The objective of this study was to examine the association between the oral microbiome and pregnancy outcomes, specifically healthy or preterm low birth weight (PLBW) in individuals with and without periodontal disease (PD).In this prospective clinical trial, we recruited 186 pregnant women, 17 of whom exhibited PD and delivered PLBW infants (PD-PLBW group). Of the remaining women, 155 presented PD and delivered healthy infants; 18 of these subjects with similar periodontal condition and age matched to the PD-PLBW group, and they became the PD-HD group. From the total group, 11 women exhibited healthy gingiva and had a healthy delivery (HD) and healthy infants (H-HD group), and 3 exhibited healthy gingiva and delivered PLBW infants (H-PLBW group). Periodontal parameters were recorded, and subgingival plaque and serum were collected during 26–28 gestational weeks. For the plaque samples, microbial abundance and diversity were accessed by 16S rRNA sequencing.Porphyromonas, Treponema, and Filifactor, whereas women with healthy gingiva showed an enrichment in Streptococcus, Actinomyces, and Corynebacterium, independently of the birth status. Although no significant difference was found in the beta diversity between the 4 groups, women that had PLBW infants presented a significantly lower abundance of the genus Neisseria, independently of PD status.Women with PD showed an enrichment in the genus Neisseria align with preterm low birth weight in pregnant women, whereas a higher abundance of Treponema, Porphyromonas, Fretibacterium, and Filifactor and a lower abundance of Streptococcus may contribute to periodontal disease during pregnancy.Lower levels of Neisseria have potential in the prediction of PLBW.The oral commensal  The oral microbiome is a complex microbial community with up to 1,000 total microbial species, comprised of bacteria, fungi, viruses, archaea, and protozoa that live in the oral cavity . CurrentPorphyromonas gingivalis and Treponema denticola, have been described as keystone periodontal pathogens and have been used as diagnostic markers of periodontitis, as assessed by traditional culture and PCR methods [P. gingivalis, Fusobacterium nucleatum, Prevotella intermedia, and Tannerella forsythia, in saliva/dental plaque samples were related to PLBW [P. gingivalis and F. nucleatum were frequently detected in feto-maternal units, such as placenta and amniotic fluid in mothers with full-term and PLBW neonates [P. gingivalis infection induced preterm birth and low birth weight in pregnant mice. P. gingivalis was also observed in the placenta of the infection group by immunohistchemistry [Over the past several decades, significant evidence has emerged that supports an association between periodontal pathogenic bacteria and preterm low birth weight (PLBW) , 11, 12. methods , 13, 14. to PLBW . Moreoveneonates , 17. Exphemistry .Neisseria, Porphyromonas, and Treponema were over-represented in the pregnant subjects, while Streptococcus and Veillonella were more abundant in the non-pregnant women [With the recent development of metagenomic sequencing technologies, a growing number of studies have revealed a greater degree of complexity in the oral microbiome than was previously appreciated , 20. Prent women . The conTherefore, in this study, our objective was to determine the relationships between the oral subgingival microbiome, gingival/periodontal inflammation, and PLBW.This study was approved by the Institutional Ethics Committee of West China Hospital of Stomatology, Sichuan University (No WCHSIRB-OT-2016-053) in agreement with the 1964 Helsinki declaration and its later amendments and comparable ethical standards. Subgingival plaque and serum were collected from patients in the Department of Periodontology, West China Hospital of Stomatology, Sichuan University. Written informed consent was obtained from all human subjects who participated in the investigation.In this prospective study, a total of 186 subjects were recruited from the Department of Periodontology, West China Dental Hospital, Sichuan University, from May 2016 to May 2018 including patients and clinical staff. Among these subjects, 17 were diagnosed with periodontal disease (PD) during pregnancy, and they delivered a PLBW infant. These were recruited into the PD-PLBW group. From the total subjects, 155 were diagnosed with PD during pregnancy, and they had a healthy delivery (HD) and delivered a healthy infant (PD-HD-155 group); 18 of these were age and periodontal condition matched with the PD-PLBW group, and they became the PD-HD group (these were selected as the first 18 that were recruited). Among the total subjects, 11 were diagnosed with healthy gingiva during pregnancy, and they had a healthy delivery (HD) and delivered a healthy infant (H-HD group), and 3 subjects were diagnosed with healthy gingiva, and they delivered an PLBW infant (H-PLBW group).th percentile for gestational age based on Chinese standards [Periodontal examinations were performed during the second trimester of pregnancy. Subgingival plaque and serum samples were collected during the periodontal examination visits. All subjects selected for the study had a minimum of 20 teeth and did not receive any periodontal or antibiotic treatment 3 months before the periodontal examinations. Subjects with any other systemic disease and/or multiple gestations were excluded from the study. Preterm birth (PB) was defined as a gestational age less than 37 weeks. Small for gestational age (SGA) was defined as a birth weight of less than the 10tandards . In thisAll 186 women underwent a full-mouth periodontal examination during 26 to 28 weeks of gestation. Initially, an interview about confounding factors, such as oral hygiene behavior , alcohol consumption, smoking behavior, education level, income level, and health insurance status, was obtained.Periodontal examinations were then performed by a periodontal specialist with the use of a manual probe . Participants were instructed to abstain from eating and performing oral hygiene procedures for at least 2 h before plaque collection. During the examination, periodontal parameters, including periodontal probing depth (PPD), clinical attachment loss (CAL), and bleeding on probe (BOP), were recorded in six different sites for each tooth, including the mesio-buccal, mid-buccal, disto-buccal, mesio-lingual, mid-lingual, and disto-lingual sites.Pregnancy seems to have a dramatic effect on the periodontium. Since the 1960s, it has been reported that there is increasing prevalence and severity of gingival inflammation in pregnant women compared to non-pregnant women . StudiesWithin the PD group, subjects who had CAL>0 were diagnosed with periodontitis (P) and those with CAL=0 were diagnosed as gingivitis (G). Subjects who did not fulfill these criteria were diagnosed as clinically healthy (H).g for 10 min at 4°C to separate the serum. Then, the serum samples were immediately stored at −80°C until further analysis.Subgingival plaque samples were collected by inserting a sterile #40 paper point for 30 s into the deepest pocket of the Ramfjord teeth , which iSerum hs-CRP levels were assessed with the Helica™ C-reactive protein assay ELISA kit  according to the manufacturer’s instructions.Total DNA was extracted from subgingival plaque samples using the QIAamp® DNA Mini kit  according to manufacturer’s instructions, and then DNA concentration and purity were determined using a Nano-Drop 2000 UV-vis spectrophotometer . DNA samples were then sent to Majorbio Bio-Pharm Technology Co. Ltd. (China) for amplification of the hypervariable V3-V4 regions of the bacterial 16S rRNA gene and sequencing using an Illumina MiSeq  platform.http://www.mothur.org). Chimeric sequences were identified and removed to reduce the effects of PCR artifacts, and each OTU representative sequence was classified taxonomically using RDP Classifier (http://rdp.cme.msu.edu), using a confidence threshold of 0.7. Then, alpha diversity  was obtained using Mothur (http://www.mothur.org). Principal component analysis (PCA) using unweighted UniFrac distance metrics was carried out, and the R package was used to visualize interactions among bacterial communities in different samples.The raw 16S ribosomal rRNA gene reads were demultiplexed and filtered by Trimmomatic trimming tool and merged by FLASH . OperatiThe 16S rRNA gene sequencing from plaque samples produced more than 2 million raw sequences, and after pre-processing, 2020149 usable sequences with an average of 41,227 ± 8,808 sequences per sample remained in the dataset. The average length of the sequence was 441 bp without the primers, ranging from 388 to 426 bp. After removing the lower credibility OTUs, taxonomic assignment of the sequences resulted in the identification of a total of 1001 OTUs with more than 97% similarity in the subgingival microbiota. Our analysis showed that 99.0% of the oral microbiota sequenced aligned into 12 phyla. Additionally, 95.6% of the oral microbiota were clustered into 99 families and 91.0% aligned with 192 genera.All statistical analyses were performed using STATVIEW software . The Friedman’s two-way ANOVA tests were applied to analyze the statistical differences in age, brushing frequency, brushing time, birth weeks, birth weight, mean PPD, mean CAL, percentage of PPD ≥ 5 mm, percentage of BOP-positive sites, and serum hs-CRP concentration between the H-HD, H-PLBW, PD-PLBW, PD-HD, and PD-HD-155 groups and between H, G, and P groups. Chi-square test was performed to compare the dental floss and rinse using rate, education level, income level, health insurance status, and cesarean section rate between the groups. Kruskal–Wallis rank sum test was used to detect comparative taxonomic profiles of different groups at phylum, family, and genus levels, while Spearman correlation coefficients were used to assess the bacterial association with periodontal parameters, hs-CRP, and birth results.The demographic characteristics of all subjects enrolled in this trial and categorized by their groups  are listed in Table Next, we evaluated the subjects’ periodontal clinical characteristics, serum hs-CRP status, and delivery characteristics, and the results are listed in Tables Also, in Table Firmicutes, Proteobacteria, Fusobacteria, Actinobacteria, Bacteroidetes, and Spirochaetes, were dominant in all subjects. Nonetheless, Spirochaetes and Bacteroidetes were significantly higher in both P and G groups compared to both gingivally healthy groups. Actinobacteria was lower in both P and G groups compared to the H group  compared to the H-HD group  of the genus level data, and the results can be seen in Fig. Capnocytophaga, Haemophilus, and Streptococcus genera were negatively correlated with periodontal parameters, such as PPD and BOP, whereas Filifactor, Fretibacterium, Peptostreptococcus, Selenomonas, TM7, and