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Objective To determine the dose-dependent effect of contemporary marijuana exposure on female menstrual cyclicity and reproductive endocrine physiology in a nonhuman primate model. Design Research animal study. Setting Research institute environment. Animals Adult female rhesus macaques (6–12 years of age; n = 8). Intervention(s) Daily delta-9-tetrahydrocannabinol (THC) edible at medically and recreationally relevant contemporary doses. Main Outcome Measure(s) Menstrual cycle length (MCL), anti-Mullerian hormone, prolactin, basal follicle-stimulating hormone (FSH), estradiol (E2) and progesterone, luteinizing hormone (LH), and thyroid-stimulating hormone. Result(s) The average before THC weight was 6.9 kg (standard deviation, 0.8), and at the highest THC dosing, the average weight was 7.2 kg (standard deviation, 0.8). With increasing THC dosing, MCL and FSH concentrations increased, while basal E2 concentration was stable. The average MCL concentration increased 4.0 days for each mg/7 kg/day of THC (95% CI, 1.4–6.6 days). Follicle-stimulating hormone concentration increased significantly with increasing THC dose, 0.34 ng/mL for each mg/7 kg/day of THC (95% CI, 0.14–0.57 ng/mL). No significant trends were observed between THC dosing and average basal progesterone, anti-Mullerian hormone, prolactin, LH, or thyroid-stimulating hormone concentrations. Conclusion(s) In rhesus macaques, a dose response toward increased MCL and basal FSH concentrations but plateau of basal E2 and LH concentrations was observed with increasing THC dosing, suggesting ovulatory dysfunction. Further studies are needed to determine the effects of a longer duration of exposure and whether the significant increase in MCL and FSH concentrations results in reduced fecundity.

We previously demonstrated decreased placental perfusion, reduced amniotic fluid protein content, and increased pregnancy loss in a nonhuman primate model of gestational protein restriction. Here, our objective was to link these detrimental findings with a functional placental assessment. As blood flow is critical to maternal-fetal exchange, we hypothesized that a protein-restricted diet would impair placental taurine uptake. Pregnant rhesus macaques were maintained on either control chow (CON, n = 5), a 33% protein-restricted diet (PR33, n = 5), or a 50% PR diet (PR50, n = 5) prior to and throughout pregnancy. Animals were delivered on gestational day 135 (G135; term is G168). Taurine activity was determined in fresh placental villous explants. Taurine transporter (TauT) protein expression, placental growth factor (PLGF), and insulin-like growth factor (IGF)-1 and IGF-2 protein concentrations were measured, and histological assessment was performed. Fetal body weights and placental weights were comparable between all three groups at G135. Placental taurine uptake was decreased in PR33- and PR50-fed animals compared to CON, yet TauT expression was unchanged across groups. PLGF was significantly increased in PR50 vs. CON, with no change in IGF-1 or IGF-2 expression in placental homogenate from PR-fed animals. Accelerated villous maturation was observed in all PR50 cases, three of five PR33, and was absent in CON. We demonstrate conserved fetal growth, despite a decrease in placental taurine uptake. Increased expression of PLGF and expansion of the syncytiotrophoblast surface area in the severely protein-restricted animals suggest a compensatory mechanism by the placenta to maintain fetal growth.

Prenatal exposure to marijuana may lead to epigenetic alterations in the placenta and fetal brain, affecting short- and long-term offspring health. This Viewpoint addresses the critical need to study and characterize the impact of maternal marijuana use and consequences of in utero exposure on later development and health. We highlight the development of new PET imaging tools and the opportunity for longitudinal in vivo non-human primate studies to help elucidate epigenetic changes resulting from prenatal marijuana exposure throughout gestation.

Since the explosive outbreak of Zika virus in Brazil and South/Central America in 2015–2016, the frequency of infections has subsided, but Zika virus remains present in this region as well as other tropical and sub-tropical areas of the globe. The most alarming aspect of Zika virus infection is its association with severe birth defects when infection occurs in pregnant women. Understanding the mechanism of Zika virus pathogenesis, which comprises features unique to Zika virus as well as shared with other teratogenic pathogens, is key to future prophylactic or therapeutic interventions. Nonhuman primate-based research has played a significant role in advancing our knowledge of Zika virus pathogenesis, especially with regard to fetal infection. This review summarizes what we have learned from these models and potential future research directions.

