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Although results from studies of first-trimester influenza vaccination and congenital heart defects (CHDs) have been reassuring, data are limited for specific CHDs.We assessed associations between reported maternal influenza vaccination, 1 month before pregnancy (B1) through end of third pregnancy month (P3), and specific CHDs using data from a multisite, population-based case-control study. Analysis included 2,982 case children diagnosed with a simple CHD (no other cardiac involvement with or without extracardiac defects) and 4,937 control children without a birth defect with estimated delivery dates during 2006-2011. For defects with ≥5 exposed case children, we used logistic regression to estimate propensity score-adjusted odds ratios (aORs) and 95% confidence intervals (CIs), adjusting for estimated delivery year and season; plurality; and maternal age at delivery, race/ethnicity, low folate intake, and smoking and alcohol use during B1P3.Overall, 124 (4.2%) simple CHD case mothers and 197 (4.0%) control mothers reported influenza vaccination from 1 month before through the third pregnancy month. The aOR for any simple CHD was 0.97 (95% CI: 0.76-1.23). Adjusted ORs for specific simple CHDs ranged from 0.62 for hypoplastic left heart syndrome to 2.34 for total anomalous pulmonary venous return (TAPVR). All adjusted CIs included the null except for TAPVR.Although we cannot fully exclude that exposure misclassification may have masked risks for some CHDs, findings add to existing evidence supporting the safety of inactivated influenza vaccination during pregnancy. The TAPVR result may be due to chance, but it may help inform future studies.

Vitamin D deficiency is associated with adverse pregnancy events. However, its role in the etiology of congenital anomalies remains unclear. We examined the association between vitamin D status, measured through prepregnancy diet, UV exposure, season of conception, and congenital anomalies.We used data from the National Birth Defects Prevention Study, a U.S. population-based case-control study (1997-2011). Prepregnancy dietary vitamin D was calculated from food frequency questionnaires and evaluated using tertiles, based on the distribution in controls. We used the National Oceanic and Atmospheric Administration Weather Service to assign UV indices based on location and estimated date of conception, then dichotomized UV exposure (low vs. high). Seasons of conception was categorized as fall/winter spring/summer. We used logistic regression to estimate adjusted odds ratios (aOR) and 95% confidence intervals (CI).Lower prepregnancy dietary vitamin D intake (65.21 IU/d vs.107.55 IU/d) was associated with increased odds of anencephaly (aOR = 1.28, 95% CI 1.01, 1.63), hypospadias (aOR = 1.21, 95% CI 1.04, 1.40), septal defects (aOR = 1.16, 95% CI 1.05, 1.30), diaphragmatic hernia (aOR = 1.42, 95% CI 1.13, 1.79), and gastroschisis (aOR = 1.27, 95% CI 1.07, 1.52). Findings were consistent when we stratified by UV exposure and season of conception.Our findings suggest lower dietary intake of vitamin D may be associated with increased risk of select congenital anomalies. Further investigations are warranted to evaluate the effects of other nutrients and appropriate thresholds and sources of vitamin D using serum.

We examined the relationship between trimester of SARS-CoV-2 infection, illness severity, and risk for preterm birth.We analyzed data for 6336 pregnant persons with SARS-CoV-2 infection in 2020 in the United States. Risk ratios for preterm birth were calculated for illness severity, trimester of infection, and illness severity stratified by trimester of infection adjusted for age, selected underlying medical conditions, and pregnancy complications.Pregnant persons with critical COVID-19 or asymptomatic infection, compared to mild COVID-19, in the second or third trimester were at increased risk of preterm birth. Pregnant persons with moderate-to-severe COVID-19 did not show increased risk of preterm birth in any trimester.Critical COVID-19 in the second or third trimester was associated with increased risk of preterm birth. This finding can be used to guide prevention strategies, including vaccination, and inform clinical practices for pregnant persons.

Obstructive heart defects (OHDs) share common structural lesions in arteries and cardiac valves, accounting for ~25% of all congenital heart defects. OHDs are highly heritable, resulting from interplay among maternal exposures, genetic susceptibilities, and epigenetic phenomena. A genome-wide association study was conducted in National Birth Defects Prevention Study participants (N

To assess associations between influenza vaccination during etiologically-relevant windows and selected major structural non-cardiac birth defects.We analyzed data from the National Birth Defects Prevention Study, a multisite, population-based case-control study, for 8233 case children diagnosed with a birth defect and 4937 control children without a birth defect with delivery dates during 2006-2011. For all analyses except for neural tube defects (NTDs), we classified mothers who reported influenza vaccination 1 month before through the third pregnancy month as exposed; the exposure window for NTDs was 1 month before through the first pregnancy month. For defects with five or more exposed case children, we used logistic regression to estimate propensity score-adjusted odds ratios (aORs) and 95% confidence intervals (CIs), adjusting for estimated delivery year and season; plurality; maternal age, race/ethnicity, smoking and alcohol use, low folate intake; and, for NTDs, folate antagonist medications.There were 334 (4.1%) case and 197 (4.0%) control mothers who reported influenza vaccination from 1 month before through the third pregnancy month. Adjusted ORs ranged from 0.53 for omphalocele to 1.74 for duodenal atresia/stenosis. Most aORs (11 of 19) were ≤1 and all adjusted CIs included the null. The unadjusted CIs for two defects, hypospadias and craniosynostosis, excluded the null. These estimates were attenuated upon covariate adjustment (hypospadias aOR: 1.25 (95% CI 0.89, 1.76); craniosynostosis aOR: 1.23 (95% CI: 0.88, 1.74)).Results for several non-cardiac major birth defects add to the existing evidence supporting the safety of inactivated influenza vaccination during pregnancy. Under-reporting of vaccination may have biased estimates downward.

BACKGROUND: Critical congenital heart defects (CCHDs) are one of the most common types of birth defects and can lead to significant morbidity and mortality along with surgical or catheter interventions within the first year of life. This report updates previously published estimates of CCHD prevalence with the latest population-based surveillance data from 19 birth defect surveillance programs. METHODS: The U.S. population-based surveillance programs submitted data on identified cases of 12 CCHDs and co-occurring cardiovascular and chromosomal birth defects from 2014 to 2018. We estimated prevalence by program type and maternal and infant characteristics. Among nine programs with active case ascertainment that collect more than live births, we estimated the percentage of co-occurring cardiovascular and chromosomal birth defects for the 12 CCHDs. RESULTS: We identified 18,587 cases of CCHD among all participating programs. Overall CCHD prevalence was 19.6 per 10,000 live births among all 19 programs and 20.2 per 10,000 live births among active programs. Among maternal racial/ethnic groups, infants/fetuses born to American Indian/Alaska Native mothers showed the highest overall prevalence for all CCHDs (28.3 per 10,000) along with eight of the 12 individual CCHDs. Among 7,726 infants/fetuses with CCHD from active case ascertainment programs, 15.8% had at least one co-occurring chromosomal birth defect. CONCLUSION: Our study provides prevalence estimates for CCHDs by maternal and infant characteristics along with co-occurrence with cardiovascular and chromosomal birth defects among infants/fetuses with CCHD using one of the largest and most recent cohorts since the implementation of widespread CCHD screening. These data can provide a basis for future research to better understand risk factors for these defects.

