Streptomyces, but the regulatory roles of the Streptomyces AdpA family on the biosynthesis of the natural product ε-poly-l-lysine (ε-PL) remain unidentified, and few studies have focused on increasing the production of ε-PL by manipulating transcription factors in Streptomyces.AdpA is a global regulator of morphological differentiation and secondary metabolism in Streptomycesalbulus NK660 by heterologously expressing adpA from S.neyagawaensis NRRLB-3092 (adpASn). First, we identified a novel AdpA homolog named AdpASa in S.albulus NK660 and characterized its function as an activator of ε-PL biosynthesis and morphological differentiation. Subsequently, four heterologous AdpA homologs were selected to investigate their phylogenetic relationships and regulatory roles in S.albulus, and AdpASn was demonstrated to have the strongest ability to promote both ε-PL production and sporulation among these five AdpA proteins. The ε-PL yield of S.albulus heterologously expressing adpASn was approximately 3.6-fold higher than that of the control strain. Finally, we clarified the mechanism of AdpASn in enhancing ε-PL biosynthesis and its effect on ε-PL polymerization degree using real-time quantitative PCR, microscale thermophoresis and MALDI-TOF–MS. AdpASn was purified, and its seven direct targets, zwf, tal, pyk2, pta, ack, pepc and a transketolase gene (DC74_2409), were identified, suggesting that AdpASn may cause the redistribution of metabolic flux in central metabolism pathways, which subsequently provides more carbon skeletons and ATP for ε-PL biosynthesis in S.albulus.In this study, we revealed the regulatory roles of different AdpA homologs in ε-PL biosynthesis and morphological differentiation and effectively promoted ε-PL production and sporulation in Streptomyces AdpA homologs in ε-PL biosynthesis and their effects on morphological differentiation and reported for the first time that AdpASn promotes ε-PL biosynthesis by affecting the transcription of its target genes in central metabolism pathways. These findings supply valuable insights into the regulatory roles of the Streptomyces AdpA family on ε-PL biosynthesis and morphological differentiation and suggest that AdpASn may be an effective global regulator for enhanced production of ε-PL and other valuable secondary metabolites in Streptomyces.Here, we characterized the positive regulatory roles of The online version contains supplementary material available at 10.1186/s12934-022-01785-6. Streptomyces species are the ability to produce multitudinous valuable secondary metabolites possessing diverse biological activities and a complex life cycle, including the generation of vegetative mycelium, aerial mycelium and spores during development [Two notable characteristics of elopment –3. The pelopment .Streptomyces, is able to amplify the A-factor signal and is a global regulator of morphological differentiation and secondary metabolism in Streptomyces [Actinobacteria [Streptomyces species [Streptomyces species [Streptomycesgriseus, such as ssgA encoding a protein that forcefully influences septum formation, σAdsA, the ECF sigma factor required for aerial mycelium formation, and amfR, which encodes a protein essential for aerial mycelium formation, and AdpA can also regulate chromosome replication by binding to the region close to OriC [Sx from Streptomycesxiamenensis 318 was reported to negatively regulate morphological differentiation [adpA is subject to a noteworthy regulation of the translation level for the UUA codon within the adpA transcript, in addition to being controlled by the repressor protein ArpA, the hormone-like molecule and asRNA [Actinobacteria and is present in approximately 2–3% of gene transcripts in any one Streptomyces, typically, based on the information from four sequenced genomes [LeuUAA that is capable of reading the UUA codon efficiently is only encoded by bldA, and its delayed occurrence limits the expression of TTA-containing genes during the cell cycle [The AraC family transcription factor known as AdpA is universally present in ptomyces –10. AdpAbacteria , 11. It  species , 13. Adp species –18. On t to OriC , 19, 20.ntiation , 21, 22 ntiation –27 and w genomes , 29. Thell cycle , 28, 30.l-lysine (ε-PL) is a kind of amino acid homopolymer composed of 25–35 l-lysine residues with isopeptide bonds between the α-carboxyl and ε-amino groups and is mainly produced by the Streptomycetaceae family [The extracellular metabolite ε-poly-e family –34. ε-PLe family , 35. Thee family , 37. More family , 39.vgb (the Vitreoscilla hemoglobin gene) [amtB (the ammonium transporter gene) [pls (the ε-PL synthetase gene) [dapA (the dihydrodipicolinate synthase gene) [Streptomyces, but few studies have focused on increasing the production of ε-PL effectively by manipulating transcription factors in Streptomyces thus far.Although ε-PL has been industrially produced, the low fermentation efficiency and high cost still need to be resolved . The nutin gene) , 53, amter gene) , pls (thse gene) , 55, andse gene)  have beeStreptomyces AdpA homologs in ε-PL biosynthesis and morphological differentiation and effectively promoted ε-PL production and sporulation in S.albulus NK660 by sieving out AdpASn from five AdpA homologs, including the novel AdpA homolog (AdpASa) identified here. Subsequently, we clarified the mechanism by which AdpASn affected ε-PL biosynthesis and its effect on the ε-PL polymerization degree in S.albulus. Furthermore, we identified seven target genes directly regulated by AdpASn in the central metabolic pathways and acetate metabolism pathway of S.albulus NK660. We hope this study will provide a reference for the enhanced production of ε-PL and other valuable secondary metabolites in Streptomyces.In this work, we revealed the regulatory roles of S.albulus NK660 and its derivatives were as described previously [Escherichiacoli DH5α and E.coli BL21 (DE3) were used as the cloning host and the expression host, respectively. We used E.coli WM6026 [S.albulus NK660. E.coli strains were grown in Luria–Bertani medium at 37 °C. When necessary, 30 μg/mL apramycin, 50 μg/mL kanamycin and 19 μg/mL diaminopimelic acid were added.All the bacterial strains and plasmids used in this study are listed in Table eviously . Escherii WM6026  as a nonadpASa, the DNA fragment containing the adpASa encoding region was amplified from the genome DNA of S.albulus NK660 with the primer pair adpA-F/-R. This DNA fragment was joined with the synthesized promoter PermE*  by overlap-PCR. Under the catalysis of Exnase II , the generated DNA fragment was ligated into the vector pSET152 [adpASa overexpression vector pSET152-adpASa, which was then transformed into S.albulus NK660 to construct the adpASa overexpression strain S.albulus NKA. To construct the heterogeneous expression vectors, DNA fragments containing the promoter PermE* and one of the genes encoding AdpASd (AFX97763.1), AdpA-SH (WP_018531726.1), AdpASn (WP_055538474.1) or AdpA-C (WP_007264197.1) were synthesized . Similarly, the obtained DNA fragments were ligated into the integrative vector pSET152 digested with XbaI by Exnase II , successively generating the vectors pSET152-adpASd, pSET152-adpA-SH, pSET152-adpASn and pSET152-adpA-C, which were then transformed into S.albulus NK660 to construct the heterogenous overexpression strains S.albulus SDA, S.albulus SHA, S.albulus SNA and S.albulus SCA. Furthermore, to investigate the effect of pSET152, the empty plasmid pSET152 was also integrated into the S.albulus NK660 genome, generating S.albulus SET. All the constructed mutants were verified by PCR and DNA sequencing with the primer pair SET-F/-R. DNA manipulations were carried out using standard procedures for Streptomyces and E.coli [For overexpression of  pSET152  that was E.coli , 60.S.albulus NK660 and its derivatives after 29 h of growth in M3G medium using a SPARKeasy Improved Bacteria RNA kit according to the manufacturer’s protocol . Isolated RNA was reverse transcribed using HiScript® II Reverse Transcriptase . For RT–PCR, the Genome DNA clearance column -treated RNA samples without reverse transcription were used as the negative controls to verify the absence of DNA contamination. The transcription levels of genes were determined by RT–qPCR  agar plates at 30 °C were observed by scanning electron microscopy (SEM). For specimen preparation, coverslips were embedded in agar inoculated with the strains at an angle and lifted out gently after 2 or 3 days of incubation for follow-up operation. Samples were fixed with 2.5% glutaraldehyde solution overnight, washed three times with phosphate buffer, subsequently dehydrated by an ethanol concentration gradient , freeze-dried , coated in gold, and then examined by SEM .albulus NK660 and its derivatives were inoculated in M3G medium at 30 °C with shaking (180 rpm) for 24 h as seed cultures, and then 5% (v/v) of the inocula were inoculated into M3G medium and cultured for 5 days at 30 °C with shaking (180 rpm). Precipitates of samples were harvested at specific time points and washed twice with distilled water. Then, they were dried by a freeze dryer  and subsequently weighed for the measurement of dry cell weights (DCWs). The supernatant of each sample was harvested at certain time points and used to determine the concentration of ε-PL on the basis of the procedures described by Itzhaki [ Itzhaki . All culzwf,pyk2 and pepc were amplified and labeled using the FAM-labeled primer pairs zwf-F/-R, pyk2-F/-R and pepc-F/-R, and they were subsequently recovered by the Multifunctional DNA Purification and Recovery Kit . The purified His-tagged AdpASn  was concentrated by centrifugal filters . For each dissociation constant measurement, the 32 μM initial concentration of AdpASn underwent a 16-step serial twofold dilution with PBS buffer, and the 16 diluted protein solutions were mixed with equal labeled DNA fragments. The mixtures were incubated for 30 min at 25 °C and transferred to standard Monolith NT.115 capillaries for follow-up measurements. The experiments were run at 40% excitation with high microscale thermophoresis (MST) power at room temperature on a Monolith NT.115 instrument  [The DNA fragments corresponding to the promoter regions of genes Germany) . The datThe operations to separate and purify ε-PL from fermentation cultures were performed as described previously . The purhttps://blast.ncbi.nlm.nih.gov/Blast.cgi). The multiple sequence alignment of the AdpA homologs was executed using the online available tool CLUSTALW  and analyzed by ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Phylogenetic analyses were conducted in MEGA-X using the maximum parsimony method [The homologous sequence database search was performed using BLASTp  shared 80% and 89% amino acid identities with the AdpA homologs of S.griseus and S.coelicolor, respectively, and designated it AdpASa. AdpASa was found between the upstream gene encoding a putative universal stress protein and the downstream gene ornA that encodes a putative oligoribonuclease in S.albulus NK660 based on computer-assisted analysis  separately. On the one hand, when