The intestinal microbiome in preterm infants differs markedly from term infants. It is unclear whether the microbiome develops over time according to infant specific factors.We analysed  metadata - to identify the main factors influencing the microbiome composition development - and the first meconium and faecal samples til the 4th week via 16 S rRNA amplican sequencing.Escherichia and Bacteroides  were associated with vaginal delivery while Staphylococcus (skin microbiome representative) was associated with CS. Secondly, irrespective of the week of sampling or the mode of birth, a transition was observed as children children gradually increased in weight from a microbiome dominated by Staphylococcus (Bacilli) towards a microbiome dominated by Enterobacteriaceae (Gammaproteobacteria).We included 41 infants  was associated with placental insufficiency during pregnancy and lower BW. In meconium samples and in samples from weeks 2 and 3 the abundance of Our data show that the mode of delivery affects the meconium microbiome composition. They also suggest that the weight of the infant at the time of sampling is a better predictor for the stage of progression of the intestinal microbiome development/maturation than postconceptional age as it less confounded by various infant-specific factors.The online version contains supplementary material available at 10.1186/s12866-021-02279-y. Clostridium perfringens in many studies, while staphylococci appear to be associated with a decreased risk for NEC development [Extreme prematurity involves high mortality and morbidity . Diseaseelopment –13.Previous studies on the intestinal microbiome of preterm infants, using 16 S rRNA-based sequencing technologies, have revealed remarkable differences with the microbiome of term infants, including higher abundances of bacilli and Gammaproteobacteria –26. The Besides host biology, such as low birth weight (BW) and immaturity of organs including the gastrointestinal tract as a result of low gestational age (GA), there are multiple other exogenous factors that could affect the intestinal microbiome maturation of preterm infants  environment and peri- and postnatal antibiotic exposure) , 19, 29.While interest in this subject is growing, the focus of research is typically, with a few exceptions –19, 23, p < 0.01), yet were of lower BW . Underlying causes of prematurity for all vaginally delivered infants (13/13) were intra-uterine infection, (prolonged) premature rupture of membranes and/or cervix insufficiency while most infants delivered by CS were born preterm because of placental insufficiency (22/27) . Table We analyzed 142 samples . These samples included one faecal sample per week, starting with the first meconium (after birth), and afterwards the first faecal sample of every week during the first four weeks after birth.Staphylococcus genus, mostly consisting out of S. epidermidis, was frequently the most dominant bacterial group (40 % of reads on average) followed by 4 members from the Enterobacteriaceae family; Enterobacter cloacae/hormaechei (11 %), Escherichia/Shigella (10 %), Klebsiella oxytoca/michiganensis (5.9 %) and Klebsiella pneumoniae/variicola (5.7 %). In week 2 an increase in the abundance of Staphylococcus was observed in this cohort (50 % of reads on average). A decline however of Staphylococcus is observed in weeks 3 and 4  with the Enterobacteriaceae family becoming dominant in most samples . Bifidobacteria and lactobacilli were groups of very minor importance within this cohort. Whilst several of the main species such as S. epidermidis, K. oxytoca/michiganensis & K. pneumonia/variicola can be identified down to the species level using V4 16 S rRNA amplicon sequencing to a reasonable degree this is not the case for Escherichia coli as methods other than 16 S rRNA amplicon sequencing are required to tell the genera Escherichia and Shigella apart. However, from both culture [E. coli. Shigellosis rarely affects infants during the first month of life whilst infections with E. coli are common [Escherichia/Shigella reads are hence referred to as Escherichia. For purposes of clarity and avoiding unsubstantiated taxonomic depth representation most other bacterial groups have been merged together at the genus level in most analyses. Detailed information on abundances per bacterial group per sample, including representative sequences & metadata are provided in Supplementary Information File An overview of the abundances (% of reads per sample) of the most important bacterial groups (species/genera) in these 4 timepoints is shown in Fig.  culture  and whol culture  the idenStaphylococcus, a genus of typical skin bacteria, was significantly associated with CS delivery in samples from the first 3 weeks of life . On the other side of the coin Escherichia and Bacteroides were the most strongly correlated with vaginal delivery. The combined prevalence the facultative anaerobe Escherichia and members of the aero-tolerant anaerobic Bacteroides genus, typical maternal faecal representatives, was significantly associated with vaginal delivery during these first 3 weeks .One of the most striking patterns in the data, alluded to in Fig. p = 0.04, p = 0.03, p = 0.02 and p = 0.04 for weeks 1–4, respectively), as no difference was present in weight gain in g/week of infants delivered vaginally and by CS . Z-scores from BW were strongly correlated with absolute weight in all 4 weeks .In this cohort the mode of delivery was significantly associated with BW and GA Fig. . The assEnterobacteriaceae in weeks 3 and 4 appeared associated with absolute weight at sampling time . This particular association was however partially indirect, as infants delivered by CS had a lower median BW and were more frequently colonized during delivery by Staphylococcus . More specifically, at a body weight of > 1100 g nearly all samples were dominated by Enterobacteriaceae. In general, the absolute weight of infants, irrespective of the week of sample collection, appeared to be (independently) associated with the shift from a Staphylococcus dominated gut towards one dominated by Enterobacteriaceae.While the mode of delivery was the most important determinant for the infants’ initial microbiome Figs.  and 3, tStaphylococcus and Enterobacteriaceae (% Enterobacteriaceae - % Staphylococcus) but that this prevalence is not linked with postconceptional age  but not with postconceptional age in this dataset.A simple linear regression analysis of this dataset shows that weight is significantly correlated with the prevalence of age Fig. a. A Mixeage Fig. b-d; the p = 0.01) as the level of microbial biomass increases steadily.Microbiome data is compositional and as such does not take into account any differences in the levels of microbial biomass that exists between samples. A clear example of this can be found when analyzing the first meconium samples in regards to the day of obtaining such a first meconium sample when looking at reagent contamination levels. Directly after delivery meconium samples will be largely devoid of microbial biomass. You might be able to detect some initial colonizers, like those derived from the mode of delivery, but these initial colonizers will not have proliferated much making it dubious whether we’re dealing with microbiomes where competition between species (based upon suitability) plays a large role yet. As the amount of bacterial DNA derived from samples from the earliest days will be lower there will be more room for reagent contaminants to be amplified, sequenced and measured. Resultingly, when looking at samples obtained from the first meconium there is a significant negative correlation between the day of obtaining the first meconium after delivery and the % of reagent contamination reads  as another replaces it (Enterobacteriaceae) as a result of competition, but simply be the result of e.g. Enterobacteriaceae becoming much more prevalent on its own irrespective of Staphylococcus levels . To circumvent bias in comparisons as the result of differences in microbial load one should instead look at the ratio’s of the main bacterial groups of interest [Staphylococcus towards one dominated by Enterobacteriaceae as infants gain weight/mature. The log value of the Enterobacteriaceae: Staphylococcus ratio is found to be positively correlated with weight  whilst postconceptional age is not . By excluding meconium samples which are extremely low biomass (where competition is questionable) the association of this ratio with weight becomes even stronger .Changes in microbiome composition might thus for example not be due to one group of bacteria becoming less prevalent  the amount of weight gain during any single week did not correlate with the gut microbiome composition  at the start of that week and (2) their previously reported negative association with necrotizing enterocolitis development was similarly found within this cohort in meconium samples (p = 0.034). Overall infant mortality in this cohort, in part caused by