Effects of smoking cessation before pregnancy were statistically insignificant for the studied outcomes. Smoking in the first trimester was associated with a higher risk of birth weight <10th percentile , LBW , and FGR . Smoking cessation in the second/third trimester was associated with a higher odds ratio of birth weight <10th percentile , FGR , and LBW , p = 0.229), to a similar degree to smoking in the first trimester. The odds ratios were higher in the subgroup of pre-pregnancy body mass index ≥25 kg/m2 for the risk of birth weight <10th percentile  and FGR . The length of cigarette smoking time was also the risk factor for studied outcomes. Conclusions: Smoking in the first trimester increased the studied risks, and the coexistence of excessive maternal weight increased the effects. Smoking cessation during the second/third trimester did not have a protective effect.Many studies have shown that neonates of smoking mothers have a lower birth weight, but several issues remain poorly studied, e.g., the effects of giving up smoking or the combined effects of smoking and maternal obesity. Therefore, we evaluated a prospective cohort of 912 mothers in a single pregnancy, recruited in Poland, in 2015−2016. In the cohort, we recorded 72 (7.9%) newborns with birth weight <10th percentile, 21 (2.3%) fetal growth restriction (FGR) cases, and 60 (6.6%) low birth weight  newborns. In the cohort, 168 (18.4%) women smoked before pregnancy; the mean number of cigarettes/day was 10.8 (1–30), and the mean number of years of cigarette smoking was 8.5 (1–25). Among smokers, 57 (6.3%) women smoked in the first trimester. Adjusted odds ratio (AOR) of newborn outcomes  was calculated in multi-dimensional logistic regressions. Compared to participants who had never smoked, smoking before pregnancy was associated with a higher odds ratio of birth weight <10th percentile (AOR = 1.93, CI: 1.08–3.44,  In recent decades, attention has been paid to factors impacting the fetal environment as important elements of the concept of the development of health and disease (DOHaD) ,2. AdverCigarette smoking is a modifiable environmental factor associated with many adverse pregnancy results, such as low birth weight, preterm birth, and higher newborn mortality ,4,6,7,8.Mechanisms of the relations between smoking during pregnancy and inappropriate weight at birth have not been explained yet. The effect of multi-direction impacts of different components of cigarette smoke on complex multi-factor processes associated with the fetal growth are difficult to assess ,13. In aAlthough many studies have shown that neonates of smoking mothers have a lower birth weight ,17, not The purpose of our study was a detailed analysis of nine categories of smoking (reported by the mother), including smoking before or during pregnancy and smoking reduction or cessation during pregnancy. We assessed smoking as categories and the amount of smoking as a continuous variable. We assessed relationships between various smoking categories and a risk of abnormal birth weight  and other adverse birth outcomes. We assessed the whole cohort and the subgroups of various categories of pre-pregnancy BMI, as well as the subgroup of women who did not develop hypertension or diabetes in the current pregnancy. We conducted this analysis in a prospectively collected cohort.We calculated odds ratios  of the birth outcomes in multidimensional logistic regression. Analyses like ours have not been reported very often. To our knowledge, this is one of the first such studies in Poland.In this analysis, we evaluated a cohort of 912 mother and newborn pairs. It was a prospectively collected cohort of pregnant women, and the study was conducted at the Obstetrics and Gynecology Hospital of the Medical University in Poznań (Poland); it is the center of the highest (third degree) reference for obstetrics and neonatology. The recruitment of women who were at the end of the first trimester of pregnancy was carried out in 2015–2016, and the pregnancy results of all the participants were recorded in 2016–2017.The objectives and design of the study were developed in accordance with the Helsinki Declaration, and were adopted by the Bioethics Committee operating at the Medical University of Poznań, Poland (769/15 and 1092/15). All the women were informed about the voluntary participation in the study and the aims of the study. All of them expressed and signed their informed consent.The recruitment of the participants for the presented study was carried out among pregnant women reporting to the hospital laboratory for routine tests. Brief information about the study was posted in the laboratory to be visible to all patients.The recruitment criteria for the presented cohort included women aged 18–45 (at the conception), healthy single pregnancy (without aneuploidy), gestational age during recruitment of 10–14th week, and delivery in ≥25th gestational week. Women without any chronic diseases were recruited, the exception being abnormal body mass index values. The use of multi-vitamin-micronutrient or folic acid preparations typical for pregnancy was not used as an inclusion or exclusion criterion.During the recruitment period, a total of 1300 women declared their readiness to participate in the study in question over 12 months. After delivery, 388 women were excluded due to the development of hypertension or diabetes before 20th and 18th week (respectively), delivery of a newborn with a birth defect and/or delivery before 25th week, as well as due to incomplete data. Finally, a cohort of 912 mother and child pairs was qualified for further analysis.The first stage of the study (during recruitment) consisted in completing a questionnaire containing questions about sociodemographic and economic data, about the course of the current pregnancy and the history of previous pregnancies, about previous diseases and diseases in the family, as well as about lifestyle, including smoking. The women completed the questionnaire themselves, in the presence of the midwives who clarified any possible doubts. All the participants declared no use of drugs, electronic cigarettes, and alcohol during pregnancy.The next stage of the study was collecting information about the results of pregnancy, which was taken from medical records. The data included child outcomes and maternal complications, as well as information about addictions (including smoking). Each participant was contacted after the end of puerperium (on the phone or by mail) to obtain additional information regarding possible change in the smoking habit in pregnancy and the course of puerperium .N = 912), we recorded the following results: 72 (7.9%) women gave birth to newborns with birth weight <10th percentile, 741 (81.2%) women gave birth to newborns between 10th and 90th percentile, 99 (10.9%) women gave birth to newborns >90th percentile, 60 (6.6%) women gave birth to newborns with birth weight <2500 g, 755 (82.8%) women gave birth to newborns between 2500 and 4000 g, 97 (10.6%) women gave birth to newborns >4000 g, 65 (7.1%) women gave birth to a child before 37th week, and 382 (41.9%) women gave birth by Cesarean section (regardless of the reason). Fetal growth restriction (FGR) was diagnosed in 21 (2.3%) newborns, and preeclampsia was diagnosed in 24 (2.6%) women.In the presented cohort . Birth weight <10th percentile (n = 72) included both constitutionally small newborns  (n = 56) as well as newborns diagnosed with fetal growth restriction (n = 16) confirmed after birth.Low birth weight (LBW) included newborns <2500 g (n = 21) were included in these analyses, including 16 cases with placenta dysfunction being the cause (and with confirmation of post-birth FGR diagnosis and birth weight <10th percentile) as well as five cases with birth weight > 10th percentile (post-birth). Detailed information on criteria used for diagnosis is unknown and likely varied by physician [Fetal growth restriction (FGR) was diagnosed based on ultrasound in pregnancy  ,15,16. Fhysician .Measurement of gestational age was based on ultrasound examination in the first trimester; the basis for assessment at 10 (+0)–13 (+6 days)th week was crown-rump length (CRL), and above the 14th week, a combination of different measurements was taken into account.Major pregnancy complications in the mother were noted from medical records. Hypertension developing in pregnancy  before pregnancy, giving up smoking before pregnancy, smoking in the 1st trimester, giving up smoking in the 2nd/3rd trimester, reduction of smoking during pregnancy in the 2nd/3rd trimester, smoking unchanged throughout pregnancy. Additionally, the value of pack-years was calculated for each woman .Clinical factors of LBW, birth weight <10th percentile, and FGR risk were identified based on the literature .The vari2. Underweight included index <18.5 kg/m2, overweight 25−29.99 kg/m2, and obesity ≥30 kg/m2. The information on the mother’s height and pre-delivery weight was taken from medical records. The difference between pre-delivery weight and pre-pregnancy weight was the gestational weight gain (GWG). GWG categories were defined in line with the Institute of Medicine (IOM) from 2009.In order to conduct subgroup analyses, body mass index (BMI) categories were designated and established for each woman according to the guidelines provided by the World Health Organization. BMI for pre-pregnancy weight was calculated as the quotient of weight (kg) (self-reported) and height (in meters) squared. Normal weight included index 18.5−24.99 kg/mMaternal characteristics were compared for the women who never smoked and those who smoked in the 1st trimester, as well as for the women who gave birth to newborns with birth weight <10th percentile and newborns between 10th and 90th percentile. The characteristics of categorical variables are presented using numbers and percentages of individual categories. The characteristics of continuous variables were presented using median (and quartiles) and/or mean values and standard deviation (SD). The normality of the data distribution was tested by the Shapiro–Wilk test. Due to the lack of compliance of continuous variables with the normal distribution, the comparison of two groups was made using the Mann–Whitney test. The Cochran–Armitage test was used to detect a trend in variables representing the division of a continuous variable into ordered categories. Dichotomous variables, depending on the fulfillment of the Cochran condition regarding the number of individual categories, were compared by the Pearson chi-square test when the Cochran condition was met or the Fisher exact if not.Several analyses were carried out which compared women with the following pregnancy outcomes: fetal growth restriction (FGR) cases vs. no FGR, newborns <10th percentile vs. newborns between 10th and 90th percentile, LBW newborns (<2500 g) vs. newborns 2500–4000 g, newborns >90th percentile vs. newborns between 10th and 90th percentile, newborns >4000 g vs. newborns 2500–4000 g, birth <37th week vs. birth ≥37th week, Cesarean section vs. other deliveries, as well as women who developed preeclampsia (PE) vs. normotensive women.Statistical analyses assessing the effects of smoking on the risk of birth outcomes were performed using one- and multi-dimensional logistic regression; crude odds ratios (OR) and adjusted odds ratios (AOR) were calculated. The odds ratios of birth outcomes for smoking categories was given in relation to the reference category “women who have never smoked” or “smokers in the 1st trimester” as well as “never smokers with normal BMI”. To describe the risk size, the odds ratio together with the 95% confidence interval, CI, was