SARS-CoV-2 infection during pregnancy has been linked to preterm birth, but this association is not well understood.To examine the association between SARS-CoV-2 infection and spontaneous and provider-initiated preterm birth (PTB), and how timing of infection, and race/ethnicity as a marker of structural inequality, may modify this association.We conducted a retrospective cohort study among pregnant people who delivered singleton, liveborn infants (22-44 weeks gestation) from 1 March 2020 to 31 March 2021 (n = 68,288). We used Cox proportional hazards models to compare the hazard of PTB between pregnant people with and without laboratory-confirmed SARS-CoV-2 infection during pregnancy. We evaluated this association according to the trimester of infection, timing from infection to birth, and timing of PTB. We also examined the joint associations of SARS-CoV-2 infection and race/ethnicity with PTB using the relative excess risk due to interaction (RERI).Positive SARS-CoV-2 tests were identified for 2195 pregnant people (3.2%). The prevalence of PTB was 7.2% (3.8% spontaneous, 3.6% provider-initiated). SARS-CoV-2 infection during pregnancy was associated with an increased risk of PTB overall (adjusted hazard ratio [HR] 1.53, 95% confidence interval [CI] 1.34, 1.74), and provider-initiated PTB (HR 1.79, 95% CI 1.50, 2.12) but not spontaneous PTB (HR 1.09, 95% CI 0.89, 1.36). Second trimester infections were associated with an increased risk of provider-initiated PTB, and third trimester infections were associated with an increased risk of both PTB subtypes. A joint inverse association between White non-Hispanic race/ethnicity and SARS-CoV-2 infection and spontaneous PTB (HR 0.56, 95% CI 0.34, 0.94; RERI -0.6, 95% CI -1.0, -0.2) was also observed.SARS-CoV-2 infections were primarily associated with an increased risk for provider-initiated PTB in this study. These findings highlight the importance of promoting infection-prevention strategies among pregnant people.

BACKGROUND Both short and long interpregnancy intervals (IPIs) have been associated with adverse birth outcomes. We undertook a multistate study to describe the prevalence of selected birth defects by IPI. METHODS We obtained data from nine population-based state birth defects registries for singleton live births in 2000-2009 among mothers with a previous live birth identified through birth certificates. IPI was calculated as the difference between prior birthdate and start of the current pregnancy (conception date). We estimated prevalence of selected defects per 10,000 live births and prevalence ratios (PRs) with 95% confidence intervals (CIs) overall and stratified by maternal age at previous birth and race/ethnicity. Primary analyses focused on short IPI

Previous studies found consistent associations between pregestational diabetes and birth defects. Given the different biological mechanisms for type 1 (PGD1) and type 2 (PGD2) diabetes, we used National Birth Defects Prevention Study (NBDPS) data to estimate associations by diabetes type.The NBDPS was a study of major birth defects that included pregnancies with estimated delivery dates from October 1997 to December 2011. We compared self-reported PGD1 and PGD2 for 29,024 birth defect cases and 10,898 live-born controls. For case groups with ≥5 exposed cases, we estimated adjusted odds ratios (aORs) and 95% confidence intervals (CIs) for the association between specific defects and each diabetes type. We calculated crude ORs (cORs) and 95% CIs with Firth's penalized likelihood for case groups with 3-4 exposed cases.Overall, 252 (0.9%) cases and 24 (0.2%) control mothers reported PGD1, and 357 (1.2%) cases and 34 (0.3%) control mothers reported PGD2. PGD1 was associated with 22/26 defects examined and PGD2 was associated with 29/39 defects examined. Adjusted ORs ranged from 1.6 to 70.4 for PGD1 and from 1.6 to 59.9 for PGD2. We observed the strongest aORs for sacral agenesis (PGD1: 70.4, 32.3-147; PGD2: 59.9, 25.4-135). For both PGD1 and PGD2, we observed elevated aORs in every body system we evaluated, including central nervous system, orofacial, eye, genitourinary, gastrointestinal, musculoskeletal, and cardiac defects.We observed positive associations between both PGD1 and PGD2 and birth defects across multiple body systems. Future studies should focus on the role of glycemic control in birth defect risk to inform prevention efforts.

BACKGROUND: Down syndrome is the most common chromosomal disorder at birth and is often accompanied by structural birth defects. Current data on major structural defects in this population are limited. METHODS: States and territorial population-based surveillance programs submitted data on identified cases of Down syndrome and identified structural birth defects during 2013–2017. We estimated prevalence by program type and maternal and infant characteristics. Among programs with active case ascertainment, we estimated the prevalence of birth defects by organ system and for specific defects by maternal age (

Objectives: Pregnant people infected with SARS-CoV-2, the virus that causes COVID-19, are at increased risk for severe illness and death compared with nonpregnant people. However, population-based information comparing characteristics of people with and without laboratory-confirmed SARS-CoV-2 infection during pregnancy is limited. We compared the characteristics of people with and without SARS-CoV-2 infection during pregnancy in Massachusetts. Methods: We compared maternal demographic characteristics, pre-pregnancy conditions, and pregnancy complications of people with and without SARS-CoV-2 infection during pregnancy with completed pregnancies resulting in a live birth in Massachusetts during March 1, 2020–March 31, 2021. We tested for significant differences in the distribution of characteristics of pregnant people by SARS-CoV-2 infection status overall and stratified by race and ethnicity. We used modified Poisson regression analyses to examine the association between race and ethnicity and SARS-CoV-2 infection during pregnancy. Results: Of 69 960 completed pregnancies identified during the study period, 3119 (4.5%) had laboratory-confirmed SARS-CoV-2 infection during pregnancy. Risk for SARS-CoV-2 infection was higher among Hispanic (adjusted risk ratio [aRR] = 2.3; 95% CI, 2.1-2.6) and non-Hispanic Black (aRR = 1.9; 95% CI, 1.7-2.1) pregnant people compared with non-Hispanic White pregnant people. Conclusions: This study demonstrates the disproportionate impact of SARS-CoV-2 infection on Hispanic and non-Hispanic Black pregnant people in Massachusetts, which may widen existent inequities in maternal morbidity and mortality. Future research is needed to elucidate the structural factors leading to these inequities.

Congenital diaphragmatic hernia (CDH) occurs when abnormal diaphragm development allows herniation of abdominal organs into the thoracic cavity. Its etiopathogenesis is not well understood, but cigarette smoking and alcohol exposure may impact diaphragm development. Using data from a large, population-based case-control study, we examined associations between maternal cigarette smoking and alcohol consumption and CDH in offspring.We analyzed maternal interview reports of cigarette smoking and alcohol consumption during early pregnancy for 831 children with CDH and 11,416 children without birth defects with estimated dates of delivery during 1997-2011. Generalized linear mixed effects models with a random intercept for study site were used to estimate associations between measures of exposure to smoking (any, type, frequency, duration) and alcohol (any, quantity, frequency, variability, type) for all CDH combined and selected subtypes (Bochdalek and Morgagni).Mothers of 280 (34.0%) case and 3,451 (30.3%) control children reported early pregnancy exposure to cigarette smoking. Adjusted odds ratios for all CDH were increased for any (1.3; 95% confidence interval 1.1-1.5), active (1.3, 1.0-1.7), and passive (1.4, 1.1-1.7) smoking. Early pregnancy alcohol consumption was reported by mothers of 286 (34.9%) case and 4,200 (37.0%) control children; odds were near the null for any consumption (0.9, 0.8-1.1) and consumption with (0.9, 0.7, 1.1) or without (0.9, 0.8, 1.1) binging. Estimates for smoking and alcohol tended to be higher for Bochdalek CDH and Morgagni CDH than those for all CDH.Findings suggest that maternal early pregnancy exposure to cigarette smoking, but less so to alcohol consumption, contributes to CDH. These findings need to be replicated in additional large studies that use systematic case ascertainment and classification, detailed exposure assessment, and examine subtype-specific associations.

