Maternal smoking during pregnancy (MSDP), driven by nicotine crossing the placenta, causes lifelong decreases in offspring pulmonary function and vitamin C supplementation during pregnancy prevents some of those changes. We have also shown in animal models of prenatal nicotine exposure that vitamin C supplementation during pregnancy improves placental function. In this study we examined whether vitamin C supplementation mitigates the effects of MSDP on placental structure, function, and gene expression in pregnant human smokers. Doppler ultrasound was performed in a subset of 55 pregnant smokers participating in the “Vitamin C to Decrease the Effects of Smoking in Pregnancy on Infant Lung Function” (VCSIP) randomized clinical trial (NCT01723696) and in 33 pregnant nonsmokers. Doppler ultrasound measurements showed decreased umbilical vein Doppler velocity (Vmax) in placebo-treated smokers that was significantly improved in smokers randomized to vitamin C, restoring to levels comparable to nonsmokers. RNA-sequencing demonstrated that vitamin C supplementation to pregnant smokers was associated with changes in mRNA expression in genes highly relevant to vascular and cardiac development, suggesting a potential mechanism for vitamin C supplementation in pregnant smokers to improve some aspects of offspring health.

Introduction: Our goal was to evaluate the potential utility of magnetic resonance imaging (MRI) placental volume as an assessment of placental insufficiency. Methods: Secondary analysis of a prospective cohort undergoing serial placental MRIs at two academic tertiary care centers. The population included 316 participants undergoing MRI up to three times throughout gestation. MRI was used to calculate placental volume in milliliters (ml). Placental-mediated adverse pregnancy outcome (cAPO) included preeclampsia with severe features, abnormal antenatal surveillance, and perinatal mortality. Serial measurements were grouped as time point 1 (TP1) <22 weeks, TP2 22 0/7-29 6/7 weeks, and TP3 >= 30 weeks. Mixed effects models compared change in placental volume across gestation between cAPO groups. Association between cAPO and placental volume was determined using logistic regression at each TP with discrimination evaluated using area under receiver operator curve (AUC). Placental volume was then added to known clinical predictive variables and evaluated with test characteristics and calibration. Results: 59 (18.7 %) of 316 participants developed cAPO. Placental volume growth across gestation was slower in the cAPO group (p < 0.001). Placental volume was lower in the cAPO group at all time points, and alone was moderately predictive of cAPO at TP3 (AUC 0.756). Adding placental volume to clinical variables had moderate discrimination at all time points, with strongest test characteristics at TP3 (AUC 0.792) with sensitivity of 77.5 % and specificity of 75.3 % at a predicted probability cutoff of 15 %. Discussion: MRI placental volume warrants further study for assessment of placental insufficiency, particularly later in gestation.