ZIKV infection during pregnancy causes microcephaly, but not all neonates are affected. This study shows that features of the maternal antibodies correlate with the risk of ZIKV-associated microcephaly., Zika virus (ZIKV) infection during pregnancy causes congenital abnormalities, including microcephaly. However, rates vary widely, and the contributing risk factors remain unclear. We examined the serum antibody response to ZIKV and other flaviviruses in Brazilian women giving birth during the 2015–2016 outbreak. Infected pregnancies with intermediate or higher ZIKV antibody enhancement titers were at increased risk to give birth to microcephalic infants compared with those with lower titers (P < 0.0001). Similarly, analysis of ZIKV-infected pregnant macaques revealed that fetal brain damage was more frequent in mothers with higher enhancement titers. Thus, features of the maternal antibodies are associated with and may contribute to the genesis of ZIKV-associated microcephaly.

Preserving male fertility Before chemotherapy or radiation treatment, sperm from adult men can be cryopreserved for future use. However, this is not possible for prepubertal boys. Fayomi et al. grafted cryopreserved testicular tissue from castrated pubertal rhesus macaques, placing each animal's own testis sections under the skin of the back or scrotum (see the Perspective by Neuhaus and Schlatt). Grafts grew, produced testosterone, and were able to generate sperm that could fertilize oocytes, in one case resulting in a successful pregnancy. The results hold promise for preserving human fertility, for example, after childhood cancer treatments. Science , this issue p. 1314 ; see also p. 1283

Geometry of the placental villous vasculature is a key determinant of maternal-fetal nutrient exchange for optimal fetal growth. Recent advances in tissue clarification techniques allow for deep high-resolution imaging with confocal microscopy; however, the methodology lacks a signal:noise ratio of sufficient magnitude to allow for quantitative analysis. Thus, we sought to develop a reproducible method to investigate the 3D vasculature of the nonhuman primate placenta for subsequent data analysis. Fresh placental tissue was dissected, formalin fixed, clarified using a modified Visikol® protocol and immunolabeled for CD31 (fetal endothelium) and cytokeratin-7 (villous trophoblast) for confocal imaging of the microanatomy. We present a detailed clarification and staining protocol augmented for imaging of nonhuman primate placental tissue. The image stacks generated by this refined staining method and our data acquisition parameters can be analyzed quantitatively to provide insights regarding the villous and vascular micro-anatomy of the placenta., METHOD SUMMARY We present a protocol for the preparation, immunolabeling and clarification of the nonhuman primate placenta refined for confocal microscopy and subsequent quantification of the microanatomic s tructures.

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.

We reported that consumption of a western-style diet (WSD) with and without hyperandrogenemia perturbed placental perfusion and altered levels of glucose transporter proteins in rhesus macaques. Based on that result, we hypothesized that placental glucose uptake would be dysregulated in this model. In this study, female rhesus macaques were assigned at puberty to one of four groups: subcutaneous cholesterol implants + standard chow diet (controls, C); testosterone implants + chow (T); cholesterol implants + a high-fat, WSD; and T+WSD. After ~6 years of treatment, animals were mated, and pregnancies were delivered by cesarean section at gestational day (G) 130 (the term is G168). Placental villous explants were immediately prepared for radiolabeled glucose assay. Linear glucose uptake was observed between 0 and 30 s. At 20 s, glucose uptake in placental villous explants did not differ across the four treatment groups with values as follows: C: 25.5 ± 6.33, T: 22.9 ± 0.404, WSD: 26.9.0 ± 3.71, and T+WSD: 33.0 ± 3.12 (mean ± SD expressed in pmol/mg). Unlike our prior experiment, glucose transporter expression was reduced in WSD placentas, and our in vitro functional assay did not demonstrate a difference in glucose uptake across the transporting epithelium of the placenta. Notably, maternal blood glucose levels were significantly elevated in animals chronically fed a WSD. This disparity may indicate differences in glucose utilization and metabolism by the placenta itself, as glucose transporter expression and circulating fetal glucose concentrations were comparable across all four groups in this pregnancy cohort.

The use of contrast agents as signal enhancers during ultrasound improves visualization and the diagnostic utility of this technology in medical imaging. Although widely used in many disciplines, contrast ultrasound is not routinely implemented in obstetrics, largely due to safety concerns of administered agents for pregnant women and the limited number of studies that address this issue. Here the microbubble characteristics that make them beneficial for enhancement of the blood pool and the quantification of real-time imaging are reviewed. Literature from pregnant animal model studies and safety assessments are detailed, and the potential for contrast-enhanced ultrasound to provide clinically relevant data and benefit our understanding of early placental development and detection of placental dysfunction is discussed.