cultured for 100 h, S.albulus NKA  separately had a 1.5-fold and 2.1-fold increase in ε-PL production and specific ε-PL formation rates compared with S.albulus SET   required for S.albulus SET and S.albulus NKA to obtain their own peak ε-PL production and the fact that the overexpression of adpASa caused an earlier time to start producing ε-PL , Streptomyces sp. HmicA12 (adpA-SH), Streptomycesneyagawaensis NRRLB-3092 (adpASn) and Streptomyces sp. C (adpA-C). Similar to the UUA codon of adpASg, the UUA codon of adpASd is located in the ʻclassicalʼ position between the ThiJ/PfpI/DJ-1-like dimerization domain and HTH domains , 65.2% (0.38 g/L) and 34.8% (0.31 g/L), respectively , pentose phosphate pathway (PPP), anaplerotic metabolic pathway, tricarboxylic acid (TCA) cycle, diaminopimelic acid pathway (DAP) and ε-PL assembly in Streptomyces  in S.albulus SNA was 20.8% lower than that in S.albulus NK660  links glycolysis, gluconeogenesis and the TCA cycle and plays a central part in the generation of adenosine triphosphate (ATP) and precursors for specialized metabolites such as acetyl-CoA, amino acids, and organic acids [pyk1 and pyk2 in S.albulus NK660. As shown in Fig. pyk2 in S.albulus SNA was 1.9-fold that in S.albulus NK660, while no distinct difference was found in the transcription level of pyk1 between S.albulus SNA and S.albulus NK660, indicating that a larger amount of the direct precursor for acetyl-CoA and more ATP might be produced by the heterologous expression of the adpAsn gene. As one of the enzymes included in the phosphoenolpyruvate-pyruvate-oxaloacetate node, which is a major branch in central carbon metabolism, phosphoenolpyruvate carboxylase (PEPC) is capable of catalyzing the carboxylation of phosphoenolpyruvate to generate oxaloacetate for the TCA cycle and other metabolic processes necessary for growth [pepc in S.albulus SNA was 2.8-fold higher than that in S.albulus NK660  are cotranscribed with zwf, and they are all under the control of the promoter located in front of the gene encoding transketolase. The upstream genes encoding phosphate acetyltransferase  and acetate kinase  are cotranscribed with pyk2, and these three genes share a common promoter  of 5.5 ± 0.3 µM, 4.9 ± 0.5 µM and 8.4 ± 0.9 µM, respectively , AdpA-SH (91.59%), AdpASn (49.07%) and AdpA-C (88.83%). The amino acid sequences of the ThiJ/PfpI/DJ-1-like dimerization domain and AraC/XylS-type DNA-binding domain possess identities among the AdpA proteins studied in this paper, and these five AdpA proteins all retain the arginine residues at their positions corresponding to Arg-262 and Arg-266 that directly recognize the target DNA sequences in AdpASg  encoding a putative pirin-like protein caused by the integration of pSET152 into the putative ϕC31 attB site within this gene. It was reported that some phenotypic changes and a significant drop in spiramycin production were found in Streptomycesambofaciens ATCC 23,877 because the integration of the ϕC31-based integrative plasmid caused the inactivation of the gene SAM23877_RS18305 (pirA) encoding pirin [In general, morphological differentiation and secondary metabolism in nditions . It was is paper . TherefopASn 49.0% and Adpl growth . Conside species –82, the dpA-C 88.%. The amng pirin .Sn affects ε-PL biosynthesis in S.albulus, we studied the influences of AdpASn on the key genes involved in ε-PL biosynthesis and the degree of ε-PL polymerization. The results of RT–qPCR and MST indicate that AdpASn activates the transcription of pyk2 and pepc but represses the transcription of zwf by directly binding to their promoter regions with similar dissociation constants. ε-PL biosynthesis is involved in EMP, PPP, the anaplerotic metabolic pathway, the TCA cycle, DAP and ε-PL assembly in Streptomyces  with no influence on mycelium growth in Streptomyceslividans [Streptomycesrimosus M4018 strain obtained a higher production of OTC by the disruption of zwf1 or zwf2 [Streptomyces, which indicates that the influence of AdpASn on the ratio of carbon flux between the PPP and EMP might be one of the reasons for the enhanced ε-PL biosynthesis. Second, the effect of AdpASn on the transcription level of pyk2  may cause more provision of carbon skeletons for the TCA cycle and a higher level of ATP for the regulation of Pls catalytic function, which may contribute to increasing ε-PL production. It was confirmed that the pyk2::Tn5062 transposon insertion mutant showed reduced polyketide coelimycin and Red yields in S.coelicolor, which indicates that the perturbation of central metabolism by adjusting the expression of genes encoding PK affects specialized metabolite biosynthesis [Sn may prompt the replenishment of more TCA cycle intermediates for L-lysine biosynthesis and cause the generation of more carbon skeletons and ATP for ε-PL biosynthesis by directly activating the transcription of pepc. Finally, combined with the fact that the transcription levels of ask, pls and hrdD showed no significant differences between S.albulus SNA and S.albulus NK660  and acetate kinase (ack) are cotranscribed with pyk2, AdpASn directly activates the transcription of pta and ack as well. The enzymes phosphate acetyltransferase and acetate kinase form the key pathway that is responsible for the interconversion between ADP, acetyl-CoA, orthophosphate and ATP, acetate, CoA in many bacteria [tal) are cotranscribed with zwf, AdpASn directly represses the transcription of the upstream gene encoding transketolase and tal. The nonoxidative branch of the pentose phosphate pathway in carbohydrate metabolism can provide precursors for the synthesis of aromatic amino acids, nucleic acids and fatty acids [Sn, which indicates that the direct regulation of AdpASn involves both the oxidative part and nonoxidative branch of the pentose phosphate pathway, glycolytic pathway, anaplerotic metabolic pathway and Ack-Pta pathway of acetate metabolism; thus, AdpASn plays an important role in the central metabolism and acetate metabolism pathways. Because of the importance of AdpASn in the regulation of central metabolism and acetate metabolism pathways and the fact that AdpASn promotes ε-PL biosynthesis mainly by regulating the transcription of the target genes in primary metabolism pathways but not the key genes (ask and pls) in the diaminopimelic acid pathway and ε-PL assembly, we hope this study will provide a reference for enhancing the production of ε-PL and other valuable secondary metabolites in Streptomyces.Moreover, we identified seven target genes in which AdpAays Fig. . Becausebacteria , 91. It bacteria , 92, 93,bacteria , 95. Becty acids . As impoty acids , 97. ConStreptomyces AdpA homologs and effectively promoted ε-PL biosynthesis and morphological differentiation in S.albulus by selecting AdpASn from five AdpA homologs to be heterologously expressed. In addition, we clarified the mechanism by which AdpASn affected ε-PL biosynthesis and its effect on the ε-PL polymerization degree in S.albulus and indicated that AdpASn promotes ε-PL biosynthesis mainly by regulating the transcription of the target genes  in primary metabolism pathways. Thereafter, we revealed the importance of AdpASn in the regulation of central metabolism and acetate metabolism pathways by the identification of its seven target genes, which provides a reference that AdpASn may possess the ability to promote the production of other valuable secondary metabolites in addition to ε-PL. These findings provide valuable insights into the regulatory roles of the Streptomyces AdpA family on ε-PL biosynthesis and morphological differentiation and supply an efficient strategy to promote the production of ε-PL and other valuable secondary metabolites in Streptomyces.In summary, we characterized the positive regulatory effects in ε-PL biosynthesis and diversified regulation of the morphological differentiation of Additional file 1: Figure S1. The upstream and downstream genes of different adpA genes and phylogenetic analysis of AdpA homologs. (A) Different adpA genes and their upstream and downstream genes in three Streptomyces species. The sequence identities between the encoded proteins of homologous genes with same color are indicated. (B) Phylogenetic analysis of the novel AdpASa, four heterologous AdpA homologs AdpASd, AdpA-SH, AdpASn, AdpA-C with NCBI BLASTP hits and well-studied ones. AdpASa is marked with the red hollow circle, while the four heterologous AdpA homologs AdpASd, AdpA-SH, AdpASn, AdpA-C are all marked with the blue hollow diamond. Different clades or subclades are indicated with different colors, and some bootstrap values of the phylogenetic tree are also displayed. Figure S2. Construction of S.albulus NKA and effect of AdpASa on specific ɛ-PL formation rate. (A) Schematic method for overexpressing adpASa in S.albulus. (B) Confirmation of the integration of adpASa gene into the genome of S.albulus by PCR. Lane M, DNA marker III; Lanes1-6, amplification products using primers SET-F/SET-R with gDNA from S.albulus SET, plasmid pSET152 DNA, gDNA from S.albulus NKA, plasmid pSET152-adpASa DNA, gDNA from S.albulus NK660 and ddH2O as templates, respectively. (C) Transcription levels of adpASa gene in S.albulus NK660 and S.albulus NKA by RT-qPCR analysis. ***P < 0.001 (Student's t-test). Error bars stand for the SD for three biological replicates. (D) Specific formation rates of ɛ-PL in S.albulus NKA and control strains  cultured in fermentation medium for 100 h. Error bars stand for the SD for three biological replicates. (E) Phenotypes of S.albulus NK660 and S.albulus SET grown on MSF agar plates for 4 days. Figure S3. Construction of S.albulus SDA, S.albulus SHA, S.albulus SNA and S.albulus SCA. (A) Schematic method for expressing heterologous adpA genes in S.albulus. The blue arrow stand for heterologous adpA genes . (B) Confirmation of the construction of mutants expressing heterologous adpA genes by PCR. Lane M, DNA marker III; Lanes1, 3, 5, 7, 9, 11, amplification products using gDNA from S.albulus SET, S.albulus SDA, S.albulus SHA, S.albulus SNA, S.albulus SCA and S.albulus NK660 as templates, respectively; Lanes 2, 4, 6, 8, 10, 12, amplification products using plasmid pSET152, pSET152-adpASd, pSET152-adpA-SH, pSET152-adpASn, pSET152-adpA-C and ddH2O as templates, respectively. And all the amplification products use the primers SET-F/SET-R. (C) RT-PCR results among S.albulus NK660, S.albulus SET and the four heterologous adpA genes expression mutants. Lane M, DNA marker III; Lanes D1-D10, amplification products using primers DadpA-F/DadpA-R with RNA from S.albulus NK660, cDNA from S.albulus NK660, RNA from S.albulus SET, cDNA from S.albulus SET, RNA from S.albulus SDA, cDNA from S.albulus SDA, gDNA from S.albulus SDA and ddH2O as templates, respectively. Lanes H1-H10, Lanes N1-N10 and Lanes C1-C10 are the amplification products respectively using primers HadpA-F2/HadpA-R, NadpA-BF/NadpA-BCR and CadpA-F/CadpA-R with the same templates and template orders as Lanes D1-D10. Table S1. Primers used in this study. Table S2. Accession number and length of the whole candidates used for phylogenetic analysis.


