,d, thouThere were no significant treatment by GA interactions in any white or grey matter ROI DTI diffusion values a.p < 0.0001) (Treatment-related differences in clustering coefficients were significantly moderated by GA b. The ri 0.0001) c.Neither white nor grey matter DTI diffusion values were associated with BSID-III cognitive, motor, or language composite scores at age 2 a.p = 0.005) (p = 0.009) (p = 0.002) and right paracentral lobule area  were positively associated with cognitive scores on the BSID-III (p = 0.002) (p = 0.008)  b,c and i= 0.009) b,d. IncrBSID-III b–d. EachBSID-III c. BSID-I= 0.002) b,c and i= 0.008) b,d. NoneEpo is an important trophic factor during fetal brain development and has robust neuroprotective effects in preclinical models of brain injury ,61,62,63Our hypothesis, that Epo treatment would improve MRI indicators of myelination and connectivity in extremely preterm infants, was not supported. We anticipated an increase in white matter FA, and decreased MD, both of which are associated with increased myelination. Indeed, we observed the opposite, with no difference in FA, and Epo-treated infants showing increased white matter MD. This is in contrast to findings from the Swiss EPO Neuroprotection Trial in which 165 infants  with mean GA at birth of 29 weeks underwent MRI at term equivalent age and showed fewer areas of gross white matter injury and increased FA in the group treated with Epo ,71. ThatWe speculate that iron deficiency, and not Epo itself, may have contributed to the increased white matter MD values seen in our extremely preterm Epo-treated patients. Iron is required for normal brain maturation as it is essential for synaptogenesis, myelination, and dopamine synthesis, and iron deficiency during critical windows of development  may lead to irreversible developmental deficits ,75,76. WInjury to cerebral white matter from cytotoxic edema and ischemia have also been shown to cause demyelination and oligodendrocyte death during early development . These cAside from acute inflammatory complications, extremely premature infants also remain particularly vulnerable to sustained inflammatory states such as those caused by recurrent hypoxic insults, oxidative stress, hypotension, and CLD . SeveralWe found it notable, however, that infants with decreased clustering coefficients in specific brain regions tended to have worse neurodevelopmental outcomes. In our study, BSID-III motor scores positively associated with increased clustering coefficients in the left middle occipital lobe and in the right paracentral lobule area. While these regions serve multiple functions, they each play a significant role in motor function ,91. SimiThere were several limitations to this study. First, scanner gradient and scanner RF imperfections cause DTI measurements to be imperfect, resulting in greater signal noise in the more peripheral, cortical regions of the brain . HoweverIn summary, extremely preterm infants remain at high risk for neurodevelopmental impairment. Early treatment with Epo did not provide structural protection of cerebral white matter as assessed by DTI. While commonly used DTI measures of white matter integrity  were not linked to neurodevelopmental outcomes, changes in cerebral clustering coefficients at 36 weeks’ PMA were positively associated with BSID-III motor, cognitive, and language scores at 2 years of age, a link which warrants further study. Investigation into neuroprotective therapies is ongoing; advanced DTI techniques may provide insight into connectivity-directed therapies to optimize neurodevelopmental outcomes in infants born extremely preterm.


Birth parameters have long been reported to have a role in human intelligence. However, the causalities reported in previous observational studies were controversial. Our study aims to provide an unbiased investigation of the causal associations between birth parameters and human intelligence using the Mendelian randomization (MR) approach.N = 10,768), birth length , and birth weight . Data for intelligence were obtained from a meta‐analysis of genome‐wide association studies of 269,867 individuals of the European ancestry. Primary MR analysis was performed using the standard inverse‐variance weighted method, and sensitivity analyses were performed using the weighted median, MR‐Egger, and MR‐PRESSO methods.Genetic instrumental variables for MR analyses were extracted from large genome‐wide association studies of infant head circumference (IVW=2.05 × 10–9). The causal relationship was robust when sensitivity analyses were performed. However, birth length and birth weight had no significant associations with intelligence.Using 10 single nucleotide polymorphisms as instrumental variables, we found that 1 standard deviation increase in infant head circumference was associated with 0.14‐fold higher scores in intelligence tests (β = 0.14, 95% confidence interval: 0.09 to 0.18, POur findings suggested infant head circumference, but not birth weight and length were associated with intelligence, which might indicate that brain development rather than general fetal growth was responsible for the development of intelligence. We performed a Mendelian randomization study to investigate the causal effects of infant head circumference, birth length, and birth weight on human intelligence by extracting genetic association datasets from large‐scale genome‐wide association studies of birth parameters and human intelligence. Our findings suggest that that head circumference, but not birth weight and length, is associated with intelligence, which might indicate that that brain development rather than general fetal growth was responsible for the development of intelligence. We extracted 10 independent single nucleotide polymorphisms (SNPs) that were strongly (p < 1 × 10−5) associated with infant head circumference from the GWAS  by Taal et al.  by Valk et al.  by Warrington et al. reported 146 (of which 8 were not available in the GWAS summary dataset of intelligence) independent SNPs  according to standardized procedures and data were standardized (data were transformed into sex‐ and age‐adjusted standard deviation scores) using Growth Analyser . The percentage of variance explained (R2) was calculated using the formula 2×minor allele frequency ×  × (β estimate)2, whereas the F statistic could be calculated from the R2 statistic as F = (N‐K‐1)/K × R2/(1‐R2), where N is the sample size and K is the number of SNPs .We obtained data on genetic associations with human intelligence from a large‐scale meta‐analysis of GWASs of 14 cohorts, which were comprised of 269,867 individuals of European ancestry .Firstly, we extracted SNP effects and corresponding standard errors for selected IVs from the GWASs of both exposure and outcome. Datasets were then harmonized aligning allele information. Primary MR analyses were conducted using the standard inverse‐variance weighted (IVW) method. Generally, the IVW method entails the basic assumption that each SNP is a valid instrumental variable  increase in infant head circumference was associated with 0.14‐fold higher scores in intelligence tests : 0.09 to 0.18, PIVW = 2.05 × 10–9). The weighted median  and MR‐PRESSO  analyses showed similar results with the IVW method. Notably, analysis with the MR‐Egger method showed a non‐significant association between infant head circumference and intelligence. This is because the MR‐Egger method is out of work when all variants showed the same direction of causal link . These non‐significant results were also observed after statistical analyses . For birth weight, we found that a 1‐SD increase in birth weight was associated with better performance in intelligence tests . However, the statistical P value did not reach the Bonferroni‐corrected threshold of p <.016. The MR‐PRESSO method produced a similar result (PMR‐PRESSO = 0.024), but analyses using the weighted median and MR‐Egger methods showed no evidence of association . Thus, our study results showed that the genetic determinants of birth weight were not strongly associated with intelligence.Our study results provided no evidence that indicate the presence of a causal relationship between birth length and intelligence  presented significant associations with intelligence. SBNO1, the corresponding gene of rs7980687, is associated with intellectual disability ; more samples should be collected for future GWASs to generate more genome‐wide significant SNPs. Second, our study captured multiple genetic variants that were associated with both infant head circumference and intelligence; however, their molecular mechanisms needed to be studied further. Finally, the GWASs for birth parameters were performed using quantitative traits, which cannot capture the causal effects of specific birth characters  on human intelligence.The present study also had some limitations. First, the SNPs for infant head circumference and birth length were selected using a relatively relaxed threshold (5The present MR study suggests that increase in genetically determined infant head circumference is associated with better performance in intelligence tests and that the causal relationship is independent of environmental factors. However, our results provided no evidence that indicates the presence of causal relationships between birth length, birth weight, and intelligence. These findings might indicate that that brain development rather than general fetal growth was responsible for the development of intelligence.The authors have no conflicts of interest to disclose.LQ and JY conceptualized and designed the study, conducted the data analysis, and drafted the manuscript. FG and BY collected data and carried out the initial analyses. LY and WW contributed to the interpretation of results. LB, XM, and JY critically reviewed and revised the manuscript. All authors approved the final manuscript as submitted and agreed to be accountable for all aspects of the work.https://publons.com/publon/10.1002/brb3.2183.The peer review history for this article is available at Table S1Click here for additional data file.


