Magnetic resonance imaging of placental intralobule structure and function in a preclinical nonhuman primate model†.

Although the central role of adequate blood flow and oxygen delivery is known, the lack of optimized imaging modalities to study placental structure has impeded our understanding of its vascular function. Magnetic resonance imaging is increasingly being applied in this field, but gaps in knowledge remain, and further methodological developments are needed. In particular, the ability to distinguish maternal from fetal placental perfusion and the understanding of how individual placental lobules are functioning are lacking. The potential clinical benefits of developing noninvasive tools for the in vivo assessment of blood flow and oxygenation, two key determinants of placental function, are tremendous. Here, we summarize a number of structural and functional magnetic resonance imaging techniques that have been developed and applied in animal models and studies of human pregnancy over the past decade. We discuss the potential applications and limitations of these approaches. Their combination provides a novel source of contrast to allow analysis of placental structure and function at the level of the lobule. We outline the physiological mechanisms of placental T2 and T2* decay and devise a model of how tissue composition affects the observed relaxation properties. We apply this modeling to longitudinal magnetic resonance imaging data obtained from a preclinical pregnant nonhuman primate model to provide initial proof-of-concept data for this methodology, which quantifies oxygen transfer and placental structure across and between lobules. This method has the potential to improve our understanding and clinical management of placental insufficiency once validation in a larger nonhuman primate cohort is complete.

Rhesus macaque fetal and placental growth demographics: A resource for laboratory animal researchers.

Rhesus macaques (Macaca mulatta) are amongst the most common nonhuman primate species used in biomedical research. These animals provide a precious resource for translational studies and opportunities to maximize rhesus data use are encouraged. Here we compile data produced from 10 years of investigator-driven pregnancy studies conducted at the Oregon National Primate Research Center (ONPRC). All pregnancies were generated within the consistent and reproducible protocols of the ONPRC time-mated breeding program. The data included are from control animals who did not experience in utero perturbations or experimental manipulations. A total of 86 pregnant rhesus macaques were delivered by cesarean section over a range of gestational days (G) 50 to G159 (where term is G165 ± 10 days in the rhesus macaque), with subsequent immediate tissue harvesting following standardized protocols. Fetal and placental growth measures, and all major organ weights are reported. All data are presented relative to gestational age for the entire cohort and in addition, data are stratified by fetal sex. The outcome is a large reference resource for use by laboratory animal researchers in future comparative fetal development studies.

Ultrasound evaluation of normal rhesus macaque fetal biometry and uteroplacental hemodynamics.

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.

Utilizing Contrast-Enhanced Ultrasonography with Phosphatidylserine Microbubbles to Detect Placental Inflammation in Rhesus Macaques.

The ability to comprehensively monitor physiological and detect pathophysiologic processes early during pregnancy can reduce maternal and fetal morbidity and mortality. Contrast-enhanced ultrasound (CEUS) is a non-invasive imaging technology that utilizes the acoustic detection of microbubbles to examine vascular spaces. Furthermore, microbubbles conjugated to specific compounds can focus studies on precise physiological pathways. We hypothesized that CEUS with phosphatidylserine microbubbles (MB-PS) could be employed to monitor placental inflammation. We tested this hypothesis in rhesus macaques (Macaca mulatta), a translational and relevant animal model of human placental health. As placental inflammation impacts many at-risk pregnancies, we performed CEUS with MB-PS in pregnant macaques fed a high-fat diet (e.g., a western-style diet, WSD) in the presence or absence of testosterone (T) to mimic the increased risk of polycystic ovary syndrome and subfertility. We have previously demonstrated a placental inflammation phenotype in this model, and, thus, we related the MB-PS CEUS signal intensity to placental inflammation markers: selectin p and angiopoietins. Testosterone exposure increased the MB-PS signal in the placental microcirculation on the maternal side compared to control animals. We found that T increased placental weight and decreased angiopoietin 2 (ANGPT2) immunoreactivity. Furthermore, a significant inverse correlation was found between MB-PS signal and ANGPT2. This indicated that CEUS with MB-PS can be used to monitor placental parameters. We propose that CEUS with MB-PS could aid in the identification of pregnancies at risk of placental vascular compromise.

Effects of early daily alcohol exposure on placental function and fetal growth in a rhesus macaque model.

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 level-dependent 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.

