Findings on the role of Mediterranean-style diet (MSD) on duration of pregnancy and birth weight have been inconsistent and based largely on Non-Hispanic white populations, making it unclear as to whether they could extend to African Americans who are at a higher risk of unfavorable birth outcomes. Our study addresses this gap using a large urban, multiethnic, predominantly low-income cohort of mother-infant dyads from Boston, MA, USA. Dietary information was obtained via food frequency questionnaires; health information including birth outcomes were extracted from medical records. A Mediterranean-style diet score (MSDS) was formulated based on intake history, and linear and log-binomial regressions were performed to assess its association with birth outcomes. After adjustment, the lowest MSDS quintile from the overall sample was found to be associated with an increased relative risk (RR) of overall preterm birth , spontaneous preterm birth , late preterm birth , and low birth weight , compared to the highest quintile. The findings were similar for the African American sample. Our study adds to the current understanding of the diet’s influence on birth outcomes by demonstrating that adherence to MSD may improve birth outcomes for African American women. Preterm birth (PTB), or birth before 37 completed weeks of pregnancy, is the leading cause of death among children under the age of five years and affects more than fifteen million infants annually worldwide . DespiteMany socioeconomic and behavioral risk factors for PTB and LBW have been identified. These include extreme young or advanced maternal age, non-optimal weight status prior to pregnancy, low income or educational attainment, primiparity, and tobacco use ,10,11,12There is growing evidence that maternal dietary patterns can affect the risk of pregnancy outcomes. For example, the risk may decrease with a high intake of vegetables, fruits, and whole grains, or low intake of fast food and added sugars during pregnancy ,25. ThisThe parent study, the Boston Birth Cohort (BBC), has been described in previous publications ,38. BrieAn assessment of dietary intake during pregnancy was completed during early postpartum via a qualitative food frequency questionnaire (FFQ), which is a closed-ended dietary history collection method used to assess how often the respondent ate over a specific period . A reforThe primary exposure was MSDS assigned to each mother, based on their FFQ responses. Calculating the MSDS requires food groups to be either positively or negatively scored based on the mother’s report of her average weekly intake frequencies during pregnancy. All foods were positively scored except meat, which was negatively scored. For positively-scored foods, we gave a score of 0 to “none”, 1 to “less than one day”, 2 to “one to two days”, 3 to “three to five days”, and 4 to “six to seven days”, and did the reverse for meat . MissingTwelve covariates were selected because of their known association with our birth outcomes of interest based on the past literature as explained earlier. These included the maternal age in years, pre-pregnancy body mass index or BMI (body weight in kilograms divided by height in meters squared), parity (number of prior live births), race or ethnicity , education level , and smoking status during pregnancy . Overall, 90.9% of the respondents fully completed the demographic questionnaire. Clinical variables included the child’s sex and presence or absence of each major maternal complications: diabetes , chronic hypertension prior to pregnancy, mild or severe preeclampsia, eclampsia, and HELLP syndrome. The research staff successfully abstracted all of the clinical items for 98.0% of the respondents. We imputed the missing data for these covariates (the proportion of which for each covariate did not exceed 6.8% across the entire study sample) with the modal frequency of response to each respective covariate for the respondent’s age and racial groups . Other potentially relevant variables such as the annual household income and gestational hypertension were not considered due to their proportion of missing responses at greater than 10%.The gestational age in weeks and birth weight in grams from medical records were used to determine each newborn’s specific birth outcomes. PTB was defined as less than 37 completed weeks of gestation, further categorized as spontaneous or medically-induced, and as late PTB (34–36 weeks) or early PTB (less than 34 weeks). LBW was defined as birth weight under 2500 g. The criteria for intrauterine fetal growth—AGA , SGA , and LGA —were also identified in accordance with the previously published protocols ,44. The 2 test were performed to compare the baseline characteristics of mothers by the individual MSDS quintiles. The primary analyses were based on the sample with imputed data. We assessed the robustness of data by running two parallel analyses with samples comprised of (1) “original data” and (2) “complete-case data” (in which any participant with “I don’t know” or missing response was dropped). Maternal age was re-categorized as: “less than 21”, “between 21 and 30”, and “greater than 30” years of age, and primiparity was identified (y/n). Women were classified based on their pre-pregnancy BMI as: “less than 18.5” (underweight), “between 18.5 and 24.9” , “between 25 and 29.9” (overweight), and “greater than or equal to 30” kg/m2 (obese). For analyses, “African American” also included “Haitian”, and those identified as “Asian”, “Cape Verdian”, “Pacific Islander”, “Mixed-race”, and “Other” were grouped as “Other”. Finally, maternal education was grouped as (1) “no school/elementary school” or “some secondary school”, (2) high school or equivalent, and (3) “some college” or “college degree and above”.Univariate analyses using xFor the primary analyses, we assessed the associations between individual MSDS quintiles and birth outcomes—gestational age, overall and categories of PTB, birth weight, LBW, and categories of fetal growth—with the fifth quintile treated as reference. For the subgroups of PTB and fetal growth, term birth and AGA were treated as reference, respectively, to assess the risk of unfavorable birth outcomes relative to their optimal counterparts while allowing for the comparison of the risks between the MSD adherence quintiles. Linear regression was used for continuous outcomes, and log-binomial regression was used for the categorical outcomes. The covariates were adjusted for in the analyses unless the initial univariate analyses revealed that they were not confounders; gestational age was also adjusted for when assessing the birth weight outcomes. For the purpose of adjustment, we collapsed pre-pregnancy BMI to consider overweight/obesity (y/n) and created a binary variable based on the presence of any blood pressure-related complication . The analyses were performed for the overall, African American, and non-African American samples.p < 0.05 and all tests were 2-tailed.Using the overall sample, the unadjusted association between raw MSDS and overall PTB, LBW risks was visualized using kernel density plots. Associations between raw MSDS and spontaneous PTB and LBW risks were visualized using a locally weighted scatterplot smoothing method, stratified by African American and non-African American samples. Finally, adjusting for other covariates as applicable, stratified analyses were performed to assess a covariate’s potential as an effect modifier on four birth outcomes whose associations with MSDS were statistically significant . Forest plots were produced for two of these as visual aids. All analyses were performed using Stata Version 14.2 . Statistical significance was set at n = 8507) were compared between the MSDS quintiles (n = 2173) were more likely to be younger, non-Hispanic white, and to have less education overall compared to those in any other quintile. They were also more likely to report entering pregnancy with BMI of 30 kg/m2 or more, to be nulliparous, have diabetes, and to have smoked during pregnancy.The baseline characteristics of mothers  and 11% greater risk of LBW when compared to those in the fifth quintile. For the African American sample, the adjusted excess risk for overall PTB and was at 20% ; it was 14% for LBW. These figures were not significantly different from those of the non-African American sample, which were 17% for overall PTB ; the excess risk of LBW for the non-African American group was 5%. For all three sample groups, associations between MSD adherence and fetal growth were statistically nonsignificant, though the direction of the associations was generally consistent and away from growth restriction. These results are generally consistent with prior research indicating that greater adherence to MSD is associated with reduced risk of adverse birth outcomes, particularly preterm delivery ,33,34,35These clear associations from our study are unsurprising given that the maternal nutritional status and diet are long recognized for their influence on duration of pregnancy and size at birth ,45,46. AHeterogeneity in the results of associations of MSD and birth outcomes in the literature may be due to a number of factors including: (1) sample characteristics, (2) covariates controlled for in each respective article’s statistical modeling, and (3) dietary assessment techniques. The BBC is a cohort of mothers with mostly low-income background and a diverse racial and ethnic profile. As such, BBC is unique amongst the aforementioned studies which are based predominantly on healthy Non-Hispanic white populations. Our analyses based on the African American demographic group comprising a large proportion of the cohort may have produced more profound effect sizes, given that this population is typically at an elevated risk of the outcomes of interest to our study. This, in turn, highlights the importance of furthering the research into the underlying factors that may be related to the greater vulnerability of African American women, though the above studies are also from outside the US and make a direct comparison difficult . In addition, differences between the aforementioned studies in choosing which variables to adjust for in their models are likely contributing to the lack of research consensus on this subject. For instance, pre-pregnancy BMI was adjusted for in many studies while the use of prenatal vitamin supplements was considered infrequently. Of note, this type of inconsistency in adjustment strategies was also recognized by a separate systematic review that recently assessed the literature on the association between MSD and birth outcomes . Lastly,Our study has several strengths. We found consistent and significant associations between MSD adherence and various birth outcomes in the overall sample and across racial groups. Given the high proportion of African American sample in BBC, our findings might be extendable to low-income African Americans in other US settings. Our study is also bolstered by relatively large sample size and by the high quality of data obtained by trained research staff using standardized protocols. In addition, to mitigate unmeasured confounding, we adjusted for many known sociodemographic and clinical variables that are pertinent to our study. Lastly, we improved the credibility of our single imputation method for the missing values and “I don’t know” responses by using each item’s mean or modal frequency of items that were matched for respondent’s age and racial groups.Our study has some limitations as well. Firstly, this is an observational study and study participants were asked about their antenatal intake pattern after delivery. This raises the question about whether the mothers correctly recalled their general intake pattern spanning the previous ten months, especially given that surveys are subject to social desirability bias. Secondly, the FFQ gathered data on intake frequency but not quantity. Thirdly, because of limitations in the diet questionnaire, we were unable to use a validated method for scoring, and we did not adjust our food groups or scores for energy intake. In addition, some of the other potentially relevant information, including the diet prior to pregnancy and physical activity history, was not available. Finally, as BBC, by design, had a relatively high proportion of PTB compared to the general US population, caution is needed to generalize our findings to other study populations with different characteristics.In conclusion, in our multi-ethnic and predominantly low-income sample, low adherence to MSD was significantly associated with elevated risk of various categories of PTB, LBW, and the subgroups. The excess risk was also significant and similar in magnitude for the African American sample. Our findings add to the literature that maternal dietary patterns influence birth outcomes and suggest the need for further research on how to improve maternal diet during pregnancy, especially, in high-risk vulnerable populations and in low-resource communities.