necrotizing enterocolitis , was not aligned with the gut microbiome composition or with BW (p = 0.36) but it was negatively associated with GA at birth (p = 0.005) and positively but not significantly (p = 0.08) with prolonged premature rupture of membranes (PPROM).While Exogenous factors such as antibiotics use and/or feeding regime were found to be of ancillary importance in comparison with patterns associated with the mode of delivery or with absolute body weight. Associations of bacterial groups with antibiotics use were either found to be non-significant or disappeared when adjusting the analyses for mode of delivery. The current number of subjects was insufficient to unravel significant associations between microbiome development with antibiotics and feeding regimes.This study, which prospectively investigated the development of the microbiome of extremely preterm infants during the first four weeks of life, has two main findings. First, confirming current data , differeSecond, this study reveals that current absolute weight is a better marker for the maturation of the infants’ intestinal microbiome than postconceptional age as it less confounded by various infant-specific factors. Our results support the hypothesis that the intestinal microbiome of the preterm infant appears to follow a patterned progression which is indicative of the microbiome developing as a result of infant development measured in weight, not the weight developing as a result of the microbiome. We did not find that the microbiome composition at any taxonomic level in paired analyses was predictive for weight gain in subsequent weeks. Important in this regard is to again note that bifidobacteria or lactobacilli are generally absent in extremely premature children. The abundance, or the diminished abundance in the case of antibiotics , of thesEnterobacteriaceae) to clostridia (and Negativicutes). In this hypothesis, the place where preterm infants step into this progression is mainly dependent on their GA at birth [Bifidobacterium (and/or Bacteroides). Our study, which focused on the first 4 weeks of life of extremely and very preterm infants, confirms the first part of this transition, namely the progression of a staphylococci (bacilli) dominance to an Enterobacteriaceae dominated microbiome in extremely preterm infants. We however found in this dataset that postconceptional age was not a meaningful descriptor of maturity/host biology with regard to the development of the gut microbiome as various complications can hamper infant development which are not taken into account by age yet which are represented by weight. A reason for this inaccuracy is highlighted by our comparison of infants delivered vaginally or by CS and the underlying causes of prematurity. In our cohort CS delivered infants had on average a higher GA at birth but a lower BW (Z-score) than their vaginally delivered counterparts  of samples starting off with a high abundance of staphylococci which in time gave way to an increased abundance of Enterobacteriaceae and the other cluster (II) starting off with a high abundance of Enterobacteriaceae that declined slowly as clostridia became more prominent [Enterobacteriaceae was particularly evident around 1100 g  in this study is the cause of prematurity; placental insufficiency was typically accompanied by CS and was logically negatively associated with lower BW Z-scores due to fetal growth restriction of the infant. Furthermore, some infants thrive and concomitantly gain weight after birth while others do not, although both increase equally in gestational/postnatal age. We observed that absolute weight, as a logical key marker for host biology, does not suffer from these confounders and could be used to visualize the patterned progression of staphylococci (bacilli) to eae Fig.  in this Exogenous factors other than the mode of delivery such as antibiotics, neonatal feeding regime were also analyzed with regard to their association with the intestinal microbiome but no significant correlations were found in this cohort. While such external factors have been found to be associated with certain aspects of the gut microbiome development, they do not appear to represent the main drivers of gut maturation in preterm infants. More samples would be required to study these factors. Maternal data, such as the results of vaginal swabs and/or the use of maternal antibiotics, were not available for this study but could be of possible relevance for initial colonization.K. oxytoca or K. pneumonia, which belong to the family Enterobacteriaceae, have been frequently associated with, amongst other things, sepsis in newborns, but they appear to be a ‘normal’ part of the patterned progression of the bacteria composition as the infant gut matures [Klebsiella strains are to be associated with disease directly or indirectly. Colonization with Klebsiella appears to be normal whereas sepsis with Klebsiella might merely be a symptom of other things going wrong in the preterm infants’ gut [This study highlights that the intestinal gut microbiome development in extremely and very preterm infants is mainly driven by absolute weight but is initially strongly affected by the mode of delivery. It also stresses the importance of pregnancy complications with diminished placental function as it directly affects the actual physical maturity level of the infant in which in turn directly affects the stage of progression , 39 from matures , 26. Trints’ gut , 42. OurThe main limitation of this study is that sample numbers are still relatively limited leading to insufficient power to say anything for example about exogenous factors such as antibiotics use and/or feeding regime. To retain as much power and sensitivity as possible we did not exclude samples with a low but still adequate sequencing depth nor did we rarify. Alpha or beta diversity analyses, which can be affected by differences by in sequencing depth , were noEnterobacteriaceae. This study improves the patterned progression hypothesis [During the first four weeks of life the gut microbiome of extremely and very preterm infants (birth weight < 1000 g) undergoes, or starts to undergo, a transition from a gut microbiome with a high abundance of staphylococci towards one dominated by pothesis  by usingThis study is part of a prospective observational trial , which studied infants for developing NEC , 44–47. Data from each sample day were used. Variables consisted of mode of delivery, BW, GA at birth, z-score BW (which represents the standard deviation in SD units from the Dutch reference growth curves) , bodyweiWe intended to analyze one faecal sample per week, starting with the first meconium, and afterwards the first faecal sample of every week during the first four weeks after birth. Faecal samples were stored at – 80 °C prior to the start of this study. The first sample of each week was used for analyses of the current sub-study.Faecal DNA was extracted from a 0.25 g faecal sample by double bead beating in combination with the QIAamp DNA Mini kit , as described by a study that used the same technique . PolymerThe 16 S rRNA amplicon data was processed using MOTHUR  and the Undibacterium oligocarboniphilum, Acinetobacter guillouiae/iwoffii, Curvibacter lanceolatus, Sphingomonas echinoides, Ralstonia pickettii, Delftia acidovorans, Ralstonia insidiosa, Sphingomonas kyeonggiensis, Methylorubrum extorquens, Phyllobacterium myrsinacearum, Sphingomonas panni, Sphingomonas faeni, Sediminibacterium salmoneum, Rhodococcus erythropolis and Pelomonas saccharophila were removed before further analysis. The number of reads per sample before reagent contamination removal had an interquartile range of 35,190–58,046  with 5 outliers above 100,000 reads and similarly 5 outliers below 10,000 reads (minimum: 2145). After reagent contamination removal the interquartile range was 33,968–57,180  with 5 outliers above 100,000 reads and 9 samples below 10,000 reads (minimum: 913). Analyses strongly influenced by sequencing depth  were not performed in this study; only relative abundances of the most abundant microbial groups are analyzed in detail.As meconium samples in particular, but also many later samples have low bacterial biomasses, reagent contamination needs to be accounted for. Reagent contamination recognition analyses were subsequently performed as described by de Goffau et al. , using tn – Weightn+1), in an infant-specific paired fashion, with the abundance of bacterial groups at the start of each week. The 4 timepoints resulted in 3 of such analyses and we performed these analyses on the OTU level, the genus level and the family level yet we did not find a single significant association (or even something resembling a trend) in any of these 3 analyses at any phylogenetic level. Microbial composition did not appear to be predictive in regards to weight gain.Statistical analyses were conducted with IBM SPSS Statistics 21.0, Past3.14 , PRISM 8Additional file 1.Additional file 2.Additional file 3.