quoted. The statistical significance of the results in the logistic regression was checked using Wald’s test.The one-dimensional logistic regression model was rebuilt and also assessed after dissection of the entire cohort into the pre-pregnancy BMI categories and in the subgroup of the women who did not develop hypertension or diabetes in the current pregnancy .The adjusted odds ratio (AOR) was given in multi-dimensional analyses. Four correcting models were used. Correcting variables were chosen from among risk factors provided by the literature (as above) and on the basis of characteristics of women with newborn <10th percentile and 10–90th percentile . Model-a (AOR-a) included the following variables: maternal age, pre-pregnancy BMI, maternal height, and gestational age at birth. Model-b (AOR-b) included model-a and prior hypotrophy. Model-c for FGR (AOR-c) included maternal age, pre-pregnancy BMI, maternal height, prior hypotrophy, and preeclampsia in the current pregnancy. Model-c for birth weight <10th percentile and LBW (AOR-c) included maternal age ≥40 years, child birth number ≥3, prior hypotrophy, maternal height, gestational age, hypertension in the current pregnancy. Model-d (AOR-d) included primiparous women, pre-pregnancy BMI, maternal age, gestational weight gain outside the range of the recommendations regardless of the BMI category (for FGR) plus fetal sex, gestational age, maternal height, preeclampsia, and gestational diabetes mellitus (for birth weight <10th percentile and LBW). Only “model-a” (without pre-pregnancy BMI) was used in BMI category analyses .1/2. Power 0.5–0.75 is accepted to be the average, whereas power 0.75–1.0 high.Due to the relatively small sizes of the studied groups/subgroups, the analyses also included statistical power (observed power and expected power) . The power was determined for two situations describing the effect size (odds ratios). In the first situation, the observed power—that is, the power for odds ratios obtained (OR) from the study—was calculated. In the second situation, the expected power was the power determined with an assumption that OR is 2 for risk increasing factors and for risk decreasing factor it is The PQStat v1.8.0 software  was used for the statistical calculations. A significance level of 0.05 was adopted in all analyses. In addition, the results of the analyses, i.e., the odds ratios and 95% CI, were visualized on the charts.2) before pregnancy. In the cohort, 137 women developed hypertension in pregnancy, 146 women developed diabetes in pregnancy, and 654 (71.7%) women did not develop hypertension or diabetes.Basic characteristics of the women who smoked in the first trimester are presented in The median age, pre-pregnancy BMI, and gestational weight gain (GWG), as well as the number of primiparous women, did not differ between these groups. In the women who smoked in the first trimester, the median newborn weight was statistically significantly lower, the number of newborns <10th percentile was higher (26.3% vs. 7.0%), the number of newborns <2500 g was higher (17.5% vs. 6.2%), and the number of FGR cases was higher (7.1% vs. 2.2%), compared to the women who had never smoked . Table S2) and the results were statistically significant (yellow points). The results for preeclampsia risk (for comparison with FGR risk) were also presented.Smoking before pregnancy , compareSmoking in the first trimester , compareSmoking cessation before pregnancy , compareSmoking cessation in the second/third trimester , compareSmoking reduction during the second/third trimester (decrease in cigarettes/daily) intensified the risk of the studied newborn outcomes . SmokingSmoking throughout pregnancy , compare2) had higher adjusted odds ratios of birth weight <10th percentile , FGR , and LBW  for smoking in the first trimester compared to never smokers .The coexistence of maternal underweight and smoking in the first trimester increased the FGR risk, compared to never smokers with normal BMI , the results for birth weight <10th percentile, FGR, and LBW were similar to the results in the whole cohort .p = 0.001) and LBW by 13% . The results for FGR were not statistically significant.p = 0.068). The relationships with preterm birth <37th week  or Cesarean section  were insignificant.In this prospective study, we assessed a cohort of 912 mother–child pairs, in order to examine the relations between nine smoking categories reported by mothers and the risk of birth weight <10th percentile, fetal growth restriction (FGR), and LBW (birth weight <2500 g). Many of the results of this detailed analysis were found to have a high statistical power . The studied groups/subgroups, however, were often small and some confidence intervals were wide; therefore, our conclusions need to be cautious.Firstly, our results can suggest that smoking in the first trimester involves a risk for lower birth weight. Secondly, it is possible that smoking cessation or reduction in the second/third trimester has no protective effect or can even intensify the analyzed risks. At the same time, our results can imply a protective effect of smoking cessation before pregnancy, particularly in comparison with those who smoked in the first trimester. Thirdly, combined effects of smoking in the first trimester and obesity/overweight are also possible. Analyses in various BMI categories and differences in the odds ratios (for smoking in the first trimester), as compared to the reference category of never smokers or those without obesity, can suggest a strong impact of obesity/overweight on the studied risks, as well as a strong impact of underweight on FGR risk. Fourthly, we have found that a longer time of smoking increased the considered risks. Importantly, high odds ratio values in some analyses and their high statistical power means that our results cannot be accidental.Our results for birth weight are in line with previously published studies. Many studies, including the meta-analyses by Abraham et al., showed relationships of smoking during pregnancy with lower fetal measurements, including low birth weight (LBW), small-for-gestational age birth weight (SGA), and FGR ,18,30,31In our study, the number of smokers was low. This may suggest the beneficial effects of various government campaigns to reduce this addiction . In our n = 21), and the statistical power  was small for all the results. Confidence intervals in FGR risk were wide, indicating the role of the sample small size. Importantly, in this study, we have examined all the FGR (n = 21) cases diagnosed based on ultrasound (USG) examination during pregnancy, 16 (76%) of which were identified after birth as growth potential restriction of a fetus with birth weight <10th percentile. Our approach could have contributed to the lack of statistical significance of some results for FGR. As compared to the results for FGR, smoking in the first trimester also increased the risk of preeclampsia (PE), but in a weaker and statistically insignificant way  and we treat this part of the analysis as a pilot study that requires further investigation  to a similar degree to smoking in the first trimester . Contrartigation .p < 0.0001) gave birth to newborns with FGR diagnosed during pregnancy. In our cohort, the number of PE cases was also small (n = 24 (2.6%)), and it is consistent with the recruitment criteria for the cohort; during recruitment, we excluded chronic diseases  and multiple pregnancies that increase the risk of preeclampsia.The comparison of our results from this and our previous research is important, because FGR often coexists with PE, and in the pathogenesis of both cases, the role of placental ischemia resulting from abnormal spiral arterial transformation in early pregnancy is taken into consideration ,10,15. I2) increased the risk for birth weight <10th percentile and FGR  are not fully understood. The main factors affecting fetal growth include the placenta, which guarantees the flow of nutrients to the fetus ,15,16. FTobacco smoke contains several thousand chemicals, including many toxic substances that exacerbate inflammation and oxidative stress, which can be harmful to placental development and/or be passed through the placenta to the fetus ,14. DiffOn the one hand, in in vitro tests, nicotine disturbs the transformation of spiral arteries, which increases the risk of pregnancy complications ,9,10. OnThe mechanisms of the combined effect of obesity and smoking are not fully explained. Relations between obesity and FGR, low birth weight (LBW), or preeclampsia were found in literature studies; this can suggest that obesity contributes to placental blood flow impairment . The strThe major clinical implications of this study relate to the adverse effects of smoking in the first trimester and the result which confirms that a longer time of smoking increases the risk of lower birth weight. In the current study, only smoking cessation before pregnancy was associated with lower chances of studied outcomes; results for this smoking category compared to smokers in the first trimester had high statistical power and statistical significance. Confirmation (in other studies) of the paradoxical intensification of the risk of adverse fetal outcomes in women who reduced their smoking during pregnancy implies caution in making such decisions. Smoking, however, has many adverse effects; therefore, even a lack of protective effect in women who gave up smoking in the second/third trimester can turn out to be beneficial.An advantage of the current analysis is the prospective model of the study . We have assessed several smoking categories. We adjusted our results for many risk factors; however, it is possible that there are other confounders that affect our results. The participants came from one region, which means that study and control groups were matched in terms of diet (common to the region) and level of prenatal care.The limitation of our study was the lack of information about passive smoking. The reduction in the frequency of smoking among women meant that in a large enough prospective cohort, there were few women in subgroups, e.g., women who reduced their smoking during pregnancy. However, despite the small quantity of some subgroups, the statistical power of many results was high. In our study, smoking was self-reported by pregnant women but the information obtained during recruitment was verified after the end of pregnancy (and puerperium). Additionally, the information obtained by means of questionnaires was compared with the information included in medical documentation; ultimately, there was no discrepancy between information from the three sources.Our results can suggest that smoking in the first trimester involves a risk for birth weight <10th percentile, fetal growth restriction (FGR), and LBW (birth weight <2500 g). It is possible that smoking cessation or reduction in the second/third trimester has no protective effect or can even intensify the analyzed risks. Nevertheless, other aspects of the harmful effects of smoking on fetal development need to be remembered. At the same time, our results suggest that smoking cessation before pregnancy could be the only safe strategy for reduction of studied risks. We have also found that a longer time of smoking increased the considered risks.All women should avoid smoking during the first trimester, with special attention and support for the high-risk group of women with non-optimal BMI.Our studied groups/subgroups were often small; however, high odds ratio values in some analyses and their high statistical power mean that our results cannot be accidental. Future prospective studies in larger groups are recommended.


