To probe into the associations between maternal personal cosmetics use during pregnancy and risk of adverse outcomes, and explore the potential dose-response relationships, we carried out a prospective cohort study involving 9710 pregnant women in Zhuzhou City and Xiangtan City in Hunan province during 2016–2017. A structured questionnaire was used to collection information for the pregnant women and their pregnancy outcomes. Odds ratios (OR) and 95% confidence intervals (CI) were calculated by binary or multinomial logistic regressions. The study population included 4652 (47.9%) cosmetics non-users and 5058 (52.1%) cosmetics users. Cosmetics use was associated with an increased risk of small for gestational age (SGA) , compared with cosmetics non-users. A positive dose-response relationship between frequency of cosmetics use and SGA was observed, although a borderline association was found at low use frequency  and moderate use frequency . High-frequency of cosmetics use (≥5 times per week) was significantly correlated with a higher risk of SGA . No significant association between personal cosmetics use and the risk of preterm birth, low birth weight, macrosomia, or large for gestational age was observed. The present study suggests that personal cosmetics use will increase the risk of SGA, but further research is required to determine which cosmetic products may account for the higher risk of SGA. Cosmetics products contain various chemical substances  and heavy metals , which are either added purposely as efficacy components to enhance the skin care effect of cosmetic products or brought in from the raw materials, auxiliary materials or technical problems during the production process7. Cosmetics use is reportedly related with increased personal exposure to parabens, phthalates and benzophenone9.Cosmetic products are necessities in our daily life and mostly used by young women, including pregnant women. Recent cross-sectional studies show 60–80% of pregnant women also use cosmetic products, including facial cleaner, day cream, foundation, mascara, lipstick, eye pencil, eye shadow and make-up remover10, and two studies suggest pregnant exposure to phthalates is associated with preterm birth or pregnancy loss12.Since the majority of cosmetic products are used directly onto skins, the hazardous chemicals can directly pass the epidermal barrier to the dermis of the human body and thereby enter the systemic blood circulation, which potentially damage the fertility and reproductive health of women. Some of these chemicals are related to adverse pregnancy outcomes. For example, a French case-control study shows maternal prenatal exposure to phenols  is inversely correlated with male birth weight17. A retrospective cohort study in New York State implies that licensed cosmetologists are prone to have low birth weight babies compared to licensed realtors15. Two registry-based studies indicate an elevated risk of SGA for cosmetologists compared to other working women16. A recent meta-analysis confirms cosmetologists are under higher risk of infertility, fetal death, and preterm birth than the general population17. However, these studies focus on adverse pregnancy outcomes of occupational exposure to cosmetic products, but not personal exposure.Previous studies show occupational exposure of pregnant women to cosmetic products  is associated with miscarriage, perinatal mortality, preterm birth, low birth weight, and small for gestational age (SGA) and other adverse pregnancy outcomesSo far as we know, there is no study reporting the relationship between maternal cosmetics use  during pregnancy and risks of adverse pregnancy outcomes. This large Chinese prospective cohort study was aimed to examine such associations and potential dose-response relationships. The adverse pregnancy outcomes concerned included preterm birth, low birth weight, macrosomia, SGA and large for gestational age (LGA).Pregnant women at 16 ± 2 weeks of gestation were recruited from local midwifery hospitals  in Zhuzhou City and Xiangtan City, Hunan Province, China, between September 2016 and August 2017. The follow-up period lasted from the first antenatal care visit to 42 days after delivery.Inclusion criteria were: (1) singleton pregnancy; (2) residence in the study areas during the follow-up period; (3) explicit cosmetics use status and frequency; (4) provision of informed consent. Exclusion criteria were: (1) miscarriage or termination of pregnancy; (2) infants with congenital anomalies; (3) infants with missing gestational age, birth weight or sex; (4) gestational age older than 42 weeks; (5) profession as hairdresser or cosmetologist.During prospective follow-up, the data concerning the pregnancy outcomes of pregnant women and the birth of infants were acquired by research midwives who had underwent a unified training. The status of cosmetics exposure was determined by directly asking each pregnant woman on the recruitment day if she used cosmetics during pregnancy. According to the self-reported use or no-use of cosmetic products at any time during pregnancy, the pregnant women were divided into two groups: cosmetics users and cosmetics non-users. The data of pregnancy outcomes were acquired by using a structured questionnaire. At the first antenatal care visit, research midwives interviewed every recruited pregnant woman face-to-face to collect demographic and clinical characteristics, including maternal age, residence, ethnicity, education level, occupation, parity, abnormal reproductive history and pre-pregnancy body mass index (BMI), pregnancy comorbidity and date of last menstrual period (LMP). After delivery, all infants were examined within the first 24 h by neonatologists in these hospitals. Information regarding infants was recorded opportunely, including birth weight, gestational age at birth, sex, and occurrence of congenital anomaly.Cosmetics use was defined as having used any personal face hygiene care products  or make-up products  during pregnancy. Only personal use of cosmetic products was considered, but not occupational exposure. The use of cosmetics was evaluated according to general use habits. Cosmetics users were asked about the use frequency, which was divided into low, moderate and high frequency with 1–2, 3–4, and ≥5 times per week, respectively.2 were classified as underweight, normal weight and overweight, respectively (BMI = weight just before pregnancy divided by height squared)18. Pregnancy comorbidity was defined as pregnant women had suffered from acute or chronic disease during pregnancy, such as hypertension, diabetes, heart or liver or kidney disease, hyperthyroidism, hypothyroidism, anemia, etc. These covariates were considered as potential confounders probably influencing the relationship between cosmetics use and adverse pregnancy outcomes and should be controlled in the analysis.Maternal age was classified into five scales: ≤20, 21–25, 26–30, 31–35 and >35 years. Residence was divided into urban and nonurban residences. Ethnicity was classified into Han and minorities (the other 55 ethnicities in China except Han). Education level was classified (1) primary school and below, (2) middle school, and (3) college and above. Occupations included farmer, worker, administrative staff, and business/company staff and else. History of abnormal reproduction covered miscarriage, stillbirth, neonatal death, preterm birth, low birth weight or congenital anomaly. Women with pre-pregnancy BMI <18.5, 18.5–25.0, and ≥25.0 kg/m19, and birth <37 gestational weeks was regarded as preterm birth15. Birth weight <10th, 10th–90th, and >90th percentile were defined as SGA, appropriate for gestational age (AGA) and LGA, respectively18, adjusted for gestational age and sex using Chinese standards (enacted by Chinese neonatologists as a reference for Chinese population)20.Birth weight was defined as the weight of each infant measured within 24 hours of birth. Gestational age was defined as the duration of pregnancy from the first day of LMP to birth. If LMP was uncertain, gestational age was estimated by sonography. Birth weight <2500 g and ≥4000 g were considered as low birth weight and macrosomia respectively21. Statistical analyses were performed on SPSS 19.0 . All statistical tests were two-tailed at the significance level of P < 0.05.Proportions of maternal characteristics and prevalence rates of adverse pregnancy outcomes were calculated among different groups. Differences of maternal characteristics between cosmetics non-users and users were determined using Chi-square test. Unadjusted and adjusted odds ratios (OR) and 95% confidence intervals (CI) were calculated by binary logistic regression for preterm birth, and by multinomial logistic regression for low birth weight/macrosomia and SGA/LGA . In order to control the potential confounders, we used multivariable logistic regression models to adjust maternal age, residence, ethnicity, education level, occupation, parity, abnormal reproductive history, pre-pregnancy BMI and pregnancy comorbidity. Dose-response relationship was tested by exploring the associations between frequency of cosmetics use and adverse pregnancy outcomes. The above results were referred to as relative risk (RR), because OR is a good approximation for the risk ratio in the case of rare outcomesThis study was conducted in accordance with the Declaration of Helsinki with ethical approval from the Ethics Committee of Hunan Provincial Maternal and Child Health Care Hospital. Written informed consents were obtained from all the pregnant women.From the initial 10920 pregnant women screened for participation, 262 (2.4%) refused to be enrolled in the study, and 10658 (97.6%) agreed to be enrolled in the study; out of the enrolled pregnant women, 253 (2.3%) had no information available for maternal cosmetics exposure mainly due to an ambiguous frequency of cosmetics use , 892 (17.6%) and 3816 (75.4%) of the 5058 pregnant women reported cosmetics use ≥5, 3–4 and 1–2 times per week, respectively  were lost. Thus, this study was not largely affected by selection bias or follow-up bias. In addition, the large sample size, reliable data collection and strong demonstrability for causality can accurately reflect the influence of maternal cosmetics use during pregnancy on the adverse pregnancy outcomes.17. A report from New York State shows maternal occupational exposure to cosmetic products is correlated with a higher risk of low birth weight15. A meta-analysis conducted by Kim concludes that cosmetologists are under higher risk of preterm birth than the general population17. The inconsistency in these studies may be attributed to the differences in cosmetics diversity, intensity and duration between personal and occupational exposures. Our findings on SGA are consistent with recent studies that maternal occupational exposure to cosmetic products during pregnancy will increase the risk of SGA16. A retrospective population-based study suggests the risk of SGA among cosmetologists’ offspring was raised by 40% compared to other working women16. Though we focused on the personal exposure to cosmetic products, our findings were similar to the research on occupational exposure. However, to date, there is no study exploring the relationship between macrosomia or LGA and cosmetic products exposure.However, our results on preterm birth and low birth weight are inconsistent with previous literatures focused on occupational exposure22. Toxic metals can freely pass the placental barrier, transferring from the mother to the embryo, and accumulate in fetal tissues. These metals can alter the placental blood flow and endocrine function and injure the placental tissues, thereby hindering the nutrient transport to the fetus23. Since the fetal period is very critical for growth and development, the effect of any toxic substances including heavy metals is the most sensitive in this period and will lead to intrauterine growth retardation26.In our study, high-frequency use of maternal cosmetics was associated with SGA, but not with preterm birth or low birth weight. The reason may be that SGA is a general consideration of the effects of gestational age and sex on birth weight, and is more sensitive than preterm birth and birth weight and is likely to show statistical differences between cosmetics users and non-users. The