Organophosphate esters (OPEs) are synthetic chemicals used as flame retardants and plasticizers in a variety of goods. Despite ubiquitous human exposures and laboratory evidence that prenatal OPE exposures may disrupt offspring metabolism, perinatal studies of OPE health effects are limited. The objectives of this study were to: 1) Determine predictors and reproducibility of urinary OPE biomarker concentrations during pregnancy, and 2) Estimate the relation of prenatal OPE exposures with birth outcomes and cord blood adipokine and insulin concentrations.We analyzed five OPE metabolites in urine samples collected at up to three visits during pregnancy from 90 women enrolled in the ORigins of Child Health And Resilience in Development (ORCHARD) pregnancy cohort in Baltimore, MD from 2017 to 2019. To quantify the variability of metabolite concentrations during pregnancy, we calculated intraclass correlation coefficients (ICCs) for each metabolite using mixed effects regression models. Using self-reported questionnaire data collected during gestation, we assessed possible sociodemographic and environmental/behavioral predictors of each OPE metabolite using generalized estimating equations to account for repeated exposure measures. We ascertained birth outcomes of 76 offspring from medical records, including weight-for-gestational age, length, ponderal index, and gestational age. In a subset of 37 infants, we measured cord blood concentrations of leptin, adiponectin, and insulin. To account for repeated exposure measures, we used linear structural equation models to assess the relations of standard deviation (SD) increases in prenatal OPE metabolite factor scores with continuous birth outcomes and cord blood biomarker concentrations.3  greater ponderal index. A SD increase in BDCIPP was associated with a 0.37  SD lower insulin concentration and 0.24  SD lower leptin concentration. Other OPEs were not associated with infant outcomes.ICCs ranged from 0.09 for isopropylphenyl-phenyl phosphate (ip-PPP) to 0.59 for bis phosphate (BDCIPP). We observed little consistency in environmental or behavioral predictors of OPE exposures, although concentrations were generally lower for samples collected in the afternoon compared to morning and winter compared to other seasons. In adjusted analyses, a SD increase in BDCIPP concentration was associated with a 0.06 g/cmThese findings suggest some OPEs may be metabolic disruptors warranting investigation in larger studies. Organophosphate esters (OPEs) are a class of synthetic chemicals primarily used as additive flame retardants that are found in a variety of products including clothing, furniture , electronics, and baby products –3. OPEs Despite the omnipresence of OPEs, studies identifying predictors and health effects of OPE exposures are limited—especially prospective studies of prenatal exposures. Of the few studies which have assessed predictors of OPE exposures during pregnancy, greater maternal pre-pregnancy BMI , 2, resiIt has recently been hypothesized that OPEs are metabolic disruptors given their abilities to interfere with multiple biologic mechanisms related to adiposity and obesity risk such as gonadocorticoids –30 and iHuman studies assessing health effects of gestational OPE exposures are very limited. To-date, only two epidemiologic studies investigated associations of OPE exposures during pregnancy with offspring birth outcomes , 46. OneGiven the lack of information regarding predictors, reproducibility, and health effects of prenatal OPE exposures, we performed a pilot study among pregnant women in Baltimore, MD to: 1) characterize the extent and variability of OPE metabolite concentrations during pregnancy, 2) identify predictors of OPE exposures, including environmental and behavioral sources, 3) elucidate the relation of prenatal OPE exposures with birth outcomes, and 4) explore the potential for prenatal OPE exposures to act as metabolic disruptors using cord blood adipokine and insulin concentrations.The ORigins of Child Health And Resilience in Development (ORCHARD) cohort enrolled pregnant women from the Baltimore, MD region during 2017–2019. Women were eligible to participate if attending their first prenatal care visit at Johns Hopkins Hospital and intended to seek further prenatal and pediatric care at that facility; were between 16 and 55 years of age; and less than 20 weeks gestation. A total of 603 women were approached at their first prenatal care visit (8.6 to 20.6 weeks) and 483 refused to enroll into to the cohort . This pilot study includes the first 90 women enrolled into ORCHARD who provided a urine specimen at their first visit. Among the 90 women with urine samples at their first visit, 69 had provided a second visit sample and 53 had provided a third visit sample at the time specimens were sent for OPE analysis. The ORCHARD protocol was approved by the Johns Hopkins Medicine Institutional Review Board and women provided informed consent at enrollment.We developed and administered two questionnaires during this study. The first was completed at the first visit and included sociodemographic information , medical history , information on prior pregnancies (including miscarriages and abortions), social history , and home environmental exposures . The second questionnaire was administered within 96 h before or after delivery (except for three participants who completed the questionnaire 27–56 days after delivery) and included alcohol, drug, and tobacco use; illnesses and injuries; and medications used during pregnancy.10-BDCIPP, 8.8 ng of d10-DPHP; 25 ng of d12-TCEP); sodium acetate  and enzyme solution  was added; and resulting mixtures were vortexed and incubated at 37 °C in a water bath overnight. Electrospray ionization (ESI) liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to analyze the extracts ), and gestational age at delivery (weeks). We calculated ponderal index as a measure of fetal growth (g/cmgthcm3.) . We estigthcm3.) . Of the In a subset of the first 37 delivered infants that provided cord blood samples, we analyzed cord blood for leptin, adiponectin, and insulin. Cord blood was chosen since it provides a more direct estimate of the fetal condition as compared to measures in placental tissue . AdiponeWe used participant self-reported responses from both questionnaires to operationalize covariates believed to predict OPE exposures or birth outcomes, or to confound associations of OPE exposures with birth outcomes. We centered continuous variables  at the sample mean; combined maternal race and ethnicity (due to few observations in most categories) and dichotomized as non-white race/ethnicity or white/Caucasian; categorized maternal education as Bachelor’s degree or higher, some college (less than a Bachelor’s degree), or high school diploma or less; and dichotomized parity (parous or nulliparous). Gestational ages 0–12 weeks, 13–27 weeks, and 28 weeks to delivery defined trimesters of pregnancy. We categorized time of day of sample collection as before 11 am, between 11 am and 1:59 pm, and 2 pm or later. We based season of sample collection on meteorological seasons and categorized as: fall , winter , spring , and summer . While we considered including maternal smoking history during pregnancy as a covariate, it has been shown not to confound the association of prenatal OPE exposures and birth outcomes , 46. FurTo correct for hydration status, we adjusted metabolite concentrations using specific gravity as previously reported , except 2-transformed concentrations (uncorrected and specific gravity-corrected) and linear mixed effects regression models with a random-intercept for subject and REML, using all available samples from subjects with at least two samples. The general formula used to calculate ICCs was: γ represents within-individual variance for linear mixed models. Since BCIPP was dichotomized, we adjusted the formula for ICC to use the variance of the standard logistic regression distribution (π2/3) as an estimate of γ [2-transformed then z-standardized prior to estimation due to non-normal distributions  using logate of γ . We did relation ).2), maternal race/ethnicity, maternal education level, parity, trimester of sample collection, time of day at sample collection, season of sample collection, and home product usage and cleaning behaviors. We estimated associations of potential predictors of urinary OPE metabolite concentrations using generalized estimating equations (GEE) with unstructured covariance and Huber-White robust standard errors, to account for the repeated sample measures among subjects [p-values < 0.20 along with the base-model predictors, to form the “fully-adjusted” models. We estimated adjusted coefficients of determination (R2) in the base-models and fully-adjusted models to determine the explanatory power of the covariates. To determine the contribution of the environmental/behavioral variables specifically, we first calculated the R2 in the base models, then again in the fully-adjusted models which included these variables. We used Efron’s [R2 for all metabolites, except for the dichotomous BCIPP variable, in which we used McKelvey and Zavoina’s [Potential predictors of OPE exposure included maternal age (in years), maternal pre-pregnancy BMI  then used these z-standardized concentrations as manifest variables of a continuous latent factor variable (F) in a Bayesian structural equation model (SEM) [F. Second, using the parameters from step one, we incorporated the indirect effects (γ) of time of day of sample collection  and season  in the models, allowing greater precision in the estimates of the factor F. To determine adequacy of model fit, we used posterior predictive checking (PPC) with the associated posterior predictive p-value (PPP) as well as the Gelman-Rubin potential scale reduction factor [F was the primary predictor of the birth outcomes and models were adjusted for other hypothesized predictors  and confounders . We fit these MIMIC models using robust full-information maximum likelihood (FIML) [For associations of repeated urinary OPE metabolite concentrations with birth outcomes, we used a structural equation modeling (SEM) framework  , estimatn factor , 84. Lasd (FIML)  to accouWe used the same SEM framework as the birth outcomes analyses to estimate the associations of each specific gravity-adjusted urinary OPE metabolite