Neonatal mortality (over the first three weeks of life) is a major concern in canine breeding facilities as an economic and welfare issue. Since low birth weight (LBW) dramatically increases the risk of neonatal death, the risk factors of occurrence need to be identified together with the chances and determinants of survival of newborns at-risk.Data from 4971 puppies from 10 breeds were analysed. Two birth weight thresholds regarding the risk of neonatal mortality were identified by breed, using respectively Receiver Operating Characteristics and Classification and Regression Tree method. Puppies were qualified as LBW and very low birth weight (VLBW) when their birth weight value was respectively between the two thresholds and lower than the two thresholds. Mortality rates were 4.2, 8.8 and 55.3%, in the normal, LBW and VLBW groups, accounting for 48.7, 47.9 and 3.4% of the included puppies, respectively. A separate binary logistic regression approach allowed to identify breed, gender and litter size as determinants of LBW. The increase in litter size and being a female were associated with a higher risk for LBW. Survival for LBW puppies was reduced in litters with at least one stillborn, compared to litters with no stillborn, and was also reduced when the dam was more than 6 years old. Concerning VLBW puppies, occurrence and survival were influenced by litter size. Surprisingly, the decrease in litter size was a risk factor for VLBW and also reduced their survival. The results of this study suggest that VLBW and LBW puppies are two distinct populations. Moreover, it indicates that events and factors affecting intrauterine growth (leading to birth weight reduction) also affect their ability to adapt to extrauterine life.These findings could help veterinarians and breeders to improve the management of their facility and more specifically of LBW puppies. Possible recommendations would be to only select for reproduction dams of optimal age and to pay particular attention to LBW puppies born in small litters. Further studies are required to understand the origin of LBW in dogs. The mortality rate of puppies during the first two months after birth is around 15% of the total number of puppies born . As puppLow birth weight (LBW) is known to be an important risk factor for puppy mortality and morbidity during early life –6. NewboA definition of LBW based on the birth weight threshold associated with an increased risk of neonatal mortality, has recently been proposed . In thisA better understanding of the determinants of LBW and factors influencing survival is crucial. Intrauterine growth regulation is complex and many factors  can affect foetal growth rate and, ultimately, birth weight –14. AvaiThe first aim of the present study was to define low and very low birth weight (LBW and VLBW) in different dog breeds. Secondly, to investigate the association between the occurrence of LBW or VLBW and different maternal and foetal factors. Finally, to explore the factors identified in each category of at-risk puppies (LBW and VLBW) that influence survival during the neonatal period.The population, after selection Fig. , consistAmong the 10 represented breeds, 48.7%  of the puppies were of normal birth weight (NBW), 47.9%  were in the low birth weight (LBW) category and 3.4%  in the very low birth weight (VLBW) category  to 32% (Bichon Frise). Distribution of the puppies in the different birth weight categories was influenced by litter size  but no statistically significant difference was found for the two other comparisons (LBW vs VLBW and NBW vs VLBW).The results of the three binary logistic regressions are given in Table P = 0.027).Neonatal mortality rates were 4.2% for NBW puppies , 8.8% for LBW , and 55.3% for VLBW . The proportion of deaths occurring between the early (0–2 days) and late (3–21 days) neonatal periods differed significantly according to the birth weight category  revealed that survival was influenced by litter size, but not gender. The odds of dying decreased significantly with the increasing litter size  showed that survival was influenced by dam age at whelping, the presence of a stillborn in the litter and breed, but not by litter size  . The breIn this study, ROC and CART analyses were combined to define the birth weight thresholds within each breed and to identify puppies with an increased risk of neonatal mortality . The pupMultiple factors  can cause intrauterine growth retardation, leading to low birth weight . Some foIn many species, including dogs, males are consistently heavier at birth than females , 18, 19.Previous studies of the influence of litter size on birth weight reported lower birth weights in large litters than in small litters , 5. LittThe consequences of intrauterine growth restriction, including its relationship with neonatal mortality, have already been studied both in dogs and in other polytoccous species like pigs , 14, 21.Dam age at whelping had no significant influence on the occurrence of LBW or VLBW Table  but had In this study, litter size influenced not only the occurrence of VLBW puppies but also their survival which was reduced in smaller litters. The factors affecting the occurrence of VLBW born in small litters might be the same as those explaining their reduced survival. For example, as in humans, placental insufficiency leading to foetal malnutrition might be one such factor . It alsoNot all the parameters that may influence the occurrence of LBW and VLBW and subsequent survival can be discussed here. Further investigations involving data collection for other factors associated with neonatal health and described in other species are required .Early detection of at-risk puppies with low birth weight is essential to reduce the neonatal mortality rate in canine species. This work confirms the necessity to conduct breed-specific analyses, with appropriate thresholds, when working on canine species. Litter size and dam age at whelping were found to be important factors affecting the occurrence of LBW or VLBW and subsequent survival. Particular attention needs to be paid to LBW and VLBW puppies born in small litters and/or from a dam more than 6 years old to ensure increased survival and thus improve animal welfare and kennel performance. Moreover, beyond the neonatal period, for surviving puppies, further research is required to study the mid-term and long-term consequences of reduced birth weight.The data used in this study were collected through a questionnaire , using two thresholds. One was determined by Receiver Operating Characteristics (ROC) analysis, and the other by Classification and Regression Tree (CART) analysis (detailed in ). Since P < 0.001; Kruskal-Wallis rank sum test). Before analysis, these three parameters were transformed to allow between-breed comparisons. The birth weight values were normally distributed and were standardised by breed (formula: Y = (X – mean) ÷ SD). Litter size and litter heterogeneity were normalised by breed (formula: Y = (X – min) ÷ (max – min)).The age (in years) of the dam at whelping was categorised into four groups: min-2; > 2–4; > 4–6; > 6-max, the reference category being “dam between 2 and 4 years old”. Litter heterogeneity represented the within-litter variation in birth weight and was expressed as the coefficient of variation (CV = standard deviation (SD) ÷ mean × 100 [Three binary logistic regressions, allowing two-by-two comparisons, were applied to identify factors associated with the likelihood of belonging to a given category . For each model, puppies belonging to the two categories involved in the paired comparison were extracted from the full dataset and their category represented the outcome . The candidate explanatory variables were: dam age at whelping, litter size , stillborns in the litter (absence or presence), puppy sex and breed.Factors related to the survival of LBW and VLBW puppies were analysed by constructing two binary logistic regressions. Puppies in the category under consideration (LBW or VLBW) were extracted from the full dataset and the binary variable  represented the outcome. The candidate explanatory variables were dam age at whelping, litter size, stillborn in the litter, sex, breed and litter heterogeneity.P of 0.1 or less in the univariable analysis were included in the corresponding multivariable logistic regression [For each model, preliminary bivariate analyses were applied to select candidate predictors . All candidate explanatory variables associated with the outcome with a gression .P and odds ratio for each parameter, AUROC and P for the Pearson residuals test) were combined using the median.The multivariable analyses were conducted by randomly dividing the selected subdataset into a training set and a testing set containing 70 and 30% of the data, respectively. The model was fitted on the training set and classification performances were evaluated on the testing set. The area under the ROC curve (AUROC) was then estimated to assess the ability of the model to differentiate puppies from the two considered categories . The appropriateness of the model was assessed by Pearson residuals test. This process was repeated 1000 times for each comparison and the results . The results from descriptive models were considered if the classification accuracy was acceptable (i.e. median AUROC over 0.65).Statistical analyses were performed with R version 3.4.2  and the nd rpart , 29. TheAdditional file 1. Location of the breeding kennels included in the analysis.Additional file 2. Survey.


The field of droplet electrohydrodynamics (EHD) emerged with a seminal work of G.I. Taylor in 1966, who presented the so-called leaky dielectric model (LDM) to predict the droplet shapes undergoing distortions under an electric field. Since then, the droplet EHD has evolved in many ways over the next 55 years with numerous intriguing phenomena reported, such as tip and equatorial streaming, Quincke rotation, double droplet breakup modes, particle assemblies at the emulsion interface, and many more. These phenomena have a potential of vast applications in different areas of science and technology. This paper presents a review of prominent droplet EHD studies pertaining to the essential physical insight of various EHD phenomena. Here, we discuss the dynamics of a single-phase emulsion droplet under weak and strong electric fields. Moreover, the effect of the presence of particles and surfactants at the emulsion interface is covered in detail. Furthermore, the EHD of multi-phase double emulsion droplet is included. We focus on features such as deformation, instabilities, and breakups under varying electrical and physical properties. At the end of the review, we also discuss the potential applications of droplet EHD and various challenges with their future perspectives. In recent years, electrohydrodynamics (EHD) of emulsion droplets has evolved in many ways as the research gained pace in this field. The field emerged with the seminal works of Taylor in 1966, who presented his leaky dielectric model (LDM) and predicted the single-phase droplet steady deformation . SubsequThis review has emphasis on the recent advancements for the essential physical insights of the droplet EHD problems. Since droplet EHD is a very broad area of research and it is not possible to cover all the aspects in a single review, here we mainly focus on the behavior of a single-phase droplet with particle-free or particle-covered interface and multi-phase (compound or double) emulsion droplets within the perspective of their deformation, instabilities, and breakups under weak and strong external stimuli of electric fields, considering only the most prominent works See . Here, wThe approaches to study the fundamentals of droplet EHD are twofold: (i) theory and/or numerical method, and (ii) experiments. The basic analytical model is the LDM ,2,53. ThIt was a general perception before the seminal work by Taylor that the liquid droplets submerged in another immiscible liquid behave as a perfect dielectric and that their exposure to an electric field will always result in prolate deformation  of the interface . This coa, under an unperturbed external electric field strength For a droplet of radius, The droplet EHD problem depends on the non-dimensional parameters based on the liquid properties, such as conductivity ratio (Oh), and controls the transient modes of deformation, such as monotonic and oscillating,The ratio of the viscous to the inertial force and surface tension is called the Ohnesorge Number , which is the ratio of the electric capillary number and electric Reynolds number and decides the droplet breakup mode transitions,Another important non-dimensional number is the Saville number  shows that the electric field is irrotational and thus, Gauss’s law is given as:Conservation of charge is another important governing equation given as: The above equation shows that the charge movement is owing to the Ohmic conduction and the convection, respectively. Here, The above equation shows that the volumetric charge decays by time exponentially and becomes zero in the bulk. Where, As the electric field is irrotational and sults in:6∇2V=0,wnditions ,(17)V1 and (19), respectively.The Maxwell stress tensor is given as :(20)TM=εThe divergence of Maxwell stress tensor determines the electric force per unit volume of the liquid  gives:As discussed above, if the charge instantly accumulates at the interface, for example, for a leaky dielectric system, the volumetric charge density This shows that the electric force affects the solution only through the momentum jump boundary condition at the interface. The solution of the resulting Stokes equation can be obtained by introducing a stream function at the two sides of the droplet interface .