relationship between cosmetics exposure and SGA is biologically plausible. The physiologic mechanism may be related to the known harmful effects of hazardous chemical substances in cosmetic products  during fetus development. Two population-based birth cohort studies confirm that maternal exposure to cadmium, mercury, and arsenic during pregnancy would increase the risk of SGAWorld Health Organization Global Survey on Maternal and Perinatal Health 27. A recent large-scale study involving 28164 women in Shanxi Province from China shows the prevalence rate of low birth weight has decreased from 4.1% in 2010 to 2.6% in 201328. Therefore, the prevalence rates of preterm birth and low birth weight in our study are similar to these studies. The prevalence rate of SGA in our study is close to the European level (4.6–15.3%)29, and the prevalence rates of LGA and macrosomia are close to the levels in developed countries30.Moreover, the prevalence rates of preterm birth and low birth weight are slightly lower than reported by a recent 17. Therefore, cosmetics use during pregnancy needs to be cautious. Antenatal care providers should provide women of childbearing age, especially pregnant women or women planning pregnancy, with relevant health and safety education to minimize personal exposure to hazardous chemical substances in cosmetic products. Women should reduce the frequency of cosmetics use as much as possible during pregnancy.The present study presents no association between personal cosmetics use and preterm birth, low birth weight, macrosomia, or LGA, but shows that high-frequency cosmetics use is related to an increased risk of SGA. Nevertheless, maternal occupational exposure to cosmetic products would increase the risk of other adverse pregnancy outcomes, such as perinatal death, miscarriage, preterm birth, and low birth weight36. Second, due to the difficulty in obtaining exact information about exposure variables, the frequency of cosmetics use throughout the pregnancy was evaluated according to self-reported general use habits of the pregnant women. During the cosmetics exposure measurement, we only focused on the average times of cosmetics use per week, but not the types or amount of cosmetic products per use. The information acquisition approach was simple and convenient, but self-reported exposure measurement inevitably had some inherent limitations. On one hand, the measurement accuracy of cosmetics exposure was decided by the subjective cooperativeness of the participants, which was not as objective as instrumental measurement; on the other hand, the recall of past cosmetics exposure might result in recall bias in the exposure data. Such measurement bias might affect the risk assessment of relationship between cosmetics use and adverse pregnancy outcomes to some extent. In addition, since the type of cosmetic products was not analyzed, we did not clarify which cosmetic products increased the risks of SGA, which should be clarified by more research and more human data. Third, the cosmetics ingredients and concentrations both differ among countries because of the differences in cosmetics hygienic criterias and provisions. Usage patterns of cosmetic products  also significantly differ among people in different countries. Hence, the generalizability of our findings may be limited by the homogeneity of this cohort, which contained only Chinese women.This study has several limitations. First, the observational nature of the study design made it subject to some residual confounding. Our estimates were adjusted for several variables, which, however, excluded maternal stature, weight gain during pregnancy, nutritional intake, folic acid supplementation, or exposure to environmental pollutants, since information on these factors was unavailable in our study. These factors are reportedly related to an increased risk of low birth weight, SGA, or preterm birthIn conclusion, personal cosmetics use is not related to preterm birth, low birth weight, macrosomia, or LGA, but high-frequency cosmetics use will increase the risk of SGA. Further research is required to determine which cosmetic products may account for the higher risk of SGA.


Klebsiella and Staphylococcus) and antibiotic administration (Streptococcus and Bifidobacterium). At 2 and 4 years of age, the microbiota of these VLBW infants became similar to the mothers’ microbiota. The number of microbial taxa shared between the infant or toddler and the mother varied, with least the overlap between infants and mothers. Overall, there was a significant association between the diversity and structure of the microbial community and infant weight and length gain in an at-risk childhood population.Preterm infants are at greater risk of microbial insult than full-term infants, including reduced exposure to maternal vaginal and enteric microbes, higher rates of formula feeding, invasive procedures, and administration of antibiotics and medications that alter gastrointestinal pH. This investigation of the VLBW infant microbiome over the course of the neonatal intensive care unit (NICU) stay, and at ages 2 and 4 years, showed that the only clinical variables associated with significant differences in taxon abundance were weight gain during NICU stay ( The microbiomes of 83 preterm very-low-birth-weight (VLBW) infants and clinical covariates were analyzed weekly over the course of their initial neonatal intensive care unit (NICU) stay, with infant growth as the primary clinical outcome. Birth weight significantly correlated with increased rate of weight gain in the first 6 weeks of life, while no significant relationship was observed between rate of weight gain and feeding type. Microbial diversity increased with age and was significantly correlated with weight gain and percentage of the mother’s own milk. As expected, infants who received antibiotics during their NICU stay had significantly lower alpha diversity than those who did not. Of those in the cohort, 25 were followed into childhood. Alpha diversity significantly increased between NICU discharge and age 2 years and between age 2 years and age 4 years, but the microbial alpha diversity of 4-year-old children was not significantly different from that of mothers. Infants who showed improved length over the course of their NICU stay had significantly more volatile microbial beta diversity results than and a significantly decreased microbial maturity index compared with infants who did not; interestingly, all infants who showed improved length during the NICU stay were delivered by Caesarean section. Microbial beta diversity results were significantly different between the time of the NICU stay and all other time points (for children who were 2 or 4 years old and mothers when their children were 2 or 4 years old).IMPORTANCE Preterm infants are at greater risk of microbial insult than full-term infants, including reduced exposure to maternal vaginal and enteric microbes, higher rates of formula feeding, invasive procedures, and administration of antibiotics and medications that alter gastrointestinal pH. This investigation of the VLBW infant microbiome over the course of the neonatal intensive care unit (NICU) stay, and at ages 2 and 4 years, showed that the only clinical variables associated with significant differences in taxon abundance were weight gain during NICU stay (Klebsiella and Staphylococcus) and antibiotic administration (Streptococcus and Bifidobacterium). At 2 and 4 years of age, the microbiota of these VLBW infants became similar to the mothers’ microbiota. The number of microbial taxa shared between the infant or toddler and the mother varied, with least the overlap between infants and mothers. Overall, there was a significant association between the diversity and structure of the microbial community and infant weight and length gain in an at-risk childhood population.Author Video: An author video summary of this article is available. This early microbial exposure begins a dynamic process, activating the immature immune system and leading to the selection of particular microbes. At birth, gut-associated lymphoid tissue is “uneducated” and the innate immune response to the presence of bacteria is crucial for the transition from fetal to postnatal life  infants and to determine the associations between the variance in microbial profile and infant health outcomes. Subsequently, 25 of the original 83 children were reexamined at ages 2 and 4, and the stool microbial profile was assessed with respect to health, development, and growth (weight and length) outcomes. We hypothesized that microbial community structure present in the first 6 weeks of the NICU stay is statistically associated with mode of delivery, gestational age, weight and length gain, type of feeding, human milk cytokines, fecal calprotectin, and adverse prenatal events and that the microbial community structure of the infant gut microbiome can be used to predict the microbial community structure in the same child at 2 and 4 years of age.Preterm infant clinical variables . Therefore, chronological age was not employed as the independent variable; instead, we used postmenstrual age  for all subsequent analyses. A total of 91.6% of the infants started antibiotics on first day of life . The mean postmenstrual age for starting antibiotics was 28.2 ± 2.5 (standard deviation [SD]) weeks. Most infants were on antibiotics for 3 to 4 days at a time, with a median course of antibiotics lasting 3.5 days. Only 7 infants did not receive any antibiotics. The mean total number of days on antibiotics was 11.88 ± 18.8 .In total, 6,383,544 16S rRNA amplicon reads were generated across all 425 samples, with a range of ∼1,000 to 40,483 reads . Sequence depth was rarified to 1,275 reads per sample, resulting in 415 samples used in subsequent analysis. Reads were classified into exact sequence variants (ESVs) using DADA2, creating a total of 2213 ESVs, with a range of 1 to 1,844,477 reads per sample . All ESVs were annotated to known microbial taxa.R2 = 0.146, P value = 0.001703); however, this relationship was not significant for birth weight and weight gain between 6 weeks and discharge (P = 0.8141). No significant relationships were observed between weight at discharge and weight gain with feeding .Our analyses were statistically underpowered for each morbidity category ; therefore, we focused on growth (increased weight and length) as a primary clinical outcome during the NICU stay. We tested the independence of the measured growth variables . Birth weight (in grams) was significantly correlated with growth rate over the first 6 weeks of life (grams/week)  correlates with growth per week initially (first 6 weeks). Copyright © 2019 Yee et al.2019Yee et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the R2, 0.09221; P = 1.28e−08)   . Values for Shannon alpha diversity difference per infant between the first NICU stool sample and the last stool sample ranged from −3.3520 to 3.1720, with a median of 0.8259. There were no significant differences with respect to delta Shannon alpha diversity between children who showed improved length and those who did not or by mode of delivery .The relationship between weight and length gain outcomes and microbial alpha diversity over the course of the NICU stay was determined. Shannon index values increased significantly with increasing postmenstrual age ; the Shannon index values were also significantly different in comparisons of 2 and 4 years of age. While the alpha diversity during the NICU stay and at 2 years of age was significantly lower than the alpha diversity of the mother’s stool-associated microbiota , the alpha diversity at 4 years of age was not significantly different from that of the mother, suggesting that the diversity normalizes to that of an adult by 4 years of age.A significant increase in Shannon index values was also observed for each child in comparisons between their last time point of NICU stay and 2 and 4 years of age . This suggests that infants with a less-volatile microbiota over time may have reduced catch-up growth (length) during the NICU stay. While the mode of delivery alone did not correlate with volatility, all the infants with improved length (n = 6) had been delivered by Caesarean section.The distribution of between-sample unweighted UniFrac distances was calculated