Treponema were positively correlated the same parameters. These data indicate that different microbial groups may be modulating the health/disease status of the periodontal tissues, with Capnocytophaga, Haemophilus, Neisseria, and Streptococcus genera possibly protecting the host whereas Filifactor, Fretibacterium, Peptostreptococcus, Selenomonas, TM7, and Treponema genera possibly aggravating/degrading hosts tissues and promoting periodontitis. However, TM7 was also negatively correlated with the general inflammation marker hs-CRP.After determining the alpha and beta diversities, we evaluated the association of the top 20 genera with general inflammation, and periodontal and delivery parameters and the results can be found in Fig. Neisseria is positively associated with birth weight, while being negatively associated with the periodontal disease parameter BOP. This result suggests a possible protective role for Neisseria in both periodontitis and during delivery. This finding is in agreement with the lower abundance of the Neisseria genus and of an unclassified Neisseria species in the PBLW samples.These data further indicate that Firmicutes, Tenericutes, Proteobacteria, Bacteroidetes, and Fusobacteria phyla. This profile is distinct from that of the vagina but similar to that of the oral microbiome [Intrauterine infection is the primary cause of APO. Evidence indicates two possible origins of intrauterine infections: ascending infection from the lower genital tract and hematogenous transmission from the oral microbiome. Although current paradigms suggest that most intrauterine infections originate in the lower genital tract and ascend into an otherwise “sterile” intrauterine environment, recent evidence showed that the placental microbiome was composed of nonpathogenic commensal microbiota from the crobiome , 31.P. gingivalis, are strongly associated with APO, such as preterm birth and low birth weight. Clinical evidence shows that higher amounts of P. gingivalis in subgingival plaque increase the risk of preterm birth, including our previous studies [P. gingivalis induce preterm birth and low birth weight [Findings from the last 2 decades supported a long-standing strong association between maternal periodontal disease and increased risk of pregnancy outcomes , 33. The studies , 36. Anih weight , 37.An important key point of this study is that we were able to match the age and the periodontal status of 18 subjects, which would assist us in understanding how the oral microbiome might differently contribute to the birth results, without these other confounding variables.Neisseria genera and an unclassified Neisseria species in patients with gingival/periodontal inflammation and preterm low birth weight. We specifically found the genus to be negatively related with bleeding on probe (BOP) and positively related to birth weight, whereas the Neisseria genus and an unclassified Neisseria species were significantly lower in subjects with PLBW delivery. Neisseria is one of the most predominant genera among the Proteobacteria, which itself is one of the four most abundant phyla colonizing the oral mucosa of humans along with Firmicutes, Bacteroidetes, and Actinobacteria [A significant finding of this study is the possible correlation of the bacteria .Neisseria species in oral diseases. Yamashita et al. [Neisseria in oral microbiome indicates healthy periodontal conditions. A study in children between 3 and 18 years of age revealed that Neisseria is highly prevalent  and Neisseria flavescens has been associated with a caries-free status [Neisseria as part of the healthy “core microbiome” of the human oral cavity. In contrast, Colombo et al. reported that Neisseria species were related to periodontal inflammation and tissue destruction [Several studies have evaluated the protective role of a et al.  and Meura et al.  reportede status . Zaura ee status  identifitruction .Neisseria genera have also been studied in relation to systemic diseases. For instance, Said et al. [Neisseria and Gemella genera in the salivary microbiota of inflammatory bowel disease patients compared with healthy ones. Interestingly, Peters et al. [Neisseria and the Streptococcus pneumoniae species were associated with lower esophageal cancer risk, whereas Farrell et al. [Neisseria elongata, along with Streptococcus mitis, were significantly less abundant in pancreatic cancer patients compared to healthy patients. In contrast, the Neisseria genus was reported to be enriched in HIV+ subjects regardless of sampling site and PD level [Neisseria gonorrhoeae is one of the most well studied pathogenic Neisseria species. The species can colonize the oropharynx, causing an infection, but without symptoms. Yet, this infection can be transmitted during sexual activities to other sites of the body that are more typically associated with infectious complications, such as pelvic inflammation, epididymitis, and adverse pregnancy outcomes. Interestingly, the nonpathogenic Neisseria commensal bacteria can negatively affect N. gonorrhoeae colonization by bacterial interspecies competition. Kim et al. reported that nonpathogenic Neisseria commensal bacteria, such as Neisseria elongata can inhibit N. gonorrhoeae by a DNA uptake-dependent manner and pathogen clearance in vivo [The d et al.  reporteds et al.  reportedl et al.  reportedPD level , which m in vivo .Neisseria on pregnancy outcomes is still uncertain. Lin et al. reported an increased abundance of Neisseria, Porphyromonas, and Treponema in the supra-gingival plaque samples of pregnant women, whereas Streptococcus and Veillonella genera were more abundant in the non-pregnant group [Neisseria and Aggregatibacter when compared to the non-pregnancy group [Neisseria genus in pregnancy. Nonetheless, this study addresses the role of the nonpathogenic Neisseria species in adverse pregnancy outcomes.However, the influence of nt group . Converscy group . TherefoP. gingivalis, T. denticola, P. intermedia, T. forsythia, Campylobacter rectus, A. actinomycetemcomitans, and Fretibacterium sp. HOT360 in the oral microbiome was positively correlated with gingival inflammation during pregnancy [Firmicutes, Proteobacteria, Bacteroidetes, Fusobacteria, and Actinobacteria were the five dominant phyla in the mouth. A higher abundance of Spirochaetes and Bacteroidetes and a lower abundance of Actinobacteria were seen in groups with gingival/periodontal inflammation compared with gingival/periodontal healthy groups. Nonetheless, at the genus level, Treponema, Porphyromonas, Fretibacterium, Filifactor, and Peptostreptococcaceae were associated with gingival/periodontal inflammation. Periodontal therapy, including non-surgical periodontal therapy or probiotics may be warranted before or during pregnancy to reduce periodontal inflammation and correct the maternal oral microbiome dysbiosis.Our data also supports that the intrinsic composition of bacterial taxa changes between pregnant women with or without gingival/periodontal inflammation and between women with healthy delivery and preterm low birth weight delivery. Prospective studies have shown that the level of regnancy –52. In tWe found no significant changes in microbiome diversity between the four groups, which is in agreement with the literature. For instance, Yang et al.  indicateThis study has some limitations. One of the limitations of this study was the small sample size, especially in the H-PLBW group. This study was performed at a single institution, which prevented us from having a larger sample size. The oral microbiome during pregnancy appears to be complex, and this fact by itself warrants further large-scale longitudinal studies to understand the role of oral bacteria in PLBW outcomes. Additionally, single-variate statistical analyses were used in this study, which could have limited the overall results of the study.Neisseria genus and of an unclassified Neisseria species in women who had preterm low birth weight deliveries. Moreover, our analysis demonstrated that Neisseria genera were positively correlated to birth weight in these women. Therefore, correcting oral microbiome dysbiosis, such as improving Neisseria genera abundance before or during pregnancy by periodontal therapy, may be useful in preventing PLBW deliveries. Thus, the oral commensal Neisseria may have a clinical potential for predicting PLBW.In this study, we found a lower abundance of the


Endogenous erythropoietin (EPO) concentrations vary widely in preterm infants and may be associated with perinatal risk factors and neurological outcomes. Erythropoietin is elevated in fetal hypoxia but is also a potential neuroprotectant.In a prospective study of 27 infants ≤ 30 weeks gestation, serum erythropoietin concentrations were measured during the first month of life, on day 1 and weeks 1, 2, and 4, and related to perinatal risk factors and outcomes including retinopathy of prematurity and cerebral injury evaluated near term-equivalent post menstrual age using magnetic resonance imaging with quantitative scoring.Lower birth weight was associated with higher EPO concentrations throughout the first 2 weeks of life . Higher day 1 and week 1 EPO concentrations were associated with lower Apgar score at 1 minute (r = - 0.5) and 5 minutes (r = -0.7), respectively (p < 0.01). Higher day 1 EPO concentrations and 2-week area under the curve were associated with increased risk (p = 0.01) and severity  of retinopathy of prematurity. Higher EPO concentrations at 2 weeks were associated with increased total brain injury score .Elevated endogenous erythropoietin concentrations in the first two weeks of life are associated with lower birth weight and increased risk of adverse outcomes. Preterm birth is a major public-health challenge. Although survival has improved over the past two decades, children born preterm are at increased risk for cerebral palsy and cognitive and neurobehavioral deficits including lowered IQ, autism spectrum disorders, attention deficit hyperactivity disorder, anxiety disorders, and learning disabilities , 2. ErytThe biological role of EPO is complex and extends well beyond hematopoiesis; EPO and its receptor (EpoR) are expressed early in fetal development in the human brain  and EPO Independent of the beneficial effects of EPO, its concentration may also be a biomarker of stressful perinatal conditions. Among preterm newborns, elevated concentrations of endogenous EPO have been associated with elevated inflammation-related proteins  and withThe commonest neuropathology documented in the very preterm infant occurs in the cerebral white matter, and along with other forms of brain injury, has been shown to be best evaluated using magnetic resonance imaging (MRI) . AlthougThe factors that regulate endogenous EPO production in the preterm infant, as well as the influence of perinatal