(27)ur=The solution using the above boundary conditions at a steady state gives:The velocity field is obtained from Equations (27) and (28).From these equations, it is evident that the flow direction is from the poles to the equator if The deformation or distortion of the droplet interface is caused by the electric and the hydrodynamic fields being discontinuous at the interface, resulting in the interfacial stress jumps countered by the interfacial capillary stresses.For small deformations:R and S of the system. If Substitution from Equations (22), (39) and (41) in Equation (38) gives a deformation of droplet in a uniform electric field:fd=0 See A. The deAjayi  used a dgiven as ,(43)DDCThe transient deformation, given as :(45)DDC See C. At ω=ωTo evaluate how much oscillatory deformation (Obtained from Equations (47) and (50). The dimensionless parameter D). Taylor’s LDM predicts the deformation of droplets within low-deformation limit, i.e., when As discussed in re found ,110,111.Oh) and the Reynolds number (Re) [The transient deformation of a droplet essentially depends on two dimensionless numbers, Ohnesorge number (ber (Re) . When Reions See A,B. FengAlthough the sense of steady-state flow circulation pattern in an AC electric field is the same as that in a DC electric field, the sense of ion (49) ,101,112.)a/γ See D. Additiycle See E. EsmaeeThe dynamics of a droplet under a strong electric field when In most of the studies related to single-phase droplets, the droplet generally settles to an equilibrium shape under a relatively weak electric field. However, if the strength of the electric field is high and  regimes ,61,113.  regimes  used an  regimes  theoreti regimes . For the0.25 See B. HoweveTwo important modes of breakups are reported, such as end pinching and tip streaming ,115. In ecreases . It is aThe droplet undergoes electro-rotation under a strong electric field if ects See A [18,90,given as ,120:(53)The equation is obtained by the balancing of electrical and viscous torques and is independent of the size of sphere. The equation is often used to estimate the threshold of electro-rotation for the liquid droplets and comparison with experimental results shows a relatively good approximation . ResultsA colloidal particle or Pickering droplet is formed when the particle-laden droplet or suspension, for which ic field ,134,135.ic field ,134. Higic field   and two interfaces (core/shell and shell/ambient), have gathered special attention due to their vast applications in various scientific areas, such as in targeted drug delivery , artificThe dynamics of a double-emulsion droplet within a weak electric field or small deformation limit is extensively reported and well understood ,150,151.nterface ,93,143. derstood ,149,150.derstood  conductederstood  showed tBehjatian and Esmaeeli  studied nd S See A. They ation See C. While This feature of multi-phase droplets is relatively less explored. Only a few studies have explored the breakups within a certain range of electric and hydrodynamic properties ,95,152. EHD-based electric shear stress is used to produce tiny droplets by cutting off the liquid interface formed under the nozzle, which is called electrospray . Also, iThe electric field can be used to produce and manipulate various types of droplets that are not spherical. Brosseau et al. used cross-linking UV curable polymer  drop to make spheroidal shapes via EHD deformation . On the When rotation ,166. Manrotation ,167, motrotation , pump [1rotation , and so rotation . The eleThe double emulsion type capsulation technique is an important technique in the process of drug delivery or producing functional particles, but the resulting double emulsion may be too unstable or too stable for these purposes. However, if an electric field is used, the core droplet can be moved to the center ,148 or eIn this review, we discussed the droplet EHD in detail. We discussed the features such as droplet deformation, instabilities, and breakups for single-phase and multi-phase droplets. The single-phase droplet deforms prolate or oblate and undergoes instabilities above a threshold value of the electric field. The low limit deformation is well-predicted by so-called Taylor’s leaky dielectric model, which is the most established theoretical model to date for the droplet electrohydrodynamics, neglects the effects of diffuse charge layers near the interfaces, and thus is incomplete to explain the electro-migration of droplets under a direct current electric field. Therefore, an effort to derive a complete model incorporating the electro-kinetic theory for liquid-liquid interface is required. Interesting phenomena, such as tip streaming or equatorial streaming, can be used to produce many tiny droplets. Particles at the emulsion interface can be manipulated to produce intriguing assemblies, such as belts, chains, and caps, by controlling the physical and electrical parameters. High particle concentration can produce colloids undergoing rotating flows under a strong electric field. Use of surfactant molecules alter the deformation and droplet breakup dynamics. Development of simulation models are required to understand the electrohydrodynamics of more complex interfaces, and those laden with surfactant or coated with particles merit further investigations. A double-emulsion droplet shows different breakups and can be controlled to produce Janus and ternary droplets. Simulating three-phase emulsion droplets under an electric field is likely to display further interesting dynamics.The applications of droplet EHD can be broad, such as mass production of micro droplets, functional particle synthesis, development of micro-rotors, rigid ellipsoids, or complex shapes, and so on. Such a method could then be implemented on a microfluidic platform, if a large quantity of rigid, deformed complex shapes is to be produced. However, unfortunately the demonstration of droplet EHD on a mass scale to produce species or development of new products has still not been fully demonstrated. Still, there remain many issues and challenges that are either less explored or not explored at all and warrant further investigations.


Varanus and the pythonid snake Python and adds to the so far rather poorly known squamate fossil record from Africa. On the basis of the new remains, Moghra marks the oldest so far described co-occurrence of Varanus and Python in the African continent. The close sympatry of these two genera in the African fossil record is thoroughly analyzed and discussed, a co-existence, which is still widespread in the extant herpetofauna of the continent. Situated rather close to the so called “Levantine Corridor” and dated at the Burdigalian, practically when Afro-Arabia collided with Eurasia, the Moghra squamate assemblage offers the potential of important insights in the biogeography and dispersal events of vertebrate groups during the early Miocene.Lizard and snake remains from the early Miocene  of the Moghra Formation, Egypt, are described herein. This material comprises the first fossil remains of squamates recovered from the otherwise rich and well known vertebrate assemblage of Moghra. The material pertains to two different genera, the varanid lizard  Varanus and Python are among the most iconic squamates. They are both almost immediately recognizable even to the general public, commonly known as monitor lizards and pythons respectively. Varanus comprises the largest extant species of lizards, while certain species of Python rank among the longest and heaviest species of snakes  of the Moghra Formation, Egypt. These are the first squamates described from this locality, which is otherwise well known for its fossil mammals and has proven pivotal for our understanding of vertebrate biogeography and diversity in the African Miocene  is located at in the northern Western Desert, Matruh Governorate, Egypt, around 60 km south of El Alamein  are derived from the lower–middle part of the section (fossil horizon F1), dated between 19.6–18.2 Ma, however, a few specimens are derived from deposits approaching 17 Ma .Description—Both vertebrae are large and incomplete , and the presence of striae on the neural arch .Description—The vertebrae are all moderately large with centrum lengths ranging between 6 and 11 mm. CUWM 137 (centrum length = 8.8 mm) is nearly complete, though still lacks most of the neural spine. CUWM 160 (centrum length = 8.0 mm) lacks the dorsal part of the neural spine and part of the left prezygapophysis, while its paradiapophyses, cotyle, and condyle are eroded. DPC 14560 (centrum length = 8.9 mm), lacks part of the neural spine and the posteriormost portion of the neural arch. DPC 14600 is the largest vertebra (centrum length = 10.9 mm) and lacks its right postzygapophysis and the dorsal portion of the neural spine. DPC 14530 (estimated centrum length around 7 mm) lacks much of the cotyle, right prezygapophysis, neural spine, and the anteroventral and posteroventral portions of the centrum. CUWM 9 (centrum length = 6.1 mm) lacks most of the posterior portion of the neural arch, neural spine, and both postzygapophyses, while its condyle and paradiapophyses are strongly eroded. In all vertebrae, the centrum is distinctly wider than long. The zygosphene is thick, massive, and with a zygosphenal tuberosity  or the African species (P. maurus). Morocco , by its Varanus and Python, occur sympatrically throughout much of the continent and the fossil record demonstrates that this sympatry may have occurred much earlier   of Rusinga Island, Kenya  . Additioabonian) . The eard, Kenya , anotherd, Kenya , and at d, Kenya . No othes Africa .Python is not well documented in the African fossil record. Paleogene records of large “booids” in Africa do exist  of Beni Mellal, Morocco, which represents the sole named extinct pythonid taxon from Africa (Python. Outside Africa (sensu stricto), Python has been reported also from the early Miocene of Saudi Arabia  mark the oldest co-occurrence of the genera Varanus and Python in Africa and document that this close sympatry between these two large squamates occurred in Africa already by the early Miocene  and outside the modern geographic extent of their co-distribution , denoting a rapid geographic expansion of the genus during that time  of Subpiatră 2/1, Romania  age, with the genus becoming ultimately extinct in the continent shortly thereafter, during the middle Miocene  and diversification to Africa  from Fayum, Egypt (Varanus remains are known from all major continents of the Old Word (Europe, Africa, Asia, and Australia; see Varanus and Python from Moghra, renders this Egyptian locality as one of only a few among the whole Mediterranean area that yielded remains of these large squamates (The geographic location of Moghra, being situated rather close to the “cord see . Neverthand Asia . The currope see —note thadigalian . On the o Africa ; howeverm, Egypt . The facquamates .Varanus and Python across the Neogene of Africa, continuing also up to today, is reminiscent of other such cases of large lizards and snakes coexisting together across large geographic distances and stratigraphic spans during the Cenozoic. Such is the case of Palaeovaranus and Palaeopython found together in multiple localities across the Eocene of Western and Central Europe (see Varanus and Python.The sympatry of rope see , a case Varanus and Python in Africa. Furthermore, Moghra marks the earliest co-occurrence of Varanus and Python in the African fossil record. The sympatric occurrence of Varanus and Python, a case that is widespread in the extant herpetofauna of Africa, is thoroughly discussed. The geographic location of Moghra, along with its stratigraphic position, seems to have played a pivotal role for dispersal events between African and Eurasian squamates. Furthermore, the identification of a strongly thermophilous reptile, such as Python, in the Moghra fossil assemblage provides temperature and climatic constraints and enables a better understanding of the palaeoenvironment of this locality, which is crucial for our understanding of the African Miocene. The new Moghra material adds to the so far poorly known fossil record of squamates from Egypt, so far confined to few but nevertheless rather important descriptions of agamid and varanid lizards and palaeophiid, madtsoiid, “booid”, russellophiid, and colubroid snakes all from the world famous Eocene of Fayum (New varanid and pythonid fossil remains from the early Miocene of Moghra expand the known fossil record of of Fayum , as wellof Fayum .