pairwise to determine the volatility of microbial community structure over time during the NICU stay. This was then associated with the clinical variables, including both categorical variables  and binary variables . Beta diversity volatility was significantly correlated with improved length only during the NICU stay, with results indicating that infants who showed improved length had significantly more intersample variability in beta diversity and hence had greater microbial volatility over the course of their NICU stay (P < 0.0018). As maturity is inversely correlated with beta diversity volatility, this result agrees with the results of the beta diversity volatility analysis described above.We ran a “maturity” analysis , which uses a regression model to predict postmenstrual age as a function of the composition of the microbiota. Therefore, if a sample had a microbiota which fell outside the confidence interval based on the average microbiota composition for infants of each postmenstrual age, it was considered immature. We correlated the maturity index with improved length during their NICU stay . Those iP < 0.05 after FDR).We also examined the beta diversity distribution (unweighted UniFrac) to determine the microbial community structure variance between the final NICU time point, age 2 years and age 4 years, and maternal samples. The maternal microbiota and those of the 2-year-old and 4-year-old children also clustered together and were significantly different from those seen with the samples collected at the end of the NICU stay -mediated inflammation. The most abundant exact sequence variant (ESV) in infants during the NICU stay was closely related to the genus Klebsiella, which belongs to the Enterobacteriaceae. Analysis of composition of microbiomes (ANCOM) was applied against all categorical and binary clinical variables to determine which bacteria were significantly differentiated in relative abundance. Only two clinical variables showed bacteria that were significantly differentially abundant. Infant weight gain from birth to discharge was significantly negatively correlated with the relative abundance of two ESVs, one associated with Klebsiella and the other related to Staphylococcus (P < 0.01). Additionally, antibiotic administration was associated with significant enrichment in Proteus levels and a significant proportional decrease in the levels of Streptococcus and Bifidobacterium compared to infants who did not receive antibiotics (P < 0.01). The predominance of Proteobacteria was greater in infants with lower beta diversity volatility than in those with higher volatility.The stool samples collected during the NICU stay maintained a predominance of 10.1128/mSystems.00229-18.2FIG S2FIG S2, PDF file, 0.3 MB.Relative abundances of taxa at the family level. Each bar represents a single sample. Bars are sorted by patient identifier (ID) and ordered by age for each patient. Download Copyright © 2019 Yee et al.2019Yee et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Enterobacteriaceae, Citrobacter, Escherichia, and Peptostreptococcaceae. For continuous clinical variables, we also ran the random forest regressor model , which identified only the duration of antibiotic administration as significantly predictable . The top most important features for the regressor-model accuracy were ESVs corresponding to Veillonella, Enterobacteriaceae, Proteus, and Escherichia (importance scores of 0.15 to 0.05).The random forest classifier was used to determine whether the microbiota was predictive of categorical clinical variables , with results which suggested that only the use of antibiotics (presence/absence) could be accurately predicted, with a baseline-to-random ratio of >2 (2.26) suggesting a marginal relationship. The random forest features identified as having the most predictive value for the presence or absence of antibiotics were ESVs corresponding to Enterococcus and Staphylococcus , samples from toddler at both 2 and 4 years old maintained ESVs associated with Clostridiales and Bacteroidales that were not detected in the infant samples for the respective individuals. However, the infants as a group shared 26 ESVs in total with 2-year-old toddlers as a group, 26 ESVs in common with 4-year-old toddlers, and 18 ESVs in common with the mothers as group  of the participants. Some infants maintained the H.parainfluenzae ESV over multiple NICU time points, but it was not found in the corresponding 2-year-old and 4-year-old samples for those infants. However, it was found in 3 children at both 2 years of age and 4 years of age and also in their mothers. As mothers’ stool samples were collected at the same time as the stool samples collected from the 4-year-olds, it is possible that this H.parainfluenzae ESV represented endemicity in these 3 families during that time.While the infant samples at 6 weeks of life had a significantly greater abundance of ESVs related to as group . Interesellaceae . Interes10.1128/mSystems.00229-18.4TABLE S2Table S2, DOCX file, 0.1 MB.Taxonomy of ESVs in common between all age groups . Download Copyright © 2019 Yee et al.2019Yee et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Proteobacteria . Infants with more volatility had reduced prevalence of these organisms and as such may have had reduced dysbiosis and improved growth. This report is among the first to document a relationship between microbiome and VLBW infant growth. Previous work found that preterm infants with prolonged microbial transitions had smaller changes in weight-for-age z-scores across time  was correlated with increased beta diversity volatility (between-sample beta diversity distance) and decreased maturity (predictive power of regressor modeling) during the NICU stay. Catch-up growth is an important consideration for preterm infants: VLBW infants are vulnerable to extrauterine growth restriction, and caloric and protein deficits result in lower growth velocity and are associated with major neonatal morbidities, including chronic lung disease and retinopathy of prematurity . Clinicaoss time . Howeveross time , suggestDue to the limited size of this cohort, it is not possible to determine if this trend is absolutely significant, but the data suggest that further studies using larger cohorts would be useful, as would studies involving the humanization of murine models to demonstrate reduced growth as a transferable phenotype. Those infants who demonstrated reduced microbial maturity also showed improved catch-up growth, likely as a result of the choice of population against which these infants were compared. Our maturity analysis is based on the average maturity data determined for a population of VLBW infants who were essentially quite sick, as infants in that category who showed improved catch-up growth would be statistical outliers against this group. Having a healthy control population might provide a more accurate interpretation of the standard maturity analysis data.In our cohort, we followed the same children to ages 2 and 4 years. Our analyses had insufficient statistical power to identify clinical outcomes that correlated with microbiome composition, but we did find that the these VLBW infants did develop a microbiome comparable to that of their adult mothers by ages 2 to 4 years. As previously demonstrated, the microbiome of children undergoes patterned progression to become more similar to the maternal microbiome as the children increase in age, with an adult-like microbiome established by 4 years . In our 29–We have demonstrated a significant association between the diversity and structure of the microbial community and infant growth in a significantly at-risk childhood population. While the population had inherent variability, which limits the potential to identify associations between the microbiota and clinical outcomes, compelling correlations between microbial structure, volatility, maturity, and composition and infant weight and length increases were determined that suggest potential biomarkers of dysbiosis in this at-risk population.The study was approved by both the hospital and the university Institutional Review Board, and parents gave informed consent for the study in the NICU and agreed to be followed up at a later time for further research.A total of 83 preterm, very-low-birth-weight (VLBW) and extremely low-birth-weight (ELBW) infants  were enrolled as soon as possible after admission to the neonatal intensive care unit (NICU) at Tampa General Hospital, an academic level III center with a single-patient-room floor plan, during the period May 2012 to December 2013. Mothers who were drug abusers or HIV positive were excluded, as were infants who had major congenital anomalies or who were moribund. Maternal and neonatal clinical information was obtained from electronic medical records and an investigator-developed demographic form. Long-term morbidity data were extracted from the Vermont Oxford Network database. Data were collected for a total of 6 weeks after entry into the study unless participants were discharged or transferred earlier. Exact volumes of the mother’s own milk (MOM), formula, fortifier, and donor milk (DM) were collected from the electronic medical record.www.earthmicrobiome.org). The V4 region of 16S rRNA gene was amplified using PCR with modified 515F and 806R primers, followed by amplicon sequencing using a MiSeq platform  based on existing protocols to generate ∼100,000 250-bp paired-end reads per sample , stored in a sterile tube, brought to the laboratory, and frozen at −80°C until processing. DNA was extracted using a MoBio PowerFecal DNA kit  with modifications based on the Earth Microbiome Project protocols  between subgroups. To identify the predictive value of subgroups for microbiome community composition, we applied random forest machine learning  and Analysis of Composition of Microbiomes (ANCOM)  and permutational multivariate analysis of variance (PERMANOVA). We performed two-sample  (ANCOM) . For con (ANCOM) . UpSet p (ANCOM) .Infant weight and length were measured at birth and weekly until discharge. Raw weight and length measurements were standardized against the 2013 revision of the Fenton preterm growth chart using the actual-age calculator tool . This prGrowth (weight gain and length-for-age z-score) over the NICU stay was assessed with the following measurements: (i) total average growth rate (difference between birth weight and discharge weight divided by length of stay), growth rate between birth and 6 weeks of life (difference between birth weight and weight at 6 weeks divided by weight at 6 weeks), and growth rate between 6 weeks of life and discharge (difference between weight at 6 weeks and discharge weight divided by length of stay minus weight at 6 weeks); (ii) weight for gestational age , and (iii) improved length (binary variable corresponding to whether or not the infant had greater-than-expected length-for-age z-scores during the NICU stay). Complete growth metadata were available for only 78 of the total of 83 infants.phs001578.v1.p1. Deidentified metadata and raw forward and reverse sequence reads were associated with each sample via SRA. Raw sequence reads are available through the Sequence Read Archive under accession number SRP171050 (BioProject number PRJNA449987).The study was registered in dbGaP under accession number