To assess whether the severity of cases of spina bifida changed after the institution of mandatory folic acid fortification in the US.Six active population-based birth defects programs provided data on cases of spina bifida for 1992-1996 (prefortification period) and 1999-2016 (postfortification period). The programs contributed varying years of data. Case information included both a medical record verbatim text description of the spina bifida diagnosis and spina bifida codes (International Classification of Diseases, Clinical Modification, or a modified birth defects surveillance coding system). Comparing the prefortification and postfortification periods, aORs for case severity (upper-level lesions [cervical, thoracic] vs lower-level lesions [lumbar, sacral]) and prevalence ratios (PRs) were estimated.A total of 2593 cases of spina bifida (out of 7 816 062 live births) met the inclusion criteria, including 573 cases from the prefortification period and 2020 cases from the postfortification period. Case severity decreased by 70% (aOR, 0.30; 95% CI, 0.26-0.35) between the fortification periods. The decrease was most pronounced for non-Hispanic White mothers. Overall spina bifida prevalence declined by 23% (PR, 0.77; 95% CI, 0.71-0.85), with similar reductions seen across the early, mid, and recent postfortification periods. A statistically significant decrease in upper-level lesions occurred in the postfortification period compared with the prefortification period (PR, 0.28; 95% CI, 0.22-0.34), whereas the prevalence of lower-level lesions remained relatively similar (PR, 0.94; 95% CI, 0.84-1.05).The severity of spina bifida cases decreased after mandatory folic acid fortification in the US. Further examination is warranted to better understand the potential effect of folic acid on spina bifida severity.

Background: Pregnant persons with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection are at increased risk of preterm birth, and evidence suggests this risk may be higher among pregnant persons with severe coronavirus disease 2019 (COVID-19) or among those infected later in pregnancy. However, the relationship between trimester of SARS-CoV-2 infection, severity of COVID-19, and preterm birth is not fully understood.Objective: This study examined the relationship between trimester of SARS-CoV-2 infection, illness severity, and risk for preterm birth after adjusting for maternal age, selected underlying conditions, and pregnancy complications.Study Design: Using a cohort of 6,396 pregnant persons with SARS-CoV-2 infection in 2020 identified through the Surveillance for Emerging Threats to Mothers and Babies Network, we analyzed data for those with infection at Results: Pregnant persons with critical COVID-19, compared to mild COVID-19, in the second (aRR 3.9; 95% CI: 1.7-9.0) or third (aRR 4.6; 95% CI: 3.2-6.6) trimester were at increased risk of preterm birth. Among persons infected in the second or third trimester, those with critical COVID-19 delivered sooner after infection compared with persons with mild COVID-19 (pConclusion: When infection occurred in the second or third trimester, critical COVID-19 was associated with increased risk of preterm birth, and those with critical COVID-19 delivered sooner after infection compared to those with mild COVID-19. These findings can be used to guide prevention strategies, including vaccination, and inform clinical practices for pregnant persons, particularly those presenting with critical COVID-19 later in pregnancy.

Multiple reports have described neonatal SARS-CoV-2 infection, including likely in utero transmission and early postnatal infection, but published estimates of neonatal infection range by geography and design type.To describe maternal, pregnancy and neonatal characteristics among neonates born to people with SARS-CoV-2 infection during pregnancy by neonatal SARS-CoV-2 testing results.Using aggregated data from the Surveillance for Emerging Threats to Mothers and Babies Network (SET-NET) describing infections from 20 January 2020 to 31 December 2020, we identified neonates who were (1) born to people who were SARS-CoV-2 positive by RT-PCR at any time during their pregnancy, and (2) tested for SARS-CoV-2 by RT-PCR during the birth hospitalisation.Among 28,771 neonates born to people with SARS-CoV-2 infection during pregnancy, 3816 (13%) underwent PCR testing and 138 neonates (3.6%) were PCR positive. Ninety-four per cent of neonates testing positive were born to people with infection identified ≤14 days of delivery. Neonatal SARS-CoV-2 infection was more frequent among neonates born preterm (5.7%) compared to term (3.4%). Neonates testing positive were born to both symptomatic and asymptomatic pregnant people.Jurisdictions reported SARS-CoV-2 RT-PCR results for only 13% of neonates known to be born to people with SARS-CoV-2 infection during pregnancy. These results provide evidence of neonatal infection identified through multi-state systematic surveillance data collection and describe characteristics of neonates with SARS-CoV-2 infection. While perinatal SARS-CoV-2 infection was uncommon among tested neonates born to people with SARS-CoV-2 infection during pregnancy, nearly all cases of tested neonatal infection occurred in pregnant people infected around the time of delivery and was more frequent among neonates born preterm. These findings support the recommendation for neonatal SARS-CoV-2 RT-PCR testing, especially for people with acute infection around the time of delivery.

About 20%-30% of children with birth defects have multiple major birth defects in more than one organ system, often referred to as multiple congenital anomalies (MCAs). Evaluating the patterns of MCAs can provide clues to the underlying causes, pathogenic mechanisms, and developmental pathways. We sought to explore selected patterns of MCAs within the National Birth Defects Prevention Study (NBDPS), a population-based, case-control study that excluded cases attributed to known chromosomal or single-gene abnormalities.We defined MCAs as having two or more NBDPS-eligible birth defects and calculated the adjusted observed-to-expected ratio for all observed MCA patterns using co-occurring defect analysis.Of the 50,186 case infants eligible for NBDPS, 2,734 (3.7%) had at least two eligible birth defects. We observed 209 distinct 2-way combinations of birth defects, 297 distinct 3-way combinations, 179 distinct 4-way combinations, and 69 distinct 5-way combinations. Sacral agenesis had the largest proportion of cases with MCAs (70%), whereas gastroschisis had the lowest (3%). Among the cases with MCAs, 63% had a heart defect, 23% had an oral cleft, and 21% had anorectal atresia/stenosis. Of the patterns with adjusted observed-to-expected ratios in the top 20%, most were consistent with the known associations or syndromes, including VATER/VACTERL association and CHARGE syndrome.Most but not all patterns that had the highest adjusted observed-to-expected ratios were instances of known syndromes or associations. These findings highlight the importance of considering birth defect combinations that suggest syndromic patterns in the absence of a formal syndromic diagnosis. New approaches for screening for sequences and associations, and VATER/VACTERL in particular, in surveillance systems with limited resources for manual review may be valuable for improving surveillance system quality. The observed MCA patterns within NBDPS may help focus future genetic studies by generating case groups of higher yield.

