To estimate county‐level associations between in utero exposure to threatened evictions and preterm birth in the United States.Complete birth records were obtained from the National Center for Health Statistics (2009‐2016). Threatened evictions were measured at the county level using eviction case filing data obtained from The Eviction Lab (2008‐2016). Additional economic and demographic data were obtained from the United States Census Bureau and Bureau of Labor Statistics.z‐score of average case filings over the pregnancy and by trimester. Our primary outcome was an indicator for preterm birth (born < 37 completed weeks of gestation). Secondary outcomes included a continuous measure for gestational length, a continuous measure for birth weight, and an indicator for low birth weight (born < 2500 g). We estimated within‐county associations controlling for individual‐ and time‐varying county‐level characteristics, state‐of‐residence‐year‐and‐month‐of‐conception fixed effects, and a county‐specific time trend.We conducted a retrospective cohort analysis using 7.3 million births from 1,633 counties. We defined threatened eviction exposures as the We merged birth records with threatened eviction data at the county‐month‐year level using mother's county of residence at delivery and month‐year of conception. We supplemented these data with information on county‐level annual 18‐and‐over population, annual poverty rate, and monthly unemployment rate.Increased levels of eviction case filings over a pregnancy were associated with an increased risk of prematurity and low birth weight. These associations appeared to be sensitive to exposure in the second and third trimesters. Associations with secondary outcomes and within various population subgroups were, in general, imprecisely estimated.Higher exposure to eviction case filings within counties, particularly in the latter stages of a pregnancy, was associated with an increased risk of adverse birth outcomes. Future research should identify the causal effect of threatened evictions on maternal and child health outcomes. In 2016, approximately 2.4 million eviction cases were filed in the United States.A developing literature links eviction to harmful effects on health. Several studies have documented associations between increased eviction prevalence and elevated risk of experiencing stress, depression, anxiety, psychological distress, and drug use.Despite mounting evidence of a link between health and eviction, there is less evidence on the relationship between threatened evictions and key maternal and child health outcomes. Evidence from the housing literature suggests that families with small children may be particularly likely to be threatened with evictions and, ultimately, be evicted.We hypothesized that increased prenatal exposure to threatened evictions would increase the likelihood of preterm birth and other adverse birth outcomes. This is because the threat of evictions is a key source of stress and other poor mental health outcomes, and a large literature demonstrates that high levels of prenatal stress are strongly associated with worse birth outcomes.22.1We conducted a retrospective cohort analysis by combining the largest county‐level dataset on legal eviction case filings in the United States to date with restricted‐use national birth records from the National Center for Health Statistics (NCHS).Data on eviction filings were provided by The Eviction Lab at Princeton University and contained county‐month‐year‐level counts of the number of eviction cases filed in local court for 1633 counties between 2008 and 2016.Live birth records from the NCHS represented the universe of live births in the United States between 2009 and 2016. These data contained individual‐level information on each woman's demographics, delivery payment method, self‐reported county of residence at delivery, completed weeks of gestation, birth weight, and day, month, and year of delivery.We supplemented our analysis by using county‐level data from two other sources. From the United States Census Bureau, we used data on annual, county‐level 18‐and‐over population and annual, county‐level poverty rate.2.2For each pregnant woman, we constructed two exposure variables using data on county‐month‐year eviction case filings. The first exposure was defined over the duration of the pregnancy from the month of conception to the month of delivery. The second exposure was defined separately for the first trimester (month of conception to third month of gestation) and together for the second and third trimesters (fourth month of gestation to the month of delivery). We label the first exposure as EP  and the second exposure as ET .To construct EP and ET, we estimated each woman's date of last menstrual period (LMP) using information on the obstetric/clinical estimate of gestational length and the day of week, month, and year of delivery. Next, we assigned a date of conception for each woman in our sample by assuming that conception occurs two weeks after the estimated date of LMP. Finally, we identified the number of eviction cases filed in each month of a woman's pregnancy by using information on her month‐year of conception and county of residence at delivery.casesi,tzscore) ascasesi,tavg,norm represents the average normalized case filings for each pregnant woman i over duration of pregnancy/trimester t, casestavg,norm¯ represents the mean of the average normalized case filings in the entire analytic sample over time period t, and SD(·) represents the standard deviation operator.Having assigned eviction cases to each pregnant woman for each month of their pregnancy, we constructed EP and ET in three steps. First, we normalized the number of cases filed for each county‐month‐year by the county's 18‐and‐over population for the same year. Next, we estimated the average of the population‐normalized eviction case filings over the duration of each woman's pregnancy as well as separately for the first trimester and second and third trimesters combined. Finally, we standardized the average, normalized cases to define a z‐score  completed weeks of gestation defined as a continuous variable and measured using the obstetric/clinical estimate; (2) birth weight (in grams) defined as a continuous variable; and (3) low birth weight defined as an indicator variable which equaled one if birth weight was less than 2500 g and zero otherwise.2.4We constructed our analytic sample by restricting live birth observations based on the following inclusion criteria: (a) had obstetric/clinical estimate of gestation reported; (b) mother resided in a county for which eviction case filing data were available; (c) eviction case filing data were available for each month of gestation; (d) delivery occurred in a state that had adopted the 2003 revised birth certificate in the year of delivery; and (e) the live birth was singleton.To prepare the data for analysis, we merged birth records with the eviction case filing data using information on the estimated month‐year of conception associated with each live birth and each pregnant woman's county of residence at delivery. We similarly merged supplementary datasets on county‐level population, unemployment, and poverty. Finally, for each observation in our data, we estimated eviction exposures EP and ET using Equation . We thenWe computed descriptive statistics of the outcome and covariates of interest in the analytic sample by tertiles of exposure EP. We assumed that covariate data were missing at random and accounted for them by constructing five imputed datasets under the assumption that the observed and unobserved data together followed a multivariate normal distribution. In each imputed dataset, we winsorized exposures EP and ET at the 1st and 99th percentile to reduce the influence of extreme values of the exposure.β represents the association between eviction filings and adverse birth outcomes, Xi,c,t represents individual‐ and county‐level covariates, δc represents county fixed effects defined based on each woman's self‐reported county of residence at delivery, θs(c),t represents state‐of‐residence‐year‐and‐month‐of‐conception fixed effects, and timec represents a county of residence–specific linear time trend. Equation . Since the effective exposure variation is constrained by our primary specification—that is, we analyze the variation that remains after accounting for county‐level fixed effects, state‐of‐residence‐year‐and‐month‐of‐conception fixed effects, and a linear county‐level time trend—an association of 1.09 percentage points corresponds to a 0.08 percentage point increase, on average, in the risk of preterm birth . Given the effective variation we are working with, a 0.7 percentage point increase in risk corresponds to a 0.05 percentage point increase in the risk of newborns being born low birth weight . After adjusting for the effective variation, this risk difference represented a 0.08 percentage point increase in the risk of preterm birth . The estimated association corresponds to a 0.06 percentage point difference in the risk of low birth after accounting for the effective variation (Figure In terms of the secondary outcomes, we estimated a positive association between threatened evictions in the second and third trimesters and risk of low birth weight (n Figure . The assTable Results from analyses checking for association heterogeneity by racial subgroups and Medicaid payment status were largely consistent with the primary results Tables . We esti4In this study, we investigated the relationship between prenatal exposure to threatened evictions and adverse birth outcomes across 1633 counties in 39 states and the District of Columbia between 2008 and 2016. We also studied if these associations varied by pregnancy trimester. We found that increased exposure to eviction filings during a pregnancy was associated with an increased risk of prematurity and being born low birth weight. We also found some evidence to suggest that risk of preterm birth and low birth weight were particularly sensitive to eviction filings in the second and third trimesters of a pregnancy.Results from various robustness checks largely support our primary results. Coefficients on the prepregnancy exposure variable, which we used as a negative control to conduct a falsification analysis, were small and imprecise. Although prepregnancy threatened eviction exposure may not be the ideal negative control because of its potential direct effect on birth outcomes through prepregnancy health, the falsification check results do provide suggestive evidence to support the claim that our primary results are not driven by spurious correlations. Results from estimating Equation  across tTo the best of our knowledge, our study is one of the first to demonstrate that increased within‐county exposure to eviction filings increases the risk of preterm birth. The positive associations between eviction filings—a source of prenatal stress—and risk of prematurity and low birth weight are consistent with studies that have investigated the impact of prenatal stressors on pregnancy outcomes. For instance, Gemmill et alHowever, the associations we estimate for preterm birth risk and low birth weight risk have relatively wide confidence intervals. For the association between exposure EP and preterm birth, for example, our results are compatible with a 0.05‐2.13 percentage point increase in the risk of prematurity. This relatively wide range of estimates may in part reflect the ecological nature of our analysis which analyzes an exposure at the county level, that is, our analysis treats all pregnant women as exposed to a given level of eviction filings regardless of whether they personally received an eviction notice or were affected by one indirectly. This means that our associations likely capture some combination of the exposure of living in a community where evictions are common combined with the direct experience of receiving an eviction filing. Individual‐level data on eviction filings would allow for the estimation of these associations with a higher degree of precision and could consequently find substantially larger effects for pregnant women who were directly affected by the threat of evictions over the study period. The potential for community‐level effects of housing instability resulting from increased eviction filings would also be consistent with the housing and social epidemiology literatures which have shown the impact of neighborhood quality on health outcomes.Another strength of our study is that we analyze the relationship between threatened evictions and physical health outcomes using data that have wide geographic and temporal coverage. Prior studies looking at associations between threatened evictions, actual evictions, and health outcomes have either come from other countries such as Spain or from very localized geographies within the United States.A final strength of our analysis is that it adds to the literature on the importance of the timing of in utero stressors on the risk of preterm birth and other pregnancy outcomes. In this regard, our finding regarding the relative importance of eviction case filing exposure in the second and third trimesters is consistent with some recent studies such as that by Gemmill et al.A key limitation of our analysis is that we did not have complete geographic and temporal coverage in terms of the eviction case filing data. The United States has over 3000 counties, but we only had data from 1633. Lack of data from all counties is explained by the fact that collection of data on eviction cases is more difficult in some areas due to incomplete or nonstandardized electronic case management systems, limitations on access to paper case records, and restrictions on bulk records requests. It is unclear how these barriers to record collection are associated with case filing volumes, which limits the generalizability of our results. Furthermore, generalizability of our results may also be limited because we could only use approximately 57 percent of the possible 176 364 county‐month‐year observations, not only due to a lack of county‐level data but also because we restricted our analysis to those state‐years in which the 2003 birth certificate revision had been implemented.Another limitation of our analysis is that we only observe residence at time of delivery, and we are therefore unable to determine whether a pregnant woman moved across counties over the course of her pregnancy. Movement across counties would mean that our exposure variable would be subject to measurement error that we expect would attenuate our results. However, there are reasons to believe that movement across counties may be limited in scope: The literature on evictions suggests that individuals who are evicted generally tend to move into worse quality neighborhoods and not necessarily across counties.A final limitation of our analysis is the possibility of selection bias due to exposure to higher levels of threatened evictions resulting in greater loss of fetuses that would have been born premature relative to lower levels of threatened eviction exposure.5The threat of evictions has been increasing over the past two decades, particularly for low‐income Americans. Our analysis shows that for pregnant women, higher levels of threatened evictions are associated with increased risk of adverse birth outcomes in general and premature deliveries in particular. Across the United States, several policies are currently being enacted to offset the threat of evictions—our study suggests that evaluating the causal impact of these policies on parental well‐being and child health is an important area of inquiry.Supplementary MaterialClick here for additional data file.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Figure S5Click here for additional data file.Appendix S1Click here for additional data file.