Necrotizing enterocolitis (NEC) is a devastating disease of intestinal inflammation that primarily affects premature infants. A potential risk factor for necrotizing enterocolitis is exposure of the premature neonatal intestine to environmental bacteria and their proinflammatory products such as lipopolysaccharide. The metalloenzyme alkaline phosphatase (ALP) has been shown to reduce lipopolysaccharide-mediated inflammation. Additionally, premature rat pups have reduced alkaline phosphatase activity and expression as compared to full term pups. To explore the possibility that the human premature neonatal intestine has a paucity of alkaline phosphatase activity, we measured endogenously produced intestinal alkaline phosphatase activity in meconium as a function of gestational age. To test whether breast milk could serve as a source of exogenous alkaline phosphatase to the neonatal intestine through ingestion, we measured alkaline phosphatase activity in breast milk across a range of time points post-birth.Alkaline phosphatase activity was quantified in 122 meconium samples from infants of gestational ages ranging from 24 to 40 weeks and in 289 breast milk samples collected from 78 individual mothers between days 2–49 post-birth.We observed a strong positive correlation between the meconium alkaline phosphatase activity and gestational age, with preterm infants having lower meconium alkaline phosphatase activities than early term or term infants. Breast milk alkaline phosphatase activity was highest in the first week post-birth, with peak alkaline phosphatase activity at day 2 post-birth, followed by relatively low alkaline phosphatase activity in weeks 2–7.Our results are consistent with the two major risk factors for necrotizing enterocolitis development, preterm birth and lack of breast milk feeding, both contributing to a paucity of alkaline phosphatase activity and impaired capacity to detoxify proinflammatory bacterial products such as lipopolysaccharide. Infants born prematurely are at risk of developing necrotizing enterocolitis (NEC), a multifactorial disease characterized by overly exuberant inflammatory responses in the immature intestine and a leading cause of late mortality and morbidity in very preterm infants , 2. As tAlkaline phosphatases (ALPs) are conserved metalloenzymes that hydrolyze the release of inorganic phosphates from a variety of substrates . ALPs haALP is also a known component of breast milk –27, and clinicaltrials.gov .The use of meconium samples for this study was reviewed by the University of Oregon Institutional Review Board and Research Compliance Services and determined to qualify for an exemption as per Title 45 CFR Part 46.101 (b). A total of 122 meconium samples from infants of gestational ages ranging from 24 to 40 weeks (except 30 weeks) were obtained from the Peacehealth Neonatal Intensive Care Unit and the Peacehealth Nurse Midwifery Birth Center . Samples were frozen at − 80 °C upon collection and subsequently analyzed. A total of 289 frozen milk samples were collected on post-birth days 2–49 from 78 individual mothers who had given birth to term infants enrolled in the UC Davis FFHI Lactation Study , 33, 34.Meconium samples were homogenized in double distilled water and centrifuged at 16,000 g for 15 min at 4 °C to collect the supernatants. The supernatants were diluted and then assayed for protein concentrations using the Bio-Rad protein assay kit (Bio-Rad Laboratories Inc.) and for ALP activities using the PNPP substrate kit (Thermo Fisher Scientific Inc.). ALP activities were compared to standard shrimp alkaline phosphatase (SAP) (Thermo Fisher Scientific Inc.) and normalized to meconium protein concentrations. Data were grouped by weeks of completed gestation at birth, and analyzed using the Prism software (GraphPad software). Correlation between the meconium ALP activity and gestational age was analyzed using one-way ANOVA, posttest for linear trend. Meconium ALP activities in preterm newborns , early term (37–38 weeks) and term (39–40 weeks) were compared using one-way ANOVA, followed by Bonferroni’s Multiple Comparison Test.p < 0.001 level. This model is a better fit for the data than a baseline model having only the random effect for mother . A Levene’s Test of equality of variance in the samples collected in the four sampling windows was statistically significant, F = 18.882, p < 0.001, indicating that the variances are not equal. A second linear regression model analyzed ALP content by day during the first week, with ALP activity as the dependent variable and day of sample collection as a categorical predictor. Only one sample per mother was collected during the first week, so this model does not include a random effect for participant. The intercept  represents the level of ALP activity in day 2.To assay for ALP activity in breast milk, we modified a fluorometric detection method already published , 36. BriP < 0.0001, R2 = 0.3416; one-way ANOVA and posttest for linear trend) . Linear hypothesis tests comparing week 1 with weeks 3–4 and weeks 6–7 were also statistically significant . ALP activity remained within 25% of the week 2 average through weeks 3–4 (2.57 units) and weeks 6–7 (3.08 units)  than breast milk sampled in week 2 (2.50 units). A linear hypothesis test on the regression model coefficients showed that the difference between week 1 and week 2 is statistically significant . All individuals for whom we had samples in all 4 time intervals displayed the same trend of decreasing ALP activity over time (Fig. Within the first week, the earliest samples, collected at day 2 post-birth, had higher ALP activity than those collected on days 3, 4, and 5 (Fig. In this study, we discover for the first time that premature infants have reduced intestinal ALP activity at birth as compared to early term or term infants Fig. . These rIn addition to endogenously produced ALP, infants who are breast fed or fed non-pasteurized donor breast milk may receive exogenous ALP. ALP is a reported component of breast milk, and a review of literature in which ALP activity is quantified in breast milk suggests a decreasing trend in ALP activity over time post-birth –25. In aOur data demonstrate high levels of ALP activity in breast milk in the first few days post-birth, with a rapid decrease after the first week Fig. . This eaOur findings suggest that there is a coordinated mother-infant program of defense against the pro-inflammatory insults of intestinal bacterial colonization that occurs after birth. We hypothesize that the higher ALP activity in the full-term neonatal intestine, combined with high ALP activity of breast milk within the first few days post birth, provides adequate capacity to detoxify the LPS of initially colonizing bacteria. We suggest that paucity of this activity in the preterm intestine and in the absence of early post-birth breast milk feeding, increases the risk of excessive inflammation and progression to NEC. A limitation of our study is that we did not have meconium and breast milk samples from mother-infant pairs. We anticipate that our initial findings will motivate prospective studies on the interplay between intestinal ALP, breast milk ALP, gut microbiota, and NEC development in premature infants. Such studies may generate support for prophylactic ALP supplementation to premature infants as an effective therapeutic for NEC prevention.


Preterm deliveries were responsible for 3.1 million neonatal deaths that occurred globally in 2010, of them 35% were directly related to preterm. This study was aimed to assess the determinants of preterm birth among women delivered of Central zone. A facility-based cross-sectional study was carried out from March to May 2018, in four randomly selected hospitals of Central zone. A total of 413 participants were interviewed using a pre-tested structured questionnaire. Data were entered into Epi-info Version 3.5.3 and then exported to SPSS Version 21 for analysis. Bivariate and multivariate logistic regression analysis was carried out to assess the association. Statistical significance was declared by 95% confidence interval of the odds ratio.From the total (413) participants, 78.7% of participants were in the age group of 20-34 years and in this study the prevalence of preterm birth was 12.8%. Factors like, marital status of unmarried , mid upper arm circumference (MUAC) (< 11 cm)  were independent predictors of preterm birth among women delivered in the study area. Preterm birth defined as live births before 37 completed weeks of gestational age –3. From In Ethiopia 320,000 preterm births are happening every year . Under-5Even though, many women have a premature birth, its risk factor is idiopathic. But it can be occurred due to many risk factors. These could be: history of premature birth, multiple pregnancies, short spacing between pregnancies (mainly less than 6 months), troubles with the uterus, substance use like drug or cigarette, low intake of nutrients, in adequate increasing of weight during the period of pregnancy, complication due to infections and some of the non-communicable diseases .From the total, about 85% of preterm babies are delivered between 32 week and 37 weeks of gestation. These babies do not need intensive care for their survival. In Ethiopia, which is one of the developing country, about 320,000 babies are born very early each year and around 24,400 less than 5 year children die due to direct effect of preterm . The maiThe study was carried out at public hospitals of Central zone which is found in Tigray regional state. It is located at 1040 km far from Addis Ababa, capital city of Ethiopia to the North. The total population of the zone is 1,347,212, of which about 686,161 are female . Administratively, it is divided into 8 districts and has nine hospitals.This study was done using a facility-based cross-sectional study design from March to May 2018. The respondents of this study were women who gave births a baby in the hospitals and who lived in the central zone for at least 6 months were included in the study. Those women who did not responses appropriately the interview due to physical or mental illness were excluded from the study.The sample size of this study was calculated using a single proportion formula as follow:y  mothers participated in the study. Three hundred six (74.1%) were in the age group between 20 and 34 years. About 390 (94.4%) of the participants were Orthodox Christian followers and 397 (96.1%) were married. Regarding maternal educational status 258 (62.4%) were secondary and above and 198 (48%) were house wife. About 187 (45.3%) of the participants their family monthly income were in the range of 89–178.5$  were multipara. Most of the mothers 332 (80.4%) received ANC four times and above, 330 (80%) initiated ANC at first trimester of the current pregnancy, 374 (90.6%) were taken iron supplementation, 290 (77.5%) were taken iron supplementation for less 3 months and from the pregnancies 383 (92.7%) were planned. Three hundred fifty-five (86%) were gave birth between 37 and 42 weeks of gestational age, where as 53 (12.8%) and 5 (1.2%) were gave birth in < 37 weeks and > 42 weeks, respectively. Majority of mothers 287 (69.5%) were counseled to take additional diet, 286 (69.3%) were took additional diet during current pregnancy. Four hundred two (97.3%) mothers were not faced any pregnancy related complication during current pregnancy. Of the total births 400 (96.9%) were live births and 13 (3.1%) were stillbirths. Regarding birth weight 59 (14.7%) newborns were low birth weight and related to maturity 51 (12.3%) were preterm. Besides 9 (2.2%) of all births were with visible birth defect  , mid upper arm circumference MUAC (< 11 cm)  were found to be significantly associated with preterm birth among mothers delivered in the hospitals Table .Table 3FPreterm birth remains a significant cause of morbidity and mortality among neonates and children. According to the 2016 Ethiopian Demographic health survey (EDHS), neonate mortality was 29 per 1000 live births. Among these causes of mortalities, preterm births were the major factor . This faIn the multivariable analysis marital status and MUAC measurement were significantly associated with preterm births. The study found that being unmarried increased the risk of preterm birth by four folds than the married. This finding is consistent with the findings done at Michigan . This miThis study also showed that MUAC less than 11 cm was significantly associated with preterm birth (PTB). The proportion of preterm birth was significantly higher (2.42 times) among mothers who had MUAC less than 11 cm compared to mothers with MUAC greater than or equal to 23 cm. This finding is consistent with the findings of studies done at Bangladeshi . This miThe prevalence of preterm birth in this study was substantially high (12.8%). Factors like marital status that are unmarried and MUAC with less than 11 cm were associated with preterm birth. The factors identified in this study can be prevented and managed easily by providing appropriate care before pregnancy, antepartum and intrapartum period.The cross sectional nature of the data, which makes it impossible to draw inferences about the direction of relations among study variables and the data are retrospective and thus are subjected to recall bias.