We did not find a statistically significant correlation between Fenton or IGW-21 z-scores and scalar data of NEPSY-II subtasks. Conclusion: In our series, neonatal growth influence on neuropsychological tests at the beginning of primary school does not seem robust, except for RIST test. New EUGR criteria do not improve the predictive ability of the old ones.Background: Controversy between short-term neonatal growth of very low birth-weight preterm (VLBW) and neurodevelopment may be affected by criteria changes of extrauterine growth restriction (EUGR). Objective: to determine if new EUGR criteria imply modifications in the relationship between old criteria and results of neuropsychological tests in preterm children. Patients and methods: 87 VLBW at 5–7 years of age were studied. Neuropsychological assessment included RIST test  and NEPSY-II  tests. The relationships between these tests and the different growth parameters were analyzed. Results: RIST index was correlated with z-score Fenton’s weight ( Brain development of newborns with very low birth weight (VLBW) depends largely on correct nutritional intake during the immediate postnatal period. Nutrition and, therefore, body and brain growth in the first weeks of life may be of great importance for the neuropsychological development of these children .Postnatal growth rate of VLBW and its relationship with neuropsychological development has been studied by many authors with different conclusions ,6,7,8,9.Nevertheless, several studies show that the neuropsychological development of preterm infants can affect functions, for instance, memory ,14, visuWhen we analyse growth of premature infants in the first weeks of life, two important questions arise: what is the ideal growth of these infants and what measure is the most appropriate to verify it? ,20,21.Since 1977, the American Academy of Pediatrics recommended using foetal growth as an ideal postnatal growth in preterm infants . For thiWith these curves, neonates are classified in percentiles or z-scores, allowing to label neonates as small (IUGR), normal, or large for their GA, in weight, length, and cranial perimeter. Additionally, such curves are used to label children’s growth from birth, during their stay in neonatal units, until 28 days, 36 weeks of GA or discharge, in order to assess so-called extrauterine growth restriction (EUGR).However, in recent years, several changes in these concepts have been postulated. It is advocated not to use curves created with data from cross-sections at birth, but rather ones that define how a healthy premature infant should grow, without prenatal or postnatal complications throughout their first months of life ,26. ThisArticles have been published comparing both strategies, recalculating percentages of IUGR preterm infants (weight below the 10th percentile at birth) and static EUGR , differing according to graphs/standards used .Another change that arises with this publication is whether we should call EUGR to all neonates who are below the 10th percentile at discharge or at 36 weeks of corrected GA, or only those who are born above this percentile (non-IUGR) and are below it at discharge, what some authors call true EUGR . To end In any case, debate remains open and discussion on the classification of premature children, with static or dynamic criteria, creates new fronts in the effect of growth on the neuropsychological development of these children .For all the above, our aim was to analyse neuropsychological functions at 5–7 years, (beginning of primary education) in a cohort of children under 1500 g at birth, and to determine if new EUGR criteria imply modifications in the relationship between old criteria and results of neuropsychological tests in preterms. That is, if new criteria are more precise than the previous ones in labelling those cases with the lowest neuropsychological outcomes at 5–7 years of age.An observational study was conducted, including 87 VLBW (less than 1500 g) who were born in the Neonatology Service of the Central University Hospital of Asturias  between 1 January 2009 and 1 January 2012.In this period, 181 initial cases were born in our hospital, of which 34 died before discharge and 147 survivors were invited to participate in this study, responding affirmatively 87 cases . All of them were set a date for a clinical assessment a neuropsychological study in our hospital between February 2016 and 6 May 2017. Comparing the studied cases (87) and those that were not studied (60 lost cases), we did not find statistically significant differences neither in neonatal variables, nor in neurological evolution at medium-long term .In the 87 studied cases, the assessment protocol was completed with anthropometry at birth, anthropometry at discharge from neonatal admission, and anthropometry at 2 years of chronological age corrected for GA, all of them obtained from medical records. The birth and discharge data were analysed with Fenton and IGW-21 graphs and standards, while 2 years data was analysed with WHO graphs.We have considered as IUGR those cases born below the 10th percentile for GA. We have defined static  EUGR as cases that were below the 10th percentile for GA at neonatal discharge. We have considered as true EUGR the cases that, have being born above the 10th percentile (not IUGR), were discharged below the 10th percentile for their GA. Finally, we have defined dynamic  EUGR those cases that lost more than 1 or more than 2 SD from birth to discharge, regardless of their initial or final percentiles.During the clinical assessment (study visit), a neuropsychological evaluation was performed by psychologists, including an estimation of IQ and an assessment of the inhibitory control capacity, the verbal and visuospatial memory and the visuospatial abilities, using the RIST test and four task from NEPSY-II battery.The RIST  provides an IQ estimation score for children and adults between 3 and 94 years old by employing two tasks: “guess what” for the verbal score and “odd-item” for the non-verbal score. On “guess what” , the examiner reads some definitions, and the child has to answer with the appropriate word. On “odd-item” , the child is shown several pictures, and s/he has to point to the different or incongruent one. Its reliability coefficient varies between 0.89 and 0.91 depending on the age of child and the task .The NEPSY II battery ,33 allowThe “inhibition” subtest was used to measure inhibitory control. In this task, the child is shown several black and white geometric shapes (circle and square) or black and white arrows, and s/he has to say the correct (denomination) and opposite (inhibition) shape or direction of the arrow.The “memory for designs” subtest was employed to assess short- and long-term visual and spatial memory. In the short-term assessment, the child is shown a grid containing from 4 to 10 meaningless picture cards per page. Then, the examiner removes the page, and the child must select the designs s/he has seen from a set of target cards and distractors and place them on a grid in the same location s/he was previously shown. This procedure is repeated 15–25 min later for the long-term evaluation.The “memory for names” subtest was used for short- and long-term verbal memory evaluation. To assess short-term memory, the child is shown 6–8 cards containing pictures of children while the examiner reads the name of each child on the cards. Then, the cards are shown again, and the examinee is asked to remember the name of the child on each card. To assess long-term memory, the same test is applied after about 25–35 min.The “route finding” subtest was employed for directionality, spatial relation, and map interpretation assessment. The child is shown a schematic map indicating how to reach a house. The child is then asked to find the house on a larger map with more roads and houses available. For the NEPSY-II battery, scalar scores from all these tests are reported.t test. We used non-parametric test for Birth Weight , and for neuropsychological test comparations, because these tests were value like scaled scores (Mann–Whitney U test). Comparisons of percentages were made with the Chi-square test (using Fisher’s exact test if expected were less than 5). To establish correlations between z-scores, Spearman Rho test was used. Finally, Kappa concordance test was used to compare Fenton and IWG-21 classifications. Statistical significance was maintained throughout the study for p-value < 0.05.Variables with normal distribution were compared with Student’s Parents were informed about the aims of the study and provided their written informed consent before the study began. The study was conducted in accordance with the Helsinki declaration for research in human subjects, and it was approved by the regional ethics committee .Anthropometric data at birth, at discharge from neonatal admission, and at 2 years of age are shown in Median (interquartile range) IQ for RIST was 91 (78.7–101). Median scalar score (interquartile range) for Inhibition was 9 (7–11), for Memory for names was 10 (7–11), for Memory for designs was 10 (8–12), and for Route finding was 2 (1–4). In Comparing scalar scores or IQ scores of different tests between cases with and without clinical neurological alterations diagnosed during development, we found statistically significant differences in Inhibition and in Route finding .There were no significant associations between growth disturbances and to have a clinical neurological development disorder, except for the loss of 2 SD in length between birth and neonatal discharge for Fenton.We found statistically significant correlations between RIST index and z-score for Fenton at birth , for IGW-21 at birth (length), for Fenton at discharge (weight and length), and for IGW-21 at discharge (weight and length) . We did Comparing RIST index values between patients with or without IUGR or EUGR, we found some statistically significant differences .Finally, we found a statistically significant relative risk for having HC IUGR by Fenton and for obtaining RIST scores lower than 89 (RR 1.8 (CI 95% 1.2–2.9), and for having IUGR weight by Fenton or IUGR weight for IGW and to obtaining a percentile of 25 or lower in the Route finding subtask (RR 1.5 (CI 95% 1.1–2.0) and RR 1.5 (1.1–2.1), respectively).VLBW may be due to prematurity and/or IUGR of different etiologies. Later, growth of these neonates can be influenced by multiple genetic, epigenetic, neonatal , and post-neonatal  factors. On the other hand, the neurodevelopment of these children is also influenced by different factors, some of them coinciding with the abovementioned and some of them being related to other matters . In published observational studies, it has been analysed whether growth is related to neurodevelopment in the short, medium and long term, although the conclusions are not uniform, and debate remains open ,6,7,8,9.Beyond major neurological alterations diagnosed clinically in the follow-up , VLBW may present neuropsychological alterations at the school age, only detectable by specific tests for this purpose, such as the RIST test and the NEPSY-II battery at 5–7 years. For this reason, our study aimed to know the relationship between the result of these tests and initial growth of VLBW, comparing different definitions of neonatal growth, without entering the influence of other neonatal factors that are already present in previous publications .One of the main problems that arises when assessing the relationship between neonatal growth and subsequent neurodevelopment is which growth parameters and anthropometry to use. Classically, Fenton’s 10th percentile was cut-off at birth and neonatal discharge for classifying children as IUGR and EUGR, but this classification is changing. First, IGW-21 standards seem much more interesting as basic data, given the way they are obtained , to classify children , althougOur study aimed to evaluate these aspects, analysing static anthropometry data , but also some dynamic ones (true EUGR and changes less or greater than −1 and −2 SD between birth and discharge), both for Fenton and IGW-21. Our results support that the percentage of VLBW classified as IUGR and EUGR varies greatly depending on the criteria used.Thus, IGW-21 is slightly less selective at birth in IUGR classification  but, at the same time, is stricter at discharge in the static EUGR classification for weight (IGW-21 labels less cases than Fenton as static EUGR). Thus, some true Fenton-tagged EUGRs are not correctly detected as such (30.4% in our series) by IGW-21, while Fenton detects all the IGW-21-tagged ones. However, this situation only happens with the weight measurement, but not with length nor with HC, which do not present a uniform trend, contrary to what appears in other series .In our series, RIST scores, as a measurement of IQ, were correlated with z-score at birth  and at neonatal discharge (Fenton and IGW-21 weight and length), but not with z-score differences between birth and neonatal discharge, nor with the z-score at 2 years. In addition, IUGRs in weight, length, and HC, and static EUGRs in length and HC show lower