Background: About 85–90% of children born small for gestational age (SGA) experience a catch-up growth that occurs mostly during the first year of life and results in a full stature recovery by the age of 2.Objective: To investigate the relation between bone maturation (BM) and catch-up growth during the first year of life in SGA infants.Method: Newborns whose weight and/or length was <-2 SD for gestational age were classified as SGA (group A). The study included a group of 32 SGA, 21 of which are full term  and 11 preterm . Control group (B) consisted of 19 full-term and adequate-for-gestational-age (AGA) newborns. All the participants were born in the same hospital and period (January–December 2017). Chromosomal disorders, congenital defects, and maternal chronic diseases were criteria of exclusion. The study population underwent longitudinal evaluation of growth parameters and BM at 0, 3, 6, and 12 months. Assessment of BM was performed by an ultrasonographic (US) study of Béclard's nucleus (NB) .Results: Significantly higher height velocity (HV) was observed in subgroup A2 vs. A1 ; nevertheless, more subjects in subgroup A2 had height <-2 SD at year 1 than had subgroup A1 . Intrauterine delay of BM was more common in group A vs. B  and in subgroup A2 vs. A1 . In group A, HV over the first year of life negatively correlates with NB diameter assessed at birth  but positively correlates with NB growth . Moreover, SGA babies with intrauterine delay of BM showed higher HV and better height gain at 12 months' evaluation than did SGA with adequate BM .Conclusion: Neonatal BM should be regarded as a predictive factor of SGA height gain during the first year of life. US evaluation of NB is a useful noninvasive technique to identify intrauterine delay of BM, which positively correlates with early postnatal catch-up growth of SGA infants. The term small for gestational age (SGA) describes neonates whose weight and/or length at birth is below the cutoff value of −2 SD for gestational age. In clinical practice, the definition of SGA requires rigorous evaluations: (a) gestational dating, assessed by first-trimester ultrasound exam; (b) accurate anthropometry at birth, including measurements of weight, length, and head circumference; and (c) comparison with data coming from adequate reference population –3.Instead, the concept of intrauterine growth restriction (IUGR) refers to slow fetal growth due to maternal, fetal, and/or placental causes, on the basis of at least two ultrasound exams performed during pregnancy. This process may result in a SGA newborn, even if it is worth noting that a SGA baby is not necessarily IUGR. Indeed, the SGA definition can also include a percentage of neonates (18–22%) who are only constitutionally small .The recognition of SGA condition raises public health issues, as it could imply a variety of short- and long-term consequences including increased perinatal morbidity and mortality, higher risk of short stature, metabolic disorders later in life, and neurocognitive vulnerabilities , 5, 6.With respect to growth, it is well documented that about 85–90% of SGA babies experience a catch-up growth that occurs mostly during the first 12 months of life and results in a full stature recovery by the age of 2 years. The remaining 10–15% of SGA children do not undergo compensatory growth and will remain permanently <-2 SD for height. Indeed, 20% of short-stature adults were born SGA. Finally, the preterm infants born SGA are regarded as a special case, as they may take up to 4 years to achieve a height within the normal range for population , 8. The To the best of our knowledge, bone maturation (BM) assessment in SGA newborns has never been reported to now. As a consequence, there is a lack of specific literature data about the potential influence of BM progression on compensatory growth in this population.Our prospective study aims to detect the factors that may be involved in early catch-up growth and to investigate the link between BM and postnatal growth during the first year of life in SGA infants.The study population consisted of 32 SGA neonates who were born in the same hospital and period (January–December 2017), fulfilling the following inclusion criteria: (a) birth weight and/or length <-2 SD for gestational age; and (b) gestational dating assessed by first-trimester ultrasound exam.Sick newborns and those with genetic syndromes, chromosomal disorders, congenital defects, neonatal screening positivity, or maternal chronic diseases were excluded from the study.The entire series of SGA newborns  was divided into two subgroups, according to gestational age: 21 full-term SGA [37–41 gestation weeks (GW)] were included in subgroup A1  and 11 SGA preterm (30–36 GW) in subgroup A2 . According to BM assessment at birth, group A was divided into two further subgroups: 13 SGA with adequate BM were part of subgroup A3 and 19 SGA with delayed BM of subgroup A4. The SGA population was compared with controls  represented by 19 full-term and adequate-for-gestational-age (AGA) neonates born in the same hospital and period.Newborns who fulfilled the above reported inclusion criteria were enrolled for this prospective one-center study.The study population underwent longitudinal evaluation of growth parameters  and BM at birth and at 3, 6, and 12 months. Bone age was assessed at 12 months of age.The study design was approved by the local ethical committee of the hospital participating to the study. Informed consent was obtained earlier by the children's parents.2, which is very similar to the cutoff value  used by Glorieux et al. . As recommended by the Italian Society of Pediatric Endocrinology and Diabetology (ISPED), the reference population standards were assessed according to the INeS  charts from birth to term , 13 and x et al. . This mex et al. . An echox et al. .t-test  and the Mann–Whitney U-test (non-parametric data) were used. Frequency rates were compared by a chi-squared test. Correlations between quantitative variables were assessed using Pearson's correlation analysis. Statistical analyses were performed using IBM SPSS Statistics for Windows, Version 21 . A p-value smaller than 0.05 was considered statistically significant.Numerical data were expressed as mean, SD, and range; categorical variables were expressed as absolute frequency and percentage. For comparisons of two means, Student's p = 0.0078) and in subgroup A2 than A1 .The clinical features of the entire study population at birth are shown in p = 0.01); nevertheless, subjects in subgroup A2 presented more frequently with height <-2 SD than did subgroup A1 . HV was overall higher in group A, if compared with controls, but without reaching statistical significance . The main features of growth pattern for each group and subgroup during follow-up are shown in p < 0.0005).Mean first-year height velocity (HV) of the entire population (group A + B) was 25.5 ± 13.2 cm. Significantly higher HV was observed in subgroup A2 vs. A1  than group A during the entire follow-up period, without statistically significant difference between A1 and A2 subgroups.r = −0.6, p < 0.001) but positively correlated with NB growth during follow-up . This relation was even stronger in subgroup A2 .US evaluation recorded a progressive NB growth during 12 months' follow-up in group A. Instead, group B showed this growth only until the ninth month of follow-up, with no further NB increase . In grouGrowth parameters, BM, and bone age at the end of 12 months' follow-up are presented in p = 0.003) at 12 months' evaluation (SGA infants with documented intrauterine delay of BM (subgroup A4) exhibited a pattern of progressive catch-up growth during the follow-up period. If compared with SGA infants with adequate BM (subgroup A3), subgroup A4 showed higher HV and better height gain .Moreover, the frequency of a height outcome <-2 SD at the age of 1 year did not significantly differ in these two subgroups .At the end of follow-up, bone age evaluation showed similar scores in both subgroups  and with higher HV than observed in SGA full term, who exhibited instead an earlier and more gradual stature recovery.In addition, our study showed that there is a positive relationship between BM at birth and HV during the first year of life in SGA infants. Indeed, intrauterine delay of BM positively correlates with catch-up growth, allowing higher HV and better height gain at 12 months' evaluation. These findings are even more evident in SGA preterm. Despite higher HV recorded among the above-mentioned subgroup, SGA preterm exhibited more frequently <-2 SD height outcome at the end of follow-up.In conclusion, our results suggest for the first time that neonatal BM should be regarded as a predictive factor of SGA height gain during the first year of life. US evaluation of NB turned out to be a useful noninvasive technique to identify intrauterine delay of BM, which seems to positively correlate with early postnatal catch-up growth of SGA infants.Limitations of the present study were represented by the small sample size; however, this weakness is counterbalanced by the high level of homogeneity exhibited by our population. In addition, our control group included only AGA full term, so it could be considered as only partially representative for the total study population (SGA full term and preterm).Taking into consideration the lack of specific literature data on neonatal BM and its relationship with gestational age, we could not exclude that the delayed BM of SGA preterm infants in the present study could be related to prematurity. Overall, our data seem to encourage the use of US evaluation of NB, especially among neonates <-2 SD of length at birth. Anyway, to further characterize the influence of BM on SGA catch-up growth, future studies on a wider population are needed.All datasets generated for this study are included in the article/supplementary material.The studies involving human participants were reviewed and approved by Ethics Committee of Messina. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.MW and MC conceived the manuscript. GP, MC, and DC were involved in data collection. GP, MW, and LM were involved in writing of the manuscript and carried out data analysis. MV, GBP, and TA were involved in literature search. All authors approved the submitted version of the manuscript.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.