Senescent cells have been demonstrated to release High Mobility Group Box 1 (HMGB1) which induces labor through an inflammatory pathway. This research is aimed at demonstrating whether telomere shortening, proinflammatory HMGB1, and oxidative damage marker 8-OHdG play a role in the placenta of preterm birth in comparison to term birth. μL, or abnormal vaginal discharge) and other pregnancy complications were excluded. Real-time polymerase chain reaction was performed to measure T/S ratio and ELISA quantification to measure the amount of HMGB1 and 8-OHdG.  A cross-sectional study on 67 full thickness of the placenta obtained from mothers with term and preterm birth. Mothers with clinical signs of infection , HMGB1 (p = 0.119), and 8-OHdG (p = 0.144) between the preterm and term groups. HMGB1 was moderately correlated with 8-OHdG (r = 0.314). Telomere T/S ratio of the placenta did not differ between preterm and term labor despite difference in gestational age, suggesting earlier shortening in the preterm group. It is possible that critical telomere length has been achieved in both term and preterm placenta that warrants labor through senescence process. The result of our study also showed that HMGB1 was not correlated to telomere length, due to the fact that HMGB1 is not upregulated until the critical length of telomere for senescence is exhibited.  A total of 34 placentas from preterm and 33 placentas from term birth were examined. Maternal characteristics were comparable between the two groups. There were no statistical difference of T/S ratio ( Similar telomere length might be exhibited due to early telomere shortening in preterm birth that mimics the term placenta. The relationship between placental telomere shortening and HMGB1 release remains to be uncovered. Further research is needed to discover the factors leading to early telomere shortening in the placenta of preterm birth. In 2018, globally, 15 million babies were born preterm despite advancing research and technology. Indonesia is ranked 5The role of senescence in preterm labor has been proposed . The preHigh oxidative stress leads to DNA damage shown by increased 8-hydroxy-2′-deoxyguanosine (8-OHdG) which may induce senescence , 8. The β and IL-6 and activates matrix metalloproteinase (MMP9) and collagen remodeling of chorioamnionitis membranes [High Mobility Group Box 1 (HMGB1) functions as a proinflammatory mediator in response to infection and stress. Senescent amniotic membrane HMGB1 expression is possibly linked to preterm birth and pPROM . Studiesembranes . The eviembranes .Research on the etiology of preterm labor had extensively focused on infection, which unfortunately does not decrease the rate of preterm birth. This study explores at other preterm pathomechanisms besides infection. Currently, no data are available on placental expression of proinflammatory HMGB1, oxidative damage marker 8-OHdG, and senescence marker telomere length in spontaneous preterm birth in the absence of infection. We aim to elucidate whether proinflammatory HMGB1 and oxidative damage marker 8-OHdG are upregulated in the placenta obtained from preterm birth and whether they are correlated with telomere shortening in inducing spontaneous preterm birth.We conducted a cross-sectional study at Cipto Mangunkusumo Hospital and Budi Kemuliaan Hospital, Jakarta, Indonesia, from February to August 2019. This study obtained ethical approval from the ethical committee of Faculty of Medicine Universitas Indonesia ethical approval number KET-126/UN2.F1/ETIK/PPM.00.02/2019, protocol number 20-06-0624.μL, or abnormal vaginal discharge) were also excluded. We documented maternal characteristics and routine laboratory results to further control confounding factors.A total of 68 samples were recruited consecutively following informed consent. Inclusion criteria were women with singleton pregnancy presenting with labor. Term labor was defined as delivery at ≥37 weeks of gestation, otherwise preterm. Gestational age was calculated based on the last menstrual period, further confirmed by the 1st trimester ultrasound record. Preterm labor was defined as signs of labor  upon presentation, with <37 weeks of gestation. Women with multiple pregnancy, intrauterine growth restriction, congenital malformation, premature rupture of the membrane, maternal hypertension, or diabetes were excluded from this study. Patients with clinical signs of infection . Samples were homogenized using Precellys 24-Dual Homogenizer  three times for 2 × 11 seconds at 5000 rpm speed with 10 minutes pause between the homogenization steps. Sample tubes were placed on ice during each pause to avoid overheating.Placental tissue was thawed at room temperature just prior to processing. Visible villi were removed from the samples. Samples were rinsed three times with Phosphate-Buffered Saline (PBS) pH 7.0 and then dried with Whatman® filter paper. 2 mg of tissue was weighed and cut into small pieces. 1000 100 mg of tissue homogenates was used for DNA extraction. The rest was lysed using an ultrasonicator for 30 minutes. Samples were then centrifuged at 8500 for 15 minutes. The supernatant was collected for determination of total protein, 8-OHdG, and HMBG1 concentration. Dilution was applied prior to each analysis if required.Total protein concentration was measured by the Bradford method using commercially available dye reagent . 8-OHdG concentration was measured using a commercially available ELISA kit , Cat No. 21026. HMGB1 concentration was measured using a commercially available ELISA kit , Cat No. ST51011.100 mg of tissue homogenates was used for DNA extraction. DNA was extracted from placental tissues using High Pure PCR Template Preparation Kit  according to the manufacturer's protocols for isolation of nucleic acids from mammalian tissue.T-test or Mann–Whitney U test accordingly. Correlation was analyzed using Pearson and Spearman analysis. Preterm birth was classified further for subgroup analysis by birth before certain gestational age: early preterm group (<32 weeks) and moderate-late preterm group (32–<37 weeks) [The minimal sample size for unpaired numerical comparative analysis was 21 for each group for two-tailed hypothesis, while the minimal sample size to analyze numerical correlation between variables was 31 for each group. Effect size for comparative analysis was set as 2, and standard error was also set as 2. Effect size for correlation analysis was set as 0.5. Significance was set at 5%, and power was set at 90%. Samples with any missing data were excluded from the analysis. Data were analyzed using the SPSS IBM version 20.0 . Data were considered as homogenous if the coefficient of variation (COV) ≤ 33% and are presented as mean ± standard deviation (SD), otherwise median (interquartile range). Numerical analysis was performed using independent sample 7 weeks) .μL for the term group and 15100 (11355–17100) μL for the preterm group . Other maternal characteristics were comparable between the two groups.A total of 67 samples were collected, 34 in the term group and 34 in the preterm group. Characteristics of the subject are presented in p = 0.053) for all subjects. Further correlation analysis on telomere ratio showed no correlation with gestational age for the term and preterm groups . HMGB1 was moderately correlated with 8-OHdG . Placental 8-OHdG and telomere ratio, as well as HMGB1 and telomere ratio, were not significantly correlated .The distribution of data is shown in n = 68; Pearson's correlation 0.238, p = 0.053), the term group , and the preterm group .Subgroup analysis on the preterm subject also found no statistical difference in terms of 8-OHdG, HMGB1, and telomere ratio between the early preterm and moderate-late preterm groups . In addiThis study demonstrates that markers of oxidative damage (8-OHdG), proinflammatory HMGB1, and senescence (telomere) are expressed comparably between the term and preterm groups. Even so between preterm subgroups, these markers show comparable results. We also found a significant correlation between 8-OHdG and HMGB1, but neither correlates with the telomere ratio.Shortening of telomere length (TL) has been linked to senescence; TL is influenced by DNA repair mechanisms, oxidative stress, and inflammation. TL tends to be highly correlated across tissues at birth, but this correlation diminishes as individuals age . As humaOur data reveals that earlier telomere shortening occurs in preterm labor, as shown by the equal telomere T/S ratio of the placenta from preterm and term labor. Senescence occurs when the telomere reaches its critical length that stopped the cell from further division , 16. GivHMGB1 from the placenta of both preterm and term labor has no significant difference despite the absence of intrauterine infection. Subgroup analysis of preterm birth occurring before or after 33 weeks of gestational age also reveals no significant difference between these groups. The labor process begins when there is a switch from uterine quiescent to contractile state which is marked by a proinflammatory condition, such as infection or sterile inflammation . A studyHowever, HMGB1 was not correlated to the telomere ratio in both groups. The causative role of HMGB1 in inducing spontaneous preterm labor and birth has been previously reported in the animal model. Our study is concurrent with this finding in terms of high HMGB1 in spontaneous preterm labor, comparable to term labor. However, we show that HMGB1 is not correlated to telomere length. We propose that this is because HMGB1 is not upregulated until the critical length of telomere for senescence is exhibited. However, unlike in term labor where the increase in HMGB1 is due to senescence-induced inflammation , other fThe marker of oxidative DNA damage 8-OHdG shows a similar quantity in both groups. Previous findings have shown that HMGB1 is increased in oxidative DNA stress , which mSimilar level of T/S ratio, HMGB1, and 8-OHdG is observed in placental sample from preterm and term birth despite difference in gestational age. Similar TL might be exhibited due to early telomere shortening in preterm birth that mimics the TL of term placenta. However, causative effect within the variables remains debatable. In addition, there was no correlation between 8-OHdG and telomere length. Further research is needed to discover the factors leading to early telomere shortening in the placenta of preterm birth.


