Jamie O. Lo

Quantitative assessment of placental perfusion by contrast-enhanced ultrasound in macaques and human subjects

BACKGROUND The uteroplacental vascular supply is a critical determinant of placental function and fetal growth. Current methods for the in vivo assessment of placental blood flow are limited. OBJECTIVE We demonstrate the feasibility of the use of contrast-enhanced ultrasound imaging to visualize and quantify perfusion kinetics in the intervillous space of the primate placenta. STUDY DESIGN Pregnant Japanese macaques were studied at mid second trimester and in the early third trimester. Markers of injury were assessed in placenta samples from animals with or without contrast-enhanced ultrasound exposure (n = 6/group). Human subjects were recruited immediately before scheduled first-trimester pregnancy termination. All studies were performed with maternal intravenous infusion of lipid-shelled octofluoropropane microbubbles with image acquisition with a multipulse contrast-specific algorithm with destruction-replenishment analysis of signal intensity for assessment of perfusion. RESULTS In macaques, the rate of perfusion in the intervillous space was increased with advancing gestation. No evidence of microvascular hemorrhage or acute inflammation was found in placental villous tissue and expression levels of caspase-3, nitrotyrosine and heat shock protein 70 as markers of apoptosis, nitrative, and oxidative stress, respectively, were unchanged by contrast-enhanced ultrasound exposure. In humans, placental perfusion was visualized at 11 weeks gestation, and preliminary data reveal regional differences in intervillous space perfusion within an individual placenta. By electron microscopy, we demonstrate no evidence of ultrastructure damage to the microvilli on the syncytiotrophoblast after first-trimester ultrasound studies. CONCLUSIONS Use of contrast-enhanced ultrasound did not result in placental structural damage and was able to identify intervillous space perfusion rate differences within a placenta. Contrast-enhanced ultrasound imaging may offer a safe clinical tool for the identification of pregnancies that are at risk for vascular insufficiency; early recognition may facilitate intervention and improved pregnancy outcomes.

Functional imaging of the nonhuman primate Placenta with endogenous blood oxygen level–dependent contrast

To characterize spatial patterns of T2* in the placenta of the rhesus macaque (Macaca mulatta), to correlate these patterns with placental perfusion determined using dynamic contrast‐enhanced MRI (DCE‐MRI), and to evaluate the potential for using the blood oxygen level–dependent effect to quantify placental perfusion without the use of exogenous contrast reagent.

Vitamin C supplementation ameliorates the adverse effects of nicotine on placental hemodynamics and histology in nonhuman primates

OBJECTIVE We previously demonstrated that prenatal nicotine exposure decreases neonatal pulmonary function in nonhuman primates, and maternal vitamin C supplementation attenuates these deleterious effects. However, the effect of nicotine on placental perfusion and development is not fully understood. This study utilizes noninvasive imaging techniques and histological analysis in a nonhuman primate model to test the hypothesis that prenatal nicotine exposure adversely effects placental hemodynamics and development but is ameliorated by vitamin C. STUDY DESIGN Time-mated macaques (n = 27) were divided into 4 treatment groups: control (n = 5), nicotine only (n = 4), vitamin C only (n = 9), and nicotine plus vitamin C (n = 9). Nicotine animals received 2 mg/kg per day of nicotine bitartrate (approximately 0.7 mg/kg per day free nicotine levels in pregnant human smokers) from days 26 to 160 (term, 168 days). Vitamin C groups received ascorbic acid at 50, 100, or 250 mg/kg per day with or without nicotine. All underwent placental dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) at 135-140 days and Doppler ultrasound at 155 days to measure uterine artery and umbilical vein velocimetry and diameter to calculate uterine artery volume blood flow and placental volume blood flow. Animals were delivered by cesarean delivery at 160 days. A novel DCE-MRI protocol was utilized to calculate placental perfusion from maternal spiral arteries. Placental tissue was processed for histopathology. RESULTS Placental volume blood flow was significantly reduced in nicotine-only animals compared with controls and nicotine plus vitamin C groups (P = .03). Maternal placental blood flow was not different between experimental groups by DCE-MRI, ranging from 0.75 to 1.94 mL/mL per minute (P = .93). Placental histology showed increased numbers of villous cytotrophoblast cell islands (P < .05) and increased syncytiotrophoblast sprouting (P < .001) in nicotine-only animals, which was mitigated by vitamin C. CONCLUSION Prenatal nicotine exposure significantly decreased fetal blood supply via reduced placental volume blood flow, which corresponded with placental histological findings previously associated with cigarette smoking. Vitamin C supplementation mitigated the harmful effects of prenatal nicotine exposure on placental hemodynamics and development, suggesting that its use may limit some of the adverse effects associated with smoking during pregnancy.