with each cord blood biomarker, individually. While OPE metabolite factors were still adjusted for time of sample collection and season, we only included key confounders  in these models due to limited sample size.As an additional exploratory analysis, we used quantile g-computation  to estimFor descriptive analyses and calculating the CV, we singly-imputed OPE metabolite concentrations below the LOD as ment}LOD2. For allment}LOD2, 89. ForWe assessed the impact of assay variability on estimated ICCs by incorporating the within-subject variance used in the CV calculations as an additional component of variance in the denominator of our ICC formula. We compared our SEM approach with a simpler method for incorporating repeated exposure data, in which we assigned exposure to each OPE metabolite as the average concentration based on available samples . We z-trAll null hypothesis testing used an α = 0.05 for statistical significance. All analysis was performed using STATA/MP v15.1 , except for SEM models which were performed using MPlus v8.2 (Muthén and Muthén 2012–2017).Of the 90 women included in the study, 64% were 30 years old or greater, 34% were obese, and 80% had completed some college education (60% had at least a Bachelor’s degree) Table . BCIPP wR2 values slightly increased for all OPEs with the greatest impact on BDCIPP (27.4 to 36.8%)  and a 0.06 g/cm3  increase in ponderal index, respectively  approach  in China which observed sex-specific effects of greater DPHP concentrations and odds of low birth weight at delivery [Only three prior epidemiologic studies have assessed relations of prenatal OPE exposures with birth outcomes , 46, 100In this first study to assess ponderal index in relation to prenatal OPE exposures, we found evidence of associations between BDCIPP and greater ponderal index but generally did not observe associations of individual OPE metabolites with birth weight for gestational age or length. Though, there was suggestive evidence of asymmetric fetal growth with respect to greater DPHP concentrations, given the trend towards reduced birth length but not birth weight. In our exploratory analysis of OPE mixtures, we observed a trend towards reduced birth length and BW-GA as well as higher ponderal index with greater exposure to the mixture of OPEs, though confidence intervals were wide. To aid the interpretation of our birth outcome findings, we assessed adipokine and insulin concentrations from cord blood samples in a small subset of participants. Overall, we observed a trend towards decreased insulin concentrations with all metabolites (except ip-PPP), which were strongest for BDCIPP and tb-PPP, and lower leptin with increased BDCIPP concentration. This finding is contrary to the classic paradigm of adiposity in which leptin is markedly increased and adiponectin is reduced in overweight individuals ; though,This study has several strengths including repeated urine samples collected throughout the gestational period to reduce exposure misclassification of these non-persistent chemicals. We used a statistically rigorous approach to modeling repeated exposures in relation to birth outcomes. This study was also the first to investigate OPE exposure in relation to ponderal index and cord blood biomarkers, allowing us to explore possible prenatal programming of metabolic dysregulation reported in prior toxicological studies, and the first to assess the joint effect of OPE mixtures on birth outcomes.Our study also had important limitations, however. This was a highly exploratory pilot study and should be viewed as hypothesis generating. Our limited sample size underpowered our ability to detect meaningful associations in many of our analyses. CVs for the metabolites are somewhat high and the resultant measurement error could have biased ICCs downward. We believe the apparent high CVs may, in part, be due to several replicates having values near or below the LOD for multiple metabolites. Also, while prior work has noted important sex differences with respect to OPE exposures and birth outcomes , we wereOverall, we observed low to moderate reproducibility in urinary OPE metabolite concentrations during gestation with few consistent predictors of exposure. Maternal pre-pregnancy BMI, parity, and increased frequency of mopping were associated with higher urinary metabolite concentrations while winter season and afternoon sample collection were associated with lower concentrations. Higher BDCIPP concentrations were associated with greater infant ponderal index and lower cord blood insulin and leptin concentrations, suggestive of potential metabolic dysregulation among infants with greater gestational exposures. Larger prospective studies are needed to replicate these findings and determine whether associations at birth persist into childhood.Additional file 1: Table S1. Overall within-subject coefficients of variation (%CV) for urinary organophosphate ester metabolite concentration (ng/mL) duplicate samples, median %CV, and range of %CVs. Table S2. Specific gravity-corrected urinary organophosphate ester metabolite concentrations (ng/mL). Table S3. Intraclass correlation coefficients (ICCs) of urinary organophosphate ester metabolite concentrations measured up to three times during pregnancy and incorporating assay variability. Table S4. Repeated measures Pearson correlations between log2-transformed specific gravity-corrected urinary organophosphate ester metabolite concentrations. Table S5. Unadjusted odds ratios or percent differences of specific gravity-corrected urinary organophosphate ester metabolite concentrations. Table S6. Birth outcome and biomarker characteristics of infants included in study. Table S7. Adjusted differences  in birth outcomes per standard deviation increase in visit-averaged urinary organophosphate ester metabolite concentrations estimated using OLS regression models. Table S8. Adjusted joint-exposure effects of OPEs (Ψ) on z-standardized birth outcomes  and expected outcome z-scores at each quartile (Q) of the OPE mixture estimated using quantile g-computation. Table S9. Adjusted differences  in z-standardized cord blood biomarker concentrations per standard deviation increase in covariate-adjusted urinary organophosphate ester metabolite latent factors estimated using structural equation regression models. Figure S1. Multiple-indicators-multiple-causes (MIMIC) model of urinary organophosphate ester metabolite concentrations and infant birth outcomes. Model loadings (λ) and indirect effects (γ) estimated among all 90 women with available urine samples. Associations (β) of the latent factor (F) with birth outcomes estimated using robust full-information maximum likelihood. Model error represented by ε0. Figure S2. OPE metabolite-specific weights indicating proportion contributions to the OPE mixture effect in the negative or positive direction. Shading of bars indicates strength of directional association. Birth length (A), ponderal index (B), gestational age at delivery (C), and birth weight for gestational age (D). Figure S3. Expected birth outcome z-score associated with quartile increases in the OPE mixture. Birth length (A), ponderal index (B), gestational age at delivery (C), and birth weight for gestational age (D).


SLE combined with antiphospholipid syndrome (APS) was associated with the risk of miscarriage . The positivity of multiple antiphospholipid antibodies (aPL) was associated with APO overall . IgG anti-cardiolipin during pregnancy resulted in a higher risk of preterm delivery . Hypocomplementaemia was associated with several APO, but only in the first pregnancies. We conclude that, despite the close follow-up provided, a majority of pregnancies resulted in ≥1 APO, but a few of them were severe. Our study confirms the importance of previous lupus nephritis as a main risk factor for APO in patients with SLE.While the management of pregnant patients with systemic lupus erythematosus (SLE) has improved over the last decades, the risk of maternal, foetal, and neonatal complications is still substantial. We evaluated the occurrence of adverse pregnancy outcomes (APO) occurring in 2002–2018 among patients with SLE from the catchment area of the Department of Rheumatology in Lund, Sweden. Longitudinal clinical and laboratory data were collected and analysed. Results were stratified according to the sequence of conception. We investigated a total of 59 pregnancies in 28 patients. Prior lupus nephritis was the clinical feature that, in a multivariable regression analysis, displayed the strongest association with APO overall (OR 6.0,  Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that often affects fertile women. Adverse pregnancy outcomes (APO), commonly grouped into maternal and foetal/neonatal complications, occur in the obstetric general population. Pregnant SLE patients are at increased risk of both maternal and foetal/neonatal APO ,2. In thMaternal APO observed in SLE include increased disease activity, pre-eclampsia, eclampsia and Haemolysis, Elevated Liver enzymes and Low Platelet count (HELLP) syndrome, as well as obstetrical complications, such as preterm labour, unplanned Caesarean delivery, and conditions related to pre-eclampsia ,4,5. FoeThe study aimed to contribute to the field of knowledge with a report of real-world longitudinal data from one single tertiary referral rheumatology centre in southern Sweden, where a multi-professional follow-up of pregnant SLE patients has been carried out since 2002. In particular, we aimed to assess the occurrence of APO in our cohort and how some established and putative SLE-related risk factors may predict the pregnancy outcomes.In patients affected by SLE, from the catchment area of Skåne University Hospital, Sweden, we investigated the outcome of all the pregnancies occurred between 2002 and 2018. Each included patient fulfilled the 1997 American College of Rheumatology (ACR) and/or the 2012 Systemic Lupus International Collaborating Clinics (SLICC) classification criteria ,24 and wWhenever pregnant, the patients underwent multi-professional follow-up, with recurrent visits at the local antenatal clinic, often in presence of one of the health professionals responsible for the regular follow-up at the rheumatology department. After informed consent from the pregnant patients, we gathered the relevant clinical features and laboratory data