Multivariate analysis found being born very or late to moderate preterm was independently associated with long-term infectious morbidity (adjusted hazard ratio (aHR) 1.5, 95% confidence interval (CI) 1.27–1.77 and aHR 1.23, 95% CI 1.17–1.3, respectively, p < 0.001). A comparable risk of long-term infectious morbidity was found in the two groups of premature births prior to 32 weeks gestation. In our population, a cutoff from 32 weeks and below demarks a significant increase in the risk of long-term infectious morbidity of the offspring.In this study, we sought to ascertain a relationship between gestational age at birth and infectious morbidity of the offspring via population-based cohort analysis comparing the long-term incidence of infectious morbidity in infants born preterm and stratified by extremity of prematurity . Infectious morbidity included hospitalizations involving a predefined set of International Classification of Diseases 9 (ICD9) codes, as recorded in hospital records. A Kaplan–Meier survival curve compared cumulative incidence of infectious-related morbidity. A Cox proportional hazards model controlled for confounders and time to event. The study included 220,594 patients: 125 (0.1%) extreme preterm births, 784 (0.4%) very preterm births, 13,323 (6.0%) moderate to late preterm births, and 206,362 term deliveries. Offspring born preterm had significantly more infection-related hospitalizations (18.4%, 19.8%, 14.9%, and 11.0% for the aforementioned stratification, respectively,  Preterm birth refers to deliveries occurring prior to 37 completed weeks of gestation. Most preterm births are spontaneous, while up to 30% may be due to iatrogenic causes such as preeclampsia, fetal growth restriction, or multiple gestation . The gloThe risk of an offspring contracting an infectious disease during infancy or childhood is dependent on many factors including, but not limited to, exposure to infections via siblings or other children during daycare or school  and enviThe increased morbidity and mortality rates of offspring born prematurely are often due to complications associated with the incomplete development of organ systems and the need for invasive care and life-support measures, which contribute to the pathogenesis of many complications including infections . Of impoWhile much study has been devoted to the identification of the short-term consequences of preterm delivery and the development of interventions to limit morbidity and mortality, significantly less research has focused on the consequences extending beyond the neonatal and infant periods. One population-based study examined the association between gestational age and growth measurements with infection-related admissions to hospital throughout childhood , observiThis retrospective population-based study included all singleton deliveries occurring between the years 1991 and 2014 at the Soroka University Medical Center (SUMC). SUMC is the largest birth center in the country and the sole tertiary hospital in the Negev area, serving a population of over 1.2 million inhabitants. The area has experienced positive immigration over the last two decades, with increasing annual birth rates from 10,000 to roughly 15,000 by the end of the study period. This study is based on nonselective population data.Cases were divided into four groups according to the extremity of prematurity as defined by the WHO —extreme Cases of multiple pregnancies and congenital malformations, which could be potentially confounding due to the relationship between both factors with preterm delivery, were excluded, as were cases of perinatal mortality . Outcomes included infection-related hospitalizations of all subtypes of the offspring up to the age of 18 years, excluding the infections occurring in the hospitalization immediately following delivery. Infectious morbidity was predefined in a set of ICD9 codes detailed in the Follow-up time was defined as time to event , and follow-up ended at first relevant hospitalization, or when censored for death of child , when the child reached 18 years of age (on the basis of date of birth), or at the end of the study period, whichever preceded.Data were merged from two databases: the computerized hospitalization database of SUMC (“Demog-ICD9”) which includes demographic information and ICD-9 codes for all medical diagnoses made during hospitalization in any of the departments at SUMC including pediatric departments, and the computerized perinatal database of the obstetrics and gynecology department at SUMC, which consists of information recorded immediately following delivery by an obstetrician and reviewed by experienced medical secretaries for accuracy and completeness prior to entering it into the database. Coding is performed after assessing medical prenatal care records, as well as routine hospital documents.p-value <0.05 was considered statistically significant.Statistical analysis was performed using the SPSS package 23rd edition . Quantitative normally distributed variables were compared by analysis of variance (ANOVA), and categorical variables were compared using the chi-square test. Kaplan–Meier survival curves were used to compare cumulative infection-related hospitalization incidences over time according to gestational age at birth, divided into the four subgroups of gestational age as detailed above. The differences between the four cumulative morbidity curves  were assessed using the log-rank test. A Cox proportional hazards model was used to establish an independent association between gestational age at birth and pediatric infection-related hospitalization risk while controlling for time to event, maternal age, birthweight, hypertensive disease, diabetes mellitus, and mode of delivery. Deliveries occurring at term (37 + 0 or more completed weeks of gestation) were considered as reference. All analyses were two-sided and a During the study period 220,594 patients met the inclusion criteria, of which 125 (0.1%) were extreme preterm birth, 784 (0.4%) were very preterm birth, 13,323 (6.0%) were moderate to late preterm birth, and 206,362 were term deliveries. p < 0.001, p < 0.001).Offspring born preterm had significantly more hospitalizations due to infectious morbidity compared to term offspring .A subanalysis of induced versus spontaneous deliveries  shows thp < 0.001). Extremely early preterm deliveries at 24 + 0–27 + 6 weeks were also found to increase the risk of long-term infectious morbidity , but this association did not achieve statistical significance.p < 0.001). The two groups with the earliest preterm deliveries showed a similar curve. The Kaplan–Meier survival curve  demonstrPediatric infections are a major cause of mortality and morbidity in both developed and developing countries, particularly in neonates and preterm babies with an estimated one million neonatal deaths annually worldwide attributed to infectious causes . While iThis large retrospective cohort study examined the long-term incidence of infectious-related hospitalizations stratified by extremity of prematurity and compared to offspring born at term. We showed that infectious morbidity is increasingly common with decreasing gestational age at birth, as shown in both the Kaplan–Meier survival curve and the Cox regression analysis model. From these models, it can be seen that, below 32 weeks gestation, there was no significant difference between the two groups with regard to the long-term infectious morbidity of the offspring; therefore, 32 weeks is the threshold below which the risk of morbidity significantly increases. Although this was not found to be statistically significant for extremely early preterm deliveries, Among the possible confounders controlled for in the multivariate analysis, hypertensive disease was not found to be independently associated with long-term infectious morbidity of the offspring. Interestingly, in our study, the incidence of hypertensive disease decreased with increasing gestational age. While it is known from previous studies that the incidence of hypertensive diseases of pregnancy increases with increasing gestational age , this stIn our study, the rate of cesarean delivery increased significantly with decreasing gestational age. As shown in previous studies, newborns delivered via cesarean delivery are not exposed to the vaginal and perianal flora of the mother; rather, their initial exposure comprises nosocomial pathogens found in the operating room and dermal microbial colonization . The offAfter controlling for the confounding effect of mode of delivery, decreasing gestational age at delivery was still significantly associated with increasing risk for long-term infectious morbidity of the offspring. The innate immune system, which provides the first line of defense against potential infection, develops and matures during fetal life and further during childhood ,26. It iPerhaps due to this continuum of immune system maturity which continues throughout gestation and indeed throughout infancy, the univariate analysis in our study showed a general trend toward an inverse relationship between gestational age and long-term infectious morbidity. Beyond 32 weeks, this trend in clear, although, below this threshold, the relationship is less linear. This can also be seen in the Kaplan–Meier survival curves, which show a clear decrease from 32 weeks gestation in long-term infectious morbidity, but similar curves for earlier gestational ages.The main limitation of this study is the retrospective design, with the analysis providing only evidence of association and not causation. Data regarding several potential confounding factors, such as trends in microorganisms and antimicrobial treatments over years were not available for further statistical analysis. An additional limitation is the inclusion of only hospital-related infectious morbidity, which likely includes only acute and severe infectious diseases, with no consideration of infectious morbidity diagnosed and treated in ambulatory settings; therefore, external validity of our findings is limited. As a single-center study, the results have limited generalizability compared to a multicenter study. However as SUMC is the largest birth center in the country and the sole tertiary hospital in the Negev area with little negative immigration, it is assumed that the majority of offspring would be followed up in our center, allowing true long-term follow-up. If immigration occurred, it can be assumed that the incidence was equal in all groups and, therefore, would not significantly impact the conclusions of this study. The large sample size is an additional strength of the study. Further studies to accurately identify the immune response in the preterm infant and studies to define the role of possible confounders such as breastfeeding, antibiotic use, and exposure to infections from siblings, amongst other maternal and pediatric characteristics, may help to clarify the relationship. In addition, more research is needed regarding interventions to improve the immune system function of these preterm infants and decrease the risk of long-term infectious morbidity. Several mechanisms have been proposed including probiotic supplementation  and mateIn conclusion, a critical threshold of 32 weeks gestation was noted in our study, below which the risk of long-term infectious morbidity of the offspring was significantly increased. Interestingly, the risk of infectious diseases of the offspring in our study does not appear to be increased further below 28 weeks gestation. Despite this threshold, it can also be seen that each additional week of gestation further decreased the risk of long-term infectious morbidity, highlighting the need to consider the timing of medically indicated preterm delivery and even early-term delivery with regard to the long-term health of the offspring. Efforts should be made in clinical practice to prevent preterm delivery where possible and to optimize the timing of medically indicated induced deliveries as close to full term as medically possible without surpassing an unacceptable increase in the risk to both mother and fetus. Offspring born prematurely, specifically those who were born prior to 32 weeks of completed gestation, may benefit from increased surveillance during childhood for signs of infectious disease. Fulfilment of recommended vaccination programs, adherence to preventative strategies including general hygiene , and a high risk of suspicion allowing for early treatment of infectious disease may all contribute to reduce the number of children hospitalized due to infectious disease.