Low placental volume (PV) as assessed by ultrasound has been linked to adverse pregnancy outcomes (APOs), but the predictive power and test characteristics of PV in the second or third trimester remain poorly understood. We aimed to assess the relationship between PVs and APOs in a longitudinal cohort study. Secondary cohort analysis of the Placenta Imaging Study, a prospective study of pregnant individuals enriched for risk factors for preeclampsia and fetal growth restriction (FGR). Participants underwent up to 3 serial MRI studies during pregnancy and acquired MRI data was used to calculate the PV. Severe placental mediated APO composite (cAPO) included preeclampsia with severe features, preterm delivery due to FGR, perinatal death, need for delivery due to FGR/oligohydramnios/abnormal fetal testing, 5 min Apgar < 7, and/or neonatal intubation. Logistic regression predictive models were built containing a) PV only, b) clinical variables only, c) clinical variables + PV. These were built at time windows 22-29 6/7 (T2) and 30+ weeks gestation (T3). Model performance and test characteristics were reported after bootstrapping to reduce optimism. We chose a cutoff of 10% predicted probability for positive test given goal of minimizing missing individuals at risk. Data were available for 316 people for the clinical model, 269 for T2, and 240 for T3. 65 (20.6%) experienced cAPOs. Those with cAPOs were more likely to smoke, have chronic HTN, diabetes, or asthma, have less education, be unpartnered, and use government insurance. Median (interquartile ratio) PV was 454.7ml (357.4-559.6) at T2 with range 68.7-1139.3ml and 684.7ml (582.5-817.0) at T3 with range 231.6-1332.9ml (Figure 1). Both the PV only models and the clinical variables + PV models outperformed the clinical only model at T2 and T3 time points with regards to discrimination and specificity, although sensitivity was lower (Table 1). Incorporating PV measurements into assessment of pregnancy risk for APOs improves specificity and reduces false positives.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Maternal malnutrition increases fetal and neonatal morbidity, partly by affecting placental function and morphology, but its impact on placental hemodynamics are unknown. Our objective was to define the impact of maternal malnutrition on placental oxygen reserve and perfusion in vivo in a rhesus macaque model of protein restriction (PR) using advanced imaging. Animals were fed control (CON, 26% protein), 33% PR diet (17% protein), or a 50% PR diet (13% protein, n = 8/group) preconception and throughout pregnancy. Animals underwent Doppler ultrasound and fetal biometry followed by MRI at gestational days 85 (G85) and 135 (G135; term is G168). Pregnancy loss rates were 0/8 in CON, 1/8 in 33% PR, and 3/8 in 50% PR animals. Fetuses of animals fed a 50% PR diet had a smaller abdominal circumference (G135, p < 0.01). On MRI, placental blood flow was decreased at G135 (p < 0.05) and placental oxygen reserve was reduced (G85, p = 0.05; G135, p = 0.01) in animals fed a 50% PR diet vs. CON. These data demonstrate that a 50% PR diet reduces maternal placental perfusion, decreases fetal oxygen availability, and increases fetal mortality. These alterations in placental hemodynamics may partly explain human growth restriction and stillbirth seen with severe PR diets in the developing world.

Nonhuman primates are important preclinical models for translational, reproductive, and developmental science. Clinical evaluation of human fetal development is performed using standard sonographic-derived fetal biometry, assessments of amniotic fluid, and uteroplacental hemodynamics. These noninvasive in utero measurements provide important information regarding fetal growth and pregnancy well-being. Abnormalities in fetal growth, amniotic fluid volume, or placental vascular function are associated with placental insufficiency and adverse perinatal outcomes including stillbirth. The fetal biometric parameters most commonly assessed are biparietal diameter, head circumference, abdominal circumference, and femur diaphysis length. Evaluation of amniotic fluid volume includes measuring the fluid in four quadrants of the uterus to generate an Amniotic Fluid Index. Measures of uteroplacental hemodynamics typically include doppler assessment of the umbilical artery and ductus venosus, but can also include interrogation of the uterine artery and umbilical vein. In this study, we compile prenatal ultrasound data of fetal biometry, amniotic fluid measurements, and uteroplacental hemodynamics obtained from pregnancy studies conducted at the Oregon National Primate Research Center. The data included are from control unperturbed pregnant animals who have not undergone in utero experimental manipulations. This is the first report of comprehensive sonographic measurements following standardized clinical obstetric protocols utilized in rhesus macaques. The outcome is a large, prenatal ultrasound resource to be used by laboratory animal researchers in future nonhuman primate pregnancy studies for antenatal assessment.

Placental T2* (PT2*) measurements and serum placental growth factor (PlGF) are associated with adverse pregnancy outcomes (APOs) due to placental insufficiency. We aimed to determine the correlation between these two predictors and their performance in predictive models of APOs. Secondary cohort analysis of the Placenta Imaging Study, a prospective study of pregnant individuals enriched for risk factors for placental insufficiency. Participants had serial blood draws and up to three MRIs. Severe placental mediated APO composite (cAPO) included preeclampsia with severe features, perinatal death, delivery due placental insufficiency, and 5 min Apgar < 7. We used Pearson correlation to evaluate PlGF and PT2*. Logistic regression models were built containing clinical variables only, PlGF, PT2* z-score, and combinations of these. Measurements from T2 (22-29 6/7 wks) and T3 (30+ wks) were used. Model performance and test characteristics were reported after bootstrapping to reduce optimism and optimized model was chosen using these. Predicted probability of 10% was considered a positive test. 316 people were in the clinical model, 146 in T3 PlGF, and 207 in T3 PT2*. 65 (20.6%) experienced cAPO. Those with cAPOs were more likely to smoke, have chronic hypertension (cHTN), pregestational diabetes (DM), or asthma, have less education, be unpartnered, and use government insurance. Correlation between PlGF and PT2* was r=0.3473 (medium strength), p=< 0.001. PlGF and PT2* both had higher specificity for cAPOs than the clinical model but lower sensitivity (Table 1). The optimized model included cHTN, DM, tobacco use, body mass index < 18.5 kg/m2, post-term, T2 PT2*, and T3 PlGF measurements, with c-statistic of 0.787 after shrinkage, sensitivity of 83.3%, specificity of 55.7%, positive predictive value of 29.9%, and negative predictive value of 93.7% (Fig. 2). Incorporating PlGF and PT2* into predictive models of APOs improves discrimination, calibration, and test characteristics, particularly with regards to specificity. Such models may prove useful in risk stratification.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