Most cases of hydatid cysts form in the liver and lung and other tissues are considered as unusual locations in hydatid cysts. The current study aimed to find out the rate and features of hydatid cysts in uncommon locations in Fars Province, Southern Iran, over a 15-year period.  The hospital records of patients who underwent surgery for hydatid cysts in university-affiliated hospitals in Fars Province, Southern Iran, from 2004 to 2018, were retrospectively reviewed. For each patient, clinical and demographical data were recorded.  During a 15-year period, a total of 501 patients were surgically treated for hydatid cysts, and out of these, 46 (9.2%) were presented with the unusual locations of hydatid disease. Males constituted 28 (60.9%) of these patients while 18 (39.1%) of the patients were females. The patients' age ranged from 5 to 80 years . The size of the cysts ranged from 2 to 20 cm . The most common unusual location for the hydatid cyst was the spleen with 30.4% of cases, followed by the pelvic cavity (15.2%). Out of 46 cases with unusual location of the hydatid cyst, 10 (21.7%) cases had lung, 22 (47.8%) cases had liver, and 5 (10.9%) cases had both liver and lung hydatid cysts, simultaneously with cysts in unusual locations.  In cystic echinococcosis- (CE) endemic areas, hydatid disease can affect any organ, from head to toe, in humans. The disease should be considered in the differential diagnosis of any cystic entities anywhere in the body. Echinococcus granulosus. The disease is widespread worldwide and is very common in the Middle East countries, including Iran, Iraq, and Turkey [Cystic echinococcosis (CE) or hydatid cyst is a zoonotic parasitic disease caused by the larvae of d Turkey –3. Dogs d Turkey . Once ind Turkey . Tissuesd Turkey –8. Out od Turkey –11.Other tissues that are rarely affected by hydatid cysts include the perianal area, infratemporal region, or neck , 12–14. The hydatid cyst of the brain is relatively unusual, and only 2% of CE cases are reported to be in the cerebral location .The development of hydatid cysts in an uncommon location of the body creates a major diagnostic dilemma, and it may mimic other entities that make it challenging to differentiate from other diseases. Above this, the lack of specific radiological findings makes it difficult to differentiate the disease in such cases. Therefore, in any CE-endemic area, a diagnosis of hydatid disease should be suspected in all cystic masses in the body. The current study aimed to find out the rate and features of CE in uncommon locations in Fars Province, Southern Iran, by evaluating the hospital records over a 15-year period.The hospital records of patients who underwent surgery for hydatid cysts in university-affiliated hospitals in Fars Province, Southern Iran, for 15 years, from 2004 to 2018, were retrospectively reviewed. Cases were considered as hydatid cyst when the pathological examination confirmed the hydatid cyst by examining the surgically resected specimens postoperatively. Only cases with a definitive diagnosis of hydatid cysts located outside the hepatopulmonary areas, with the unique disease code for hydatid cyst , were included in the data study. For each patient, clinical and demographical data, history of previous hydatid cyst, and cyst features, along with laboratory data and applied treatments were recorded in a data sheet. Hydatid cyst in patients with no history of hepatopulmonary hydatid cyst was considered as primary cases while cases with the previous history of hepatic and/or pulmonary hydatid cyst were considered as secondary cysts.The study was approved by the Ethics Committee of Shiraz University of Medical Sciences (SUMS).SPSS  was used for data analysis. Demographics characteristics were analyzed by descriptive data analysis and presented either as mean and standard deviation (SD) or median and interquartile (IQR) as appropriate, based on normality of distribution. Chi-square or Fisher's exact test was utilized to find out the differences among the categorical variables.During a 15-year period, between 2004 and 2018, a total of 501 patients were surgically treated for hydatid cysts in two main referral hospitals in Fars Provinces, Southern Iran. Out of these 501 cases, 46 (9.2%) were presented with the unusual location of hydatid disease.Males constituted 28 (60.9%) of unusual location cases while 18 (39.1%) of the patients were females. The patients' age ranged from 5 to 80 years , with the highest frequency (20%) among 20 to 29 years and also 50–59 years of age. The patients' hospital admission duration ranged from 3 to 55 days .2) of the area of cysts based on the area of the oval/circle formula; largest radius  multiplied by the smallest radius  multiplied by Pi (3.14). The size of the cysts ranged from 2 to 20 cm  for the largest diameter of the cyst, while the area of the cysts ranged from 3 to 314 cm2 . To evaluate the area of the cyst in our study, we decided to calculate the square centimeters , followed by the pelvic cavity (15.2%). Out of 46 cases with unusual locations of hydatid cyst, 10 (21.7%) cases had pulmonary, 22 (47.8%) cases had hepatic, and 5 (10.9%) cases had both hepatic and pulmonary hydatid cyst, simultaneously with cysts in unusual locations. Among the patients, 2 (4.3%) had the following occurrence of a pulmonary hydatid cyst after being treated for a hydatid cyst of an extrapulmonary location (one cardiac and the other spleen). Also, 3 (6.5%) had the following occurrence of hepatic hydatid cyst after treating the cyst in an unusual location . Also, 3 (6.5%) of the patients  had a previous history of hydatid cyst of the liver.Based on the radiological evaluations for hydatid cyst, out of 19 computed tomography (CT) scans conducted for the evaluation of hydatid cyst in other locations, all were positive and were characteristic and highly suspected of hydatid cyst (sensitivity 100%). However, based on the sonography evaluations, 25 out of 27 (92.6%) were characteristic and highly suspect of hydatid cyst. Also, in two cases  hydatid cyst), magnetic resonance imaging (MRI) had characteristic and highly suspected of hydatid cyst.Regarding the surgical treatment of the cases in our study, 17 (37%) underwent radical surgery while 29 (63%) underwent conservative surgery. Regarding the surgical procedures, 24 (52.2%) underwent laparotomy, 16 (34.8%) evacuation of hydatid cyst, 8 (17.4%) unroofing, 6 (13%) excision, 5 (10.9%) resection, 3 (6.5%) drainage, and 13 (28.3%) underwent organ removal. Among the primarily unusual location hydatid cysts, during the mentioned period of our study, only one case of relapse in another unusual location (other than the lung and liver) was observed which developed primarily in the liver and subdiaphragmatic area and relapsed after 3 years in the mesothelium of the terminal ileum.Hydatidosis is considered a re-emerging disease even in regions that used to have a low prevalence of the disease. This may be due to the lack of preventive measures caused by economic problems and also lack of resources, subsequently increasing the concern for public health issues . HydatidIn a systematic review by Gramizadeh in 2013, from 463 published cases of the hydatid cyst during 20 years, in the unusual location from Iran, the most common locations were reported to be the CNS, musculoskeletal system, heart, and kidney, whereas some less common locations were the spleen, pancreas, appendix, thyroid, salivary gland, adrenal gland, breast, and ovary . A studyAfter the liver and lungs, the most common organ involved in hydatid cysts is usually the spleen. Spleen infestation usually occurs by the arterial route if the parasite passes through pulmonary and hepatic filters or via a retrograde venous path in patients with portal hypertension, bypassing the liver and lung , 21. FurIn our study, out of 46 hydatid cysts in unusual locations, 30.4% of cases were belonging to the spleen which is 2.8 percent of all reported hydatid cysts (501) in this region. Splenic hydatid cyst may present as a secondary cyst as a result of the rupture of a previous cyst, yet primary are frequently reported , 24, 25.Spinal hydatid cysts often have unusual clinical presentations sometimes imitating malignant tumors. Such cases have been misguided as a spinal tumor on radiological examination and were diagnosed as hydatid cyst on fine-needle aspiration and histopathological examination .Bone hydatid cyst, which mostly affects the spine, accounts for 0.5% to 2.5% of hydatid cyst cases , 28, 29.Cardiac hydatid cyst accounts for about 0.5–2.5% of all hydatid cyst cases in humans , 30. It Soft tissue hydatid disease is rare and occurs in 1% to 4% of hydatid cyst patients. Muscles are not suitable for hydatid cysts to grow, yet hydatid cyst of Psoas muscle, inguinal area, and proximal muscles of the upper and lower limbs are reported in the literature –34.In the current study, a kidney hydatid cyst was seen in 2.1% of cases. In Geramizadeh's study, hydatid cyst of the kidney and the urinary tract was reported in 31 out of 463 (6.7%) cases, reported in the literature .In our series, there were some very rare locations of hydatid cysts including the common bile duct, diaphragm, and pancreas.In a systematic review by Sokouti et al.  comparinThe center in which our study was performed is a referral center in Southern Iran which results in a high number of patients visiting due to various complaints. Since hydatidosis is endemic in our country, this differential diagnosis should not be neglected in patients with complaints addressing other locations than the lung and liver, in which the cyst usually occurs and should especially be suspected in patients with radiological features in favor of cystic structures. Furthermore, in cases of uncommon hydatid cyst, evaluating other locations should be taken into consideration for any infestations of hydatidosis.In CE-endemic areas, hydatid disease can affect any organ, from head to toe, in humans. The disease should be considered in the differential diagnosis of any cystic entities anywhere in the body. Spleen and pelvic cavity should be considered as the most unusual locations of hydatid cyst in any CE-endemic area. Besides, hospital records lack information about hydatid cyst imaging details, which must be carefully noted in each patient's record.