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 would improve placental function. Methods Female Japanese macaques were divided in to three groups: diet reversal animals (n=5) were switched from a chronic WSD (36% fat) to a low fat, control diet (14% fat) prior to conception and throughout pregnancy. The control (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 non-invasive imaging was performed at mid-gestation (gestational day 90, G90 or 0.5 of gestation, where full term is G175), and G129, one day prior to C-section delivery at G130 (0.75 of gestation). Imaging studies comprised Doppler ultrasound (US), Contrast-Enhanced US (CEUS) and Dynamic-Contrast-Enhanced Magnetic Resonance Imaging (DCE-MRI) 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 control fed animals. Dietary reversal ameliorated these concerning derangements, restoring these hemodynamic parameters to control levels. Conclusion Preconception dietary modification has beneficial effects on the maternal metabolic phenotype, and results in improved placental hemodynamics.

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.

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

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.

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.

Background While blood-brain barrier integrity is intact under normal pregnancy conditions, animal studies suggest that blood-brain barrier impairment occurs in preeclampsia. Yet, human data is 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 neuroinflammation is increased, in women with preeclampsia. Study Design We performed an observational case-control study in pregnant women >24 weeks gestation, undergoing spinal anesthesia for elective cesarean delivery or combined spinal epidural analgesia for labor. Cases were women with preeclampsia and controls 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, TNF-α, and IL-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 (Q alb ) was used as a surrogate for maternal blood-brain barrier integrity. Cerebrospinal fluid concentrations of C5a, C5b-9, TNF-α, and IL-6 were used as surrogate markers of neuroinflammation. Differences in Q alb and cerebrospinal fluid protein concentrations between groups were assessed by non-parametric test of medians. Results Forty-eight subjects were enrolled, including 16 cases with preeclampsia, 16 controls with healthy pregnancy and 16 controls with either chronic or gestational hypertension. Q alb values were not increased in preeclampsia cases compared to healthy or hypertensive controls [Q alb median (interquartile range): 3.5 (2.9-5.1) vs. 3.9 (3.0-4.8) vs. 3.9 (3.0-4.8), P=0.78]. Moreover, Q alb values were not increased in the subset of women with preeclampsia with severe features (n=8) compared to those without severe features (n=8) [Q alb median (interquartile range): 3.5 (3.3-4.9) vs. 3.7 (2.3-5.5), P=0.62]. Cerebrospinal fluid concentrations of C5a, C5b-9, TNF-α and IL-6 were not increased in cases of preeclampsia, compared to controls with either healthy pregnancy, chronic hypertension, or gestational hypertension (P>0.05, all comparisons). In contrast to the negative findings in cerebrospinal fluid, plasma concentrations of both C5b-9 and IL-6, and urine concentrations of C5a and C5b-9, were increased in subjects with 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 investigating a wider range of proteins are suggested to validate our findings.

The risk of adverse perinatal outcomes with maternal polycystic ovary syndrome may differ among hyperandrogenic and nonhyperandrogenic phenotypes and is likely modulated by maternal obesity and diet. The relative contribution of maternal hyperandrogenism and nutritional status to placental dysfunction is unknown. Female rhesus macaques (N = 39) were assigned at puberty to one of four treatment groups: subcutaneous cholesterol implants and a standard chow diet (controls); testosterone (T) implants and a normal diet; cholesterol implants and a high-fat, Western-style diet (WSD); and testosterone implants in combination with a high-fat diet. After 3.5 years of treatment, contrast-enhanced and Doppler ultrasound analyses of placental blood flow were performed for a representative subset of animals from each treatment group during pregnancy, and placental architecture assessed with stereological analysis. Placental growth factors, cellular nutrient sensors, and angiogenic markers were measured with ELISA and Western blotting. WSD consumption was associated with a 30% increase in placental flux rate relative to that in animals receiving a normal diet. T and WSD treatments were each independently associated with increased villous volume, and T also was associated with an ∼ 40% decrease fetal capillary volume on stereological analysis. T treatment was associated with significantly increased mTOR and SOCS3 expression. WSD consumption was associated with decreased GLUT1 expression and microvillous membrane localization. Hyperandrogenemic and nonhyperandrogenemic phenotypes are associated with altered placental angiogenesis, nutrient sensing, and glucose transport. WSD and T appear to have distinct effects on vascular impedance and capillary angiogenesis.