exposures, are not fully understood. This study investigates the impact of perinatal risk factors on endogenous EPO concentrations during the first month of life in preterm infants and the potential correlations between EPO concentrations and outcomes, including retinopathy of prematurity (ROP) and brain injury using MRI at term-equivalent post menstrual age.The Washington University Institutional Review Board approved this research, protocol 08–0233. Written consent was obtained.Prospective data were collected between June 2008 and March 2009 at St Louis Children’s Hospital neonatal intensive care unit (NICU) in Saint Louis, Missouri, USA  after IRB approval. Mothers and fetuses were not living at high altitude (>2500m). All preterm infants with gestational age (GA) ≤ 30 weeks expected to survive > 72 hours (as determined by the attending neonatologist) were eligible for recruitment. Exclusion criteria included infants with chromosomal abnormalities, major congenital anomalies, hemolytic blood disorders and suspected or proven congenital infections . Patients missing both cord and day 1 EPO concentration values were also excluded. During periods of active enrollment, parents or guardians of consecutive eligible infants were approached by the investigators or a trained NICU research nurse to obtain written informed consent as soon as possible after delivery. All infants were treated according to the accepted care standards of the NICU.Perinatal data were collected by review of maternal and infant charts by the investigators and a NICU research nurse. Information on perinatal risk factors and neonatal morbidities was collected. Prenatal factors included sex, race, gestational age, intrauterine growth restriction (IUGR), pregnancy-induced hypertension (PIH), preterm labor, maternal illnesses or drugs, multiple birth and antenatal steroids. Postnatal factors included birth weight, birth weight Z-score , mode oCranial ultrasound (US) was obtained according to standard clinical practice, which included a scan within the first 7–14 days of life to evaluate for intraventricular hemorrhage (IVH). Infants were scanned using MRI using a 3T Tim Trio system  without sedation near term gestational age or prior to discharge from the NICU. MR images were evaluated using a modified scoring system to assess brain injury as previously described  showed wide variation with median 6.5 (range 2.7–174) mU/ml. Erythropoietin (EPO) and hemoglobin (Hgb) concentrations at each time point, and their interrelationship, are shown in [EPO]0-2weeks  (0-2 weeks)  . EPO con < 0.01) . After a2 weeks) . EPO con2 weeks) .Higher early EPO concentrations were associated with lower Apgar scores. First EPO negatively correlated with 1 minute Apgar score , and 1 week EPO negatively correlated with 5 minute Apgar score  . These a[EPO]0–2 weeks were significantly higher for patients with any (grade 1–3) ROP than for patients without ROP   also correlated with stage of ROP (Early EPO concentrations were higher for subjects with ROP. First EPO and AUC = 0.01) . Week 1  = 0.01) . First Ee of ROP . The pose of ROP  Tables.A negative correlation between EPO and Hgb concentration was observed at 2 weeks   that wasThe number of transfusions received during NICU stay correlated with stage of ROP , all with p < 0.0001 . The numMRI imaging was completed for 24 subjects at 37 ±2  weeks post menstrual age. A positive correlation of 2 week EPO with total brain injury assessed by MRI was observed   but not Endogenous Erythropoietin (EPO) concentrations in premature infants may both be a biomarker for hypoxic stress and have an influence on the response to brain injury. This study investigated whether EPO concentrations in the first month of life were driven by individual perinatal risk factors and were associated with subsequent outcomes and brain injury as assessed by MRI prior to NICU discharge.EPO concentrations varied widely at birth, with a decline in point estimates to a nadir at about 1 to 2 weeks of life and recovery by week 4. This initial decline in EPO is consistent with early studies . EPO conSubjects with higher EPO concentrations at birth and 1 week had increased risk and severity of ROP. Higher initial and 1 week EPO concentrations were also associated with lower Apgar scores, and there was a trend towards higher initial EPO in subjects with perinatal stressors such as emergent caesarian section , IUGR , and early sepsis  . First EWe interpret these results to indicate that these associations of early elevations in EPO with adverse outcomes are due to increased EPO production in response to fetal/neonatal hypoxia. Early studies have shown that fetal hypoxemia stimulates erythropoiesis and that it is a very responsive system; plasma EPO peaked less than 12 hours after the onset of hypoxemia in late-gestation fetal sheep . EmergenTeramo et al  hypothesAt birth through the first 2 weeks of life, EPO concentrations also negatively correlated with Hgb concentrations and were higher in infants that received transfusion than those that did not. Furthermore, EPO concentrations increased between weeks 2 and 4 only in the non-transfused group and by week 4 were higher in the non-transfused group than in the transfused group. These findings are consistent with appropriate physiological regulation of EPO production in preterm infants.The number of transfusions received during NICU stay correlated with increased white matter injury by MRI. A reasonable hypothesis is a “double hit” to white matter: the hypoxia of low Hgb (leading to the transfusion in the first place) and the inflammation associated with transfusion . Increas[EPO]0–2 weeks], The number of transfusions received during NICU stay also correlated with stage of ROP . This finding may be relevant to current controversies over transfusion thresholds. Although no significant differences in severe ROP were observed between liberal and restrictive transfusion groups in the PINT randomized trial , a recenRegarding possible etiologies for the potential impact of transfusion on injury, the lower EPO at 1-month observed in RBC-transfused patients may translate into loss of a neuroprotective growth factor that attenuates inflammation . DecreasThis study is unique in that it investigated endogenous EPO concentrations throughout the first four weeks of life and subsequent brain injury as assessed by MRI with systematic scoring of grey/white matter injury near term gestation. EPO concentrations at 2 weeks were associated with increased total brain injury score . However, this association lost significance  after correcting for GA or birth weight Z-Score. In contrast, the correlation of 2 week EPO concentration with grey matter injury was present only after adjusting for birth weight Z-Score . In comparison to other studies, pre-discharge MRI findings of increased brain injury in association with elevated EPO levels at 2 weeks would be consistent with and could presage the long-term neurodevelopmental deficits reported by the ELGAN study investigators. In ELGAN study subjects, endogenous EPO concentrations at 2 weeks of life, regardless of intermittent or sustained systemic inflammation, were associated with low mental and/or psychomotor development indices and microcephaly at 2 years  and withThese results showing endogenous EPO concentration as a biomarker for hypoxic injury have no bearing on the potential protective effects of exogenous recombinant human EPO (rhEPO). Exogenous rhEPO is neuroprotective in animal experiments , and hasIndependent of the potential benefit of rhEPO, endogenous EPO elevations within the first 2 weeks correlated in this study with morbidities associated with hypoxic injury including ROP and brain injury. Of interest, this association was not observed for EPO concentrations at 1 month of age, and anecdotally the lowest 1-month EPO concentration was observed in a single subject with NEC who died on day of life 38.This study has limitations, including its small sample size with resulting low statistical power, and no adjustment was made for multiple comparisons. It is also limited to analysis of clinically discarded blood samples, which restricts both the number and volumes available for analysis. Furthermore, as in any observational study, confounding variables may complicate interpretation of findings. The MRI scans were performed at a range of postmenstrual ages, although the presence of injury should not influence the later MRI definition of injury presence. Finally, longer term clinical outcome data were not collected.Endogenous Erythropoietin (EPO) concentrations in the first month of life vary widely in preterm infants and may both be a marker for perinatal hypoxic injury and affect the healing response. Elevated EPO concentrations in the first two weeks are associated with lower birth weight, increased risk of ROP, and are suggestive of higher brain injury scores by MRI that may presage long term neurodevelopmental outcomes. RBC transfusions were associated with increased risk and severity of ROP and white matter injury. Additional studies are warranted to further asses the risks of transfusion and the potential role of early endogenous EPO as a biomarker for hypoxic injury in preterm infants.S1 TableSpearman partial correlation coefficient estimate adjusted for gestational age; p-value, and r are presented for association of ln(EPO) at each time point with continuous variables.(PDF)Click here for additional data file.S2 TableSpearman partial correlation coefficient estimate adjusted for birthweight Z-Score; p-value, and r are presented for association of ln(EPO) at each time point with continuous variables.(PDF)Click here for additional data file.S3 TableInjury scores by MRI at near term gestational age are presented as mean (SD) with two-sample t-test comparing subjects with transfusion (yes) vs. without transfusion (no) in gender subgroups.(PDF)Click here for additional data file.S4 TableSpearman correlation and partial correlation coefficients for association of number of transfusions with ROP and brain injury scores by MRI at near term-equivalent post-menstrual age, adjusted for ln[EPO] or [Hg] at week 1 or 2; coefficient estimate, p-value and r are presented.(PDF)Click here for additional data file.S5 TableLinear regression correlating ln(EPO) over time with continuous variables; coefficient estimate, p-value and R-square are presented.(PDF)Click here for additional data file.S6 TableLinear regression correlating Ln(EPO) over time with continuous variables adjusted for gestational age; coefficient estimate, p-value and r are presented.(PDF)Click here for additional data file.S7 TableLinear regression correlating Ln(EPO) over time with continuous variables adjusted for birthweight Z-Score; coefficient estimate, p-value and r are presented .(PDF)Click here for additional data file.