Associations between the latent chemical construct, non-chemical stressors and birth outcomes were assessed using path models. The results showed a significant direct relationship between perceived stress and birthweight (β = −0.17), however even though the relationship between perceived stress and depression was significant in all three path models (β = 0.61), the association between depression and birth outcomes was not significant. Perceived stress was significantly associated with community engagement (β = −0.12) and individual resilience (β = −0.12). BMI (β = 0.12) was also significantly directly associated with birthweight. The latent chemical construct did not show an association with the birth outcomes. Our data indicate the need for the development of a support system for pregnant women by involving them in prenatal care programs to reduce maternal stress, which may also influence depression and (in)directly improve the birth outcomes. Interventions regarding weight management for women of childbearing age are necessary to halt obesity and its negative effects on birth outcomes.The cumulative exposure to chemical and non-chemical stressors may have an impact on birth outcomes. The aim of this study is to examine the cumulative exposure of a mixture of chemicals  and non-chemical stressors  on birth outcomes . The study population is a subset ( Chemical and non-chemical stressors have been found to be independently associated with adverse birth outcomes such as low birthweight (LBW), low gestational age at birth and low Apgar score ,2,3,4,5.While there is evidence of an association between either some non-chemical stressors or chemicals and birth outcomes, only a few studies have been conducted that study the cumulative and combined exposure of non-chemical stressors and chemicals on birth outcomes ,12. In aSuriname, a middle-income country in South America, has a multi-ethnic population, consisting of people of Asian , African , Indigenous (3.8%), “Other” descent , and unknown (0.6%) ,18,19. TDespite the potential exposure to high levels of Hg, perceived stress, and probable depression, and high rates of LBW and PTB in Suriname, to date no studies have examined the combined effect of the cumulative exposure of chemicals and non-chemical factors on birth outcomes in Suriname. The aim of this study is to examine the cumulative exposure of a mixture of chemicals  and non-chemical stressors  on birth outcomes in Suriname.This study is part of the Caribbean Consortium for Research in Environmental and Occupational Health (CCREOH)–MeKiTamara study, which is a prospective environmental epidemiological cohort study. Pregnant women were recruited during the first or second trimester of pregnancy in three regions of Suriname: Paramaribo, Nickerie and the Amazonian Interior. The types and levels of exposures are expected to be different among these regions, with respect to both non-essential and essential elements, which may be explained by their diets and metal-based pesticides .n = 1189). The subset of 384 participants from the CCREOH cohort, is a statistically acceptable representation of the entire study cohort (n = 1190). Women were eligible if they were 16 years or older, spoke Dutch, Saramaccan, or Trio, had a singleton gestation, were planning to give birth at one of the study sites and provided written informed consent/assent. Pregnant women were recruited at four hospitals, prenatal clinics and midwife facilities of the Regional Health Department, and at multiple health care clinics of the Medical Mission Primary Health Care Suriname (MMPHCS) in the Interior [The study population is a subset of the MeKiTamara study that recruited participants from December 2016 to July 2019 , the Edinburgh Depression Scale (EDS), and the Social Support List-Interactions-12 (SSL-I-12), all self-report questionnaires, were administered by trained recruiters through face-to-face interviews during the first or second trimester of pregnancy using encrypted iPads. Data on height and weight were also collected at inclusion and used to calculate BMI. BMI calculations were based on body weight during pregnancy. Birth outcomes were collected after birth from parturition records. These data were uploaded in a Research Electronic Data Capture (REDCap) project site database for data cleaning and analysis purposes. REDCap is a secure web application for building and monitoring online surveys and databases to collect data for research purposes and can be used online or offline ,24.Maternal blood was collected at enrollment during either the first or second trimester. Whole blood was collected by venipuncture into 10 mL trace element vacutainers containing K2EDTA. Samples were kept cold (4 °C) for no more than 24 h prior to processing. Two mL aliquots of each sample were transferred to plastic cryovials and then stored at −80 °C.Chemicals: Solubilization and analysis of the whole blood samples for the chemicals  and Cadmium (Cd)) by magnetic-sector ICPMS (SF-ICPMS) were performed by the Trace Element Research Group of the Wisconsin State Laboratory of Hygiene (WSLH) at the University of Wisconsin-Madison, United States. Sample preparation and analysis were carried out in a Class 1000 ISO 6 trace element clean lab at the WSLH. Critical sample handling steps were performed under HEPA-filtered (CLASS 100) all-polypropylene biosafety cabinets.Perceived stress: This was assessed using the Cohen’s Perceived Stress Scale (PSS). The PSS is a widely used measure of perceived stress that has been validated in a number of populations and languages, including Dutch, which is the formal language in Suriname and, in line with our inclusion criteria, the questionnaire was thus administered in Dutch ,26,27,28Probable depression: This was assessed using the Edinburgh Postnatal Depression Scale. This questionnaire has also been validated for assessing prenatal depression. For prenatal purposes, the scale is called the Edinburgh Depression Scale (EDS) . The EDSSocial support: Social support was assessed using the Social Support List-Interactions-12 (SSL-I-12), which includes twelve statements about support, affection, and attention from family and friends. There are four response options: 1 for rarely or never, 2 for occasionally, 3 for regularly and 4 for very often. Before data collection, one question (“Does it ever happen that people confide in you?”) was deleted, because of concerns regarding subjectivity based upon Suriname’s cultural context. Since the SSL-12-I scale was modified, an exploratory factor analysis was implemented, resulting in a two-factor solution: the Individual Resilience subscale  and the Community Engagement subscale (affection/attention from the community) .Demographic Data: Demographic variables were categorized into the following groups: age at intake , household income ), educational level ; race/ethnicity: African descent , Asian descent , or other ); BMI (continuous); and region , rural  and the Interior .Birth Outcomes: Information on gestational age, birthweight and Apgar score was obtained from parturition books completed by midwives in the hospitals, prenatal clinics or midwife facilities where the baby was born.p ≥ 0.05). The data were log transformed because the normality assumption was not met. Even after the log transformation the data were slightly skewed. To account for the skewness the ULS method was used for exploratory factor analysis (EFA). Measurements were done on Hg, Pb, Se, Sn, Mn and Cd. The data were randomly divided into the test and the validation sample. The EFA was conducted on the test sample to reduce the six chemicals into a smaller number of latent construct. The EFA resulted in a one factor solution with high factor loadings of Hg, Pb, Se, and Sn on this factor. Evaluation of the EFA models was based on eigenvalues, amount of variance explained, factor loadings higher than 0.35 and no cross loading greater than 0.25. We used confirmatory factor analysis (CFA) to confirm the latent constructs using the validation sample. The EFA resulted in a one factor solution with no cross loadings , Root Mean Square Error of Approximation (RMSEA) of 0.05, and the adjusted goodness-of-fit index (AGFI) with values greater than 0.95 indicating good fit (Bentler–Bonett Normed Fit Index (NFI ≥ 0.90), and Bentler’s Comparative Fit Index (CFI ≥ 0.90)). Lagrange Multiplier (LM) test was used to identify new causal paths if the fit criteria were not met. The best fit model included the following variables: the latent construct , non-chemical stressors  and the birth outcomes . Covariates such as age, ethnicity, income, education and region were used to describe the sample, but were not part of the path model. The statistical package that was used to analyze the data is SAS 9.4.1 .Furthermore, the t-test was used for significance of the computed Pearson correlation coefficient. We then built a path model using structural equation modelling (SEM) to estimate the associations between the latent chemical construct and non-chemical stressors in predicting birthweight, gestational age and Apgar score. The path model was based on the fit criteria for the SEM models, which included several tests: the Chi-square  of Tulane University (number: 839093) and the Medical Ethical Commission of Suriname’s Ministry of Health (VG 023-14). All women 18 or older gave written informed consent, and assent was obtained from women who were 16 or 17 years old.The median levels of perceived stress, probable depression, community engagement and individual resilience were respectively 17.0 (IQR 13.0–20.0), 7.0 (IQR 4.0–11.0), 4.0 (IQR 4.0–6.0) and 25.0 (IQR 21.0–28.0). The median age of the participants was 28.2 years (IQR 24.2–32.7). The majority of the participants were of African descent (50.0%), followed by Asian descent (29.7%) and other/mixed ethnicities (20.1%). The majority