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 (10(7) 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 (P1.1) than in those with normal blood flow (P1.6, P

ObjectivesMaternal protein malnutrition is associated with impaired fetal growth, and lifetime consequences for the offspring. Our group has previously developed a model of protein-restriction in the non-human primate, which was associated with fetal growth restriction, stillbirth, decreased placental perfusion, and evidence of fetal hypoxia, suggesting perturbed vascular development. Our objective was to histologically characterize the micro-anatomic alterations associated with adverse pregnancy outcomes taking an approach that permits investigation of the 3D vascular structure and surrounding histology without the requirement for 3D vascular casting or relying on 2D stereology which both have methodological limitations.MethodsRhesus macaques were assigned in the pre-gestational period to a control diet that contained 26% protein, or study diet containing 13% protein (50% PR diet). Placental tissue was collected at delivery and processed using a clarification, immunohistochemistry, and confocal microscopy protocol published previously by our group. Three dimensional reconstructions and quantitative assessment of the vascular micro-anatomy was performed using analysis software (Imaris®) and statistical analysis accounted for maternal and fetal confounders.ResultsIn unadjusted analysis, when comparing those pregnancies on a 50% PR diet (n = 4) with those on a control diet (n = 4), protein-restriction diet was associated with decreased maternal pre-pregnancy weight (difference of -1.975kg, 95% CI -3.267 to -0.6826). When controlling for maternal pre-pregnancy weight, fetal sex, and latency from tissue collection to imaging, a gestational protein-restriction diet was associated with decreases in total vascular length, total vascular surface area, total vascular volume, and vascular density.ConclusionIn this pilot study, a gestational protein-restriction diet altered the placental micro-vasculature with decreased vascular caliber and density, which may be related to the observed adverse pregnancy outcomes and perturbed placental perfusion previously demonstrated in this model.

During pregnancy, high fat diet (HFD) induces maternal obesity, insulin resistance, and placental inflammatory responses that compromise placental and fetal development. Whether maternal HFD would adversely affect amniotic fluid volume (AFV) has not been explored. Vascular endothelial growth factor (VEGF) is expressed in the amnion and has been proposed as a regulator of AFV. Our aim was to investigate the effects of HFD on AFV and the associated changes in VEGF and soluble VEGF receptor 1 (sFlt‐1) expression profiles in three amnion regions of a nonhuman primate model. Further, we examined the relationships between VEGF expression and HFD‐induced changes in maternal metabolic status. Japanese macaques were maintained on control or HFD and amniotic fluid index (AFI) was measured as an ultrasonic estimate of AFV. Amniotic fluid VEGF concentrations were determined by ELISA and amnion VEGF and sFlt‐1 mRNA levels by real‐time RT‐qPCR. HFD increased maternal plasma triglyceride while glucose levels were unchanged. Maternal weight gain was found in diet‐sensitive animals whereas amniotic fluid VEGF concentration was reduced in diet‐resistant animals. HFD did not alter AFI and there was no correlation between AFI and maternal weight or amniotic fluid VEGF concentrations. VEGF mRNA levels were lowest in secondary placental amnion while sFlt‐1 mRNA were lowest in the primary placental amnion. HFD did not affect amnion VEGF or sFlt‐1 mRNA expression. These findings suggest that although maternal HFD increased maternal weight in diet‐sensitive and reduced amniotic fluid VEGF concentrations in diet‐resistant phenotype, AFV as indicated by the AFI, was not significantly affected.

Placental function is essential for the development of the fetus, and is – in part – related to the 3D arrangement of the villous and vascular geometry. Recent advances in tissue clarification techniques allow for deep high-resolution imaging with confocal microscopy without altering the spatial characteristics of the tissue. These image stacks can be analyzed quantitatively to provide insights regarding the villous and vascular micro-anatomy as well as the interrelationships between the two. However, such analyses require optimization of the tissue preparation, immuno-labeling, and clarification protocol in order to provide reliable results suitable for the detection of subtle differences in pathologic pregnancies. Placental and fetal development are similar between human and non-human primate pregnancies, with the latter serving as a reliable, validated, highly-controlled, well-characterized translational model for the former.We present a protocol for the preparation, immuno-labeling, and clarification of the non-human primate placenta optimized for confocal microscopy and subsequent quantification of the micro-anatomic structures.