While according to the United Nations birth registration is a human right, in sub-Saharan Africa (SSA) only half of new-born children currently have their birth registered. To gain insight into the reasons behind this low registration rate, we study the role of determinants at the household, sub-national regional and country level, using self-reported birth registration data on 358,842 children in 40 SSA countries. While most of the variation in reported birth registration is due to factors at the household level, context factors are found to play an important role as well. At the household level, poverty, low education, restricted autonomy of women, and belonging to a traditional religion are associated with lower odds of being registered. Lack of professional care during pregnancy, delivery, and early life also decrease the odds of being registered. Important factors at the context level are the average number of prenatal care visits in the local area, living in an urban area, the kind of birth registration legislation, decentralization of the registration system, fertility rates, and the number of conflicts. To improve registration, the complex dynamics of these factors at the household and context level have to be taken into account. Birth rThe consequences of not having a birth registration are enormous. Research shows that unregistered children have limited access to services, like health care, and cannot be protected by the law –8. ThereUntil around 1995, most research on birth registration was focused on developed countries –17. SincHowever, recent research for India indicates that studying factors at the household and context level simultaneously can significantly improve our understanding of birth registration problems . It therIn this way, we aim to contribute to the literature in important ways. First, by studying the determinants of birth registration at the household and context level simultaneously, the relative contribution of risk factors at the different levels can be estimated. Second, by using a very large sample of children, more precise estimates of the effects of those risk factors can be obtained. Third, by using data for 40 SSA countries, the role of factors at the national level can be studied better than in earlier research focusing on only one or a few countries.Birth registration is defined as: “the continuous, permanent, and universal recording, within the civil registry, of the occurrence and characteristics of births in accordance with the legal requirements of a country” . Birth rWhen the registration procedure is completed the child is legally existent and has documentation as proof, enabling the protection of other child’s rights as well, such as access to healthcare and education and legal protection from crimes like child labour , 21, 22.Although the benefits of having a birth registration are substantial, only 46% of the children in sub-Saharan Africa is registered , 7. ThisThe context in which the birth registration decision is made has basically three relevant levels: the household, local/regional and national level , 14. ThiThe birth registration decision is made at the household level, generally by the parents or caregivers of the child involved , 19. TheBesides the proper resources, knowledge about the importance and procedures of birth registration is needed , 14, 19.Demographic factors like the age of the child or family structures may be important too. Identity documents become more important if children grow older, as they may be needed for entering or graduating from school or to access health services , 31, 34.The ethnic or religious group to which a household belongs can also influence the birth registration decision , 19, 37.Besides socio-economic factors, care-related factors are important. According to Fagernäs & Odame , “registAvailability of primary care, provided by health institutions, in the first years of a child’s life is important as well , 28. AccAlthough the birth registration decision is made at the household level, the situation in the regional and national context can influence the outcome of this decision, as the availability and efficacy of services, policies, and infrastructures vary considerably within and across countries , 20, 34.The availability of services, such as health care and education, is also important , 39. FolNational context factors can be expected to be more important than local factors, as birth registration legislation is made at the national level. In low income countries, civil registration systems are often underdeveloped due to the lack of economic resources while these resources are needed for the creation and maintenance of good systems –14, 17. Another consequence of lack of resources is that the formation of appropriate institutions for birth registration is problematic . Due to With regard to demographic factors at the national level, both fertility and child mortality may be important. If fertility rates are high, registering every child may be problematic because of the substantial direct and indirect costs of registration , 19, 29.A final relevant factor might be the history of the country. The colonial era and periods of war and conflict may have long-lasting consequences for institutional arrangements , 17, 44.www.dhsprogram.com) and Multiple Indicator Cluster Surveys  have been used, which were derived from the Global data Lab  [For this study, combined datasets from the Demographic and Health Surveys  . DHS andOur combined dataset contained information on 442,433 children aged 0 to 4 years old who were living in 809 sub-national regions within 40 sub-Saharan African countries. For an overview of the countries and the numbers of subnational regions, see Context factors at the sub-national regional level were constructed by aggregating data from the household surveys to the local area level, using the regional codes present in the datasets , 49. ForA three-level multilevel logistic analysis is used to address the clustering of households within sub-national regions and countries , 27. LogFollowing Mohanty & Gebremedhin , first tHousehold wealth was measured by the International Wealth Index (IWI), which indicates the standard of living of households based on their possession of durable goods, the quality of their housing and access to basic services . Mobile Regarding demographics, age was measured in years. Missing of one or both parent(s) was measured by two dummy variables, indicating whether (1) or not (0) the father or mother was missing from the household. The position of women in the households was indicated by two variables, a dummy indicating whether (1) or not (0) the mother has given birth before the age of 18  and whetThe variables ethnicity and religion were based on pre-coded questions with the option to add additional categories. Religion consists of 7 categories, namely: (1) Catholic, (2) Protestant, (3) Christian, not specified, (4) Muslim, (5) no religion, (6) other, and (7) Traditional. For ethnicity a three-category variable was constructed indicating whether the ethnicity of the household is a (1) majority group, a (2) normal sized group, or a (3) minority group. These values were given based on the percentage distribution of the existing groups within the country. If a group concerned 0–10% it was seen as a minority, if it concerned 10–30% it was seen as a regular group, and if it concerned 30+% it was seen as a majority.Prenatal care personnel indicates who has performed the prenatal check: (1) no prenatal check was performed, (2) a traditional health care worker, (3) another person, (4) or skilled health personnel. The place of delivery is indicated by a dummy variable with value 1 when the birth took place at (someone’s) home while value 0 when at an institution. Assistance during delivery was computed to show whether the birth was assisted by: (1) no one, (2) a traditional birth attendant, (3) another person, (4) or skilled health personnel. The variable postnatal check reflects whether (0) or not (1) a postnatal check has been performed within 2 months after the birth. The variable vaccination shows whether children have ever received a vaccination (0) or not (1) while the variable vitamin A reflects whether a child received vitamin A in the last 6 months (0) or not (1).Context factors at the regional level were aggregated from the household data, following Smits & Huisman . AvailabThe economic situation of the country was measured by the Gross Domestic Product per capita (in current US dollars). The variable birth registration legislation indicates whether a country has (1) a legislation for birth registration or not (0). In turn, the variable no update in legislation represents whether there has been an update in birth registration legislation over the years (0) or not (1). Time allowed for registration is a dummy variable that indicates that a birth must be registered within a month (1) or not (0). Registration fee is a dummy variable indicating whether (1) or not (0) a fee was involved in birth registration. The organizational structure indicates whether the procedure is decentralized (0) or centralized (1). The level of governance of the country is proxied by the variable rule of law, in which -2.5 is the lowest score and 2.5 the highest . The demIn Looking at the map as a whole, it becomes clear that the variation among countries is larger than the variation within countries. This observation is confirmed if we look at the intraclass correlation coefficients (ICC) of intercept only models . Of the Among the care variables at the household level, we see that children born at a health institution, with help of skilled health personnel, who were checked within 2 months after birth, who have a vaccination, and who have received vitamin A in the last 6 months all have significantly higher odds of being registered. Receiving prenatal care is also important, but whether it is given by a professional or a traditional health care worker does not make much difference.Regarding context factors at the sub-national regional level, both the average number of prenatal care visits in the region and urbanization show significant effects. As expected, registration rates are higher among children living in urban areas and in areas in which the average number of prenatal care visits is higher. If the average number of prenatal care visits increases by 1, or one lives in an urban area, the odds of having a birth registration increase by about 10%. No significant effects are found for the age at first birth of women in the region, the educational level and the percentage of households with a phone in the region.With respect to the variables at the national level, GDP per capita shows a significantly positive relationship with birth registration. Moreover, the organization of the birth registration system is important. In countries with a centralized system, the odds of having a birth registered are 72% lower, and in countries where a fee has to be paid they are 67% lower. Next to the content of birth registration legislation, the fertility rate in the country and the number of conflicts experienced in recent decades are significantly associated with