of the participants had lower household incomes (59.1%) and were less educated (54.2%). The median BMI of the participants was 25.9 (IQR 22.6–30.8). The largest group (69.0%) of the participants resided in an urban area. Median concentrations of Hg, Pb, Se and Sn were 2.9 ug/L (IQR 1.7–4.6), 2.0 ug/dL (IQR 1.3–3.1), 191.2 ug/L (IQR 167.4–217.7) and 0.7 ug/L (IQR 0.5–1.0), respectively .p < 0.001), gestational age and birthweight , gestational age and Apgar score , and birthweight and Apgar score . A negative correlation was found between perceived stress and individual resilience , community engagement  and birthweight . A correlation also was observed between community engagement and individual resilience , and between pregnancy-BMI and birthweight   .To examine the cumulative exposure of the non-chemical stressors and the latent chemical construct, three path models were constructed. There was a significant direct relationship between perceived stress and birthweight (β = −0.17); however, even though the relationship between perceived stress and depression was significant in all three path models (β = 0.61), the association between depression and birth outcomes was not significant. Perceived stress was significantly associated with community engagement (β = −0.12) and individual resilience (β = −0.12) see . BMI  . The model predicting the associations between the latent chemical construct, social support, perceived stress, probable depression, BMI and Apgar score also resulted in good model fit .Results of the structural equation path models  showed g = 0.95) . The modModeling exposures cumulatively using a path model may explain how different levels of chemicals together may affect birth outcomes . The anaThe lack of a statistically significant association between the combined chemical latent factor and birth outcomes may be explained by the concentrations of the chemical contaminants, which may be too low to find an association, or that the non-chemical factors outweighed any effects of the chemical exposure. The latter phenomenon was also observed in a study by Pao et al. (2019) in which socioeconomic variables played a dominant role and outweighed other effects, including environmental exposure . Thomas Our results are also in line with our previous study of nearly the entire study population (n = 1143) where we did not find an interaction between depression, stress and Hg, but observed associations between socio-demographic factors, perceived stress, and birth outcomes . The stuThe (in)direct association of social support with perceived stress, depression and birthweight is in line with previous studies ,5. A proPerceived stress was directly significantly associated with birthweight. In the birthweight model, higher levels of perceived stress were associated with low birthweight. This was also seen in a study of 279 pregnant women in a suburban area in the United States, with some similarities to our study regarding demographic variables such as age and education. This U.S.-based study showed that latent pregnancy-specific stress could predict adverse birth outcomes better than other latent factors such as state of anxiety, perceived stress, life event stress and a combined latent factor constructed from all stress measures. Pregnancy-specific stress contributed directly to preterm birth and indirectly through its association with smoking to LBW . There wp = 0.042). Women in our study with a high maternal BMI (55.9% overweight or obese) compared to a normal or underweight BMI were over six times more likely to give birth to a macrosomic infant. A systematic review and meta-analysis found that mothers that were obese (as measured by BMI) prior to conception were more likely to give birth to a child with obesity with increasing child age [In the birth outcome model, the pregnancy BMI of the mother showed a positive direct association with birthweight. A systematic review and meta-analysis of the risk of maternal BMI and neonatal adverse outcomes in China revealed that high maternal BMI compared to normal maternal BMI was associated with fetal overgrowth, defined as macrosomia ≥4000 g, and increased risk of preterm birth ,37,38,39hild age ,42,43.There are some limitations to the study. The psychosocial questionnaires were not specifically validated for the Surinamese setting before data collection. However, these questionnaires are standardized and are widely utilized in research settings, including in low- and middle-income countries (LMICs) . In addiThis study is the first to examine cumulative exposure to environmental contaminants and concentrations of Se as an essential element and non-chemical stressors in Suriname and the entire Caribbean region. Results indicate that combined exposures may influence birth outcomes, with the influence of non-chemical stressors being of particular importance. Our data indicate the need for the development of a support system for pregnant women by involving them in prenatal care programs to reduce maternal stress, which may also influence depression and (in)directly birth outcomes. Interventions regarding weight management for women of childbearing age are necessary to halt obesity and its negative effects on birth outcomes. We suggest further research on the cumulative exposure of chemicals and non-chemical factors, including a larger sample size, more socio-demographic risk factors, and a better assessment of any history of diseases of maternal health and mental health.


Microbes benefit plants by increasing nutrient availability, producing plant growth hormones, and protecting against pathogens. However, it is largely unknown how plants change root microbial communities.Rhizoctonia solani AG8 (hereafter AG8) to examine how plants impact the rhizosphere bacterial community and recruit beneficial microorganisms to suppress soilborne fungal pathogens and promote plant growth. Successive plantings dramatically enhanced disease suppression on susceptible wheat cultivars to AG8 in the greenhouse. Accordingly, analysis of the rhizosphere soil microbial community using deep sequencing of 16S rRNA genes revealed distinct bacterial community profiles assembled over successive wheat plantings. Moreover, the cluster of bacterial communities formed from the AG8-infected rhizosphere was distinct from those without AG8 infection. Interestingly, the bacterial communities from the rhizosphere with the lowest wheat root disease gradually separated from those with the worst wheat root disease over planting cycles. Successive monocultures and application of AG8 increased the abundance of some bacterial genera which have potential antagonistic activities, such as Chitinophaga, Pseudomonas, Chryseobacterium, and Flavobacterium, and a group of plant growth-promoting (PGP) and nitrogen-fixing microbes, including Pedobacter, Variovorax, and Rhizobium. Furthermore, 47 bacteria isolates belong to 35 species were isolated. Among them, eleven and five exhibited antagonistic activities to AG8 and Rhizoctonia oryzae in vitro, respectively. Notably, Janthinobacterium displayed broad antagonism against the soilborne pathogens Pythium ultimum, AG8, and R. oryzae in vitro, and disease suppressive activity to AG8 in soil.In this study, we used a multi-cycle selection system and infection by the soilborne fungal pathogen Our results demonstrated that successive wheat plantings and pathogen infection can shape the rhizosphere microbial communities and specifically accumulate a group of beneficial microbes. Our findings suggest that soil community selection may offer the potential for addressing agronomic concerns associated with plant diseases and crop productivity.Video AbstractThe online version contains supplementary material available at 10.1186/s40168-020-00997-5. It is widely accepted that the rhizosphere microbial communities are tightly associated with plant roots , 2. The Arabidopsis, cucumber, bean, Brachypodium, maize, barley, and wheat ) Hoagland’s solution once a week. After 4 weeks, the wheat seedlings were removed from the cones and roots were shaken to remove adhering soil. Roots were cut and placed in a 250-ml sterile flask. Soil slurries were prepared with 1 g fresh roots and 6 ml of sterile double-distilled water at a ratio of 1:6, then vortexed for 30 s until the roots were visibly clear from adhering soil. The plants were evaluated for Rhizoctonia disease root rot severity on a scale of 0 to 8 as described previously ) Hoagland’s solution once a week. After 3 weeks, the seedlings were removed from the cones, the roots were washed free of soil, and the fresh weight, length of the shoots, and fresh weight of root were measured. Each treatment had 6 replicates, and the experiment was conducted three times.Bacteria that showed antagonistic activity in dual culture assay were tested against R. solani AG8 infection on bacterial community structure using the metaMDS and “Adonis” functions of the vegan package (version 2.4.1) in R (version 3.6.3) [x)-transformed sequence counts of abundant  were used to examine significant differences in bacterial families among treatments (p ≤0.05) and heatmap was generated using ggplot2 package in R [H] and inverse Simpson’s [1/D]) were estimated and compared among treatments using analysis of variance (ANOVA) followed by Tukey’s honest significant difference (HSD) post hoc tests (Tukey’s test). Differences in the relative abundances of bacterial genera in the rhizosphere soil between “good” (G) (the least wheat root disease) and “bad” (B) treatments (the worst wheat root disease) of successive planting cycles were assessed using DESeq2 analysis started with unrarefied OTU tables [p <0.1, and estimated log2-fold change > 1. Multiple comparisons were performed using Tukey test in JMP  to identify differentially abundant bacterial taxa that were impacted by planting cycles and R. solani