BACKGROUND: Prenatal alcohol exposure is the most common cause of birth defects and intellectual disabilities and can increase the risk of stillbirth and negatively impact fetal growth. OBJECTIVE: To determine the effect of early prenatal alcohol exposure on nonhuman primate placental function and fetal growth. We hypothesized that early chronic prenatal alcohol would alter placental perfusion and oxygen availability that adversely affects fetal growth. STUDY DESIGN: Rhesus macaques self-administered 1.5 g/kg/d of ethanol (n=12) or isocaloric maltose-dextrin (n=12) daily before conception through the first 60 days of gestation (term is approximately 168 days). All animals were serially imaged with Doppler ultrasound to measure fetal biometry, uterine artery volume blood flow, and placental volume blood flow. Following Doppler ultrasound, all animals underwent both blood oxygenation leveledependent magnetic resonance imaging to characterize placental blood oxygenation and dynamic contrast-enhanced magnetic resonance imaging to quantify maternal placental perfusion. Animals were delivered by cesarean delivery for placental collection and fetal necropsy at gestational days 85 (n=8), 110 (n=8), or 135 (n=8). Histologic and RNA-sequencing analyses were performed on collected placental tissue. RESULTS: Placental volume blood flow was decreased at all gestational time points in ethanol-exposed vs control animals, but most significantly at gestational day 110 by Doppler ultrasound (P<.05). A significant decrease in total volumetric blood flow occurred in ethanol-exposed vs control animals on dynamic contrast-enhanced magnetic resonance imaging at both gestation days 110 and 135 (P<.05); moreover, a global reduction in T2*, high blood deoxyhemoglobin concentration, occurred throughout gestation (P<.05). Similarly, evidence of placental ischemic injury was notable by histologic analysis, which revealed a significant increase in microscopic infarctions in ethanol-exposed, not control, animals, largely present at middle to late gestation. Fetal biometry and weight were decreased in ethanol-exposed vs control animals, but the decrease was not significant. Analysis with RNA sequencing suggested the involvement of the inflammatory and extracellular matrix response pathways. CONCLUSION: Early chronic prenatal alcohol exposure significantly diminished placental perfusion at mid to late gestation and also significantly decreased the oxygen supply to the fetal vasculature throughout pregnancy, these findings were associated with the presence of microscopic placental infarctions in the nonhuman primate. Although placental adaptations may compensate for early environmental perturbations to fetal growth, placental blood flow and oxygenation were reduced, consistent with the evidence of placental ischemic injury.