Mallomonas ampla. Fusion of identifiable components of the living cell post cyst formation is unknown in modern investigations. The identification of the cyst structure for M. ampla could be valuable in determining cyst morphotypes for other species in the lineage.Chrysophyte algae produce a siliceous stage in their life cycle, through either asexual or sexual reproduction, known as a cyst. Cysts form in response to shifts in environmental conditions, population density, or predation pressure, and upon germination provide a seed source for future populations. Cysts are morphologically distinct for each species, and since their remains become part of the sediment or fossil record cysts are valuable tools in ecological and paleolimnological investigations. However, their value as biological indicators is limited because the vast majority of cyst morphotypes have not been linked to specific vegetative species. In the current work, an exquisitely preserved and morphologically complex cyst type is described from a 48 million year old early Eocene fossil site. This finding is remarkable since many of the cysts were still associated with components of the living vegetative cells that produced them, enabling the morphotype to be immediately linked to the synurophyte,  Species are mostly microscopic, planktonic or attached, autotrophic, heterotrophic or mixotrophic, naked or with a cell covering, motile or non-motile, and the class embraces numerous vegetative forms5. Synurophytes are a monophyletic clade of chrysophytes that construct a highly organized covering around the cell composed of distinctive siliceous scales3.The Chrysophyceae, commonly referred to as golden-brown algae, is a diverse, cosmopolitan, and ecologically significant group of heterokont algae that is especially important in freshwater ecosystems7, synurophytes are capable of forming a siliceous stage known as a stomatocyst, statospore, or more commonly a cyst, that serves as a resting stage in the life cycle of the species8. Cysts are presumably produced as a result of either asexual or sexual reproduction9, and their formation is often triggered by sudden changes in environmental conditions, predation pressure10, or in the case of sexually-produced cysts, population density4. Cysts form a seed bank and when conditions once again become favorable for growth, they germinate to initiate a new population.As is true with all members of the Chrysophyceae3. The SDV takes the shape of the cyst, enclosing a large percentage of the cell cytoplasm, including the nucleus and other vital organelles. The cyst wall forms within the SDV in what is thought to be a two-step process12. The first step involves the deposition of an inner wall, which is unornamented, morphologically similar for many species, and often different in appearance from the mature cyst11. Additional outer wall layers comprise the second step of cyst formation. In some cases, the final layer simply results in a smooth outer surface. In other cases, additional wall ornamentation is added and, if present, a collar constructed. The collar is a specialized structure that surrounds the cyst pore, the latter of which becomes plugged with an organic material during the final stages of cyst maturation.Cysts are hollow structures, often more or less spherical in shape, with a single germination pore, that are formed endogenously within a silica deposition vesicle (SDV)et al.8 and Wilkinson et al.13. Briefly, cysts have anterior and posterior hemispheres, divided by an equator, with the pore usually being situated atop of the anterior hemisphere. Although the majority of cyst types are spherical, there is a wide diversity of shapes, including oval, ovate, oblong, as well as flattened pancake forms. Pore openings are almost always circular, but the inner sides can be straight, conical or concave. Pores may be simple or surrounded by a thick and continuous rim of silica called the collar. Collars may be simple or complex, the latter consisting of two or more separate collars surrounding the pore in a concentric fashion. The outer wall of the cyst can be smooth, or consist of a multitude of different types of elements, including papillae, nodules, spines, ridges, reticulation designs and depressions. The ornamentation of the mature outer wall and the pore-collar complex is of taxonomic importance13.Details describing cyst morphology are given in Duff 5, the remains of their cysts can serve as valuable paleoindicators9. Cysts have been used to reconstruct a range of environmental parameters, including nutrient conditions14, pH17, specific conductivity19 and climate21. Since cysts are also found in the fossil record as far back as the Late Triassic22, they also represent potential proxies for reconstructing the geologic past. Hundreds of cyst morphotypes are known, but the vast majority have not been linked to specific clades or actual species6, inhibiting their full use as bioindicators.Since the morphology of a mature cyst is specific for a given species, the remains of a cyst type can indicate the presence of that species in a given waterbody. Since many chrysophyte species are found under specific environmental conditionsMallomonas ampla Siver & Lott was described from an early Eocene Arctic deposit, the Giraffe Pipe locality23. Although this taxon is extinct, a closely related modern species, Mallomonas neoampla Gusev & Siver, was recently described from tropical Vietnam24, and both of these taxa are closely related to another modern species, M. multisetigera Dürrschmidt25. The cyst is not known for either of the living species. Recently, numerous cysts still bearing scales belonging to M. ampla were uncovered from the Giraffe Pipe fossil locality. The purpose of this study is to document and describe the cyst produced by M. ampla, and to discuss the remarkable and unique conditions under which these fossils were formed.The fossil synurophyte species 28 and briefly summarized here. The Giraffe Pipe locality is a kimberlite diatreme that was emplaced into the Slave Craton in the Northwest Territories of Canada  approximately 48 million years ago during the Eocene. The diatreme crater filled with water, becoming a maar lake, and subsequently infilled over thousands of years with a sequence of lacustrine and eventually paludal sediments. The sediment strata were later capped by Neogene glacial deposits, entombing them within the crater. A 163 m long drilled core, collared at a 47° angle, was uncovered from the Giraffe maar in 1999 by BHP Billiton Inc. A 113.1 m portion of the core contains well preserved stratified organic sediment, including 68.3 m of lacustrine mudstones, overlain with 44.8 m of peaty and terrestrial remains. An air-fall tephra bed located near the end of the aquatic phase indicates that the lake sediments were all deposited during the Eocene28.Details of the Giraffe Pipe fossil locality are given in previous works e.g.28. The entire lacustrine phase of the core contains an extensive array of microfossils which reflect a lake teaming with organisms, including chrysophytes, synurophytes, diatoms, euglyphids, heliozoans and sponges33. Shifts in the complement of organisms indicate changing lake conditions, including between alkaline and acidic periods, and shallow versus deep phases32. The presence of synurophytes, diatoms and sponge lineages found today largely in tropical regions reflect the warm climate of the region26. In addition, the remains of palm trees indicate ice-free conditions and winter temperatures above freezing34. The current hypothesis is that post phreatomagmatic kimberlite emplacement, a waterbody formed within the crater, varying in depth and physical attributes over time, slowly infilling, and eventually transitioning to a terrestrial environment.This region of the Arctic was warm and wet compared to today, with reconstructed mean annual temperature and mean annual precipitation values 17 °C higher and 4 times greater, respectively, than present, and the region supported a warm mixed forest29. The first number represents the core box in which the sample is stored. Core boxes are numbered sequentially starting with box 1 (closest to the land surface), and increasing with depth in the core. The larger the number, the deeper the section is within the core. Box 11 represents the top and end of the lacustrine phase within the sequence. Each box contains three 1.5 m core lengths, stored in channels 1, 2 and 3. The second number represents the channel. The third number is the measurement in cm down from the top of a core length within the channel. For example, sample 19-1-100 represents a sample taken from 100 cm down along the core length positioned in channel 1 from box 19. This study includes samples from 17-1-15, 17-2-40, 17-2-94, 17-2-138, 19-1-100, 19-2-98 and 19-2-100.Samples for this study were obtained from seven sections of a drilled core taken from the Giraffe Pipe locality. Samples from this core are identified using a three-part number2O2 under low heat for a minimum of an hour, and longer if rock fragments remained mostly intact29. Samples were rinsed with distilled water a minimum of five times with centrifugation, and the final slurries stored in glass vials at 4 °C. For analysis with scanning electron microscopy (SEM), an aliquot of each slurry was air dried onto a piece of heavy duty aluminum foil, trimmed, and attached to an aluminum stub with apiezon wax. Samples were coated with a mixture of gold and palladium for 2 min with a Polaron Model E sputter coater and observed with a Leo (Zeiss) 982 FESEM, or a FEI Nova NanoSEM 450, field emission scanning electron microscope29.From 50-100 mg of mudstone was retrieved from each section and oxidized using 30% H35. Measurements of the diameters of cysts, pores, collars and spines were made directly from SEM micrographs. Measurements of the pore and collar were made on specimens where these structures were facing straight up. Specimens were not tilted. Spine lengths were estimated from ones along the midsection positioned parallel with the stub surface.Details of the cyst are as described by the guidelines prepared by the International Statospore Working GroupMallomonas ampla Siver & Lott.Large concentrations of an unidentified cyst were uncovered in seven samples from the Giraffe Pipe core, with especially abundant numbers of specimens found in samples from stratum 19-1-100. Many of the cysts from 19-1-100 were unique in that they still contained attached scales, allowing identification as M. ampla are oval with a perforated base plate, posterior rim, V-rib and a shallow dome structure , 29 were between 19–21 µm Fig. . The diaM. ampla were abundant in the rock samples, only broken pieces were found on cysts . As a result of the current project, material from the GP 19-1-100 stratum and two permanent glass slide mounts were also deposited at the Canadian Museum of nature (CANA 128453). Each slide has numerous specimens of the cyst for M. ampla. They also contain specimens of the rare diatom genus Ambistria which is detailed in a separate publication31.The type specimen for M. ampla, this section of the core is also dominated with microfossils of the planktonic diatom, Aulacoseira giraffensis32. Remains of the synurophytes