Fourier-transform infrared (FT-IR) spectrometry revealed that the antioxidants and HDPE were uniformly mixed with noncovalent bonding. In addition, the differential scanning calorimetry (DSC) test revealed that the change in melting point and initial extrapolation temperature of the antioxidant active caps was not significant. Sensory evaluation and removal torque tests validated the suitability of the antioxidant active plastic bottle caps in industrial application. The antioxidant activity increased with a greater concentration of BHA and BHT incorporated in both antioxidant active caps (p < 0.05) and with more impact on the BHA cap compared to BHT cap in terms of antioxidant activity. Migration experiments for 10 days at 40 °C and 2 h at 70 °C showed that active antioxidants in the plastic bottle cap were more easily released into fatty foods and milk products that are highly sensitive to oxidation, and the migration of BHA and BHT did not exceed the maximum amount specified in (EC) No 1333/2008 (<200 mg/kg). As such, the antioxidant active plastic bottle caps inhibited oxidation, thereby ensuring higher food quality.In this study, we prepared new antioxidant active plastic bottle caps by incorporating butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT) and 2% ( Improvements in living standards and conscious eating have revolutionized the food industry. For instance, polyethylene terephthalate (PET) bottles have been popular in beverage packaging . HoweverThe major challenge associated with adopting food antioxidant packaging materials is an effective and practical combination of the antioxidant and packaging material without changing the packaging line or the characteristics of the materials ,12. MoreThe most common synthetic phenolic antioxidants used in food preservation include butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). These antioxidants bind free radicals, remove oxygen, inhibit oxidases, and block oil and fat oxidation ,14. BHA This study was aimed at developing antioxidant active plastic bottle caps using BHA and BHT. The effects of different BHA and BHT concentrations on the functional and antioxidant properties of caps were also tested.s-triazine (TPTZ), (purity ≥ 97%), and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) (purity ≥ 98%) were purchased from Macklin . Analytically pure (AR) potassium persulfate (K2S2O8) was purchased from Xilong Scientific Co., Ltd. , while AR sodium acetate trihydrate (C2H3NaO2·3(H2O)) was purchased from Guangdong Guangtry reagent Technology Co., LTD, . AR Ferric chloride (FeCl3) (99% pure) was purchased from Shanghai Macklin Co., Ltd. AR hydrochloric acid (HCl) was purchased from Guangdong Guangtry reagent Technology Co., LTD.HDPE  and masterbatch  were obtained from Jinfu Technology Co., Ltd. . BHA (≥99%), while BHT (≥99%) was purchased from Shanghai Macklin Co., Ltd. . High-performance liquid chromatography (HPLC)-grade acetic acid, ethyl acetate ethanol, methanol, 2-diphenyl-1-picrylhydrazyl (DPPH) , vitamin C (Vc) (purity ≥ 99%), 2,4,6-Tris(2-pyridyl)-w/w) were separately mixed with HDPE granules and 2% (w/w) masterbatch to obtain white granules. The mixture was transferred to a twin-screw extruder  using a screw diameter of 20 mm and 100 rpm screw speed. Temperatures in the nine heating zones of the rotating twin-screw extruder were 160 °C, 165 °C, 165 °C, 170 °C, 175 °C, 175 °C, 175 °C, and 175 °C. Molten extruded material was ejected as thin strings, passed through a cold-water basin, and then cut into small granules. The screw was set to rotate at 20 rpm, under temperatures of 190 °C, 200 °C, 210 °C, and 230 °C. The remaining granules were added into an injection molding machine  to produce plastic bottle caps. The injection molding machine was purchased from Jinfu Technology Co., Ltd. The injection pressure and speed were set to 0.30 s and 80 bar. Temperatures of the injection molding machine were 200 °C, 220 °C, 220 °C, and 220 °C. The schematic flow of the extrusion molding process for the production of the active antioxidant active plastic bottle caps is shown in Synthetic antioxidants  . The machine was calibrated using a standard white tile, according to Equation (1).−1 nitrogen atmosphere. The first heating was performed from 25 °C to 250 °C at a rate of 20 °C·min−1. The materials were cooled to 25 °C at a rate of 20 °C·min−1 before a second heating stage to 250 °C. The degree of crystallinity (cX), glass transition temperature (gT), and melting temperature (mT) were obtained in the second heating stage  radical by the antioxidant active caps . Compare 1% BHT) .p < 0.05). We found that the antioxidant activities of BHA and BHT were almost similar, although BHA exhibited a slightly stronger oxidation capacity than BHT, consistent with a previous report [Comparison of the antioxidant effects of the antioxidant active caps showed a significant difference for all the antioxidant assays (s report . For the2r), and linear ranges of BHA and BHT antioxidant activities are shown in 2r) was greater than 0.999.The LODs, LOQs, linear equation, correlation coefficient (The recoveries and RSD of BHA and BHT at three spiked levels are shown in 4 mg/kg) and 2% (2 × 104 mg/kg), respectively. This shows that BHT underwent greater thermal decomposition. The extraction capacity of BHA and BHT using the three solvents was also investigated. In the independent experiments, the three methods presented significant differences (p < 0.05). The dichloromethane solution exhibited the highest extraction performance, whereas methanol had the worst. Ethyl acetate had a moderate extraction capacity. Methanol has a low polarity that is not suitable for completely extracting BHA and BHT. Methylene chloride and ethyl acetate have good extraction capacities for antioxidants and other phenolic substances. However, methylene chloride extraction is recommended over ethyl acetate.Initial antioxidant concentrations in the sample were also assessed . It was Antioxidant activities of BHA and BHT have been extensively studied ,42. It h3)3 groups) [The migration potentials of BHA and BHT in 50% and 95% ethanol at 70 °C are presented in  groups) . As showUsually, 95% ethanol is used as a simulant for fats, oil, and fatty foods due to their comparable hydrophobicity. Ethanol is completely miscible in water and provides a more hydrophobic environment. Since all the antioxidants used in this study were hydrophobic, they were rapidly released. Additionally, graphs for the rate of antioxidant release with time in comparison to the initial antioxidant amount in HDPE caps were plotted to elucidate on antioxidant release behaviors. The rate of BHA and BHT release in 95% ethanol at 40 °C is shown in The EU Commission specifies 10% ethanol for simulating alcoholic foods and 3% acetic acid for acidic foods; no migration in these conditions was detected for BHA and BHT, possibly due to the smaller molecular volume of both compounds, which impeded their release into the highly polar simulants. In contrast, more polar food simulants such as water or 3% acetic acid conferred lower release rates. Therefore, the release of antioxidants is often observed in fatty foods such as olive oil, 95% ethanol, or isooctane, unlike aqueous food simulants.According to (EC) No 1333/2008, BHA and BHT residues in edible oils, fried foods, dried fish products, biscuits, instant noodles, canned fruits, and pickled meat products should not exceed 200 mg/kg. In this study, the specific migration limit (SML) of BHA and BHT at 40 °C for 10 days or 70 °C for 2 h was lower than 0.2 g/kg. Therefore, the antioxidant active plastic bottle caps meet the safely thresholds for food packaging.Diffusion of BHA and BHT from HDPE caps to simulants exhibited Fick’s behavior. The higher migration in oil simulant is also a positive feature for the protection of foodstuff with a high fat content, since this kind of food needs better protection from oxidation. In addition, incorporation of BHA and BHT into the antioxidant plastic bottle caps and films had no significant effect on the original excellent HDPE properties. They exhibited good antioxidant properties that could meet the minimum production thresholds. It is very important to control BHA and BHT within a specific migration limit. The antioxidant bottle cap should have good antioxidant properties and ensure the safety of food contact materials. Accordingly, BHT and BHA are promising compounds in the manufacture of active packaging materials. Nonetheless, more studies using typical foods should be performed to validate the suitability of active antioxidant plastic bottle caps in food packaging.


