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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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

P2X(4) receptors are activated by extracellular ATP to raise intracellular calcium, thus altering cell signalling. ATP release occurs under pathophysiological, stress and adverse cell conditions; these are all increased in preeclampsia. Although P2X(4) is abundantly expressed in normal placenta neither the differences in the amount of protein nor its post-translational modifications have been studied in placentae from pregnancies complicated by preeclampsia. Thus we examined P2X(4) protein expression, localization and post-translational modifications in normotensive controls, term and preterm preeclamptic placentae. Densitometric analysis of Western blots showed a significant increase in P2X(4) protein expression in both term (p=0.002) and preterm preeclamptic (p=0.0008) placental samples compared to normotensive controls however the tissue localization of this receptor subtype was unaltered across the groups. Our data showed that P2X(4) is a nitrated protein in the placenta and this nitration is upregulated in preterm preeclamptic placenta compared to normotensive controls (p=0.03). We also demonstrated that P2X(4) is heavily glycosylated in the placenta by deglycosylation with PNGase F which reduced the protein product size by 23 kDa. We propose that P2X(4) acts within the syncytiotrophoblast to alter intracellular calcium and subsequent signalling pathways thereby restoring placental cell homeostasis following ATP-induced changes during pathophysiological conditions such as preeclampsia. We also propose that the post-translational modifications of nitration and glycosylation are required for the normal functioning of P2X(4).

Intracellular calcium concentration ([Ca(2+)](i)) is an important signalling molecule in the human placenta and regulation of [Ca(2+)](i) must be tightly controlled to ensure normal cell function and in order to meet the changing demand for calcium with increased fetal growth over gestation. Little is known about the receptors and mechanisms involved in intracellular calcium signalling in the human placenta but in isolated cytotrophoblast cells members of the P2 purinergic receptor family have been shown to mediate an ATP-stimulated rise in [Ca(2+)](i). In this study we examined activation and expression of several of the purinergic receptor subtypes in human placental villous fragments at two stages of gestation, first trimester and term. We demonstrate mRNA and protein expression of the P2X(4), P2X(7) and P2Y(2) subtypes but found no evidence of P2Y(4) protein in the placenta. Using fluorescent calcium imaging we demonstrate that 300 microM ATP, 450 microM UTP and 300 microM BzATP significantly elevate [Ca(2+)](i) in villous fragments with a significant increase in agonist-induced response seen in the term compared to the first trimester fragments (ATP, P<0.0001; UTP, P=0.018; BzATP, P=0.015). The roles of the purinergic receptors within the human placenta are not known but it seems likely for this study that calcium handling through these receptors is altered with advancing gestation. This may be due to the need to meet increased fetal Ca(2+) requirements due to growth or as a secondary function to alterations in placental [Ca(2+)](i) signalling.