AG8 infection. Significance was accepted at an alpha level of ≤ 0.05.Nonmetric multidimensional scaling (NMDS) and PERMANOVA were performed on Bray-Curtis dissimilarities to assess the significance of planting cycles and n 3.6.3) . ANOVA oage in R . BacteriU tables . BrieflyRhizoctonia solani AG8 infection in the first planting cycle  and non-infested controls (CK) in cycle 1, and the “good” (G) and “bad” (B) treatments and controls (CK) in cycles 2 and 9. A total of 2,674,677 sequences were obtained and represented by 7658 OTUs. Bacterial communities were dominated by phyla Proteobacteria , Bacteroidetes (19.87% ± 0.50%), and Actinobacteria (9.54% ± 0.47%) , Sphingobacteriaceae (7.23% ± 0.29%), Oxalobacteraceae (7.21% ± 0.53%), Xanthomonadaceae (7.17% ± 0.56%), Pseudomonadaceae (7.07% ± 0.40%), and Enterobacteriaceae (6.33% ± 0.53%)  Fig. a. The moaceae 7.2% ± 0.29%R. solani AG8 infection . Moreover, the bacterial richness and diversity tended to be higher in control (CK) and AG8 infection (R) in cycle 1. However, the richness and diversity did not display significant differences between the “good” (G) and “bad” (B) treated bacterial communities . Moreover, the planting cycles and R. solani AG8 infection were found to influence soil microbial communities at the family level  than AG8-infected wheat rhizosphere  . Among them, the abundance of families Planococcaceae, Cryptosporangiaceae, and Bacillaceae_1 in the wheat rhizosphere without AG8 infection (CK) increased over cycles. In contrast, families Pseudomonadaceae, Rhizobiaceae, Rhodospirillaceae, and Cytophagaceae were more abundant in the rhizosphere of R. solani AG8-infected wheat. In addition, family Oxalobacteraceae in AG8-infected wheat dramatically decreased over planting cycles, whereas Xanthomonadaceae increased over planting cycles both with and without AG8 infection. Intriguingly, the rhizosphere soils of wheat from “good” (G) treatment were more enriched in Flavobacteriaceae than those from “bad” (B) treatment . Moreover, the abundance of Chitinophaga from AG8-infected wheat rhizosphere and Bacillus from the control samples significantly increased with the planting cycles. In addition, some genera of bacteria which are not known to have antagonistic functions also responded to successive wheat plantings and AG8 infection. Four genera  were found to be more abundant in AG8-infected wheat rhizosphere, whereas the abundance of six genera  increased in R. solani AG8 non-inoculated soils.After five successive plantings, wheat root damage and shoot stunting were relieved, indicating that disease suppression had developed. Similar results were reported in our previous greenhouse study and a few antagonistic bacteria were successfully isolated from the test samples . Similarudy Fig. . For exaFlavobacterium consistently increased in rhizospheres from “good” (G) treatment in both cycle 2 and cycle 9, while OTU17 (Sphingomonas) was more abundant in “bad” (B) treatment in both cycles. However, OTU1 (Azospirillum) and OTU18 (family Caulobacteraceae) showed an opposite pattern between “good” (G) and “bad” (B) treatments in both cycles, and most OTUs varied with planting cycles. Interestingly, the abundance of a group of plant growth-promoting (PGP) microbes, including Rhizobium, Pedobacter, and Variovorax, increased in the rhizosphere from the “good” (G) treatment in cycle 9  treatment (the least wheat root disease) and “bad” (B) treatment (the worst wheat root disease) in both cycle 2 and cycle 9 Fig. . NineteeRhizoctonia solani AG8, R. oryzae, and Pythium ultimum. Eleven of 35 bacterial species exhibited antagonistic activities to AG8 at different levels. These were Pantoea (OTU951), Pseudomonas (OTU163), Streptomyces (OTU22), Chryseobacterium (OTU993), Pseudomonas (OTU118), Pseudomonas (OTU245), Sphingomonas (OTU2657), Cupriavidus (OTU162), Asticcacaulis (OTU29), Rhodococcus (OTU854)  , Chryseobacterium (OTU993), Pseudomonas (OTU118), Pseudomonas (OTU245), Sphingomonas (OTU2657)  , inhibited the growth of Pythium ultimum in ¼ TSA medium  was used for further study because it exhibited broad antagonistic activities to all three soilborne pathogens. Janthinobacterium is a gram-negative bacterium able to produce violacein, a dark purple-violet compound with antimicrobial properties [Janthinobacterium (OTU131) were more abundant in wheat rhizospheres from “good” (G) treatment  than those from “bad” (B) treatment (relative abundance: 0.52 ± 0.01%) in planting cycle 9. Furthermore, in dual culture assays, the percent inhibition of radial growth (PIRG) values for R. solani AG8, R. oryzae, and Pythium ultimum were 49.63% ± 0.56%, 13.33% ± 1.51%, and 20.12% ± 1.84%, respectively  Fig. , and Jan57) Fig. , and Janoperties   before planting. After 3 weeks growth, compared with the controls , three-week-old wheat seedlings were stunted in all R. solani AG8-infested soils compared with non-infested, but grew marginally better following the Janthinobacterium bacterial treatment. The fresh weight of wheat roots treated with Janthinobacterium significantly increased compared with AG8 inoculation only, although it was still significantly less than the controls  treatment (the least wheat root disease) gradually separated from those from “bad” (B) treatment (the worst wheat root disease) over the planting cycles. Most notably, some bacterial species were isolated from the wheat rhizospheres over multi-cycle growth and displayed antagonistic activities to soilborne fungal pathogens. Among them, a species of Janthinobacterium exhibited broad-spectrum antagonism against R. solani AG8, R. oryzae, and Pythium ultimum in a dual culture assay and against AG8 in soil. Overall, these findings suggest that repeated monocultures and AG8 infestation could change the root microbiome structure and recruit beneficial microbiota which promote plant growth and reduce soilborne pathogens, and eventually might induce disease-suppressive soils.A growing body of research indicates host plants impact root-associated microbial communities , 51, 52.R. solani AG8 and the root disease suppression was enhanced over successive growth cycles. Soils suppressive to Rhizoctonia were similarly reported in our previous study [Pantoea agglomerans, Exiguobacterium acetylicum, and Microbacteria [Asaia spp. and Paenibacillus borealis [Flavobacterium, Chryseobacterium, and Chitinophaga [Bacillus significantly increased with plant growth cycles without AG8 infection. It is well documented that Bacillus spp. secrete several metabolites not only to trigger plant growth but to inhibit pathogenic microbial growth in soil or kill pathogens through degrading the cell walls [Chitinophaga was significantly higher in AG8-infested wheat rhizosphere in cycle 9 than those in cycle 2 and cycle 1 -triggered root immune responses and protect Arabidopsis against pathogens [Duganella was more abundant in diseased plant rhizospheres, indicating Duganella may have different behavior in specific conditions. Lysobacter is a chitinolytic bacterium and has potential antagonistic activity against Rhizoctonia and nematodes [Kaistia strain, suppressed the fungal phytopathogen Fusarium oxysporum and morphological observations showed the formation of swollen F. oxysporum cells in the presence of these bacterial pairs. It demonstrated that complex interactions among apparently non-antagonistic bacteria can result in antagonism against pathogens. Thus, these uncharacterized emergent functions of bacterial consortia may also contribute to suppression activities but require further investigation. Taken together, our results provide further evidence that under the pressure of pathogen attack, plants can enrich beneficial microorganisms to suppress pathogens in the rhizosphere.Interestingly, us study : Chitinon agents . Most ren agents  recovereoccurred . The genompounds –72 and ws of tea . In contathogens . In a prathogens  Duganellematodes –77. Collematodes  reportedR. solani AG8 were screened from each cycle and used as rhizosphere inoculants for the following cycle, thus forming the two groups, “good” (G) (the least wheat root disease) treatment and “bad” (B) (the worst wheat root disease) treatment. In the first two cycles, wheat displayed severe stunting. Starting from cycle 3, suppressiveness to AG8 gradually developed and was notable by cycle 5, but the disease severity was still variable among four replicates from “good” or “bad” treatments. To achieve uniform disease symptoms among replicates, the planting was continued and by the 9th cycle all wheat plants of two treatments were tolerant to AG8 infection, but the “good” treatment showed slightly less disease symptoms than the “bad” treatment. Consistent with the disease phenotype, sequence analysis found that the microbial communities separated gradually over the growth cycles. The different quantities of diseased roots likely affect the microbial community composition, but other factors may also contribute. Although care was taken to water the plant containers equally, those with more severe root disease had reduced root systems and typically retained more moisture towards the end of the cycles. Thus, water content may influence soil microbiomes and the development of suppressive soils, something which should be addressed further. Furthermore, DESeq2 analysis found that some OTUs were differentially abundant in the rhizosphere soil between “good” (G) and “bad” (B) treatment in both cycle 2 and cycle 9. Interestingly, the “good” treatment in cycles 2 and 9 was highly enriched the antagonistic microbe OTU 19 belonged to genera Flavobacterium [Rhizobium, Pedobacter, and Variovorax, were highly abundant in the rhizosphere soil from “good” (G) treatment in cycle 9. PGP microbes have shown potential to promote plant growth at different stages via a wide variety of mechanisms [Rhizobium is a gram-negative soil bacterium and promotes