Existing methods for evaluating in vivo placental function fail to reliably detect pregnancies at-risk for adverse outcomes prior to maternal and/or fetal morbidity. Here we report the results of a prospective dual-site longitudinal clinical study of quantitative placental T2* as measured by blood oxygen-level dependent magnetic resonance imaging (BOLD-MRI). The objectives of this study were: 1) to quantify placental T2* at multiple time points across gestation, and its consistency across sites, and 2) to investigate the association between placental T2* and adverse outcomes. 797 successful imaging studies, at up to three time points between 11 and 38 weeks of gestation, were completed in 316 pregnancies. Outcomes were stratified into three groups: (UN) uncomplicated/normal pregnancy, (PA) primary adverse pregnancy, which included hypertensive disorders of pregnancy, birthweight <5th percentile, and/or stillbirth or fetal death, and (SA) secondary abnormal pregnancy, which included abnormal prenatal conditions not included in the PA group such as spontaneous preterm birth or fetal anomalies. Of the 316 pregnancies, 198 (62.6%) were UN, 70 (22.2%) PA, and 48 (15.2%) SA outcomes. We found that the evolution of placental T2* across gestation was well described by a sigmoid model, with T2* decreasing continuously from a high plateau level early in gestation, through an inflection point around 30 weeks, and finally approaching a second, lower plateau in late gestation. Model regression revealed significantly lower T2* in the PA group than in UN pregnancies starting at 15 weeks and continuing through 33 weeks. T2* percentiles were computed for individual scans relative to UN group regression, and z-scores and receiver operating characteristic (ROC) curves calculated for association of T2* with pregnancy outcome. Overall, differences between UN and PA groups were statistically significant across gestation, with large effect sizes in mid- and late- pregnancy. The area under the curve (AUC) for placental T2* percentile and PA pregnancy outcome was 0.71, with the strongest predictive power (AUC of 0.76) at the mid-gestation time period (20–30 weeks). Our data demonstrate that placental T2* measurements are strongly associated with pregnancy outcomes often attributed to placental insufficiency. Trial registration: ClinicalTrials.gov: NCT02749851.

Objective Delta-like homolog 1 (DLK1) is a growth factor that is reduced in maternal sera in pregnancies with small for gestational age neonates. We sought to determine if DLK1 is associated with stillbirth (SB), with and without placental insufficiency. Study Design A nested case-control study was performed using maternal sera from a multicenter case-control study of SB and live birth (LB). SB and LB were stratified as placental insufficiency cases (small for gestational age <5% or circulatory lesions on placental histopathology) or normal placenta controls (appropriate for gestational age and no circulatory lesions). Enzyme-linked immunosorbent assay (ELISA) was used to measure DLK1. The mean difference in DLK1 was compared on the log scale in an adjusted linear regression model with pairwise differences, stratified by term/preterm deliveries among DLK1 results in the quantifiable range. In exploratory analysis, geometric means were compared among all data and the proportion of “low DLK1” (less than the median value for gestational age) was compared between groups and modeled using linear and logistic regression, respectively. Results Overall, 234 SB and 234 LB were analyzed; 246 DLK1 values were quantifiable within the standard curve. Pairwise comparisons of case and control DLK1 geometric means showed no significant differences between groups. In exploratory analysis of all data, adjusted analysis revealed a significant difference for the LB comparison only (SB: 71.9 vs. 99.1 pg/mL, p = 0.097; LB: 37.6 vs. 98.1 pg/mL, p = 0.005). In exploratory analysis of “low DLK1,” there was a significant difference between the odds ratio of having “low DLK1” between preterm cases and controls for both SB and LB. There were no significant differences in geometric means nor “low DLK1” between SB and LB. Conclusion In exploratory analysis, more placental insufficiency cases in preterm SB and LB had “low DLK1.” However, low DLK1 levels were not associated with SB. Key Points