Pudendal nerve block analgesia (PNB) is used as pain relief in the final stage of childbirth. We hypothesized that PNB is associated with higher rates of postpartum urinary retention.We performed a cohort study among primiparous women with a singleton, cephalic vaginal birth at Oslo University Hospital, Norway. Women receiving PNB were included in the exposed group, while the subsequent woman giving birth without PNB was included in the unexposed group. We compared the likelihood of postpartum urinary retention, defined as catheterization within 3 h after birth. Logistic regression analysis stratified by mode of delivery was performed adjusting for epidural analgesia, episiotomy and birth unit.Of the 1007 included women, 499 were exposed to PNB and 508 were unexposed. In adjusted analyses, women exposed to PNB did not differ in likelihood of postpartum urinary retention compared to women unexposed to PNB in either spontaneous  or instrumental  births. Furthermore, no differences between the groups were observed with excessive residual urine volume or catheterization after > 3 h.PNB was associated with neither risk of postpartum urinary retention nor excessive residual urine volume and is therefore unlikely to hamper future bladder function.The online version contains supplementary material available at 10.1007/s00192-021-04768-0. Postpartum urinary retention (PUR) is a common condition following birth. PUR may lead to short- and long-term complications such as bladder dysfunction, recurrent urinary tract infection and, very rarely, bladder rupture , 2. RepoThe known risk factors for urinary retention include advanced age, nerve dysfunction and bladder outlet obstruction . Among oVoluntary control of the lower urinary tract requires interaction between autonomic (sympathetic and parasympathetic nerves) and somatic, afferent (sensory nerve) and efferent (motor nerve) pathways. Somatic efferent function deterioration leads to an attenuated contractile force with insufficient urine evacuation as a result . The pudDuring the first stage of birth (from 3 to 4 cm to 10 cm dilatation), the most efficient pain relief is epidural analgesia, whereas pudendal nerve block (PNB) is an option during the second stage of birth (from 10 cm to birth) during the final descent of the fetal head and expulsion of the baby. PNB is an effective method of pain relief, providing analgesia to the vulva and anus –18 by trThis study was conducted at the Department of Obstetrics at Oslo University Hospital (OUH). OUH has two delivery locations: Rikshospitalet with 2500 births/year (named Unit 1) and Ullevål with 6950 births/year (named Unit 2). The two units share clinical guidelines and management. Unit 1 reintroduced PNB as part of quality improvement in 2014, aiming to ensure uniform pain relief availability for all women. In this unit, women with a request for more pain relief may be provided PNB regardless of whether the midwife alone, or in collaboration with the attending doctor, handles the birth. Primiparous women having delivered a singleton live newborn in cephalic position at term, during the period from January 1, 2017, until June 1, 2019, were eligible for inclusion. All women who were exposed to PNB during birth were invited to participate . For each invited woman exposed to PNB, the subsequent woman at the same unit unexposed to PNB was invited to participate . The eligible women received written study information 4 weeks postpartum. The women were informed that a response to the attached questionnaire about childbirth experiences was considered a written informed consent. The childbirth experience is the focus of an ongoing study. In accordance with patient consent, we collected clinical data from the electronic medical records. In addition, we thoroughly reviewed the electronic medical records for reported hematoma or abscess.We excluded women transferred during birth from the low-risk midwife-led birth unit , women with allergy to local anesthetics and women with uncertainty of timing (before or after birth) or status (categorization) of PNB.This study was originally designed to explore the association between PNB and childbirth experience aimed at including 1000 women. With the same sample size available, we may detect an absolute difference of 10% in overt PUR among women exposed compared to unexposed to PNB among women with spontaneous births, assuming a baseline rate of 25%, a power of 80% and a significance level of 5%. We considered a difference of 10% in overt PUR to be clinically relevant. For instrumental deliveries the available sample was smaller, and thus the power was < 80%.The exposure in this study was PNB provided during birth, whereas overt PUR was the primary outcome. We defined overt PUR as the inability to void within 3 h postpartum, equaling the need for at least one catheterization within 3 h postpartum according to the department’s guideline.We considered the proportions of two postpartum catheterization volumes, namely ≥ 750 ml and ≥ 1000 ml. Other secondary outcomes were catheterizations after > 3 h postpartum, Apgar score < 7 at 5 min of age and anal sphincter injury.2), gestational length at delivery (days) and birth weight (grams). Epidural analgesia, spinal analgesia, instrumental birth (vacuum and/or forceps extraction), episiotomy and oxytocin used during birth were dichotomous variables. Prolonged second stage of birth was defined as > 3 h and long duration of birth as > 12 h (start of birth defined as 3–4 cm dilation and with regular contractions). Local anesthetics used in PNB were bupivacaine, lidocaine or bupivacaine with epinephrine. PNB duration was defined in minutes from administration to the birth of the baby. Birth unit was either Unit 1  or Unit 2 (Ullevål).Clinical data included maternal age at birth (years), marital status (married/cohabiting or not), higher maternal education (> 13 years or not), body mass index  for normally distributed continuous variables. Comparison of demographic and obstetric characteristics between the groups exposed and unexposed to PNB was performed by chi-square test, Fisher’s exact test and t-test, as appropriate. Primary and secondary outcomes are presented as numbers and proportions with 95% confidence intervals (CI) according to group. The association between PNB and instrumental delivery is complex, and sometimes the first is chosen when planning the latter, and therefore we chose to stratify by mode of birth  in our analysis of PNB’s association with PUR.We explored the association between PNB and overt PUR by performing logistic regression analysis, stratified by mode of birth. Both unadjusted and adjusted odds ratios (aOR) with 95% CI are presented. Due to the restricted number of observations in the stratified analysis, four variables were included in the multivariable analyses. Adjustments were made for epidural and/or spinal analgesia, episiotomy and birth unit, as these were pre-defined based on a potential association with PNB and overt PUR. During birth, epidural (continuous infusion) and spinal (single injection) analgesia are provided in the same anatomical segments, and we therefore merged epidural and spinal analgesia for the purpose of adjustment in the regression analyses. Prolonged birth [both total duration of birth (> 12 h) and prolonged second stage of birth (> 3 h)] was strongly correlated to epidural analgesia and thus omitted from the analyses. Interaction effects between the adjusting variables and PNB were tested by adding product terms, one at a time, into the models.We used the same logistic regression frameworks for the secondary outcomes, residual urine volume ≥ 750 among women with overt PUR and catheterization after > 3 h. Furthermore, in a sensitivity analysis, missing information about residual volume was considered as < 750 ml if no value was registered, based on the assumption that lack of registrations was likely lack of a large volume. Because of few observations of residual urine volume ≥ 1000 ml when overt PUR was diagnosed, Apgar score < 7 at 5 min of age and sphincter injury, we did not perform multivariate analyses on these secondary outcomes.p value < 0.05 as statistically significant. We conducted all analyses using SPSS version 25 .We considered an association with a www.clinicaltrials.gov (NCT04391075). The user council of the Division of Obstetrics and Gynecology at Oslo University Hospital was involved in the design and the development of the study.All enrolled women provided informed consent. The local Data Personal Officer at OUH approved the study, 2016/18884. This quality study was exempt from approval by the Regional Committee for Medical and Health Research Ethics system in Norway according to the Norwegian act on health research. The study is registered in A total of 1760 primiparous women were invited to participate in the study. The final study sample comprised 1007 women, 499 women exposed to PNB and 508 women not exposed to PNB. In the PNB group 46% had an instrumental birth, whereas the proportion was 20% in the non-PNB group, while the proportion of overt PUR was 36% (PNB group) and 30% (non-PNB group) . There was no difference in newborn rates of Apgar score < 7 at 5 min of age between the groups. The anal sphincter injury rate was low overall, without any difference between the groups Table .Table 1When stratifying also for mode of birth, differences between women exposed and unexposed to PNB varied similarly to those shown in Table In this cohort, there were some differences in obstetric characteristics between the units: Unit 1 had shorter PNB duration than Unit 2. Unit 1 had more overt PUR and more epidural and spinal analgesia, but the rates of episiotomy and instrumental birth did not differ  and were more often catheterized after > 3 h (18% vs. 9%) compared to women with spontaneous birth. The rates of excessive residual urine volume (≥ 1000 or ≥ 750) did not differ with mode of delivery (Table 4% and wep = 0.03) (data not shown). Thus, stratified analyses by birth unit were performed in spontaneous birth  in adjusted analyses stratified by mode of birth. Nor did we find any significant association between PNB and rates of excessive residual urine volume or between PNB and rate of catheterization after > 3 h postpartum. No adverse effects of PNB on newborn (Apgar score) or maternal (abscess or hematoma) outcomes were observed. There was a difference in frequency of postpartum catheterization between the units despite their shared management and guidelines. The lacking association between PNB and overt PUR was however not affected by this difference.n = 163) investigated the association between PNB and urinary retention [To our knowledge, the association between PNB and postpartum urinary retention has not been investigated and published previously. Only one previous study on hemorroidectomy in both women and men ; this is likely due to our definition of overt PUR , which is a shorter time than that of Yip et al. . Yip et The strengths of this study include the prospective design and the large sample size. Another strength is the adjustment for obstetric risk factors of PUR , epiduraOur data are observational and cannot demonstrate causality. We considered a randomized clinical trial but found it unethical to randomize women to pain relief or placebo during birth. Furthermore, bias due to unknown and unmeasured confounders may be another limitation of observational studies such as ours.Even though overt PUR may be of multifactorial origin, the results of our study clearly show that PNB is not associated with an increased risk of postpartum urinary retention or excessive residual urine volume after spontaneous vaginal birth. To evaluate the safety concerning PUR in instrumental vaginal births, low Apgar score and anal sphincter injury, a larger sample size is required because of the low incidence, but we found no indication of such risks.In conclusion, we suggest the results of our study can provide women with a better basis for making informed choices regarding the use of PNB as pain relief during birth. Whether PNB provides a better overall childbirth experience is yet to be determined, but it has been shown to provide good pain relief during birth and for suturing lacerations after birth . Our stuESM 1(DOCX 30 kb)