registration. Birth registration rates are higher in countries with higher fertility rates and in countries that experienced less conflicts.Lack of birth registration is a major problem in many LMICs . Most reAt the household level, both socio-economic, demographic and care-related factors are important. Children from wealthier households, older children, children with more highly educated parents, children with a mother who was over age 18 when her first child was born, and children from families with a non-traditional religion have significantly higher odds of being registered than other children. Although the variable phone is not significant in the current model, its role may become more important in the coming years given the increasing penetration of mobile phones with access to internet.Regarding the role of (reproductive) health care related factors, most findings are in line with the expectations. Children from households that make less use of health facilities and of skilled health personnel have lower odds of being registered. Not receiving prenatal care during pregnancy is especially problematic. In line with these findings, the average number of prenatal care visits at the sub-national regional level is associated with more birth registration as well. Other relevant context factors are urbanization, GDP per capita, centralization of the birth registration system, a fee for birth registration, the fertility rate, and the number of conflicts. Urbanization, GDP per capita, and the fertility rate all increase the odds of having a birth registration, while a centralized birth registration system, a fee for birth registration, and the number of conflicts all decrease the odds of having a birth registration.Given the large number of involved factors at different levels of analysis, we can conclude that the birth registration problem is complex and highly context specific. Underestimation of this complexity and the role of the context might be important reasons for the limited and slow progress made in achieving universal birth registration , 7, 64. Factors at the household level are only part of the story, and are probably the most difficult to change. Policies should therefore also focus on context factors, like those related to the local health care system and the national birth registration system and regulations. According to Muñoz et al. , to imprA limitation of our study is that the birth registration question in the DHS and MICS surveys does not ask the parents for evidence of registration, which means that the answers may suffer from misreporting due to desirability bias and misremembering , 14, 31.In sum, our simultaneous analysis of the major risk factors at the household, sub-national regional and national level and our finding that 29% of the variation in birth registration is due to factors at the context level constitute major steps forward in the birth registration literature. In particularly the identified associations between context factors and birth registration may have important implications for policy-makers.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Background: A major contributor to disability after hemorrhagic stroke is secondary brain damage induced by the inflammatory response. Following stroke, global increases in numerous cytokines—many associated with worse outcomes—occur within the brain, cerebrospinal fluid, and peripheral blood. Extracellular vesicles (EVs) may traffic inflammatory cytokines from damaged tissue within the brain, as well as peripheral sources, across the blood–brain barrier, and they may be a critical component of post-stroke neuroinflammatory signaling. Methods: We performed a comprehensive analysis of cytokine concentrations bound to plasma EV surfaces and/or sequestered within the vesicles themselves. These concentrations were correlated to patient acute neurological condition by the Glasgow Coma Scale (GCS) and to chronic, long-term outcome via the Glasgow Outcome Scale-Extended (GOS-E). Results: Pro-inflammatory cytokines detected from plasma EVs were correlated to worse outcomes in hemorrhagic stroke patients. Anti-inflammatory cytokines detected within EVs were still correlated to poor outcomes despite their putative neuroprotective properties. Inflammatory cytokines macrophage-derived chemokine (MDC/CCL2), colony stimulating factor 1 (CSF1), interleukin 7 (IL7), and monokine induced by gamma interferon (MIG/CXCL9) were significantly correlated to both negative GCS and GOS-E when bound to plasma EV membranes. Conclusions: These findings correlate plasma-derived EV cytokine content with detrimental outcomes after stroke, highlighting the potential for EVs to provide cytokines with a means of long-range delivery of inflammatory signals that perpetuate neuroinflammation after stroke, thus hindering recovery. Cerebrovascular accident (CVA), or stroke, is the worldwide leading cause of long-term disability, occurring at any age and across all demographics . HemorrhA major contributor to disability after stroke is secondary brain damage induced by the inflammatory response . After sCytokine diffusion, and thus their signaling influences, are functions of cellular release, cellular uptake (via specific cytokine receptors), and extracellular degradation ,13,14. AIn this study, we quantified the abundance of 80 cytokines in the plasma of hemorrhagic stroke patients. Further, we quantified the same 80 cytokines on patient plasma EVs, assessing both EV membrane-bound and internally sequestered cytokines. We found EV-associated cytokines to correspond to worse patient outcome compared to cytokine profiles from plasma alone. We report here that EV-associated cytokine profiles are largely correlated to worse acute (GCS) and chronic (GOS-E) neurological conditions regardless of the cytokines′ individual predicted effects on neuroinflammation or neuroprotection.p = 0.66, r = 0.08; p = 0.49, r = −0.13, respectively). The average BMI was 25.8 ± 4.3 SD, which was not correlated to either GCS or GOS-E . Multiple types of hemorrhage were recorded, including subarachnoid hemorrhage (SAH) (n = 7); subdural hemorrhage (SDH) (n = 6); intracranial hemorrhage (ICH) (n = 3); intraparenchymal hemorrhage (IPH) (n = 4); intraventricular hemorrhage (IVH) (n = 3); and a combination of up to 3 hemorrhage types (n = 8). Upon admission, the average GCS score was 10.54 overall, 10.62 ± 4.33 SD for males, 10.40 ± 2.91 SD for females, 13.75 ± 0.96 SD for IPH, 7.60 ± 3.27 SD for IVH, 9.40 ± 4.30 SD for ICH, 11.44 ± 4.42 SD for SAH, and 9.30 ± 3.34 SD for SDH. The average hemorrhage volume was 32,855.49 mm3 ± 24,403.33 SD, which was negatively correlated to the GCS  and not significantly correlated to the GOS-E . The type of hemorrhagic stroke was not observed to affect GCS or GOS-E. Healthy subjects include 8 males and 6 females averaging 53 years old ± 13.42 SD. A healthy pooled plasma sample was also included to provide an “averaged” cytokine level for healthy plasma. Pooled healthy donor plasma averaged 52 years old ± 10.96 SD. These and other demographic data, as well as outcome scores, are shown in In total, 31 patients were included in this analysis . Four patients were African American, 3 were Hispanic, and 24 were Caucasian. The average age was 51.17 ± 15.88 SD. Age was not significantly correlated to either GCS or GOS-E , followed by Eotaxin-3/CCL26  and hepatocyte growth factor (HGF) . Insulin-like growth factor binding protein-4 (IGFBP-4)  was correlated to a more negative GCS score, and tissue inhibitor of metalloproteinases 2 (TIMP-2)  was correlated to a more positive GCS score. Cytokines considered to be inflammatory  were correlated to more negative GCS than those considered neuroprotective   and GOS-E scores (30+ day outcome measure). We correlated the concentrations of these plasma cytokines to patient GCS, recorded at the time of blood draw—5–7 days after admission—and to their GOS-E scores obtained at least 3 months after stroke; these were then compared to plasma cytokine concentrations of the healthy controls. Of the cytokines correlated with GCS from the blood plasma only, granulocyte chemotactic protein 2 (GCP-2/CXCL6) was shown to correlate to the most negative GCS ( TIMP-2) .p = 0.0494, r = 0.414), RANTES/CCL5 , neutrophil-activating peptide 2 (NAP-2/CXCL7) , and osteopontin/SPP1  were correlated to positive GOS-E scores , HGF , and EGF . Of the inflammatory cytokines identified, the highest correlations to negative GCS include macrophage-derived chemokine (MDC/CCL22) , interleukin 7 (IL7) , and GRO/CXCL1   . Unlike p = 0.03, r = −0.389) and fractalkine/CX3CL1  were shown to correlate to the most negative regression values concerning GOS-E outcome score, followed by growth factor PlGF/PGF  (We identified 10 anti-inflammatory and 14 inflammatory cytokines from EV surfaces that correlated to GOS-E. The chemokines MCP-4/CCL13 ( −0.376) . EV surfp = 0.086, r = −0.313), which indicated a negative GCS (not shown). Four anti-inflammatory intra-EV cytokines were correlated to GOS-E outcomes, including fibroblast growth factor 6 (FGF6) , oncostatin M , stem cell factor (SCF/KITLG) , and vascular endothelial growth factor (VEGF)   was identified as having the most negative affect on GOS-E, followed by IL15 , RANTES/CCL5 , and IFN-γ . Inflammatory cytokine MIP-3 alpha/CCL20  was the only cytokine correlated to a positive GOS-E regression score  ( −0.383) . The cyton score .P) = −2.2) (P) = −2.9) and its regulation (Log10 (P) = −3.0) (P) = −2.25) and herpes simplex infection (Log10 (P) = −3.85). The regulation of phagocytosis (Log10 (P) = −3.3) was also correlated. No cytokines identified from plasma alone or from lysed vesicles were shown to overlap between GCS and GOS-E. The resultant protein networks and enrichment clusters we found would be influenced by our choice to use cytokines with correlations up to p = 0.1 and their correlations to GCS or GOS-E; any bias would be assuaged by using the cytokines that did not correlate to GCS/GOS-E for background subtraction.The Metascape platform provides insight and understanding of common and unique protein–protein interactions and pathways achieved by the analysis of target proteins against 40 independent “omic” databases . This pl = −2.2) . No addi = −3.0) , along w = −3.0) . These fTo assess potential cellular implications of cytokine levels in the plasma compartment versus the different EV compartments, we employed Core Analyses and Comparison Analyses in the Ingenuity Pathway Analysis (IPA). The cytokine array scores from each patient or healthy donor from plasma, EV lumen, and EV surface cytokines were quantitatively analyzed to generate log(2) fold change data of stroke patient values compared to healthy donor values. The algorithm generated heat maps from activation z-scores listing the top 50 diseases and biofunctions , where wp values) indicating the probabilities of random protein associations. Both networks or interactomes had scores of 25, and each had 13 focus molecules or nodes from which the networks initiate. Higher cytokine scores are indicated by red shading and lower values by green shading, which are again relative to their representation in stroke patients vs. healthy