P value less than 0.05. A total of 240 mothers (60 cases and 180 controls) were included in the study with a 98.3% response rate. Factors like attending secondary educational and above [adjusted odd ratio (aOR) = 0.07 (0.08-0.65)] and attending antenatal care [aOR = 0.41 (0.18-0.93)] were protective whereas having urinary tract infection [aOR = 3.6 (1.1-11)], having human immune virus diseases [aOR = 4.2 (0.9-18)], having a history of abortion [aOR = 2.3 (1.1-5)], having a history of preterm delivery [aOR = 5 (1.6-15)], and having hypertensive disorders of pregnancy [aOR = 5 (1.9-13)] were significantly associated risk factors for preterm birth. The main determinant factors for preterm birth are having antenatal care follow-up, attending secondary education and above, hypertensive disorders of pregnancy, having HIV/AIDS, and history of abortion. This shows a need to strengthen female education; screen mothers for HIV/AIDS, urinary tract infection, and hypertension; and strengthen nutritional counseling, during ANC visits.Globally, every year, 1.1 million newborns die due to prematurity. In Ethiopia, 320,000 preterm births occur each year; out of these, 24,400 deaths were due to preterm complications. However, there is little evidence about preterm birth in the study area. Therefore, this study provides an important direction for health professionals, health programmers, and researchers. A facility-based unmatched case-control study design was employed among 244 women (61 cases and 183 controls) who gave birth in Dilla University Referral Hospital and were selected with purposive sampling. The bivariate and multivariable logistic regression model was used to select independent predictors of preterm birth. The multivariate analysis was used, and the results were interpreted using an adjusted odds ratio at 95% confidence interval and statistically significant level at a  Preterm birth is defined as all births before 37 completed weeks of gestation or less than 259 days since the first day of a woman's last normal menstrual period. It is classified as medically indicated (iatrogenic) and spontaneous (idiopathic) preterm birth. And according to gestational age, it is also classified as extremely preterm (<28 weeks), very preterm (28 to <32 weeks), and moderate to late preterm (32 to <37 weeks). It is the leading cause of prenatal morbidity and mortality in both developed and developing countries including Ethiopia. In most cases, preterm deliveries occur spontaneously and are associated with long-term complications in surviving infants .The majority of global preterm births occur in Asia and Africa where health systems are weak and access to utilization of health services is limited . The lowThe global community committed to the Sustainable Development Goals and Every Woman and Every Child initiatives to prevent and improve survival of preterm births; different interventions have been implemented which need huge financial capacity . Some ofThe national-level profile provides the most current national-level information on the status of prevention and cares for preterm birth and low birth weight in Ethiopia. More effort is needed to identify women at risk of preterm labor and support them to give birth in a health facility that can offer extra care when needed, such as support for adequate feeding with breast milk, continuous skin to skin contact, antibiotics, and antenatal corticosteroids . To do tThe preterm birth causes significant health consequences to the infant and economic costs for families and communities. In advances, prenatal and neonatal care have improved the survival of preterm infants, but those infants who do survive have a risk of developmental disabilities, health and growth problems, chronic diseases, and mortality later in life  than infAccording to the Gedeo Zone maternal and child health (MCH) report in 2017, preterm births were 122 (0.6%); from these, 103 (84%) were alive and 19 (16%) were dead . This study is aimed at identifying the determinants factors of preterm birth in the Gedeo Zone, Dilla University Referral Hospital.th to July 18th, 2018. The study was conducted at Dilla University Referral Hospital which is situated in Dilla town, Gedeo Zone, South Ethiopia. Dilla town is located 360 km away from Addis Ababa, the capital city of Ethiopia. This referral hospital has seven departments: surgical, gynecologic/obstetric, medical, radiology, dermatology, psychiatry, and pediatric wards. Taking into account the better curative services provided for, it is one of the major important destinations for patients coming from all directions of the zone, including other bounding regions like the Oromia region and Sidama and Amaro Kelle zones [A facility-based unmatched case-control study was employed from May 5le zones .Women who gave birth less than 37 weeks of gestation were cases, and women who gave birth greater than or equal to 37 weeks of gestation were controls. The gestational age was calculated based on the last normal menstrual period (LNMP) or ultrasound examination (for those who have early ultrasound examination). All the cases and controls were recruited during the time of child birth.The sample size was computed using a formula for two population proportions and calculated by OpenEpi version 3.03 statistical software package by considering 23.6% of cases and 7.5% of controls exposed taken from a study done in Addis Ababa public hospitals [The outcome of the study was preterm birth, and the independent variables were sociodemographic characteristics , obstetrics-related variables , medical condition , and maternal nutritional factor (mid-upper arm circumference (MUAC)).Data collection instruments were adapted from the Ethiopia Demographic and Health Survey (EDHS) and different kinds of literature –17. It iThe outcome variable preterm birth and independent variables like sociodemographic, obstetric, and medical history were collected from the mother by using a structured questionnaire by direct interview, and data on medical factors were extracted from medical records. Gestational age was measured by clinicians either through the last normal menstrual period (LNMP) or ultrasound examination of those women who had preterm birth.Mid-upper arm circumference (MUAC) was measured by using flexible nonstretchable tape taken at the midpoint of the left upper arm at the relaxed position without any clothing and with optimal tape tension between the acromion process on the shoulder blade and tip of olecranon process of the ulna.The questionnaire was prepared in English and translated to the local language and then backtranslated to English to check the consistency. Data collection was done by trained diploma nurses. Two-day training was given for data collectors on the objectives of the study, the contents of the questionnaire, issues related to the confidentiality of the responses, and the rights of the respondents. Pretest was done at the Yirgacheffe District Hospital on 10% (24 women), which is 24 km away from Dilla town. The collected data was checked daily by the principal investigator, supervisors, and data collectors to reduce incomplete data.P value of less than 0.25 in a bivariable analysis were considered as candidates for multivariable logistic regression. In multivariable analysis, a variable with a P value of ≤0.05 was considered as having statistically significant association with preterm birth. The level of the association was measured by using an odds ratio (OR) with corresponding 95% confidence interval (CI).First, the data were checked for their completeness and consistency and entered into OpenEpi version 3.03 exported to SPSS statistical software version 20 for analysis. Descriptive statistics were used to measure the characteristics of the study participants. Both bivariable and multivariable logistic regression analysis were computed to identify factors associated with preterm birth. Variables with a The study was approved by the institutional review board (IRB) of Dilla University College of Health Science and Medicine. For the purpose of data collection from the participants, an explanation was given to them regarding to the purpose of the study, the importance of their participation, and true responses. It was also explained that the study had no connection with the individual affairs of respondents. All sampled populations were encouraged to participate in the study, while at the same time, their right not to participate was also respected. Volunteer participants signed written informed consent. For those participants less than 18 years old, we got written consent from their parents and guardians and assent from them. Women who have a preterm baby in the data collection period were counseled about nutrition and provision of care to their child.From a total of 244 women, 240 (60 cases and 180 controls) responded, and the response rate was 98.3% in both cases and controls. The case and control groups have comparable sociodemographic characteristics. The mean maternal age in both groups was 29 (SD ± 5.4) years. In both study groups, more than 86.6% of the women were married. Among cases, 6 (10%) attended tertiary education while the proportion of mothers who attended above tertiary education among controls was 56 (31%) .Regarding the ANC service utilization during pregnancies, 35 (58%) of the cases and 145 (81%) of the controls had ANC follow-up. Twenty-three percent of the cases and 31% of the controls had 4 or more ANC visits during pregnancy. Around 27% of the cases and 11% of the controls had hypertensive disorders of pregnancy (HDP). About 11 (18%) of the cases and 8 (4%) of the controls have a history of preterm birth. Nine (15%) of the cases and 5 (2.7%) of the controls were HIV positive. Thirty-eight percent of the cases and 23.3% of the controls had a history of abortion in the previous pregnancy. Regarding medication use, 20% of the cases and 14.4% of the controls use medication during pregnancy. Fifty-eight percent of the cases and 52.8% of the controls had more than 5 family sizes .P value of less than 0.25 were moved to multivariable logistic regression analysis to determine the predictors of preterm birth. Based on the final multivariable analysis model, variables that were identified as independent predictors of preterm birth were as follows: attending education secondary and above level, having ANC follow-up, HIV-positive serostatus, having urinary tract infection, history of abortion, history of preterm birth, and hypertensive disorder of pregnancy.Variables that have a In this finding, women who had attended tertiary education were 93% less likely to have preterm birth as compared to those who had not attended education . Women who had ANC follow-up were 59% less likely to have preterm birth as compared to those who had no ANC follow-up . Women who had urinary tract infection during pregnancy were 3.6 times more likely to have preterm birth as compared to those who have no urinary tract infection during pregnancy . HIV-positive women were 4.2 times more likely to have a preterm baby as compared to their counterparts . Those women who had a history of preterm birth were 5 times more likely to have preterm birth as compared to their counterparts . Women who had a history of abortion were 2.3 times more likely to have preterm birth as compared to those who had no history of abortion  .This study was an unmatched case-control study on 240 mothers who gave birth at Dilla University Referral Hospital intending to identify the determinant of preterm births. According to this study, the variables like educational status, receiving antenatal care, having hypertensive disorders of pregnancy, HIV/AIDS-positive serostatus, having urinary tract infection, having a history of preterm birth, and history of abortion have a statistically significant association with preterm birth.Women who had attended tertiary and above education were 93% less likely to have preterm birth as compared to those who cannot read and write. This finding is in agreement with a study done in Jimma ; Bengal,The other factor found to be associated with preterm birth in this study was ANC visit. Mothers who had ANC visit had a 59% decreased risk of preterm birth than mothers who had no ANC visit. This finding is supported by a study done in Debre Markos ; Bengal,This study revealed that women who had urinary tract infections during pregnancy were 3.6 times more likely to have preterm birth as compared to those who had no urinary tract infection during pregnancy. This result agreed with the study done in Tanzania  and KenyThe history of abortion is another significant risk factor associated with preterm birth. Women who had a history of abortion were 2.3 times more likely to have preterm birth as compared to those who had no history of abortion. This finding is similar to the studies conducted in Jimma  and KenyWomen who had a history of preterm birth were 5 times more likely to have preterm birth as compared to those who had no history of preterm birth. This finding agreed with studies done in Jimma ; WesternAnother determinant of preterm birth is hypertension during pregnancy. Women who had hypertension during pregnancy were 5 times more likely to have preterm birth as compared to those who had no hypertension during pregnancy. This study is in agreement with the study done in Gondar , Ghana , Kenya , and NigBeing HIV/AIDS positive is also significantly associated with preterm birth. Women who are HIV positive were 4.2 times more likely to have preterm birth as compared to their counterparts. This finding is similar to studies done in Addis Ababa  and TanzFinally the limitation of the study was recall bias, being a case control study; it can only tell us the presence of association but not causal relationship. To alleviate such recall biases, the data collectors cross-checked the data from medical records of the participants.This study showed the determinant factors like education level secondary and above and having antenatal care visit were protective factors, whereas having hypertension during pregnancy, HIV/AIDS-positive serostatus, having urinary tract infection, history of preterm birth, and history of abortion were risk factors for preterm birth. This shows a need for strengthening of female education; early screening and treatment of maternal HIV/AIDS, urinary tract infection, and hypertension; strengthening of antenatal care follow-up; nutritional counseling during ANC visit; and prevention of the occurrence of abortion is recommended to reduce the preterm birth.