Early developmental success and school readiness strongly influence future skill development, occupational opportunities, and health. Therefore, it is critical to identify and address early determinants of school readiness for supporting children’s overall well-being and success. In this retrospective cohort study, we examined the effects of pre-birth household challenges, such as homelessness or experiences of intimate partner violence, on children’s early school readiness. We linked data from the Alaska 2009–2011 Pregnancy Risk Assessment Monitoring System (PRAMS) to administrative and education records through 2019. Education records included kindergarten developmental scores, third grade reading assessments, and attendance records. Generalized linear models with Quasi-Poisson distributions for each outcome of interest examined the predictive value of pre-birth household challenges on the risks of not meeting school readiness expectations. We found that experiencing higher numbers of pre-birth household challenges was related to higher risk of the child not meeting developmental and reading proficiency and having chronic absenteeism. These results suggest that it is imperative support systems for pregnant persons and their families be introduced as soon as possible in pre-natal care routines to address current pre-birth household stressors and prevent future challenges. Such early prevention efforts are needed to ensure the best possible developmental start for children. The pre-natal period, including the health and well-being of the pregnant parent, is crucial for a child’s early developmental success . High prBefore children enter kindergarten, they have already experienced a plethora of early educational experiences that shape their cognitive and socio-emotional development . The ongPrior research demonstrates that both mutable and immutable pre-birth factors are linked to early childhood outcomes. In a predictive risk model for school readiness developed by Camacho and colleagues , the mosAdditional research has connected the accumulation of the above and other types of pre-birth stressors to school readiness. Children born to birthing parents who self-reported experiencing higher levels of pre-natal stress through natural disaster exposure  demonstrated lower general intellectual and language abilities at age 2 , prior tWhile prior research has identified risk factors for not meeting early school readiness related to perinatal stressors, no study to these authors’ knowledge has directly studied the effect of pre-birth household challenges experienced by the birthing parent around pregnancy on multiple measures of school readiness and early academic achievement of the child in a representative statewide longitudinal cohort. To fill this knowledge need, the current study examined whether the number of pre-birth household challenges experienced by the birthing parent predicted the child’s school readiness and early academic achievement, as measured by performance on the Alaska kindergarten developmental profile assessment, third grade reading assessments, and average school attendance.This project used retrospective data from the Alaska 2009–2011 Pregnancy Risk Assessment Monitoring System (PRAMS). Alaska PRAMS is a population-based weighted sample of birthing parents delivering live births in Alaska. Birthing parents are surveyed about factors related to pre-pregnancy, pregnancy, and post-birth experiences. The 2009–2011 PRAMS phase sampled 5578 of the 33,709 eligible Alaska resident births, with oversampling on birthing parents who identified as Alaska Native and who had newborns with low birthweight status (<2500 g). The average weighted response weight was 66%. The complete PRAMS survey methodology is described elsewhere .https://education.alaska.gov/assessments/developmental, accessed on 14 March 2022).Leveraging the Alaska Longitudinal Child Abuse and Neglect Linkage Project (ALCANLink), which links PRAMS survey responses with multiple administrative records  data were used to identify household challenges experienced by the birthing parent during the pre-birth period . Household challenges were identified from Centers for Disease Control and Prevention (CDC) standardized PRAMS questions regarding stressful life events [The 2009–2011 PRAMS cohort . We created a bivariate  ADP variable consistent with DEED’s developmental gold standard definition. The second outcome of interest was the child’s 3rd grade PEAKS ELA score. We created a bivariate PEAKS variable by categorizing Advanced and Proficient (A/P) scores together as “met proficiency” and Below Proficient and Far Below Proficient (BP/FBP) as “did not meet proficiency.” The final outcome of interest was chronic absenteeism, defined as missing at least 10% of the days in which a student was enrolled in school. The child’s school attendance records were averaged across the 2015–2019 school years, and we created a bivariate variable from the resulting average to indicate the presence of chronic absenteeism .a priori covariates in multivariate analyses. It is important to note that the above demographic variables do not represent causal factors or biological predispositions toward certain early childhood outcomes, but likely represent a population experiencing differential distribution of underlying modifiable risk factors .Prior research working with Alaska population data ,32 identUsing the PRAMS post-stratification population weights, we derived the estimated proportion of the birth population reflective of each component of the demographic, exposures, and outcome classifications.We separately examined the bivariate association of each individual pre-birth household challenge component and risk of not meeting PEAKS proficiency, not meeting the 11-goal threshold on ADP, and being chronically absent by calculating risk ratios with “no exposure” as the reference category. We then constructed two generalized linear models with Quasi-Poisson distributions for each outcome of interest to examine the predictive value of pre-birth household challenges on the risks of not meeting school readiness expectations. We used Quasi-Poisson models to account for overdispersion, which relaxes the assumption that the variance is equal to the mean and instead assumes the variance is a linear function of the mean. First, we separately modeled the association of the total number of pre-birth household challenges  on ADP scores, PEAKS scores, and attendance records. Outcome reference categories were A/P (PEAKS), ≥11 goal met (ADP), and ≥90% attendance (attendance), respectively. Secondly, we constructed a multivariate model adjusting for Alaska Native race , maternal education , and maternal age  at the time of the birth to understand the individual association of each component. We used backward elimination stepwise regression to remove nonsignificant covariates to establish our final most parsimonious prediction models. Covariates were retained in the final model if their removal produced a >10% change in the effect estimate.All analyses were conducted using R version 4.1.0 and either the survey  or srvyrThe PRAMS, DEED, and administrative records used in this study were examined retrospectively under routine public health surveillance. Full details on the PRAMS institutional review board (IRB) approval are found in the Institutional Review Board Statement subsection at the end of this article. The remainder of the current study involved the linkage of existing legally authorized administrative databases housed within the Alaska Department of Health and Social Services (DHSS) and DEED. Under these circumstances, IRB approval was not required or sought for the current study.A total of 3549 birthing parents responded to the PRAMS survey, which represents 33,417 (±233) children born in Alaska during 2009–2011. Overall, 69% of the population fell below the developmental profile gold standard . Chronic absenteeism was significantly associated with the birthing parent facing homelessness, being jailed or their partner being jailed, divorce or separation, death of a close friend or family member, increased frequency of arguments with partner or spouse, or drug or alcohol abuse by someone close to the birthing parent during the pre-birth period . Only chThis study investigated the relationships between pre-birth household challenges and children’s school readiness and academic achievement within an Alaska statewide representative birth cohort. Indicators of school readiness and achievement included performance on kindergarten developmental profiles, third grade reading evaluation scores, and average school attendance across five years. We discovered that children born into households that experienced high levels of pre-birth household challenges  were at an increased risk of not meeting future kindergarten developmental goals and experiencing chronic absenteeism compared to children born in households that experienced one or no pre-birth challenges. The level of pre-birth household challenges experienced also had a dose–response relationship with the child’s third grade reading evaluation scores, with higher levels of pre-birth household challenges  being associated with a stepwise increase in the risk of the child not meeting reading proficiency compared to children born in households who experienced 1 or no pre-birth challenges. Considering that early developmental success and school readiness influences future achievement, these results suggest that it is imperative support systems for pregnant persons and their families are introduced as soon as possible in the normal pre-natal care routine to address current pre-birth household stressors and prevent future challenges. Such early prevention efforts are needed to ensure the best possible developmental start for children.Results from this study align with and expand upon prior research highlighting links between individual pre-natal stressor exposure and negative education outcomes of offspring. Consistent with prior research linking economic disadvantage during the pre-natal period with the future children not meeting school readiness expectations ,15,16, iThere are several potential reasons why a parent’s experiences of stress or challenges prior to the birth of their child would affect the child’s eventual school readiness and early academic achievement. Parents experiencing financial-based challenges, such as homelessness or losing their job(s), may not be able to afford basic pre-natal care or, if the challenge continues post-birth, to enroll their child in beneficial early success programs such as preschool. Poor or absent pre-natal care can negatively affect the child’s physical development , which in turn can have negative effects on school readiness . In addiPerson- or family-level prevention efforts and programs may mitigate the effects of pre-birth household challenges on children’s school readiness and early learning development. In the pre-birth window, primary care physicians and related health care providers can screen pregnant individuals for the presence of these household challenges. If significant challenges are present, health care providers can guide the pregnant individual and their family toward public health resources and care coordinators aimed at helping families navigate and connect with financial, social, or other supports services needed. Comprehensive programs that address family-specific challenges are effective. Children whose families participated in a nurse home visiting program from the pre-natal period through the child’s infancy demonstrated higher intellectual functioning and fewer clinical behavioral issues . Other iwww.stressinamerica.org, accessed on 19 February 2022). However, it should be noted there are individual differences in subjective feelings of stress following challenging experiences. This study is also limited by the lack of potential heterogeneity in education outcomes, with over 50% of Alaskan children not meeting the developmental profile standard or third grade reading score proficiency levels. Additional research that accounts for the pre-birth and early childhood experiences are critical, especially considering the length of time between the stressors being experienced in this study and the school outcomes being measured. However, the current study indicates that despite this limitation, initiating and supporting families in some key areas may have impacts on school readiness of offspring years later. Finally, there is a limitation in the calculation of the household challenge total scores used in the bivariate analyses. In constructing these scores, any missing response was counted as a “no” response, which could lead to underestimated counts.There are some limitations to the current study. One limitation is that the study was conducted using an Alaska-based population, making the population demographic distribution different from the general United States population. However, this study can serve as a generalizable platform for other states and areas to adopt when examining pre-birth effects on children’s school readiness. Another limitation is that PRAMS responses are self-reported and may reflect social-desirability bias toward not reporting on sensitive experiences. However, results from the PRAMS survey show 19% of our cohort reported high levels of household challenges (4+), which is comparable to the U.S. percentage of adults who reported feeling high levels of stress over the years during which PRAMS was conducted (24% in 2009 and 2010, 22% in 2011; Future research should focus on ways in which public health can target and mitigate the effects of pre-birth household stressors on children’s school readiness and development and the impact of care coordination to assist families dealing with multiple challenges. Several intervention strategies were mentioned above, but none specifically addressed the relationship between pre-birth household challenges and targeted comprehensive school readiness measures as outcomes of interest. It is important to note that the relationship between pre-birth household stressors and school readiness is likely mediated by several factors, such as parent−child attachment, that were beyond the scope of the current project. These factors and intervention efforts to target them can be addressed by future public health and education researchers.Education readiness and early school performance are strong predictors of future education success and graduation. Education success and graduation are strong predictors of health and economic attainment. Understanding these relationships is critical for developing a cross-sector approach including education, public health, and medical professionals. Thus, while efforts are made to restructure and improve the educational system, we also need to focus on supporting families early on and to recognize them as the first “educational institution” that establishes the learning foundation. This would increase the number of children entering school ready to learn.The current study examined whether the number of pre-birth household challenges experienced by the birthing parent predicted the child’s early school readiness, as measured by kindergarten developmental profiles, third grade reading assessments, and average school attendance. We found that experiencing higher numbers of pre-birth household challenges was related to higher risk of the child not meeting developmental and reading proficiency and having chronic absenteeism. Experiencing homelessness was consistently the highest risk pre-birth challenge for poor school readiness outcomes. These results demonstrate how important the pre-birth window is for providing familial support programs, particularly financial, early on in an individual’s pregnancy to start children on a path to educational success immediately at birth.