Mallomonas insignis Penard, M. asmundiae (Wujek & Van der Veer) Nicholls, and Synura recurvata Siver & Wolfe are also common, along with a wide diversity of chrysophyte cyst morphotypes. Other diatoms included Ambistria spp., Fragilariforma virescens  Williams & Round, and Nupela mutabilis Siver, Wolfe & Edlund. Scales of the heterotrophic chrysophyte genera Paraphysomonas and Clathromonas are also abundant, and the stratum contained scales of several heliozoan species, siliceous plates of the testate amoebae genera Scutiglypha and Euglypha, and a variety of sponge spicules.The 19-1-100 stratum from the Giraffe Pipe core contains an extensive diversity of siliceous microfossils. Along with numerous scales and cysts of 6. This fact severely limits the usefulness of cysts in ecological, biogeographic and paleolimnological investigations. The limited number of cysts that have been successfully linked to specific vegetative states were done so through direct observation of living organisms in field collections, or induced from cultures in laboratory experiments6. Cysts uncovered from sediments are no longer associated with the living cells that produced them, making it impossible to assign them directly to specific organisms8. No cyst morphotypes described from sediment samples have ever been found still associated with siliceous scales from the cell covering. This fact is what makes the findings reported here for the Giraffe locality truly remarkable.Of the vast number of cyst morphotypes described from studies around the world, only 5% or fewer are linked to specific speciesMallomonas ampla also had isolated scales of this species, but only specimens from the 19-1-100 stratum still had attached scales allowing for a positive identification. This is extraordinary since specimens were initially treated with oxidizing acids in order to remove them from the rock matrix. Even after treatment with acids, scales remained fused to the cysts in their original orientations. This indicates that not only did the cells forming cysts remain intact upon death, but that the scales became fused together and with the cyst wall during the fossilization process. This phenomenon is unique for fossil remains from the Giraffe locality, but it is not unique for M. ampla. Cysts of other species with intact scales have also been uncovered from the Giraffe locality, indicating that the conditions in the sediments and overlying water column of this maar lake that resulted in the cells remaining intact were unusual, possibly highly anoxic and undisburbed. Scales still attached to the cysts of another species, Mallomonas aperturae Siver, were so well preserved that the original arrangement of the scales on the cell covering could be critically examined36. The same situation is now documented for M. ampla from a different section of the extensive core, and in this case with a significantly greater number of specimens.All samples from the Giraffe locality containing cysts of M. ampla, despite the presence of many other cyst morphotypes in the sample rock. A second explanation is that after formation, what would be the outer cyst “germinated” internally forming a cell with scales, which, in turn, produced the inner cyst. This phenomenon has never been observed and seems unlikely, yet could explain those specimens where more than half of the circumference of the inner cyst is surrounded by the outer cyst. It is also interesting as remarkable that it is to have uncovered cysts with scales still attached in their original overlapping patterns, it is important to note that these “triple” fossil specimens depicting an inner cyst-attached scale coat-outer cyst also remain attached and fused to each other. Does this indicate that the three units were indeed formed together, supporting the internal germination idea? None of these specimens were found attached to a third outermost cyst which would seem likely under the compression hypothesis.The specimens of cysts partially surrounded by a second cyst are curious and perplexing. The most logical explanation is that during the fossilization process the inner cysts became compressed against the outer cyst, breaking through the outer cyst, and ending up within the rock matrix appearing to be within the outer cyst. However, in some cases the outer cyst appears to encircle more than half of the diameter of the inner cyst, which could not be explained by this compression hypothesis. In addition, the inner cysts were never found surrounded by ones other than those representing 37 reported a range in diameter from 2–30 µm, with the majority of cyst types between 5–10 µm. This matches nicely cyst diameter estimates of ca. 3–35 μm given by previous researchers38. In addition, the range in diameter for cyst specimens formed by the same species was remarkably small with 85% of the species forming cysts with a range in diameter of approximately 3 µm or less37. This was especially true for taxa with scale forming cell coverings. Some of the variation in cyst diameter is likely due to differences in the sizes of vegetative cells at the time of formation6. Compared to the results reported by Siver37, the cyst formed by Mallomonas ampla is on the larger end of the spectrum, and the range in diameter is consistent with the results reported in that study. In addition, the diameters of the collar and of the pore were both remarkably consistent, yielding a mean ratio of 3.5 ± 0.5. The ratio of cyst diameter to pore diameter was large with a mean of 16 ± 0.8. The small size of the pore relative to the cyst is remarkable since when the cyst germinates, the newly formed (and naked) cell must squeeze out of the pore.In a recent literature survey that included 1409 records of modern cysts (chrysophytes and synurophytes), Siver39. Most cysts are spherical and when they form the anterior hemisphere usually faces the anterior end of the cell1. This means that if the cell has an oval shape, that the distance between the developing cyst and scale covering would be greater extending from the anterior hemisphere, and less between the sides and posterior portions of the cyst. Thus, there would be more space for development of longer spines and more complex and projecting collars on the anterior hemisphere, yielding longer spines with no or less splayed apices.Compared to the structure of the collar and pore, there was a wide range of variation in the character of the spines, both within and between specimens. Most of the variation was in the length of the spine and the degree to which the apex was splayed into the finger projections. Shorter spines with more complex apices are the most common morphology and these spines typically cover the majority of the cyst wall. Longer and more slender spines with less splayed apices were restricted to the anterior hemisphere and usually near the collar complex. A hypothesis explaining the variability in spine morphology is that spine length and the degree of lateral splaying of the spine apex for chrysophytes with a siliceous scale covering is dependent on the distance from the developing cyst to the confining cell covering. Formation of spines begins from the cyst wall and progresses outwards. As spines lengthen in an organism with a confining outer scale covering, they come up against the scale coat causing the silica deposition process to bend laterally, resulting in the splayed structure of the apex. Specimens with scales still intact showing splayed apices in contact with the scales clearly supports this hypothesis. Under this hypothesis, the shorter the distance from the cyst wall to the scale covering, the smaller would be the length of the spine. The majority of synurophytes have oval or obovate-shaped cellsMallomonas are known to produce cysts with various types of splayed apices. These include Mallomonas muskokana (Nicholls) Siver & Wolfe, M. acaroides var. acaroides Perty emend. Ivanov, M. pseudocoronata Prescott, and M. crassisquama (Asmund) Fott. Siver40 reported an almost identical spine structure for M. muskokana as reported here for M. ampla. Kristiansen25 noted the spines of M. acaroides var. acaroides to be “irregularly curved with blunt disc-like tips, which are surrounded by a whorl of short, thin twisted projections”. This describes spines referred to herein as those with splayed apices. Kristiansen25 further notes that the cyst formed by M. crassisquama has spines similar to those of M. acaroides var. acaroides. Spines of M. pseudocoronata are described as slightly curved with flat splayed tips41. Interestingly, a similar type of spine spaying is found on the ends of linking spines of some species of the diatom genus Aulacoseira (e.g. A. crassipunctata and A. lirata42). In this case, developing spines become splayed laterally into free spaces as they come in contact with confining silica structures on adjoining valves.Although the structures of cysts are unknown for many species bearing scale coverings, there is support for the confinement hypothesis resulting in splayed spine apices. At least four other species of M. ampla cysts could prove useful in discovering the cyst morphotypes for the two closely related species in this lineage, M. multisetigera and M. neoampla. Collections containing these species, or surface sediments from waterbodies with active populations, could be examined for cysts with similar spine and collar characters as those for M. ampla. Although matches made using this technique would only provide indirect evidence, it would nonetheless be worthwhile evidence.Knowing the morphological structure of M. ampla cysts with attached scales from the cell covering in the Giraffe Pipe core are remarkable and further emphasize the uniqueness of this fossil locality. The morphology of the cyst, especially the complex collar, is unique among known cyst types, and the splayed nature of the spine tips is likely a function of forming within a space confined by the scales of the cell covering.In summary, the numerous specimens of