donors.Using IPA-extracted pathway data, in We note in Network 2 . We did not define cutoff values to make specific determinations, as this may require subjective interpretation of individual patients’ clinical status, particularly for longer term outcomes. This would be an ideal area of future investigation.Extracellular RNAs, such as microRNAs, presumably from plasma EVs, have been proposed to distinguish ischemic from hemorrhagic stroke . In geneA recent investigation by Fitzgerald et al.  concludeThe use of plasma EVs as reservoirs of predictive biomarkers for stroke outcome provides a cytokine-rich diagnostic source that is relatively easy to retrieve and minimally invasive compared to the spinal tap for CSF. Secondly, EV sequestration of cytokines may veil the true extent of their influence in post-stroke neuroinflammation. Failure to detect encapsulated cytokines through traditional assays leaves a substantial gap in the understanding of how neuroinflammation may be perpetuated, affecting CVA recovery. Curiously, we have shown similar correlations to negative outcomes in both pro-inflammatory and anti-inflammatory cytokines; however, the populations of inflammatory cytokines correlated to a poor outcome outnumber non-inflammatory cytokines when measured as bound to the surface of the extracellular vesicles as well as protected within. The impacts of cytokine localization in/on EVs may have profound influences on recipient cells, which may be determined by cytokine receptors on those cells, or modes of internalization and intracellular release of EV luminal cytokines. While this topic goes beyond the coverage of this study, further research is clearly needed to assess the potentially differential effects of EV cytokine localization both at the level of recipient cells and in overall systemic impacts.Diagnostically, the true inflammatory nature of these EVs may not be understood through traditional cytokine measurements of EVs, and other methods may need to be employed. The Metascape platform was used to gain insight into the interactions of cytokines identified as significant or trending toward significant in correlation to GCS and GOS-E. Among the 80 cytokines measured, those that were not correlated significantly or trending toward significance were used as background subtraction in an effort to detect cytokine interactions and biological pathways mediated only by those cytokines correlated to GCS or GOS-E. For instance, insulin-like growth factor signaling is well recognized in the context of ischemic stroke , suggestGCS has been shown to correlate to overall patient outcome after brain injury . We idenp = 0.038)  cells, which are responsible for inflammatory responses to infection, or T-helper 2 (TH2) cells, which aid in adaptive immunity and are considered neuroprotective ,46. Afte= 0.038) . This coThe involvement of lymphocytes and myeloid/monocytic cells in terms of development and migration is also implied by the IPA data . We noteTying into the IPA data, the chemokine RANTES/CCL5 was identified to significantly correlate to patient outcomes in both patient plasma and within EVs. Interestingly, the concentrations of RANTES were correlated to a more positive outcome as a free soluble form in the plasma and a more negative outcome within the EV lumen . RANTES p = 0.003, r = −0.516), IL15 , RANTES/CCL5 , and IFN-γ   . Each cyhttp://www.microvesicles.org/, accessed on 20 May 2021) and ExoCarta  databases. Our own work identified APOA1 and APOA4 in the proteomic analyses of EVs derived from human medulloblastoma cell lines and murine glioma cells [There are certainly caveats associated with this work. One assumption made is that EVs are inherently stable and are not spontaneously lysing to release internalized cytokines. Previous work  demonstrma cells ,63, withma cells ,65. WhilWe also note that the relatively small number of patients in the study precludes a robust multivariable analysis, particularly with the number of variables we already describe, along with many that we do not. For instance, individualized treatments, ranging from aspirin to surgical intervention, were not accounted for in this study (and we did not necessarily have access to such information). A detailed study involving the effects of anti-inflammatory agents or other clinical mediation in plasma/EV cytokine profiles and patient outcomes is beyond the scope of this work but would be a worthy effort.Edema is another clinical aspect of hemorrhagic stroke with important clinical implications  and a clFurther work is necessary to determine the cellular/tissue recipients of EV-delivered cytokines, as these will likely be points of therapeutic interventions. The effects of cytokine presence on EVs as well as within EVs is a new area of research; in conjunction with free plasma cytokines, there is great potential to alter the landscape of inflammatory responses during and following CVA. We point out that this is not a definitive biomarker study, given the complexity of the study, but it provides a novel springboard for future work.Study subjects were admitted to the University of Colorado Hospital within 8 h after onset of a stroke event. Thirty-one hemorrhagic stroke patients and 15 healthy non-stroke subjects were included in the study. This was the first stroke event for all stroke subjects, and the course of clinical care was similar across all stroke subjects as per standard of care suggested by AHA/ASA guidelines . All expg)  for 10 min, and plasma was collected. Plasma was collected from healthy subjects  as well as a pooled healthy donor plasma sample . Plasma was stored at −80 °C until analyzed.Whole blood was collected from the patients at 5–7 days after the stroke event. Ten milliliters of whole blood were collected in EDTA (K2) blood tubes . Whole blood was centrifuged at 1850 RPM (780× Patient non-contrast head computed tomography (CT) was performed within 12 h of hemorrhage and prior to surgical intervention per standard of care. Volumetric analysis of CT images was conducted through manual segmentation of the visualized edema region, using either BrainLab  or Insight Toolkit (ITK)-SNAP (ver. 3.80) .® Ultra EV Isolation Kit for Serum and Plasma  for the isolation of EVs by precipitation per kit instructions. This includes an initial centrifugation of 3000× g for 15 min for further removal of platelets. We utilized ExoQuick® due to the small volumes of materials available as in [In total, 250 μL of plasma was used with the ExoQuickle as in , and we le as in . Note beg centrifugation of the whole blood to collect plasma, per ExoQuick® instructions the plasma was spun at 3000× g for 15 min; this step should remove platelets. Further, in studies where cytokines associated with platelet EVs were measured, the vesicles were from isolated platelets that were activated to release larger particles . EVs isolated from 2 healthy donor individuals and from two CVA patients were assessed for EV protein markers using ExoCheck™ arrays  . AdditioFurther characterization of extracellular vesicles was performed through transmission electron microscopy . For eac® Ultra were stripped of surface proteins with 2 mg/mL proteinase K  in NEB buffer 2  and 50 mg/mL of BSA  for 1 h at 37 °C with gentle shaking. Whole EVs were then ultra-centrifuged at 150,000× g in a Beckman Ti 70.1 rotor (k factor = 150) for 2 h at 4 °C  to sepaCytokines in plasma, cytokines bound to membranes of isolated unlysed extracellular vesicles, and cytokines packaged within the isolated vesicles were measured. The total protein concentrations of these 3 preparations from subject samples were quantified using BCA assay and adjusted to 25 μg/mL for cytokine quantification via array measuring abundance of 80 cytokines . Samples were analyzed per the manufacturer’s instructions with primary antibody cocktail probing performed overnight at 4 °C and secondary antibody probing for 2 h at room temperature. The final cytokine arrays were imaged with a Cell Biosciences FluorChemQ Multiimage III chemiluminescence imaging cabinet . Fluorchem imaging software was used to quantify array signal and normalized to internal controls included on the array. Quantification results for cytokines from the different plasma/EV fractions are found in w/v) bovine serum albumin. Proteins were detected by incubation with primary antibodies against APOE  and APOB  in blocking solution overnight at 4  °C. Following 3 × 5 min washes of TBS with 0.1% Tween-20 (TBST), membranes were incubated in HRP-labeled rabbit anti-goat IgG secondary antibody (R&D Systems HAF017) diluted 1:1000 in blocking solution for 1 h at RT. The blots were washed 5 times in TBST for 5 min and washed a final time in TBS for 5 min. Blots were developed with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (ThermoFisher 34579) and were imaged with a Cell Biosciences FluorChemQ Multiimage III.The presence of apolipoprotein B (APOB) and apolipoprotein E (APOE) was evaluated by Western blot, adapted from Brennan et al. 2020  Figure . After lPatient physical and neurological status was evaluated by various stroke etiology-dependent scales, including the Glasgow Coma Scale (GCS) , Nationap ≤ 0.1 were evaluated using the Metascape platform [p ≤ 0.1 were analyzed against each other. The remaining cytokines of the 80 cytokines measured that were quantified, but not correlated to GCS or GOS-E, were used as background subtraction. Protein–protein interactions were considered significant at values of Log10 (P) = −2.0 or less by the Metascape platform, with significance increasing as the values decreased.Protein–protein interactions between all cytokines that correlated to GCS or GOS-E outcome up to platform . Cytokinhttps://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis, accessed on 14 February 2021)) [p values) indicating the probabilities of random protein associations.Per Qiagen format, we state that data were analyzed through the use of IPA ) . We usedThe statistical analysis was performed using the R suite, version 3.6 . All aveThe inflammatory response to stroke involves a series of highly complex interactions of cytokine signaling. Further heightening this complexity is the role of extracellular vesicles in delivery of these cytokines. This investigation sought to provide a comprehensive analysis of cytokine profiles delivered by EVs after hemorrhagic stroke. We identified several EV-contained cytokines that persistently correlate to poor neurological condition throughout stroke recovery. Our results also implicate the cytokine-enriched EV as a highly inflammatory agent detrimental to patient recovery after hemorrhagic stroke. Development of future therapies for hemorrhagic stroke should consider profiling these cell–cell communicators as a comprehensive unit, with EVs as a vehicle in which cytokines may be transported beyond their typical diffusion range. The impact of cytokines assisted by EV transport after CVA could prove to be a critical component in understanding the neuroinflammatory response after hemorrhagic stroke. Continued study of the potential inflammatory impact of EV-trafficked cytokines is necessary to further clarify their role in hemorrhagic stroke recovery.