Administration of antenatal corticosteroids (ACSs) is an effective strategy for managing preterm infants, which improves neonatal respiratory distress syndrome (NRDS) and attenuates the risk of neonatal mortality. However, many preterm infants are not exposed to a complete course of ACS administration, and the effects of different ACS-to-delivery intervals on NRDS and respiratory support remain unclear. Therefore, this study explored the relationship between ACS-to-birth intervals and NRDS and respiratory support in preterm infants.  In this retrospective cohort study, the preterm infants born between 240/7 and 316/7 wks of gestation were recruited from January 2015 to July 2021. All participants were categorised based on the time interval from the first ACS dose to delivery: <24 h, 1-2 d, 2–7 d, and >7 d. Multivariable logistic regression analysis examined the relationships between the ACS-to-birth interval and primary or secondary outcome while adjusting for potential confounders.  Of the 706 eligible neonates, 264, 83, 292, and 67 received ACS-to-delivery intervals of <24 h, 1-2 d, 2–7 d, and >7 d, respectively. After adjusting these confounding factors, the multivariable logistic analysis showed a significantly increased risk of NRDS , neonatal mortality , the need for surfactant use , endotracheal intubation in the delivery room , and mechanical ventilation  in the ACS-to-delivery interval of <24 h group when compared with the ACS-to-birth interval of 2–7 d group.  Neonatal outcomes such as NRDS, neonatal mortality, the need for surfactant use, intubation in the delivery room, and the risk of mechanical ventilation are higher when the neonates are exposed to an ACS interval for less than 24 h before delivery. Neonatal respiratory distress syndrome (NRDS) is a common problem in premature neonates due to inadequate surfactant activities in the immature fetal lungs, which is the most common cause of respiratory failure in the first few days after birth resulting in neonatal death. Other complications such as bronchopulmonary dysplasia, intraventricular haemorrhage (IVH), and necrotising enterocolitis (NEC) are also at increased risk for preterm infants. Administration of antenatal corticosteroids (ACS) is an effective treatment strategy for pregnant women at high risk of preterm birth. It accelerates lung maturation and reduces the incidence of NRDS, IVH, NEC, and neonatal death risk after a complete course of ACS. Therefore, a complete course of ACS between 24 and 34 wks of pregnancy is recommended for women at risk of premature labour –4. PreviIn this retrospective cohort study, the preterm infants born between 240/7 and 316/7 wks of gestation were recruited from January 1, 2015, to July 31, 2021, in Shaoxing Maternity and Child Health Care Hospital, a tertiary hospital in Zhejiang Province. Gestational age (GA) was described by ultrasonography before 20 weeks of pregnancy or the first day of the woman's last menstrual period. The inclusion criteria were infants who were inborn and transferred to the neonatal intensive care unit (NICU) within 2 h of birth and had GA <32 wks. Infants who died in the delivery room or were not admitted to the NICU or discharged due to the high-cost burden on family members were not included in this study. Those admitted to the NICU after birth, 2 hrs after birth, who had major congenital malformations, had not been exposed to ACS before delivery, or had repeated use of ACS before delivery were also excluded from this study. The ethical approval for this study was obtained from the Hospital Research and Ethics Committee.A complete course of ACS was defined as an intramuscular administration of 6 mg of dexamethasone every 12 h for a maximum of four doses. The interval was determined from the first ACS dose to delivery. All infants were categorised into four groups: <24 h, 1-2 d, 2–7 d, and >7 d. Maternal and infant health data were acquired from electronic medical records. The maternal data include maternal age, ACS-to-birth interval (hours from the first of ACS dose to delivery), mode of delivery, maternal complications such as gestational diabetes mellitus, gestational hypertension , placental abruption, placenta previa, preterm prelabour rupture of membranes (PPROM) > 18 h, oligohydramnios, and fetal intrauterine distress. Neonatal data, such as GA at birth, gender, birth weight, multiple births, the need for endotracheal intubation in the delivery room, BPD  , NRDS . The secondary outcomes included the need for pulmonary surfactant use, invasive mechanical ventilation, BPD, intubation at birth, surfactant use or invasive mechanical ventilation, and neonatal mortality.H test for continuous variables with nonnormal distribution or one-way ANOVA for those with normal distribution. Categorical variables were compared with the chi-square test. Univariate and multivariate logistic regression analyses assessed the relationships between ACS-to-delivery intervals and respiratory outcome rates. For multivariate analysis, the covariates used were as follows: birth weight, GA at birth, delivery mode, premature rupture of membranes >18 h, placenta previa, and pregnancy-induced hypertension disorders. The infants exposed to ACS within 2–7 d before birth were regarded as the reference group. And the secondary outcome of ventilation duration was not included in the multivariate analysis. All statistical tests were conducted using STATA 15.0. Odds ratio (OR) and 95% confidence interval (CI) were estimated for binary outcomes. A two-sided p value of <0.05 was considered statistically significant.Data were expressed as median  for variables that did not follow a normal distribution, as mean ± standard deviation (SD) for normally distributed data, and as frequencies for categorical variables. The baseline characteristics were compared with the Kruskal–Wallis n = 2), admitted to the NICU after 2 hrs of birth (n = 11), not admitted to the NICU (n = 53), and discharged due to family financial difficulties or concerns about prognosis (n = 43) were excluded. Moreover, 6 infants received multiple courses of ACS, 40 infants did not receive ACS before birth, and 1 infant with a maternal diagnosis of systemic lupus erythematosus was also excluded. Finally, 706 infants were included in the study: 264, 83, 292, and 67 infants were born at ACS-to-delivery intervals of <24 h, 1-2 d, 2–7 d, and >7 d, respectively . The intervals from the first corticosteroid dose to delivery were remarkably different among the 4 groups, while the length of hospitalisation was quite similar among the 4 groups  compared to those exposed <24 h (37.1% and 18.9%), 1-2 d (18.1% and 10.8%), and >7 d (22.4% and 13.4%) before delivery, respectively. Unadjusted cumulative incidence rates and ORs testing the associations between ACS intervals and neonatal outcomes are shown in After adjusting these confounding factors, multivariable logistic regression analysis revealed a significantly increased risk of NRDS  observed in the ACS-to-birth interval of <24 h group compared with the ACS-to-birth interval of 2–7 d group. The secondary outcomes include neonatal mortality rate , the need for surfactant use , invasive mechanical ventilation rate , and surfactant use or invasive mechanical ventilation rate  which were significantly higher in the ACS-to-birth interval of <24 h group compared with the ACS-to-birth interval of 2–7 d group . The risn = 706). Approximately 49.15% of preterm infants did not receive a complete course of ACS, while 37.39% of preterm infants received ACS at an interval of <24 h. Our findings demonstrated that the infants with the ACS-to-delivery interval of <24 h had a higher risk of NRDS, neonatal mortality, the need for surfactant use, and mechanical ventilation when compared to those with an ACS-to-delivery interval of 2–7 d. Administration of ACS >7 d before delivery was not significantly associated with an increased risk of these complications compared with the infants exposed to ACS 2–7 d before delivery.In the present study, we assessed the effects of different ACS-to-delivery intervals on NRDS incidences and respiratory support in preterm infants  and secondary (>24 h) phases compared with the first dose. Studies showed an increase in fetal heart rate (FHR) variability for both dexamethasone and betamethasone at the primary phase, in which antenatal betamethasone increased the expression of Nas of ACS –24. This h and seThere is conflicting evidence on the effect of the ACS-to-birth interval of >7 d in neonatal death. In a large retrospective cohort study of infants born at 24–33 wks of gestation from the Canadian Neonatal Network, neonatal morbidity and mortality rates were significantly higher in the ACS-to-delivery interval of >7 d . A seconn = 66) was relatively small in our study. Moreover, BPD occurs almost exclusively in premature infants (born at 28 wks or less) as they are more vulnerable to lung anomalies in the postnatal environment. The most substantial risk factors for BPD are prematurity and low birth weight [The rates of BPD did not differ significantly among the 4 groups. This could be explained by that the sample size of infants delivered at <28 wks of gestation  of preterm infants were acquired without data loss. We assessed the effects of different ACS intervals of <24 h on neonatal mortality, which were sparsely investigated in previous studies. The existing evidence on the association between ACS administration and neonatal outcomes focuses mainly on high-income countries and hospital settings where antenatal betamethasone is primarily applied for perinatal care. However, antenatal dexamethasone is mainly used for perinatal management in developing countries. Therefore, the findings of the antenatal dexamethasone-to-delivery interval and neonatal respiratory support are more applicable to preterm birth in developing countries.Several limitations should be noted. First, the ACS used in our hospital was dexamethasone; thus, the results of this study should be interpreted with caution. Second, there is a potential postnatal bias in which some infants did not receive intensive care. To reduce this effect, the infants did not receive delivery room resuscitation. Those admitted to the NICU after 2 h of delivery were excluded from the analysis because of the impact of hypothermia on neonatal morbidity and mortality –30. ThirThis study demonstrates that preterm infants exposed to ACS <24 h are associated with a higher risk of NRDS and neonatal mortality in preterm infants born at 240/7–316/7 wks of gestation compared to those with the ACS-to-birth interval of 2–7 d. This study also points out that the severity of NRDS could be reduced by ACS administration at least 24 h before delivery in preterm infants. A multicenter, prospective study is needed to assess the differences in ACS administration rates among preterm infants in the future. Well-designed randomised controlled trials should be conducted to verify the effects of ACS administration on NRDS and respiratory support in larger population-based cohorts.