Obstructed labor had different maternal outcomes such as uterine rupture, postpartum hemorrhage, puerperal sepsis, Vesico-Vaginal fistula (VVF), recto-vaginal fistula can leads to death. Besides fetal outcomes including birth asphyxia, still birth, neonatal jaundice and umbilical sepsis can occur. Identifying maternal and fetal outcomes of obstructed labor among women who gave birth at Suhul general Hospital, Shirie town, Tigray, Ethiopia has been done using a retrospective review of delivery charts and registration book.Majority of mothers 69 (75.8%) came from rural areas and 74.7% were married. Cephalo pelvic disproportion occurs in 59 (64.8%) and mal-presentation in 28 (30.8%) of obstructed labor. Fetal congenital anomaly  occurs in 3 (3.3%) of cases and pelvic mass constituted 1 (1.1%) of cause of obstructed labor. Above half of mothers delivered with obstructed labor had sepsis 23 (25.3%), post-partum hemorrhage 10 (11%), Vesico Vaginal Fistula 5 (5.5%) and anemia 15 (16.5%). From the well-known causes of obstructed labor; mal-presentation, Cephalo pelvic disproportion, fetal congenital anomaly, and pelvic mass were found to the common outcomes of obstructed labor in our study area. Besides Still birth, birth asphyxia, and birth injury were the others. Obstructed labor is defined as failure of the fetal presenting part to descent in the birth canal due to mechanical reasons, adequate, and strong uterine contraction it leads to various maternal and fetal outcomes –3.In trends of maternal mortality from 1990 to 2013, there is the reduction of maternal mortality ratio (MMR) by 45% in all Millennium Development Goal (MDG) regions of the world. But there is still high amount of MMR, which is 210 per 100,000 live births or 289,000 maternal deaths in 183 countries globally. From this, developing countries account for 99% maternal deaths and 62% maternal deaths occurred in Sub Saharan region [Even though, Ethiopia reduced MMR to 676 per 100,000 live births in 2010 from 871 per 100,000 live births in 2000, still it had the highest maternal mortality ratio in Africa. And the country is clearly off the track in goal 5 because of unable to reduce MMR to 267 per 100,000 live births in 2015 based on its plan. Delays in seeking skilled emergency obstetric care; delays in reaching the health facility, delays in receiving a timely intervention after reaching the facility, low skilled birth attendant and large proportions of unmet family planning needs among girls in child-bearing ages are mentioned as major factors for this unsuccessful achievement towards MDG5. Obstructed or prolonged labor, postpartum hemorrhage, infections, ruptured uterus, severe preeclampsia and unsafe abortion were the major direct causes of maternal death and from these causes, except sever preeclampsia and unsafe abortion the others were direct complications of obstructed labor [Obstructed labor has different magnitude in different developing countries ranging from 2 to 8%. When we come to Africa some research finding showed that the magnitude of obstructed labor was more than the above determined once; In Uganda and Ethiopia the magnitude of obstructed labor was described as 10.5% and 12.2% respectively , 8.About 80% of global maternal deaths were contributed by pregnancy induced hyper tension and Obstructed labor with its direct complications such as hemorrhage, sepsis and uterine rupture . AccordiApart from maternal deaths, obstructed labor had different maternal outcomes such as uterine rupture, postpartum hemorrhage, puerperal sepsis, Vesico-Vaginal fistula (VVF), recto-vaginal fistula (RVF) and fetal outcomes including birth asphyxia, still birth, neonatal jaundice and umbilical sepsis –13. WomeThe study was conducted in Suhul General Hospital in 2015. Maternal charts for those mothers who gave birth at Suhul general hospital from September 2012 to August 2015 were studied. There were 91 obstructed labor maternal charts that had the diagnosis of obstructed labor for those mothers who gave birth at suhul general hospital. We have included maternal charts, for those mothers, who gave birth in normal vaginal and operative deliveries during the study period satisfied the inclusion criteria. Besides, maternal charts for those mothers who gave birth with elective c/s and charts without medical record number were excluded from the study.Secondary data was collected primarily by reviewing parturient chart with prepared checklist. If adequate data was not obtained from the parturient chart, delivery registration book and operation registration book were used to fulfill the remaining information.The data quality was controlled by doing pretest in about 10% of the sample and providing training for every data collectors before the actual data collection was conducted. Close supervision of the data collectors during the data collection period was another means of data quality control and each checklist was checked for its completeness daily. Data collectors were four Degree midwife staffs who work at Suhul general hospital delivery unit in different shifts.A total of 91 maternal charts with obstructed labor were studied. The mean age and standard deviation were 25.4 ± 7.7, while majority of mothers (25.3%) were found in the age range of 25–29 years old. With regard to maternal marital status majority of mothers 68 (74.7%) were married, but about 19 (20.9%) had no registration that can describe whether they are married or not and they were described as unknown marital status  whereas, grand multipara mothers were accounts for 19 20.9%). With regard to ANC follow up about three-fourth of mothers had three or less ANC follow up, which was 67 (73.6%). Only 24 (26.4%) of mothers had regular ANC visit among cases. In related to gestational age 43 (47.3%) of the women were found in term gestational age, (37 weeks–40 weeks). The mean duration of labor among mothers was 26.9 ± 15.2 h with the range of 11–72 h. About 13 (14.3%) of mothers had ever different obstetrical problems such as, anti-partum hemorrhage, premature rupture of membrane, pre term labor, twin pregnancy, hypertension problems and others , mal-presentation, congenital anomaly and pelvic mass were assessed as the causes of obstructed labor in this study. From these CPD and mal-presentations were the commonest one. CPD occurs in 59 (64.8%) and mal-presentation occurs in 28 (30.8%) cases. Fetal congenital anomaly  occurs in 3 (3.3%) of cases and pelvic mass constituted 1 (1.1%) of cause of obstructed labor.Following obstructed labor there were many perinatal outcomes and from this study around 49 (52.1%) neonates had birth asphyxia with APGAR score of less than four at 5th minutes after birth, 34 (36.2%) were born as still birth and 1 (1.1%) neonate had birth injury  during operative delivery. Only 10 (10.6%) of neonates had an APGAR score of ≥ 7 at 5th minutes after birth from these obstructed laboring mothers.Different maternal outcomes were observed among obstructed laboring mothers. From these, about 8 (8.8%), 15 (16.5%) and 10 (11%) of them had bladder trauma, ruptured uterus and hysterectomy respectively. Additionally sepsis, post-partum hemorrhage, VVF and anemia were developed in 23 (25.3%), 10 (11%), 5 (5.5%) and 15 (16.5%) mothers respectively. Around 20 (22%) of women from obstructed labor did not develop any complication.In this study, majority of the cases 75.8% were rural resident, nearly similar with the study result of Hawassa in which about 65.3% of the cases were from rural areas . AdditioIn this study about three-fourth of the mothers (73.6%) had irregular ANC follow up, almost similar with the study conducted in west Wollega, that revealed around half of the mothers 48.3%) had irregular visit and around 12% of them had unknown frequency of visit . But thi.3% had iIn this study majority of the cases (44%) were primiparas. This result was concurred with the study result of Hawassa, Pakistan, Bangladesh and Uganda in which most of the cases were primiparas , 13, 14.Obstructed labor with different maternal outcome was assessed; from this sepsis, uterine rupture and anemia were the commonest in about 25.3%, 16.5% and 17% of the cases respectively. This study was almost similar with the study results of Adigrat, Jimma and Zaire in which most of the cases develop at least one complication , 14.With regard to perinatal outcome, about 32.6% of the newborns had still birth. It is relatively low when compared with the study results of Adigrat and Jimma, in which about 50.3% and 54.2% of the newborns had still birth respectively , 8. ThisIn this study we have identified different maternal and perinatal outcomes of obstructed labor.From the well-known causes of obstructed labor; Mal-presentation, Cephalo pelvic disproportion, fetal congenital anomaly and pelvic mass were found to the common outcomes of obstructed labor in our study area. Besides Still birth, birth asphyxia and birth injury were another perinatal outcomes following obstructed labor.Furthermore, different outcomes such as uterine rupture, maternal sepsis, anemia, post-partum hemorrhage, bladder injury and fistula were assessed from mothers chart as the maternal problems that come up following obstructed labor.We recommend the Health institution to give immediate feedback for those health institutions that refer obstructed laboring mothers and supervise them in outreach programme. Health professionals have better to give emphasis about obstructed labor complications for each pregnant mother during ANC visits. Furthermore, health extension workers have to adequately work on teaching the community about the adverse maternal and fetal outcome of obstructed labor.Because of using secondary data some variables were missed.