Maternal hypothyroidism has been reported to have concerns over neonatal outcomes, not only in the context of neurocognitive development but also in the short term as birth weight and neonatal jaundice.We conducted a cross-sectional retrospective study on 638 cases who delivered live births in the Aga Khan University Hospital after ethical approval. Data were collected on hypothyroid pregnant females who were diagnosed before conception or during their antenatal visits during the year 2008–2016. Neonatal outcomes were noted for birth weight, maturity, and neonatal jaundice, neonatal hypothyroidism, neonatal respiratory distress syndrome, sepsis, hypocalcaemia, congenital anomalies, need for intensive care admission, and neonatal death. Subgroup analysis was performed on the timing of diagnosis of maternal hypothyroidism. Data analysis was performed on Statistical Package for the Social Sciences version 20.0.p ≤ 0.05) and congenital anomalies  with women diagnosed with hypothyroidism before pregnancy.Neonatal jaundice was the most common neonatal outcome (37.6%) in our cohort of 662 live births. Nearly 15% required intensive care unit admission, however, neonatal death was very rare. The most common clinically significant congenital anomalies were cardiovascular defects, whereas Mongolian spots were the commonest congenital condition to report. There is a statistically significant association between low birth weight (OR 1.86, 95% CI 1.0–3.2, Overall, none of the neonates of hypothyroid pregnancies developed congenital hypothyroidism.Cardiovascular defects in these neonates imply extensive screening and monitoring during pregnancy.Low birth weight and congenital anomalies are associated with the timings of diagnosis of hypothyroidism in pregnancy.We report the neonatal outcomes and spectrum of congenital anomalies of hypothyroid pregnancies diagnosed before and during conception for the first time from the region of Pakistan. Overt hypothyroidism occurs in 2–3% of pregnancies  and is wWe therefore aim to report the neonatal outcomes and congenital anomalies of pregnancies affected by hypothyroidism from a tertiary care centre for the first time from the region of Pakistan.The study was approved by The Aga Khan University’s ethical review committee (ERC number: 3977-Med-ERC-15). The study was performed under Helsinki’s ethical principle. To preserve confidentiality, we coded each patient and removed their original identifications. Informed consent was based on hospital consent policy at the time of admission or clinic visit. Moreover, the principle of Justice, Beneficence and Non-Maleficence was maintained and followed throughout the conduct of this study.We conducted a cross-sectional retrospective chart review of neonates of hypothyroid pregnant patients delivered at the Aga Khan University Hospital. Data was collected by trained medical students. It was randomly double-checked and corroborated by the principal investigator. We reviewed the medical record files of neonates of the mothers who were either a known case of hypothyroidism  or were diagnosed during their antenatal visits, during the year 2008–2016. We determined the timing of diagnosis of hypothyroidism (newly diagnosed as well as previously diagnosed) through data recorded by their primary physicians (endocrinologist or obstetricians) in relation to the gestation during the study period. We started the file review of the most recent case. We noted the data from neonatal files including neonatal TSH level of 2nd or 3rd day of life (whichever available).We selected neonates born to mothers who were diagnosed hypothyroid according to the following inclusion criteria: All pregnant women attending endocrine and obstetric clinic who had been diagnosed overt [thyrotropin (TSH) > 2.5 mIU/l and low free tetra-iodothyronine (FT4) OR TSH > 10 mIU/l only] or subclinical  hypothyroidism either before or during pregnancy. Patients were either on levothyroxine replacement or newly started according to their presentation. Maternal characteristics and maternal outcomes of these neonates have already been published in a separate article (MHPO-1) . PregnanBirth Weight: Defined as “Low (<2500 g)”, “Appropriate (2500–4000 g)” and “Large (>4000 g)”. Low APGAR score at 5 min: Assessment of physical health is noted in the one-minute APGAR score (scored from 0 to 10) and then again at five minutes to assess how the baby has responded to any resuscitation attempts. A score below 7 is considered low at 5 min. Respiratory complications : As indicated by the physician’s notes. Neonatal jaundice: Total bilirubin levels above 17 µmol/L checked within 48–72 h of birth. Hypocalcaemia: Serum calcium levels below the reference range defined for neonates in our hospital, that is, 146.2–173.4 µmol/L checked at any time after birth till hospital discharge. NICU  admission: Neonates requiring ICU admission for 24 h or more. Neonatal death: Classified further as early; if occurs in less than 7 days or late; if occurs between 7 and 28 days of life. Congenital anomalies are defined using International Classification of Diseases (ICD-10) codes Q00-Q99 but excludes “inborn errors of metabolism” (codes E70-E90). Conditions of clinical importance like cardiovascular defects, nervous system defects and urogenital anomalies were grouped separately. Rest of the conditions of less clinical importance were also noted and detailed in the results.We divided the gestational age at birth into “extremely preterm (<28)”, “very preterm (28–31)”, “late preterm (32–36)”, “term (37–42)” and “post-term (>42)”. The results of demographic and clinical features are presented as mean ± standard deviation for quantitative variables and number (percentage) for qualitative variables. Proportional differences were assessed by using the Chi-square test or Fisher exact test where appropriate. For the purpose of subgroup analysis, variables of birth weight and gestational age at delivery were recoded dichotomously into low birth weight and premature birth. Unadjusted Odds ratio were calculated for the effect of timing of diagnosis of hypothyroidism on various neonatal outcomes and congenital anomalies. We sought ethical approval from the university’s ethical review committee (ERC number: 3977-Med-ERC-15).Among 708 hypothyroid women recruited, 638 had live births . The meaSupplementary File, Table 1). Neonates with total bilirubin <255 µmol/L, requiring phototherapy were total 82 out of overall 98. Out of these, 25 had appropriate birth weight and born at term and 7 had low birth weight at term. Only one neonate had large birth weight at term whose mother had diabetes as well as gestational hypertension. Now out of those with appropriate birth weight at term, 1 had Glucose-6-Phosphate dehydrogenase (G6PD) deficiency, 1 had Down's syndrome and high TSH of 34.3 mIU/l. Eight mothers of these neonates had gestational diabetes (GDM), 3 had diabetes mellitus (DM), 3 had GH/preeclampsia and 2 had chronic hypertension. One mother also had past obstetric history of Trisomy 18. Since none of our neonates were diagnosed with congenital hypothyroidism in the short-term (within a month), we did not analyse the association of this level of hyperbilirubinemia with neonatal TSH levels. Although majority of the respiratory complications were not detailed in the charts, transient tachypnoea of newborn accounted for the most (0.9%) of them, and only three cases (0.5%) required ventilatory support. Sepsis was found amongst 6.5% of the neonates, out of which 1.2% were presumed to have sepsis, with only one case each for bacterial pneumonia, acute gastroenteritis, urosepsis, and meningitis. Out of 662 cases, 12% required NICU admission, but neonatal death was 1.6%. Amongst our cohort , 92% of the neonates had birth weight appropriate for gestational age at term; however, 56% were small for gestational age at even late preterm. There were five cases of large for gestational age, with only one of them being born to a diabetic mother who also had gestational hypertension. The median TSH of the neonates was 4.18 mIU/l at 48 h of birth and only nine neonates had a TSH of more than 20 mIU/l. The highest TSH recorded was 57.6 mIU/l.Supplementary File, Table 3). So, major congenital anomalies were classified into cardiovascular defects, nervous system defects and urogenital anomalies and accounted for 10.3% only. Overall, 48 neonates had two congenital defects and nine of 662 had three defects. Mongolian spots were the commonest congenital condition, but cardiovascular defects were the highest clinically significant congenital anomaly. Out of two cases of Down syndrome, one had a TSH of 34.3 uIU/L and the other had 11.0 uIU/L. One child was found to have learning difficulty at 6.5 years of age.Twenty-two percent (147/662) of the neonates had some forms of congenital anomalies, defects and conditions   with only 29 (4.4%) cases having no record. Upon subgroup analysis , although the sample size was relatively small [Neonates of hypothyroid mothers in our hospital had similar rate (12%) of