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.
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The blood supply to the placenta is a critical determinant of maternal-fetal nutrient exchange throughout pregnancy. In the primate placenta, establishment of uteroplacental blood flow in the first trimester is largely dependent on trophoblast invasion of the maternal spiral arteries ; inadequate remodeling of this vascular space has been demonstrated to underlie fetal growth restriction, preeclampsia, and stillbirth. As gestation progresses, plasticity within the developing placenta allows for vascular adaptations to meet fetal growth demands. Unfortunately, the lack of safe, noninvasive imaging modalities that facilitate the in vivo study of both normal and abnormal placental vascular perfusion and blood volume impedes our understanding of placental vascular function. Specifically, currently available imaging modalities are limited in their ability to assess uteroplacental blood flow quantitatively.
Contrast-enhanced ultrasound scanning (CE-US) is a noninvasive technique that permits imaging of microvascular perfusion with the use of acoustic detection of gas-filled, lipid-encapsulated microbubble contrast agents. This method has been used extensively in cardiac diagnostic imaging, with microbubbles used as flow tracers. Thus, safety studies have examined the rheology of microbubbles in the microcirculation and demonstrated that they are similar in size to red blood cells and do not interfere with hemodynamics. Microbubbles emit a high acoustic signal because of either stable or inertial cavitation. This established technique originally was used to highlight the ventricular endocardial borders and to assess liver tissue vasculature and has now advanced to being used to assess tissue perfusion as a targeted delivery system for local drug/agent distribution. Side-effects of CE-US have been reported with severe allergic reaction that has occurred in approximately 1 in 10,000 patients and a 1 in 200 rate of flank/back pain thought to be due to complement-mediated reactions. No fatal events have been reported, and in general, acute therapy has resolved severe allergic responses within 8 hours. However, the use of contrast agents during pregnancy raises appropriate safety concerns for clinical application; consequently, CE-US in pregnant women has not been trialed previously. The main concerns of CE-US use during pregnancy are (1) lodging of contrast agent in the microcirculation, (2) complement activation, and (3) micro hemorrhages as a result of cavitation.
Studies that have been performed in pregnant rats have shown that phospholipid encapsulated microbubbles that contain sulfur hexafluoride do not appear to cross the fetoplacental barrier at a mechanical index below 1.5 13 and that real-time perfusion can be measured across gestation, with blood flow increasing from days 14-17. In addition, CE-US has been used in early pregnancy implantation studies in the Rhesus macaque but not later in gestation. Importantly, Keator et al demonstrated no adverse effect on pregnancy outcome at full-term after early CE-US use in their small sample cohort of 3 animals. Limited data are available from in vitro studies that demonstrate the bioeffects of microbubbles in combination with ultrasound scans on cellular properties. Primary endothelial cells in culture that were exposed to ultrasound with and without microbubbles exhibited short-lived effects (<1 hour until restoration) on stress fibers, cytoskeletal arrangements, and oxidative stress markers. However, studies in placental tissue have not been performed previously.
In the current study, our objective was to demonstrate the feasibility of assessing perfusion kinetics of the intervillous space (IVS) at 2 time points in mid to later gestation in the nonhuman primate and in the late first trimester of human subjects. Although we and others have not observed microvascular damage that is associated with CE-US, there may be a subtle effect on the placental ultrastructure. Thus, we also sought to address whether CE-US use results in placental tissue injury or adversely effects pregnancy outcomes.
Methods
Animals
All protocols were approved by the Institutional Animal Care and Utilization Committee of the Oregon National Primate Research Center; guidelines for humane animal care were followed. The Oregon National Primate Research Center abides by the Animal Welfare Act and Regulations enforced by the United States Department of Agriculture. Japanese macaques (Macaca fuscata) were housed socially in indoor/outdoor pens with ad libitum access to food and water. Animals were allowed to breed naturally, and pregnancies were identified by routine early first-trimester pregnancy 2-dimensional ultrasound scans (Voluson 730 Expert; GE Medical Systems, Kretztechnik, Zipf, Austria); fetal biometry measurements were used for gestational dating.