A large body of research has found an association between short birth intervals and the risk of infant mortality in developing countries, but recent work on other perinatal outcomes from highly developed countries has called these claims into question, arguing that previous studies have failed to adequately control for unobserved heterogeneity. Our study addresses this issue by estimating within-family models on a sample of 4.5 million births from 77 countries at various levels of development. We show that after unobserved maternal heterogeneity is controlled for, intervals shorter than 36 months substantially increase the probability of infant death. However, the importance of birth intervals as a determinant of infant mortality varies inversely with maternal education and the strength of the relationship varies regionally. Finally, we demonstrate that the mortality-reducing effects of longer birth intervals are strong at low levels of development but decline steadily toward zero at higher levels of development. These findings offer a clear way to reconcile previous research showing that birth intervals are important for perinatal outcomes in low-income countries but are much less consequential in high-income settings.The online version of this article (10.1007/s13524-019-00798-y) contains supplementary material, which is available to authorized users. The World Health Organization (WHO) has identified birth interval length (the period between two consecutive live births) as a critical determinant of child mortality risks, recommending that women space their births between three and five years apart to reduce health risks to children and mothers WHO . This reMuch of the previous literature on the relationship between birth intervals and infant mortality has not adequately addressed the issue of residual confounding by unobservable characteristics. Endogeneity is always a concern when studying the effects of fertility behavior on children’s outcomes  and infant mortality varies across developmental contexts while applying uniform methods that can minimize residual confounding from unobserved heterogeneity. The benefit of a standardized comparative approach is that it allows us to shed light on both the average effects of birth interval length on infant mortality and also whether the importance of birth intervals varies according to contextual conditions. An international comparison may help us to reconcile the apparently discrepant findings in the literature and provide benchmarks for knowing when increasing birth spacing may or may not be a relevant intervention for reducing infant mortality.Our study addresses the aforementioned issues by using data from 77 countries and more than 200 waves of the Demographic and Health Surveys (DHS). First, we account for the probable endogenous relationship between birth spacing and infant mortality by estimating within-family linear probability models. These models can account for unobservable maternal factors, such as maternal health or shared frailty within the sibling group, which may be correlated with both interval length and infant mortality risks. Second, we explore how the relationship between birth intervals and infant mortality risks varies both within and between populations in order to identify whether specific groups of mothers drive any observed association. Finally, we link our estimates of birth intervals’ effects on infant mortality to several macro-level indicators of development in order to understand the conditions under which birth intervals are more or less important for child survival.A detailed description of the theoretical mechanisms linking preceding birth intervals to children’s outcomes can be found elsewhere , which is critical for the growth and development of the fetus and is generally replenished in the postnatal period, is less likely to return to optimal levels during shorter intervals  are also disproportionately associated with higher risks of adverse maternal outcomes, such as preeclampsia and eclampsia, which are known to be associated with fetal loss and preterm birth   and total fertility rate because these were widely available across the countries and years in the DHS data.To analyze the effects of birth spacing on infant mortality, we estimated the following linear probability model:Y, is binary and indicates whether child i of mother j died in the first year of life. Our main independent variable, S, is the length of the preceding interbirth interval . We treated it as a continuous variable with a quartic functional form in order to account for the possibility of a nonlinear relationship between interval length and mortality risks  birth year, survival status of the previous child at the time of the index child’s birth, and birth order. Summary statistics of the model’s covariates may be found in Table The dependent variable, Most previous studies on this topic in low-income countries have not addressed the probable endogeneity of birth interval length when studying its effects on infant health. Interval length may be correlated with a host of characteristics that may be unobserved, such as maternal breastfeeding preferences or health behaviors, and may themselves influence the probability of infant mortality. Recent work has called attention to the importance of accounting for unobserved factors that may bias estimates of the effect of birth spacing on child outcomes  who have not yet had three or more children, although it will not exclude women who gave birth at younger ages in older cohorts.The within-family approach is not without limitations, however. First, we were unable to control for any source of endogeneity that emerges as a result of In our analysis, we first compared the between-family estimates (ordinary least squares (OLS)) with the within-family estimates (FE) using the pooled sample of surveys to identify whether the relationship between preceding interval length and infant mortality persists after minimizing residual confounding from maternal heterogeneity. We then stratified the sample by United Nations subregion  for each country-birth cohort combination. These two indicators were chosen because they serve as good general proxies for social and economic development, and information on these indicators was also consistently available across countries and years.increase in mortality risks with increasing birth intervals, which is also consistent with another recent study using DHS data examining neonatal and under-5 mortality  in each country-cohort combination to allow for comparison across years and populations. The vertical axis can therefore be interpreted as the expected percentage change in the probability of dying before age 1 if a birth interval increased from 12 to 24 months in a specific country and cohort. All plots were fitted with a kernel-weighted local polynomial smoothed trend.The final part of our analysis compared the effects of spacing across levels of development. To do this, we estimated similar FE linear probability models as in Eq.  but inclPanel a first plots the estimated effects against the national IMR. At levels of IMR greater than about 100 infant deaths per 1,000 live births, increasing a birth interval from 12 to 24 months was associated with a reduction in the probability of dying before age 1 by about 50 %, on average. Such large effects were persistent until the IMR fell well below 100, after which the protective effect of increasing a birth interval from 12 to 24 months began to weaken. At levels of IMR around 50 per 1,000 and lower, the effect of increasing intervals from 12 to 24 months clearly approached zero.Panel b of Fig. In addition to our main results, we conducted several supplementary analyses to further explore heterogeneity in our findings and to check the robustness of our results. First, we stratified the models by a woman’s number of children ever born (CEB) and by index children’s birth cohorts to identify how consistent our findings are in small versus large families and to explore whether the patterns observed until now were driven exclusively by older birth cohorts. Because women with higher fertility will disproportionately contribute to the number of birth intervals and infant deaths in a population, we stratified our models by CEB to assess whether our findings are also generalizable to women with relatively low fertility. We also restricted our analysis to a subsample of births that occurred within the 10 years preceding the survey in order to account for the possible displacement or omission of births from women’s self-reported birth histories Potter . The disWhen we stratified our models by a woman’s total CEB, the relationship between interval length and infant mortality was stronger in larger families  index children born within 10 years preceding the survey, (2) children from five-child families or larger born at even parities, and (3) those born at odd parities , on the risk of infant mortality. In our view, defining birth spacing in terms of the interpregnancy interval has two advantages: (1) it may provide a slightly better representation of women’s recuperative potential, and (2) it may also help to avoid misattributing the effects of preterm birth to those of short birth intervals. Nevertheless, the measure certainly would have drawbacks if it is self-reported, as it is in the DHS. If systematic differences exist in the misreporting of pregnancy durations or miscarriages, this would introduce greater uncertainty into our main exposure of interest. Furthermore, although the DHS includes information on time and length of pregnancies for a subset of children listed in the birth histories , its use would exclude many cases from our analysis, and its reliability is less clear. In addition, its use would eliminate the possibility of controlling for sibling FE, which was a central goal of this study. Second, because our estimates are based on within-family models, we were able to show that the relationship between birth intervals and infant mortality in low-income contexts is not attributable to time-invariant compositional differences between women. Nevertheless, our approach cannot remove the influence of within-family Nevertheless, our study also has important strengths. To our knowledge, this study is the first to apply a methodology that can account for unobserved heterogeneity in a comparative framework to identify the effects of birth spacing on infant mortality. In doing so, we confirmed many of the findings of previous research while also uncovering new details that can help revise general recommendations for birth spacing practices. By adopting a comparative approach, our study helps to reconcile some of the supposed inconsistencies in the current body of literature.The findings presented here also offer several promising paths for future research. First, future research ought to focus more explicitly on identifying the causal mechanisms connecting birth interval length to infant mortality. Although our study sought to identify whether the relationship between birth spacing and mortality holds when adopting a robust control strategy, it was beyond its scope to identify which mechanisms facilitate this relationship. To explicitly identify the relative importance of the mechanisms linking short intervals to infant mortality, longitudinal data that include detailed information on factors such as biomarkers, household spending, and medical care would be required. Second, future work should also consider potentially different associations between birth spacing and mortality occurring at various times during infancy . Because the DHS is based on retrospective birth histories, deaths occurring in the early neonatal period may be especially prone to misreporting, and it is for this reason that we focused our study on infant mortality as a whole. Third, more comparative work that also includes wealthier populations would help to fill in the gaps regarding why birth intervals seem to matter a great deal in low-income contexts but much less in high-income contexts. Finally, it would be worthwhile to investigate whether the relationships between birth spacing and other outcomes are similarly moderated by the level of population health or other development indicators. Further comparative research may therefore help us to understand the conditions under which birth intervals matter for child health.ESM 1(PDF 1792 kb)