BACKGROUND: Many previous studies have identified risk factors for stillbirth, but few examine stillbirth among pregnancies affected with birth defects. Because many hypothesized etiologies of stillbirth work through vascular pathologies of the placenta, we examined maternal use of vasoactive medications in relation to stillbirth among pregnancies affected with birth defects. METHODS: Data were analyzed from the National Birth Defects Prevention Study (1997–2011). We examined use of nonsteroidal anti-inflammatory drugs (NSAIDs), decongestants, short- or long-acting beta-agonists (SABA/LABA), and antihypertensive medications in relation to pregnancies affected by birth defects ending in stillbirth compared to live birth. Associations were measured with odds ratios (ORs) for early pregnancy use and hazard ratios (HRs) for time-varying late pregnancy use. RESULTS: Among all birth defects (n = 12,394), the risk of stillbirth was associated with use of antihypertensive medications in early (odds ratio [OR]: 1.8; 95% confidence interval [CI]: 1.0, 3.1) and late pregnancy (HR: 2.0; 95% CI: 1.1, 3.6). Other vasoactive medications were not associated with increased risk of stillbirth. Of 27 specific defect groups, increased risks were observed for only one medication/defect pair: early decongestant use was more common among mothers of stillbirth versus live birth cases with spina bifida (OR: 2.4; 95% CI: 0.9, 6.5). CONCLUSION: This exploratory analysis of vasoactive medication use suggests that use of NSAIDs, decongestants, and SABA/LABA is not associated with increased risk of stillbirth among pregnancies affected with birth defects. Our finding of increased risks associated with antihypertensive medication use raises questions of confounding by indication, which we were not able to fully address.

We investigated factors associated with maternal consent to use residual newborn dried bloodspots (DBS) in a national case-control study of birth defects.A subset of sites in the National Birth Defects Prevention Study (NBDPS; 1997-2011) asked participants to provide consent for investigators to retrieve DBS from local newborn screening programs to use for research on risk factors for birth defects. We assessed whether consent differed by factors including maternal age, education, parity, body mass index, language of interview, country of birth, and case-control status.Of 5,850 mothers of cases and 2,534 mothers of controls, 57% provided consent for the DBS component. Mothers of cases were more likely to participate than mothers of controls (61% vs. 52%), as were mothers who self-reported white race,12 years of education, and born in the United States.Retrieval of DBS can be integrated into retrospective studies of neonatal outcomes including birth defects. In NBDPS, participation in the DBS component was moderate and varied by some sociodemographic factors. Further research is needed to better understand families' perspectives on using residual DBS for secondary research. Representative participation is important to reduce the potential for selection bias in future studies using DBS for children's health research.

During the Centers for Disease Control and Prevention’s Zika Virus Response, birth defects surveillance programs adapted to monitor birth defects potentially related to Zika virus (ZIKV) infection during pregnancy. Pregnancy outcomes occurring during January 2016-June 2017 in 22 U.S. states and territories were used to estimate the prevalence of those brain and eye defects potentially related to ZIKV. Jurisdictions were divided into three groups: areas with widespread ZIKV transmission, areas with limited local ZIKV transmission, and areas without local ZIKV transmission. Prevalence estimates for selected brain and eye defects and microcephaly per 10,000 live births were estimated. Prevalence ratios (PRs) and 95% confidence intervals (CIs) were estimated using Poisson regression for areas with widespread and limited ZIKV transmission compared to areas without local ZIKV transmission. Defects with significantly higher prevalence in areas of widespread transmission were pooled, and PRs were calculated by quarter, comparing subsequent quarters to the first quarter (January – March 2016). Nine defects had significantly higher prevalence in areas of widespread transmission. The highest PRs were seen in intracranial calcifications (PR=12.6, 95% CI [7.4, 21.3]), chorioretinal abnormalities (12.5 [7.1, 22.3]), brainstem abnormalities (9.3, [4.7, 18.4]), and cerebral/cortical atrophy (6.7, [4.2, 10.8]). The PR of the nine pooled defects was significantly higher in three quarters in areas with widespread transmission. The largest difference in prevalence was observed for defects consistently reported in infants with congenital ZIKV infection. Birth defects surveillance programs could consider monitoring a subset of birth defects potentially related to ZIKV in pregnancy.

Background: Multiple reports have described neonatal SARS-CoV-2 infection, including likely in utero transmission and early postnatal infection. Most neonatal infections reported to date have been asymptomatic or mild disease; however, severe cases, including respiratory failure requiring intensive care unit admission, have been described.Objectives: To describe maternal, pregnancy and infant characteristics among neonates born to women with SARS-CoV-2 infection during pregnancy by neonatal SARS-CoV-2 testing results.Methods: Using aggregated data from the Surveillance for Emerging Threats to Mothers and Babies Network (SET-NET) from March 29, 2020–August 6, 2021, we identified neonates who were: 1) born to women who were SARS-CoV-2 positive by RT-PCR at any time during their pregnancy, and 2) tested for SARS-CoV-2 by RT-PCR during the birth hospitalization. Results: Among 25,896 neonates of mothers with SARS-CoV-2-infection, 3,381 (13%) underwent PCR testing. One hundred thirty-six neonates (4%) were PCR-positive. Neonates testing positive were born to both symptomatic and asymptomatic women, and 95% were born to women with infection identified ≤ with 14 days of delivery.Conclusions: While perinatal SARS-CoV-2 infection was uncommon among neonates born to women with SARS-CoV-2 infection during pregnancy, nearly all cases of neonatal infection occurred in pregnant women infected around the time of delivery. These findings underline the need for infection prevention and control measures in delivery and outpatient pediatric settings, as well as counselling for persons who acquire COVID-19 during pregnancy about potential risk to their neonates. Moreover, pregnant people and those wanting to become pregnant should be vaccinated against COVID-19 in order to protect themselves and their infants.

In May 2018, a study of birth defects in infants born to women with diagnosed human immunodeficiency virus (HIV) infection in Botswana reported an eightfold increased risk for neural tube defects (NTDs) among births with periconceptional exposure to antiretroviral therapy (ART) that included the integrase inhibitor dolutegravir (DTG) compared with other ART regimens (1). The World Health Organization* (WHO) and the U.S. Department of Health and Human Services† (HHS) promptly issued interim guidance limiting the initiation of DTG during early pregnancy and in women of childbearing age with HIV who desire pregnancy or are sexually active and not using effective contraception. On the basis of additional data, WHO now recommends DTG as a preferred treatment option for all populations, including women of childbearing age and pregnant women. Similarly, the U.S. recommendations currently state that DTG is a preferred antiretroviral drug throughout pregnancy (with provider-patient counseling) and as an alternative antiretroviral drug in women who are trying to conceive.§ Since 1981 and 1994, CDC has supported separate surveillance programs for HIV/acquired immunodeficiency syndrome (AIDS) (2) and birth defects (3) in state health departments. These two surveillance programs can inform public health programs and policy, linkage to care, and research activities. Because birth defects surveillance programs do not collect HIV status, and HIV surveillance programs do not routinely collect data on occurrence of birth defects, the related data have not been used by CDC to characterize birth defects in births to women with HIV. Data from these two programs were linked to estimate overall prevalence of NTDs and prevalence of NTDs in HIV-exposed pregnancies during 2013-2017 for 15 participating jurisdictions. Prevalence of NTDs in pregnancies among women with diagnosed HIV infection was 7.0 per 10,000 live births, similar to that among the general population in these 15 jurisdictions, and the U.S. estimate based on data from 24 states. Successful linking of data from birth defects and HIV/AIDS surveillance programs for pregnancies among women with diagnosed HIV infection suggests that similar data linkages might be used to characterize possible associations between maternal diseases or maternal use of medications, such as integrase strand transfer inhibitors used to manage HIV, and pregnancy outcomes. Although no difference in NTD prevalence in HIV-exposed pregnancies was found, data on the use of integrase strand transfer inhibitors in pregnancy are needed to understand the safety and risks of these drugs during pregnancy.