scores, statistically significant, compared to neonates who were not, with differences of 10–20 points between them .These results coincide partially with the previous literature, finding that some neonatal and early postnatal measures, which indicated a lower growth status, could be related with later and lower IQ achievements .However, it is also worth mentioning some major methodological differences of our study with some previous publications, such as the use of different definitions of EUGR, with Fenton and IGW-21 graphs and standards. Therefore, looking at our results, we can say that the new approaches of growth classifications (the use of the IGW-21 standards or the new dynamic definitions) do not modify the findings of the classical criteria. IGW-21 standards do not improve the performance of Fenton graphs in terms of RIST prognosis, and the new EUGR concepts (dynamic or true) do not either.Another issue to consider would be the different importance given to the IQ in these previous publications. While previous articles are focused on IQ assessment as the only measurement of cognitive development, our aim was to consider a wider neuropsychological profile, and due to that, the RIST test was included only as a screening measure of IQ. With these results, we must admit that IQ index is mathematically related with static somatometric values at birth and discharge, but not with dynamic ones. In any case, this relationship, as we discussed previously, can be influenced by many other prior or intermediate factors that surely play important roles.In the NEPSY-II tests, we did not find a relationship with the growth parameters analysed. Perhaps this is because these subtasks are excessively specific, or because we have not gotten enough power with our sample size to find possible true relationships. It is striking, for example, that Memory for names and Memory for design tasks, related to verbal and visuospatial memory, respectively, did not present any type of relationship  with any growth parameter. This may be since the memory scores reported in the present study are composite indexes of both short- and long-term memory, which are related but independent cognitive functions and their addition could decrease their ability to differentiate between growth statuses. Besides, it should be noticed that in Memory for designs, any of our participants scored below expected, and then, this can limit the discriminatory capacity of such a task. We also did not find correlations between scalar scores of other studied NEPSY-II subtasks (Inhibition and Route finding subtasks) and the z-scores of different measures. These findings could point to the possibility that slower growth does not affect specific neuropsychological functions, but rather a more global pattern of cognitive development.Among the strengths of our study, we believe that we can point out that it is a prospective study, specifically designed to assess RIST and NEPSY-II in a cohort of children under 1500 g, in a university hospital. It is also a strength that the field study was carried out by only two psychologists and one paediatrician, with little or no variability in the assessment of the tests and the variables under study. Among the limitations, we can point out that the study was limited to the age of 5–7 years, an age in which these premature infants can still present significant intra-group variations in their brain maturation.In the future, the advantages of using some graphs over others (IGW-21 vs. Fenton) and of some EUGR criteria over others  should be evaluated in further development studies, in order to determine which graph and which criteria are the best when it comes to predicting the neuropsychological future of these premature infants.In summary, classification IUGR and EUGR  with Fenton and IGW-21 show very varied percentages according to the criteria used in the classification. Besides, growth influence on neuropsychological tests at 5–7 years of age does not seem robust, except for IQ measurement (RIST test), which is clearly correlated with some static measures, and which shows clinically important differences between some groups classified according to these measurements. However, the new criteria do not improve the predictive ability of the old ones, nor does IGW improve Fenton for this purpose.Reviewing growth importance in the first weeks of life in later neurodevelopment is a topic of great interest to neonatologists, endocrinologists, neurologists, and psychologists. New IUGR and EUGR concepts should be evaluated in future studies in this field to know exactly its prognostic capacity in long-term neurodevelopment.


To evaluate the self-reported pain scores as a predictor of preterm birth (PTB) in symptomatic twin pregnancy and to develop a nomogram for the prediction model.We conducted a retrospective study of 148 cases of symptomatic twin pregnancies before 34 weeks of gestation visited at Seoul national university hospital from 2013 to 2018. With other clinical factors, self-reported pain score was evaluated by the numerical rating scale (NRS) pain scores for pain intensity. By multivariate analyses and logistic regression, we developed a prediction model for PTB within 7 days. Using the Cox proportional hazards model, the curves were plotted to show the predictability of the PTB according to NRS pain score, while adjusting the other covariates.P < 0.05), shorter cervical length  and positive fibronectin results  affect PTB within 7 days. Using the variables, the area under the receiver operating characteristic curve (AUROC) of the prediction model was 0.917. In addition, we developed a nomogram for the prediction of PTB within 7 days.Twenty-three patients (15.5 %) delivered preterm within 7 days. By a logistic regression analysis, higher NRS pain score (OR 1.558, 95 % CI 1.093–2.221, Self-reported pain scores combined with cervical length and fetal fibronectin are useful in predicting impending PTB in symptomatic twin pregnancy. Preterm birth (PTB) is one of the most important problems in twin pregnancy. In spite of developments in the management of preterm labor, such as antenatal corticosteroids or tocolytics, the risk of preterm birth in twin pregnancy is still high, with a reported incidence of 19.8 % for PTB < 34 weeks and 59.9 % for PTB < 37 weeks [Preterm labor is one of the most frequent problems for antenatal admission to the hospital. It made up about one-third in US hospitals , 3. HoweAs good candidates for predictive markers, sonographic cervical length measurement and fetal fibronectin test are well studied in asymptomatic and symptomatic twin pregnancy –8. But tFor the evaluation of pain intensity, self-reported pain scales are the most commonly used method to perform mathematical calculations in a variety of areas, including postoperative pain, orthopedic pain, and obstetric pain . Among sThe purpose of this study was to demonstrate whether self-reported pain scores can predict impending preterm birth in twin pregnancy with preterm labor and to develop a nomogram for the prediction of preterm birth.It is a single-center retrospective cohort study. The institutional review board of Seoul National University Hospital Clinical Research Institute approved this study  and has waived the requirement of the informed consent for this study. All methods were performed in accordance with the relevant guidelines and regulations.This study population consisted of consecutive twin pregnancies who visited Seoul National University Hospital with symptomatic preterm uterine contractions from March 2013 to January 2018 and met the following inclusion criteria: (1) gestational age < 34 weeks at the time of visit; (2) without evidences of ruptured membranes; (3) both alive fetuses.At the time of the visit, physical examinations were performed including digital vaginal examinations for cervical effacement and dilatation. Patients with ruptured membranes were excluded, and rupture of the membrane was diagnosed by speculum examinations with a combination of the presence of amniotic fluid leakage or pooling, positive nitrazine or amnisure test. A swab was taken in the posterior fornix and analyzed for a fetal fibronectin test. Transvaginal ultrasonography was performed for the evaluation of cervical length and funneling.In our institution, it is a routine practice to obtain clinical information regarding the frequency of uterine contractions and the numerical rating scale (NRS) pain score during uterine contractions in patients with threatened preterm birth. The frequency of uterine contractions was determined based on the cardiotocography. NRS pain score was estimated by patients for pain intensity to be expressed as 0 to 10. These data was collected from the electrical medical records. Patients were classified into two groups according to the interval-to-delivery. Interval-to-delivery was determined as the time interval between the date of the visit for symptomatic uterine contractions and the date of delivery. Clinical factors were compared between women who delivered within 7 days and those who delivered 7 days or later.The use of tocolytics therapy or corticosteroid administration was decided at the discretion of the attending physician. Antibiotics are not routinely used in patients with preterm labor, especially without the evidence of chorioamnionitis.p < 0.05) according to the interval-to-delivery. We performed 3-fold cross-validation to compute the area under the receiver operating characteristic curves (AUC) of the nomograms for prediction of preterm birth from stepwise selection by AUC using leave-one-out cross validation. As results, the nomogram for prediction of preterm birth within 7 days was built with the variables that remained significant in logistic regression analysis. Additionally, cut-off value of NRS pain score was estimated by the receiver operating characteristic (ROC) curve. Using the Cox proportional hazards model, the curves were plotted to show the predictability of the preterm birth according to NRS pain score, while adjusting the other covariates. A prior sample size calculation was performed to determine how many pregnant women would be needed to detect an increased risk for preterm birth from 10 to 30 % in women with high NRS score. In the literature, the risk of preterm birth was reported as 20 % in twin preterm labor [P < 0.05 was considered significant.Proportions of categorical variables were compared using the Fisher’s exact test, and Mann-Whitney U test analyzed comparisons of continuous variables. There were described by median with interquartile range for continuous variables and by numbers and percentages for categorical variables. To develop the prediction model for preterm birth within 7 days, multiple logistic regression with backward elimination was performed with clinical variables selected from univariate analysis (rm labor . With 80p < 0.001). In addition, patients who were delivered at preterm within 7 days had more frequent uterine contractions, shorter cervical length, a more dilated cervix, and a higher rate of positive fibronectin test.Between March 2013 and January 2018, a total of 148 twin pregnancies met the inclusion criteria. Among these 148 women, 23 women (15.5 %) delivered preterm within 7 days. Table P < 0.05), shorter cervical length  and positive fibronectin results  affect preterm delivery within 7 days in symptomatic twin pregnancies. Using final clinical variables retained in the prediction model of preterm birth within 7 days, the AUC of the prediction model was 0.917 in Fig. The association between the risk of preterm birth within 7 days and a high NRS pain score remained significant even after adjusting for confounding variables, as noted in Table p < 0.01).In addition, an NRS pain score was analyzed for estimated cut-off value by ROC curve. The cut-off value for prediction of preterm birth within 7 days was an NRS pain score of 4. Table http://lsy.io/nomogram/painscore) for easy access to promote clinical use.From the above results, we developed a static nomogram for the prediction of preterm birth within 7 days in Fig. In the present study, patients who delivered within 7 days had a higher NRS pain score during uterine contractions, more frequent uterine contractions, shorter cervical length and a higher rate of positive fibronectin test. In logistic regression analysis, NRS pain score, cervical length, and positive fibronectin were significant risk factors for preterm birth within 7 days. And by using three factors, we developed a static nomogram for prediction of preterm birth within 7 days.Many papers were published about the prediction of preterm birth. Research on singleton pregnancy accounts for the majority, but there are some studies on twin pregnancy. Out of many factors, cervical length measured by ultrasound and fetal fibronectin test are suggested as a promising tool for purpose of the prediction of preterm birth. Some studies have commented that positive fetal fibronectin and short cervical length are associated with impending preterm birth in both symptomatic and asymptomatic twin pregnancies , 16. TheAmong clinical factors during