Animal Models Evaluating the Impact of Prenatal Exposure to Tobacco and Marijuana.

Within this review, the literature and outcomes from animal models of maternal marijuana use and cigarette smoking are summarized. The existing data demonstrate that prenatal marijuana and nicotine exposure both have multifaceted adverse effects on maternal, gestational, placental, and fetal outcomes. These include placental function and development, fetal growth and birth weight, and offspring neurodevelopment.

DLK1: A Novel Biomarker of Placental Insufficiency in Stillbirth and Live Birth.

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: · Maternally circulating DLK1 is correlated with placental insufficiency.. · Maternally circulating DLK1 is not correlated with SB.. · DLK1 is a promising marker for placental insufficiency..

Transcriptomic analysis of primate placentas and novel rhesus trophoblast cell lines informs investigations of human placentation.

BACKGROUND: Proper placentation, including trophoblast differentiation and function, is essential for the health and well-being of both the mother and baby throughout pregnancy. Placental abnormalities that occur during the early stages of development are thought to contribute to preeclampsia and other placenta-related pregnancy complications. However, relatively little is known about these stages in humans due to obvious ethical and technical limitations. Rhesus macaques are considered an ideal surrogate for studying human placentation, but the unclear translatability of known human placental markers and lack of accessible rhesus trophoblast cell lines can impede the use of this animal model. RESULTS: Here, we performed a cross-species transcriptomic comparison of human and rhesus placenta and determined that while the majority of human placental marker genes (HPGs) were similarly expressed, 952 differentially expressed genes (DEGs) were identified between the two species. Functional enrichment analysis of the 447 human-upregulated DEGs, including ADAM12, ERVW-1, KISS1, LGALS13, PAPPA2, PGF, and SIGLEC6, revealed over-representation of genes implicated in preeclampsia and other pregnancy disorders. Additionally, to enable in vitro functional studies of early placentation, we generated and thoroughly characterized two highly pure first trimester telomerase (TERT) immortalized rhesus trophoblast cell lines (iRP-D26 and iRP-D28A) that retained crucial features of isolated primary trophoblasts. CONCLUSIONS: Overall, our findings help elucidate the molecular translatability between human and rhesus placenta and reveal notable expression differences in several HPGs and genes implicated in pregnancy complications that should be considered when using the rhesus animal model to study normal and pathological human placentation.

Maternal azithromycin therapy for Ureaplasma parvum intraamniotic infection improves fetal hemodynamics in a nonhuman primate model.

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 ρ=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.

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

BACKGROUND: In 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. METHODS: Female 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. RESULTS: Dietary 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. CONCLUSIONS: Preconception dietary modification has beneficial effects on the maternal metabolic phenotype, and results in improved placental hemodynamics.

Assessment of blood-brain barrier integrity and neuroinflammation in preeclampsia.

BACKGROUND: Although blood-brain barrier integrity is intact under normal pregnancy conditions, animal studies suggest that blood-brain barrier impairment occurs in preeclampsia. Yet, human data are limited, and the integrity of the blood-brain barrier has not been assessed in women with preeclampsia. OBJECTIVE: We sought to test the hypothesis that the integrity of the blood-brain barrier is impaired and that neuroinflammation is increased in women with preeclampsia. STUDY DESIGN: We performed an observational case-control study in pregnant women >24 weeks gestation who underwent spinal anesthesia for elective cesarean delivery or combined spinal epidural analgesia for labor. Cases were women with preeclampsia, and control subjects were women with either healthy pregnancy, chronic hypertension, or gestational hypertension. Paired samples of blood, urine, and cerebrospinal fluid were collected from each subject before delivery. We measured albumin, C5a, C5b-9, tumor necrosis factor-α, and interleukin-6 concentrations in plasma and cerebrospinal fluid, and albumin, C5a, and C5b-9 concentrations in urine, using colorimetric or enzyme-linked immunosorbent assays. The ratio of albumin in cerebrospinal fluid to plasma (Qalb) was used as a surrogate for maternal blood-brain barrier integrity. Cerebrospinal fluid concentrations of C5a, C5b-9, tumor necrosis factor-α, and interleukin-6 were used as surrogate markers of neuroinflammation. Differences in Qalb and cerebrospinal fluid protein concentrations between groups were assessed by nonparametric test of medians. RESULTS: Forty-eight subjects were enrolled, which included 16 cases with preeclampsia, 16 control subjects with healthy pregnancy, and 16 control subjects with either chronic or gestational hypertension. Qalb values were not increased in preeclampsia cases compared with healthy or hypertensive control subjects (Qalb median, 3.5 [interquartile range, 2.9-5.1] vs 3.9 [interquartile range, 3.0-4.8] vs 3.9 [interquartile range, 3.0-4.8]; P=.78]. Moreover, Qalb values were not increased in the subset of women with preeclampsia with severe features (n=8) compared with those without severe features (n=8; Qalb median, 3.5 [interquartile range, 3.3-4.9] vs 3.7 [interquartile range, 2.3-5.5]; P=.62]. Cerebrospinal fluid concentrations of C5a, C5b-9, tumor necrosis factor-α and interleukin-6 were not increased in cases of preeclampsia, compared with control subjects with either healthy pregnancy, chronic hypertension, or gestational hypertension (P>.05, all comparisons). In contrast to the negative findings in cerebrospinal fluid, plasma concentrations of both C5b-9 and interleukin-6 and urine concentrations of C5a and C5b-9 were increased in cases of preeclampsia. CONCLUSION: Through measurements of albumin, complement proteins, and cytokines in paired samples of blood and cerebrospinal fluid at the time of delivery, we found no evidence of blood-brain barrier impairment or neuroinflammation in preeclampsia. Larger studies that will investigate a wider range of proteins are suggested to validate our findings.