OBJECTIVE: To estimate the risk of stillbirth (fetal death at 20 weeks of gestation or more) associated with specific birth defects. METHODS: We identified a population-based retrospective cohort of neonates and fetuses with selected major birth defects and without known or strongly suspected chromosomal or single-gene disorders from active birth defects surveillance programs in nine states. Abstracted medical records were reviewed by clinical geneticists to confirm and classify all birth defects and birth defect patterns. We estimated risks of stillbirth specific to birth defects among pregnancies overall and among those with isolated birth defects; potential bias owing to elective termination was quantified. RESULTS: Of 19,170 eligible neonates and fetuses with birth defects, 17,224 were liveborn, 852 stillborn, and 672 electively terminated. Overall, stillbirth risks ranged from 11 per 1,000 fetuses with bladder exstrophy (95% CI 0–57) to 490 per 1,000 fetuses with limb-body-wall complex (95% CI 368–623). Among those with isolated birth defects not affecting major vital organs, elevated risks (per 1,000 fetuses) were observed for cleft lip with cleft palate (10; 95% CI 7–15), transverse limb deficiencies (26; 95% CI 16–39), longitudinal limb deficiencies (11; 95% CI 3–28), and limb defects due to amniotic bands (110; 95% CI 68–171). Quantified bias analysis suggests that failure to account for terminations may lead to up to fourfold underestimation of the observed risks of stillbirth for sacral agenesis (13/1,000; 95% CI 2–47), isolated spina bifida (24/1,000; 95% CI 17–34), and holoprosencephaly (30/1,000; 95% CI 10–68). CONCLUSION: Birth defect-specific stillbirth risk was high compared with the U.S. stillbirth risk (6/1,000 fetuses), even for isolated cases of oral clefts and limb defects; elective termination may appreciably bias some estimates. These data can inform clinical care and counseling after prenatal diagnosis.

Introduction We examined the association of congenital diaphragmatic hernia (CDH) with maternal dietary intake, using semi-Bayes hierarchical models and principal components analysis to consider intake of nutrients that contribute to one-carbon metabolism and oxidative stress pathways, and a diet quality index. Methods We included data on 825 cases and 11,108 nonmalformed controls born from 1997-2011 whose mother participated in the National Birth Defects Prevention Study (NBDPS), a multisite, population-based case-control study. Exposure data were from maternal telephone interviews, which included a food frequency questionnaire. Adjusted odds ratios (aOR) and 95% confidence intervals (CI) were generated from logistic regression models that included nutritional factors as continuous variables and were adjusted for maternal energy intake, race-ethnicity, parity, and vitamin supplement intake. Results In the semi-Bayes hierarchical model that included all nutrients and confounders, riboflavin was the only nutrient for which the 95% CI excluded 1.0; the aOR for a 1 SD increase was 0.83. The aORs were 0.79 (95% CI 0.69-0.91) for the one-carbon metabolism pathway score, 0.90 (95% CI 0.80-1.01) for oxidative stress, and 0.85 (95% CI 0.77-0.93) for diet quality (the aORs correspond to a 1 SD increase). Conclusions The findings from this study provide some support for the hypothesis that better prepregnancy nutrition is associated with reduced risk for CDH. These results provide etiologic clues but should be interpreted with caution given the novelty of the investigation.

Background Although autopsy is a key component of the etiologic evaluation following fetal and early neonatal death, and traditionally has been the preferred method to determine the cause of death, an alternative may be suitable when traditional autopsy by a perinatal pathologist is not available or declined. Methods Among 3137 cases evaluated through the Wisconsin Stillbirth Service Program (WiSSP), a community-based program for etiologic evaluation of second trimester miscarriage, stillbirth, and early neonatal death, most diagnoses are based on multiple types of data including placental pathology, clinical examination, photographs, maternal records, radiographs, and laboratory testing. Results Cases in the WiSSP cohort without autopsy have nearly the same overall rate of diagnosis as those with traditional autopsy (56% vs. 58%). Review of the literature shows that although recent systematic protocols including autopsy, placental pathology and genetic studies yield a definite or probable diagnosis in 70% or more, both healthcare providers and families desire less invasive options. Several minimally invasive protocols substituting imaging, primarily MRI, for traditional autopsy have been proposed, but the numbers of deaths evaluated are still very small. Conclusion We join others who have promoted the benefits of a targeted or less invasive protocol to study perinatal deaths, and emphasize integration of clinical data, selective imaging, genetic testing, and parental counseling. Birth Defects Research, 2017. © 2017 Wiley Periodicals, Inc.

BACKGROUND/OBJECTIVES: In this report, the National Birth Defects Prevention Network (NBDPN) examines and compares gastroschisis and omphalocele for a recent 5-year birth cohort using data from 30 population-based birth defect surveillance programs in the United States. METHODS: As a special call for data for the 2019 NBDPN Annual Report, state programs reported expanded data on gastroschisis and omphalocele for birth years 2012–2016. We estimated the overall prevalence (per 10,000 live births) and 95% confidence intervals (CI) for each defect as well as by maternal race/ethnicity, maternal age, infant sex, and case ascertainment methodology utilized by the program (active vs. passive). We also compared distribution of cases by maternal and infant factors and presence/absence of other birth defects. RESULTS: The overall prevalence estimates (per 10,000 live births) were 4.3 (95% CI:4.1–4.4) for gastroschisis and 2.1 (95% CI: 2.0–2.2) for omphalocele. Gastroschisis was more frequent among young mothers (40 years). Mothers of infants with gastroschisis were more likely to be underweight/normal weight prior to pregnancy and mothers of infants with omphalocele more likely to be overweight/obese. Omphalocele was twice as likely as gastroschisis to co-occur with other birth defects. CONCLUSIONS: This report highlights important differences between gastroschisis and omphalocele. These differences indicate the importance of distinguishing between these defects in epidemiologic assessments. The report also provides additional data on co-occurrence of gastroschisis and omphalocele with other birth defects. This information can provide a basis for future research to better understand these defects.

BACKGROUND: While Roux-en-Y gastric bypass (RYGB) is one of the most effective and durable treatment options for obesity and its comorbidities, it is complicated by long-term weight regain in over 20% of patients. AIMS: We sought to determine the metabolite signatures of serum samples of patients with weight regain (RYGB-WR) after RYGB and features distinguishing these patients from patients with sustained weight loss (RYGB-SWL). METHODS: We prospectively analyzed serum samples from 21 RYGB-WR patients, 14 RYGB-SWL patients and 11 unoperated controls. The main outcome measure was their serum metabolite profile. RESULTS: Weight regain after RYGB was associated with a unique serum metabolomic fingerprint. Most of the statistically different metabolites were involved in amino acid metabolism, one-carbon metabolism and related nucleotide metabolism. A principal component analysis identified groups of metabolites that correlate with weight regain. Specifically, weight regain was associated with lower serum levels of metabolites related to the serine, glycine and threonine pathway, phenylalanine metabolism, tricyclic acid cycle, alanine and glutamate metabolism, and higher levels of other amino acids. CONCLUSIONS: Weight regain after RYGB is associated with unique serum metabolite signatures. Metabolite profiling may eventually help us to identify markers that could differentiate the patients who will regain weight vs. those who will likely sustain weight loss.