Toxoplasma gondii infection is maintained in the central nervous system by thick-walled cysts. If host immunity wanes, cysts recrudesce and cause severe and often lethal toxoplasmic encephalitis. Currently, there are no therapies to eliminate cysts, and little biological information is available regarding cyst structure(s). Here, we investigated cyst wall molecules recognized by succinylated wheat germ agglutinin (s-WGA), a lectin that specifically binds to N-acetylglucosamine-decorated structures. N-Acetylglucosamine regulates cell signaling and plays structural roles at the cell surface in many organisms. The cyst wall and cyst matrix were heavily stained by s-WGA in mature cysts and were differentially stained during cyst development. The relative accumulation of molecules that bind to s-WGA in the cyst wall was not dependent on the expression of GRA2. Our findings suggest that glycosylated cyst wall molecules gain access to the cyst wall via GRA2-dependent and GRA2-independent mechanisms and colocalize in the cyst wall.Chronic  Toxoplasma gondii cyst wall glycoprotein CST1 is heavily stained by Dolichos biflorus agglutinin, a lectin that binds to N-acetylgalactosamine. The cyst wall is also heavily stained by the chitin binding lectin succinylated wheat germ agglutinin (s-WGA), which selectively binds to N-acetylglucosamine-decorated structures. Here, we tracked the localization of N-acetylglucosamine-decorated structures that bind to s-WGA in immature and mature in vitro cysts. s-WGA localization was observed at the cyst periphery 6 h after the differentiation of the tachyzoite-stage parasitophorous vacuole. By day 1 and at all later times after differentiation, s-WGA was localized in a continuous staining pattern at the cyst wall. Coinciding with the maturation of the cyst matrix by day 3 of cyst development, s-WGA also localized in a continuous matrix pattern inside the cyst. s-WGA localized in both the outer and inner layer regions of the cyst wall and in a continuous matrix pattern inside mature 7- and 10-day-old cysts. In addition, s-WGA colocalized in the cyst wall with CST1, suggesting that N-acetylglucosamine- and N-acetylgalactosamine-decorated molecules colocalized in the cyst wall. In contrast to CST1, GRA4, and GRA6, the relative accumulation of the molecules that bind s-WGA in the cyst wall was not dependent on the expression of GRA2. Our results suggest that GRA2-dependent and GRA2-independent mechanisms regulate the trafficking and accumulation of glycosylated molecules that colocalize in the cyst wall.The glycosylated mucin domain of the IMPORTANCE Chronic Toxoplasma gondii infection is maintained in the central nervous system by thick-walled cysts. If host immunity wanes, cysts recrudesce and cause severe and often lethal toxoplasmic encephalitis. Currently, there are no therapies to eliminate cysts, and little biological information is available regarding cyst structure(s). Here, we investigated cyst wall molecules recognized by succinylated wheat germ agglutinin (s-WGA), a lectin that specifically binds to N-acetylglucosamine-decorated structures. N-Acetylglucosamine regulates cell signaling and plays structural roles at the cell surface in many organisms. The cyst wall and cyst matrix were heavily stained by s-WGA in mature cysts and were differentially stained during cyst development. The relative accumulation of molecules that bind to s-WGA in the cyst wall was not dependent on the expression of GRA2. Our findings suggest that glycosylated cyst wall molecules gain access to the cyst wall via GRA2-dependent and GRA2-independent mechanisms and colocalize in the cyst wall. Toxoplasma gondii is a protozoan pathogen that chronically infects one-third of the global human population  that is breached when tachyzoites egress to invade new host cells , 11. In ro cysts . In addiro cysts , 15. Genxoplasma –19, suggin vitro cysts, GRA1, GRA4, GRA6, and GRA9 established a continuous matrix pattern, while GRA2 and GRA12 were isolated in distinct puncta in the cyst matrix  and succinylated wheat germ agglutinin (s-WGA)  ablated cyst wall recognition by DBA as well as s-WGA  , 24. DBAructures . DBA preructures . The CST and -T3 . Deletioas s-WGA , suggestas SRS13 . Howeveras SRS13 , which ias SRS13 . Geneticas SRS13 .N-acetylgalactosamine specificity of DBA, the chitin binding lectin s-WGA selectively binds to N-acetylglucosamine-modified structures  test whether the accumulation of the s-WGA-binding N-acetylglucosamine-modified molecules in the cyst wall is regulated by GRA2. Our results show that s-WGA recognized N-acetylglucosamine-modified molecules at the cyst periphery/wall throughout cyst development and in the cyst matrix by day 3 after differentiation. The deletion of GRA2 did not affect the trafficking or cyst wall accumulation of the molecules that bind s-WGA in the cyst wall, and the cyst wall molecule(s) that binds s-WGA was colocalized with GRA4, GRA6, and CST1.Exposure of tachyzoite PVs to low COntiation , 32, andus cysts  that posted mice . Here, wN-acetylglucosamine-modified molecules that bind to s-WGA in 6-h-, 1-day-, 2-day-, 3-day-, 7-day-, and 10-day-old in vitro cysts. Green fluorescent protein-positive (GFP+) bradyzoites were visible within developing cysts  or Δgra2 (B) strain were differentiated for 1 day or 2 days. Cysts were located using differential interference contrast (DIC) microscopy and imaged by confocal microscopy. The presence of bradyzoites inside cysts was verified by locating parasite nuclei with 4′,6-diamidino-2-phenylindole (DAPI) staining and verifying that each parasite nucleus was surrounded by the expression of cytosolic GFP (GFP+ bradyzoites). DAPI stains parasite and host nuclei. Cysts were stained with streptavidin and goat anti-mouse IgG antibody (A) or biotinylated s-WGA and anti-GRA2 antibody (B) . Bars = 5 μm. Download FIG S1, TIF file, 1.7 MB.Streptavidin labels biotinylated proteins inside the developing cyst, and PruΔCopyright © 2020 Guevara et al.2020Guevara et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the gra2 cysts  of the s-WGA peak, confirming a reduced localization of GRA4  of GRA4, GRA6, and s-WGA, we measured the cyst fluorescence intensity profiles of GRA4 and GRA6 relative to s-WGA using the Fiji macro . The flu of GRA4  to C and of GRA4  to F in yst wall . However10.1128/mSphere.00031-20.2FIG S2ku80 and Δgra2 strains. Dotted black lines define the cyst wall region. The dotted red line indicates the middle of the cyst wall, which corresponds to the peak s-WGA fluorescence intensity. Download FIG S2, TIF file, 1.9 MB.s-WGA accumulation at the cyst periphery is GRA2 independent in immature cysts. Fluorescence intensity profiles of representative cysts, shown in Copyright © 2020 Guevara et al.2020Guevara et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the + bradyzoites were visible in s-WGA-stained cysts. In parental PruΔku80 cysts, GRA4 (gra2 cysts. The cyst periphery/cyst interior fluorescence intensity ratios of s-WGA (0.48)  , GRA4 (0A (0.48) , and GRAA (0.48)  were simizations . The fluizations  and GRA6izations  in 10-daizations  was incr of GRA4 A and B w of GRA4 C and D p10.1128/mSphere.00031-20.3FIG S3ku80 and Δgra2 strains. Dotted black lines define the cyst wall region. The dotted red line indicates the middle of the cyst wall, which corresponds to the peak s-WGA fluorescence intensity. Download FIG S3, TIF file, 1.3 MB.s-WGA accumulation at the cyst wall and distribution in the cyst matrix are GRA2 independent in mature cysts. Fluorescence intensity profiles of representative cysts, shown in Copyright © 2020 Guevara et al.2020Guevara et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the + bradyzoites   were visible inside GFPdyzoites . As expedyzoites . To confdyzoites , 7-day-odyzoites , and 10-dyzoites  cysts. Wof <1.0) .Toxoplasma cyst formation within distal neuronal processes  by forming cyst structures , 39. Neurocesses , 41. Brarocesses –44, whicocesses –, 46 and ocesses –. The cysocesses –. Glycosyocesses –, which iocesses –.Toxoplasma cyst wall is recognized by a variety of lectins, including concanavalin A (α-d-mannose or α-d-glucose), wheat germ agglutinin (WGA) , succinylated WGA (s-WGA) (N-acetylglucosamine), soybean agglutinin , and Dolichos biflorus, Vicia villosa, Artocarpus integrifolia, and Helix pomatia agglutinins  (Dolichos biflorus agglutinin (DBA), which intensely stains the cyst wall of in vitro  , 24, 26.in vitro  and in v (CST1) and the cyst wall components GRA1, GRA2, GRA4, GRA5, GRA6, GRA7, GRA9, and GRA12  that binds s-WGA traffics to and accumulates at the cyst wall through GRA2-independent mechanisms.The genetic deletion of GRA2 disrupted the organization of the cyst matrix, altered the staining patterns of GRA4 and GRA6 in the cyst matrix, and delayed the accumulation of GRA4, GRA6, and DBA-stained CST1 in the cyst wall . In the 2 sensing, nutrient storage, and in vitro growth (O-linked N-acetylglucosamine transferase (OGT) that mediates O-linked β-N-acetylglucosaminylation. O-linked β-N-acetylglucosaminylation is a posttranslational modification of cytosolic, nuclear, and mitochondrial proteins by a single residue of N-acetylglucosamine which is transferred from UDP-N-acetylglucosamine and is hypothesized to be involved in the localization of nuclear proteins (O-linked β-N-acetylglucosaminylation is reversible by O-linked β-N-acetylglucosaminyl hydrolase (OGA), which removes the N-acetylglucosamine residue  recognized by s-WGA and chitinase is essential for the stability of the cyst wall and cysts. Thus, the molecules recognized by s-WGA and chitinase and the biological functions of these cyst wall-localized molecules are likely to be potential targets for cyst disruption and cyst elimination.The chitin-binding s-WGA lectin specifically recognizes A lectin . PreviouA lectin  and (ii)A lectin , 30. TheN-acetylglucosamine that is present in chitin and chitin-like oligosaccharide structures. The Toxoplasma cyst wall as well as the oocyst wall most likely do not contain chitin in view of the fact that Toxoplasma as well as its mammalian hosts do not express a chitin synthase  monolayers (ATCC SCRS-1041.1) cultured in Eagle’s modified essential medium (EMEM) (Lonza) containing 1% fetal bovine serum (FBS) (Life Technologies), 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 36°C in 95% air and 5% CO2. HFF cells were maintained in EMEM containing 10% FBS (HyClone), 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in 95% air and 5% CO2. The parental Pru strain PruΔku80 was previously made transgenic for green fluorescent protein (GFP) under the control of the LDH2 bradyzoite-stage-specific promoter (gra2 strain was previously developed using the PruΔku80 knockout strain of the type II Pru strain (Type II Prugniaud (Pru)-background promoter . The Δgru strain , 76.in vitro into bradyzoites within cysts essentially as previously and elegantly described by Tobin and colleagues (l-glutamine (HyClone), 20 mM HEPES-free acid (IBI Scientific), 1% XL-glutamine , 1% FBS, and 1% penicillin-streptomycin. The pH of differentiation medium was adjusted to 8.1 with sodium hydroxide and the medium was filter sterilized. HFF cells were cultured on circular micro cover glass until confluent (Electron Microscopy Sciences), and confluent monolayers were infected with type II Pru parasites at a multiplicity of infection (MOI) of ∼0.5. Infected HFF cells were washed once 3 h after infection in Dulbecco’s phosphate-buffered saline (DPBS) supplemented with Ca2+ and Mg2+ and incubated in differentiation medium for 6 h, 1 day, 2 days, 3 days, 7 days, or 10 days at 37°C in ambient air. Medium was changed on day 3 and day 7.Tachyzoites were differentiated lleagues . Differe2+ and Mg2+ and incubated for 1 h at room temperature (RT) with a 1:1,000 dilution of secondary goat anti-rabbit IgG(H+L) (Thermo Fisher) and goat anti-mouse IgG(H+L) antibodies conjugated to Alexa Fluor 647 . All samples were incubated with a 1:200 dilution of DyLight 594 streptavidin (Vector Laboratories) for 1 h at RT. Samples were mounted in SlowFade gold antifade with 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies) and then imaged with a Nikon A1R SI confocal microscope  using an Apo total internal-reflection fluorescence (TIRF) 100× oil differential interference contrast (DIC) N20 objective. Cysts were randomly selected for analysis by locating cysts using DIC microscopy. Bradyzoite differentiation in cysts was confirmed by GFP+ bradyzoites. The focal plane (from a z-stack) selected for quantification was from the middle of the cyst, where the cyst size is maximal. Raw .nd2 files of cyst images were imported into Fiji for processing. Images were minimally processed for brightness  in Fiji ; a 1:25 dilution of primary mouse monoclonal anti-CST1 antibody ; a 1:1,0 in Fiji . The colRaw .nd2 image files were imported into Fiji to measure fluorescence intensity parallel to the cyst wall, as previously described . Images The cyst wall region was identified and defined as previously described . The cysku80 and Δgra2 strains. To measure background fluorescence, a circle was drawn using the freehand selection tool, and fluorescence was measured outside the cyst on three different sides. All data were imported into Excel to be further analyzed, as previously described  and P values were performed using GraphPad Prism software version 5.0c.Unpaired