preterm labor, the patients’ subjective symptom (uterine contraction or labor pain) has not been evaluated by objective parameters in previous studies on preterm birth. However, labor is related to physiological changes of the myometrium and cervix which are divided into four phases of parturition , 20. In In the symptomatic uterine contractions or threatened preterm labor, patients may express pain characteristics  and intensity (as a self-reported pain score). Self-reported pain scores are widely used in pain assessment . The useAs a result, we showed that NRS as pain intensity was critical factor in progression of preterm labor. Self-reported NRS pain score with known risk factors of cervical length and fetal fibronectin test improves the diagnostic accuracy of preterm delivery within 7 days up to 91.7 %. In the clinical management of patients with threatened preterm birth, the prediction of impending preterm birth within 2 or 7 days is the most important to optimize pregnancy and neonatal outcomes. Based on this prediction, the physicians must make decisions regarding several interventions, such as transfer to other hospitals and the use of corticosteroid, magnesium sulfate or tocolytics , 24.Although we used cross-validation for indirect verification of our model, our study has some limitations. It was based on retrospective and single center data with a limited number of cases. As a result, several critical factors were significantly different between women with high NRS and those with low NRS . In the study population, the use of tocolytics or steroid was at the discretion of attending physician. As a results, NRS was a significant risk factor for preterm birth even after adjustment for these factors. However, we could not split the study population into training and test set.Larger studies in prospective and multicenter settings are needed to confirm the results of the current study. In addition, the findings of the current study should be further evaluated in other study populations such as in singleton pregnancy. Moreover, a higher NRS pain score may be related to other obstetric or gynecologic conditions . Furtherhttp://lsy.io/nomogram/painscore) for easy access to promote clinical use. Further prospective studies are needed to verify the clinical usefulness of this suggested normogram.Lastly, we suggested a static nomogram for the prediction of preterm birth within 7 days in Fig. The accurate prediction of preterm birth will result in reduced neonatal complications by proper therapeutic strategies at the appropriate time. The developed nomogram will enable the accurate estimation of preterm birth by physicians. It might help twin pregnancies receive proper management of preterm labor. As a result, unnecessary hospitalization and the overuse of tocolytics or antibiotics will be reduced and neonatal outcomes will be improved.


Conclusion: Promoting healthy weight amongst women of child bearing age is important in preventing childhood obesity, and increasing daily infant PA is as important as a healthy infant diet.Background: The growth and maturation of infants reflect their overall health and nutritional status. The purpose of this study is to examine the associations of prenatal and early postnatal factors with infant growth (IG). Methods: A data-driven model was constructed by structural equation modelling to examine the relationships between pre- and early postnatal environmental factors and IG at age 12 months. The IG was a latent variable created from infant weight and waist circumference. Data were obtained on 274 mother–child pairs during pregnancy and the postnatal periods. Results: Maternal pre-pregnancy BMI emerged as an important predictor of IG with both direct and indirect (mediated through infant birth weight) effects. Infants who gained more weight from birth to 6 months and consumed starchy foods daily at age 12 months, were more likely to be larger by age 12 months. Infant physical activity (PA) levels also emerged as a determinant. The constructed model provided a reasonable fit ( The growth and maturation of infants reflect their overall health and nutritional status. The first 1000 days of life, including gestation and the first two years of age, is a critical period of infant growth that may shape the adulthood health and well-being of individuals . UnderstHowever, despite the identification of several determinants in the development of childhood obesity including biological, social, and behavioral factors , the majAs an emerging approach to multivariate analysis of causal relations amongst observed and unobserved (latent) variables, SEM  offers aMother–child pairs who had participated in a prospective birth cohort study—Growing Up in Wales: Environments for Healthy Living (EHL) ,13, wereMaternal height, weight, skinfold thickness  and mid-arm circumference were measured during pregnancy. The mean gestation of women was 24.4 weeks  when data collection occurred.Birth weight, gestational age and Apgar scores (at 1 and 5 min) were obtained from medical notes recorded by a health visitor. These pieces of information were also available from clinical records. Average weight gain per week (from birth to 6 months) was calculated by taking a subsequent measure of weight from clinical records. At 12 months postpartum, follow-up questionnaires provided; details on any changes in maternal living circumstances, infant feeding practices , sleep patterns, developmental stages , physical activity patterns and TV viewing time. An experienced and trained researcher obtained infant anthropometric measures which also included length, and mid-arm circumference (as measured in the mother). Using such a data-driven approach, our analyses can produce a relatively simple model displaying causal pathways. Some studies have indicated the associations of infant weight and waist circumference with adverse health outcomes, such as future obesity and cardiovascular risk ,17,18. AInfants vary in size and shape as much as adults do. Different anthropometric parameters were developed , weight p ≤ 0.05) (X2), the Root Mean Square Error of Approximation (RMSEA), the Standardized Root Mean Square Residual (SRMR) and the Comparative Fit Index (CFI), and followed the cut-offs for good fitting models suggested by Hu et al. [A two-step statistical analyses approach was adopted for the present study: first, a series of regression analyses  . To examu et al. . MaximumWeight, waist circumference and skinfold measurements at age 12 months were available for 270 (141 boys and 129 girls), 225 (122 boys and 103 girls) and 202 (111 boys and 91 girls) infants respectively. Eighteen per cent (n = 49) of the sample did not have an outcome variable available and were excluded from the main SEM analyses.p < 0.0001) and to have received higher education , less likely to smoke during pregnancy  and be primiparous . No significant differences were shown between ethnicity groups, pre-pregnancy BMI values, the proportion of mothers in full-time employment or those reporting an annual household income of less than £15,000.Comparing maternal characteristics between those mothers who did (n = 274) and did not (n = 269) participate in the 12 month visit, mothers who did participate were; more likely to be older  were available for 63 infants (25.5%) of which we examined the relationships between hours spent sedentary and intensity of time spent active with infant size. Although advised to wear the device for 7 days, only 6 days with 24 h data were used . The average wear time was 4.5 days with 55% of infants wearing the device for the full 6 days. On average infants were shown to be sedentary for 10.3 h per day (range 1.5–18.2 h). Of the infants with physical activity data, 49% had taken their first steps. No significant differences in the number of hours spent sedentary or the intensity of active periods were shown between the two groups. Examining the data for each gender, females spent less time being sedentary (mean 9.13 h (SD 5.2) vs. Mean 10.05 (SD 5.5)) and males showed a higher intensity during active periods  vs. 255,825 ) however, neither finding reached statistical significance.p < 0.05, signifying that each variable made a meaningful contribution to the model. The relationship noted between maternal weight (pre-pregnancy BMI) and infant size was of modest size (β = 0.12). An indirect effect of pre-pregnancy BMI on infant size was evident with infant birth weight acting as a mediator (β = 0.08). Self-reported infant consumption of carbonated drinks (β = −0.18) and lower levels of infant play (β = −0.14) were related to smaller infant size. Weight gain was not related to birth weight or any infant feeding measure. Notably, no pathway involving maternal physical activity or maternal diet was apparent within our final model.For the ease of interpretation, The final model displays an improved goodness of fit ; howeverp ≥ 0.1). Only carbohydrate consumption, for both genders and birth weight for boys (p < 0.05) remained significant predictors within the model. The goodness of fit indices of SD and SRMR (girls model only) reveals a poor fit for both genders whilst further analyses showed that infant carbohydrate consumption was significantly different between the two groups . Using MLMV with all 225 mother–child pairs produced a similar model as that of the true data, demonstrating similar path coefficients and negative effects of infant play and carbonated drink consumption. Effect sizes for each variable within the latent construct remain unchanged. A noticeable difference is that infant-play no longer demonstrates a significant association with infant size. Modelling by gender revealed the modest path coefficients, as the significance levels of most pathways dropped  when compared to those reporting no consumption. With approximately 43% of an infant’s diet attributed to beverage intake at age 1 year , it is pNo statistically significant differences in the number of hours spent sedentary or the intensity of active periods were found between females and males. Lower levels of infant physical activity were also associated with smaller infant size, that is, playing physically active games less than once a day was associated with lower weight and a smaller waist circumference. The observed effect size was comparative to the effect shown of starchy foods but in a counteractive fashion, revealing an inverse effect. A potential explanation for this association lends to previous research on bone properties. Research involving both children and adolescents has shown the benefits of physical activity on bone density , which cThe present study innovatively adopts an SEM approach to examine longitudinal data with prenatal and postnatal variables measured from early pregnancy to 1 year postpartum, providing a platform for analysis based on a life course approach. Using a data-driven approach, our analyses produced a relatively simple model displaying causal pathways of infant size. Modelling infant size as a latent variable, we were able to account for both increased weight and larger waist circumference, evading problems associated with collinearity. The majority of infant size did, however, consist of infant weight.There are however some limitations with this analysis. First, due to the incompleteness of data, our model was based on a significantly reduced sample of the total study population. This could be a contributing factor to the model’s goodness of fit estimations and also limited our ability to produce gender specific models. Nonetheless, the observed pathways were all significant and our model does provide a greater understanding of pre- and postnatal interactions. The high variation observed amongst infant sizes and measured factors also warrant the need for larger datasets to strengthen our observations and identify further pathways. Second, our studied population was predominantly white and well educated, with two thirds of women presenting as first time mothers at study enrolment. These characteristics limit the generalization of our findings to the wider population with diverse ethnic groups and lower education levels. Third, as with all cohort studies, we cannot rule out any potential effects of unmeasured variables within our model. Throughout our study design, we were unable to account for factors relating to the non-shared family environment, such as interactions with siblings and the influence of childcare arrangements. Additionally, no consideration was given to paternal factors. It is therefore possible that infant size is mediated by other factors which are not included within our study design.Moreover, the reliance on mothers to provide all dietary data relating to child-feeding practices, infant dietary content, and their own diet during pregnancy and at 12 months is subject to limitations. Even though every effort was made to encourage mothers to record dietary data on a day-to-day basis, mothers often reported recall problems and completion of their diet diary over several days as opposed to daily. Within the infant dietary data, it was difficult to distinguish between infant specific