Nonhuman primate CE-US
Six pregnant animals underwent imaging studies at 90 days gestation (G90) and 129 days gestation (G129), where full-term in the Japanese macaque is 175 days gestation. After an overnight fast, animals were sedated initially with 3-5 mg/kg Telazol (tiletamine HCl and zolazepam HCl) intramuscularly. Once intubated and maintained under anesthesia with 1-2% isoflurane gas, Flumazenil (0.01 mg/kg, intravenously) was administered to reverse the effects of Telazol. CE-US was performed with a multiphase amplitude-modulation and phase-inversion algorithm on a Sequoia system (Siemens Medical Systems, Mountain View, CA) that was equipped with a 15L8 transducer at a transmit frequency of 7 MHz with a 0.18 mechanical index and a 55 dB dynamic range. Lipid-shelled octofluoropropane microbubble contrast reagent (Definity; Lantheus Medical Imaging, Billerica, MA) was prepared in 0.9% saline solution at a final concentration of 5%. Intravenous infusion via a cephalic catheter was performed initially at a rate of 10 mL/hr to obtain baseline measurements of the maternal blood pool within the brachial artery of the catheterized arm. Digital video clips were recorded for 10-second durations to measure video intensity (VI) of the blood pool (VI pool ). Three replicates of all recordings were obtained during each study. For visualization of uteroplacental blood flow, the infusion rate was increased to 60 mL/hr. The acoustic beam was centered over individually identified maternal spiral artery sources, and the microbubbles within the path of the beam were destroyed by a brief (2-second) increase in mechanical index to 1.9. Microbubble reentry in the spiral artery and the IVS were recorded at 1 frame per 75 msec (rapidly refilling vessels) or 1 frame per 125 msec (slower refilling vessels) until the area of interest reached signal saturation (VI max ).
Human CE-US studies
This protocol was approved by the Institutional Review Board at Oregon Health and Science University (IRB#10744); written consent was obtained from all study participants. Women who were scheduled for elective termination of pregnancy were approached about participation in this study. White women (n = 4) underwent ultrasound examinations at 11-13 weeks gestation while in a reclined position with normal oxygen saturation on room air. The ultrasound protocol was the same as the animal studies with the following exceptions: A 4C1-S transducer set at a frequency of 3 MHz was used to obtain all images. The resting mechanical index was 0.11. All other parameters were consistent with the nonhuman primate studies. At this early gestational age, perfusion of the entire placenta can be visualized in 1 field of view. In addition, acquisition parameters can be adjusted to focus in and selectively view individual maternal spiral artery sources by adjustment of the depth.
Data analysis
Digital imaging data were analyzed with a custom-designed CE-US analysis program. The VI pool was analyzed with the use of recordings that were obtained from the brachial artery measurements. Three overlapping regions of interest were drawn to facilitate calculation of the VI pool . For placental assessment, regions of interest were drawn over the area of each cotyledon. The data were fit to the function y = A(1 – e –βt ), where y is the VI at the pulsing interval t, A is the VI plateau, and β is the flux rate constant. The β-value determines the rate constant of the postdestructive VI recovery and reflects the microvascular blood flux rate.
Placental tissue collection
The animals used in this cohort were part of a larger study in which pregnancies were delivered by cesarean section at 130 days gestation. Maternal serum was collected at the time of surgery, processed, and stored at –80°F for later analysis of complement activation. After delivery, fetal and placental weights were recorded, and placental tissue was collected. Full-thickness sections (maternal decidua to chorionic plate) were collected in cassettes and fixed in 10% zinc formalin for paraffin embedding. Villous tissue was dissected from each individually identified and mapped cotyledon and flash frozen in liquid nitrogen. Tissue was collected from animals from the imaging studies described earlier (n = 6) and from a control group who were not exposed to CE-US during pregnancy (n = 6).
Placental tissue analysis
Placental villous tissue was homogenized in lysis buffer that contained protease inhibitors and was centrifuged at 20,000 g for 5 minutes, as previously described. Supernatant from at least 3 individual cotyledons per placenta was pooled in to 1 representative sample for protein expression analysis. Complement activation, apoptosis, and nitrative stress levels were assessed with the use of commercially available enzyme-linked immunosorbent assay kits (Monkey Terminal Complement Complex C5b-9 and Caspase 3 ELISA: NeoBioLab, Woburn, MA; Nitrotyrosine ELISA: Millipore, Billerica, MA). Assays were performed according to manufacturers’ instructions, and data were corrected for protein content.