Prevalence of neonatal microcephaly in populations without Zika-epidemics is sparse. The study aimed to report baseline prevalence of congenital microcephaly and its relationship with prenatal factors in an area at risk of Zika outbreak.This study included singletons born after 24 gestational weeks in 2017–2018 at four hospitals in Guangzhou, China. Microcephaly was defined as a head circumference at birth >3SD below the mean for sex and gestational age. Prevalence of microcephaly was estimated by binomial exact method. Multivariable logistic regression was used to examine the associations of microcephaly with prenatal factors. The population attributable fraction (PAF) for associated risk factors was calculated.Of 46,610 live births included, 154  microcephalies were identified. Maternal hepatitis B virus carriers  and primipara  had higher risk of having a microcephalic baby. Higher prevalence of microcephaly was observed in women who had premature labor  and had a baby with fetal growth restriction . Four identified factors  contributed to 66.4% of the risk of microcephaly.The prevalence of microcephaly in Guangzhou was higher than expected. This study identified four prenatal risk factors that, together, contributed to two-thirds of the increased risk of microcephaly. This is the first reported association between maternal HBV carrier status and microcephaly. Congenital microcephaly is characterized as a smaller head compared to others of the same sex and gestational age. Infants with microcephaly have a significantly increased risk of developmental delay, intellectual disability, long-term disability, and even mortality –5. The WRelatively well-known causes of microcephaly include maternal infections, such as Zika virus and cytomegalovirus, genetic factors, and teratogens. These understandings are mainly based on evidence from recent Zika-epidemic areas –10. The Guangzhou, located in South China, is an area with frequent epidemics of dengue virus, which is a flavivirus of the same genus as Zika. It is possible that maternal immunity to dengue virus promotes Zika infection and Zika virus–induced microcephaly in fetuses . Its natAs head circumference (HC) measurement is not routinely collected and recorded in clinical practice in China, a surveillance study for HC at birth was introduced in February 2017 in four hospitals in Guangzhou, China. The four hospitals were selected using a cluster sampling from 13 municipal and district-level Maternal and Child Care Service Centers in Guangzhou, including two municipal tertiary healthcare centers , one district-level tertiary centers  and one district-level secondary centers , located in the central, north and west areas of Guangzhou, China, respectively . GWCMC pThe HC of the newborns was measured at each study center by a uniform instrument, namely the Seca HC measuring band 212, made of non-stretching Teflon. The midwives were trained to follow a standard protocol before the study was started. The definition of HC is the widest possible circumference of the head around the back of the head with the measuring band held above eyebrow and ears. The HC measurements of the newborns were completed immediately after birth. All measurements were read to the nearest millimeter and then recorded. Each newborn was measured twice and the average value was recorded. The maximum difference accepted between the two measurements was 4 mm.The Z-scores of HC at birth was calculated according to INTERGROWTH-21st newborn size standards by gestational age and sex . MicroceData on maternal age, parity, gestational diabetes mellitus (GDM), hypertensive disorders of pregnancy (HDP), birth weight, gestational age at delivery, and newborn sex were obtained from the Guangzhou Perinatal Health Care and Delivery Surveillance System, which covers more than 99% of deliveries in Guangzhou . GDM diaBirth weight was measured by midwives immediately after delivery. Birth weight Z-scores were calculated according to INTERGROWTH-21st newborn size standards by gestational age and sex . GestatiRobust regression with the iteratively reweighted least square procedure was used to identify implausible HC values either caused by misclassification of gestational age or by invalid HC measurements. HC values with residual out of range of − 3.89 SD and + 3.89 SD were considered as outliers , 29.The prevalence of microcephaly and its subtypes was estimated and the 95% CI was calculated using the binomial exact method. Data were summarized as means (standard deviations) for continuous variables and frequency (percentage) for categorical variables.Multivariable logistic regression models were applied to estimate the unadjusted and adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for the association between prenatal characteristics and the risk of microcephaly or microcephaly subtypes, with non-microcephaly (HC Z-score at birth ≥ − 3.0) as the reference. In models for the associations with maternal age, GDM, HDP, and parity, these characteristics entered the model simultaneously and were mutually adjusted for each other. The association with maternal carrier of HBV was assessed among births in GWCMC, adjusted for maternal age at conception, parity, HDP, GDM, year of birth, and place of birth. Based on the year when routine hepatitis B immunization for newborns was implemented, the year of birth was grouped into a binary variable (“before 1992” and “in 1992 or after”) , 31. MatP < 0.05 was considered significant.All analyses were done with SAS version 9.3 . n = 3) and missing (n = 328) or implausible HC data (n = 145), stillbirths (n = 222), and those with brain and central nervous system (CNS) abnormalities (n = 25) and/or chromosome abnormalities (n = 36), 46,610 newborns were included in the final analysis.A total of 47,369 singletons were eligible, with 17,798 births from Zhujiang Newtown Campus of GWCMC, 12,270 births from Maternal-Infant Campus of GWCMC, 13,217 from Guangzhou Huadu Maternal and Child Care Service Center, and 4084 from Guangzhou Liwan Maternal and Child Care Service Center. After excluding newborns with unknown sex . Of these, 14 were disproportionate, with a prevalence of 0.3 , and the remaining 140 were proportionate, with a prevalence of 3 .n = 24,927) of the newborns. The mean HC Z score, birth weight Z score and difference between the HC Z-score and birth weight Z-score were − 0.3 (SD 1.0), 0.0 (SD 0.9), and 0.3 (0.8), respectively.Table Associations between prenatal factors and microcephaly are shown in Fig. Associations between prenatal factors and microcephaly subtypes are shown in Table The PAF associated with identified risk factors for microcephaly and its subtypes are presented in Table This multi-center study found that the prevalence of microcephaly was 0.33%, which is higher than the expected risk of 0.13% in a population with normally distributed HC and using a definition of HC Z score smaller than − 3. Microcephaly was associated with maternal HBV carrier status, primiparity, FGR, and preterm birth, which together contributed to two-thirds of the risk of microcephaly. The risk factors associated with microcephaly varied between proportionate and disproportionate microcephaly.Although the estimated prevalence of microcephaly in the present study is lower than pre-Zika virus epidemic estimate in two cities in Brazil (5 ~ 7 per 1000 births), where the diagnosis of microcephaly was also based on the INTERGROWTH-21st standard , it was Considering that early neurodevelopmental interventions recommended by the World Health Organization could improve microcephalic children’s quality of life , identifIn the present study, we found that infants with proportionate microcephaly included more infants with SGA, 18–22% of whom were reported constitutionally small but healthy in the previous studies . As the The definition of disproportionate microcephaly in the present study represents a head growth being significantly lagged behind body growth. Previous studies defined disproportionate microcephaly as HC Z-score < − 3 and weight or height Z-score ≥ − 3, which will include a large portion of infants whose head growth being little lagged behind body growth , 34. It This study is the first to report the association between maternal HBV carrier status and a higher risk of microcephaly. Previous studies regarding maternal virus infection and microcephaly focused mainly on the Zika virus and were conducted mainly in Zika-epidemic areas. Some previous studies have found that Hepatitis B surface antigen (HBsAg) positivity during pregnancy increased the risk of congenital malformation . HoweverMajor strengths of this study include the large sample size, which allows for the identification of novel risk factors of conditions with low prevalence, and the high-quality of HC data that was ensured by standardized measurements. Importantly, using high quality data we provide approximate population-based baseline prevalence estimations for future surveillance, which will become very important in the case of outbreaks of Zika or other infectious diseases in this region of China. This study has some limitations. First, we only collected the data in one developed city in China, which limits its generalization to other regions. However, Guangzhou is a megalopolis with a large migrating population moving in from other areas of China. Second, the association between HBV and microcephaly should be interpreted with caution because potential unrecognized confounders within populations with higher hepatitis B rates may drive this association. However, in a subgroup of pregnant women who participated in both the current study and the Born in Guangzhou Cohort Study , the ORsIn summary, based on this multi-center study in an area not yet experiencing, but at risk of, Zika outbreaks, the estimated prevalence of microcephaly was higher than expected. Four factors, namely maternal HBV carrier status, primiparity, SGA, and preterm birth, are associated with 66.4% of the increased risk of microcephaly, and the risk factors varied between different subtypes of microcephaly. This study contributed to the better understanding of microcephaly subtypes characteristics. In addition, as infection is a main cause of microcephaly, the association between maternal HBV carrier status and microcephaly, which was first reported in this study, warrants further investigations.


