Early first trimester uteroplacental flow and the progressive disintegration of spiral artery plugs: new insights from contrast-enhanced ultrasound and tissue histopathology.

STUDY QUESTION: Does the use of a vascular contrast agent facilitate earlier detection of maternal flow to the placental intervillous space (IVS) in the first trimester of pregnancy? SUMMARY ANSWER: Microvascular filling of the IVS was demonstrated by contrast-enhanced ultrasound from 6 weeks of gestation onwards, earlier than previously believed. WHAT IS KNOWN ALREADY: During placental establishment and remodeling of maternal spiral arteries, endovascular trophoblast cells invade and accumulate in the lumen of these vessels to form 'trophoblast plugs'. Prior evidence from morphological and Doppler ultrasound studies has been conflicting as to whether the spiral arteries are completely plugged, preventing maternal blood flow to the IVS until late in the first trimester. STUDY DESIGN, SIZE, DURATION: Uteroplacental flow was examined across the first trimester in human subjects given an intravenous infusion of lipid-shelled octofluoropropane microbubbles with ultrasound measurement of destruction and replenishment kinetics. We also performed a comprehensive histopathological correlation using two separately archived uteroplacental tissue collections to evaluate the degree of spiral artery plugging and evaluate remodeling of the upstream myometrial radial and arcurate arteries. PARTICIPANTS/MATERIALS, SETTING, METHODS: Pregnant women (n = 34) were recruited in the first trimester (range: 6+3 to 13+6 weeks gestation) for contrast-enhanced ultrasound studies with destruction-replenishment analysis of signal intensity for assessment of microvascular flux rate. Histological samples from archived in situ (Boyd Collection, n = 11) and fresh first, second, and third trimester decidual and post-hysterectomy uterine specimens (n = 16) were evaluated by immunohistochemistry (using markers of epithelial, endothelial and T-cells, as well as cell adhesion and proliferation) and ultrastructural analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Contrast agent entry into the IVS was visualized as early as 6+3 weeks of gestation with some variability in microvascular flux rate noted in the 6-7+6 week samples. Spiral artery plug canalization was observed from 7 weeks with progressive disintegration thereafter. Of note, microvascular flux rate did not progressively increase until 13 weeks, which suggests that resistance to maternal flow in the early placenta may be mediated more proximally by myometrial radial arteries that begin remodeling at the end of the first trimester. LIMITATIONS REASONS FOR CAUTION: Gestational age was determined by crown-rump length measurements obtained by transvaginal ultrasound on the day of contrast-enhanced imaging studies, which may explain the variability in the earliest gestational age samples due to the margin of error in this type of measurement. WIDER IMPLICATIONS OF THE FINDINGS: Our comprehensive in situ histological analysis, in combination with the use of an in vivo imaging modality that has the sensitivity to permit visualization of microvascular filling, has allowed us to reveal new evidence in support of increasing blood flow to the IVS from 6 weeks of gestation. Histologic review suggested the mechanism may be blood flow through capillary-sized channels that form through the loosely cohesive 'plugs' by 7 weeks gestation. However, spiral artery remodeling on its own did not appear to explain why there is significantly more blood flow at 13 weeks gestation. Histologic studies suggest it may be related to radial artery remodeling, which begins at the end of the first trimester. STUDY FUNDING/COMPETING INTEREST(S): This project was supported by the Oregon Health and Science University Knight Cardiovascular Institute, Center for Developmental Health and the Struble Foundation. There are no competing interests.

Functional imaging of the nonhuman primate Placenta with endogenous blood oxygen level-dependent contrast.

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.

Restriction of placental vasculature in a non-human primate: a unique model to study placental plasticity.

The limits of placental plasticity, i.e., the ability of the placenta to adapt and alter its growth trajectory in response to altered fetal requirements, are not known. We report fetal and placental hemodynamic adaptations in a novel non-human primate model in which the fetal inter-placental bridging vessels were surgically ligated. Doppler ultrasound studies showed that the rhesus placenta compensates for an approximate 40% reduction in functional capacity by increased growth and maintenance of umbilical volume blood flow. This unique experimental animal model has applications for mechanistic studies of placental plasticity and the impact on fetal development.

Effect of increasing maternal body mass index on oxidative and nitrative stress in the human placenta.