Although autopsy is a key component of the etiologic evaluation following fetal and early neonatal death, and traditionally has been the preferred method to determine the cause of death, an alternative may be suitable when traditional autopsy by a perinatal pathologist is not available or declined.Among 3137 cases evaluated through the Wisconsin Stillbirth Service Program (WiSSP), a community-based program for etiologic evaluation of second trimester miscarriage, stillbirth, and early neonatal death, most diagnoses are based on multiple types of data including placental pathology, clinical examination, photographs, maternal records, radiographs, and laboratory testing.Cases in the WiSSP cohort without autopsy have nearly the same overall rate of diagnosis as those with traditional autopsy (56% vs. 58%). Review of the literature shows that although recent systematic protocols including autopsy, placental pathology and genetic studies yield a definite or probable diagnosis in 70% or more, both healthcare providers and families desire less invasive options. Several minimally invasive protocols substituting imaging, primarily MRI, for traditional autopsy have been proposed, but the numbers of deaths evaluated are still very small.We join others who have promoted the benefits of a targeted or less invasive protocol to study perinatal deaths, and emphasize integration of clinical data, selective imaging, genetic testing, and parental counseling. Birth Defects Research 109:1430-1441, 2017.© 2017 Wiley Periodicals, Inc.

Alterations in intestinal function, often characterized as a "leaky gut," have been attributed to children who are on the autism spectrum. Disaccharidase activity, intestinal inflammation, and permeability were analyzed in 61 children with autism and 50 nonautistic individuals with gastrointestinal symptoms.All patients had duodenal biopsies assayed for lactase, sucrase, maltase, and palatinase activity. Intestinal permeability was evaluated by rhamnose/lactulose test and measured by high-performance liquid chromatography-mass spectrometry. Intestinal inflammation was evaluated by fecal calprotectin and lactoferrin levels using enzyme-linked immunosorbent assay and histology.Some children with autism had mild levels of mucosal inflammation on intestinal biopsy. Disaccharidase activity was not different in autistic and nonautistic individuals. Fecal calprotectin and lactoferrin were similar in both groups. Differences between lactulose and rhamnose recovery and lactulose/rhamnose ratio in urine were not statistically different in patients with and without autism.The present study supports the observation that children with autism who have symptoms of gastrointestinal disorders have objective findings similar to children without autism. Neither noninvasive testing nor endoscopic findings identify gastrointestinal pathology specific to autism, but may be of benefit in identifying children with autism who have atypical symptoms.

All available treatments directed towards obesity and obesity-related complications are associated with suboptimal effectiveness/invasiveness ratios. Pharmacological, behavioral and lifestyle modification treatments are the least invasive, but also the least effective options, leading to modest weight loss that is difficult to maintain long-term. Gastrointestinal weight loss surgery (GIWLS) is the most effective, leading to60-70% of excess body weight loss, but also the most invasive treatment available. Sleeve gastrectomy (SGx) and Roux-en-Y gastric bypass (RYGB) are the two most commonly performed GIWLS procedures. The fundamental anatomic difference between SGx and RYGB is that in the former procedure, only the anatomy of the stomach is altered, without surgical reconfiguration of the intestine. Therefore, comparing these two operations provides a unique opportunity to study the ways that different parts of the gastrointestinal (GI) tract contribute to the regulation of physiological processes, such as the regulation of body weight, food intake and metabolism.To explore the physiologic mechanisms of the two procedures, we used rodent models of SGx and RYGB to study the effects of these procedures on body weight, food intake and metabolic function.Both SGx and RYGB induced a significant weight loss that was sustained over the entire study period. SGx-induced weight loss was slightly lower compared with that observed after RYGB. SGx-induced weight loss primarily resulted from a substantial decrease in food intake and a small increase in locomotor activity. In contrast, rats that underwent RYGB exhibited a substantial increase in non-activity-related (resting) energy expenditure and a modest decrease in nutrient absorption. Additionally, while SGx-treated animals retained their preoperative food preferences, RYGB-treated rats experienced a significant alteration in their food preferences.These results indicate a fundamental difference in the mechanisms of weight loss between SGx and RYGB, suggesting that the manipulation of different parts of the GI tract may lead to different physiologic effects. Understanding the differences in the physiologic mechanisms of action of these effective treatment options could help us develop less invasive new treatments against obesity and obesity-related complications.

The effectiveness of Roux-en-Y gastric bypass (RYGB) against obesity and its comorbidities has generated excitement about developing new, less invasive treatments that use the same molecular mechanisms. Although controversial, RYGB-induced improvement of metabolic function may not depend entirely upon weight loss. To elucidate the differences between RYGB and dieting, we studied several individual organ molecular responses and generated an integrative, interorgan view of organismal physiology. We also compared murine and human molecular signatures. We show that, although dieting and RYGB can bring about the same degree of weight loss, post-RYGB physiology is very different. RYGB induces distinct, organ-specific adaptations in a temporal pattern that is characterized by energetically demanding processes, which may be coordinated by HIF1a activation and the systemic repression of growth hormone receptor signaling. Many of these responses are conserved in rodents and humans and may contribute to the remarkable ability of surgery to induce and sustain metabolic improvement.

Both paired homeo box-2 (Pax-2) and N-myc genes play pivotal roles in renal morphogenesis via their effects on cell proliferation and differentiation, but whether and how they interact have not been addressed. In the present study, we investigated such a potential interaction using embryonic renal cells in vitro. Mouse embryonic mesenchymal (MK4) cells stably transfected with Pax-2 cDNA in sense (+) or antisense (-) orientation were used for experiments. Pax-2 promoter activity was monitored by luciferase assay. Reactive oxygen species (ROS) generation, cell proliferation, and cell apoptosis were evaluated. We found that Pax-2 and N-myc gene expression were upregulated and downregulated in Pax-2 (+) and Pax-2 (-) stable transformants, respectively. ROS generation and apoptosis were significantly reduced both in Pax-2 (+) transformants compared with Pax-2 (-) transformants and in naïve MK4 cells cultured in either normal- (5 mM) or high-glucose (25 mM) medium. Transient transfection of N-myc cDNA into Pax-2 (-) stable transformants restored Pax-2 gene expression and prevented ROS generation induced by high glucose. Our data demonstrate that Pax-2 gene overexpression prevents hyperglycemia-induced apoptosis, and N-myc appears to provide a positive autocrine feedback on Pax-2 gene expression in embryonic mesenchymal cells.