Birth asphyxia is a prominent and avoidable cause of infant illness and death worldwide, particularly in underdeveloped countries such as Ethiopia. Early identification and control of the underlying contributory factors would help to alleviate the situation. As a result, the goal of this study was to assess the magnitude and determinants of neonatal asphyxia among live newborns at the northern Gondar public Hospitals in northwest Ethiopia.From April 1 to May 2, 2020, 357 newborns were studied in an institution-based cross-sectional study. The sample size was proportionally distributed among three public hospitals, namely Gondar referral teaching hospital, Debark general hospital, and Kola-Diba District Hospital, which was chosen at random. The number of deliveries given at each hospital six months prior to the data collecting period was used to allocate the hospitals. To get all participants, a systematic random sampling approach was adopted based on hospital delivery registration. The physicians’ evaluation of an APGAR score of 7 in the first and fifth minutes of birth was used as the confirmation of birth asphyxia. Data was collected using a standardized and pretested questionnaire. Variables having p-values less than 0.25 were entered into a multivariable logistic regression analysis in the bivariable analysis. At a p-value of 0.05, a statistically significant level was reported.As per the study, the total prevalence of neonatal asphyxia was found to be 27.1 . In a multivariable logistic regression analysis, neonates born to rural mothers , primiparity , premature rupture of membrane,  and low birth weight (< 2.5kg)  were all found to be independent predictors of birth asphyxia.This study identified that rural residence, primiparity, premature rupture of membrane, and birth weight were found to be the independent predictors of birth asphyxia. The majority of variables that cause birth asphyxia can be controlled. The first four weeks of a child’s life are the most susceptible for survival, with more than one-third of all child fatalities occurring during this period . The neoThe immature newborn brain is highly susceptible to dangerous situations . In mostVarious international studies found that multiple risk factors: socio-demographic , antepartum , intrapartum , neonatal  from April 1 to May 2, 2020. The hospitals are 727, 812, and 757 kilometers from Ethiopia’s capital city, Addis Ababa, respectively. These hospitals serve a total of over 2 million people in the catchment regions.All neonates born with a gestational age of ≥ 28 weeks and with their mother were evaluated for eligibility. Newborns with life-threatening abnormalities such as hydrops and cyanotic congenital heart defects were excluded as are already predisposed for asphyxia. Furthermore, preterm babies <28 weeks, those who were seriously ill during the study period, and those neonates who were transferred to advanced care before assessment of APGAR score were also excluded.A single population proportion formula was used to calculate the sample size. Based on a previous study done in Jimma, Southwest Ethiopia , the preWhere: n = the required sample size, Z α/2 = the standardized normal distribution curve value for the 95% confidence level, P = the proportion of birth asphyxia among the general population, and d = degree of precision (the margin of error between the sample and population).Three hospitals were chosen at random from the zone’s eight hospitals for this study. The number of study participants was proportionally assigned to each hospital based on the monthly average number of deliveries. In each hospital’s delivery unit, a systematic sampling technique with every fifth interval was used to enroll study participants.Data were collected by using structured questionnaires from both primary and secondary (chart review) sources. A pre-tested structured interviewer based questionnaire was used to collect data on maternal sociodemographic , antepartum , intrapartum , and neonatal related factors  were abstracted using a pre-tested structured checklist from the medical records of pregnant women who gave birth during the data collection period. In each hospital, data was collected by qualified BSc holding midwives and supervised by an MSc holder nurse. The data collection process was evaluated on a daily basis by the primary investigator and supervisor to ensure data quality and to check for missing information or potential errors. The physicians’ evaluation of an APGAR score of 7 in the first and fifth minutes of birth was used as the confirmation of birth asphyxia. The APGAR score was obtained in the birth ward and operation room throughout both day and night.Data collectors received two days of training on how to gather and record data. The questionnaires were translated into Amharic and then retranslated into English for consistency under the supervision of a pediatrician, and the supervisors and principal investigator checked them daily for accuracy, consistency, and completeness. One week before the actual data collection, the questionnaire was pretested on 17 eligible mother-newborn dyads (5% of the sample size) in Alfa primary hospital.The data was cleaned up and double-checked before being input into Epi data version 3.1 and exported to SPSS version 20 for analysis. The outcomes of the maternal socio-demographic, antepartum, intrapartum, and neonatal studies were described using descriptive statistics. The degree of relationship between independent and dependent variables was determined using the odds ratio (OR) and confidence interval (CI). Using odds ratios, bivariate logistic regression was used to rank the relative relevance of each exposure variable with the outcome variables. Variables with a P value of less than 0.25 in the bivariate logistic regression analysis were chosen and fitted to the multiple logistic regression analysis to determine the independent effects of each variable. Finally, the Adjusted Odds Ratio with 95% confidence intervals (CI) was used to assess the existence and strength of relationships, with statistical significance reported if p ≤ 0.05.In this study, all 357 mothers agreed to participate, with a 100% response rate. More than half of the respondents, 238(66.67%) were rural inhabitants. The mean age was 26.92 years (SD ± 4.7) of whom 122 (34.17%) belonged to the age category of 20–24 years. Three hundred (84.1%) women were married. In addition, 123 (34.45%) were housewives and only 102 (28.6%) of mothers attended tertiary education. A small number of mothers 23 (6.4%) had a history of adverse pregnancy outcomes .322(90.2%)] had attended antenatal care at either public hospitals or public health centers. However, only 196(54.9%) moms had four and above antenatal care visits. Of the total respondents, 83(23.2%) of women had Obstetric complications during pregnancy. About 61(17.1%) mothers ever used substances during their gestation .In our study, 303 (84.87%) of newborns were of vertex presentation, 20 (5.6%) were born by assisted vaginal delivery. The majority 253(70.9%) of the mothers had intrapartum rupture of fetal membranes whereas 104 (29.1%) premature rupture of the membrane was reported among mothers. Finally, 32 (9%) mothers had complicated labor and 103 (28.9%) of births were meconium-stained amniotic fluid following membrane rupture .189 (53%) of the newborns were males. The mean gestational age at birth was 38.5 (±2.4) weeks and the majority of the newborns 242(67.8%) were term. Moreover, 58(16.3%) of the newborns had low birth weight. At birth, there were 71(19.9%) newborns with health problems. There were 15 (4.2%) twin newborns .The magnitude of birth asphyxia was found to be 97 (27.2%)  based on APGAR scoring less than 7 persistently for more than 5 minutes after birth. Most of the asphyxiated neonates had moderate asphyxia 76 (78.4%) whereas 21 (21.2%) neonates had severe birth asphyxia.The Bi-variable logistic regression analysis showed that 7 factors namely, residence, gestational age, parity, premature rupture of membrane, birth weight of neonates, antenatal obstetrics complication, mode of delivery were crudely associated with birth asphyxia. However, after statistical adjustments in the final model, gestational age, Antenatal obstetric complications, and mode of delivery were not significant.Neonates of women from the rural residences had 2.4 more likelihood of being asphyxiated at birth as compared to the urban residence. Neonates born from primipara mothers were 