Glial cell-derived neurotrophic factor (GDNF) promotes survival of neurons. In stillbirths, low levels of GDNF are associated with placental causes of stillbirth. Thus, our objective was to assess the association between serum GDNF during pregnancy and adverse pregnancy outcome (APO) due to placental disease. Nested case-control study of the Placenta Imaging Project. Primary APO cases were defined as hypertensive disorders of pregnancy, birthweight below the 5th %ile, and stillbirth. Controls were term pregnancies with birthweight 5th-95th %ile, without hypertensive disorders, chorioamnionitis, placental abruption, or fetal anomaly. The following APOs were defined as severe: preeclampsia with severe features, indicated delivery < 37 weeks, and stillbirth. GDNF levels were assessed via ELISA at three visits during pregnancy and at delivery and compared between groups with ANOVA. Proportion of GDNF values falling below, within, and above the standard ELISA curve were analyzed by c2 test. Mixed effects modeling was used to assess change in GDNF over gestational age by group. Serum was available for n=141-159 controls and n=40-57 cases, depending on visit number (visit 1 early second trimester, visit 2 late second trimester, visit 3 mid third trimester). Controls delivered at a mean of 39.3 weeks and APO cases at 36.2 weeks. Serum GDNF levels did not change throughout gestation for cases or controls (Figure 1). Although there was statistically significant variation in proportion of ELISA levels below, within, and above the standard ELISA curve by outcome group when cases were separated by severity (p=0.012), mean values were not different (Table 1), and there was not a logical dose response. Severe adverse cases had the highest levels, control groups had levels in the middle, and adverse non-severe cases had the lowest mean levels of GDNF across gestation, but there was significant overlap of values and groups were not statistically different. Serum GDNF values during pregnancy do not predict occurrence of APO related to placental disease.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Background Excessive gestational weight gain has been associated with increased total body fat (TBF), metabolic syndrome, and abdominal obesity. However, little is known about the relationship of gestational weight gain with changes in metabolically active visceral or ectopic (hepatic and skeletal muscle) lipid stores. Objectives In a prospective study of 50 healthy, pregnant women, we assessed whether changes in weight were associated with changes in total, visceral, and ectopic lipid stores. Methods Participants (ages 19-39) were primarily White (84%). The mean preconception BMI was 25.8 kg/m(2) (SD, 4.5 kg/m(2); min-max, 17.1-35.9 kg/m(2)). Measurements were completed at visits 1 and 2 at means of 16 and 34 weeks gestation, respectively, and included TBF using BOD POD; abdominal subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) using MRI; and intrahepatic lipids (IHL), intramyocellular lipids (IMCL), and extramyocellular lipids (EMCL) using magnetic resonance spectroscopy. We used paired t-tests to examine changes in adipose tissue and Pearson's correlation to examine associations of adipose tissue changes and weight changes. We also examined whether changes in adipose tissue stores differed by preconception BMI (normal, overweight, and obese), using 1-way ANOVA. Results The TBF (mean change, +3.5 kg; 95% CI: 2.4-4.6 kg), SAT (mean change, +701 cm(3); 95% CI: 421-981 cm(3)), VAT (mean change, +275 cm(3); 95% CI: 170-379 cm(3)), and IHL (percentage water peak; median, +0.15; IQR = -0.01 to 0.32) values increased significantly; the IMCL and EMCL values did not change. Changes varied by BMI strata, with the least increase (or, for SAT, net loss) among women with obesity. Weight change was positively correlated with changes in TBF (r = 0.83; P < 0.001), SAT (r = 0.74; P < 0.001), and VAT (r = 0.63; P < 0.001) but not significantly correlated with changes in ectopic lipids (IHL, IMCL, and EMCL; -0.14 r < 0.26). Conclusions Preferential deposition of adipose tissue to the viscera in pregnancy, as seen in our sample, could serve an important metabolic function; however, excessive deposition in this region could negatively affect maternal health.

The placenta is a remarkable organ that coordinates and regulates maternal-fetal interactions during pregnancy to optimize fetal development. A host of obstetric complications are associated with placental dysfunction, and existing methods for evaluating in vivo placental function fail to reliably detect at-risk pregnancies prior to maternal or fetal morbidity. Although routinely used as a monitoring tool, the predictive power of ultrasound for identifying compromised pregnancies is poor. Recent preclinical studies performed in our laboratory, using blood oxygen-level dependent magnetic resonance imaging (BOLD-MRI) in the pregnant nonhuman primate (NHP), established a strong correlation between placental T2* values and maternal-fetal oxygen transport. Here we extend this work to a large, longitudinal, two-site study of quantitative in vivo T2* mapping in human pregnancies across 11 to 38 weeks of gestation to characterize the evolution of placental oxygenation in uncomplicated pregnancies and to elucidate the relationship between aberrant placental T2* and adverse obstetric outcomes attributable to placental dysfunction. This methodology has high discriminatory power and strong potential diagnostic utility.