items; therefore, it is possible that infants were consuming specific tailored brands and not adult products as assumed in the present study. As a result, our finding of smaller sizes with higher consumptions of carbonated drinks could be indicative of infants consuming carbonated drinks suitably lowered in sugar; however, this is speculative. Moreover, our ability to comment on the causal pathways involving infant dietary predictors is somewhat limited as infant dietary measures were obtained at the same time point as our study outcome. Finally, our measures of size did not look at body composition. High carbohydrates are associated with larger (overweight infants) but higher physical activity is associated with larger (more muscle) infants. This is speculative and is not based on body composition measurements. In addition, our measurements of infant physical activities did not consider some situations, such as the infants being picked up or carried.The results of the present study suggest that future efforts aiming to prevent childhood obesity should focus on promoting healthy weights amongst women of childbearing age, by encouraging healthy diets and active lifestyles. In addition, public health programmes, such as the Healthy Child Programme, should include interventions that facilitate the adoption of healthy family lifestyle approaches. Further longitudinal, large-scale studies, involving in-depth dietary and physical activity assessments are needed to confirm our findings.This study presented an SEM approach to evaluate the impact of prenatal and early postnatal factors as predictors of growth of infants at age 12 months. The research findings supported that promoting healthy weights amongst women of childbearing age is important in preventing childhood obesity, and increasing daily infant physical activity is just as important as the emphasis placed on the need for a healthy infant diet.Some machine learning methods have emerged to identify the causal features from the large dataset ,42. Thes


Adolescent birth (10–19 years old) declined slowly, from 84% in 1996 to 71% in 2017. Compared with adult mothers (≥20 years old), young adolescent mothers (10–15 years old) were more likely to be underweight (+11 pp), have lower education (−24 pp), have less decision‐making power (−10 pp), live in poorer households (−0.9 SD) with poorer sanitation (−15 pp), and have poorer feeding practices (10 pp), and were less likely to access health and nutrition services (−3 to −24 pp). In multivariable regressions controlled for known determinants of child undernutrition, children born to adolescents had lower height‐for‐age Z‐scores (−0.29 SD for young and −0.10 SD for old adolescents (16–19 years old)), weight‐for‐age Z‐score  as well as higher stunting (5.9 pp) and underweight (6.0 pp) than those born to adults. In conclusion, birth during adolescence, a common occurrence in Bangladesh, is associated with child undernutrition. Policies and programs to address poverty and improve women's education can help delay marriage, reduce early childbearing, and improve child growth.Adolescent birth is a major global concern owing to its adverse effects on maternal and child health. We assessed trends in adolescent birth and examined its associations with child undernutrition in Bangladesh using data from seven rounds of Demographic and Health Surveys (1996–2017,  Adolescent birth is a major global concern owing to its adverse effects on maternal and child health. We assessed trends in adolescent birth and examined its associations with child undernutrition in Bangladesh using data from seven rounds of Demographic and Health Surveys. Our analysis demonstrates that birth during adolescence, a common occurrence in Bangladesh, is associated with child undernutrition. A growing number of countries have recognized the importance of addressing adolescents’ needs in the “leave no one behind” Sustainable Development Goals (SDGs) era.It is well established that adolescence is a critical phase of social, physical, and cognitive development.We recently reported that giving birth during adolescence is associated with poorer outcomes for Indian women—in terms of women's nutrition, access to health services, child feeding practices, living conditions, and education—and with undernutrition in their children.The objectives of the current analysis were (1) to assess trends in adolescent birth and child undernutrition in Bangladesh in the last two decades; (2) examine how adolescent birth explains variability in known determinants of child undernutrition, including maternal nutritional status, education, living conditions, access to health services, and complementary feeding; and (3) examine associations between maternal age at first birth and child undernutrition outcomes accounting for known determinants of child undernutrition.https://www.dhsprogram.com/) after obtaining permission from the DHS program. Full details of the survey protocol, sampling procedures, and questionnaires are provided in the BDHS reports.This study used data from seven rounds of data from the Bangladesh Demographic and Health Surveys (BDHS 1996–2017).n = 12,006).The sample selection process for this study is presented in Table The outcome variables of interest in this study were child anthropometric measures. Enumerators were trained to measure height/length and weight according to the internationally recommended standard protocol.Women's age at first birth was calculated as the difference between the birth date of the first‐born living child and the birth date of the woman. Adolescent birth was defined as women giving first birth between the age of 10–19 years old.2. Women's status was measured using completed years of education and bargaining power . Living condition was measured by improved sanitation facility and household socioeconomic status , four rounds , or three rounds . In case data were not available for all rounds, the sample size was smaller for the models containing those variables.To determine the extent to which adolescent birth is associated with child nutritional status, we conducted multivariable regression analyses using full‐information maximum likelihood for estimation while accounting for missing data among the controlled variables under the assumption of missing at random and without having to do imputation.P < 0.05, P < 0.01, and P < 0.001.All analyses were conducted using Stata® 16 . Statistical significance was considered at three levels: At the national level, the prevalence of first births occurring in women aged 10–19 years old has decreased slowly in Bangladesh from 84% in 1996 to 71% in 2017 (by 13 percentage points (pp); Fig. Stunting decreased from 59.5% in 1996 to 26.6% in 2017 by 32.9 pp) at the national level .There were significant associations between maternal age at first birth and various determinants of child nutritional status Table . AdolescAdolescent birth was also negatively associated with living conditions, women's education, and bargaining power. Compared with adult women, women who gave birth during young adolescence were more likely to live in a household with lower SES (−0.9 SD) and poorer sanitation (−14.9 pp); and they had lower education (−24.1 pp for secondary school education) and less household decision‐making power (−9.8 pp).Adolescent birth was negatively associated with access to services during pregnancy, delivery, and early postpartum. Compared with women who first gave birth as adults, those who gave first birth during young adolescence reported poorer use of antenatal care , less service use during delivery  and postnatal care (−16.6 pp), and less access to full immunization (−6.3 pp) and child vitamin A supplementation (−6.5 pp). Finally, adolescent birth was negatively associated with complementary feeding practices (−8.5 pp for adequate diet). Similar associations were found for the comparison of old adolescent versus adult births, with a smaller magnitude of the differences.Children of women who had their first child during adolescence had lower HAZ and WAZ, as well as higher stunting and underweight prevalence, compared with children of women who had their first child during adulthood. The multivariable regression that adjusted for known determinants of child undernutrition showed that children born to old adolescent mothers (16–19 years old) had lower HAZ (−0.10 SD) and WAZ (−0.06 SD) than children born to adults (20–49 years old)  but remained high (at 71.4%). Child undernutrition improved over time (by 23 pp for stunting and 36 pp for underweight), but poor growth was more prevalent among children born to adolescent mothers. The risk of being stunted and underweight increased 5.9 and 6.0 pp, while HAZ and WAZ reduced by 0.29 and 0.18 SD, respectively, even after controlling for known determinants of undernutrition, such as maternal nutritional status, education, living conditions, access to health services, and complementary feeding practices.The multiple linkages between adolescent birth and child undernutrition observed in this Bangladesh study align well with a previous study conducted in India.Adolescence is a period of rapid physiological, sexual, neurological, and behavioral changes; the growth spurt in adolescence requires increased energy and nutrients.Beyond increased nutritional needs, adolescent mothers face several other barriers to achieving optimal pregnancy outcomes. Consistent with previous studies,A major cause of adolescent pregnancies in Bangladesh is child marriage—that is, marriage before the legal age of 18 years old, which is highly prevalent in the country (∼70%).Drawing lessons learned from global evidence,For girls who are already married, the focus should be on delaying pregnancy. To this end, massive social campaigns, similar to what Bangladesh has very successfully done for child immunization, could be effective for encouraging family planning to delay pregnancy for adolescent girls. While many interventions show modest impacts on delaying age at marriage, few programs have been able to enable adolescent mothers to continue their education or increase their access to reproductive health services,In order to achieve the SDG target to end child marriage by 2030, the pace of early marriage reduction would need to be eight times faster than what was observed in the last decade.Our study has multiple strengths, including large sample size from multiple nationally representative surveys, advanced statistical methods, and the disaggregation of results for younger and older adolescent mothers. By using multiple rounds of DHS data between 1996 and 2017, we present trends over the last two decades and show robust associations between adolescent birth and child undernutrition as well as determinants of child undernutrition across survey rounds. Our study is one of very few studies that have disaggregated outcomes for adolescent mothers by age, which provides in‐depth insights into the detrimental effects of very early childbearing (by 15 years of age). We acknowledge some limitations of the study, including the use of repeated cross‐sectional data, which prevents us from drawing causal inferences. We are also not able to examine how social norms have changed over time and the direct or indirect roles of these unobserved factors.In conclusion, adolescent birth is still the norm in Bangladesh, and it has implications for the health, nutritional, and SES of mothers as well as children. These findings call for effective convergence of interventions across multiple sectors at the right time in life to address these persistent problems. Targeting meaningful reductions in the number of women marrying and having children as adolescents will contribute to the attainment of several SDGs, including those addressing poverty, hunger, good health and wellbeing, inclusive and quality education, and gender equality.P.H.N. conceived the idea for the manuscript and led the overall synthesis of the manuscript, conducted the statistical analysis, and wrote significant sections of the manuscript. S.S. helped conceive the idea for the manuscript, conducted literature review, reviewed the statistical analyses, and wrote significant sections of the manuscript. L.Q.K. conducted the statistical analysis and prepared tables and figures for the manuscript. P.P. conducted literature review and edited the manuscript. A.A., S.F.R., and K.A. supported data interpretation and reviewed and edited the manuscript. P.M. reviewed the statistical analyses, supported data interpretation, and reviewed and edited the manuscript. All authors read and approved the final submitted manuscript.The authors declare no competing interests.Table S1. Descriptive characteristics of participants in seven BDHS 1996–2017.able S2. Trends in adolescent birth and child undernutrition in seven BDHS 1996–2017.TTable S3. Association between mother's age at first birth and child stunting, underweight, and wasting by survey year, BDHS 1996–2017 .Figure S1. Weight‐for‐age Z‐score, weight‐for‐height Z‐score, underweight, and wasting by mother's age at first birth and survey year, BDHS 1996–2017 .Click here for additional data file.