5.5times  more prone to be asphyxiated at birth when compared to Multiparous mothers. Furthermore, Neonates born to mothers with premature rupture of fetal membranes were 3.2 times  more prone to be asphyxiated at birth as compared to those with intrapartum rupture of membranes. Birth asphyxia was 3.8 times  higher among low-birth-weight neonates than those normal weight neonates .In this study, the prevalence, and predictors of birth asphyxia amongst live births at public hospitals in the south Gondar zone are explored in depth. Birth asphyxia was found to be significantly associated with rural residency, primiparity, preterm ruptures of the membranes, and low birth weight.In the study, the Perinatal asphyxia was found to be lower (27.1%) than in a study conducted at Dilla Referral Hospital in Southern Ethiopia (32.8%) . HoweverDespite the fact that prenatal asphyxia contributes significantly to neonatal morbidity and mortality in underdeveloped countries, evidence is scarce on the factors that predict perinatal asphyxia. There isn’t enough evidence to say whether maternal or neonatal variables play a role in the occurrence of birth asphyxia , 24.In an analysis of associated factors, newborns born to women who reside in rural areas had a 2.5 higher risk of having birth asphyxia than those born to women who live in the city. This finding is in line with research was undertaken in Nepal , Gondar In comparison to multiparous women, neonates delivered to primiparous mothers were shown to be five times more likely to be asphyxiated at delivery. The finding is consistent with research conducted in Pakistan, Nigeria, and central Tigray, which found that being primipara is one of the independent factors of newborn asphyxia , 29. TheIn this study, low birth weight was also founded to be a major factor of birth asphyxia. Low—birth—weight neonates were 3.72 times more likely than normal birth weight neonates to suffer from birth asphyxia. These findings matched those of research undertaken at Gondar Referral Hospital , Jimma hFurthermore, compared to those delivered to mothers with intrapartum rupture, neonates born to mothers with premature rupture of fetal membranes (PROM) were 3.2 times more likely to be asphyxiated at birth. This finding is in line with studies in Cameroon , Uganda In conclusion, the prevalence of birth asphyxia is comparable to previous studies in underdeveloped nations. Rural inhabitants, primiparity, preterm rupture of membranes, and low birth weight were all found to be independent predictors of birth asphyxia. As a result, health care personnel, particularly those in labor and delivery wards, must pay more attention to complex labors to foresee and prevent birth asphyxia. 13 Jan 2022 <div> PONE-D-21-24852 </div>  <div> The magnitude of Neonatal Asphyxia and its Associated Factors among newborns; A cross-sectional study </div>  <div> PLOS ONEDear Dr. Dejenie  </div>  <div> Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. 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During our internal evaluation of the manuscript, we found significant text overlap between your submission and the following previously published works, some of which you are an author.https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0226891https://bmcpregnancychildbirth.biomedcentral.com/articles/10.1186/s12884-020-03348-2We would like to make you aware that copying extracts from previous publications, especially outside the methods section, word-for-word is unacceptable. In addition, the reproduction of text from published reports has implications for the copyright that may apply to the publications.Please revise the manuscript to rephrase the duplicated text, cite your sources, and provide details as to how the current manuscript advances on previous work. 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Nevertheless, authors should clarify some points and improve the discussion, as suggested below.Authors should consider the following recommendations:- Manuscript should be further revised in order to correct some typos and improve style.- I would recommend to highlight clearly other conditions that may cause adverse obstetric outcomes, including neonatal asphyxia, such as advanced maternal age (PMID: 25027820), pre-eclampsia  and obstructed labor .Reviewer #2: This is a cross sectional study analyzing cases of fetal asphyxia in northwest Ethiopia.The article has some inconsistencies. The most evident are:- In the abstract you say " The diagnosis of birthasphyxia was confirmed based on the physician’s diagnosis of an APGAR score < 7 inthe 1 st and 5 th minutes of birth". IN the text you say "The diagnosis of birth asphyxia was made based on the fifth-minute APGAR scores of < 7 ". Please correct- Regarding the antepartal visits, you reported in the text that 59.1% of patients had 4 or more visits, while in the table they are 54.09%- Married women are 325 (91.1%) in the text and 32 (8.9%) in the tableI recommend reviewing all correspondences between text and tables.In table 4 you wrote about "Neonatal medical problem at birth" that 286 babies had no problems, 41 sepsis, 19 birth injury and 11 congenital malformation. It is not clear where the 97 cases of childbirth asphyxia fit in.In lines 192-193 In lines 192-193 you divide fetal asphyxia into moderate and severe but it is not defined by what criteria this subdivision is carried out.https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. 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Below are our responses to each point raised by the academic editor and reviewers.Best regards!Tadesse Asmamaw Dejenie, on behave of all authorsResponse to academic editor’s commentComment 1: Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file namingAuthor response: Thank you so much for sending the link to the PLOS ONE style templates; we have taken your advice and updated our manuscript to meet the style requirements of PLOS ONE. Please double-check it in the revised manuscript.Comment 2: Thank you for submitting the above manuscript to PLOS ONE. During our internal evaluation of the manuscript, we found significant text overlap between your submission and the following previously published works, some of which you are an author. Author response: We'd like to thank you for your input. We've taken your advice and thoroughly reread our amended manuscript. Please see the revised manuscript for further information.Comment 3: We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.Author response: We'd want to thank you one more for alerting us. Please correct, as the datasets used in this work are available upon reasonable request from the corresponding author.Response to Reviewers’ commentReviewer #1: I read with great interest the manuscript, which falls within the aim of this Journal. In my honest opinion, the topic is interesting enough to attract the readers’ attention. Nevertheless, authors should clarify some points and improve the discussion, as suggested below.Authors should consider the following recommendations:- Manuscript should be further revised in order to correct some typos and improve style.- I would recommend highlighting clearly other conditions that may cause adverse obstetric outcomes, including neonatal asphyxia, such as advanced maternal age (PMID: 25027820), pre-eclampsia  and obstructed labor .Author response: We are really grateful for your suggestions. We have acknowledged and adjusted your helpful suggestions. Tables 3 and 4 of the revised manuscript address some of your issues. For further information, please kindly check the revised manuscript.Reviewer #2: Comment 1: The article has some inconsistencies. The most evident are:- In the abstract you say " The diagnosis of birth asphyxia was confirmed based on the physician’s diagnosis of an APGAR score < 7 in the 1st and 5th minutes of birth". IN the text you say "The diagnosis of birth asphyxia was made based on the fifth-minute APGAR scores of < 7 ". Please correctAuthor response: We'd like to express our gratitude for your insightful remarks and suggestions. We've taken note of the suggestions and made the necessary revisions.Comment 2: Regarding the antepartal visits, you reported in the text that 59.1% of patients had 4 or more visits, while in the table they are 54.09%Author response: We appreciate your thoughtful and helpful remarks once again. We are perplexed as to why this issue has arisen. This is most likely due to a technical glitch. In any case, we took your