Echinococcus granulosus. In human CE, the parasites develop and form cysts in internal organs. The differentiated cysts can be classified into five types based on WHO-IWGE standard CE1-5 representing different developmental stages. Infection with E. granulosus triggers hosts’ humoral and cellular response, displaying elevated serum antibodies and Th1 and Th2 cytokines, which are presumed to be in association with the disease outcome. Identification of immunological markers for evaluation of disease progression has been a growing concern. However, the distinctive profile of cytokines and antibodies associated with the cyst progression has not been ascertained.Cystic echinococcosis (CE) is a worldwide parasitic zoonosis caused by infection of the larval stage of tapeworm To better understand the interaction between host immune response and disease outcome, the present study followed-up four CE patients over three years by yearly measuring serum level of 27 cytokines, total IgG and isotypes, and ultrasound scanning, beginning in year 1 for all patients with CE1 and CE2 cysts before treatment and continued in year 2 with CE4 and in year 3 with CE3-CE5 post-treatment.Nine cytokines including Th1-type IL-2, Th17-type IL-17A, and inflammatory cytokines IL-1β, IL-1Rα and TNF-α, chemokines IL-8, MIP-1α, MIP-1β, and growth factor G-CSF were significantly elevated in patients with cyst type CE1, compared to the normal controls, and then declined to a normal level at CE4 and CE5. Examining the antibody production, we found that serum specific IgG was significantly increased in patients with active and transitional cysts, specifically the total IgG at CE1/CE3/CE4-CE5, IgG4 at CE1 and IgG1 at CE1/CE3 cyst status, in comparison with the normal controls, but showed no significant changes between the cyst stages.Our findings provide new information on the profile of multiplex cytokines and serum antibodies associated with cyst stages in cystic echinococcosis patients through a three-year follow-up, implying that further studies using an approach combining cyst-associated immune parameters may aid in identifying immunological markers for differentiation of disease progression. Echinococcus granulosus. The disease is prevalent in China, Central Asia, the Middle East, South America and some parts of Europe [E. granulosus in humans triggers humoral and cellular response, displaying elevated serum antibodies and T helper cell 1 (Th1) and T helper cell 2 (Th2) cytokines. Most of the earlier studies on CE cytokines were based on in vitro experiments, to examine cytokine production by stimulation of peripheral blood mononuclear cell or T helper cells of patients with crude or B hydatid antigen. Experimental infection studies in mice with viable protoscoleces, found that cytokine response shows a biphasic kinetics: an early predominant induction of Th1-type cytokines , followed by a shift toward a Th2-type profile  [Cystic echinococcosis (CE), also called hydatid disease, is a serious parasitic zoonosis with a worldwide distribution, caused by the larval stage of the cestode f Europe , 2. In hf Europe –6. Clinif Europe , 8. The d IL-13) , 10. It d IL-13) . A very d IL-13) .Given that the host immune response against the parasite has been recorded and analysed, it is assumed that the CE cytokines are possibly associated with the outcome of the disease after clinical interventions. Thus, identification of serum immunological markers for evaluation of therapy effectiveness of CE draws increasing concerns. Naik et al.  detectedCE patients and normal donors participating in this study were from the Huan county, Gansu Province in China. Four CE patients were selected for the study from those receiving routine medical care, and three normal donors as a control. To meet the aim of this study, the selection inclusion criteria are defined as: (i) the participants agree to be enrolled in the clinical follow-up programme for three consecutive years with no interruption; (ii) a patient diagnosed with active or transitional cyst (CE1-CE3), indicating a need for immediate treatment; (iii) the cyst stage shows progressive changes over three consecutive years; and (iv) the patient has no other confirmed diseases which may affect the immune reactions. Venous blood was taken from all participants, 5 ml serum was obtained and divided into two aliquots and stored at − 80 °C until analysis. One aliquot was tested as part of routine CE specific IgG antibody monitoring and the second aliquot was used for cytokine detection. The clinical and laboratory checks were monitored once a year for three consecutive years (2014–2016) by ultrasound (US) examination and serological assays, initiating from the time before treatment in year 1. The normal controls were examined in year 1 to obtain the normal level of cytokines and antibodies defined in the study.E. granulosis IgG antibody was performed using a commercial enzyme-linked immunosorbent assay (ELISA) kit . After diagnosis, the CE patients were treated with oral albendazole at of 10–15 mg/kg/day for at least 3 months. When the cyst showed characteristics of type CE4 or CE5 (inactive stages), the chemotherapy was ended, entering into the watch-and-wait approach; however, if the cystic lesion returned to CE1-CE3 (active stages), a new course of drug treatment was delivered.The diagnosis and classification of CE was made using the standard developed by the WHO-IWGE . CE was We simultaneously analyzed 27 immune cytokines using a commercial Bio-Plex Pro Human Cytokine Group 1 Panel 27-plex Assay kit  in accordance with the manufacturer’s instruction. This bead-based immunoassay allows simultaneous detection of multiple immune mediators from one sample . In the To evaluate the antibody response during the three-year follow-up, the levels of total IgG, IgG subclasses  and IgE in CE patients’ sera against hydatid cyst fluid (HCF) antigen were quantified by ELISA. Briefly, microtiter plates  were coated with HCF (10 μg/ml); the wells were blocked with 1% bovine serum albumin; and then washed with washing buffer before application of CE patient sera . Total IgG, IgG subclasses and IgE were detected with HRP-conjugated mouse anti-human IgG antibodies  at 1:10,000, IgG subclass antibodies and IgE antibodies , both at 1:1000. Optical densities (OD) were measured at 450 nm using a microplate autoreader .P < 0.05 were considered statistically significant. The statistical analysis was performed using GraphPad Prism version 5.01 .One-way analysis of variance (ANOVA) with Bonferroni correction was used to compare the differences between quantitative variables. Values of n = 2; type CE2, n = 2) when examined prior to treatment in Year 1. In Year 2 (post-treatment), all four patients presented type CE4 (inactive stage), thus no treatment was delivered. In Year 3, only one patient showed the inactive type CE5, whilst the other three participants were at the transitional stage CE3, which is an indication for treatment; thus a new treatment course will be followed after the study ; among these cytokines, IL-1β, IL-8, MIP-1α and G-CSF presented the highest concentration  and compared cytokine levels with the normal controls (Table We further assessed the dynamic changes of cytokine levels in relation to the cyst stages during the three years monitored. Each patient (P1-P4) presented a disease progress path  during the three-year period, exhibiting corresponding cytokine responses. To examine the cyst stage-related cytokine response, we analyzed the level of each of 9 highly expressed cytokines of individual patients by plotting the cyst stage (CE1-CE5) against cytokine concentration (pg/ml) to show a dynamic change. Figure P < 0.05)  and IgE by ELISA. We found that serum-specific IgG against HCF significantly increased in patients with active and transitional cysts, specifically, the total IgG at CE1/CE3/CE4-CE5, IgG4 at CE1 and IgG1 at CE1/CE3 cyst stage when compared to the normal controls. However, there were no significant changes between the CE4 and CE5 cyst stages (E. granulosus protoscoleces demonstrated that the Th1-type cytokine profile was predominant at the early post-infection phase (3–4 weeks); then a shift to Th2-type cytokine took place in week 4 [Cystic echinococcosis is a chronic and complex zoonosis. The CE cysts can parasitize in the intermediate host for a long time, establishing an immune microenvironment in the peripheral immune system in subtle and complicated ways. Ample evidence suggested that cytokines play a crucial role in the immune response process as both Th1 and Th2 cytokines are coexisting during hydatid disease. Published data indicate that Th1 cytokines are related to the protective immunity whereas Th2 cytokines are associated with the chronic stage, clinical complications and secondary episodes , 18, 19.n week 4 . Howevervia cytokine Th1-type IFN-γ and Th2-type IL-4 [Our findings present several noticeable facets: (i) our findings are in agreement with the general understanding that the immune response to hydatid cysts in intermediate hosts is a complex and contradictory issue, the imbalance of Th1/Th2 plays an important role in promoting the immunopathogenesis change of this disease ype IL-4 , 21;  response with varied intensity and specificity. In the chronic phases of CE, the level of IgG, IgM and IgE are frequently elevated, with IgG1 and IgG4 being predominant. Furthermore, the IgG responses induced in patients with CE1-CE3 cysts are mainly of the IgG4 isotype, but not in patients with inactive cysts. Published data indicate that the IgG4 response is associated with cystic development, growth and disease progression, whereas IgG1, IgG2 and IgG3 responses occur predominantly when cysts become inactive [E. granulosus induces the host humoral and cellular immune responses and releases molecules to modulate the host immune responses favoring a strong anti-inflammatory response and perpetuating parasite survival in the host [Echinococcus multilocularis metacestode-specific IgG1, IgG3, and IgE responses progressively diminished with regression from active to stable and cured AE. IgG2 and IgG4 reactivity remained similarly high in stable and progressive cases, and lessened only in cases with cured AE. Interestingly, the reactivity against E. multilocularis vesicle antigen enabled differentiation between cured, stable, or progressive AE. It is proposed that combined and longitudinal examination of several cytokines and chemokines with the evaluation of vesicle-specific antibody responses may be of value for differentiation between the distinct states of AE [In helminth infections, distinctive antibody responses are known to correlate with the clinical state of helminth infection, with IgG4 and IgE indicative of a Th2-type immune response, particularly in chronic infections , 24. Echinactive , 26. In inactive . Placingthe host . HoweverThis is a preliminary study examining the profiles of multiple cytokines and antibodies in CE patients, using a limited number of patients and normal controls. Although we performed statistical analysis, the statistical power is weakened due to the small number of participants. Thus, although we found the cytokine and antibody responses at the early stage CE, the follow-up could not detect profile shifts of Th1/Th2 cytokines and antibodies between cyst stage groups. The data analysis of this study was individual-based rather than group-based. For further study, an appropriate sample size is essential for gaining an insight into the interplay between immune mediators and antibodies with disease progression.The present results indicate significantly elevated serum levels of a panel of nine cytokines, particularly inflammatory cytokines IL-1β and IL-1Rα, chemokine MIP-1α and MIP-1β screened from 27 cytokines, in CE patients with active cysts compared with those in the normal controls, and significantly increased specific antibody response including total IgG, IgG4 and IgG1, in CE patients with active and transitional cysts in comparison with the controls. Our findings provide new information on the profiles of multiplex cytokines and IgG antibodies associated with cyst stages in cystic echinococcosis patients through a three-year follow-up, implying that further studies using combination of cyst-associated immune parameters may aid in identifying immunological markers for differentiation of disease progression.Additional file 1: Table S1. Serum cytokine concentrations of the participants followed-up over three years.