already recorded in the local SLE database, to define the clinical phenotype of each patient , as wellDisease activity and acquired organ damage were assessed using the SLEDAI-2000 (SLEDAI-2K)  and the To account for systematic bias, we analysed all the first pregnancies occurring after the diagnosis of SLE apart from the remaining pregnancies. Women who had been pregnant up to 12 months before the time-point of SLE diagnosis were not suitable for the “first pregnancy group” but for the “subsequent pregnancy” group. Identical statistical analyses were employed separately in these two subgroups, as well as in the pooled data of all pregnancies.We collected data concerning the following maternal complications, documented during the investigated pregnancies: gestational hypertension, pre-eclampsia (or eclampsia or HELLP syndrome), preterm delivery , and gestational diabetes.We assessed the occurrence of the following foetal/neonatal complications: foetal loss, defined as early miscarriage (occurring before 10th week of gestation), or late miscarriage (occurring between the 10th and 24th week of gestation), or intrauterine foetal death (IUFD) ; stillbirth; IUGR ; SGA newborns ; low birth weight ; congenital heart block and NLE.We investigated the role of SLE-related clinical and immunological features as putative risk factors for the development of APO. The following clinical features were assessed and gathered as binary variables (yes/no): concomitant APS (recognized before the time of conception) and history of LN . In addition, acknowledged risk factors for APO in the general obstetric population were studied, such as smoking habits , obesity (body mass index >30) and age ≥35 years at the time of conception .The following immunological findings were gathered as binary variables (yes/no) and grouped as “ever present”, “present 1-year before conception” and “present during pregnancy”: presence of anti-dsDNA, anti-Ro/SSA, anti-La/SSB, anti-cardiolipin , anti-β2-glycoprotein-I , lupus anticoagulant (LA) test and hypocomplementaemia (decreased levels of complement proteins: C3 and/or C4 and/or C1q).The assessments were made at the local clinical immunology laboratory, according to the validated assays and methods in current use at the time of evaluation.p-value of <0.05 was considered significant. Potential risk factors for developing any APO in each studied pregnancy were examined also by logistic regression analysis (univariate and multivariable). All the significant associations in the univariate model were included in the multivariable analysis. No corrections for multiple comparisons were made, but by reporting the exact p-values, we enable this by any preferred method [Statistical analyses were performed with SPSS Statistics, version 25.0  and GraphPad Prism, version 6.07 . Median values were calculated for continuous variables. Other variables were presented as binary categorical variables, apart from the SLEDAI-2K and SDI scores, collected as continuous variables and presented (mean ± SD) in a Box-whisker plots graph. To handle with repeated measurements during each gestation and with data collected from recurrent gestations in the same patient, we used Generalized Estimating Equation (GEE) to assess the association between potential risk factors and outcomes (APO). A d method .Oral and written informed consent was obtained from all participants. The study protocol was approved by the regional ethics board in Lund (Dnr: LU 378–02).Twenty-eight patients affected by SLE experienced 1–5 gestations each during the study period. We totally investigated 59 pregnancies, whereof 26 were categorised as “1st pregnancies”. The remaining 2 patients had had ≥1 gestation before the onset of SLE; therefore, only their subsequent pregnancies were recorded. Twenty-one patients accounted for the 33 “subsequent pregnancies”. Overall, 61 embryos were conceived, being 2 out of the 59 gestations multiple (3.4%) with twins.All recorded demographic data, clinical manifestations, immunological features, and pharmacological therapy concerning the included patients are summarized in At least 1 APO complicated 33 pregnancies (56%) in 18 patients (64%). A total of 44 gestations (75%) ended with delivery, whereof 8 (18%) were preterm. No post-term deliveries were recorded. As shown in Regarding foetal/neonatal adverse outcomes, we observed 13 cases (22%) of early foetal loss, but no cases of late miscarriage, IUFD, or stillbirth. Restricted foetal growth  was recorded in 13 pregnancies (30%). One case (1.7%) of NLE and no congenital heart block was observed.Among the 26 “1st pregnancies”, 22 (85%) ended with delivery and 4 with early miscarriage. A total of 11 of these 22 deliveries (50%) were complicated with ≥1 APO. The 33 “subsequent pregnancies” resulted in 22 (66%) deliveries, 9 (27%) early miscarriage and 2 (6%) induced abortions. Seven deliveries (32%) were complicated with ≥1 APO. Further details concerning types of APO observed are summarized in p = 0.04) of early miscarriage. Furthermore, the presence of aPL was associated with increased risk  of any APO. In particular, the presence of aCL during pregnancy resulted in a higher risk  of preterm delivery, regardless of APS. A history of LN was associated with increased risk of any APO , particularly any kind of restricted foetal growth .The analysis of all the 59 gestations showed that APS diagnosed before the time of conception in SLE patients may enhance the risk . Restricted foetal growth was associated with presence of different immunological features and clinical manifestations, such as low levels of C3  and C4  up to 1-year before conception, as well as presence of aPL during pregnancy , concomitant APS diagnosis  and previous LN . In addition, increased risk of pre-eclampsia/HELLP was associated with low C4 levels detected up to 1-year before conception . At first pregnancy after the onset of SLE, the detection of aCL during the gestation was associated with an increased risk of preterm delivery , concomitant APS diagnosis  and a history of LN  indicated higher risk of miscarriage. Ever documented presence of aPL  and previous LN  were also associated with increased risk for any APO. In contrast, ever documented low C3 levels  showed an inverse association with pre-eclampsia/HELLP. The associations between risk factors and APO are summarized in In the subgroup of 33 subsequent pregnancies, a positive aCL test ≤1-year before the time of conception , particularly in the subsequent pregnancy group . In the subgroup of 26 first pregnancies, none of the investigated risk factors showed any significant associations with maternal or foetal APO in the univariate model.Next, we performed a multivariable logistic regression analysis, emphasizing that previous LN was the only significant risk factor in our study population for the occurrence of any APO , as compared with the incidence recorded between 2002 and 2009 (60%). We speculate that the accurate use of effective and safe drugs, as well as the implementation of a multi-professional follow-up program, has contributed to achieving a lower rate of APO among patients with SLE, despite the growing number of recorded gestations in our centre.Renal involvement in SLE has previously been reported as a risk factor for developing several APO, mainly foetal loss, preterm delivery, restricted foetal growth, and pre-eclampsia ,14,34,35Concomitant APS and the presence of aPL have already been reported as potential risk factors for the development of APO ,21,36,37The results of our investigation show that hypocomplementaemia, or activation of the complement system, during the first pregnancy after the presumptive onset of SLE, may have a pathological role in the occurrence of some APO, such as pre-eclampsia and restricted foetal growth . This isOn the other hand, any decreased level of C3 ever documented in our patients showed an inverse association with the occurrence of pre-eclampsia/HELLP syndrome during the subsequent pregnancies. These results were unexpected and should be interpreted with caution. However, it may reflect the tailored interventions usually undertaken to minimize the risk of APO in patients with a history of hypocomplementaemia . In lineMany reports have emphasized the importance of low disease activity in women with SLE at the time of conception and throughout the entire pregnancy period, to minimize the risk of APO ,50,51. KSome limitations should be acknowledged. Firstly, we could not investigate the effect of medication on the major APO. Moreover, the small number of studied pregnancies in a limited number of patients from one single centre may have led to uncertain results, and conclusions could thus be difficult to generalize to a broader population of SLE patients. Finally, one must also consider the risk of observation and treatment bias; patients so closely followed might run the risk of being delivered at the first signs of a developing APO, such as pre-eclampsia or restricted foetal growth, thereby increasing the number of abnormal parameters observed but limiting the number of serious APO. On the other hand, a major strength is the Swedish healthcare system, which is public, tax-funded, and offers universal access. This significantly reduces the risk of selection bias and ensures a very high coverage of cases, especially at a tertiary referral centre, offering high-specialized health-care services with longstanding experience of SLE care . A clearTo conclude, the results of the present investigation highlight the risks of APO in SLE. No new predictors could be identified in this study. Nonetheless, our data indicate the importance of planning pregnancy and organizing a multi-professional follow-up during pregnancy, with regular visits, to minimize the prevalence of APO and/or the consequences of APO. The early detection of unfavourable conditions may help to step up surveillance and to provide the patients with the best treatment to prevent and avoid serious adverse outcomes. The sequence of conception may play an important role in the risk stratification and prevention. We confirm the importance of some clinical phenotypes  and immunological factors  as risk factors for the occurrence of APO. Larger multicentre studies would be needed to identify further reliable predictors of APO and to investigate the actual impact of pharmacological treatments.


Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease. However, no reliable biomarkers have been identified to represent the clinical status. This study aimed to investigate whether diffusion tensor imaging (DTI) findings are useful imaging biomarkers to indicate the clinical status of ALS patients. Ninety-six probable or definite ALS cases and 47 age- and sex-matched, normal controls were enrolled. Demographic and clinical data were collected at the time of DTI. DTI data were acquired using a 3-Tesla magnetic resonance imaging scanner and analysed by voxel-wise statistical analyses for fractional anisotropy, axial diffusivity, radial diffusivity, mean diffusivity, and mode of anisotropy. Compared with the healthy control group, the ALS group had significant differences in DTI scalars in the diffuse tracts of the brain, which was predominant in the corticospinal tract at the brainstem and cerebellar peduncle area. Furthermore, the DTI values correlated with the ALS functional rating scale-revised (ALSFRS-R) scores and the delta ALSFRS-R score representing the rate of disease progression. The subgroup analysis revealed a more severe and widespread brain degeneration was observed in rapidly progressive ALS. Therefore, our results suggest that DTI findings are useful as imaging biomarkers for evaluating the clinical severity and rate of disease progression in ALS. The identification of genes linking ALS with frontotemporal dementia (FTD) and the emerging concept of multisystem proteinopathies have changed the widely held belief that ALS affects only the motor neuron system3. This heterogeneity has been recently emphasised, not only in the designing of clinical trials with stratification or enriched enrolment criteria but also in our understanding of the post-hoc analysis of responders and non-responders for the specific treatment. Therefore, stratification of patient groups according to clinical phenotypes  should be considered while designing clinical trials in heterogeneous ALS populations. In addition, objective biomarkers that can be used to reflect the disease status and predict prognosis could be valuable for the diagnosis and treatment of ALS and the design of clinical trials.Amyotrophic lateral sclerosis (ALS) is a heterogeneous disease in many aspects, including clinical phenotypes and genetics4. Multiple DTI scalars extracted from raw DTI data reflect the various pathological changes. Fractional anisotropy (FA) is the most widely used scalar in DTI and indicates various characteristics of axon fibres, including the number and size of axon fibres, and the density of crossing fibres5. Axial diffusivity (AD) has been suggested to be sensitive to axonal pathologies; radial diffusivity (RD) has been shown to be sensitive to myelination; and mean diffusivity (MD) has been shown to be related to cellularity, oedema, and necrosis, which may reflect the white matter tissue microstructure that affects functional connectivity9. In addition, the mode of anisotropy (MO) has been shown to be sensitive to crossing fibres10.With advances in brain imaging techniques, functional and microstructural changes in the central nervous system (CNS) have been investigated. Specifically, diffusion tensor imaging (DTI) is one of the most useful magnetic resonance imaging (MRI) techniques for examining microstructural changes in the CNS15. Notably, decreased FA in the corticospinal tract (CTS) is the main DTI finding in ALS. However, previous studies have mostly focused on FA. Some studies used AD, RD, and MD values along with FA values to evaluate ALS  with a large number of ALS participants (n = 87) investigated only the corticospinal tract from the primary motor cortex to the cerebral peduncle16.Previous studies using DTI have demonstrated the widespread degeneration of the brain in ALSThis study aimed to investigate whether DTI findings can be reliable imaging biomarkers for evaluating the clinical phenotype and disease severity of ALS. We examined multiple DTI scalars in ALS cases compared with those in normal healthy subjects, as well as the correlation of DTI findings with clinical parameters. In addition, we examined differences in DTI findings according to clinical manifestations, such as symptom onset regions, cognitive status, and the rate of disease progression.SOD1 mutations, and one had FUS mutation).In total, 96 ALS patients and 47 NCs were included in this study Fig. . The demp < 0.05). In particular, ALS patients exhibited significantly decreased FA values in the bilateral CST at the brainstem level (p < 0.05). The AD map revealed significantly decreased AD values in the bilateral CST at both the internal capsule and brainstem level, whereas significant increases were observed in the CC and bilateral corona radiata (CR) in ALS patients (p < 0.05). Both RD and MD maps revealed significantly increased values in the CC and bilateral CR in ALS patients (p < 0.05). In addition, ALS patients had significantly increased RD values in the bilateral CP and left CbP (p < 0.05). The MO map revealed significantly decreased MO values in the CC and left CST, whereas significantly increased MO values were observed in the bilateral internal capsule in ALS patients (p < 0.05).The TBSS analysis revealed significantly decreased FA values, increased RD and MD values, and a mixed pattern of AD and MO values for patients with ALS Fig. . The FA p < 0.05), whereas no statistical differences were observed in RD, MD, and MO values. In addition, ALS patients exhibited significantly increased RD, AD, MD, and MO values in the CR (p < 0.05), but no statistical differences were found in FA values. Significantly decreased FA values along with increased AD, RD, and MD values were observed in the CC (p < 0.05) in ALS patients. In addition, ALS patients had significantly decreased FA and AD values, but increased RD values in the CP. In the CbP, ALS patients exhibited significantly decreased FA values but increased RD and MD values (p < 0.05).In the VOI analysis , bilateral CST at the brainstem level , bilateral CP , and bilateral superior and inferior CbP . However, both the RD and MD values negatively correlated with the ALSFRS-R score at the initial evaluation in the CC , bilateral CST at the brainstem level , right superior CR , left CP , right medial lemniscus , and bilateral inferior CbP . Supplementary Table A total of 78 ALS patients without known genetic mutations were classified into three subgroups according to the symptom onset regions. One patient who exhibited axial weakness as an initial symptom was excluded. The mean age at onset and study entry was older in the bulbar-onset group than in both the upper- and lower-limb-onset groups. The mean bulbar FRS scores were significantly lower in the bulbar-onset ALS subgroup than in the other subgroups. The mean upper and lower limb FRS scores were significantly lower in the upper- and lower-limb-onset ALS groups, respectively. No differences were found between the mean respiratory FRS scores of the three subgroups. The clinical characteristics of the subgroup are summarised in Table In the VOI analysis  had a longer disease duration than those in the other two groups. The subgroup comparisons of clinical characteristics are summarised in Table In the VOI analysis  and the rate of disease progression (ΔALSFRS-R). In the subgroup analysis based on the rate of disease progression, further changes in DTI values representing more severe brain degeneration were found in the rapidly progressive ALS group (ΔALSFRS-R ≥ 1) compared with the other subgroups. Therefore, our findings suggest that DTI findings have potential as imaging biomarkers for representing the clinical and functional status in ALS.28. Moreover, recent studies have reported the alteration of cerebrocerebellar connectivity in motor neuron disease30. These findings could suggest the involvement of the cerebellum and its connectivity in ALS, although further studies are needed to elucidate the role of the cerebellum in ALS. Taken together, ALS could manifest as widespread degeneration of the CNS, including the lower brainstem and cerebellum. Thus, a whole-brain DTI protocol  would be needed when performing DTI in the study of ALS.Our study found significant differences in the DTI values between patients with ALS and NCs in the CST at the brainstem level and CbP area using multiple DTI parameters. Anatomically, the CST is the motor pathway extending from the cerebral cortex to the spinal cord, and the CbP is the connecting pathway between the brainstem and cerebellum. In our study, significantly decreased FA and AD values in the CST at the brainstem level were observed. Since the tract fibres are tightly packed in the brainstem, the DTI values at the brainstem level could be more sensitive to structural changes. Another finding of our study is the significantly decreased FA value along with increased RD and MD values at the CbP. Little is known regarding the involvement of the cerebellum and cerebrocerebellar connectivity in ALS. Few studies have demonstrated the degeneration of the cerebellum in ALS33. Therefore, our findings could imply that DTI may be used to investigate the clinical severity and the rate of disease progression in ALS. Several studies have previously reported the correlation between DTI values and clinical data. Previous studies have also reported that FA values were related to the ALSFRS score, upper motor neuron score, and rate of disease progression34, and the MD value was correlated with the disease duration11. Furthermore, longitudinal studies have shown significantly decreased FA values28 and increased RD values20. The changes in DTI values correlated with those of the ALSFRS-R score26. Taken together, DTI has an application potential as imaging markers representing the rate of disease progression and functional status in ALS.Our study showed a weak yet significant correlation between DTI values and the clinical data (the ALSFRS-R score and ΔALSFRS-R from the onset). These correlations were observed in not only FA values but also MD and RD values. Specifically, the correlations between the clinical data and the values of FA, RD, and MD were predominant in the motor tracts, especially CC, CST, and CbP areas. We also demonstrated that the rapidly progressive ALS group had more severe neurodegeneration in CST, CP, and CbP in the subgroup analysis. The ALSFRS-R score and rate of disease progression (ΔALSFRS-R) are promising predictors of prognosis in ALS20, whereas others found no significant differences between bulbar- and limb-onset ALS36. In addition, previous studies investigating distinct differences in DTI findings according to the cognitive functional status demonstrated that ALS patients with cognitive impairment had more widespread degeneration in the white matter, including not only the motor tract, CST, and CC but also the extra-motor regions, particularly the frontal lobe37. These findings may suggest that DTI seems to be a less effective tool for distinguishing the phenotypes of ALS; nonetheless, it may postulate that ALS manifests as widespread white matter degeneration in the brain, regardless of the phenotypes. Further investigations are warranted to determine the correlation between DTI and ALS phenotypes.ALS has heterogeneous phenotypes in terms of the symptom onset regions and cognitive function. However, what determines the ALS phenotypes remains unclear. In our study, no significant differences in DTI were observed between bulbar- and limb-onset ALS. We also could not identify differences in DTI findings between ALS patients with and without cognitive impairment. In previous studies investigating topographical distributions of brain degeneration according to the symptom onset regions, some demonstrated that bulbar-onset ALS had significantly decreased FA in the corticospinal tractAlthough our study was conducted retrospectively, a large number of ALS patients were enrolled from the prospectively registered patient database at two university-affiliated hospitals. In addition, only patients with clinically probable or definite ALS according to the El Escorial criteria were included in the analysis to enhance the reliability of this study.In conclusion, DTI parameters have demonstrated widespread degeneration of the brain in ALS, and the changes in DTI findings exhibited significant correlations with the ALSFRS-R score at the initial evaluation and the rate of disease progression in ALS. Our results indicate that DTI findings could be reliable imaging biomarkers for assessing the clinical severity and rate of disease progression using ΔALSFRS-R in ALS.38. Patients who did not undergo DTI and whose first symptoms manifested before 40 years of age39 were excluded from this study. Age- and sex-matched healthy volunteers were recruited as normal controls (NCs).We reviewed the medical records of all ALS patients who were prospectively enrolled in the ALS Registry of two university-affiliated hospitals since 2008. Patients with definite or probable ALS  were included in this studyData on demographic and clinical characteristics were collected. The functional status at the time of DTI scanning was examined using the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R). The rate of disease progression at the time of DTI scanning was evaluated using the delta ALSFRS-R score (ΔALSFRS-R), which was calculated