suggestions and made the necessary changes.Comment 3: Married women are 325 (91.1%) in the text and 32 (8.9%) in the tableI recommend reviewing all correspondences between text and tables.In table 4 you wrote about "Neonatal medical problem at birth" that 286 babies had no problems, 41 sepsis, 19 birth injury and 11 congenital malformation. It is not clear where the 97 cases of childbirth asphyxia fit in.Author response: We value your critical opinion. In fact, when we assessed the neonatal medical condition at birth, we did not consider neonatal asphyxia; instead, we assessed medical problems other than asphyxia.Comment 4: In lines 192-193 you divide fetal asphyxia into moderate and severe but it is not defined by what criteria this subdivision is carried out.Author response: Fetal asphyxia was classified using the Standard Treatment Protocol for Management of Common Newborn Conditions in Small Hospitals, which is based on WHO Guidelines.AttachmentResponse to Reviewers.rtfSubmitted filename: Click here for additional data file. 18 Feb 2022The magnitude of neonatal asphyxia and its associated factors among newborns in public hospitals of North Gondar Zone, Northwest Ethiopia: A cross-sectional studyPONE-D-21-24852R1Dear Dr. Tadesse Asmamaw, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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For more information, please contact Kind regards,Francesca CrovettoAcademic EditorPLOS ONE 22 Feb 2022PONE-D-21-24852R1 The magnitude of neonatal asphyxia and its associated factors among newborns in public hospitals of North Gondar Zone, Northwest Ethiopia: A cross-sectional study Dear Dr. Dejenie:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. 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It is unknown whether prenatal lipid-based nutrient supplements (LNSs) affect blood pressure (BP). Associations between hypertension and birth outcomes using recently updated BP cutoffs are undetermined.We aimed to assess the impact of LNSs on maternal hypertension and associations between hypertension and birth outcomes.n = 1320) were randomly assigned to receive daily 1) iron and folic acid (IFA), 2) multiple micronutrients (MMN), or 3) LNSs until delivery. BP was measured at enrollment and 36 weeks of gestation. We analyzed the effect of LNSs on BP using ANOVA and associations between hypertension [systolic BP (SBP) ≥130 mm Hg or diastolic BP (DBP) ≥80 mm Hg] and birth outcomes by linear and logistic regressions.Pregnant Ghanaian women at ≤20 weeks of gestation . At enrollment, higher DBP was associated with lower birth weight and shorter gestation; women with high DBP had greater risk of low birth weight (LBW)  and preterm birth (PTB) . At 36 weeks of gestation, higher SBP was associated with lower birth weight, length, and head circumference and shorter gestation; higher DBP was associated with lower birth weight and length; and women with high DBP had greater risk of LBW . Neither high SBP nor hypertension were associated with birth outcomes at either time point.Mean ± SD SBP and DBP were 110 ± 11 and 63 ± 8 mm Hg at 36 weeks of gestation and did not differ by supplementation group (SBP, Daily provision of LNSs does not affect maternal hypertension, compared with IFA and MMN. Higher SBP and DBP are associated with a shorter gestation and smaller birth size; however, only high DBP is associated with LBW and PTB. The new BP cutoffs may help identify pregnancies at risk of adverse birth outcomes.clinicaltrials.gov as NCT00970866.This trial was registered at  Hypertension is currently defined as a systolic blood pressure (SBP) ≥130 mm Hg or diastolic blood pressure (DBP) ≥80 mm Hg , compareDuring a typical pregnancy, blood pressure (BP) decreases from early (<15 weeks of gestation) to mid-pregnancy (22–24 weeks of gestation), increases in late pregnancy (36 weeks of gestation), and then returns to prepregnancy levels by delivery , nutritiIncreased dietary intake of essential fatty acids (EFAs) has been shown to decrease the risk of hypertension in nonpregnant populations . AlthougIt is unclear whether the new definition of hypertension, which uses a lower threshold, is appropriate for predicting pregnancies at risk of adverse outcomes. High BP during pregnancy, as defined by the previous cutoffs, has been associated with an increased risk of low birth weight (LBW) , small fGhana is a low-middle-income country located in Western Africa, with staple foods that include maize, cassava, rice, fish, and leafy vegetables. The country is currently experiencing a nutrition transition to more energy-dense, “Western” diets and corresponding increases in overweight and obesity. Several micronutrient deficiencies, including iron, folate, and vitamin A, are common in Ghana, and especially among lower-income households . Dietary1) evaluate the impact of prenatal lipid-based nutrient supplements (LNSs) on maternal BP; and 2) assess the association between maternal BP during early and late pregnancy and birth outcomes using the new BP cutoffs. We hypothesized that women receiving LNSs would have a lower mean SBP and DBP than those receiving iron and folic acid (IFA) or multiple micronutrients (MMN) at 36 weeks of gestation. We also hypothesized that hypertension during pregnancy would be associated with a smaller newborn birth size and lower duration of gestation than in women with normal BP.Our objectives were to 1) IFA supplements that contained 60 mg Fe and 400 μg folic acid, 2) MMN that contained 1–2 RDAs of 18 vitamins and minerals, or 3) small-quantity LNSs that contained the same micronutrients as the MMN as well as calcium, phosphorus, potassium, magnesium, EFAs, and other macronutrients from enrollment until delivery. We report on a secondary data analysis of 1 of the randomized controlled trials in the International Lipid-Based Nutrient Supplements (iLiNS) Project, the iLiNS-DYAD trial in Ghana . A totalThe small-quantity (20 g/d) LNS provided to women in this trial was a paste designed to be mixed with local foods to increase the nutrient and energy content of diets during pregnancy and lactation . NutriseEligibility for this study was specific to women attending 4 prenatal clinics in the Yilo and Manya Krobo districts of Ghana between December 2009 and December 2011, who were ≥18 y old, and at ≤20 weeks of gestation as determined by ultrasound. Reasons for exclusion were a test result that was HIV positive, residence > 20 km outside of the study area, history of peanut or milk allergies, severe illness, or the intention to move out of the study area within 2 y.A block-randomization scheme was designed and implemented by a study statistician and has been detailed previously . Each woAt enrollment and 36 weeks of gestation, trained fieldworkers interviewed participants to record sociodemographic characteristics. A study nurse measured height (Seca 217), weight (Seca 874), and duplicate BP measurements on either arm using automated BP monitors (Andon BPM model KD-595). We collected a blood sample by venipuncture and measured hemoglobin , malaria parasitemia , and the inflammatory markers C-reactive protein  and α1-acid glycoprotein   from plasma. Gestational age estimates were determined at enrollment by physicians at the antenatal clinics using ultrasound imagers . Fieldworkers distributed a 2-wk supply of the assigned supplement along with instructions on consumption methods. Fieldworkers collected data on supplement adherence, calculated as the percentage of pregnancy days after enrollment that women reported consuming the supplement, and morbidity at biweekly follow-up visits at the participants’ homes.Fieldworkers visited the home or hospital at delivery to complete anthropometric measurements of newborns. Birth weight was measured to the nearest 20 g , and length  and head circumference (Shorr Productions) to the nearest 0.1 cm. Measurements were recorded within 48 h of birth for 91% of infants and between 3 and 14 d after birth for 9% of infants. For age- and sex-standardization of weight, we used the WHO Child Growth Standards . If the z score <−2), and SGA (birth weight <10th percentile according to the Intergrowth Standards)  or high DBP (≥80 mm Hg) (30 mm Hg andards) . PTB wasP < 0.1 in univariate models) were included in an adjusted regression model. Among all the variables included in the final adjusted model, there was no evidence of collinearity [variance inflation factors (VIFs) <10] and all variables significantly associated with the outcome were