Intercostal artery pseudoaneurysm is rare and at the risk of rupture. The aetiology is always reported to be iatrogenic and traumatic injury. Embolisation is the most common therapeutic method. Here, we report a case of spontaneous intercostal artery pseudoaneurysm and cured by combining covered stent grafting and surgical management.A 60-year-old man complained of acute right back pain for 5 h. Computed tomography showed right massive hemothorax and a giant mass with distinct feeding vessel originated from the thoracic aorta within the right hemithorax. Thoracocentesis was performed, and then a covered stent was positioned across the origin of the feeding vessel. The patient was diagnosed with intercostal artery pseudoaneurysm. Finally, we successfully resected the pseudoaneurysm and ligated the proximal part of the artery. Histologic examination have proved the diagnosis. The postoperative course was uneventful, and the patient was discharged on postoperative day 10. There is no recurrence reported during follow-up.Spontaneous intercostal artery pseudoaneurysm is extremly rare. Delayed hemothorax due to rupture of the pseudoaneurysm may occur years after the formation. Early diagnosis is important and a combined treatment of endovascular intervention and surgical management is feasible, especially for the case of ruptured large tumour-like mass presentation of the pseudoaneurysm. Intercostal artery pseudoaneurysm is rare and has mostly been described through case reports –24. DiffA 60-year-old man was referred to our hospital by an ambulance because of acute right back pain for 5 h. The pain was constant and sharp. Firstly, it was limited to an area just inferior to the tip of his right scapula. But it subsequently radiated to his right chest. There was no associated fever, coughing, shortness of breath, or hemoptysis. He denied any falls or other trauma. He had a history of hypertension for about 4 years without standard treatment and highest reached 180/95mmhg. Chest computed tomography (CT) 3 years prior showed a mass between the right spine and diaphragm measuring 6.4 × 4.3 × 6.3 cm Fig. a. We diaCommunicating with the flowing blood, pseudoaneurysm is a collection of blood outside the vessel lumen . The absHemothorax was the most common presenting symptom in almost all the cases reported previously. So patients with massive hemothorax should be suspected to have an intercostal artery pseudoaneurysm, especially when they had chest trauma or underwent a surgical procedure via the intercostal space. The rupture of intercostal artery pseudoaneurysm causes brisk bleeding which may lead to shock or death. But, as reported, delayed hemothorax might occur two to 4 weeks after trauma or surgical procedures, so early diagnosis was possible and essential. Traditionally, diagnosis of an intercostal artery pseudoaneurysm was usually made by means of arteriography which allows endovascular treatment in a single procedure. But doppler ultrasound and CT are the two most primary diagnostic modality for intercostal artery pseudoaneurysm. In our case, intraoperative findings and pathology on resected tissues were also important in the diagnosis of intercostal artery pseudoaneurysm. Because, as for differential diagnosis, initially, we took pulmonary sequestration and mediastinal tumor into consideration.The ruptured intercostal artery pseudoaneurysm was a good indication for endovascular intervention. Embolisation was considered to be the preferred treatment of a ruptured pseudoaneurysm. Many successful cases have been reported , 21–24. Intercostal artery pseudoaneurysms are rare and have a risk of rupture. Diagnosis before rupture is important. Embolisation is the most common therapeutic method because of its efficacy and low invasiveness, and surgical management is always reserved for embolisation failure. We report a patient with spontaneous intercostal artery pseudoaneurysm. Delayed hemothorax due to rupture of the pseudoaneurysm occurred more than 3 years after the formation. We emphasize the significance of combined treatment of endovascular intervention and surgical management, especially for the case of ruptured large tumour-like mass presentation of the pseudoaneurysm.


The yellow fever virus vaccine, 17D, was derived through the serial passage of the wild-type (WT) strain Asibi virus in mouse and chicken tissue. Since its derivation, the mechanism of attenuation of 17D virus has been investigated using three 17D substrains and WT Asibi virus. Although all three substrains of 17D have been sequenced, only one isolate of Asibi has been examined genetically and all interpretation of attenuation is based on this one isolate. Here, we sequenced the genome of Asibi virus from three different laboratories and show that the WT strain is genetically homogenous at the amino acids that distinguish Asibi from 17D vaccine virus. Yellow fever virus (YFV) is the causative agent of yellow fever (YF), a hemorrhagic disease involving the liver, kidney and heart. Each year, up to 170,000 cases of YF are reported, with as many as 60,000 related deaths . Macaca mulatta) monkeys with periodic passages in Aedes aegypti before it was used to derive 17D [The wild-type (WT) YFV strain Asibi was isolated from the blood of a Ghanaian man with a mild case of YF. The virus was passaged 53 times in rhesus macaque (rive 17D . NeitherMacaca mulatta) and three passages in C6/36 cells [Many studies have investigated the mechanism of attenuation of 17D virus through the comparison of the vaccine to the Asibi parental strain and have involved genomic sequences of vaccine from different manufacturers of each of the three 17D substrains ,4. Thus,36 cells . Here, wThree Asibi viruses from different sources  and one vaccine ampule of 17D-204 were used in this study. The Asibi-Yale strain was obtained from the World Reference Center for Emerging Viruses and Arbovirus . The virus was received as lyophilized cell culture and was passaged one time in C6/36 cells. The Asibi-LSHTM strain was obtained from the London School of Hygiene and Tropical Medicine [Viral RNA was extracted using QiaAmp vRNA kit  and sequenced at the UTMB Next Generation Sequencing Core . cDNA was constructed using random hexamer primers  and sequenced using the NextSeq550 platform . Average coverage for Asibi-Yale, Asibi-LSHTM and Asibi-FC was 4689, 964 and 610 reads per base, respectively. Reads were trimmed of adapters and quality filtered for Q-scores greater than 30. De novo consensus sequences were generated using ABySS (v1.3.7) using paired end reads. Full-length sequences can be found on Genbank .The genomes of Asibi-Yale, Asibi-LSHTM and Asibi-FC were identical in length, 10,862 nucleotides . Asibi-Yale and Asibi-FC were very similar, differing by only three nucleotides  with no amino acid changes. Asibi-LSHTM is more distinct and differs from the other two Asibi viruses by 17 nucleotides encoding six amino acid substitutions . FurtherThe Asibi viruses were then compared to all 17D substrains . It was The Asibi viruses sequenced here differed by varying degrees. Asibi-Yale and Asibi-FC by three, synonymous nucleotides, whereas these two viruses differed from Asibi-LSHTM by 17 nucleotides, resulting in six synonymous changes. All nucleotide changes resulting in amino acid substitutions occurred in the first or second bases of the codon, suggesting that they were a result of selection pressure. The E protein contains three of these amino acid differences  at E-D155R, E-K331R, and E-V412I. Other changes were in the nonstructural proteins: NS1-R10K, NS2A-T105A, and NS4B-V98I. The non-conservative replacement of amino acids at E-155 and NS2A-105 (E-D155R and NS2A-T105A) likely have an impact on protein structure and therefore may impact protein function. E-K331R is an interesting difference between the Asibi strains as all other published genomes of WT YFV strains display an arginine at this position ,8. As eaThe sequence of the Asibi-Yale virus determined by Hahn et al. (referred to as Asibi-Hahn) is not available on Genbank; however, comparison of Asibi-Hahn et al. to Asibi-Yale revealed 11 synonymous nucleotide changes  and four non-synonymous nucleotide changes : E-T153N, NS1-K10R, NS4A-V107I and NS5-D657N) . The AsiAsibi-Yale has been the most utilized Asibi isolate and was used as the basis of the Asibi infectious clone (i.c.)  that has been used to define determinants of mosquito competence, genetic diversity and susceptibility to the antiviral Ribavirin ,17,18,19


Poor environmental conditions and hygiene practices at the time of childbirth is linked to life-threatening infections and death in mothers and babies. Improvements in water, sanitation, and hygiene (WASH) have been identified as a means through which the lives of mothers and babies could be saved. This study was carried out to explore WASH conditions and infection prevention and control (IPC) practices in traditional birth homes/centers in Abeokuta, Southwest Nigeria. A total of 50 traditional birth centers and attendants (TBAs) were enrolled in the study. Sociodemographic characteristics of the TBAs and features of the birth centers were obtained using a semi-structured questionnaire. Assessment of WASH conditions and IPC practices was based on established protocols.Findings revealed that majority of the centers operated under poor WASH conditions and IPC practices; none met with the WHO minimum standards for environmental health.Adequate WASH facilities and IPC practices remain a critical component of maternal and child health even in non-facility birth. As the transition to facility births continues in many countries, the large number of non-facility births demands their inclusion in WASH-related strategies, if global goals of reducing deaths of newborns and women deaths will be achieved. Although clean water has received the most attention, the greatest health benefits and increase in standards of living are achieved when improvements in sanitation and hygiene are made alongside access to clean water , 2. UntiThe links between hygiene and healthcare-associated infections in mothers and their babies have been established –10. CleaCompared to efforts on health system strengthening, and increased uptake of facility births , 19, lesDespite the burden of maternal and neonatal infections and the links with poor hygiene, data on clean delivery practices around birth or the status of prerequisite facilities necessary for these practices are limited. Beyond health facilities, there is paucity of information on WASH conditions and adherence to clean delivery practices in non-facility births. It is therefore imperative that strategies that are been developed to reduce maternal and neonatal mortality due to preventable causes take into consideration, deliveries that take place in traditional birth centers or homes. The objective of this study was to describe