The risk of NS increased as the gestational age decreased (p for trend < 0.001). Among the NS population, preterm births were not associated with more complications  or a requirement for secondary immunosuppressants (p = 0.61). In conclusion, preterm births were associated with subsequent NS, where the risk increased as the gestational age decreased. Our study provides valuable information for future pathogenesis studies.The pathogenesis of nephrotic syndrome is unclear. We conducted a nationwide population-based cohort study to examine the associations between preterm births and subsequent development of NS. NS was defined as ≥ 3 records with ICD-9-CM codes for NS in hospital admission or outpatient clinic visits. To avoid secondary nephrotic syndrome or nephritis with nephrotic range proteinuria, especially IgA nephropathy, we excluded patients with associated codes. A total of 78,651 preterm infants  and 786,510 matched term infants born between 2004 and 2009 were enrolled and followed until 2016. In the unadjusted models, preterm births, maternal diabetes, and pregnancy induced hypertension were associated with subsequent NS. After adjustment, preterm births remained significantly associated with NS ( Although steroids can induce remission in more than 90% of patients with NS2, the pathogenesis is not fully understood. In recent years, several studies have explored the elevation of immunoglobulin (Ig)E and interleukin (IL)-4 and -13 in children with NS3. Additionally, regulatory T (Treg) cells have been demonstrated to play a role in minimal change nephropathy4, and the numbers of Treg cells have been found to be reduced in patients with NS5. These data suggest that adaptive immune response dysfunction may play a role in NS pathogenesis.Nephrotic syndrome (NS) can affect pediatric population of any age, from infant to adolescent periods. Minimal change nephropathy is responsible for most cases of pediatric NS, followed by focal segmental glomerulosclerosis6 and might impair neonatal Treg cells7. Maternal rheumatoid diseases are associated with offspring immunological disorders9. Interestingly, Zhang et al. found that preterm births also affect the immune systems in later life10. Therefore, preterm births might influence the immune response, specifically the T cell immunity, and then predispose the onset of the NS. However, although some investigations revealed that among children with nephrotic syndrome, those born with low birth weight or premature births were associated with unfavorable clinical courses12, the relationship between preterm birth and NS development remains unknown. We hypothesized that preterm infants had a higher risk for subsequent NS and aimed to reveal their relationship in the current study. We used a nationwide population cohort to test the hypothesis due to the low incidence of NS. It should be noted that, many maternal immunologic diseases may be responsible for allergic diseases later in life9. To address this problem, we used the cohort that provided the parent–offspring linkage, which provided information on the possible impact of maternal disorders.Growing data have demonstrated that early life exposure may influence immunologic systems in later life. An early meta-analysis showed that maternal and paternal asthma is associated with offspring asthmaTo decrease the possibility of misclassifications, we first validate our definition of NS. During 2004–2016, 31 patients were born between 2004 and 2009 had ≥ 3 records with ICD-9-CM codes for NS in hospital admission or outpatient clinic visits and did not have ICD-9-CM codes for secondary nephrotic syndrome or nephritis with nephrotic range proteinuria. Among them, one had IgA nephropathy, another had IgM nephropathy, and the other 29 patients had a diagnosis of NS. The positive predictive value was 93.5%.p < 0.001). Although the maternal and paternal ages were statistically higher in the preterm cohorts, the differences in the point estimation were low and without clinical significance.In total, 78,651 preterm infants and 786,510 matched term infants were analyzed in the current study (Table p for trend < 0.001). Interestingly, in the univariable but not multivariable models, maternal diabetes was found to potentially predispose offspring to NS , and the offspring of mothers with pregnancy-induced hypertension also had a higher NS incidence . After adjusting for the possible risk of NS, the preterm births remained significantly associated with subsequent NS development . Similarly, this condition was even more evident in the lower gestational age group . When we set the gestational age as the continuous variable, the hazard ratio also consistently decreased with increases in gestational age .Overall, 50 children in the preterm cohort and 285 children in the term cohort developed NS, with incidence rate of 7.1 and 4.0 per 100,000 person-years, respectively (Table p < 0.001). In addition, if we grouped the preterm cohorts according to the different gestational ages, the condition was even more evident in the lower gestational age groups . We also examined the relationship between gestational age and the age when nephrotic syndrome diagnosed. The Pearson correlation analyses showed that the correlation coefficient is low between gestational age and the age when nephrotic syndrome diagnosed   Fig. .Figure 1p = 0.52). We further tested the correlation between gestational age and complications. The gestational ages were comparable between patients with and without complications  (Table p = 0.91).We further examined whether preterm NS patients had more complications, such as hypertension, serious infections, or ESRD. In the patients with NS during the study period, we found that 10 patients developed hypertension, 40 patients had serious infections and 5 patients had ESRD. The results showed that the odds for hypertension and serious infections were comparable in the preterm and term children  -associated cytokines and childhood nephrotic syndrome relapse21. Jacques et al. also elucidated that TH2 polarization precedes development of nephrotic syndrome in animal models22. Additionally, Treg cells have been proven to be associated with NS5. Few studies have examined the differences in long-term T cell subset development in preterm and term infants. One recent work involved the use of an animal model. The result demonstrated a more systemic TH2 skewed pattern in preterm pigs than in term pigs with the same corrected age23.The underlying mechanisms for the relationship between preterm birth and development of NS development are not known. Previous studies suggest that adaptive FSGS and podocytopenia may be the reasonreg cell proportions are higher in preterm infants at birth24. In contrast, preterm infants may be exposed to other risk factors for T cell dysfunction, including prenatal steroids, chorioamnionitis, stress, postnatal dysbiosis, and infection. Prenatal steroids can cause a reduced fetal thymus and have been shown to influence thymocyte development in both mice and humans25. A recent investigation also revealed that prenatal betamethasone may change the T cell receptor repertoire and may induce autoimmunity26. Claire et al. found that although preterm birth did not affect Treg frequency, it may cause decreased Treg functions27. Interestingly, the data from the ICESTORM project demonstrated that stress could elevate TH2 cytokine expression28. In addition, preterm infants have a higher risk of infections and exposure to antibiotics, and perinatal antibiotic exposure can influence the microbiota and is associated with allergic diseases29. Neonatal gut dysbiosis has also been explored to induce CD4+ T cell dysfunction30.T31. Likewise, a recent investigation revealed that exposure to maternal type 2 diabetes may contribute significantly to asthma later in life. This phenomenon has also been observed in exposure to gestational diabetes requiring medication but not in exposure to gestational diabetes that didn’t require medication32.Similarly, a previous study also showed that maternal diabetes is associated with the development of asthma in offspring. These authors suggested that disruption in the development of the fetal immune system may be the reason for this relationship33. In addition, the birth weights were from BRD and were previously validated34. Hypertension and ESRD may have been underreported because of the short observation periods and possible issues with misclassification. Second, because no laboratory and pathologic data are available in our database, we cannot truly know in our NS patients whether preterm births were associated with greater FSGS pathology or nephron deficits. Therefore, this requires further investigation. We also cannot determine the subtypes of NS based on steroid responsiveness. We hence used the secondary immunosuppressants usage as the surrogate of frequently-relapsing, steroid-dependent, and steroid-resistant nephrotic syndrome. Finally, because our database mainly comprised Chinese participants, whether the results can be generalized to other ethnicities requires further verification.Some limitations of the current study should be mentioned. First, we used the ICD-9 code for the definition of NS and the birth weight from the TMCHD for the definition of small for gestational age, which may have resulted in misclassifications. To decrease the possibility of misclassifications, we first carried out a validation for our definition of NS, for which the positive predictive value was 93.5%, which was higher than many studies using health claims data for identification of chronic kidney diseasesIn conclusion, we demonstrated that preterm infants have an increased risk of NS and that its occurrence is aggravated by maternal DM. However, preterm birth may not be associated with complicated clinical NS courses. The underlying mechanisms require further investigation to elucidate their causal relationship.35. All births, including both live births and stillbirths, have to be registered legally within ten days in Taiwan. Thus, the BRD provides birth information, including sex, birth weight, gestational age, and gestational numbers. It also included the parents' characteristics, such as age at delivery. The BCA contains prenatal care details and the information recorded at birth, such as Apgar scores and congenital abnormalities. The National Health Insurance (NHI) program was implemented in 1995 and provides coverage for more than 99% of the residents of Taiwan36. The database described above, therefore, covered 99% of the entire population in Taiwan, where very few patients were lost to follow up37. Most components of the TMCHD were validated37. The TMCHD uses the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) as the diagnostic codes. This study was approved by the Institutional Review Board of National Cheng Kung University Hospital (A-ER-108-245) and all research was performed in accordance with the Declaration of Helsinki. Due to privacy regulations, information on the beneficiaries was anonymized before being released to the researchers; therefore, the Institutional Review Board of National Cheng Kung University Hospital approved informed consent was not required.We used the Taiwan Maternal and Child Health Database (TMCHD) for the current investigation. The TMCHD comprises the medical claims of four Taiwanese nationwide population-based databases: the Taiwan Birth Registration Database , Birth Certificate Application , National Register of Death , and the National Health Insurance Research Database  and therefore provided the parent–offspring linkage38. The observational period was between January 1, 2004, and December 31, 2016. Our primary interest was whether prematurity was associated with NS and whether it was related to chronic diseases in the mother. To improve the diagnostic accuracy, NS was defined as ≥ 3 records with ICD-9-CM codes for NS, which were listed in Supplemental Table A nationwide retrospective cohort study design was conducted. We identified live preterm infants  born in Taiwan between January 1, 2004, and December 31, 2009, as the preterm cohort. Due to the low incidence of NS, ten randomly selected live term infants were matched by sex and birth year for each preterm infant as the comparison cohortWe also examined whether prematurity was responsible for more comorbidities in the NS population for the secondary outcomes. We defined hypertension when patients with related codes for ≥ 2 records in an inpatient or ambulatory claims diagnosis field .1. The parental sociodemographic factors include maternal and paternal age at delivery, family income, and urbanization. The family income was assessed at birth and was classified into four quartiles based on each birth year and the levels of urbanization were based on the previous published methods39.The potential confounding factors were divided into three categories: perinatal, parental sociodemographic, and maternal medical factors. The perinatal factors include small for gestational age, which was defined as birthweight below the 10th percentile for gestational age32, hypertensive diseases40, rheumatoid arthritis8, asthma6, and systemic lupus erythematosus9. We therefore carried out extended adjustments for these factors to avoid any confounding bias. Pregnancy induced hypertension was defined as hypertension that complicated pregnancy after 20 weeks of gestation.Finally, some maternal conditions were associated with allergic responses in offspring, such as diabetesWe compared the characteristics of the preterm cohort to the comparison cohort using the t-test or Mann–Whitney U test for continuous factors and the chi-square or Fisher’s exact tests for categorical variables, according to variable distributions. We used Kaplan–Meier methods to demonstrate the NS-free survival probability and log-rank test to analyze the differences between distinct gestational age groups. The Pearson correlation analyses were used to examine the relationship between gestational age and the age when nephrotic syndrome diagnosed. The univariable Cox proportional-hazards model was used to test the association between preterm births and NS. To avoid confounding factors, we conducted a multivariable model to evaluate the relationship between NS and preterm birth and adjusted for gestational age, small for gestational age, urbanization, family income, maternal and paternal age, and maternal complications. We also evaluated whether preterm birth was associated with more complications in NS patients using logistic regression models.p < 0.05.The statistical analyses were performed with SAS statistical software (SAS Institute Inc), version 9.4. Statistical significance was defined as two-tailed Supplementary Information.