Objectives: The definition of circulatory impairment in the premature infant is controversial. Current research suggests overdiagnosis and overtreatment. We aimed to analyse which biomarkers move clinicians to initiate cardiovascular treatment (CVT). The prognostic capacity for adverse outcome  of these biomarkers was evaluated.Study Design: Retrospective data analysis from preterm infants enrolled in a placebo-controlled trial on dobutamine for low superior vena cava (SVC) flow, who showed normal SVC flow within the first 24 h (not randomized). Five positive biomarkers were considered: MABP < gestational age (GA)-1 mmHg; MABP < GA-5 mmHg; lactate > 4 mmol/L; BE < −9 mmol/L; SVC flow <51 ml/kg/min.Results:Ninety eight infants formed the study cohort. Thirty six received CVT (2–95 h). Logistic regression models adjusted for gestational age showed a positive association between CVT and the risk of death or moderate-severe abnormal cranial ultrasound at term equivalent [ p = 0.002]. MABP < GA-1 mmHg and lactate > 4 mmol/L were the most prevalent biomarkers at start of treatment. Low BE, high serum lactate and low SVC flow at first echocardiography showed a trend toward being associated with adverse outcome, although not statistically significant.Conclusions: Low blood pressure and high lactate are the most prevalent biomarkers used for CVT prescription. Lactic acidosis and low SVC flow early after birth showed a trend toward being associated with adverse outcome. These findings support using a combination of biomarkers for inclusion in a placebo-controlled trial on CVT during transitional circulation. Circulatory impairment is a common complication of the transitional circulation in the preterm infant . HoweverThe literature increasingly supports the need of further work on the assessment of efficacy and safety of CVT routinely used in neonates; within the safeguards of high quality placebo-controlled trials , 10. A kHypotension is probably the most widely used biomarker to initiate CVT , 12. HowTwo cut-off values for MABP are being considered because there is considerable variability amongst clinicians in the threshold for intervention in the case of low blood pressure , 8, 20: The SVC flow cut-off value is derived from an internal (unpublished) reliability study for SVC flow measurement, to address the theoretical variability of the method , 37. TheThe proposed cut-off values of the laboratory early biomarkers to be explored, lactate and BE, are supported by previous studies , 29, 30.The primary motivation of this study was to determine which of the five chosen cut-off values would qualify as biomarkers to be used as criterion for inclusion of patients in a future phase III randomized clinical trial to demonstrate efficacy and safety for dobutamine to treat hemodynamic insufficiency during transitional circulation in the preterm infant  .ad hoc analysis was conducted on data from a pilot randomized placebo-controlled trial of dobutamine for low superior vena cava flow (SVC flow) in preterm infants reported elsewhere  scan within 12 h from birth, when the majority of infants had not received any CVT.For completeness, the remainder of this section presents a brief description of assessments and intervention for both the normal and low flow groups in the pilot trial, with full details available from Bravo et al. .2), blood pressure (either invasive or non-invasive), and urine output were recorded every 6 h during the first 96 h from birth. Biochemical parameters  were obtained when clinically indicated. Infants with SVC flow < 41 ml/kg/min at any time during the first 24 h of life (low flow group) were randomized to receive dobutamine or placebo; whilst infants with SVC flow ≥ 41 ml/kg/min in all the scans performed during the first 24 h of life  were not randomized but continued in the trial for further assessment and follow-up and are the subject of this report. The low flow group was excluded from this retrospective analysis because it had a systematic and differential intervention based on Echo-D findings that could eventually influence their outcomes.After enrolment all infants had serial Echo-D scans at baseline, 60 min after baseline  and at the following times after birth: 12, 24, 48, 72, and 96 h. Heart rate, peripheral oxygen saturation  open-label treatment with other cardiovascular support that did not include dobutamine was allowed if indicated by the attending physician and was guided by a constellation of routine clinical or biochemical data, following a standardized treatment protocol: dopamine (first line), epinephrine (second line), plus/minus volume expansion or hydrocortisone –17.Main neonatal clinical outcomes were recorded at term equivalent, together with combined adverse outcome defined as: death; or moderate-severe abnormal cranial ultrasound according to term equivalent ultrasound findings  , 17. TheQuantitative  data are described as mean (SD), median (IQR), and qualitative  data as counts and percentages.The Mann-Whitney rank-sum test and the Fisher's exact test were used for quantitative and qualitative data analysis, respectively. The CVT effect and threshold biomarkers on binary outcomes were assessed using Binary Logistics Regression Analysis (unadjusted and adjusted for GA). The odds ratio and the 95% confidence interval are reported. The CVT effect on the quantitative outcome variables was assessed using an analysis of covariance (ANCOVA) using the CVT as factor and GA age as covariate. SAS 9.3  was used for analyses.n = 17), the median postnatal age at start on treatment was 12.5 (11–24) h. Infants who received CVT were less mature, and had lower birth weight and poorer condition at birth  p = 0.001], respectively. Severe ischemic event  and posthemorrhagic hydrocephalus were observed in 2 and 3 infants, respectively, who received CVT. However, the logistic regression analysis adjusted for gestational age could not be done for them as no infants without CVT showed these outcomes.The median postnatal age at the first Echo-D (definition of baseline) of the 98 patients in the normal flow group was 4 (3) h. Among them, 36 infants were judged to suffer circulatory impairment by the attending physician and received open-label CVT  at a median postnatal age of 12.2 (5.4–23.2) h. For those receiving only catecholamines (at birth . Outcomeat birth  comparedp < 0.01).The extent to which having a positive biomarker  influenced treatment decision is shown in p = 0.002) and combined adverse outcome (p = 0.02). In addition, BE < −9 mmol/L was associated with increased risk of death (p = 0.01).High serum lactate levels at any time during the first 96 h from birth were associated with an increased risk of death  but not predictive . Another limitation of this study is the fact that a more in depth analysis to explore whether potential confounders  could have had an impact on the observed association between CVT and adverse outcome, was not done. However, CVT (any type) and CVT (only catecholamines) showed a positive association with the risk of death or moderate-severe abnormal cranial ultrasound at term equivalent adjusted for gestational age. Thus, it was considered that the most powerful confounding factor was taken in consideration and this is of great interest in itself.For this work we used the BEST  resource from the FDA-NIH Biomarker Working Group, aiming to be as consistent as possible in the use of definitions and key terms around biomarkers. We believe this is needed for true effective communication, evaluation and interpretation of scientific evidence in this field of research .This retrospective pilot analysis has found that high serum lactate levels and low blood pressure are the most prevalent biomarkers used to prescribe CVT in this cohort of preterm infants. In addition to low SVC flow, high lactate serum levels and worse BE showed a trend toward being associated with adverse outcome. The findings support the use of these prognostic biomarkers as entry criterion in randomized placebo-controlled trials on cardiovascular support for circulatory impairment during the transitional circulation in the preterm infant.The Ethics Committee of La Paz University Hospital approved the study.MB conceptualized and designed the study, drafted the initial manuscript, collected the information from the medical charts, performed the initial analyses and approved the final manuscript as submitted. PL-O and LS collected the information from the medical charts and approved the final manuscript as submitted. RM performed the statistical analysis, reviewed the manuscript and approved the final manuscript as submitted. FC and AP conceptualized and designed the study, drafted the initial manuscript and approved the final manuscript as submitted. AK participated in the design of the statistical analysis, reviewed the manuscript and approved the final manuscript as submitted. HR-A and HR conceptualized the manuscript and approved the final manuscript as submitted.Neo-Circulation Expert Advisory Board: Tonse N. K. Raju, Bethesda, USA; Nicholas Evans, Sydney, Australia; Stephanie Läer, Düsseldorf, Germany; Silke Mader, Karlsfeld, Germany; Monika Seibert-Grafe, Mainz, Germany; Adrian Toma, Bucharest, Romania.Neo-Circulation Data Monitoring Committee:Gorm Greisen,Copenhagen, Denmark; Lena Hellström-Westas,Uppsala, Sweden; Josef Högel,Ulm, Germany].We also acknowledge the SAMID network (RETICS funded by the PN 2018-2011 (Spain), ISCIII-Sub-Directorate General for Research Assessment and Promotion and European Regional Development Fund (FEDER), ref. RD12/0026 and the Spanish Ministry of Health (SAS/2481/2009) for the scientific and financial support, respectively.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.


We aimed to clarify how maternal physical characteristics explains the association between adolescent pregnancy and adverse birth outcomes, focusing on their height. We used a national multicenter-based delivery registry among 30,831 women under age 25 years with a singleton pregnancy between 2005 and 2011. Adolescent pregnancy was defined as younger than 20 years of age, and categorized into “junior adolescent” (aged ≤15 years) and “senior adolescent” (aged 16–19 years). We used multivariate Poisson regression and mediation analysis to assess the extent to which maternal height explained the association between adolescent pregnancy and risk of adverse birth outcomes. Risks for preterm birth , low birthweight , and low Apgar score  were significantly higher among adolescent women compared to women of 20–24 years of age. The mediation effect of maternal height on these outcomes were moderate for low birthweight (45.5%) and preterm birth (10.5%), and smaller for low Apgar score (6.6%). In all analyses, we did not detect significant differences between junior adolescent and senior adolescent. Adolescent women have higher risk of adverse birth outcomes. This association is partially mediated by shorter maternal height. One main reason for this large interest is that adolescent pregnancy, with its many associated social problems such as single parenthood, welfare dependency, maternal low educational attainment, and maternal disability pension, has been known to increase risk of subsequent child abuse, neglect, suicide and double suicide8. Furthermore, studies show that offspring of adolescent pregnancies are more likely to become teenage parents as well9, thus causing a generational chain of people with social and economic risk.Adolescent pregnancy has been a target for prevention15. However, it is not clear based on current evidence as to what may be explaining this association. Together with possible lifestyle behaviors associated with both adolescent pregnancy16 and adverse birth outcomes17, it is plausible that physical immaturity in adolescents can be contributing to the association. Studies focusing on gynecologic age, or uterine immaturity, have supported this hypothesis18.In addition to these potential social and economic repercussions, several studies in both low and higher income countries have shown that adolescent pregnancy is associated with an increased risk of adverse birth outcomes, including preterm birth, low birthweight, neonatal asphyxia and perinatal death22. However, to our knowledge, none of the previous studies which studied the increase in risk of adverse outcomes in adolescent pregnancies have considered the role of maternal height15.As female height continues to increase until 18 to 19 years of age, shorter maternal height would reflect such physical immaturity in adolescents. Shorter height is also known to increase risk of adverse birth outcomes such as preterm delivery, small for gestational age (SGA), and preeclampsia among mature adultsUsing a Japanese national multicenter-based delivery registry, we investigated birth outcomes in Japanese adolescent women, and estimated the extent to which shorter maternal height, a proxy for the immaturity of the maternal body, may mediate this association.22. Briefly, this database is an ongoing registry which is currently based on 149 Japanese tertiary hospitals and covers over a hundred thousand annual births. In each hospital, maternal demographics, pregnancy complications, and birth outcomes were transcribed from medical charts using a standardized format.We used data from the Japan Society of Obstetrics and Gynecology Perinatal Database (JSOG-DB), which has been described elsewhere in detail23 (n = 415). In a large proportion of