plant growth through establishing nitrogen-fixing symbiosis with leguminous plants and increasing soil fertility [Pedobacter is also capable of colonizing roots of many crops, such as oilseed rape, potato, and strawberry, and could be used as a biofertilizer [Variovorax is a metabolically diverse genus of plant growth-promoting rhizobacteria which belongs to the family Comamonadaceae. Variovorax sp. promoted plant growth via producing plant growth substances and enzymes such as siderophores and ACC deaminase [In our multi-cycle selection system, wheat seedlings with roots showing relatively more or less disease to acterium . In addichanisms . For exaertility . Anotherrtilizer –83. Varieaminase . These reaminase , 86 and eaminase , 87. FurIn vitro bacterial isolation and antifungal capability testing found that eleven of 35 bacterial species inhibited the growth of R. solani AG8. Six of them suppressed R. oryzae and only one for Pythium ultimum. Testing further indicated that AG8 infection is a major driver for the colonization of those antagonistic isolates. Most of them, such as Pseudomonas, Streptomyces, and Chryseobacterium, have been well-documented as pathogen suppressive [Janthinobacterium produces a dark-purple compound, violacein [Janthinobacterium was more highly associated with rhizospheres of plants more tolerant to AG8. Janthinobacterium is a gram-negative aerobic bacterium, which belongs to the family Oxalobacteraceae of the Class Betaproteobacteria, and commonly exists in soil and aquatic habitats. As a secondary metabolite, violacein has been reported to have antifungal effects [E. coli which had no significant inhibition on Fusarium graminearum growth and further observed that the fungal growth inhibition was independent of the amount of violacein. Our antifungal capabilities test showed that Janthinobacterium has a broad-spectrum antagonism against soilborne pathogens R. solani AG8, R. oryzae, and Pythium ultimum. Further greenhouse assays discovered that Janthinobacterium has antagonistic activity against AG8 in soil. Microbial communities have many potential applications in agriculture and medicine, such as pathogen suppression and environmental remediation. With more antagonistic and plant growth-promoting (PGP) microbes being discovered and isolated, synthetic microbial communities might provide plants with stronger disease resistance and growth promotion than single species, thus become more powerful biotechnological tools to improve the sustainability of agro-ecosystems [pressive , 52, 61.iolacein . More da effects , 90. How effects  revealedosystems . Our resAdditional file 1. Methodological Supplement for the V1-V3 hypervariable region of 16S rRNA gene was amplified by PCR.Additional file 2: Table S1. Bacteria isolated from rhizosphere soil in this study.Additional file 3: Table S2. Inhibition of radial growth of soilborne pathogens in dual culture by Janthinobacterium (replicates 2 and 3).Additional file 4: Table S3. The suppression activity of Janthinobaterium against Rhizoctonia solani AG8 in greenhouse assay (replicates 2 and 3).Additional file 5: Figure S1. The phenotype of wheat grown in Lind soil in the growth chamber after the cycle 1. CK: control without Rhizoctonia solani AG8 infection.Additional file 6: Figure S2. The phenotype of wheat grown in pasteurized Lind soil in greenhouse after cycle 2. CK: control plants without Rhizoctonia solani AG8 infection; G: plants with ‘Good’ treatment (the least wheat root disease); B: plants with ‘Bad’ treatment (the worst wheat root disease).Additional file 7: Figure S3. The phenotype of wheat grown in pasteurized Lind soil in greenhouse after cycle 5. CK: control plants without Rhizoctonia solani AG8 infection; G: plants with ‘Good’ treatment (the least wheat root disease); B: plants with ‘Bad’ treatment (the worst wheat root disease).Additional file 8: Figure S4. The phenotype of wheat grown in pasteurized Lind soil in greenhouse after cycle 9. CK: control plants without Rhizoctonia solani AG8 infection; G: plants with ‘Good’ treatment (the least wheat root disease); B: plants with ‘Bad’ treatment (the worst wheat root disease).Additional file 9: Figure S5. Dual culture assays in vitro for inhibition of growth of Rhizoctonia solani AG8 by bacteria isolates on ¼ TSA medium.Additional file 10: Figure S6. Dual culture assays in vitro for inhibition of growth of Rhizoctonia oryzae by bacteria isolates on ¼ TSA medium.Additional file 11: Figure S7. Violacein produced by Janthinobacterium on TSA medium.


Hyaline membrane disease (HMD) is a leading cause of morbidity and mortality in preterm newborn babies. Though, there are studies related to Hyaline membrane disease inclusive of all neonates, studies related to the burden among preterm neonates were limited. In addition, increasing neonatal mortality in Ethiopia could be related to increase in the burden of hyaline membrane disease among preterm neonates. Therefore, this study was aimed to assess the burden of hyaline membrane disease, mortality and its associated factors among preterm neonate admitted at neonatal intensive care unit, North Central Ethiopia.An institution-based retrospective follow-up study was conducted among 535 preterm neonates admitted at neonatal intensive care unit from January 1, 2014-December 30, 2017. Data were entered into EPi-data 4.2.0.0 and transferred to STATA version 14 statistical software for statistical analysis. Binary logistic regression was used for the analysis. All variables with P-value < 0.25 during bi-variable analysis were considered for multivariable logistic regression. Level of statistical significance was declared at P value ≤0.05.th minute Apgar score less than 7 , and newborn with birth weight of less than 1500 gram  were predictors of hyaline membrane disease.In the current study, proportion of hyaline membrane disease was 40%  of which 49.5% died. Preterm neonate born with Gestational age of less than 34 weeks of age ), 5The mean gestational age (±) was 33.46 (±2.55) weeks. The incidence of hyaline membrane disease among preterm admissions was high. Preterm neonate born with gestational age of less than 34 weeks of age, asphyxiated newborns and newborn with birth weight of less than 1500 gram were predictors of hyaline membrane disease. So, emphasis should be given on early screening, follow up and timely interventions for preterm neonate. More than a third of newborn deaths are related to preterm birth complications. Hyaline membrane disease  is one of the commonest health problem encounter in preterm neonates and typically worsen within the first 48 to 72 hours , 2. It iBeing born preterm increases risk of dying because of immature organs , 7. HyalAbout three-quarter of preterm neonates could survive, if they had access to early screening and care . Even thBased on different studies; gestational age , vitaminThough, there are studies related to HMD inclusive of all neonates, studies related to the burden among preterm neonates were limited. In addition, increasing neonatal mortality in Ethiopia could be related to increase in the burden of hyaline membrane disease. Therefore, thestudy was aimed atassessing the burden of hyaline membrane disease, mortality and its associated factors among preterm neonate admitted at neonatal intensive care unit, North Central Ethiopia.An institutional based retrospective follow-up study was conducted among 535 preterm neonate with a study period from January 1, 2014-December 30,201. All preterm neonates admitted at Debre Tabor General Hospital during the study period were included. Debre Tabor town is found 666 km far from Addis Ababa, capital city of Ethiopia. The hospital receives a patient with severe disease referred from the nearby Primary Hospitals and Health Centers which is also true for neonatal care. The hospital provide health care service for more than 2.4 million populations in its catchment area. The hospital has a Neonatal Intensive Care Unit (NICU) with a total of 21 neonatal beds and approximately 1,159 neonates were admitted per year. Most of the neonatal admissions were associated with prematurity .A total of 614 preterm neonates admitted during the study period. Of these, medical charts were not available for 25 neonates and 54 charts were excluded due to data incompleteness. Finally, 535 preterm neonates were involved in the study. A list of medical record number was found from Neonatal registration book. Then, charts were found from medical record office by using medical chart number.Data were collected based on the structured data abstraction sheet from medical charts. Abstraction sheet includes maternal socio-demographic, obstetric, and neonatal-related variables by considering hyaline membrane as an outcome variable.Hyaline membrane disease .