Every Newborn Birth Indicators Research Tracking in Hospitals” (EN-BIRTH) was an observational study validating selected maternal and newborn health indicators. This paper reports results for EIBF.Immediate newborn care (INC) practices, notably early initiation of breastfeeding (EIBF), are fundamental for newborn health. However, coverage tracking currently relies on household survey data in many settings. “The EN-BIRTH study was conducted in five public hospitals in Bangladesh, Nepal, and Tanzania, from July 2017 to July 2018. Clinical observers collected tablet-based, time-stamped data on EIBF and INC practices . To assess validity of EIBF measurement, we compared observation as gold standard to register records and women’s exit-interview survey reports. Percent agreement was used to assess agreement between EIBF and INC practices. Kaplan Meier survival curves showed timing. Qualitative interviews were conducted to explore barriers/enablers to register recording.Coverage of EIBF among 7802 newborns observed for ≥1 h was low . Survey-reported  and register-recorded results  overestimated coverage compared to observed levels across all hospitals. Registers did not capture other INC practices apart from breastfeeding. Agreement of EIBF with other INC practices was high for skin-to-skin (69.5–93.9%) at four sites, but fair/poor for delayed cord-clamping (47.3–73.5%) and drying (7.3–29.0%). EIBF and skin-to-skin were the most delayed and EIBF rarely happened after caesarean section (0.5–3.6%). Qualitative findings suggested that focusing on accuracy, as well as completeness, contributes to higher quality with register reporting.Our study highlights the importance of tracking EIBF despite measurement challenges and found low coverage levels, particularly after caesarean births. Both survey-reported and register-recorded data over-estimated coverage. EIBF had a strong agreement with skin-to-skin but is not a simple tracer for other INC indicators. Other INC practices are challenging to measure in surveys, not included in registers, and are likely to require special studies or audits. Continued focus on EIBF is crucial to inform efforts to improve provider practices and increase coverage. Investment and innovation are required to improve measurement.The online version contains supplementary material available at 10.1186/s12884-020-03421-w. Almost half of all deaths in children under the age of five occur in the first month of life , totalling 2.4 million deaths, with 1 million dying on their birthday .INC practices include skin-to-skin contact during the first hour of life, immediate drying, delayed cord clamping (1–3 min after birth), and early initiation of breastfeeding within 1 h of birth (EIBF) . EIBF haPopulation-based surveys, such as the Demographic and Health Survey (DHS) and Multiple Indicator Cluster Surveys (MICS) are the main source of coverage data for INC practices in low- and middle-income countries (LMICs). These are undertaken every 2 to 5 years in about 60 countries. Currently, core questionnaires for both DHS and MICS include questions to capture EIBF and skin-to-skin initiation. Other components of immediate and essential newborn care (such as drying) are in an optional module specific to newborn care  Additio. Of fiveInstitutional birth rates are increasing, with over three-quarters of births worldwide now in facilities , and manThe timing and sequencing of INC practices represents one dimension of quality of care not generally included in large-scale survey tools , but thaEvery Newborn Action Plan, endorsed by all United Nations member states, includes an ambitious measurement improvement roadmap .”-Health worker, Muhimbili TZThese findings were consistent with evidence from Bangladesh data collectors, and are reflected in the low observed breastfeeding coverage compared with high register-recorded practice in both sites. Multiple locations for documentation contributed to the complexity of the record-keeping system and these challenges were compounded when breastfeeding was initiated after discharge from the labour wards:Respondents in all five sites also reported that breastfeeding was not routinely initiated or recorded in the operation theatres, this was especially the case for Bangladesh:“Breastfeeding is not done in the operation theatre. They never do it in operation theatres.”-Data collector, Kushtia BD“They usually do not initiate it in the in the theatre, it is initiated in the post-caesarean ward.”-Data collector, Temeke TZAcross all sites, the primary midwifery or nursing carer was responsible for documentation for women having vaginal births, except Pokhara NP where labour ward registers do not include a column for breastfeeding initiation . There is compelling plausibility for the agreement between skin-to-skin and breastfeeding . We alsoOur time-to-event analysis using the Kaplan Meier curves highlights the rapid timing of skin-to-skin initiation drying, and cord clamping, but major delays in breastfeeding, especially for babies born via caesarean. Given the increasing rate of caesareans, this represents an urgent research gap . One EN-In these CEmONC hospitals, low rates of breastfeeding indicate gaps in quality of care. Given the well-evidenced, extensive benefits of EIBF, low coverage and delays are startling and may reflect separation of mother and baby. Breastfeeding initiation is crucial for establishing breastfeeding and for multiple other benefits for mother and baby ; hence oRegister design also plays a role, the Pokhara NP register did not have a column to capture EIBF. In three out of four EN-BIRTH sites with a specific column, register-recorded coverage was above 90%. In Tanzania, Temeke and Muhimbili had different register-recorded coverage (95.3% and 43.8% respectively) despite sharing the same register design and having similar observer-assessed EIBF rates (26% and 19.1% respectively). Hospitals in Bangladesh introduced revised registers during the study period, and register-recorded breastfeeding coverage in Azimpur increased from 0 to > 90%, and in Kushtia from 57.3 to 96.8%, despite a maximum observer-assessed EIBF coverage of 9.8% . These fStrengths of this study include the large sample size, and rigorous multi-country design with gold standard with direct observation by clinically trained observers. Observer data could be subject to errors, but this risk was minimised through a custom-built electronic data capture system, standardised training and refresher sessions, and quality assurance through double observation and data entry .However, there were also limitations. Observation was discontinued when women were transferred out of labour and delivery wards, so we were unable to record EIBF beyond the immediate postpartum period. As the current definition of EIBF includes a 1 h time period, the 12,701 women who were not observed for > 1 h needed to be excluded from the validation analysis. This may have introduced bias as women observed for ≥1 h were more likely to have had a vaginal birth , and even lower for women with caesarean births. Given the global epidemic of caesareans, more focus on supporting women and newborns with EIBF is crucial. Unless measurement accuracy is improved, EIBF coverage changes may be missed. Register-recorded and survey-reported coverage both over-estimated observed coverage of EIBF, demonstrating a need for further research to improve instructions and register design/survey questions. Our analysis suggests that agreement between EIBF and skin-to-skin initiation is high. However, immediate drying and delayed cord clamping are even more challenging to measure in surveys and unlikely to be captured in registers, so they will likely require special audits and studies. Renewed focus is needed to promote zero separation of women and their babies, increase coverage of EIBF and INC practices irrespective of mode of birth, and to ensure and measure INC practices including respectful care practices for every woman and their newborn at birth.Additional File 1. Definition of immediate newborn care indicators .Additional File 2. Previous studies regarding validation for measures of immediate newborn care practices.Additional File 3. National context and number of births in EN-BIRTH study hospital.Additional File 4. Data collection dates by site, EN-BIRTH study.Additional File 5. Observation, survey and register indicator definitions, EN-BIRTH study.Additional File 6. STROBE Checklist.Additional File 7. Ethical approval by institutional review boards, EN-BIRTH Study.Additional File 8.  Hospital register design and completion approaches by site, EN-BIRTH study (n = 6548).Additional File 9. Respondents for focus group discussion and in-depth interviews for EN-BIRTH Study.Additional File 10.  Individual-level validation in exit-survey report of early initiation of breastfeeding, EN-BIRTH study (n = 7802).Additional File 11. Individual-level validation of register recording for early initiation of breastfeeding, EN-BIRTH study (n = 7802).Additional File 12.  Characteristics of women observed in labour and delivery wards for < 1 h, EN-BIRTH study .Additional File 13. Assessment of routine recording responsibilities for breastfeeding, EN-BIRTH study.Additional File 14. Register recording order and prioritisation for breastfeeding, EN-BIRTH study.Additional File 15. Inter-observer agreement for early initiation of breastfeeding using Kappa, EN-BIRTH study.