using the following equation: /disease duration from onset (in months). The ALSFRS-R scores were divided into four parts, i.e., bulbar, upper limb, lower limb, and respiratory scores.SOD1, FUS, TARDBP, OPTN, AND, and C9orf72, which were regarded as ALS-FTD spectrum-related genes in all participants41. In addition, neuropsychological assessment was performed using the Seoul neuropsychological screening battery42. We defined cognitive impairment as the 16th percentile (one standard deviation) below the healthy controls. The participants were categorised into two groups according to the revised ALS-FTD diagnostic criteria43: ALS without cognitive impairment and ALS with cognitive impairment, including patients with ALS-FTD or patients with cognitive or behavioural impairment.To exclude the genetic influence on DTI analysis, we screened six genes, including This study was approved by the Institutional Review Board (IRB) of Korea University Anam Hospital (IRB number: 2015AN0334). Our ethics committee waived the requirement for written informed consent by participants because this study was conducted by reviewing retrospective data. In addition, this study was performed in accordance with the Declaration of Helsinki and the relevant institutional guidelines and regulations.0 image with b-value = 800 s/mm2, repetition time/echo time = 13898 ms/55 ms, field of view = 224 × 224, matrix = 112 × 112, number of average = 2, slice thickness = 2.0 mm, and flip angle = 90°). The SENSE acceleration factor was not applied to increase the signal to noise ratio.Brain DTI were acquired using a 3.0-T Achieva MRI system  with a 32-channel sensitivity encoding (SENSE) head and neck coil. DTI images were acquired with transverse orientation using a pulse sequence with a single shot spin-echo diffusion-sensitised echo-planar imaging sequence (15 gradient directions plus Bhttp://www.fmrib.ox.ac.uk/fsl/) was used for pre-processing raw DTI data. The voxel-wise statistical analyses of the fractional anisotropy (FA), axial diffusivity (AD), radial diffusivity (RD), mean diffusivity (MD), and mode (MO) data were performed using tract-based spatial statistics (TBSS)44. A binary brain mask image was segmented from each DTI image using the brain extraction tool. The eddy current distortions and head movements of the DTI images were corrected using FSL’s ‘eddy’ tool45, and then, a binary brain mask image was segmented from each DTI image using the brain extraction tool. Images of FA, Eigenvalue , MD, and MO were generated using FSL’s ‘DTIFIT’ tool. All FA images were nonlinearly transformed to the FMRIB58 _FA template image and were resampled into a 1 × 1 × 1 mm3 standard space  using the FMRIB Nonlinear Image Registration Tool. Subsequently, the transformed FA maps were averaged to produce a mean FA image, which was then used to generate FA skeleton white matter tracts of all subjects. The skeletonised FA maps were fed into a voxel-wise group-level analysis. Using the FA map’s transformation matrix, the AD, RD, MD, and MO images were transformed to a standard space, and skeletonised images of AD, RD, MD, and MO were then generated. The FA skeleton image was set at a threshold of FA ≥ 0.2 to exclude tracts, which could have high inter-subject variability or partial volume effects. Volumes of interest (VOIs) were defined on the basis of Johns Hopkins University International Consortium of Brain Mapping-DTI-81 White-Matter Labels in FSL  Software Library  between clinical data and DTI parameters.Comparisons of DTI findings between patients with ALS and NCs were performed using a generalised linear model with sex and age at the time of DTI as covariates. The resulting data were corrected for multiple comparisons using a false discovery rate method, with p-value of <0.05 was considered statistically significant in this study. Statistical analyses were performed using SPSS software version 20.0 .In addition, three subgroup analyses were performed. In the first subgroup analysis, ALS patients were divided into three subgroups based on the symptom onset regions: bulbar, upper limb, and lower limb. In the second subgroup analysis, ALS patients were divided into two subgroups based on the cognitive profiles: ALS with or without cognitive impairment. In the third subgroup analysis, ALS patients were classified into three subgroups according to the rate of disease progression: Group A (ΔALSFRS-R < 0.5), Group B (0.5 ≤ ΔALSFRS-R < 1), and Group C (ΔALSFRS-R ≥ 1). In the subgroup analyses, only subjects without genetic mutations were included. Repeated measures ANOVA with within-subject factor (VOIs) and between-subject factors (age at DTI and sex) was applied for comparisons between ALS subgroups and NCs. A Supplementary data.


Apolipoprotein B is considered the primary protein constituent of low-density lipoprotein. LDL contains variable quantities of cholesterol, but each lipoprotein contains a single ApoB protein. Thus, ApoB is a better index for the LDL circulation if compared to LDL cholesterol. On the contrary, apolipoprotein A-1 is a main structural protein of high-density lipoprotein. It has a major role in reversing cholesterol flow and cellular cholesterol homeostasis once detected. The aim of the study is to measure apo B/apo A-1 ratio in patients with acute coronary syndrome and assess its relationship with the severity of CAD.A total of 90 patients were enrolled in the study and subdivided into 3 groups: 30 patients of STEMI, 30 patients of NSTEMI, and 30 patients presented with unstable angina. Serum levels of apolipoprotein A-1 and apolipoprotein B were properly measured upon admission, and apo B/apo A-1 ratio was calculated.P value <0.001). High Gensini score patients had significantly high Apo B/Apo A1 ratio with the best cutoff value of 0.8 with sensitivity of 90% and specificity of 70%.Both of Apo B and Apo B/Apo A1 ratio correlated significantly with Gensini scores (Apo B is an independent risk predictor for the severity of CAD in patients with acute coronary syndromes. Moreover, the Apo B/Apo A1 ratio remains highly significant in patients with high Gensini score. Acute coronary syndrome refers to a range of different presentations which are ST-segment elevation myocardial infarction (STEMI) and non-ST-segment elevation myocardial infarction (NSTEMI) and unstable angina (UA). Pathologically speaking, ACS is commonly associated with an atherosclerotic plaque rupture that forms thrombus in the infarct-related artery that leads to severe damages . DyslipiApolipoprotein B is considered the major protein constituent of low-density lipoprotein. LDL had variable quantities of cholesterol, but each lipoprotein had only one protein which is ApoB; so ApoB is a better predictor of the number of LDL particles than LDL-C . ApoB waBoth ApoB/ApoA1 ratios were considered a better risk predictor to acute myocardial infarction than the TC/HDLc ratio . In thisThis was a cross-sectional observational study that involved 90 subjects who were eligible for coronary angiography.The patients were categorized into 3 groups according to their ACS presentation. Group I included 30 patients admitted with STEMI, group II included 30 patients with NSTEMI, and group III included 30 patients with unstable angina. The patients were further subdivided according to their Gensini score (GS) into high Gensini score > 47, intermediate Gensini score from 24 to 47, and low Gensini score < 24 .Those with a history of chronic renal failure, sepsis, severe chronic liver disease, previous coronary stenting, and CABG were excluded from the study. Informed written consent was taken from all patients in the study, and the study was approved by the local ethical committee.Studied patients underwent detailed history taking, full clinical examination, 12-lead ECG, and laboratory investigations in the form of cardiac enzymes (troponin and CK-MB), serum creatinine level, and serum levels of Apo A-1 and Apo B at the time of admission by nephelometric analysis.F test with pairwise comparison between different groups was used. Chi-squared (χ2) test was used for comparison regarding qualitative variables. Pearson’s correlation was used to show strength and direction of association between two quantitative variables. ROC (receiver operating characteristic) curves were used to show the connection between clinical sensitivity and specificity for every possible cutoff for a test or a combination of tests. P value was considered significant if <0.05.Results were statistically analyzed by statistical Package for Social Science . P value > 0.05)  Table .Table 1P value <0.05). On the contrary, hypertension did not show significant difference in the studied patients   Table .Table 2P value <0.05). Moreover, Apo B levels in patients presented with NSTEMI exceeded those with STEMI, but it did not reach significant value .In our study, we noticed that patients presented with STEMI and NSTEMI had significantly higher levels of Apo B than those with unstable angina   Table .Table 3P value <0.05). Moreover, Apo B/Apo A1 ratios in patients presented with NSTEMI were more elevated than those with STEMI, but it did not reach significant value    Table . Additio5) Table .Table 4P value <0.001). Furthermore, the patients with intermediate GS had significantly higher Apo B levels and Apo B/Apo A1 ratios than those with low GS .The patients with high GS had significantly higher Apo B levels and Apo B/Apo A 1 ratios than those with low GS  but Apo B levels did not show any significant difference between both groups    Table .Fig. 2CP value <0.001). Also, there was a significantly positive correlation between Apo B/Apo A1 ratio and Gensini scores    Fig. . On the 5) Table .Fig. 3CApo B/Apo A1 ratio was significantly high in patients with high GS with the best cutoff value (0.8) at a 90% sensitivity and a 70% specificity Fig. .Fig. 4RSeveral epidemiological and clinical studies have constantly and reliably documented that LDL-C constitutes the chief atherogenic lipoprotein for evolving ACS. Substantially, it is viewed as the principal target for lipid-lowering therapies if we targeted to cardiovascular-disease prevention and treatment , 10. SurOur study included 90 patients admitted with ACS. They were divided according to their presentation into 3 groups: STEMI, NSTEMI, and unstable angina. All the patients underwent coronary angiography and were further subclassified according to their Gensini scores into 3 groups: high Gensini score > 47, intermediate Gensini score (24–47), and low Gensini score < 24. Serum Apo B and Apo A1 levels were measured and compared with their Gensini scores.P value <0.001). Patients with NSTEMI and STEMI had significantly higher Apo B/Apo A 1 ratio and Apo B levels than those with UA . On the contrary, no significant correlation between Apo A and Gensini scores was found . It is known that each particle of the atherogenic lipoproteins like low-density lipoprotein, very-low-density lipoprotein, intermediate-density lipoprotein, and lipoprotein (a) carries a single ApoB100 molecule. Hence, the plasma ApoB100 concentrations notably reflect proatherogenic potentials. On the other hand, ApoA is the key apolipoprotein component of high-density lipoprotein which represents the ApoA serum content whereby antiatherogenic potentials are witnessed. Thus, the ApoB100/ApoA1 ratios represent the balance between the harmful and beneficial potentials. The higher the ApoB100/ApoA1 ratio, the more developed atherogenic potentials we get and/or the less antiatherogenic potentials we attain [We found a strong positive correlation of both Apo B and ApoB/ApoA1 in predicting high Gensini scores (scores ≥ 47) in patients who suffered from acute coronary syndromes  showed that Apo B/Apo A-1 ratios were identified as a single variable which was closely associated with attributable risk of fatal MI, predominantly when lipid levels fall within the range of desirable values .Additionally, the INTERHEART study examined about 30 thousand individuals in 52 countries and concluded that the Apo B/Apo A-1 ratios were associated strongly with the prediction of MI than other traditional risk factors as hypertension, smoking, and diabetes regardless of gender, age, and ethnic group .P value <0.05). These findings come in agreement with a previous study by Du et al. [Moreover, Gensini scores presented in this study were significantly elevated in diabetic than non-diabetic patients . Similarly the Quebec study and the Northwick Park Heart study did not find any significant association between ApoA and CAD risks in multivariable analysis [In our study, no significant difference in Apo A1 levels between the studied patients was found  the small number of study population, the full lipid profile was not taken to compare the predictive power of Apo B, and Apo B/Apo A1 ratios with LDL and triglycerides in detection of severe coronary artery disease, we failed to exclude patients who were already on statin therapy before presentation with ACS and lastly there was no follow-up for the patients with higher levels of ApoB and ApoB/ApoA ratios in order to detect early and late cardiovascular events.Apo B is an independent risk predictor of severity of CAD in patients presented with ACS. Moreover, the Apo B/Apo A1 ratio remains highly significant in patients with high GS with the best cutoff value 0.8 that may add a benefit in risk prediction.