Maternal obesity is an increasing problem in obstetrics associated with adverse pregnancy outcomes and delivery complications. As an inflammatory state, where elevated levels of pro-inflammatory cytokines are found, obesity can lead to the increased incidence of oxidative and nitrative stress. These stresses may result in protein oxidation and protein nitration respectively, which are post- translational covalent modifications that can modify the structure and subsequently alter the function of a protein. The objective of this study was to examine whether placental oxidative and nitrative stress increase with increasing maternal body mass index. Placental tissue was collected from three groups of patients categorized as lean, overweight and obese. The presence of nitrotyrosine residues, a marker of nitrative stress, and antioxidant enzymes, as markers of oxidative stress, were assessed by immunohistochemistry, Western blot and ELISA. Protein carbonyl formation, a specific measure of protein oxidation, was measured by OxyBlot kit. Nitrotyrosine residues were increased in obese compared to lean and overweight groups although localization was unaltered across the three groups. Superoxide dismutase enzyme expression, localization and activity was unaltered between the groups. Protein carbonyl formation was greater in the lean compared to the overweight individuals. This study demonstrates that with increasing maternal body mass index there is an increase in placental nitrative stress. There does not appear to be a corresponding increase in oxidative stress and indeed we demonstrate some evidence of a decrease in oxidative effects in these placenta samples. Potentially the formation of peroxynitrite may be consuming reactive oxygen species and reducing oxidative stress. There may be a shift in the balance between nitrative and oxidative stress, which may be a protective mechanism for the placenta.

Protein nitration in placenta - functional significance.

Crucial roles of the placenta are disrupted in early and mid-trimester pregnancy loss, preeclampsia, eclampsia and intrauterine growth restriction. The pathophysiology of these disorders includes a relative hypoxia of the placenta, ischemia/reperfusion injury, an inflammatory response and oxidative stress. Reactive oxygen species including nitric oxide (NO), carbon monoxide and superoxide have been shown to participate in trophoblast invasion, regulation of placental vascular reactivity and other events. Superoxide, which regulates expression of redox sensitive genes, has been implicated in up-regulation of transcription factors, antioxidant production, angiogenesis, proliferation and matrix remodeling. When superoxide and nitric oxide are present in abundance, their interaction yields peroxynitrite a potent pro-oxidant, but also alters levels of nitric oxide, which in turn affect physiological functions. The peroxynitrite anion is extremely unstable thus evidence of its formation in vivo has been indirect via the occurrence of nitrated moieties including nitrated lipids and nitrotyrosine residues in proteins. Formation of 3-nitrotyrosine (protein nitration) is a "molecular fingerprint" of peroxynitrite formation. Protein nitration has been widely reported in a number of pathological states associated with inflammation but is reported to occur in normal physiology and is thought of as a prevalent, functionally relevant post-translational modification of proteins. Nitration of proteins can give either no effect, a gain or a loss of function. Nitration of a range of placental proteins is found in normal pregnancy but increased in pathologic pregnancies. Evidence is presented for nitration of placental signal transduction enzymes and transporters. The targets and extent of nitration of enzymes, receptors, transporters and structural proteins may markedly influence placental cellular function in both physiologic and pathologic settings.

Purinergic receptor expression and activation in first trimester and term human placenta.

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.

Post-Translational Modifications of the P2X(4) purinergic receptor subtype in the human placenta are altered in preeclampsia.

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).

Purinergic receptors in human placenta: evidence for functionally active P2X4, P2X7, P2Y2, and P2Y6.

Appropriate regulation of ion transport by the human placental syncytiotrophoblast is important for fetal growth throughout pregnancy. In nonplacental tissues, ion transport can be modulated by extracellular nucleotides that raise intracellular calcium ([Ca2+]i) via activation of purinergic receptors. We tested the hypothesis that purinergic receptors are expressed by human placental cytotrophoblast cells and that their activation by extracellular nucleotides modulates ion (K+) efflux and [Ca2+]i. P2X/P2Y receptor agonists 5-bromouridine 5'-triphosphate (5-BrUTP), ADP, ATP, 2',3'-O-(4-benzoyl-benzoyl)adenosine 5'-triphosphate (BzATP), and UTP stimulated 86Rb (K+ tracer) efflux from cultured cytotrophoblast cells at early (mononuclear) or later (multinucleate syncytiotrophoblast-like) stages of differentiation, with ATP and UTP particularly potent. 2-Methylthioadenosine 5'-triphosphate (2-MeS-ATP), and UDP elevated 86Rb efflux only from multinucleated cells. All agonists caused a significant peak and plateau increase in [Ca2+]i, although the magnitude of responses was variable. The effect of BzATP, UTP, and UDP in multinucleated cells was unaffected, and that of ATP partially inhibited, by removal of extracellular Ca2+, implicating P2Y receptor activation. mRNA encoding P2X1, P2X2, P2X4, and P2X7 and P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 were identified in mono- and multinucleated cells, whereas P2X3 and P2X5 mRNA were absent from all samples. Western blot analysis revealed P2X4, P2X7, P2Y2, and P2Y6 protein in cytotrophoblast cells, but P2Y4 was not detected. On the basis of published agonist selectivity, the data indicate the presence of functionally active P2X4, P2X7, P2Y2, and P2Y6 receptors in cytotrophoblast cells. We propose that activation of these receptors, and subsequent elevation of [Ca2+]i, modulates syncytiotrophoblast homeostasis and/or maternofetal ion exchange in response to extracellular nucleotides.