Gadolinium Chelate Safety in Pregnancy: Barely Detectable Gadolinium Levels in the Juvenile Nonhuman Primate after in Utero Exposure.

Purpose To determine whether gadolinium remains in juvenile nonhuman primate tissue after maternal exposure to intravenous gadoteridol during pregnancy. Materials and Methods Gravid rhesus macaques and their offspring (n = 10) were maintained, as approved by the institutional animal care and utilization committee. They were prospectively studied as part of a pre-existing ongoing research protocol to evaluate the effects of maternal malnutrition on placental and fetal development. On gestational days 85 and 135, they underwent placental magnetic resonance imaging after intravenous gadoteridol administration. Amniocentesis was performed on day 135 prior to administration of the second dose of gadoteridol. After delivery, the offspring were followed for 7 months. Tissue samples from eight different organs and from blood were harvested from each juvenile macaque. Gadolinium levels were measured by using inductively coupled plasma mass spectrometry. Results Gadolinium concentration in the amniotic fluid was 0.028 × 10-5 %ID/g (percentage injected dose per gram of tissue) 50 days after administration of one gadoteridol dose. Gadolinium was most consistently detected in the femur (mean, 2.5 × 10-5 %ID/g; range, [0.81-4.1] × 10-5 %ID/g) and liver (mean, 0.15 × 10-5 %ID/g; range, [0-0.26] × 10-5 %ID/g). Levels were undetectable in the remaining sampled tissues, with the exception of one juvenile skin sample (0.07 × 10-5 %ID/g), one juvenile spleen sample (0.039 × 10-5 %ID/g), and one juvenile brain (0.095 × 10-5 %ID/g) and kidney (0.13 × 10-5 %ID/g) sample. Conclusion The presence of gadoteridol in the amniotic fluid after maternal injection enables confirmation that it crosses the placenta. Extremely low levels of gadolinium are found in juvenile macaque tissues after in utero exposure to two doses of gadoteridol, indicating that a very small amount of gadolinium persists after delivery. © RSNA, 2017.

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.

Impact of maternal obesity on fetal programming of cardiovascular disease.

The in utero environment is a key determinant of long-term health outcomes; poor maternal metabolic state and placental insufficiency are strongly associated with these long-term health risks. Human epidemiological studies link maternal obesity and offspring cardiovascular disease in later life, but mechanistic studies in animal models are limited. Here, we review the literature pertaining to maternal consequences of obesity during pregnancy and the subsequent impact on fetal cardiovascular development.

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.

Gadolinium Chelate Contrast Material in Pregnancy: Fetal Biodistribution in the Nonhuman Primate.