Candida auris is a multidrug-resistant, difficult-to-eradicate pathogen that can colonize patients and health-care environments and cause severe infections and nosocomial outbreaks, especially in intensive care units. We observed an extremely low-birth-weight (800 g), preterm neonate born from vaginal delivery from a C. auris colonized mother, who was colonized by C. auris within a few hours after birth. We could not discriminate whether the colonization route was the birth canal or the intensive care unit environment. The infant died on her third day of life because of complications related to prematurity, without signs or symptoms of infections. In contexts with high rates of C.auris colonization, antifungal prophylaxis in low-birth-weight, preterm neonates with micafungin should be considered over fluconazole due to the C. auris resistance profile, at least until its presence is excluded. Candida bloodstream infections (BSIs) are the third most common cause of health care-associated BSIs, especially in extremely low-birth-weight (<1000 g) and/or preterm  neonates [-weight <000 g andCandida spp. has changed worldwide in recent years, with emergence of resistant non-albicans strains. From its first identification in 2009 in Japan [Candida auris has raised global attention due to its rapid spread and its multidrug-resistance profile [C. auris clades, and hence its use in the treatment of or prophylaxis in specific settings is limited [C. auris isolates were most often misidentified as a range of other Candida species, especially as C. haemulonii, to whom it is phylogenetically closely related [C. auris from other Candida species. Molecular methods based on DNA sequencing can also identify C. auris; sequencing of the D1-D2 region of the 28s ribosomal DNA (rDNA) or the internal transcribed spacer (ITS) region of rDNA are accepted methods. More recently, the development of polymerase chain reaction (PCR) assays specific for C. auris and for C. auris-related species using cultured colonies has shown promise for the rapid and accurate identification of C. auris, which could prove particularly useful in outbreak situations [Epidemiology of  profile , with re profile ,5. Fluco limited . In the  related ,8; howevC. auris frequently causes outbreaks, especially in intensive care units (ICU), including neonatal intensive care units (NICU). C. auris is normally identified as a colonizer, more rarely as a cause of BSI or deep site localisations, with horizontal transmission probably as the most frequent way of acquisition [C. auris has been documented [C. auris invasive disease being substantially identical to those for other Candida species  [C. auris [uisition , since ccumented . Notablymission) , colonizmission) ,8. PreveC. auris .C. auris and its management, and to discuss the possibility of using prophylaxis with micafungin in contexts of high risk of azole-resistant fungi nosocomial acquisition.The aim of this report is to describe the first case of possible vertical transmission of abruptio placentae in another Center. The mother was admitted to the ICU because of COVID-19 pneumonia necessitating intubation and mechanical ventilation. Notably, vaginal swab of the mother resulted positive for C. auris.An extremely low weight birth (800 g), preterm , female neonate was born from vaginal delivery following preterm labor complicated by maternal bleeding in C. auris were performed at time of admission following the CDC’s procedures for high-risk patients [C. auris, so the results interpretation was defined according to CDC’s tentative breakpoints [C. auris identification, strict contact isolation procedures were implemented according to the hospital policy for patients coming from other Centers to contain the spread of resistant pathogens [C. auris was observed.The newborn was transferred within a few hours after delivery to the NICU of Istituto Giannina-Gaslini, Genoa, Italy. Multiple swabs for patients . Cultureakpoints : fluconaathogens ,15. The C. auris vertical transmission, since, to the best of our knowledge, no case of colonization of a neonate born from vaginal delivery has been reported . The patient was colonized within a few hours after delivery, confirming the possibility of colonization by resistant pathogens already in the first hours of life, as observed in other contexts [C. auris was acquired from the environment and hands of healthcare workers, although acquisition through the birth canal from a colonized mother was more likely.This is the first description of contexts . HoweverC. auris in Italy was a recent event and its spread could have been facilitated by the COVID-19 pandemic, as was probably the case for our patient. Indeed, a recent a study conducted in the same hospital where the patient was delivered showed the spread of multi-drug resistant (MDR) Gram-negatives and C. auris during the SARS-CoV-2 pandemic [C. auris strains observed in the patient and her mother was consistent with nosocomial transmission of the pathogen, suggesting that protective measures used to prevent SARS-CoV-2 acquisition do not share the benefit of preventing cross-transmission of other pathogens between patients. Therefore, dedicated strategies are warranted to prevent horizontal spread and maintain effective antimicrobial stewardship programs in the setting of COVID-19 care, particularly in the case of C. auris due to the difficulty of its eradication and the very limited therapeutic options once infection has ensued.Remarkably, the introduction of pandemic . The cloC. auris infection/colonization should be isolated and should routinely be investigated for C. auris colonization because of the risk of BSI and its poor outcomes, and because of the risk of horizontal spread. Fluconazole prophylaxis is considered a standard for low-birth-weight neonates [C. auris, micafungin, with less than 10% of resistant strains described [C. auris, at least until its presence is excluded.Although further studies and more evidence is needed to understand the risk of vertical transmission, according to the recent recommendation issued by the International Society for Antimicrobial Chemotherapy Working Group on Infection Prevention and Control , we suggneonates . Howeverescribed , should C. auris, possibly acquired from the birth canal. Invasive candidiasis prophylaxis was performed with fluconazole, as per our hospital protocol, before we identified azole-resistant C. auris in surveillance cultures.We presented a brief case of a low-birth-weight preterm female neonate colonized by C. auris in hospitals, micafungin prophylaxis for invasive candidiasis should be considered as an alternative to fluconazole in low-birth-weight neonates.In light of the rising prevalence of


Echinococcus granulosus. The tapeworm larvae can create cyst in different areas of the body, especially the liver and lungs; however, the formation of the cyst in the perineal and femoral regions are very rare. The unusual location of the cyst can help us with the differential diagnosis of soft tissue mass(es) in this location, especially in endemic areas. Diagnosis of this disease is crucial because if the cysts are ruptured during surgery, the fluid inside can cause anaphylactic shock.Hydatid cyst is a zoonotic disease caused by the parasite Our case is a 55-year-old woman with the chief complaint of a painful mass in the right thigh and perineal area with progressed pain and paresthesia to the right thigh and shin. The patient had no history of fever, abdominal pain, digestive dysfunctions, or chest pain. The vital signs were normal, and there was no family history. Physical examination showed that the skin over the mass had no discoloration, and the size was around 5.7 cm long. The result of the ultrasonography examination showed a cystic mass with suspicion toward the femoral hernia. After a CT scan, the result of secondary workups was a multi-lobular cystic mass with no connection to the abdominal region, which suggested a hydatid cyst. The patient underwent spinal anesthesia and surgery, a cystic mass with ecto- and endocyst, with clinical similarity to a hydatid cyst, was removed with wide margins, and the cyst wall was kept intact. In the next step, the specimen was sent for histological examination that confirmed cystic hydatidosis. The Post-surgical Abdominal and thoracic Ultrasonography screening were used to exclude relapse, and medical therapy was given for 3–6 months. An 18-months follow-up demonstrated no reoccurrence and no newly formed cyst.Although rare, femoral hydatid cyst can occur in some cases, especially in endemic areas. We highly recommend our colleagues consider ruling out cystic hydatidosis in any patients complaining of regional mass(es), mostly painless, presenting with adjacent tissue compression with or without manifestation of an allergic reaction. Echinococcus granulosus [Hydatid cyst is a chronic parasitic disease caused by four species of a famous family of parasitic tapeworms called echinococcus, most commonly, anulosus . Cystic anulosus –4. This anulosus .from head to toe” [In some rare cases, the cysts are implanted in the areas including the inguinal canal, perineal region, spleen, skeleton, brain, kidney, peritoneum, thyroid, and parathyroid. In other words, hydatid cyst can be found “ to toe” , 7.In this report, we represent a rare case of perineal and femoral hydatid cyst. The unusual location of the cyst can help us with the differential diagnosis of soft tissue masses in this location, especially in endemic areas where unusual hydatid cyst formation is observed. Diagnosis of a hydatid cyst is crucial because if the cysts are ruptured during operation, the fluid inside the cyst can cause anaphylactic shock inside the body. Therefore, preoperative diagnosis of the hydatid cyst is necessary for proper treatment.A 55-year-old female was admitted to the hospital’s emergency department with the chief complaint of a painful mass located in the right thigh and perineal area and with progressed pain and paresthesia to the right thigh and right shin. The patient seemed ill but not toxic. No history of fever, abdominal pain, digestive dysfunctions, chest pain, cough, hemoptysis, urticarial were found. The vital signs were in a normal range, and there was no family history.Physical examination showed that the skin over the mass had no discoloration, and the size of the mass was around 5.7 cm long. The blood tests were normal, and the lab results showed no eosinophilia. The result of the ultrasonography examination was a cystic mass with suspicion toward femoral hernia. After a CT scan, the result of secondary workups was a multi-lobular cystic mass with no connection to the abdominal region Fig. , which sThe patient underwent surgery under spinal anesthesia in the next step, and the right thigh was opened up. A cystic mass with ecto- and endocyst with clinical similarity to a hydatid cyst was removed with wide margins and without penetration of the cyst wall. The removed specimen was sent for histological examination with the first impression of cystic hydatidosis.Received specimen in formalin consist of a creamy-whitish colored cyst M = 7 * 5 * 3 cm filled by multiple variable-sized creamy cysts Fig. . The cysFollowing the surgical removal of the cyst, the patient underwent recovery. Besides, Post-surgical Abdominal and thoracic Ultrasonography screening were used to exclude recurrence. Albendazole 400 mg PO BID was prescribed for 3–6 months. An 18-months follow-up demonstrated no reoccurrence or any other site of cystic hydatidosis.After a year of routine follow-up, the patient did not demonstrate any signs of recurrence of the adverse effect of surgery, and she was entirely well.from head to toe” [Hydatid cyst is a disease that can cause Public health challenges, especially in endemic areas that consist of tepid climate countries, including the Mediterranean region, Central Asia, South America, Africa, and China . Also, t to toe” , 7. This to toe” .Echinococcus granulosus larva in this location is still a mystery; however, the primary distribution of this parasite by blood is probably the most reasonable explanation [In our case, the hydatid cyst was present in the perineal and right femoral region, which is a rare area for the formation of this cyst. The exact mechanism for explaining the implantation of lanation , 13. Thelanation . The diflanation .In femoral or perineal hydatidosis, the preoperative diagnosis of the disease is crucial because the cyst contains a fluid inside it, which has specific antigens and, if ruptured during operation, can cause a type 3 hypersensitivity reaction. This reaction can vary from benign symptoms such as benign urticaria, episodic chills, or fever to more severe and fatal symptoms, such as edema and ultimately anaphylactic shock , 17. TheThe diagnosis of a hydatid cyst is mainly constructed around the patient’s history, clinical findings, and examinations. The examinations consist of Serology test, Ultrasonography, with the best efficacy for detecting cysts as small as 1 cm , 19, CT The treatment options for this disease consist of operative and non-operative methods. In this study, we prescribed albendazole alongside surgical procedures, pre-and post-operatively. The best way to treat hydatid cyst is Surgery, and medication alone is insufficient , 27. ComTo detect recurrence or secondary formation in other parts of the body at an early stage, follow-up and patients monitoring by using radiological and serologic applications are necessary. The protocol for monitoring patients consists of a complete physical examination in 3, 6, 12, 18 months and, after that, annually for 10 years. In addition, serologic test, chest X-ray, hepatic sonography, and sonography of the area where the primary mass(es) were found is necessary .Although rare, femoral hydatid cyst can occur in some cases, especially in endemic areas. Variations of patients’ manifestation depend on adjacent compressed tissue. We highly recommend our colleagues consider ruling out cystic hydatidosis in any patients complaining of regional mass(es), mostly painless, presenting with adjacent tissue compression with or without manifestation of an allergic reaction.