Placental histology
Representative macaque placental tissue samples were fixed in formalin, paraffin-embedded, and histologic sections (5 μm) that were stained for hematoxylin and eosin for evaluation by a placental pathologist (T.K.M.). In addition, sections were stained with a terminal deoxynucleotidyl transferase deoxyuridine-triphosphatase nick end labeling (TUNEL) assay (APO-BRDU-IHC kit; Phoenix Flow Systems via Biorad, Hercules, CA) as a marker of apoptosis and with anti- heat shock protein 70 (1:100; Cell Signaling, Danvers, MA) as a marker of oxidative and cellular stress.
First-trimester human samples were collected and fixed for examination within 1 hour after CE-US imaging. Representative first-trimester chorionic villous samples from 3 subjects were sectioned and assessed by routine hematoxylin and eosin stain. In addition, 2.5% glutaraldehyde-fixed samples were examined by electron microscopy. For the purposes of this pilot study, all 3 cases were compared with gestational age–matched negative control subjects (n = 6) to evaluate for trophoblast surface damage by a placental pathologist (T.K.M.) while blinded to treatment group. Notably, areas of hydropic generation that were related to chorionic membrane development were excluded from this analysis because of the fragmented nature of the specimens that were obtained after the procedure.
Statistical analysis
Contrast ultrasound parameters were compared between the G90 and G129 time points with the use of an unpaired t -test that was performed with statistical analysis software (GraphPad Prism, La Jolla, CA). Similarly, expression of markers of injury between CE-US exposed vs nonexposed maternal serum and placental tissue were assessed with the use of an unpaired t -test. A probability value of <.05 was considered to be significant.
Results
CE-US in pregnant nonhuman primates
CE-US with Definity microbubbles was used to locate and visualize maternal spiral artery sources that supplied individual placental cotyledons of the primate placenta in mid gestation. Figure 1 , A, shows an example time series of refilling of the placental IVS after a “burst” of the microbubbles until signal saturation is reached at VI max ( Video 1 ). For orientation, the maternal spiral artery and a cotyledon are outlined with the yellow and red dashed lines, respectively ( Figure 1 , B). A parametric image of IVS flux rate (β) is also shown ( Figure 1 , C).
Data were analyzed from 6 pregnant females at 2 time points in the second trimester (G90) and early third trimester (G129). Maternal blood pressure, heart rate, and oxygenation were stable and unchanged over the duration of each ultrasound examination (data not shown). We demonstrate a significant increase in maternal VI pool that was assessed in the brachial artery from G90 to G129 ( Figure 2 , A). Flux rate (β) provides a measure of microvascular resistance with the significant increase in β at G129 vs G90 demonstrating a decrease in resistance as gestation advances ( Figure 2 , B). Importantly, we observed no sequential deterioration in perfusion with replicate measurements that were obtained in each cotyledon (raw data not shown).
Safety of CE-US use during pregnancy
There was no significant difference in fetal or placental weights at 130 days gestation in animals with and without CE-US exposure ( Table ). Complement activation was assessed by measurement of C5b-9 in both maternal serum and placental protein. There was no significant difference in C5b-9 expression in maternal serum ( Figure 3 , A), and expression was below detectable levels in the placenta (data not shown). Assessment of placental tissue integrity in samples collected 1 day after CE-US compared with gestational age–matched controls (no CE-US exposure) demonstrate no significant changes in protein expression levels of caspase-3 and nitrotyrosine, as markers of apoptosis and nitrative stress, respectively ( Figure 3 , B and C). Histologic assessment of placental sections revealed no evidence of intervillous micro hemorrhage or acute inflammation ( Figure 3 , D). Additional assessment of apoptosis by TUNEL assay demonstrates minimal cell death within the placental villous tissue that resulted from CE-US exposure ( Figure 3 , E). Expression and localization of heat shock protein 70 ( Figure 3 , F), as a marker of cellular and oxidative stress, were semiquantitatively assessed by blinded grading of staining intensity and found to be unchanged by CE-US, compared with controls.