PURPOSE: To determine the extent to which gadolinium chelate is found in nonhuman primate fetal tissues and amniotic fluid at 19-45 hours after intravenous injection of a weight-appropriate maternal dose of the contrast agent gadoteridol. MATERIALS AND METHODS: Gravid Japanese macaques (n = 14) were maintained as approved by the institutional animal care and utilization committee. In the 3rd trimester of pregnancy, the macaques were injected with gadoteridol (0.1 mmol per kilogram of maternal weight). Fetuses were delivered by means of cesarean section within 24 hours of maternal injection (range, 19-21 hours; n = 11) or 45 hours after injection (n = 3). Gadolinium chelate levels in the placenta, fetal tissues, and amniotic fluid were obtained by using inductively coupled plasma mass spectrometry. The Wilcoxon rank sum test was used for quantitative comparisons. RESULTS: Gadoteridol was present in the fetoplacental circulation at much lower quantities than in the mother. At both time points, the distribution of gadolinium chelate in the fetus was comparable to that expected in an adult. The highest concentration of the injected dose (ID) was found in the fetal kidney (0.0161% ID per gram in the 19-21-hour group). The majority of the in utero gadolinium chelate was found in the amniotic fluid and the placenta (mean, 0.1361% ID per organ ± 0.076 [standard deviation] and 0.0939% ID per organ ± 0.0494, respectively). Data acquired 45 hours after injection showed a significant decrease in the gadolinium chelate concentration in amniotic fluid compared with that in the 19-21-hour group (from 0.0017% to 0.0007% ID per gram; P = .01). CONCLUSION: Amounts of gadolinium chelate in the fetal tissues and amniotic fluid were minimal compared with the maternal ID. This may impact future clinical studies on the safety of gadolinium contrast agent use in pregnancy.

Using dynamic contrast-enhanced MRI to quantitatively characterize maternal vascular organization in the primate placenta.

PURPOSE: The maternal microvasculature of the primate placenta is organized into 10-20 perfusion domains that are functionally optimized to facilitate nutrient exchange to support fetal growth. This study describes a dynamic contrast-enhanced magnetic resonance imaging method for identifying vascular domains and quantifying maternal blood flow in them. METHODS: A rhesus macaque on the 133rd day of pregnancy (G133, term = 165 days) underwent Doppler ultrasound procedures, dynamic contrast-enhanced magnetic resonance imaging and Cesarean-section delivery. Serial T1 -weighted images acquired throughout intravenous injection of a contrast reagent bolus were analyzed to obtain contrast reagent arrival time maps of the placenta. RESULTS: Watershed segmentation of the arrival time map identified 16 perfusion domains. The number and location of these domains corresponded to anatomical cotyledonary units observed following delivery. Analysis of the contrast reagent wave front through each perfusion domain enabled determination of volumetric flow, which ranged from 9.03 to 44.9 mL/s (25.2 ± 10.3 mL/s). These estimates are supported by Doppler ultrasound results. CONCLUSIONS: The dynamic contrast-enhanced magnetic resonance imaging analysis described here provides quantitative estimates of the number of maternal perfusion domains in a primate placenta and estimates flow within each domain. Anticipated extensions of this technique are to the study placental function in non-human primate models of obstetric complications.

Beneficial and cautionary outcomes of resveratrol supplementation in pregnant nonhuman primates.

Resveratrol has been proposed as a potential therapeutic to improve metabolic health during pregnancy, yet little is known about the fetal effects of this maternal dietary supplement. We hypothesized that when administered to pregnant nonhuman primates (NHPs), resveratrol would increase uterine blood flow and mitigate the harmful consequences of maternal Western-style diet (WSD) consumption. NHPs were fed a WSD (36% fat) supplemented with 0.37% resveratrol throughout pregnancy. Outcomes were compared with cohorts fed WSD alone and control chow (14% fat) to distinguish between WSD and resveratrol-specific effects in these animals. In the early third trimester, uterine blood flow was measured by Doppler ultrasound before fetal delivery and tissue collection. Resveratrol resulted in 30% maternal weight loss and improved glucose tolerance, increased uterine artery volume blood flow, and decreased placental inflammation and liver triglyceride deposition. In addition, fetal pancreatic mass was enlarged by 42%, with a 12-fold increase in proliferation by Ki67 immunohistochemistry. These results demonstrate that resveratrol use during pregnancy yields improvements in maternal and placental phenotype with beneficial effects in the fetal liver but an unexplained and concerning alteration in fetal pancreatic development, which strongly cautions against the use of resveratrol by pregnant women.

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.

Impaired placental hemodynamics and function in a non-human primate model of gestational protein restriction.

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.