Every Newborn Birth Indicators Research Tracking in Hospitals (EN-BIRTH) is an observational study to assess the validity of measurement of maternal and newborn indicators, and this paper reports findings regarding measurement of coverage and quality for uterotonics.Postpartum haemorrhage (PPH) is a leading cause of preventable maternal mortality worldwide. The World Health Organization (WHO) recommends uterotonic administration for every woman after birth to prevent PPH. There are no standardised data collected in large-scale measurement platforms. The The EN-BIRTH study took place in five hospitals in Bangladesh, Nepal and Tanzania, from July 2017 to July 2018. Clinical observers collected tablet-based, time-stamped data. We compared observation data for uterotonics to routine hospital register-records and women’s report at exit-interview survey. We analysed the coverage and quality gap for timing and dose of administration. The register design was evaluated against gap analyses and qualitative interview data assessing the barriers and enablers to data recording and use.Observed uterotonic coverage was high in all five hospitals . Survey-report underestimated coverage (79.5 to 91.7%). “Don’t know” replies varied (2.1 to 14.4%) and were higher after caesarean (3.7 to 59.3%). Overall, there was low accuracy in survey data for details of uterotonic administration (type and timing). Register-recorded coverage varied in four hospitals capturing uterotonics in a specific column . The average coverage measurement gap was 18.1% for register-recorded and 6.0% for survey-reported coverage. Uterotonics were given to 15.9% of women within the “right time” (1 min) and 69.8% within 3 min. Women’s report of knowing the purpose of uterotonics after birth ranged from 0.4 to 64.9% between hospitals. Enabling register design and adequate staffing were reported to improve routine recording.Routine registers have potential to track uterotonic coverage – register data were highly accurate in two EN-BIRTH hospitals, compared to consistently underestimated coverage by survey-report. Although uterotonic coverage was high, there were gaps in observed quality for timing and dose. Standardisation of register design and implementation could improve data quality and data flow from registers into health management information reporting systems, and requires further assessment.Supplementary information accompanies this paper at 10.1186/s12884-020-03420-x. An estimated 295 000 maternal deaths occur annually worldwide, 99% are in low and middle income countries (LMICs) . ApproxiEvery Newborn and Ending Preventable Maternal Mortality (EPMM) strategies .Fi.Fi32].Within this category, two themes emerged for uterotonic recording. Focus group participants talked about the complexity of health data systems and the specific register design for uterotonics. Across all sites, health workers identified multiple places where they were expected to document information about care during the third stage of labour, including the register, clinical records, partograph, and drug chart. Many staff reported they did not know who would be taking primary responsibility for documentation  was identified as a critical challenge throughout all the focus group discussions and was a key barrier to uterotonic data collection among other indicators:‘There is a specific place kept and arranged for documenting all the provided care … they are supposed to be there, equipment like books for recording and pens [are there], and it is not far’–Data collector, Temeke TZEvidence from Temeke TZ suggests that some of these challenges can be addressed with good organisation of workspaces to ensure that clinical environments are enabling with the required register books, computers, and stationary positioned in convenient clinical locations that facilitate health workers to remain near service users:Healthcare staff reported that they are usually completing care during the third stage of labour and documentation simultaneously. Staff from Kushtia and Muhimbili identified the location of registers as problematic, which was also identified as a register-recording barrier, across all sites, for women giving birth in the operating theatres Fig. ‘We usually buy our pen ourselves, we do not get a pen from the office.’-Health worker, Kushtia BDParticipants from Kushtia BD and Muhimbili TZ reported supply challenges with basic equipment including multiple register stock-outs in Kushtia, and the requirement for staff to supply their own pens:‘These documents show what the patient is suffering from and what medication is given … Also these documents are important for research works, planning, improving health services, helping secure you in the court, and in statistics. The documents are very important in improving health services.’–Data collector, Temeke TZRespondents from Temeke TZ and Azimpur BD reported these sites have instituted regular opportunities for staff to use and reflect on their data. Moreover, staff in these hospitals were using data for a variety of purposes in their own practice:‘Leadership in general from the lower level to the upper level should have good communication and cooperation to ensure that everything is well documented and records are kept with good quality.’–Data collector, Temeke TZFeedback was consistently valued by staff in all hospitals, and health workers suggested leadership was an enabling factor for documentation. Temeke TZ had highly accurate register reporting for uterotonics. Staff reported being well-supported by management with regular feedback, training and opportunities to use their data during budget planning, stock management, audit and monitoring:Staff from EN-BIRTH sites with more accurate register-recording of uterotonic coverage reported training as an essential component. Managerial gaps and lack of training were cited as barriers to documentation in Kushtia, the site with lowest performing register-recording.However we are not well trained’.‘–Health worker, Kushtia BDPostpartum haemorrhage remains a leading cause of preventable maternal mortality. Despite WHO recommendations for universal access to prophylactic uterotonics, there are no nationally representative data to track coverage and quality of this intervention , 7. EN-BSurvey-reported data for assessing uterotonic coverage was problematic, with high “don’t know” replies for caesarean births, and lower accuracy than the better performing registers. Our findings align with previous validation studies suggesting low individual-level accuracy for survey measures of uterotonic coverage –24. TherOur results underline that accurate report in surveys is challenging for events around the time of birth, especially for women receiving more complex clinical care (e.g. PPH management or caesarean section). This is unlikely to be recall alone; the women’s knowledge will depend on the quality of information provided by healthcare staff, and if informed consent was elicited , 24. IndRegister completion varies , 38–41. Register design is necessary but not sufficient to achieve high quality data, inclusive training and implementation strategies are also imperative. Despite sharing the same register design and layout, results differed between Temeke and Muhimbili TZ, and between Azimpur and Kushtia BD after implementation of the new national register. Our results support evidence that data collection and management processes represent a heavy workload for health workers , 44–46, Maternal mortality remains high in many settings despite good coverage of facility births ; this diUterotonic coverage was high in our study sites, although these high caseload referral centres are not representative of all facilities in LMICs. Several studies indicate that quality of care is lower in primary-level facilities, especially those with a low case-load . We usedDenominators are crucial for public health decision-making . WorldwiEN-BIRTH study strengths include use of direct observation as gold standard, the large number of participants, time-stamped data, stratification of results by mode of birth, and five differing hospitals from three LMICs. Unfortunately, even the high number of observed births were not able to mitigate statistical challenges validating indicators with high prevalence, especially those only calculated for observations with ≥10 counts in each column of the 2 × 2 tables to assess sensitivity, specificity, inflation factor and area under the curve . The golSystematic research and investment in implementation are needed to improve register design and use. Where coverage is high, a simple uterotonics coverage indicator might be insufficient to drive quality improvement. Other measures may be required such as health facility assessments regarding drug quality, and stock management, or use of specific audits. There is potential for linking databases (such as survey and facility-based data) but this may require special studies and complex analyses , 57–60.Assessment of data flow within HMIS and inter-operability with related platforms, such as supply logistics systems, are also needed. This could be undertaken as part of a feasibility assessment of maternal and newborn HMIS tool kits in a range of LMICs and humanitarian settings. It should include data quality assessments at different levels of the HMIS, including costs for data collection and assessment of usefulness to policymakers.EN-BIRTH findings for uterotonics measurement are compatible with existing evidence suggesting that asking women about clinical interventions during or immediately after birth is unreliable –24, espeAdditional file 1. Summary of previous validation for measures of uterotonic administration.Additional file 2. Ethical approval of local institutional review boards, EN-BIRTH study.Additional file 3. STROBE Checklist.Additional file 4. Data collection dates by site, EN-BIRTH study.Additional file 5. Facility register design and completion approaches for uterotonics by site, EN-BIRTH study .Additional file 6. Inter-observer agreement for uterotonic administration using Kappa, EN-BIRTH study.Additional file 7. Survey- reported uterotonic indicator combinations compared with observer-assessed coverage, EN-BIRTH study.Additional file 8. Descriptive uterotonic coverage data: observer-assessed, exit-survey reported and register-recorded findings, EN-BIRTH study .Additional file 9. Individual-level validation of exit-survey report for uterotonic administration, EN-BIRTH Study .Additional file 10. Individual-level validation of register recording for uterotonic administration, EN-BIRTH study .Additional file 11. Comparison of uterotonic coverage measurement using original and revised Bangladesh registers, EN-BIRTH study (n = 5207).Additional file 12. Association testing for timing of Oxytocin administration, EN-BIRTH Study .Additional file 13. Oxytocin dose by EN-BIRTH site and mode of birth, EN-BIRTH study .Additional file 14. Estimated Blood Loss (EBL) compared with Oxytocin coverage, EN-BIRTH Study.Additional file 15. Assessment of routine recording responsibilities for uterotonic provision, EN-BIRTH Study.Additional file 16. Register recording order and prioritisation for uterotonic provision, EN-BIRTH study.