Summary. Background: The pathogenesis of Shiga toxin (Stx)-mediated childhood hemolytic uremic syndrome (HUS) is not fully delineated, although current evidence implicates a prothrombotic state. We hypothesized that the tissue factor (TF) pathway plays a major role in the pathophysiology of HUS. Materials and methods: We measured cell surface TF activity in response to tumor necrosis factor-α (TNF-α) (20 ng mL−1, 2–144 h), Stx-1 (10−11 mol L−1, 4–144 h), or their combination (TNF-α 22 h and Stx-1 for the last 0.5–4 h of TNF-α incubation) on human glomerular (microvascular) endothelial cells (HGECs) and human umbilical vein (macrovascular) endothelial cells (HUVECs). Results and conclusions: We observed that while TNF-α caused an increase in cell surface TF activity on both cell types, the combination of TNF-α and Stx-1 differentially affected HGECs. On these cells, TF activity was increased further by 2.67 ± 0.38-fold (n = 38, P

Introduction Cytokine activation of endothelial cell monolayers is associated with cell detachment, microparticle shedding from plasma membranes, and phosphatidylserine appearance in the plasma membrane outer leaflets. While tissue factor expression on activated endothelial cells and microparticles is well documented, the contribution of detached endothelial cells to tissue factor activity is less clear. We studied tissue factor expression and the role of tissue factor pathway inhibitor on adherent and detached endothelial cells and on microparticles following endothelial cell activation with TNF-α. Materials and methods Detached endothelial cells and microparticles were obtained from cultures of human umbilical vein endothelial cells by differential centrifugation of cell culture supernatant. For microparticle capture, an antibody directed against CD146 was used. Functional tissue factor activity was measured by chromogenic assay and tissue factor antigen by ELISA. Endothelial cell and microparticle morphology was examined by light and transmission electron microscopy. Results After cell activation for 22 h, functional tissue factor activity was distributed as follows: 60%, adherent endothelial cells; 35%, detached cells; and 5%, microparticles. Tissue factor protein followed a similar distribution. Cell detachment was 47%. Electron microscopy demonstrated shedding of microparticles with a diameter of 0.1–0.6 μm. Cy3–annexin V revealed increased phosphatidylserine on activated adherent endothelial cells and microparticles. Pre-incubation of adherent and detached endothelial cells and microparticles with anti-tissue factor antibody blocked factor Xa production. Pre-incubation with anti-tissue factor pathway inhibitor antibody increased tissue factor activity of adherent endothelial cells 2.8-fold, detached cells 1.4-fold, and microparticles 45-fold. Conclusions Detached endothelial cells as well as microparticles from activated endothelial cell monolayers express tissue factor activity, and this activity on microparticles is markedly inhibited by microparticle-associated tissue factor pathway inhibitor.

Although the prevalence of obesity has increased dramatically throughout the world during the last 25 yr, its long-term control remains poor. Currently, only gastrointestinal weight loss surgery, especially Roux-en-Y gastric bypass (RYGB), is associated with substantial and sustained weight loss and resolution or significant improvement of diabetes mellitus and other metabolic obesity-induced complications. Clinical observations and recent studies have suggested that RYGB induces its effects by changing the physiology of weight regulation. Understanding the underlying mechanisms of these profound and sustainable effects could facilitate the development of novel and less invasive treatments against obesity and its complications. To study the physiological mechanisms of RYGB, we have developed a mouse RYGB model that replicates the human operation. The aims of this study were to develop a roadmap for assessing energy expenditure (EE) in animal models of weight loss surgery and to examine the effects of RYGB on EE. We first measured EE by indirect calorimetry in groups of animals that underwent RYGB or a sham operation. Calorimetry data were analyzed using three different methods: normalization by total body mass, allometric scaling, and analysis of covariance modeling. RYGB in mice induced a significant increase in EE that was independent of the method used. An energy balance analysis was then performed, which also confirmed that RYGB-treated animals have higher energy maintenance needs. Finally, we determined the EE components that account for the observed increase in EE, and we found that resting EE and postprandial thermogenesis are the major contributors to this increase.

Background Gastrointestinal weight loss surgery, especially Roux-en-Y gastric bypass (RYGB), is the most effective treatment for severe obesity. RYGB is associated with a remarkable decrease in the rate of death from obesity-related complications, such as diabetes mellitus, coronary artery disease, and cancer. Dissecting the mechanisms of RYGB effects could augment our understanding about the pathogenesis of obesity and its complications. Objectives and methods In this study, we describe in detail a mouse model of RYGB that closely reproduces the surgical steps of the human procedure. Results We show that RYGB in mice has the same effects as in human patients, proving the high translational validity of this model system. We present an intraoperative video to facilitate the widespread use of this complex and difficult method. Conclusions The study of the mechanisms of RYGB using this model system can greatly facilitate our understanding about the effects of RYGB in human patients. The reverse engineering of the physiological mechanisms of RYGB could lead to discovery of new, effective, and less invasive treatments.

Current evidence implicates a prothrombotic state in the development of Shiga-toxin (Stx)-mediated hemolytic uremic syndrome (HUS). We recently reported that Stx modulates procoagulant activity by enhancing functional tissue factor (TF) activity on cytokine-activated human glomerular endothelial cells (HGECs). Since angiotensin II (Ang II), the key effector of the renin angiotensin system (RAS), has been shown to increase TF expression in vascular tissue, we examined the possible involvement of Ang II in TF expression in HGECs. HGECs were exposed to tumor necrosis factor (TNF)-alpha +/- Stx-1 +/- Ang II. Exogenous Ang II significantly increased TF activity and TF mRNA in TNF-alpha- +/- Stx-1-activated HGECs. This increase was mediated via Ang II type I receptor (AT(1)R), as losartan, an AT(1)R inhibitor, attenuated Ang-II-induced TF activity. To study the effect of endogenous Ang II in TF expression by TNF-alpha +/- Stx-1, HGECs were incubated with losartan or an AT(2)R inhibitor (PD 123319) or an angiotensin-converting enzyme inhibitor (enalapril). Losartan but not PD 123319 decreased TF activity induced by TNF-alpha +/- Stx-1 (P0.05). Enalapril, also, dose dependently, downregulated TF expression in HGECs exposed to TNF-alpha +/- Stx-1 (P0.05). AT(1)R mRNA was upregulated in TNF-alpha- +/- Stx-1-activated HGECs (P0.05). These data indicate that TF expression in TNF-alpha- and Stx-1-activated HGECs is enhanced by exogenous Ang II and that endogenous Ang II production may be upregulated by TNF-alpha +/- Stx-1. Hence, local RAS activation may be important in the development of the thrombotic microangiopathy observed in HUS.

The role of plasma-phase risk factors for stroke in the pediatric age group is presently unclear due to the lack of sufficiently large prospective studies, and due to the fact that these risk factors do not apply uniformly to newborns, children with sickle cell disease, and older children. Available evidence indicates that factor V Leiden, prothrombin 20210A, and lipoprotein (a) are all important in the pathogenesis of arterial ischemic stroke in older children, but the role of other plasma-phase risk factors remains uncertain. The contribution of these risk factors to newborn stroke and the stroke of children with sickle cell disease is similarly unclear, likely because the ischemia in affected children is predominantly due to nonhematologic perinatal events and erythrocyte adhesion to endothelium with obstruction of flow in the cerebral microcirculation, respectively. Evaluation of childhood stroke should, in our view, always be performed from the standpoint of the presenting clinical symptoms, diagnostic imaging, and determination of plasma-phase risk factors. Therapeutic anticoagulation and use of antiplatelet agents at present focus on the older child.