the status of WASH services as well as clean delivery practices in traditional birth (TBHs). The large number of deliveries occurring in these settings necessitates understanding how environmental contexts surrounding these deliveries potentially impact maternal and newborn outcomes and setback global maternal-newborn goals.The study was carried out in Abeokuta, the largest city and capital of Ogun State. Its geographical area covers Abeokuta South and parts of Abeokuta North, Obafemi Owode, and Odeda Local Government Area. In Abeokuta, healthcare is provided by few governmental and private hospitals, primary healthcare centers, as well as a myriad of community, faith-based, herbal, and traditional practitioners. Traditional Birth Attendants (TBAs) commonly provide perinatal care in rural and low-income urban areas of the town. TBAs form traditional birth attendant association and are recognized by the government. The activities and practices of the TBAs are regulated by the Primary Health Care Development Board of the State.A mixed-methods approach was used to assess WASH services and clean delivery practices in the recruited traditional birth homes (TBHs) Table . The tooFirst, in-depth interviews were conducted with TBAs in their birth homes using a semi-structured questionnaire as a guide. The questionnaire was used to assess the status of WASH facilities and clean delivery practices (six cleans) in the birth homes. For assessing the status of WASH services, the following domains were covered: basic water supply, basic sanitation, waste management, and access to basic hygiene in the TBHs Table .Table 2Assessment of clean delivery practices was based on adherence to WHO’s six cleans including clean hands, clean perineum, clean delivery surface, clean cord cutting, clean cord tying, and clean cord care. Adherence to six cleans was recorded through observation and recording in the walkthrough checklist as well as the use of a questionnaire.Secondly, an in-depth assessment was combined with the walkthrough methodology, which consisted of recording observations in a checklist and collecting water samples for microbiological analysis. This assessment focused on visual and microbiological cleanliness of birth home environments and tools. Data was collected through observation and recording in the walkthrough checklist. Samples were collected from delivery surfaces and cord-cutting instruments as well as from water used for domestic purposes such as hand washing and bathing in the TBHsWater quality testing was conducted in all the TBHs. Samples of water available in birth homes were collected directly from sources (on-site) or storage vessels to assess the microbial quality of water used for domestic purposes and during delivery.Analyses of the TBHs interviews focused on the description of WASH facilities and clean delivery practices. Data analysis was carried out using the Statistical Package for Social Sciences (SPSS) software (version 17.0). Descriptive statistics  was used to describe WASH conditions and clean practicesVisual Inspection and Microbiological Analysis:The analysis of both the checklist and microbiological data focused on describing the cleanliness at different sites of the birth home as well as cleanliness of birth tools/equipment and the cleanliness at the different sites of the birth homes. Isolation of microorganisms from water samples was carried out on general-purpose and selective media using the membrane filtration methods. Microbiological analysis of water samples focused on the presence of Enterococcus and fecal coliforms; standard indicators for assessing water quality. Total bacterial count in the water samples was also determined.A total of 50 traditional birth/herbal homes (TBHs) were recruited through the State Birth Attendant Association into the study. Only one-fifth of the traditional birth homes were solely for maternal and newborn care, others were into other forms of herbal or general healthcare. An average of one - five deliveries in a month was reported from the homes. Many (58 %) of the homes were operated by the birth attendants with the support of family members Table . TBAs weUncovered wells were the most common source of water in the TBHs. Other sources included springs, boreholes, rain, covered well, and public tap water. Some birth homes (36 %) relied on two or more sources. Unimproved water sources were the most common (66 %), followed by improved sources (22 %) and a combination of both sources (22 %) Fig. . 64 % ofWalkthrough assessment collaborated data from the interview and revealed inadequacies in water storage as storage containers in many of the TBHs were visibly soiled. Stored water was visibly contaminated with debris or a film on the surface Fig. . In addiAbout two-thirds of the birth homes (68 %) had no toilet facility reserved for use by TBAs and women in delivery. Access to improved toilet facilities was found in 34 % of the centers Fig. . StandarThere were no provisions for sharps disposal in any of the birth homes. None of the centers had a standard protocol for healthcare waste disposal Fig. .Fig. 4WReliable handwashing points with reliable water and soap was found in only 8 % of the delivery rooms Fig. . All birClean Hands: All TBAs reported the use of gloves for vaginal exams or when handling the baby. However, correctness of glove use was not established among any of the TBAs. There was also a lot of negligence on the correct use of gloves including re-use of gloves and improper storage of gloves Fig. .Fig. 7SClean Hands: Although handwashing was reported by 76 % of the TBAs, adherence to WHO recommendations on the five moments of hand hygiene was not reported or described by any of the TBAs. Other TBAs regarded glove use as synonymous to handwashing. None of the TBAs met established guidelines for clean hands Fig. .Clean perineum: Assessing of clean perineum was limited to insertion of herbs because TBA inconsistencies with perineal cleaning and cofounding by patient perineal cleaning. Insertion of herbs during delivery was reported by 38 % of the TBAs  reported the use of ties made from threads and clothes; 16 % of these TBAs soaked cord ties in methylated spirit , chlorhexidine gel (12 %), or both (16 %) in cord care Fig. .Availability of adequate WASH services and hygiene practices during births are acknowledged as key to ensuring better outcomes during childbirth –29. The Data from our study across the 50 traditional birth homes highlights gaps in WASH services and clean delivery practices. Findings revealed majority of the birth homes operated under poor WASH conditions. None of the birth centers matched up with the WHO minimum standards for environmental health conditions with regards access to safe and sufficient WASH facilities .First, there was alarming lack of reliable and continual water supply, with majority of the centers lacking access to improved water sources. More than half of the centers had no sanitation facilities for TBAs and mothers’ use. None of the centers adhered to standard protocols on healthcare waste disposal and sterilization. Functional handwashing points were absent in majority of the birth homes. Interviews with TBAs and walkthrough observations also revealed major deficits in TBA knowledge and practice of clean delivery practice for the various components of the six cleans, especially for clean hands, clean perineum, and clean delivery surfaces.Results from this study are not unexpected and corroborate previous reports on the alarming lack of basic WASH services in healthcare facilities in different communities across Nigeria . In a stThe gaps in WASH and clean delivery practices in these traditional birth homes preclude optimal hygiene during delivery. Suboptimal WASH conditions and ICPs predispose and increase the vulnerability of women and their newborns to the risk of life-threatening infections. Although findings from the study are unsurprising due to limited resource availability and accessibility in low-income settings, they should top consideration in strategies that seek to improve newborn and maternal health at the community level.The Nigeria government has several WASH development strategies to ensure provision of safe and accessible WASH services for all of its citizens, however, many of these strategies have failed to translate into actionable plans . Also, tImproved household WASH condition is definitely a plus. Since many of these births with TBAs take place in home environments, improving the quality of WASH facilities in communities will directly impact these births. Clean births can only achievable in birth environments where the most basic level of improved water and sanitation access are present . EffortsThe state of WASH in all healthcare settings need to be continuously monitored to track decline or improvement. It is known that improvements in WASH facilities are not all that is needed , therefoIncreased availability of skilled birth attendants and a reduction in the demand for TBA services remain key strategies for improving newborn and maternal health across the globe. However, findings from this study draw attention to the need to continuously engage TBAs as important interim partners in maternal health care as the transition to skilled attendance continues. Addressing unsanitary conditions during delivery in TBHs is important in avoiding infection-related deaths and achieving global reduction in maternal and neonatal mortality rates. Appropriate policies and protocols should be instituted to ensure infection prevention and control in all maternal health care settings including non-facility settings. Enlightenment and education on clean delivery practices within the communities would also be a valuable tool in reducing maternal morbidity and mortality in these settings.This study was limited in a number of ways. First, the study included only TBAs registered with the TBA association in the capital city of the State. These TBAs do not completely represent untrained birth workers in the state. They are recognized by the state government and may benefit from training programs organized by the government or other NGOs. It is also likely that there is more awareness on general health and wellness unlike other parts of the state. Therefore, the results identified from the study may not entirely reflect what obtains in other geographic regions beyond the area studied, especially in the rural areas where TBA care for most of the pregnancies and delivery.The study does not cover practices during the postnatal period in the birth homes which is also a critical time in maternal and neonatal mortality. Although interviews were supplemented with walkthrough observations, some of the assessments may be subject to recall bias as they depended on the ability of TBAs to recall and report on WASH facilities as well as clean delivery practices.