Zika virus infection in pregnant rhesus macaques causes placental dysfunction and immunopathology

Zika virus (ZIKV) infection during pregnancy leads to an increased risk of fetal growth restriction and fetal central nervous system malformations, which are outcomes broadly referred to as the Congenital Zika Syndrome (CZS). Here we infect pregnant rhesus macaques and investigate the impact of persistent ZIKV infection on uteroplacental pathology, blood flow, and fetal growth and development. Despite seemingly normal fetal growth and persistent fetal-placenta-maternal infection, advanced non-invasive in vivo imaging studies reveal dramatic effects on placental oxygen reserve accompanied by significantly decreased oxygen permeability of the placental villi. The observation of abnormal oxygen transport within the placenta appears to be a consequence of uterine vasculitis and placental villous damage in ZIKV cases. In addition, we demonstrate a robust maternal-placental-fetal inflammatory response following ZIKV infection. This animal model reveals a potential relationship between ZIKV infection and uteroplacental pathology that appears to affect oxygen delivery to the fetus during development.Zika virus infection during pregnancy can result in birth defects, but underlying pathogenesis at the maternal-fetal interface is unclear. Here, the authors use non-invasive in vivo imaging of Zika-infected rhesus macaques and show that infection results in abnormal oxygen transport across the placenta.

First trimester alcohol exposure alters placental perfusion and fetal oxygen availability affecting fetal growth and development in a non-human primate model

Background: Prenatal alcohol exposure leads to impaired fetal growth, brain development, and stillbirth. Placental impairment likely contributes to these adverse outcomes, but the mechanisms and specific vasoactive effects of alcohol that links altered placental function to impaired fetal development remain areas of active research. Objective: Recently, we developed magnetic resonance imaging techniques in nonhuman primates to characterize placental blood oxygenation through measurements of T2* and perfusion using dynamic contrast‐enhanced magnetic resonance imaging. The objective of this study was to evaluate the effects of first‐trimester alcohol exposure on macaque placental function and to characterize fetal brain development in vivo. Study Design: Timed‐pregnant Rhesus macaques (n=12) were divided into 2 groups: control (n=6) and ethanol exposed (n=6). Animals were trained to self‐administer orally either 1.5 g/kg/d of a 4% ethanol solution (equivalent to 6 drinks/d) or an isocaloric control fluid from preconception until gestational day 60 (term is G168). All animals underwent Doppler ultrasound scanning followed by magnetic resonance imaging that consisted of T2* and dynamic contrast‐enhanced measurements. Doppler ultrasound scanning was used to measure uterine artery and umbilical vein velocimetry and diameter to calculate uterine artery volume blood flow and placental volume blood flow. After noninvasive imaging, animals underwent cesarean delivery for placenta collection and fetal necropsy at gestational day 110 (n=6) or 135 (n=6). Results: Fetal weight and biparietal diameter were significantly smaller in ethanol‐exposed animals compared with control animals at gestational day 110. By Doppler ultrasound scanning, placental volume blood flow was significantly lower (P=.04) at gestational day 110 in ethanol‐exposed vs control animals. A significant reduction in placental blood flow was evident by dynamic contrast‐enhanced magnetic resonance imaging. As we demonstrated recently, T2* values vary throughout the placenta and reveal gradients in blood deoxyhemoglobin concentration that range from highly oxygenated blood (long T2*) proximal to spiral arteries to highly deoxygenated blood (short T2*). Distributions of T2*throughout the placenta show significant global reduction in T2* (and hence high blood deoxyhemoglobin concentration) in ethanol‐exposed vs control animals at gestational day 110 (P=.02). Fetal brain measurements indicated impaired growth and development at gestational day 110, but less so at gestational day 135 in ethanol‐exposed vs control animals. Conclusion: Chronic first‐trimester ethanol exposure significantly reduces placental perfusion and oxygen supply to the fetal vasculature later in pregnancy. These perturbations of placental function are associated with fetal growth impairments. However, differences between ethanol‐exposed and control animals in placental function and fetal developmental outcomes were smaller at gestational day 135 than at gestational day 110. These findings are consistent with placental adaptation to early perturbations that allow for compensated placental function and maintenance of fetal growth.