✓ Socio-demographical characteristics of the mother ✓ Intrapartum factor ✓ Neonatal related factor Maternal complication was considered if mother had an obstetric hemorrhage, puerperal sepsis and pyrexia, prolonged labor, eclampsia and preeclampsia, mal-presentation and mal-position, premature rupture of membrane (PROM), cord prolapse, obstructed labor, cephalopelvic disproportion, emergency cesarean section, and retained placenta.Hyaline membrane disease was diagnosed based on the presence of one or more of the following signs: an abnormal respiratory rate, expiratory grunting, nasal flaring, chest wall recessions and thoraco-abdominal asynchrony with or without cyanosis .Pretest was done on 5% of the sample size. One day training was given for data collectors and supervisors on data collection tools and data collection procedures. Completeness of each data collection tool had been checked on a daily base. Double data entry was done by two data clerks and consistency of the entered data was cross-checked.Data were entered, coded, cleaned, and checked by Epi Data version 4.2.0.0 and analyzed using STATA Version 14 statistical software. Descriptive statistics of included variables were presented by using tables and texts. Binary logistic regression was used to saw the association between each independent variable with a dependent variable. All variables with a p-value≤0.25 in the bi-variable analysis were entered into multivariable analysis. Variables with p values ± 0.05 in multivariable logistic regression model analysis was considered as statistically significant.Ethical clearance was obtained from the Institutional Health Research Ethics Review Committee of Haramaya University, Ethiopia. Then, official letter was written to Debre Tabor General Hospital for permission. The neonates’ name and medical record identification information were not collected. Since the data obtained from medical charts, all data were fully anonymized. All data extracted from the chart were kept strictly confidential.The mean age with standard deviation (±SD) of mother was 27.56 (±6.54) years. Sixty-six percent, of mothers were in the age group of 20–34 years. The majority, (72.7%) of them were from the rural area. Seventy-seven percent, (77.1%) of mothers had ante-natal care (ANC) follow-up. Sixty-nine percent of preterm neonates were single births. More than half, 51.6% were male newborn with mean gestational age (±) of 33.46(±2.55) weeks .In the current study, the incidence of hyaline membrane disease was 40.0% ; among this 49.5% (106 per 214) were died.th minute Apgar score and PPROM were significantly associated with HMD. Whereas, in multivariable analysis; Gestational age, 5th minute Apgar score and birth weight were statistically significant.Both bi-variable and multivariable logistic regression were undertaken. In binary logistic regression gestational age, number of antenatal care visit, parity, birth order, birth weight, maternal complications, APH, 5th minute were 2.2 times more likely to have hyaline membrane disease in contrast with newborns with APGAR score of greater than seven .In this study, newborn with less than 34 weeks of gestation were 2.64 times more likely to develop hyaline membrane disease than neonates born greater than or equal to 34 weeks of gestation . Similarly preterm neonate born with less than 7 APGAR score of 5Those preterm neonates born with birth weight of less than 1500 gram were 2.4 times more likely to have hyaline membrane disease than newborn with birth weight of greater than 1500 gram  .Hyaline membrane disease is one of the significant predictor of neonatal morbidity and mortality. In this study proportion of hyaline membrane disease was 40%, this is consistent with study conducted in Iraq, 42.2% , in BlacIn multi variable analysis those with gestational age of less than 34 weeks of age, asphyxiated newborns and newborn with birth weight of less than 1500gram were predictors of hyaline membrane disease. In the current study, newborn with less than 34 weeks of gestation were more likely to develop hyaline membrane disease than neonates born greater than or equal to 34 weeks of gestation. This is consistent with study conducted in China , and Indth minute Apgar score of less than 7 were more likely to have hyaline membrane disease in contrast with newborns with Apgar score of greater than seven. This is similar to studies conducted in UK [2 >40%).Preterm neonates born with 5ed in UK , USA [51ed in UK , China [ed in UK , and Adied in UK . This coed in UK , 53. Thoed in UK .The ratied in UK . Hence, Since, the study is retrospective follow-up study important variables like surfactant administration, Vitamin D status of the newborn and placental abnormality were missed. Therefore, future researcher should do prospective studies by including those variables which may have key indicators for HMD.The incidence of hyaline membrane disease among preterm birth was high. Preterm neonate born with gestational age of less than 34 weeks of age, asphyxiated newborns and newborn with birth weight of less than 1500 gram were predictors of hyaline membrane disease. So, emphasis should be given on early screening, follow up and timely interventions of preterm neonates borne with low birth weight and low Apgar score.S1 File(DTA)Click here for additional data file.


We studied the demographic and clinical characteristic, risk factors, outcomes of full-term small-for-gestational-age (SGA) infants born to mothers with gestational diabetes mellitus (GDM) in China. A retrospective case-control study that included 1981 SGA infants was conducted; the demographic and clinical data between SGA infants born to mothers with and without GDM were compared. Of 383 SGA infants born to mothers with GDM, 221 (57.7%) were female, and the incidence of these infants was 1 in 155 live births. The risk of SGA siblings , low 1- and 5-min Apgar scores , early thrombocytopenia , hypoglycemia, and hypoxic-ischemic encephalopathy  were increased in SGA infants born to mothers with GDM compared to SGA infants born to mothers without GDM. SGA girls born to mothers with GDM had a significantly higher ratio of catch-up growth (CUG)  in the first year of life. These results show that genetic factors may be one of the etiologies of SGA infants born to mothers with GDM; and these infants have more adverse perinatal outcomes compared to SGA infants born to mothers without GDM. SGA girls born to mothers with GDM had accelerated CUG in the first year of life. Gestational diabetes mellitus (GDM) is a major public health problem for women in China and worldwide . FetusesGDM and SGA are associated with delayed development and poor growth of the fetuses. Epidemiological studies showed that infants born to mothers with GDM had a higher rate of adverse perinatal outcomes  than those in infants born to mothers without GDM; but the clinical characteristics of SGA infants born to mothers with GDM are poorly understood , 5. Therin-utero. Thus, we evaluated the outcomes of SGA infants born to mothers with GDM by measuring weight gain and the ratio of CUG in the first year of life.Multiple epidemiological studies have shown that SGA infants and infants born to mothers with GDM share common long-term complications  in adulthood and biological pathways leading to these long-term complications; studies also showed that faster post-natal growth in these infants appeared to be an important determinant of cardiovascular disease and type 2 diabetes –10. ThisIn this study, we sought to investigate the demographic and clinical characteristics, risk factors, and outcomes of SGA infants born to mothers with GDM in China.This study was a population-based, retrospective case-control study; written informed consent was provided by all parents. This study was approved by the research ethical committee of Guangzhou Women and Children's Medical Center, Dongguan Houjie hospital and Dongguan city maternal and child health hospital; all procedures performed in studies involving human participants were in accordance with the relevant guidelines and regulations.We performed a retrospective case-control study on demographic and clinical features of SGA infants born to mothers with GDM, and infants delivered in Guangzhou Women and Children's Medical Center, Dongguan Houjie hospital and Dongguan city maternal and child health hospital between January 1, 2008 and October 31, 2018. All mothers giving births to SGA infants who underwent regular obstetric examination in hospitals and had undergone a 75 g oral glucose tolerance test (OGTT) following the standard protocol at 24–28 weeks of gestation at the Department of Obstetrics were included. GDM was diagnosed when any one value reaches or exceeds 5.1 mmol/L at 0 h, 10.0 mmol/L at 1 h, or 8.5 mmol/L at 2 h .All included mother/infant pairs met the following inclusion criteria: (1) Chinese nationality and gestational age ≥37 weeks ; preterm were excluded from analysis because Chinese children growth trajectories are inapplicable to these infants; (2) No chromosomal aberrations and genetic syndromes in infants; (3) mothers between 20 and 40 years of age and having no disorders known to affect glucose metabolism including diabetes, polycystic ovarian syndrome and uncontrolled thyroid during pregnancy; and (4) mothers/infant pairs with complete maternal  and neonatal data . Mothers/infant pairs were excluded if they did not meet all of these inclusion criteria. Only one birth for mother was included in our study.All infants included in our study were divided into two groups. Group 1 (case group) included SGA infants born to mothers with GDM. Group 2 (control group) included SGA infants born to mothers without GDM. We performed 1:4 (case group: control group) matching according to neonatal gestational age (difference was ≤ 3 days), gender, birth weight range  and maternal age (difference was ≤ 3 years), place of residence. The definition of SGA was birth weights ≤ 10th percentile for the gestational age based upon defined standards for males and females according to Chinese Neonatal Network .9/l in the first 72 h of life. Hypoglycemia was defined as blood glucose <35 mg/dl or plasma glucose <40 mg/dl. The diagnostic criteria for hypoxic-ischemic encephalopathy (HIE) were: (1) evidence of clinical encephalopathy in the first 12 h of life; (2) history of perinatal asphyxia; (3) Apgar score ≤ 5 at 5 min; and with at least one of the following: (a) continued need for positive pressure ventilation for 10 min or history of cardiopulmonary resuscitation at birth; and/or b) pH ≤ 7.00 in arterial cord blood or other sample or base deficit ≤ 12 mmol/l. HIE was classified according to modified Sarnat criteria. The grade for ICH was classified according to Papile criteria. Late-onset sepsis, occurring after 72 h from birth, was defined as positive blood culture. Necrotizing enterocolitis (NEC) was diagnosed according to Bell criteria. Neonatal respiratory distress