NICU admission, as described in the international literature. A recent Finnish study reported 14.4% NICU admissions in a large cohort of 16,364 hypothyroid mothers which was only slightly higher (13%) than those mothers who were consistently using levothyroxine replacement amongst them  [ly small . We had ly small . Overallly small ,32.p ≤0.05) of congenital anomalies in the neonates of women diagnosed hypothyroid prior to pregnancy as compared to those diagnosed during pregnancy. A recent Spanish study showed 53.7% women were diagnosed hypothyroidism before pregnancy [A recent cross-sectional study from Bangladesh has reported maternal hypothyroidism to be present in 5.12% of babies born with congenital anomalies . Studiesregnancy , and anoregnancy . But nonp ≤0.05) [Most studies reported major congenital anomalies as a perinatal outcome of hypothyroid mothers but did not describe the details of the anomalies . Among tp ≤0.05) . We alsop ≤0.05) in hypothyroid women diagnosed before pregnancy. This is in contrast to a large Danish registry, which reported increased risk of high birth weight associated with hypothyroidism in mothers  rather than low birth weight which was actually associated with hyperthyroidism, even after adjusting for potential confounders [p ≤ 0.05) as reported in our study [Neonates in our cohort had significant association with low birth weight  and sepsis  with the timing of diagnosis of maternal hypothyroidism, with higher chance in those diagnosed prior to pregnancy. Premature birth is a known complication of maternal hypothyroidism as reported in many studies [Our study also observed marginally significant association of premature birth (OR 1.76, CI 1.00–3.09,  studies ,48,49, h studies . Differe studies . When reIn conclusion, our study described hyperbilirubinaemia as the most common neonatal outcome and cardiovascular defects as the most common major congenital anomaly. There is significant association of congenital anomalies and low birth weight with the timing of diagnosis of hypothyroidism in women. Moreover, premature birth and neonatal sepsis is also associated in this respect with marginal significance. Our study also has a limitation with regards to selection bias. We observed higher congenital anomalies and conditions in neonates born to women diagnosed during pregnancy. As this is a single hospital study, women with known hypothyroidism could have been more likely referred into our hospital for the high-risk obstetrical care with lessor congenital defects. Despite of all the limitations associated with a retrospective study design, this is the first study reporting neonatal outcomes and congenital anomalies of hypothyroid pregnancies from Pakistan. We propose to do multi-centre prospective studies to determine the association of congenital anomalies in neonates of uncontrolled hypothyroid mothers either diagnosed before or during pregnancy with adequate levothyroxine replacement. We also recommend to perform case–control studies to determine the effects of hypothyroid pregnancies on the neonatal outcomes, whether diagnosed before pregnancy or anytime during gestation.Click here for additional data file.


Case Presentation. A 36-year-old woman was diagnosed with an allantoic cyst during the first trimester. The allantoic cyst underwent spontaneous rupture during the 32nd week of gestation, and an omphalocele developed secondary to the cyst's rupture. Two days after birth, the peritoneum covering intestinal loops broke spontaneously and the newborn underwent successful urgent surgery.  We report the first case in which the onset of omphalocele was after the spontaneous rupture of an allantoic cyst. We hypothesize a causal link between the spontaneous rupture of the cyst and the herniation of the viscera.  This case may suggest that the relative benignity of the allantoid cysts may recommend a close ultrasound follow-up in order to identify the onset of any complications, as a late third trimester onset of omphalocele. Prenatal diagnosis of such complications may allow multidisciplinary management of the pregnancy with planned cesarean section, prenatal pediatric surgery consultation, and neonatal surgery. In the early stages of embryogenesis, the bladder's dome communicates with the allantoid, a structure located inside the umbilical cord, through the urachus. Patent urachus in the prenatal period has an estimated incidence of 3 cases out of 1,000,000, three times more frequent in males . During Here, to the best of our knowledge, we report the first case of omphalocele after spontaneous rupture of the allantoic cyst, highlighting the need for a close follow-up of the cystic pathologies of the umbilical cord in order to exclude the onset of this complication.A 36-year-old woman at her third pregnancy was referred to our third level prenatal ultrasound diagnostic center to undergo the first trimester screening exam during the 13th week of gestation. Fetal anatomy was regular, except for the presence of an anomalous umbilical cord cyst. It had a maximum size of 18 mm and regular margins and was adjacent to the abdominal wall. This cyst was avascular, had no internal septa or vegetations, and was within the umbilical cord, embraced by the two umbilical arteries, in communication with the bladder. Its size varied during the ultrasound examination, which lasted several hours, configuring the diagnostic suspicion of an allantoic cyst with patent urachus .Amniocentesis was performed to rule out chromosomal or genomic anomalies. The traditional karyotype study and array-based Comparative Genomic Hybridization- (CGH-) array showed no alterations related to an allantoic cyst, such as trisomies 13 and 18. Subsequently, the patient underwent a second trimester screening exam at our center during the 21st week of gestation. The biometric values appeared overall greater than a week compared to gestational age, and the allantoic cyst showed an increased size, with a maximum diameter of 46 mm; the remaining anatomy explored by ultrasound was regular . In the Gestation was carried out regularly, and delivery was by elective cesarean section in a facility equipped with neonatal intensive care and pediatric surgery unit. Two days after birth, the peritoneum covering intestinal loops broke spontaneously, and the newborn underwent successful urgent surgery . SubsequThe urogenital system derives embryologically from the intermediate mesoderm; in the beginning, the excretory ducts of both apparatuses  end inside a common cavity, called the cloaca . BetweenDuring the first trimester screening examination, a cystic anomaly of the umbilical cord could come across the sonographer. These anomalies are differentiated into true cysts, located near the abdominal wall, and pseudocysts, generally more distal and frequent . True cyClinically, these anomalies are most often asymptomatic; however, they can lead to urinary tract infections and kidney reflux hydronephrosis .The natural history of these cysts during the prenatal period is their progressive enlargement and, consequently, the rupture. It occurs more often between the 22nd and 32nd weeks of gestation .This event leads to a free communication of the bladder with the amniotic cavity and consequently its failure of repletion, with preserved amniotic fluid , 10.The prevalence of cord cysts during the first trimester screening is 3.4% , some reHere, we report the first case in which the onset of omphalocele was after the spontaneous rupture of the allantoic cyst. In particular, the diagnosis was made at 12 weeks of pregnancy. The ultrasound follow-up revealed a progressive increase in size and its subsequent rupture after the 21st week. Lastly, during the third trimester, our case was characterized by a late development of omphalocele. We hypothesize a causal link between the spontaneous rupture of the cyst and the herniation of the viscera, due to the creation of a locus minoris resistentiae of the abdominal wall, hernial gate of the omphalocele. Therefore, besides reported associations with chromosomal and anatomical abnormalities, this report highlights the necessity of a close follow-up of allantoic cysts due to the possible development of omphalocele. Early diagnosis of such complications is crucial for pregnancy management. In fact, although barring any fetal complications, preterm delivery is not recommended in neonates with omphalocele ; cesareaThe anomalies of the urachus-allantoic system usually occur during the first trimester of pregnancy. Once the presence of associated anatomical or genetic abnormalities is excluded, the relative benignity of the allantoid cysts may recommend a close ultrasound follow-up in order to identify the onset of any complications, as a late third trimester onset of omphalocele. Prenatal diagnosis of such complications may allow multidisciplinary management of the pregnancy with planned cesarean section, prenatal pediatric surgery consultation, computed tomography scan at birth for the evaluation of omphalocele or any bladder lesions, and neonatal surgery.