CAR was negatively associated with LAZ at birth  but positively associated at 13–14 months , suggesting infant catch-up growth with lower birth weights, likely related to elevated cortisol exposure, continues beyond early infancy. A negative correlation between breastfeeding and 10-month WLZ  and LAZ  suggests that breastfeeding assists in managing infant growth. WLZ and LAZ increased from birth to 6 months (ps < 0.01), though this was unrelated to interactions between prenatal cortisol and breastfeeding , suggesting that other factors played a role, which should be further investigated. Findings add to our understanding of the predictors of infant growth.Fetal/infant growth affects adult obesity and morbidities/mortality and has been associated with prenatal exposure to cortisol. Bidirectional relations between maternal stress and breastfeeding suggest that they interact to influence offspring growth. No models have tested this hypothesis, particularly regarding longer-term offspring outcomes. We used a subset of the IDAHO Mom Study ( Fetal and infant growth influence the risk of obesity, morbidity, , and mortality across the lifespan . HoweverDuring pregnancy, maternal cortisol is regulated by the placenta, uterine decidua, and fetal membrane, in addition to the hypothalamic–pituitary–adrenal (HPA) axis . SignifiMoreover, there are several indices of cortisol that have been validated in human research with perhaps the most common including area under the curve (AUC) and cortisol awakening response (CAR) . The areThe impact of maternal prenatal cortisol and resulting low birth weight has important implications for offspring outcomes, including increased risk of cardiovascular and metabolic disorders , depression, schizophrenia, and autism . ResearcFurther impacting infant growth is postnatal infant feeding practices. Whether or not an infant is breastfed, as well as the duration of breastfeeding, has been found to be associated with infant growth. Previous research has found that infants who were not breastfed had, on average, a lower weight compared to infants who were breastfed until 4 to 6 months of age ,12. SimiElevated cortisol levels are known to lead to stunted prenatal growth  and decrG) and cortisol awakening response (CAR)) and infant standardized anthropometric growth outcomes  and length-for-age z-scores (LAZ)) and that less breastfeeding at 6 months will increase the strength of this relationship. Specifically, we hypothesize that: (1) greater prenatal maternal circadian cortisol release  will be related to lower infant WLZ (a) and LAZ (b) from birth to 6 months and (2) the negative relationships between greater maternal prenatal cortisol  and lower infant WLZ (a) and LAZ (b) at 6 months will be exacerbated by less breastfeeding  at 6 months.It is hypothesized that there will be significant negative relations between maternal prenatal diurnal cortisol rhythms  Human Subjects Committee. Data for this project were drawn from the larger, longitudinal Infant Development and Healthy Outcomes in Mothers (IDAHO Mom) Study, which examined mothers’ health and behavior during pregnancy and infants’ growth and development at 6, 10, 13–14, and 18 months postpartum. Participants were recruited from local businesses, schools, and doctor’s offices with flyers, brochures, and in-person educational sessions, as well as advertisements on social media platforms and local newspapers, radio, and television stations. Potential participants were contacted and briefly screened prior to being invited to participate in the study. Following oral consent for screening, potential participants were queried on the following exclusion criteria: younger than 18 or older than 35 years of age, consumption of United States Food and Drug Administration (FDA) Category D or X medications during pregnancy , being pregnant with multiple fetuses, having a diagnosis of a severe and persistent mental illness , the anticipated involvement of Department of Health and Welfare after birth, being over 37 weeks gestation, and having a major medical condition  that would result in being considered a high-risk pregnancy. Eligible participants were scheduled for a prenatal session between 33 and 37 weeks gestation.The prenatal session consisted of trained research assistants (at least one undergraduate and one graduate) providing instructions and support to participants during written informed consent procedures, interviews, self-report questionnaires including demographic information, such as ethnicity, household income, and education level, and measurements of their height, weight, and waist circumference as part of the larger study. Participants were compensated for their participation with a monetary award of $30 per session. Following the completion of the laboratory session, participants were provided with a saliva collection kit to measure diurnal cortisol for a 3-day collection period, collecting four samples per day, including: immediately after wakening, 30 min post-awakening, 45 min post-awakening, and prior to going to bed at night. The passive drool method was used for saliva collection because it provides the purest sample possible of whole saliva . Participants recorded their saliva collection times on the instruction sheet and also sent a text message to the IDAHO Mom Study cellular phone and linked e-mail account when they collected each sample if they opted to receive automatic text message reminders. They were instructed not to eat, drink, smoke, or brush their teeth prior to collecting the samples and then to place each sample in their freezer after they were collected. Participants were compensated $5 for each day of completed saliva collection. A total of 30 out of the 125 mothers in the larger IDAHO Mom Study did not return saliva kits. Once the saliva collection kit was returned to the laboratory, saliva specimens were stored in a −20 °C freezer until sent to the ISU Molecular Research Core Facility (MRCF) for cortisol assay . The sensitivity of the Salimetrics kits was <0.007 ug/dL and the assay range for the kits was 0.012–3.00 ug/dL .G) were calculated and used for statistical analyses. CAR is calculated by computing a change score between cortisol concentration at awakening and cortisol concentration post-awakening [G is calculated by summing a series of trapezoids that reflected repeated measurements of cortisol [G values for each participant. AUC indicates the average amount of circulating cortisol, while CAR represents the morning spike in circulating cortisol.Once the ELISA assays were completed, a coefficient of variation (CV%) was calculated to determine the intra-replicate variation. Sample concentrations were eliminated if the CV% between the replicates exceeded 15% and the concentration values differed by 0.007 ug/dL. Cortisol concentrations greater than 4 ug/dL were eliminated, as they are unlikely to be physiologically plausible. From the cortisol concentration values, the cortisol awakening response (CAR) and area under the curve with respect to ground . Anthropometric data from mothers and infants were collected at each session. Infant birth weight and length were reported via a maternal report at the 6-month session. Infant length was measured using a ShorrBoard (±0.1 cm), infant weight was assessed using a Seca mother–infant scale (±100 g), and infant waist circumference was measured with a ShorrTape (±0.1 cm). The scale was calibrated monthly to ensure accurate measurements. Infant weight was measured once, whereas length and waist circumference were collected twice and averaged across the two measurements. Measurements were collected in kilogram and centimeter units. Z-scores for Weight-for-Length (WLZ) and Length-for-Age (LAZ) were calculated based upon infants’ sex and age at birth and each follow-up session via conversion software utilizing the WHO Child Growth Standards  . To calcG and CAR). Correlation analyses were also conducted to examine associations among breastfeeding duration and frequency and infant growth from birth to 18 months. Next, a series of two-way Mixed Analyses of Variance (Mixed ANOVAs) were conducted to test relations between maternal prenatal circadian cortisol release and infant growth from birth to 6 months. Time served as the within-subjects independent variable, maternal prenatal diurnal cortisol indices served as the between-subjects independent variables, and infant anthropometric variables served as dependent variables, in separate models.Sample sizes varied across analyses following data cleaning and therefore are reported in the respective analyses sections. IBM SPSS Statistics (version 24.0) was used for all analyses. Descriptive statistics for demographic variables were conducted to characterize the sample. In order to better understand the relationship between maternal prenatal circadian cortisol release and infant growth, a series of correlations were run to probe the relationship between WLZ and LAZ at birth, 6 months, 10 months, 13–14 months, and 18 months and the cortisol indices  to determine whether maternal breastfeeding frequency and duration moderate the relationship between maternal prenatal diurnal cortisol concentrations and infant growth. A moderation model was conducted for each outcome variable, Y, including WLZ and LAZ, at 6 months with separate models for each primary predictor variable, X, including AUCN = 125) years at the time of enrollment in the study. Seventy-nine percent of participants indicating that they were married, 10% reported being in a committed relationship or engaged, 8% reported being single and 2% indicated that they were divorced. Approximately 54% of participants would be considered middle class, making $30,000 to $100,000 annually with the majority (82%) having continued education beyond high school (see Participants were primarily European-American (93%) with an average age of 26.84  = −0.25, p < 0.05) and a significant positive correlation between infant LAZ at 13–14 months and maternal prenatal CAR (r(32) = 0.38, p < 