Zika virus (ZIKV) is an arthropod-borne Flavivirus that can also be transmitted vertically from infected mother to fetus. Infection of the fetus during pregnancy can lead to congenital malformations and severely impact fetal brain development causing a myriad of diseases now labeled Congenital Zika Syndrome (CZS). The mechanisms by which ZIKV crosses the placenta into the fetal circulation and the extent of ZIKV-induced changes remain unclear. We have previously shown that ZIKV infection of pregnant rhesus macaques results in abnormal oxygen transport across the placenta which may promote uterine vasculitis and placental villous damage. Changes in immune cell frequencies and activation status were also detected, as were distinct changes in the proportions of CD14+ cell subsets with an altered ratio of classical to non-classical CD14+ monocyte cells in both the maternal decidua and placental villous from ZIKV-infected animals compare to uninfected controls. In the current study, we performed single cell RNA sequencing on CD14+ cells isolated from the decidua of animals that were ZIKV infected at 31, 51, or 115 days of gestation (where term is ~168 days) compared to pregnant, time-matched uninfected controls. Bioinformatic analysis identified unique transcriptional phenotypes between CD14+ cells of infected and uninfected animals suggesting a distinct and sustained difference in transcriptomes between infected and uninfected CD14+ cells derived from the decidua. The timing of ZIKV infection had no effect on the CD14+ cell transcriptional profiles. Interestingly, ZIKV infection caused changes in expression of genes in pathways related to cellular stress and metabolism as well as immune response activation. Type 1 interferon response genes (ISGs) were among those that were differentially expressed following infection and these included members of the ISG12 family, IFI27 and IFI6. These ISGs have been recently described as effectors of the IFN response to flaviviruses. Supplementing our animal findings, in CD14+ cells isolated from human placenta, ZIKV infection similarly induced the expression of IFI27 and IFI6. Overall, our results showed that ZIKV infection during pregnancy induces the stable expression of antiviral genes within CD14+ cells of the placenta, which may provide an immune shield to protect the placenta from further infection and damage.

Introduction: Extracellular vesicles derived from the fetal central nervous system (FCNSEs) can be purified from maternal serum or plasma using the protein Contactin-2/TAG1that is expressed almost exclusively by developing neurons in the hippocampus, cerebral cortex and cerebellum. We hypothesized that fetal CNSEs could be used to non-invasively detect and quantify viral mediated in-utero brain injury in the first trimester. Materials and Methods: First trimester maternal samples were collected from a human clinical population infected with primary cytomegalovirus (CMV) and a non-human primate model of Zika (ZIKV) infection. In the CMV cohort, a nested case control study was performed comparing pregnancies with and without fetal infection. Cases of fetal infection were further subdivided into those with and without adverse neurologic outcome. ZIKV samples were collected serially following maternal inoculation or saline. All ZIKV cases had histopathologic findings on necropsy. Serum was precipitated with ExoQuick solution and FCEs were isolated with biotinylated anti-Contactin-2/TAG1 antibody-streptavidin matrix immunoabsorption. FCE Synaptopodin (SYNPO) and Neurogranin (NG) protein levels were measured using standard ELISA kits and normalized to the exosome marker CD81. Results: Fetal CNSE SYNPO and NG were significantly reduced in cases of first trimester fetal CMV infection compared to those with infection limited to the mother but could not discriminate between fetal infection with and without adverse neurologic outcome. Following ZIKV inoculation, fetal CNSE SYNPO was reduced by 48 h and significantly reduced by day 4. Discussion: These data are the first to suggest that first trimester non-invasive diagnosis of fetal viral infection is possible. Fetal CNSEs have the potential to augment clinical and pre-clinical studies of perinatal viral infection. Serial sampling may be needed to discriminate between fetuses that are responding to treatment and/or recovering due to innate defenses and those that have ongoing neuronal injury. If confirmed, this technology may advance the paradigm of first trimester prenatal diagnosis and change the calculus for the cost benefit of CMV surveillance programs in pregnancy.

