Contrast-Enhanced Ultrasound Reveals Real-Time Temporospatial Changes in Uterine and Placental Vascular Perfusion During Early Pregnancy in Rhesus Macaques.

Abstract

Classical morphological studies on preserved uterine tissue sections have provided an anatomical understanding of the uterine vascular changes that occur during early pregnancy, but little is known regarding the physiological changes (i.e. uterine blood flow) that occur in vivo during pregnancy initiation in primates. Doppler ultrasound (DUS), the prevailing technique used to detect the fetal heartbeat, lacks the sensitivity needed to measure the low velocity vascular perfusion associated with implantation and subsequent early placentation. Contrast-enhanced ultrasound (CEU) is a non-invasive method with high spatial resolution used to assess quantitative differences in vascular blood flow and volume. Thus, the goals of this study were to use CEU to characterize and quantify the temporal changes in the vascular adaptations associated with early pregnancy, and to compare the information provided by CEU and DUS. Adult female rhesus macaques (n = 5) in the time-mated breeding program at ONPRC were pair-caged with males of proven fertility for 96 hr during the window of ovulation, and mating was confirmed by video recording. DUS and CEU began 14 days after the first day of pairing (DAP) and continued every 3 days until a fetal heartbeat was detected by DUS. The uterus of sedated animals was first visualized on a Voluson Expert 730 system (GE HealthCare) with a 2D trans-abdominal probe with Doppler capability. CEU was performed with a contrast-specific imaging technique, via an Acuson Sequoia system (Siemens), during a continuous intravenous infusion of lipid microbubble contrast reagent (Definity). Vascular blood flow and blood volume were quantified for the myometrium, endometrium and placental lobes from replenishment kinetics on the intensity data obtained after a high power pulse sequence destroyed all microbubbles in the acoustical beam. A functional corpus luteum formed (P4 > 4 ng/mL) in all females and 80% (4/5) became pregnant, as confirmed by a fetal heartbeat observed on 28.0±1.2 DAP. CEU clearly delineated the primary and secondary lobes of the placenta ~2 days apart (paired t-test; P<0.05), on 19.8±1.0 and 22.0±1.2 DAP, respectively. DUS was not able to differentiate between the placental lobes and detected endometrial thickening on 22.8±1.0 DAP, 3 days after (P<0.05) CEU identified the primary placental lobe. Visualization of the primary lobe correlated with the vaginal bleeding associated with implantation. The CEU calculated vascular perfusion was greater (P<0.05) in the primary placental lobe, which exhibited a 29% increase in relative blood volume and a 40% increase in relative blood flow, compared with the secondary lobe on the day of heartbeat detection. CEU identified greater flow through the hypertrophied endometrial spiral arteries supplying the placental lobes compared with other regions of the endometrium, whereas changes in the myometrium were not seen. In sum, CEU has defined the temporospatial changes in vascular perfusion associated with the anatomical vascular remodeling during early pregnancy in primates. Thus, CEU has great potential as a tool for basic researchers examining the peri-implantation period in the primate endometrium. Furthermore, CEU has promise as a future application for obstetricians, to resolve vascular complications that arise during pregnancy, including ectopic implantation and preeclampsia. Supported by NIH grants HD055744, HD18185, HD07133 and RR000163. (platform)