Western style, high‐fat diet (HFD) and associated high lipid levels have deleterious effects on fetal and placental development independent of maternal obesity and/or diabetes. Our objectives were to determine whether HFD without development of obesity would alter amniotic fluid volume (AFV) and amnion aquaporin (AQP) expression in a non‐human primate model. Japanese macaques were fed either a control diet or HFD before and during pregnancy. The four quadrant amniotic fluid index (AFI) was used as an ultrasonic estimate of AFV at 120 days gestation. Amnion samples were collected at 130 days gestation by cesarean section and AQP mRNA levels were determined by quantitative RT‐PCR. Similar to that in human, AQP1, AQP3, AQP8, AQP9, and AQP11 were expressed in the macaque amnion with significant differences in levels among AQPs. In macaque, neither individual AQPs nor expression profiles of the five AQPs differed between control and non‐obese HFD animals. There were regional differences in AQP expression in that, AQP1 mRNA levels were highest and AQP8 lowest in reflected amnion while AQP3, AQP9, and AQP11 were not different among amnion regions. When subdivided into control and HFD groups, AQP1 mRNA levels remain highest in the reflected amnion of both groups. The HFD did not significantly affect the AFI, but AFI was positively correlated with AQP11 mRNA levels independent of diet. Collectively, these data suggest that HFD in pregnant non‐obese individuals may have at most modest effects on AFV as the AFI and amnion AQP expression are not substantially altered.

Purpose 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. Methods MRI was performed on three pregnant rhesus macaques at gestational day 110. Multiecho spoiled gradient echo measurements were used to compute maps of T2*. Spatial maxima in these maps were compared with foci of early enhancement determined by DCE-MRI. Results Local maxima in T2* maps were strongly correlated with spiral arteries identified by DCE-MRI, with mean spatial separations ranging from 2.34 to 6.11 mm in the three animals studied. Spatial patterns of R2* ( = 1/ T2*) within individual placental lobules can be quantitatively analyzed using a simple model to estimate fetal arterial oxyhemoglobin concentration [Hbo,f] and a parameter viPS/Φ, reflecting oxygen transport to the fetus. Estimated mean values of [Hbo,f] ranged from 4.25 mM to 4.46 mM, whereas viPS/Φ ranged from 2.80 × 105 cm-3 to 1.61 × 106 cm-3 . Conclusions Maternal spiral arteries show strong spatial correlation with foci of extended T2* observed in the primate placenta. A simple model of oxygen transport accurately describes the spatial dependence of R2* within placental lobules and enables assessment of placental function and oxygenation without requiring administration of an exogenous contrast reagent. Magn Reson Med 76:1551-1562, 2016. © 2015 International Society for Magnetic Resonance in Medicine.

Adequate maternal supply and placental delivery of long chain polyunsaturated fatty acids (LCPUFA) is essential for normal fetal development. In humans, maternal obesity alters placental FA uptake, though the impact of diet remains uncertain. The fatty fetal liver observed in offspring of Japanese macaques fed a high fat diet (HFD) was prevented with resveratrol supplementation during pregnancy. We sought to determine the effect of HFD and resveratrol, a supplement with insulin-sensitizing properties, on placental LCPUFA uptake in this model.J. macaques were fed control chow (15% fat, n = 5), HFD (35% fat, n = 10) or HFD containing 0.37% resveratrol (n = 5) prior to- and throughout pregnancy. At ∼ 130 d gestation (term = 173 d), placentas were collected by caesarean section. Fatty acid uptake studies using (14)C-labeled oleic acid, arachidonic acid (AA) and docosahexanoic acid (DHA) were performed in placental explants.Resveratrol supplementation increased placental uptake of DHA (P0.05), while HFD alone had no measurable effect. Resveratrol increased AMP-activated protein kinase activity and mRNA expression of the fatty acid transporters FATP-4, CD36 and FABPpm (P0.05). Placental DHA content was decreased in HFD dams; resveratrol had no effect on tissue fatty acid profiles.Maternal HFD did not significantly affect placental LCPUFA uptake. Furthermore, resveratrol stimulated placental DHA uptake capacity, AMPK activation and transporter expression. Placental handling of DHA is particularly sensitive to the dramatic alterations in the maternal metabolic phenotype and placental AMPK activity associated with resveratrol supplementation.

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.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.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).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.

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.

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. Magn Reson Med 73:1570–1578, 2015. © 2014 Wiley Periodicals, Inc.