included in adjusted models. Owing to randomization of the trial in the intervention, we followed the intention-to-treat principle when analyzing the effect of LNSs on maternal BP. During the study, some of the IFA and MMN capsules were unintentionally mislabeled, causing 92 IFA women and 85 MMN women to have mixed exposure during pregnancy. All women pregnant during the period of mixed exposure were excluded from this analysis including the 92 IFA and 85 MMN women who experienced mixed exposure, as well as 86 LNS women. After exclusion the final sample size was 1057 (n = 1320) was also conducted. Considering the protocol violation resulting in mixed exposure, the analyses included intervention groups according to the supplement treatment assignment actually received when they were enrolled and not the treatment originally assigned at enrollment, which is consistent with a previous iLiNS-DYAD publication reporting birth size  . If a mowas 1057 . A sensirth size . All tesP values < 0.05 were considered to be statistically significant. Log-binomial models were used to estimate RR, 95% CIs, and P values for categorical birth outcomes in adjusted and unadjusted models using both the old and new definitions of high SBP, high DBP, and hypertension  and birth outcomes. Multiple linear regression models were used to determine the associations of SBP and DBP with birth weight, length, head circumference, and duration of gestation. We checked all models for linearity using quadratic terms and found a lack of any U-shaped relations. Results of the regression models are presented as standardized regression coefficients (β-coefficients), which allow for standardized comparisons between the predictors and the outcome variables. Using β-coefficients, a 1-SD increase in the predictor determines the change in SDs of the outcome variable, with all other variables held constant. Interactions between maternal BMI and BP, with respect to birth outcomes, were assessed in linear regression and rtension . In a fertension . The catoutcomes . Inclusioutcomes .SAS software 9.4  was used for data analysis in addition to Microsoft Office Excel for the configuration of figures.). Only 4.4% of women dropped out of the study before delivery and the attrition rate did not significantly differ (P > 0.05) between the IFA (4.6%), MMN (3.7%), and LNS (5.1%) groups .Between December 2009 and March 2011, a total of 1320 pregnant women were enrolled in the main iLiNS-DYAD trial, with a final sample size of 1057 for this analysis after excluding women who were pregnant during the mixed-exposure period  groups . Table 2P < 0.05) and shorter pregnancy duration  in adjusted models . For each 1-SD increase in DBP at enrollment (8 mm Hg), birth weight was reduced by 0.087 SD, which translates to 37 g reduced birth weight per 1-SD increase in DBP. For each 1-SD increase in DBP at enrollment, duration of gestation was reduced by 0.069 SD, which translates to an ∼1-d reduction in gestation duration per 1-SD increase in DBP. When the model for birth weight was further adjusted for duration of gestation, the effect size was attenuated and no longer statistically significant .Higher DBP at enrollment was associated with lower birth weight  and length  in adjusted models. For each 1-SD increase in DBP at 36 weeks of gestation (8 mm Hg), birth weight was reduced by 0.095 SD, which translates to 41 g reduced birth weight per 1-SD increase in DBP. When the model for birth weight was further adjusted for duration of gestation, the effect size remained significant . Higher SBP at 36 weeks of gestation was associated with a lower birth weight , birth length , newborn head circumference , and a shorter pregnancy duration . When the model for birth weight was further adjusted for duration of gestation, the effect size was attenuated and no longer significant .Higher DBP at 36 weeks of gestation was associated with a lower birth weight  and PTB  than women with normal DBP at enrollment . Women wP < 0.05) and DBP . Higher BMI at enrollment was also associated with higher SBP  and DBP  at 36 weeks of gestation, and newborn birth weight . We also checked all models for collinearity and confirmed all VIFs were <2.We observed variation in coefficients between unadjusted and adjusted models and examined which covariates had the greatest impact on results. BMI had a large influence in adjusted models. At enrollment, higher BMI was associated with higher SBP . Women with high DBP at 36 weeks of gestation had greater risk of LBW ; however, neither high SBP nor hypertension were associated with any birth outcomes.When using the prior BP cutoff, women with high DBP (≥90 mm Hg) at enrollment had greater risk of LBW  and PTB  than women with normal DBP at enrollment. High SBP (≥140 mm Hg) at enrollment was not significantly associated with LBW in unadjusted or adjusted models  was associated with increased risk of LBW and PTB, whereas high SBP, according to the new cutoff of ≥130 mm Hg, was not associated with risk of any adverse birth outcomes examined in this study.Our findings that LNSs did not have a significant effect on maternal BP are consistent with a previous trial conducted in Bangladesh, which compared pregnant women consuming either LNSs or IFA and found no significant difference in mean SBP, DBP, or risk of hypertension . As was The associations we found in our study between BP and birth outcomes are also consistent with previous research. A 2014 systematic review/meta-analysis of 55 studies confirmed an association between hypertension and risk of LBW and PTB  and birth weight were attenuated with an adjustment for duration of gestation, which suggests that duration of gestation may be a mediating factor in the relation between DBP and birth weight. However, the association between DBP at 36 weeks of gestation and birth weight was significant even after adjusting for duration of gestation.Our findings show a positive relation between maternal BMI and birth weight, and also a positive relation between maternal BMI and BP. These associations may explain the substantial impact that maternal BMI has on adjusted results. This suggests that there are opposing pathways with regard to how maternal BMI might influence birth size. Higher BMI may lead to greater newborn birth size, but if the mother also develops high BP, that may counter the effect of higher BMI, because high BP is associated with a smaller birth size.Strengths of our study include the large prospective design with BP measurements in early and late pregnancy, a low loss to follow-up, the use of ultrasound scans to determine gestational age, and the use of the most recent cutoff definitions for hypertension. A limitation of our study was that adherence to supplement consumption was based primarily on participant self-report. However, fieldworkers also visited the houses biweekly and counted any unconsumed supplements to assist in confirming adherence. In addition, participants were able to distinguish between small-quantity LNS sachets and IFA or MMN capsules. However, anthropometrists and data analysts were blinded to group assignments. It should be noted that maternal BP was not the main outcome of the iLiNS trial and it is possible that the quality of the BP measurements could have been improved. Either the left or right arm was used to take BP measurements, and it is possible that the duplicate measurements were not appropriately timed, i.e., they may have been taken within 5 min of each other. We did not have information related to the use of antihypertensive medications during pregnancy; however, it is likely that only a limited number of women, if any, were using such medications during pregnancy given the low prevalence of hypertension in this study. Lastly, the low prevalence of hypertension may introduce bias and may be due to the exclusion of women with chronic conditions, as previously mentioned. Future studies should further explore the etiology of maternal hypertension and associations with birth outcomes using the updated BP cutoffs.Within a sample of Ghanaian women, this analysis examined the impact of prenatal supplementation on maternal hypertension risk using the new, more conservative hypertension threshold. We did not observe differential effects of daily prenatal LNS, MMN, or IFA supplementation on maternal hypertension risk. Thus, although LNS is shown to benefit maternal and newborn health in this population , our resnxab018_Supplemental_FileClick here for additional data file.