Background: Interactions between the genome and intrauterine environment can affect bone mineralization in newborns and even in adult life. Several studies show that intrauterine fetal bone mineralization or early postnatal bone condition influences the risk of osteoporosis in later life.Objectives: To determine whole body bone mineral content (WB BMC) and factors that influence neonatal WB BMC in Indonesian term newborns.Subjects/Methods: A cross-sectional study was conducted in Dr. Sardjito General Hospital, Yogyakarta, Indonesia. A total of 45 term, appropriate for gestational age (AGA) newborns were included in this study. BMC was assessed by dual-energy x-ray absorptiometry (DXA) in the first week of life. Weight (g), length (cm) and head circumference (cm) were measured at birth. Data on maternal characteristics were obtained from the maternal health records or reported by the mothers.Results: WB BMC measured in the present study (mean ± SD: 33.2 ± 9.3 g) was lower than WB BMC of similar populations in developed countries. Multiple linear regression showed that birth weight, birth length, and gestational age had a positive association with WB BMC , while maternal cigarette exposure had a negative association with WB BMC (p = 0.012). Male infants had significantly higher of WB BMC than female (p = 0.025). These determinants contribute to 55% variability of WB BMC.Conclusions: WB BMC in Indonesian term newborns is lower than populations in developed countries. Birth weight, length, gestational age, sex, and maternal cigarette exposure during pregnancy are significantly associated with WB BMC observed in Indonesian newborns. Osteoporosis has a significant impact on a person's quality of life as well as being a massive economic burden for the country. In 1990, the worldwide annual direct and indirect burden of hip fracture was estimated at US $ 34.8 billion, rising to $ 131.5 billion by 2050  , while with Lunar DXA it was 78.9 g (95% CI 78.4–79.4) .With respect of cigarette smoking, Indonesia has one of the highest prevalence of active smokers in the world, with 55.8 % of men aged 10 years and over, compared to just 1.9 % of women of the same age . In viewWe enrolled 45 term infants hospitalized in the newborn nursery of Dr. Sardjito General Hospital from March 2018 to March 2019. Inclusion criteria were gestational age ≥ 37 weeks, appropriate for gestational age (AGA), and medically stable. Infants with major congenital abnormalities, bone disorders, gastrointestinal, or renal diseases were excluded.2): underweight, <18.5; normal, 18.5 to <24.99; overweight, 25 to <29.99; and obese, ≥30 , parity, education level, and cigarette exposure during pregnancy. Information on pre-pregnancy weight and maternal height were taken from the maternal health records or reported by the mothers. Pre-pregnancy maternal body mass index (BMI) was calculated as pre-pregnancy weight (kg) divided by height (m) squared. The Institute of Medicine (IOM) classification system was used to categorize BMI  was measured using calibrated electronic digital scale  to the nearest 0.1 g. Length was measured using a standard length-measuring board  by two trained nutritionists to the nearest 0.1 centimeter (cm). Head circumference was measured with a non-stretch measuring tape to the nearest 0.1 cm. Infants were classified as AGA if their birth weight were ≥ 10th and <90th percentile for gestational age, using PediTools Fenton 2013 .WB BMC, WB BMC except head, trunk BMC and legs BMC were measured using dual-energy X-ray absorptiometry  as describe elsewhere . AssessmThe BMC was assessed within the first week of life. The mother was asked to breastfeed the baby before being transported to the scanning room to help keep the baby calm and minimize movements during scanning. Babies were placed in a portable carriage and brought to the scanning room. Most newborns became drowsy after swaddling, which facilitated their standardized positioning, thus minimizing movement artifacts in the scanning. During scanning, infants were in supine position on the scan table. The scan began at the top of the head and moved in a rectilinear pattern down the body to the feet. The scan was stopped and repeated if there was a significant movement. BMC was measured in grams (g).post hoc Tukey's test. Independent t-test was also used to detect mean difference between two groups. Significance was set at p < 0.05. Because there are no data of newborn BMC available in Indonesia, we decided not to calculate sample size. We start with observational study to evaluate BMC in Indonesian newborns.Statistical software SPSS for Windows, version 18.0  was used to perform data analyses. Data were expressed as means ± standard deviation (SD) or ratio. The associations between WB BMC at birth and the determinants  were analyzed using linear regression. The mean differences between subgroups were identified using ANOVA and The study was approved by the Medical and Health Research Ethics Committee of the Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta. Written parental informed consent was obtained from both parents of the study subjects in accordance with the appropriate Ethics Committee requirements.A total of 45 term AGA newborns consisting of 23 male and 22 female newborns were included in this study. The subjects' characteristics are shown in p = 0.028). Infants born to mothers exposed to cigarette smoke had a significantly lower WB BMC than those not exposed (p = 0.046)  .Linear regression analysis showed that sex, gestational age, birth weight, birth length and maternal cigarette exposure were significantly associated with WB BMC at birth .p < 0.25 in the simple regression analysis were included in the multiple regression model . Male infants had significantly higher of WB BMC than female (p = 0.025). On the other hand, only maternal smoking exposure was negatively associated the newborns' WB BMC (p = 0.012). Those determinants contribute to almost 50% of the variance of WB BMC (adjusted R2 = 0.55).Multiple linear regression analysis was also performed to assess which variables were independently associated with WB BMC. Variables with on model . The resThe present study investigated the association between maternal and neonatal conditions and BMC assessed with DXA in term AGA Indonesian newborns at birth. The results of our study showed that birth weight, -length, gestational age, sex, and maternal smoking exposure had significant associations with BMC.The International Society for Clinical Densitometry (ISCD) advises that spine and whole body scans be used to diagnose bone disorders in children. However, there are currently no clear guidelines for the infant. In infants, measuring the whole body except head may be preferred because of inaccuracies in the algorithm for determining the BMC of the skull. Spine vertebrae especially the lumbar is recommended because of their speed and precision of measurement, easily identified bony landmarks, and less subjected to movement artifact. However, because of its excellent precision and the fact that it measures overall bone density, the total body is still a preferred site . MoreoveThe WB BMC observed in this study was much lower than the total body BMC observed in several studies conducted in developed countries. A systematic review of term infants, mostly from developed countries, found a mean WB BMC of 68.8 g and 65.9 g for male and female infants, respectively . This syIt is well-known that vitamin D is essential for mineral-bone homeostasis. Several studies have reported that maternal serum 25-hydroxyvitamin D (25 [OH] D) concentrations are associated with offspring bone mass –23. Low The supply of calcium from maternal sources plays an important role in the normal development of fetal bones. Maternal hypocalcemia may stimulate intrauterine parathyroid hyperplasia causing poor mineralization of the fetal skeleton . PreviouAs observed in previous research, the findings of the present study found an increase in WB BMC that was consistent with rising birth weight and length , 30. ThiOur findings are consistent with the results of a previous study indicating that birth weight and birth length were strongly, positively associated with neonatal bone mass even after adjusting for sex and gestational age . In the In utero, the testes produce testosterone, which is believed to increase lean body mass in fetal life. In the first week of life, male infant testosterone levels increase rapidly and nearly match the concentrations achieved in adult males, whereas the ovaries are relatively silent regarding estrogen production, and do not release significant amounts of testosterone during perinatal development  Faculty of Medicine Gadjah Mada University-Dr. Sardjito General Hospital. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.TW performed the conception and investigation of the study, performed the statistical analysis, prepared the tables, and wrote the final version of the manuscript. NN identified the statistical methods and validated the statistical analysis results. JH reviewed and edited the final version of the manuscript and did the verification of the reproducibility results. AA helped the investigation of the study and maintained the research data. MH, MJ, and MV obtained funding for the study, advised the study design, supervised the statistical analysis, interpretation of the results, and reviewed the final version of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.