Human blood flowing over monolayers of endothelial cells (ECs) in vitro under controlled flow conditions constitutes a promising model of blood-EC interactions, especially in a system permitting real-time imaging of single platelets (1 μm resolution or better) and platelet aggregates. We have developed such a model and have used it to study platelet adhesion/aggregation on monolayers of human renal microvascular ECs grown on optically clear vinyl slides and activated with TNFα (20 ng/ml; hr 0 to 22) and Shiga toxin-1 (Stx; 10 pM; hr 18 to 22), in simulation of probable events in the childhood hemolytic uremic syndrome (HUS). Normal donor blood, collected into 4U/ml low MW heparin (dalteparin) and 10 μM mepacrine (platelet, EC, and granulocyte label), was drawn at shear rates of 270–650 sec−1 through a parallel-plate flow chamber for which one wall was one of the above slides. Such shear rates give rise to shear stresses which approximate the 20–25 dynes/cm2 estimated to exist in glomerular arterioles. Platelets, ECs, and granulocytes were imaged in real time using epifluorescence digital videomicroscopy. With activated, but not with control ECs, clusters of platelets deposit on the monolayers in strings (Fig.1, upper right, with flow from top to bottom) superposed upon ECs, for which the cytoplasm is fluorescent. The strings are tethered at the upstream end, as has been observed by others using platelet-rich plasma. Granulocytes, identifiable by size (Fig. 1, upper middle), also adhere to activated ECs. In five paired experiments, monolayer preincubation × 30 min with 50 nM active-site inactivated recombinant factor VIIa (ASIS; courtesy of Dr. Ulla Hedner, Novo Nordisk Pharmaceuticals) largely eliminated the platelet strings, while reducing the number of adherent granulocytes to zero. In control experiments, preincubation of monolayers with 100 nM of a monoclonal antibody directed against human tissue factor similarly largely eliminated both platelet strings and adherent granulocytes. In other control experiments, the use of the above concentration of low MW heparin did not by itself block fibrin formation (by immunostaining) in the boundary region near the slide-blood interface. These findings may be explained on the basis that ASIS both interrupts the tissue factor pathway of coagulation and blocks tissue factor-associated upregulation of endothelial cell E-selectin. ASIS therefore may have promise as a therapy for the prothrombotic and proinflammatory effects of childhood HUS. Studies with the TAB monoclonal antibody and ALEXA 555 are in progress to permit platelet-specific labeling and quantitation of platelet adhesion/aggregation with and without ASIS. Figure Figure

The Dutch belted rabbit constitutes a promising animal model of the childhood hemolytic uremic syndrome (HUS; A. Garcia, J. Infect. Dis. 2002). When Shiga toxin (Stx)-producing E. coli are administered orally, these rabbits develop HUS. Platelet-fibrin thrombus formation in this model was examined on unfixed renal tissue (5 μm sections) after mounting on 150-μm glass slides in test animal-control animal pairs. Normal donor blood, collected into 4U/ml low MW heparin and 10 μM quinacrine dihydrochoride, was drawn at shear rates of 270–650 sec-1 through a parallel-plate flow chamber for which one surface was one of the above slides. Such shear rates give rise to shear stresses which approach the 20–25 dynes/cm2 estimated to exist in glomerular arterioles. Platelets depositing on the sections were imaged in real time using epifluorescence digital videomicroscopy, with quantitation of adherent platelets via image processing. For 16 pairs of test and control sections, the percent of image pixels occupied by adherent platelets after 6–9 min of blood flow was 17.7 ± 3.06 (mean ± SD) for test sections vs 5.71 ± 5.18 for controls (P

Post-diarrheal hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children, is most often caused by Shiga toxin (Stx)-producing Escherichia coli infection. The most widely recognized tissue damage in HUS occurs within the kidney, most prominently with local thrombosis and platelet-fibrin accumulation, suggesting activation of the coagulation cascade. We have shown that human proximal tubular epithelial cells (HK-2 cells) exposed to Stx-1 augment their constitutive surface tissue factor (TF) activity by 3.2-fold, and that Stx-1 treatment following TNF-alpha exposure enhances TF activity by 2.7-fold on human glomerular endothelial cells (HGECs). We investigated the possibility of modulating this cellular response to Stx-1 by blocking the TF pathway with active site-inactivated recombinant factor VIIa (irFVIIa), which retains the ability to bind TF, but is enzymatically inactive, thereby inhibiting thrombin formation. Moreover, its action is limited to exposed, functional TF and does not significantly prolong the bleeding time in vivo. Monolayers of both HK-2 cells and TNF-alpha-activated HGECs were incubated at 37°C with Stx-1. Functional TF was determined chromogenically as the production rate of factor Xa. The use of 60 nM irFVIIa completely abrogated the enhanced activity of TF caused by 4 hours incubation with 10 pM Stx-1 of TNF-alpha-activated HGECs (N = 4; Figure 1). On HK-2 cells, irFVIIa (60 – 200 nM) also effectively reversed the observed TF upregulation after exposure to 1 nM Stx-1 for 22 hours as well as blocked the constitutive TF expression which is seen with this cell type (N = 3; Figure 2). The TF pathway may prove to be of major importance in the pathogenesis of HUS, and direct targeting of this pathway with irFVIIa might provide a novel therapeutic approach to this disease. Download : Download high-res image (269KB) Download : Download full-size image Download : Download high-res image (240KB) Download : Download full-size image

Up-regulation of tissue factor activity on human proximal tubular epithelial cells in response to Shiga toxin. Background The pathophysiology of hemolytic uremic syndrome (HUS) is incompletely established. Based on clinical studies demonstrating the presence of prothrombotic plasma markers in patients with HUS, we hypothesized that Shiga toxin might cause activation of the coagulation pathway by augmenting tissue factor, the major initiator of coagulation. Methods Human proximal tubular epithelial cells (PTECs) [human kidney-2 (HK-2 cells)] were exposed to Shiga toxin-1, and expression of tissue factor, cell detachment, protein synthesis, caspase-3 activity, and Shiga toxin-1 binding were examined. Results HK-2 cells expressed constitutive surface tissue factor activity and increased their tissue factor expression upon exposure to Shiga toxin-1. Shiga toxin-1 bound to HK-2 cells and inhibited protein synthesis. The up-regulation of tissue factor was dose- and time-dependent and strongly correlated with cell detachment and increase in caspase-3 activity caused by Shiga toxin-1 exposure. A general caspase inhibitor simultaneously inhibited HK-2 cell detachment and tissue factor up-regulation while mutant Shiga toxin-1 neither caused cell detachment, protein synthesis inhibition, nor increase in tissue factor activity. Tissue factor activity elicited by Shiga toxin-1 was abrogated by a monoclonal antitissue factor antibody. Calphostin C, a protein kinase C (PKC) inhibitor, partially blocked tissue factor up-regulation, indicating possible involvement of PKC-dependent mechanism. Conclusion These data, taken together, suggest a strong link between Shiga toxin–induced up-regulation of tissue factor activity, cytotoxicity, and apoptosis in HK-2 cells. The proximal tubule is a target of Shiga toxin in HUS, and it seems plausible that injured proximal tubular cells trigger the activation of the coagulation system, the formation of intrarenal platelet-fibrin thrombi, and the development of acute renal failure in HUS.