Multisystemic smooth muscle dysfunction syndrome (MSMDS) is a genetic disease that affects multiple organs. The report here concerns a patient with MSMDS, who is known so far as the youngest among all the reported patients. In addition to the typical manifestations, we observed previously unreported ocular abnormalities, including persistent anterior tunica vasculosa lentis (TVL) and early-onset retinal arteriolar tortuosity, by the fluorescein angiography (FA).ACTA2 gene missense mutation p.R179H.The patient was admitted to the neonatal intensive care unit immediately after birth for a diagnosis of urinary system dysplasia during fetal life. After a thorough examination, the patient was found with patent ductus arteriosus, pulmonary hypertension, cerebrovascular disease, hypotonic bladder, intestinal malrotation, and congenital mydriasis. The FA of the eyes undertaken in her 6-week demonstrated perfused vasculature in the persistent anterior TVL and prominent retinal arteriolar tortuosity. The whole exome sequencing revealed a de novo heterozygous The patient was diagnosed with MSMDS.Follow-up observation.At the 3-month follow-up, no change of the ocular disease was observed.ACTA2 p.R179H mutation affects not only the smooth muscle cells but also the pericytes, and further affects the TVL regression. The prominent retinal arteriolar tortuosity in this 6-week-old infant indicates that the retinal arteriolar tortuosity can present early in MSMDS.The persistent anterior TVL in this case implies that  ACTA2 gene encodes α-actin protein, which is the most abundant isoform in SMCs. SMCs are found in the wall of arteries and hollow visceral organs, lacrimal ducts, hair follicles and the iris. MSMDS, mostly caused by heterozygous missense mutation of the ACTA2 altering arginine 179, represents the most severe ACTA2 mutation-associated disease because of the early onset and highly penetrant vascular disease. It leads to aortic and cerebrovascular disease, pulmonary hypertension, hypotonic bladder, intestinal hypoperistalsis and malrotation, and ocular abnormalities.Multisystemic smooth muscle dysfunction syndrome (MSMDS) is a rare genetic disease characterized by the dysfunction of smooth muscle cells (SMCs) throughout the body. It is caused by the heterozygous mutation of –4 Hereafter we will describe a patient with typical features of MSMDS and with the most severe ocular abnormalities, including congenital mydriasis, early-onset retinal arteriolar tortuosity and a previously unreported ocular manifestation, persistent anterior tunica vascluosa lentis (TVL).The most common ocular manifestation of MSMDS is congenital mydriasis. Late-onset and progressive retinal arteriolar tortuosity, and persistent pupillary membrane have been reported in some cases.2This study was approved by the ethics committee of West China Hospital, Sichuan University. Written informed consent was obtained from the patient's parents. A female full-term born baby with a normal birth weight was admitted to the neonatal intensive care unit immediately after birth for a diagnosis of urinary system dysplasia during fetal life. Echocardiogram revealed patent ductus arteriosus, atrial septal defect, patent foramen ovale, tricuspid regurgitation, dilation of pulmonary artery, and pulmonary hypertension. Color ultrasound of abdominal demonstrated enlarged bladder, bilateral hydronephrosis, and ureter dilation. Abdominal CT scan revealed intestinal malrotation. Cranial magnetic resonance imaging was normal except for widening extracerebral spaces of the temporal lobes and left parietal lobe, and widening lateral fissures. Cranial magnetic resonance angiography demonstrated the absence of A1 segment of right anterior cerebral artery and abnormally straight course of the intracranial arteries. Ligation of patent ductus arteriosus was performed 2 weeks after birth.ACTA2 gene missense mutation p.R179H, confirming the MSMDS diagnosis along with the typical clinical manifestations. Since there is no evidence of active disease in the anterior nor the posterior of the eyes, we decided to observe and follow up the patient monthly for the first 3 months. At the latest 3-month follow-up, no change of the ocular disease was observed. The patient was asked to visit the ophthalmic outpatient clinic every 3 months during the next year.An ophthalmic examination under anesthesia was conducted at the 6th week after her birth. It demonstrated fixed dilated pupils (5 mm) bilaterally, which were unresponsive to light and cycloplegic eye drops Fig. A and B. 3To the best of our knowledge, this is the first report that describes both the anterior and the posterior ocular features of MSMDS by FA demonstrating perfused vasculature in the persistent anterior TVL and prominent retinal arteriolar tortuosity. Meanwhile this 6-week-old girl is the youngest MSMDS patient among all the reported cases so far.–4 The TVL, part of the primary hyaloid vasculature system, provides oxygen and nutrients to the lens from about 6 weeks gestation and regresses completely by the eighth month of gestation in normal eye development.,6 Persistent TVL has been described in premature infants, aniridia, and congenital myotonic dystrophy.–9 In both our case and the previously reported MSMDS cases with persistent pupillary membrane, the patients were born at full term. Pericytes are the major components of TVL walls and express α-actin in the large branches of TVL. We speculate that ACTA2 p.R179H mutation affects not only the SMCs but also the pericytes, and further affects the TVL regression.Persistent anterior TVL described here for the first time is considered to be a more severe condition of persistent pupillary membrane in previously reported cases, both of which are considered as incomplete regression of anterior TVL. Nevertheless, the prominent retinal arteriolar tortuosity in this 6-week-old infant indicates that retinal arteriolar tortuosity can present early. According to the FA, it is clear that the tortuosity is much more prominent in arterioles near the optic disc and even loops are formed. We speculate that it might result from the uneven distribution of α-actin in retina arterioles. The largest arterioles, near the optic disc, are richer in α-actin than arterioles after several branchings. The more α-actin arterioles contain, the more tortuous arterioles appear.According to the fundus examination and FA, the retinal arteriolar tortuosity was the only retinal vascular abnormality in this case. In the previous reports, retinal arteriolar tortuosity was considered to be late-onset and progressive. The absence of iris crypts and furrows, which can be seen in congenital microcoria, may be the result of the dilator muscle hypoplasia. In summary, we observed congenital mydriasis, persistent anterior TVL and early-onset retinal arteriolar tortuosity in the eyes of a 6-week-old girl with a de novo heterozygous ACTA2 gene missense mutation p.R179H causing MSMDS.Congenital mydriasis, which is common in MSMDS, is due to the reduced contractility of iris sphincter and dilator muscles, resulting in fixed dilated pupils.Conceptualization: Fang Lu.Data curation: Licong Liang.Investigation: Licong Liang.Supervision: Fang Lu.Writing – original draft: Kaiqin She.Writing – review & editing: Kaiqin She, Fang Lu.