women, data on maternal height , maternal body mass index (BMI) , and gestational weight gain  were missing; thus, we conducted our main analysis on 30,831 women with complete covariate data. In addition, we conducted analyses on the total 53,301 women using imputed values for the missing data in order to evaluate potential for selection bias and external validity of results. Multiple imputation by chained equations was used to create 100 sets of imputed datasets. For analyses of severe laceration as an outcome, the subjects were restricted to 24,631 women who had never received cesarean-section.The registry included 54,047 women under 25 years old who gave birth to a singleton with no congenital anomaly between April 2005 and December 2011. Exclusions included women with missing outcome variables (n = 331) or unreliable data on pre-pregnancy weight (<20 kg considered unreliable), height (>4 SD or <−4SD of the population), and birthweight for gestational age25.Maternal age was our exposure of interest. As our database did not have age at conception, we used age at delivery. We broadly categorized women into young adult (20–24 years of age as a reference group) and adolescent (≤19 years). We further divided adolescent into two sub-categories (junior adolescent as ≤15 years of age and senior adolescent as 16–19 years of age) because previous studies have reported junior adolescents may have even higher risk of adverse outcomesth percentile for the given gestational age using the Japanese birthweight ref.26, preterm birth as less than 37 completed weeks of gestation, very preterm birth as less than 32 completed weeks of gestation, and extremely preterm birth as less than 28 completed weeks of gestation27. Preeclampsia and severe preeclampsia were diagnosed clinically by obstetricians at each hospital according to national guideline as systolic/diastolic blood pressure over 140/90 mmHg and 160/110 mmHg, respectively, that emerges after 20 weeks gestation with significant proteinuria (≥300 mg/day)28. We also defined severe laceration at birth as three or four degree laceration, and low Apgar score at five minutes as below 7.Primary outcomes of interest were maternal and neonatal complications at birth, including preterm birth, very preterm birth, extremely preterm birth, SGA, intrauterine fetal death (IUFD), cesarean section, unplanned cesarean section, pre-eclampsia, severe preeclampsia, severe vaginal laceration at birth, and neonatal Apgar score at 5 minutes. We defined SGA as birthweight below 1015. For smoking, a separate category was created for those with missing data.We considered maternal height, as a mediator of interest. The following factors were also included in the multivariate models: parity, year of delivery, maternal smoking status, pre-existing hypertension, pre-existing diabetes or gestational diabetes, pre-pregnancy BMI, and gestational weight gain during pregnancy29.We compared baseline demographics among the three categories of maternal age using the chi-squared test for categorical variables and one-way ANOVA test for continuous variables.; we also compared the characteristics between 30,831 women with complete covariate data and 22,470 with missing data, using the chi-squared test for categorical variables and student t test for continuous variables. Poisson regression was used to estimate the effect of adolescent pregnancy on the risk of adverse birth outcomes, as well as changes in the effect after sequentially adjusting for demographics and considering maternal height as a mediator. For each outcome, we pursued two separate multivariate models; (1) including adolescent pregnancy, parity, year of delivery, maternal smoking, pre-pregnancy BMI and gestational weight gain (model 1), and (2) including all variables in Model 1 and maternal height (model 2). To confirm the generalizability of our results, we conducted sensitivity analysis on the whole sample  with missing values of the following variables imputed through multiple imputation with 100 sets of imputations: maternal height , maternal BMI , maternal gestational weight gain during pregnancy  and smoking . For all Poisson models, we conducted analyses comparing all adolescents with non-adolescents, as well as comparing junior adolescents and senior adolescents with non-adolescents. All results were based on robust variance estimates30. Theoretically this calculation is based on three models, (1) one estimating the total effect (c) of X on Y, (2) one estimating the indirect effect (c’) of X on Y with M included as a covariate, and (3) one estimating the effect of X on M30 from which the mediating effect (of X on Y through M) is calculated as a fraction of the total effect (of X on Y). We used an improved analytical method that takes into account possible exposure-mediator interactions as proposed by VanderWeele31 rather than crudely calculating the mediating effect by dividing c’ by c as proposed by Baron and Kenny30.Next, for the subset of adverse outcomes that were associated with adolescent pregnancy , we conducted mediation analysis to estimate the mediated effect of adolescent pregnancy through maternal height. Mediation analysis hypothesizes that the total effect of maternal age (X) on an adverse outcome (Y) consists of a direct effect and an indirect effect ), and calculates the proportion of the total effect that is indirect (i.e. mediated)All statistical analyses were conducted using the statistical software package Stata SE 14 . Statistical significance was set under 0.05, and all statistical tests were two tailed. The protocol for this study was approved by the Institutional Review Board of the National Center for Child Health and Development on Apr. 18, 2017 (No 1448).Women in younger categories of age were significantly shorter, thinner, and more likely to gain weight during pregnancy Table . Of 30,8When adjusted for confounders (model 1), adolescent women had significantly higher risk of adverse neonatal outcomes including preterm birth , very preterm birth , extremely preterm birth , low birthweight , and low Apgar score  compared to women aged 20–24 years  as well as unplanned cesarean section  compared to women aged 20–24 years Table . After aWe did not observe significant differences in effect on birth outcomes between junior and senior adolescent age groups , although the estimated risks of junior adolescent were larger for several outcomes such as low Apgar score and severe laceration. For all analyses, estimated effect of maternal age, as well as its change due to sequential adjusting, were similar between the main analyses results and sensitivity analyses using the dataset with imputed data  and extremely preterm birth (1.9%) compared to total preterm birth (10.5%). As limited pelvic size would only be a problem in late pregnancy when the fetus is quite large, these observations are also consistent with the interpretation that physical immaturity contributes to those adverse effects of pregnancy in adolescents.In our study, we observed that maternal height significantly mediated the association between adolescent pregnancy and preterm birth, low birthweight and low Apgar score. These results support a hypothesis that shorter height, which relates to smaller pelvic size among adolescent women33 which has been repeatedly observed among pregnancies of adolescent women34.It is also noteworthy that while the observed mediated effect was significant, the estimated effect size was quite small for all outcomes (aRR 1.01 to 1.04), with the unexplained direct effect much larger (aRR 1.04 to 1.38). These findings suggest that the majority of the adverse effect observed in adolescent pregnancies may not be due to the physical constraints of an adolescent body, but may rather be driven by socially-derived risk factors, such as inadequate prenatal or delivery care15, we found decreased risk of cesarean section (including unplanned cesarean section) associated with adolescent pregnancies. However, the explanation for this observed decrease in risk is not yet clear. A previous report showed an increased risk for cesarean section in adolescent women when restricted to those with indication for presumed cephalo-pelvic disproportion, suggesting that differences in indication for cesarean section may be confounding the results4. Another study showed increased risk of failure to progress or cephalop-pelvic disproportion in adolescent women11. Thus, we cannot rule out the possibility that differences in indication for cesarean section confounded our findings as we did not have appropriate data for a thorough evaluation. Further assessment of this issue is warranted.Consistent with previous studies25, the inability of our study to detect a clear pattern may have been due to limited statistical power in the analysis of adolescents under 15 years of age.When comparing risk of adverse birth outcomes between junior and senior adolescent women, we did not detect significant differences between the two groups, although the estimated risk was larger among junior adolescents for several of the outcomes such as preterm birth and severe laceration. As larger studies have reported junior adolescents have greater risk of preeclampsia, preterm birth, low birthweight, and neonatal asphixia5, which adolescent pregnancies are also at higher risk, were not captured in our database, thus our analysis based on pregnancies past 22 weeks may be underestimating the true adverse risk associated with adolescent pregnancies. Future prospective population based studies are encouraged to confirm the reproducibility of our findings. Third, we relied on maternal height as a proxy for physical immaturity in this study. However, adolescent women may have further biological constraints due to immaturity  which are not captured through height. Studies investigating the extent to which other constraints may account for the adverse effect of adolescent pregnancy on birth outcomes are needed. Fourth, while our study indirectly suggests that social and lifestyle factors also contribute a significant proportion of the effect of adolescent pregnancy on adverse birth outcomes  this effect may have been underestimated due to residual confounding. We were unable to account for socio-economic status, lifestyle factors including undernutrition leading to lower maternal height, as well as adverse birth outcomes34 due to of the lack of information in our database. Furthermore, we were able to adjust for smoking status only crudely using a binary categorization (smoked during pregnancy: yes/no) despite previous studies indicating that frequency and duration may influence on birth outcomes. Future studies are need to evaluate whether social factors and detailed smoking status may be responsible for the remaining unexplained increase in risk. Fifth, as the main interest of this study focused on the role of maternal height in explaining the adverse effects of adolescent pregnancy, we did not conduct detailed analysis of how the effect of adolescent pregnancy may vary by maternal characteristics and assumed the effects were similar. Observing that previous studies has reported that the adverse effects of adolescent pregnancy was greater among multiparous women compared to primiparous women36, this assumption may have be violated in our study. Future studies focusing on effect modification by maternal characteristics are encouraged.The strengths of our study include the implementation of mediation analysis to clarify the role of physical factors in the association between adolescent pregnancy and adverse birth outcomes, as well as the utilization of a large nation-wide clinical database in Japan which enabled us to conduct complex analyses on adolescent pregnancies. Nonetheless, the current study has some limitations. First, we did not have maternal age at conception and used maternal age as delivery as its proxy. However, we believe its influence on our results would be relatively small considering the pregnancy period is 7–10 months. Second, our analyses were conducted using a database based on Japanese tertiary hospitals, which may have resulted in the inclusion of more women with higher risk pregnancies compared with the general population. However, we excluded women with pre-existing clinical risk factors of adverse birth outcome, such as maternal diabetic disorder or pre-existing hypertension, multiple pregnancy, and fetal congenital anomaly, to maximize generalizability of findings. In addition, termination of pregnanciesIn conclusion, we found an increased risk of preterm birth, low birthweight, and low Apgar score associated with adolescent pregnancies in Japanese women. We provide evidence that while physical immaturity  does significantly contribute to this association as a mediator, the modest levels of estimated mediation leaves room for a potentially larger role for social and lifestyle-related factors that are associated with adolescence.supplemental Table 1–3