Store-operated Ca2+ entry in first trimester and term human placenta.

We have examined whether store-operated Ca2+ entry, a common pathway for Ca2+ entry in non-excitable tissue, is apparent in the syncytiotrophoblast of both first trimester and term human placenta. Expression of transient receptor potential (TRPC) homologues, a family of channels thought to be involved in store-operated Ca2+ entry, was also studied at the mRNA and protein levels. [Ca2+]i in syncytiotrophoblast of first trimester and term placental villous fragments was measured by microfluorimetry using the Ca2+-sensitive dye fura-2. Store-operated Ca2+ entry was stimulated using 1 microM thapsigargin in Ca(2+)-free Tyrode buffer (no added Ca2+ + 1 mM EGTA) followed by superfusion with control (Ca2+-containing) buffer. In term fragments, this protocol resulted in a rapid increase in [Ca2+]i, which was inhibited in the presence of 150 microM GdCl3, 200 microM NiCl2, 200 microM CoCl2 or 30 microM SKF96365 but was unaffected by addition of 10 microM nifedipine. It was not possible to stimulate such a rise in [Ca2+]i in first trimester fragments. Messenger RNA encoding TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6 was identified in both first trimester and term placentas. From Western blotting, TRPC3 and TRPC6 proteins were detected in term, but not in first trimester, placentas, while TRPC1 protein was not detected. By immunocytochemistry, TRPC3 and TRPC4 were localised to cytotrophoblast cells in first trimester placentas and to the syncytiotrophoblast in term placentas. TRPC6 staining was present in the syncytiotrophoblast of both first trimester and term placenta, but the intensity was much greater in the latter. We propose that store-operated Ca2+ entry may be an important route for Ca2+ entry into the syncytiotrophoblast of term, but not first trimester placentas, and that in human placenta TRPC channels may underlie this entry mechanism.

ATP-stimulated Ca(2+)-activated K(+) efflux pathway and differentiation of human placental cytotrophoblast cells.

The aim of this study was to determine whether extracellular ATP ([ATP](o)) stimulated a Ca(2+)-activated K(+) efflux in trophoblast cells that was dependent on extracellular Ca(2+) ([Ca(2+)](o)). Cytotrophoblast cells, isolated from human placenta, were examined following 18 h (relatively undifferentiated) and 66 h (multinucleate cells) of culture. Potassium efflux was measured using (86)Rb as a trace marker. Intracellular Ca(2+) ([Ca(2+)](i)) was examined by microfluorometry using fura 2. [ATP](o) significantly increased (86)Rb efflux to a peak that declined to control (18-h cells) or an elevated plateau (66-h cells) and was inhibited by 100 nM charybdotoxin. Removing [Ca(2+)](o) significantly reduced (86)Rb efflux in both groups as did application of 150 microM GdCl(3). [ATP](o) significantly increased [Ca(2+)](i) in both groups of cells. The response was reduced by removing [Ca(2+)](o) and applying 150 microM GdCl(3). For both (86)Rb efflux and microfluorometry experiments, the response to [ATP](o) was more dependent on [Ca(2+)](o) in 66-h cells compared with 18-h cells (approximately 70% greater). Cytotrophoblast cells exhibit an [ATP](o)-stimulated Ca(2+)-activated K(+) efflux. The dependency of this pathway on [Ca(2+)](o) is greater in the 66-h multinucleate syncytiotrophoblast-like cells, suggesting that the mechanism for Ca(2+) entry may be altered during differentiation of trophoblast cells.