syndrome (NRDS) was diagnosed by (1) evidence of respiratory failure, (2) administration of exogenous pulmonary surfactant (3) radiographic evidence. The criteria of maternal diseases listed in All mother/infant pairs underwent structured medical examinations and physical examinations. Data on mothers and neonates were obtained from medical records. The weight and length/height of mother/infant pairs was measured by trained nurses using standard anthropometric methods, pre-pregnancy weight obtained according to maternal self-report. Body mass index (BMI) was calculated by dividing weight in kilograms by the square of height in meters. Early thrombocytopenia was defined as a platelet count of <1,501 × 0t-tests were used for continuous variables that were described with the means ± standard deviations (x− ± s). χ2 tests were used for categorical variables that were described with frequencies and percentages. Multivariate binary Logistic regression was used to determine risk factors of SGA infants born to mothers with GDM. Pearson's correlation coefficients were conducted to determine the association between maternal factors  and birth weight. The significance level was set at p < 0.05.Sample size calculation was performed using PASS 15. With 90% power and the assumption of relative risk = 2.0, we calculated that 256 SGA infants born to mothers with GDM were needed; four comparison individuals per case, 1024 SGA infants born to mothers without GDM were needed. Statistical calculations were performed by using SPSS version 22. For the statistical analysis, Of 160,474 infants that were screened in our study from 2008 to 2018, 16,059 were SGA infants , 15,726 infants born to mothers with GDM(9.8%). Of 160,474 infants that were screened, 1,034 were SGA infants born to mothers with GDM (0.6%), which yielded an incidence for full-term SGA infants born to mothers with GDM of approximately 1 in 155 live births. Of 1,034 SGA infants born to mothers with GDM, 808 singleton births, 220 twins births, and six triplets.P >0.05)  , 2.A total of 383 eligible SGA mothers with GDM at a mean age of 31.2 (SD 4.7) years were included, 380 (99.2%) women with GDM were given dietary therapy. Significant differences were found in pre-pregnancy weight (51.67 ± 0.8 vs. 51.17 ± 0.0 kg), pre-pregnancy BMI (20.82 ± 0.8 vs 0.202 ± 0.9), increased weight during pregnancy (11.84 ± 0.6 vs. 12.83 ± 0.9 kg), and the incidence of vaginal delivery , cesarean section  between SGA mothers with and without GDM groups. Additional population characteristics were available in The rate of SGA siblings in the SGA infants born to mothers with GDM was higher than that in the SGA infants born to mothers without GDM . The risk of low 1- and 5-min Apgar scores , early thrombocytopenia , HIE , hypoglycemia , obstetric trauma , and pulmonary hemorrhage  were increased in SGA infants born to mothers with GDM compared to SGA infants born to mothers without GDM.P < 0.01) were significantly higher in the group of SGA girls born to mothers with GDM than that in the group of SGA girls born to mothers without GDM. All SGA infants with severe perinatal complications were followed for 2 years. In the group of SGA infants born to mothers with GDM group, cerebral palsy occurred in two cases (0.6%), short bowel syndrome occurred in one case with NEC (0.3%), and there was one death (0.3%).The outcomes of the infants were summarized in Pearson's correlation coefficients were conducted to determine the association between maternal factors, results are shown in P < 0.05).Multivariate binary logistic regression was used to determine the associations between maternal complications and GDM; all results were listed in We collected population-based, case-control, including risk factors, demographic and clinical characteristics, and outcomes, for full-term SGA infants born to mothers with GDM in China. To our knowledge, this is the first study to report that SGA infants born to mothers with GDM had more adverse perinatal outcomes than SGA infants born to mothers without GDM, the girls of these infants had accelerated CUG in the first year of life.It has long been understood that the pathogenesis of SGA infants includes both genetic and environmental components . The recAll environmental components are listed in Our study presented novel information about perinatal complications of SGA infants born to mothers with GDM, which had not been reported. In our study, the risks of low 1- and 5-min Apgar scores (≤3) (3.4 and 1.0%), obstetric trauma (2.1%), hypoglycemia (7.6%), HIE (1.0%), pulmonary hemorrhage (1.0%), and early thrombocytopenia (2.1%) were higher in SGA infants born to mothers with GDM than in SGA infants born to mothers without GDM. A possible association between GDM and birth defects has been reported in some studies –24, but Ninety percent of SGA children will experience CUG in the first 2 years of life, and these infants are likely to have excess central fat and abnormal adipocyte function as well as endocrine system disturbances . Recent via a population-based case-control study. Our findings are also helpful for clinicians to design more accurate postnatal care programs and follow-up programs for SGA infants born to mothers with GDM.One of the important strengths of our work is its large sample size of SGA infants born to mothers without GDM. Additionally, this study is an observational case-control study. To the best of our knowledge, this is the first study to report the risk factors, demographic and clinical characteristics and outcomes of full-term SGA infants born to mothers with GDM This study has several limitations. First, the number of GDM mothers who were given drug therapy may be small, the blood glucose of 99.2% GDM women could be controlled through dietary therapy; while in the literature it ranged from 3 to 15%; but these studies showed that there were no significant difference in maternal and neonatal outcomes between diet therapy group and insulin therapy group , 33. SecIn conclusion, the incidence of full-term SGA infants born to mothers with GDM was 1 in 155 live births in China. Genetic factors may be an etiology of SGA infants born to mothers with GDM, and other risk factors for these infants should be derived from multivariable logistic regression analysis via comparisons with AGA infants born to mothers with GDM. Compared with SGA infants born to mothers without GDM, SGA infants born to mothers with GDM have a higher risk of perinatal complications ; the girls of these infants had accelerated CUG in the first year of life.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.JC, HX, YY, and WZ designed the study, designed the data collection instruments, collected data, carried out the initial analyses, drafted the initial manuscript, and created the tables and figures. YT, XY, and ZZ conceptualized and designed the study, coordinated and supervised data collection, and helped draft the initial manuscript. WL, JY, and LH designed the data collection instruments, collected data, and conducted the initial analyses. XL conceptualized and designed the work, helped draft the initial manuscript, and provided feedback about the study design. All authors contributed to the manuscript's critical revision and read and approved the submitted version.This article was supported by Science and Technology Planning Project of Guangdong Province (2016A020215177), China.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The effect of pulsed and oscillating electric fields with different frequencies on the conformational properties of all-α proteins was investigated by molecular dynamics simulations. The root mean square deviation, the root mean square fluctuation, the dipole moment distribution, and the secondary structure analysis were used to assess the protein samples’ structural characteristics. In the simulation, we found that the higher frequency of the electric field influences the rapid response to the secondary structural transitions. However, the conformational changes measured by RMSD are diminished by applying the electrical field with a higher frequency. During the dipole moment analysis, we found that the magnitude and frequency of the dipole moment was directly related to the strength and frequency of the external electric field. In terms of the type of electric fields, we found that the average values of RMSD and RMSF of whole molecular protein are larger when the protein is exposed in the pulsed electric field. Concerning the typical sample 1BBL, the secondary structure analysis showed that two alpha-helix segments both transit to turns or random coils almost simultaneously when it is exposed in a pulsed electric field. Meanwhile, two segments present the different characteristic times when the transition occurs in the condition of an oscillating electric field. This study also demonstrated that the protein with fewer charged residues or more residues in forming α-helical structures display the higher conformational stability. These conclusions, achieved using MD simulations, provide a theoretical understanding of the effect of the frequency and expression form of external electric fields on the conformational changes of the all-α proteins with charged residues and the guidance for anticipative applications. AK values, even quite remote from the origin of the perturbation. Beyond the huge amount of studies on protein structure and stability, the inherent mechanisms and their relationships to experimental observables are still a challenge for the scientific community [With the rapid development of science and technology, the widespread applications of electronic devices play an important part of our everyday lives. The investigation of the effects of radio frequency and microwave electric/electromagnetic radiation fields on biological tissues has become a very interesting research topic ,2,3,4,5.ommunity ,11. Receommunity . Such fiommunity . Among fommunity ,15. Thesommunity . TherefoThe protein response to an external field is involved in protein folding/unfolding, protein aggregation, protein adsorption, protein recognition process, etc. ,19,20,21In addition to existing experimental studies on electric effects of protein denaturation and stability, computational biophysics has started to focus on researching the inherent mechanisms of bio-molecules at atomistic level that cannot easily be observed from experiments . RecentlEscherichia coli (PDB code: 1BBL) was investigated as a typical sample in the present work. The mutations that are in the residues at an active site or any other residue at the distant site affect the structure of proteins, substrate, and cofactor binding site. Mutations do not just provide us an understanding of novel protein design with enhanced stability and activity, but it also gives us a platform where we can design mutants with a strong understanding of the importance of a particular amino acid at a specific location, having a stability effect. The wild-type protein 1BBL has a net positive charge. The 6 types of mutants of 1BBL were achieved by replacing the charged residues. For example, aspartic acid (Asp) and asparagin (Asn) are often transformed into each other in nature. The mutant 1, protein D43N, was obtained using aspartic acid (Asp) instead of No. 43 residue asparagine (Asn) from the wild-type protein. The side chains of aspartic acid containing carboxyl groups, which give it a negative charge under physiological conditions. The uncharged asparagine has the polar side chain, which can form hydrogen bonds appearing at the corner. Then the mutant D43N has two positive charges. The chloride ions were added to the system to neutralize it. The protein configuration was enclosed in the center of the periodic cubic simulation box. The detailed charged conditions of the 1BBL and its mutants are listed in The initial configuration of object proteins  were obtained from the Protein Database Bank. The detailed information of all protein samples used in this present article are given in T = 310 K and constant pressure P = 1 atm. The time step and mesh spacing were set as 2 fs and 0.1 nm, respectively. The van der Waals interactions were calculated by the switching function, which start at a distance of 1.0 nm and reach zero at 1.2 nm. The long-range electrostatic interactions were calculated by the particle grid Ewald (PME) method. The protein configuration was enclosed in the center of a periodic cubic simulation box with TIP3 waters filled in. A multiple time stepping integration scheme was applied by NAMD. Following the equilibration stage, alternating electric fields with different forms and frequencies were applied. The pulsed and oscillatory forms were adopted to express the external electric field. All electric fields were applied along the x-direction with the strength 0.5 V/nm. The detailed parameters of various electric fields are listed in All simulation procedures were performed by molecular dynamic algorithms implemented in the NAMD 2.6 software package  using an all-atom CHARMM27 force field . First, N is the number of protein atoms. finalr(i) and initialr(i) are the coordinates of an atom i in its final structure and initial structure, respectively. RMSF is an important tool to characterize the freedom of the center ttotal is the total simulation time, and the reference coordinate refr is the average coordinate of N is the total number of protein atoms. i, In this study, we carried out MD simulations to explore the effects of alternating electric fields, varying in frequency and electric form, on the protein samples and their mutants. The conformational stability of proteins during the simulation procedure was examined by calculating the root mean square deviation (RMSD) and the root mean square fluctuation (RMSF). RMSD is a numerical measurement of the conformational changes between two structures. It is defined as:x-component dipole moment maintain the same directions and frequencies as that of the external electric fields applied on the protein. xd in these four figures are equal in magnitude, which are directly related to the same intensity of the external electric fields. Meanwhile, the y and z components of the dipole moment show the small fluctuations between positive and negative values. In addition, the average value over the simulation time is close to zero. We concluded that the magnitude of dipole moment had a positive relation with the amplitude of the external electric field [xd are the same as the external electric fields applied along the x-direction. It indicates that the general effect of applied electric fields on the dipole moment is to increase its magnitude along the same direction, simultaneously. Green and Kubo showed in serial papers that transport coefficients, such as heat conductivity and bulk viscosity, can be related to the correlation functions of the corresponding flux or tensor in thermal equilibrium [To investigate the effect of the frequency of the external electric field on the molecular protein, a series of MD simulations were carried out by applying the electric fields with the same electric intensity (0.5 V/nm) but different frequencies (1000/2000 MHz). The time evolutions of three components of a dipole moment during 10 ns simulation are shown in ic field . Figure ilibrium ,42. The The time evolutions of the RMSD of 1BBL exposed in electric fields with the same intensities but different frequencies and forms are shown in To investigate the general effect of the frequency on the all-α protein samples with different α-helix ratios, the average values of RMSD during the whole simulation procedure are presented in The effect of the frequency of the electric field on the secondary structure of the protein 1BBL was estimated by the VMD software package. The time evolutions of the RMSD of 1BBL exposed in different types of electric fields with the same intensities and frequencies were presented by comparing Besides the discussion of RMSD, RMSF also gives us some useful information to know the structural properties of the molecular chain. The RMSFs of skeleton The stride evolutions of secondary structures of 1BBL in the different types of electric fields with the frequency 1000 MHz are shown in In order to investigate the relationship between the quantity of electric charge among the molecular protein and its conformational properties, the wild-type 1BBL and its mutants in the external electric field were examined by MD simulations. The time evolutions of RMSD of 1BBL and D43N exposed in the electric fields with different frequencies and types are shown in The stride evolutions of secondary structures of 1BBL and D43N under the oscillating electric field with a frequency of 2000 MHz are shown in In order to further explore the relationship between the charge distribution and conformational properties of protein molecules, D43N and other mutants of 1BBL obtained by changing several residues were investigated. The average values of RMSD of 1BBL and six mutants exposed in the same external condition OEF2 are presented in In this work, the effects of external electric fields with different frequencies and electric types on the wild-type protein 1BBL and its mutants were investigated by MD simulations. The conformational stability and the structural transition of the protein samples were discussed according to our simulation results. The dipole moment analysis showed that the external electric field could affect the conformation of the protein by changing its axial component of the dipole moment. The magnitude and frequency of the dipole moment is directly related to the strength and frequency of the external electric field. The curve oscillographs and frequencies of the dipole moment are the same as the external electric fields applied on the protein. It also concluded that the frequency of the electric field influenced the rapid response of the conformational change. Meanwhile, the protein presented the low conformational stability when it was subjected to the electric field with a low frequency. The protein consisting of more α-helices presents higher conformational stability even when exposed in the electric fields. In terms of the type of electric fields, the average values of RMSD and RMSF of a whole molecular protein are slightly larger in the pulsed electric field which has the higher average intensity over the simulation time than the oscillating electric field. According to the analysis of the secondary structure, two alpha-helix segments transit to turns or random coils almost simultaneously when the protein was exposed in the pulsed electric field. However, two segments present different characteristic times when the transition occurs in the condition of an oscillating electric field. Moreover, the influences of the quantity of electric charge among the molecular protein on its conformational stability were analyzed by the RMSD trajectory of wild-type protein 1BBL and its mutant, D43N, exposed in the pulsed and oscillating electric fields. The net charge dependence of average RMSD of 1BBL and its six mutants showed that the charged residues receive the effect of an electric field by perturbing peptide conformation. The protein with more charged residues had the low conformational stability. The conclusions derived from MD simulation will be helpful to understand the effect of the frequency and expression form of external electric fields on the conformational changes of the protein with charged residues. These findings might potentially impact and provide insights into the mechanism of biological effects of nanosecond-scale intense electric pulses and oscillating electric fields, which are being experimentally explored for their biomedical applications.