0.05). There were no statistically significant correlations between infant LAZ at 10 or 18 months and maternal prenatal cortisol. Additionally, there were no statistically significant correlations between infant WLZ and maternal prenatal cortisol indices at any time point.Pearson’s product–moment correlations were conducted to assess relations among infant growth indices, including WLZ and LAZ, from birth to 18 months and maternal prenatal cortisol indices, including AUC CAR See . There wr(35) = −0.47, p < 0.005) and breastfeeding duration (range = 0–180 days of breastfeeding through 6 months) and infant WLZ at 10 months (r(37) = −0.34, p = 0.037). There were no statistically significant correlations between breastfeeding frequency and infant WLZ at any age or between breastfeeding duration and infant LAZ at any age. There were no statistically significant correlations between breastfeeding frequency and infant LAZ or between breastfeeding duration and infant WLZ at any time point.Pearson’s product–moment correlations were completed to examine associations among breastfeeding and infant growth indices from birth to 18 months See . There wHypothesis 1a (H1a).Greater Prenatal Maternal Circadian Cortisol Release Will Be Related to Lower Infant WLZ from Birth to 6 Months.G and infant WLZ from birth to 6 months. There was a significant main effect of age  = 10.75, p = 0.002, ηp2 = 0.164; See G and the interaction between age and AUCG were not significant (p = 0.242 and p = 0.707). Similarly, a two-way ANOVA for maternal prenatal CAR and infant WLZ from birth to 6 months revealed a significant main effect for age  = 44.82, p < 0.001, ηp2 = 0.401). The main effect of CAR and the interaction between age and CAR were not statistically significant (p = 0.061 and p = 0.252).A two-way mixed ANOVA was performed to examine the relationship between maternal prenatal AUCHypothesis 1b (H1b).Greater Prenatal Maternal Circadian Cortisol Release Will Be Related to Lower Infant LAZ from Birth to 6 Months.G and infant LAZ from birth to 6 months, a two-way mixed ANOVA revealed a significant, positive main effect of age  = 12.69, p = 0.001, ηp2 = 0.185; See G and the interaction between age and AUCG was not statistically significant (p = 0.207 and p = 0.573). A two-way mixed ANOVA was performed to examine the relationship between maternal prenatal CAR and infant LAZ from birth to 6 months. There was a significant, positive main effect of age  = 102.55, p < 0.001, ηp2 = 0.601), however, the main effect of CAR and the interaction between age and CAR were not statistically significant (p = 0.055 and p = 0.132).For maternal prenatal AUCHypothesis 2a (H2a).The Negative Relationship between Greater Maternal Prenatal Cortisol and Lower Infant WLZ at 6 Months Will Be Exacerbated by Less Breastfeeding at 6 MonthsG = X, infant WLZ at 6 months = Y, and breastfeeding frequency = W was not found to be statistically significant  = 0.41, R2 = 0.02, p = 0.749). A similar model where breastfeeding duration = W, X and Y held constant, was not found to be statistically significant  = 0.33, R2 = 0.02, p = 0.806). Moderation models where maternal CAR = X, infant WLZ at 6 months = Y, and breastfeeding frequency  = 1.08, R2 = 0.05, p = 0.366) and duration = W  = 0.76, R2 = 0.03, p = 0.519) were not found to be statistically significant.A moderation model where maternal prenatal AUCHypothesis 2b (H2b).The Negative Relationship between Greater Maternal Prenatal Cortisol and Lower Infant LAZ at 6 Months Will Be Exacerbated by Less Breastfeeding at 6 Months.G = X, infant LAZ at 6 months = Y, and breastfeeding frequency = W was not statistically significant  = 1.21, R2 = 0.07, p = 0.314). Similarly, a moderation model where maternal prenatal AUCG = X, infant LAZ at 6 months = Y, and breastfeeding duration = W was not found to be statistically significant  = 1.08, R2 = 0.05, p = 0.365). Moderation models where maternal CAR = X, infant LAZ at 6 months = Y, and breastfeeding frequency  = 0.74, R2 = 0.03, p = 0.533) and duration = W  = 0.49, R2 = 0.02, p = 0.692) were not found to be statistically significant.A moderation model where maternal prenatal AUCThe current study proposed to fill a gap in the existing literature on how maternal prenatal cortisol and breastfeeding influence growth outcomes in later infancy, as previous research has primarily focused on growth outcomes at birth . We hypoAlthough we hypothesized that we would see impacted infant growth from birth to 6 months as a result of early cortisol exposure, we did not find significant main effects. However, we did find that there was a significant effect of age for infant WLZ and LAZ. Given that both age and sex are accounted for in the standardized z-scores used for analyses, this suggests that there are other factors that influence infant growth outcomes in this sample . However, based upon the recruitment strategy and exclusion criteria in this community sample, there was limited variability in these measures, so they were not examined in relation to current study predictors and outcomes. In addition, an examination of potential covariates that might influence this relationship revealed consistently non-significant results in the study sample, including gravida, gestational age, gestational weight gain, and diet.Particularly, although our sample contained infants who were born notably smaller than average (mean WLZ was −1.02), we found that at the first postnatal follow-up session, infants were above average (average WLZ was 0.61). This change in growth could be attributed to our relatively low-risk sample. The inclusion and exclusion criteria for the current study excluded most mothers who would be considered high risk . Furthermore, our study sample had relatively high rates of breastfeeding mothers, with 64% of infants being breastfed through 6 months compared to the national average of 58% at this timepoint , which mThe findings in the current study include important implications for infant growth outcomes. We found evidence for a continued association between elevated maternal prenatal cortisol with later infant growth outcomes suggesting that continued catch-up growth may occur in the offspring of mothers with higher cortisol levels during pregnancy. Higher cortisol levels in pregnancy may be the result of dysregulation of the HPA axis secondary to anxiety, depression, posttraumatic stress disorder, schizophrenia , excessiAdditionally, the current study sample was drawn from a rural area that is classified as a Health Provider Shortage Area for primary care and mental health services, and although the sample is a relatively low-risk community sample, our participants represent a population with fewer studies describing maternal health and infant outcomes and may have fewer resources available for women and infants. Therefore, additional studies are needed to better understand the unique risk and resiliency factors in these settings in relation to offspring growth.AGEM = 26.5 years, largest percentage (27%) with annual income of USD 50,000–75,000, and mostly (68%) completed partial college or BS/BA, (94%) White, and (62%) members of Church of Jesus Christ Latter Day Saints) and without  with annual income of USD 50,000–75,000, and mostly (79%) completed partial college or BS/BA, (92%) White, and (64%) members of Church of Jesus Christ Latter-Day Saints) prenatal cortisol samples. Beyond the current sample, variations in the formulas used to calculate cortisol  may influence findings across research labs and contribute to inconsistencies in findings [Current study findings should be interpreted while considering several limitations. Since the relations between breastfeeding, maternal prenatal cortisol, and infant growth have not been previously examined, it is possible that a relationship between the three variables does not exist, however, it could be that our community sample of low-risk mothers could influence the absence of a relationship among these variables that does, in fact, exist. In order to test this possibility, studies could compare differences between normative and at-risk samples with regard to these important variable relationships. Furthermore, our sample consisted primarily of relatively high and homogeneous socioeconomic status, which could factor into lower levels of cortisol during pregnancy. Lastly, in regard to our sample, there were participants who failed to complete their saliva samples and missing data across time points led to relatively small sample sizes in several analyses, which were likely underpowered with regard to the current study’s analysis plan, particularly when examining moderation effects. It is possible that there were significant and meaningful differences in cortisol concentrations of women who did not complete saliva samples and therefore are not represented in the current data. It is likely that there is a difference between mothers who were more involved or less involved with the study though given the lack of heterogeneity in the current study sample, sociodemographic differences do not appear to exist between mothers with (findings . In futuTaken together, current study results suggest that in utero exposure to cortisol negatively predicts infants’ size at birth, but positively predicts similar anthropometric indices at 13–14 months of age. This may be due to the effects of cortisol on fetal growth restriction, which may result in infants who are born earlier and/or smaller and then engage in rapid catch-up growth. This converges with current study findings that there were significant increases in WLZ and LAZ from below average to above average from birth to 6 months of age. The current study also found significant negative associations between breastfeeding and 10-month anthropometric outcomes, which may be due to the positive effects of breastfeeding for managing a healthy weight in infants. Specifically, breastfeeding has positive benefits for infant nutrition, immune functioning and emotional development compared to formula feeding . In fact