With the ongoing COVID-19 pandemic, large numbers of people will receive one of the several medications proposed to treat COVID-19, including patients of reproductive age. Given that some medications have shown adverse effects on sperm quality, there might be a transgenerational concern. We aim at examining the association between drugs proposed to treat COVID-19 when taken by the father around conception and any pre-term birth or major birth defects in offspring in a nation-wide cohort study using Danish registry data. Offspring whose father filled at least one prescription of the following medications in the three months preceding conception were considered exposed: chloroquine, hydroxychloroquine, losartan, azithromycin, naproxen, dexamethasone and prednisone.For azithromycin and naproxen, large numbers of offspring were exposed (> 1800 offspring), and we found no association with adverse birth outcomes. For chloroquine, losartan and dexamethasone, exposure was intermediate (~900 offspring), and there was no statistically significant association with birth defects. For hydroxychloroquine and prednisone, exposure was limited (<300 offspring). Our evidence suggests that azithromycin and naproxen are safe with respect to pre-term birth and birth defects. For the other drugs investigated larger exposures are needed for conclusive statements. These drugs meant to treat non COVID-19 specific conditions are now given massively to patients affected by COVID-19.To find an effective treatment for COVID-19 several existing drugs have been assessed and approved for clinical trials through accelerated procedures worldwide4 and therefore are exposed to more proposed treatments. Importantly, while most COVID-19 patients who require treatment are beyond reproductive age, the epidemiological profile of this pandemic suggests that patients also include men and women in their respective reproductive ages. The age distribution of patients has varied across countries and over time. As for end of summer and beginning of autumn 2020, an increase of young adults affected is observed.Evidence suggests that males are more severely affected by the ongoing pandemic irrespective of age7, raising a potential transgenerational concern. In the present cross-sectional study we analyze the association between the use of COVID-19 medications used by fathers three months before conception and pre-term birth and congenital malformations, using the Danish National Birth, Patient and Prescription Registries10 from 1997 to 2017.We aimed to investigate the safety of COVID-19 drugs taken by fathers prior to conception to assess for adverse birth outcomes. A few recent studies have shown adverse effects of drugs, including chloroquine, on gametes8 records information on all children born in Denmark including the father (if known), the mother, and basic vital information, such as Apgar score, birth weight, birth length, and gestational age. We restricted the analysis to live born singletons for the time frame 1997–2016 .The Danish Birth Registry (MFR)9 provides administrative and clinical data of all patients discharged from Danish hospitals and outpatient clinics. One primary and optional secondary diagnoses are recorded according to the International Classification of Diseases for each visit. Using the CPR number of the Danish Birth Registry, data of the Danish National Patient Registry were linked at the patient level for all children. Allowing one year after birth for follow-up, we identified all newborns who were found to have at least one major birth defect as per EUROCAT Guide 1.411. Hence, the last cohort of live born singletons of 2016 is followed up to 2017 in the National Patient Registry.The Danish National Patient Registry (DNPR)10 through the CPR number of the father. This registry includes all medications prescribed by a medical doctor. We examined the medications that are being trialed as treatments for COVID-19. The list of medications was retrieved from the Danish Medicines Agency12. We included those drugs with adequate usage to have a reasonable power: chloroquine, hydroxychloroquine, losartan, azithromycin, naproxen, dexamethasone and prednisone. We examined prescriptions filled in the three months prior to conception based on a spermatogenesis cycle. Using the CPR number of the father, also the birth day, income, and the highest educational attainment of the father was retrieved from the population and education registries held at Statistics Denmark.The Danish Birth Registry can be linked to the Danish National Prescription Registry (LMDB)12).Our two primary outcomes were odds of pre-term birth (birth at less than 37 weeks of gestation) and of having at least one major birth defect in offspring  we created five imputed datasets using multiple imputation by chained equations with the R package mice (version 3.6.1) using predictive mean matching for the numeric variables and polytomous regression for the categorical variables. We performed logistic regression on each of the medications separately, adjusting for birth year, the age of both parents, smoking status of the mother, and education and income of the father. In addition, the regressions of major birth defects were adjusted for gestation age. We also performed logistic regression with all medications as independent variables simultaneously. The statistical significance threshold for all tests is set at two-side 0.05 level.All analyses were carried out at the secure server of Statistics Denmark in R (version 3.6.1)The average age of fathers was 32.3 years, while mothers were on average 29.7 years old. Of the mothers, 84.1% did not smoke at conception, 2.6% quit during pregnancy and 13.3% smoked during pregnancy. Of the fathers, 79.9% had at least upper secondary education, of which 11.8% had at least a masters degree.Medications taken by a large number of fathers (>100 fathers) were chloroquine, hydroxychloroquine, losartan, azithromycin, naproxen, dexamethasone, and prednisone.The overall birth defect rate was 3.2%, while 7.4% of all births were preterm.None of the investigated drugs taken by men up to three months before conception was statistically associated with pre-term birth or congenital malformations at 5% significant level . IncludiFor hydroxychloroquine and prednisone, exposure was limited (<300 offspring). For azithromycin and naproxen, large numbers of offspring were exposed (> 1800), and we found no association with adverse birth outcomes with 95% confidence interval of odds ratios given in Using a nationwide cross-sectional study with data over 20 years in Denmark, we found no statistical evidence of association between the COVID-19 drugs under study, taken by men up to three months before conception, and pre-term birth or major birth defects in offspring. Evidence of drugs safety before paternal conception is limited. Our findings suggest that azithromycin and naproxen can be considered safe with respect to pre-term birth and major birth defects. For chloroquine, losartan and dexamethasone, our study was more constrained in size, while for hydroxychloroquine and prednisone we would be able only to find very large effects. While reassuring, given the limitations of the current report, continued surveillance of parental exposure to experimental COVID-19 treatments remains prudent.The prescription registry does not include prescription diagnosis. It was therefore not possible to separate the effect of the medication from the effect of the disease for which the medication was given. This study will therefore tend to overestimate, but not underestimate any effect. This might (partly) explain odds ratios larger than one for chloroquine and losartan. In addition, medications that can be obtained over the counter (e.g. naproxen) may be misclassified biasing results toward the null.


Tracheal agenesis (TA) is a rare disorder usually diagnosed prenatally when a congenital high airway obstruction syndrome (CHAOS) is identified. We present a case of unexpected TA in a neonate without prenatal diagnosis of airway obstruction, with a difficult management at birth. Moreover, we discuss about differential diagnosis, classification and treatment issues.A 2280 g female neonate was born at 35 week gestational age (GA) with prenatal diagnosis of aortic coarctation, polyhydramnios and diffuse hyperechogenicity of the right lung. At birth, the neonate had no audible cry, no air entry to the lungs, and hypotonia. Tracheal intubation was unsuccessful, and no visualization of the trachea was obtained when tracheostomy was attempted. Post-mortem examination showed tracheal agenesis associated with tracheoesophageal fistula and revealed no cardiologic malformations. Aortic coarctation had been suspected prenatally because of the first portion of the descendent thoracic aorta being compressed by a fibrous band connecting the proximal and distal tracheal branches. CHAOS had not developed due to the tracheoesophageal fistula (TOF).TA is not always diagnosed in the fetus and it may present unexpectedly making the neonate’s management at birth critical. An effective rescue temporary oxygenation may be obtained with mask ventilation or oesophageal intubation in those cases of TA associated with a TOF. We suggest to consider a fetal magnetic resonance imaging (MRI) when the association polyhydramnios/lung hyperechogenicity occurs, even in the absence of CHAOS or other malformations. Once a diagnosis is provided, the mother should be transferred to selected centres where an ex-utero intrapartum procedure (EXIT) can be attempted. Moreover, despite high mortality, different surgical management are described to improve survival. Survival after delivery is dependent on prompt diagnosis and airway management . NowadayWe present a case of unexpected TA in delivery room, without prenatal diagnosis of CHAOS, but evidence of aortic coarctation, lung hyperechogenicity and polyhydramnios at fetal ultrasonography.A 2280 g female neonate was born at 35 week gestational age (GA). Aortic coarctation was suspected at the 22 week GA ultrasound and confirmed by fetal echocardiography 1 week later. The ultrasound performed at 35 of gestation weeks showed diffuse hyperechogenicity of the right lung that was misdiagnosed as cystic adenomatous malformation. At the same time, severe polyhydramnios was diagnosed and treated with amnioreduction. Following this procedure, premature rupture of membranes and contractile activity occurred. Emergency caesarean section was then performed according with the fetal aortic coarctation. The neonate presented with no audible cry, gasps, no air entry to the lungs and hypotonia. Positive pressure mask ventilation was started, that did not result in thoracic excursions. Several attempts of intubation were performed but the tube did not progress beyond the vocal cords even though the glottis could be clearly visualised. Stabilization with mask ventilation at 100% oxygen was obtained. At 10 min of life, heart rate was about 140 bpm and SatO2 was about 85–90%, while gasping, no audible cry and hypotonia persisted. APGAR score was 2–5-5, respectively at 1, 5 and 10 min. Umbilical venous blood sample, obtained at 30 min of life, showed severe respiratory acidosis: pH 6.85, PCO2 147 mmHg, and base-excess was − 11.3 mmol/L. Tracheostomy attempted by the otorynolaryngologist was impossible. Clinical conditions got worse and severe bradycardia occurred. Resuscitation was performed, consisting in thoracic compression and administration of repeated doses of IV adrenalin , that only resulted in transient rise of heart frequency to 80 bpm. Despite sustained resuscitative efforts the baby died at 150 min of life. Post-mortem examination  until ECMO is started. The surgical approach, then, is not necessarily needed immediately after birth [When TOF is present, unexpected polyhydramnios, due to defective fetal swallowing, and lung hyperechogenicity, especially when associated with other congenital malformations, should alert clinicians of potential tracheal anomalies. In those conditions, a fetal MRI might establish a definitive prenatal diagnosis allowing the physicians to inform the parents and consider treatment strategies. Multidisciplinary management may be necessary. That includes the opinions of neonatologist, obstetrician, anaesthetist, otolaryngologist and paediatric surgeon. Therefore, once a diagnosis is provided, the mother should be transferred to selected centres where an EXIT strategy could be attempted. In cases without TOF even initial stabilization is impossible and the condition is invariably fatal . Therefoer birth , but a ler birth , 11, 12.er birth , 13. A rer birth . All of er birth . An innoer birth  for the TA can be diagnosed at birth when orotracheal intubation is impossible to perform in neonates presenting with unexpected severe respiratory distress, hypotonia and no audible cry.We present this case to underline that the absence of a prenatal diagnosis makes the management of TA at birth very difficult. Moreover, the real challenge is the suspicion of TA when a complete airway obstruction not occurs and when prenatal ultrasound findings are not clear or indicative of other major malformation. This is particularly relevant because infants with this presentation are the ones who have chance of survival if a prompt management with airway stabilization with EXIT.In conclusion we suggest to perform a fetal MRI prenatally when the association of polyhydramnios and lung hyperechogenicity occurs, even in the absence of other malformations. If TA is diagnosed, multidisciplinary management should be performed in order to propose the parents the best treatment options.