BACKGROUND: Ureaplasma parvum infection is a prevalent cause of intrauterine infection associated with preterm birth, preterm premature rupture of membranes, fetal inflammatory response syndrome, and adverse postnatal sequelae. Elucidation of diagnostic and treatment strategies for infection-associated preterm labor may improve perinatal and long-term outcomes for these cases. OBJECTIVE: This study assessed the effect of intraamniotic Ureaplasma infection on fetal hemodynamic and cardiac function and the effect of maternal antibiotic treatment on these outcomes. STUDY DESIGN: Chronically catheterized pregnant rhesus monkeys were assigned to control (n=6), intraamniotic inoculation with Ureaplasma parvum (107 colony-forming units/mL, n=15), and intraamniotic infection plus azithromycin treatment (12.5 mg/kg twice a day intravenously, n=8) groups. At approximately 135 days' gestation (term=165 days), pulsed and color Doppler ultrasonography was used to obtain measurements of fetal hemodynamics (pulsatility index of umbilical artery, ductus venosus, descending aorta, ductus arteriosus, aortic isthmus, right pulmonary artery, middle cerebral artery and cerebroplacental ratio, and left and right ventricular cardiac outputs) and cardiac function (ratio of peak early vs late transmitral flow velocity [marker of ventricular function], Tei index [myocardial performance index]). These indices were stratified by amniotic fluid proinflammatory mediator levels and cardiac histology. RESULTS: Umbilical and fetal pulmonary artery vascular impedances were significantly increased in animals from the intraamniotic inoculation with Ureaplasma parvum group (P<.05). Azithromycin treatment restored values to control levels. Amniotic fluid prostaglandin F2 alpha levels were significantly higher in animals with abnormal umbilical artery pulsatility index (>1.1) than in those with normal blood flow (P<.05; Spearman r=0.6, P<.05). In the intraamniotic inoculation with Ureaplasma parvum group, left ventricular cardiac output was significantly decreased (P<.001), and more animals had abnormal right-to-left ventricular cardiac output ratios (defined as >1.6, P<.05). Amniotic fluid interleukin-6 concentrations were elevated in cases of abnormal right-to-left ventricular cardiac output ratios compared with those in normal cases (P<.05). CONCLUSION: Fetal hemodynamic alterations were associated with intraamniotic Ureaplasma infection and ameliorated after maternal antibiotic treatment. Doppler ultrasonographic measurements merit continuing investigation as a diagnostic method to identify fetal cardiovascular and hemodynamic compromise associated with intrauterine infection or inflammation and in the evaluation of therapeutic interventions or clinical management of preterm labor.

Zika virus infection during pregnancy is associated with miscarriage and with a broad spectrum of fetal and neonatal developmental abnormalities collectively known as congenital Zika syndrome (CZS). Symptomology of CZS includes malformations of the brain and skull, neurodevelopmental delay, seizures, joint contractures, hearing loss and visual impairment. Previous studies of Zika virus in pregnant rhesus macaques (Macaca mulatta) have described injury to the developing fetus and pregnancy loss, but neonatal outcomes following fetal Zika virus exposure have yet to be characterized in nonhuman primates. Herein we describe the presentation of rhesus macaque neonates with a spectrum of clinical outcomes, including one infant with CZS-like symptoms including cardiomyopathy, motor delay and seizure activity following maternal infection with Zika virus during the first trimester of pregnancy. Further characterization of this neonatal nonhuman primate model of gestational Zika virus infection will provide opportunities to evaluate the efficacy of pre- and postnatal therapeutics for gestational Zika virus infection and CZS.

Delta-like homolog 1 (DLK1) is a growth factor important for adipose homeostasis and is reduced in maternal sera in pregnancies with small for gestational age (SGA) neonates. We sought to determine if DLK1 is associated with stillbirth, with and without placental insufficiency. A nested case-control study was performed using maternal sera from a multicenter, prospective, case-control study of stillbirths (SB) and live births (LB). Sera were collected at the time of delivery and stored at -80°C. SB and LB were stratified as placental insufficiency cases (PI; SGA < 5% or circulatory lesions on placental histopathology) or normal placenta controls (NP; AGA and no circulatory lesions). ELISA was used to measure DLK1. Proportion of “low DLK1” (less than the median value for GA) were compared between groups and modeled in logistic regression. The mean difference in DLK1 was compared on the log scale in an adjusted linear regression model with pairwise differences, stratified by term/preterm deliveries among DLK1 results in the quantifiable range. 234 SB and 234 LB were analyzed; 246 DLK1 values were quantifiable within the standard curve. (Figure 1). In analysis of all data, SB and LB had similar proportions of “low DLK1” (term aOR 0.54, 95% CI 0.25-1.15; preterm aOR 0.56, 95% CI 0.27-1.16, Figure 2), and mean DLK1 (term p=0.103, preterm p=0.096). Preterm PI was more likely to have “low DLK1” than NP for SB and LB (SB aOR 2.33, 95% CI 1.12-4.84; LB aOR 5.89 95% CI 1.68-20.6, Figure 2). In DLK1 samples within the quantifiable range, pairwise comparison of DLK1 means were not significantly different in unadjusted or adjusted models. Among cases, mean DLK1 in cases with both circulatory lesions and < 5% SGA, were similar to cases meeting only one criteria (188.7pg/ml, 95% CI 114.1-312.2 vs 239.4pg/ml, 95% CI 211.8-270.5, p=0.36), adjusted for gestational age and SB/LB. More PI cases in preterm SB and LB had “low DLK1” and lower values of DLK1 compared to NP. Low DLK1 levels were associated with PI, but not SB. DLK1 deserves evaluation as a marker for placental insufficiency.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