Proteomic analysis of cervical vaginal fluid proteins among women in recurrent preterm labor

Abstract Objective: Proteomic analysis of four cervical-vaginal fluid (CVF) proteins to identify biomarkers of recurrent preterm birth (rPTB) in at-risk women prior to onset of preterm labor. Methods: Nested case control study from 2007 to 2011 of women with prior spontaneous preterm birth(s) (PTB) who underwent serial CVF sampling. Mass spectrometry analysis was used and ELISA analysis was performed to validate candidates. Results: 108 patients were enrolled and 10 cases and 20 gestational age matched controls were analyzed after exclusions. Of 748 CVF proteins identified, 72 had statistically significant (p < 0.05) expression differences and 38 were highly differentially expressed (p < 0.01). Four candidate proteins were abundant and involved in immune/inflammatory response, but ELISA analysis did not confirm altered expression patterns. Conclusion: The lack of confirmation of potential biomarkers identified by mass spectrometry and ELISA demonstrates the challenges of validating PTB biomarkers and suggests that a panel of biomarkers would improve the predictive value of CVF testing.

Novel Detection of Placental Insufficiency by Magnetic Resonance Imaging in the Nonhuman Primate

The placenta is a vital organ necessary for healthy fetal development. Placental insufficiency creates an in utero environment where the fetus is at risk of insufficient oxygen or nutrient exchange. This is primarily caused by impairment of either maternal or fetal circulation or vascular thrombosis such as placental infarction. As a result of placental dysfunction, affected fetuses may be growth restricted, neurologically impaired, and at risk of increased morbidity and mortality. In a cohort of 4 pregnant Rhesus macaques, we describe antenatal detection of naturally occurring intrauterine growth restriction (IUGR) and aberrant fetal neurodevelopment in 1 animal. Abnormal growth parameters were detected by Doppler ultrasound, and vascular insufficiency in the intervillous space was characterized by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Furthermore, placental oxygen reserve was shown to be reduced compared to control animals by measurements of placental water T 2*. To characterize the effects of IUGR on fetal brain development, T 2 and diffusion anisotropy images of the fetal brain were acquired in utero. Reduced brain volume and cerebral cortical surface area were apparent macroscopically. Microstructural abnormalities within the developing white matter and cerebral cortex were also observed through analysis of water diffusion anisotropy. After delivery by cesarean section, pathological examination confirmed placental insufficiency with hypoxia. These findings exemplify how DCE-MRI and T2 *-based measurements of blood oxygenation within the placenta can provide noninvasive imaging methods for assessing in vivo placental health to potentially identify pregnancies affected by placental insufficiency and abnormal fetal neurodevelopment prior to the onset of fetal and neonatal distress.

Adverse Placental Perfusion and Pregnancy Outcomes in a New Nonhuman Primate Model of Gestational Protein Restriction

Maternal malnutrition during pregnancy impacts fetal growth, with developmental consequences that extend to later life outcomes. In underdeveloped countries, this malnutrition typically takes the form of poor dietary protein content and quality, even if adequate calories are consumed. Here, we report the establishment of a nonhuman primate model of gestational protein restriction (PR) in order to understand how placental function and pregnancy outcomes are affected by protein deficiency. Rhesus macaques were assigned to either a control diet containing 26% protein or switched to a 13% PR diet prior to conception and maintained on this PR diet throughout pregnancy. Standard fetal biometry, Doppler ultrasound of uteroplacental blood flow, ultrasound-guided amniocentesis, and contrast-enhanced ultrasound (CE-US) to assess placental perfusion were performed mid-gestation (gestational day 85 [G85] where term is G168) and in the early third trimester (G135). Our data demonstrate that a 50% reduction in dietary protein throughout gestation results in reduced placental perfusion, fetal growth restriction, and a 50% rate of pregnancy loss. In addition, we demonstrate reduced total protein content and evidence of fetal hypoxia in the amniotic fluid. This report highlights the use of CE-US for in vivo assessment of placental vascular function. The ability to detect placental dysfunction, and thus a compromised pregnancy, early in gestation, may facilitate the development of interventional strategies to optimize clinical care and improve long-term offspring outcomes, which are future areas of study in this new model.

Trends in Obesity and Implications for the Fetus

Obesity is a worldwide pandemic. The high prevalence of obesity and the projected increasing trend have adverse implications for pregnant women including thromboembolic complications, gestational diabetes, hypertension, cesarean section, maternal hemorrhage, and infection. Maternal obesity has a measureable impact on infant and future adult health. Offspring are at increased risk for spontaneous miscarriage, fetal malformations, fetal macrosomia, stillbirth, and preterm delivery. This review focuses on the trends in maternal obesity and its implications for maternal and fetal health as well as evidence suggesting that the pre-pregnancy and the prenatal period provide a window of opportunity to intervene and curtail the intergenerational cycle of obesity.

Maternal high-fat diet reversal improves placental hemodynamics in a nonhuman primate model of diet-induced obesity

BackgroundIn a Japanese macaque model of diet-induced obesity, we have previously demonstrated that consumption of a high-fat, “Western-style” diet (WSD) is associated with placental dysfunction and adverse pregnancy outcomes, independent of an obese maternal phenotype. Specifically, we have reported decreased uterine placental blood flow and increased inflammation with maternal WSD consumption. We also previously investigated the use of a promising therapeutic intervention that mitigated the adverse placental effects of a WSD but had unexpected detrimental effects on fetal pancreatic development. Thus, the objective of the current study was to determine whether simple preconception diet reversal (REV) would improve placental function.MethodsFemale Japanese macaques were divided into three groups: REV animals (n = 5) were switched from a chronic WSD (36% fat) to a low fat, CON diet (14% fat) prior to conception and throughout pregnancy. The CON (n = 6) and WSD (n = 6) cohorts were maintained on their respective diets throughout pregnancy. Maternal body weight and composition were regularly assessed and advanced noninvasive imaging was performed at midgestation (gestational day 90, G90, or 0.5 of gestation, where full term is G175), and G129, 1 day prior to C-section delivery at G130 (0.75 of gestation). Imaging studies comprised Doppler ultrasound (US), contrast-enhanced US, and dynamic contrast-enhanced magnetic resonance imaging to assess uteroplacental hemodynamics and maternal-side placental perfusion.ResultsDietary intervention resulted in significant maternal weight loss prior to pregnancy, and improved lean to fat mass ratio. By advanced imaging we demonstrated that a chronic WSD led to decreased blood flow velocity in the intervillous space, delayed blood flow transfer through the maternal spiral arteries, and reduced total placental blood flow compared to CON fed animals. Dietary reversal ameliorated these concerning derangements, restoring these hemodynamic parameters to CON levels.ConclusionsPreconception dietary modification has beneficial effects on the maternal metabolic phenotype, and results in improved placental hemodynamics.