We aim to measure the relationship between angiogenesis related proteins in serum and adverse pregnancy outcomes due to placental insufficiency. Samples from two prospective studies (S1, a case-control study of stillbirth and S2, a prospective imaging study of the placenta in low and high risk pregnancies) were evaluated. For S1, stillbirths due to placental causes (n=86) were compared to gestational age matched stillbirths due to non-placental causes (n=86). These samples were obtained at time of delivery and cause of death was assigned using the INCODE classification system. For S2, serum samples were obtained from 26-39 weeks. Pregnancies with adverse placental outcomes such as preterm preeclampsia, birth weight < 10th percentile, or stillbirth (n=13), were compared to pregnancies with uncomplicated (n=22). For both S1 and S2, the Proteome Profiler Human Angiogenesis Array Kit, (R&D Systems, USA, ARY007) was used to assess the presence of 55 angiogenesis related proteins using a membrane-based sandwich immunoassay. Because samples were pooled for these initial exploratory studies, statistical significance testing was not performed. Angiogenesis related proteins were considered candidates for future analysis if the ratio between groups was ≤0.5 or ≥2.0 (a two-fold decrease or increase). In S1, the following had values of ≤0.5, suggesting potential down regulation in placental insufficiency: angiopoietin-2, angiostatin/plasminogen, coagulation factor III, EG-VEGF, FGF-acidic, FGF-7, GDNF, GM-CSF, IL-1β, IL-8, TGF-β1, PD-ECGF, PlGF, thrombospondin-2, uPA, vasohibin, VEGF, VEGF-C. There were no proteins with ratio ≥2.0. In S2, the following had values of ≤0.5, suggesting potential down regulation in placental insufficiency: amphiregulin, FGF-7, GDNF, IL-8, PlGF, VEGF-C. The following proteins had values of ≥2.0, suggesting potential up regulation in placental insufficiency: activin A, ADAMTS-1 (Figure 1). FGF-7, GDNF, and VEGF-C showed lower expression in both S1 and S2, suggesting they are possible biomarker candidates of placental insufficiency.

Women in low- and middle-income countries frequently consume a protein-deficient diet during pregnancy and breastfeeding. The effects of gestational malnutrition on fetal and early postnatal development can have lasting adverse effects on offspring metabolism. Expanding on previous studies in rodent models, we utilized a nonhuman primate model of gestational and early-life protein restriction (PR) to evaluate effects on the organ development and glucose metabolism of juvenile offspring. Offspring were born to dams that had consumed a control diet containing 26% protein or a PR diet containing 13% protein. Offspring were maintained on the PR diet and studied [body and serum measurements, intravenous glucose tolerance tests (ivGTTs), and dual-energy X-ray absorptiometry scans] up to 7 mo of age, at which time tissues were collected for analysis. PR offspring had age-appropriate body weight and were euglycemic but exhibited elevated fasting insulin and reduced initial, but increased total, insulin secretion during an ivGTT at 6 mo of age. No changes were detected in pancreatic islets of PR juveniles; however, PR did induce changes, including reduced kidney size, and changes in liver, adipose tissue, and muscle gene expression in other peripheral organs. Serum osteocalcin was elevated and bone mineral content and density were reduced in PR juveniles, indicating a significant impact of PR on early postnatal bone development.