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.

Hypoxia, AMPK activation and uterine artery vasoreactivity.

Genes near adenosine monophosphate-activated protein kinase-α1 (PRKAA1) have been implicated in the greater uterine artery (UtA) blood flow and relative protection from fetal growth restriction seen in altitude-adapted Andean populations. Adenosine monophosphate-activated protein kinase (AMPK) activation vasodilates multiple vessels but whether AMPK is present in UtA or placental tissue and influences UtA vasoreactivity during normal or hypoxic pregnancy remains unknown. We studied isolated UtA and placenta from near-term C57BL/6J mice housed in normoxia (n = 8) or hypoxia (10% oxygen, n = 7-9) from day 14 to day 19, and placentas from non-labouring sea level (n = 3) or 3100 m (n = 3) women. Hypoxia increased AMPK immunostaining in near-term murine UtA and placental tissue. RT-PCR products for AMPK-α1 and -α2 isoforms and liver kinase B1 (LKB1; the upstream kinase activating AMPK) were present in murine and human placenta, and hypoxia increased LKB1 and AMPK-α1 and -α2 expression in the high- compared with low-altitude human placentas. Pharmacological AMPK activation by A769662 caused phenylephrine pre-constricted UtA from normoxic or hypoxic pregnant mice to dilate and this dilatation was partially reversed by the NOS inhibitor l-NAME. Hypoxic pregnancy sufficient to restrict fetal growth markedly augmented the UtA vasodilator effect of AMPK activation in opposition to PE constriction as the result of both NO-dependent and NO-independent mechanisms. We conclude that AMPK is activated during hypoxic pregnancy and that AMPK activation vasodilates the UtA, especially in hypoxic pregnancy. AMPK activation may be playing an adaptive role by limiting cellular energy depletion and helping to maintain utero-placental blood flow in hypoxic pregnancy.

Energy status and HIF signalling in chorionic villi show no evidence of hypoxic stress during human early placental development.

Early human placental and embryonic development occurs in a physiologically low oxygen environment supported by histiotrophic secretions from endometrial glands. In this study, we compare the placental metabolomic profile in the first, second and third trimesters to determine whether the energy demands are adequately met in the first trimester. We investigated whether hypoxia-inducible factors, HIF-1α and/or HIF-2α, might regulate transcription during the first trimester. First and second trimester tissue was collected using a chorionic villus sampling-like (CVS) technique. Part of each villus sample was frozen immediately and the remainder cultured under 2 or 21% O2 ± 1 mM H2O2, and ±the p38 MAPK pathway inhibitor, PD169316. Levels of HIF-1α were assessed by western blotting and VEGFA, PlGF and GLUT3 transcripts were quantified by RT-PCR. Term samples were collected from normal elective Caesarean deliveries. There were no significant differences in concentrations of ADP, NAD(+), lactate, and glucose, and in the ATP/ADP ratio, across gestational age. Neither HIF-1α nor HIF-2α could be detected in time-zero CVS samples. However, culture under any condition (2 or 21% O2 ± 1 mM H2O2) increased HIF-1α and HIF-2α. HIF-1α and HIF-2α were additionally detected in specimens retrieved after curettage. HIF-1α stabilization was accompanied by significant increases in VEGFA and GLUT3 and a decrease in PlGF mRNAs. These effects were suppressed by PD169316. In conclusion, our data suggest that first trimester placental tissues are not energetically compromised, and that HIF-1α is unlikely to play an appreciable role in regulating transcriptional activity under steady-state conditions in vivo. However, the pathway may be activated by stress conditions.

Ascorbate prevents placental oxidative stress and enhances birth weight in hypoxic pregnancy in rats.

This study isolated the effects of maternal hypoxia independent of changes in maternal nutrition on maternal circulatory and placental molecular indices of oxidative stress and determined whether maternal antioxidant treatment conferred protection. Pregnant rats were subjected to normoxic pregnancy or 13% O2 chronic hypoxia for most of gestation with and without maternal treatment with vitamin C in the drinking water. Maternal hypoxia with and without vitamin C did not affect maternal food or water intake and led to a significant increase in maternal and fetal haematocrit. At gestational day 20, maternal plasma urate and L-cysteine concentrations, and placental levels of 4-hydroxynonenal and heat shock protein 70 were increased while placental heat shock protein 90 levels were decreased in hypoxic pregnancy. The induction of maternal circulatory and placental molecular indices of oxidative stress in hypoxic pregnancies was prevented by maternal treatment with vitamin C. Maternal hypoxia during pregnancy with or without vitamin C increased placental weight, but not total or compartmental volumes. Maternal treatment with vitamin C increased birth weight in both hypoxic and normoxic pregnancies. The data show that maternal hypoxia independent of maternal undernutrition promotes maternal and placental indices of oxidative stress, effects that can be prevented by maternal treatment with vitamin C in hypoxic pregnancy. While vitamin C may not be the ideal candidate of choice for therapy in pregnant women, and taking into consideration differences in ascorbic acid metabolism between rats and humans, the data do underlie that antioxidant treatment may provide a useful intervention to improve placental function and protect fetal growth in pregnancy complicated by fetal hypoxia.

Dietary composition programmes placental phenotype in mice.

Dietary composition during pregnancy influences fetal and adult phenotype but its effects on placental phenotype remain largely unknown. Using molecular, morphological and functional analyses, placental nutrient transfer capacity was examined in mice fed isocaloric diets containing 23%, 18% or 9% casein (C) during pregnancy. At day 16, placental transfer of glucose, but not methyl-aminoisobutyric acid (MeAIB), was greater in C18 and C9 than C23 mice, in association with increased placental expression of the glucose transporter Slc2a1/GLUT1, and the growth factor Igf2. At day 19, placental glucose transport remained high in C9 mice while MeAIB transfer was less in C18 than C23 mice, despite greater placental weights in C18 and C9 than C23 mice. Placental System A amino acid transporter expression correlated with protein intake at day 19. Relative growth of transport verses endocrine zones of the placenta was influenced by diet at both ages without changing the absolute volume of the transport surface. Fetal weight was unaffected by diet at day 16 but was reduced in C9 animals by day 19. Morphological and functional adaptations in placental phenotype, therefore, occur to optimise nutrient transfer when dietary composition is varied, even subtly. This has important implications for the intrauterine programming of life expectancy.

A quantitative analysis of transcriptionally active syncytiotrophoblast nuclei across human gestation.

The syncytiotrophoblast (STB) epithelial covering of the human placenta is a unique terminally differentiated, multi-nucleated syncytium. No mitotic bodies are observed in the STB, which is sustained by continuous fusion of underlying cytotrophoblast cells (CTB). As a result, STB nuclei are of different ages. Morphologically, they display varying degrees of chromatin compaction, suggesting progressive maturational changes. Until recently, it was thought that STB nuclei were transcriptionally inactive, with all the mRNAs required by the syncytium being incorporated upon fusion of CTB. However, recent research has shown the presence of the active form of RNA polymerase II (RNA Pol II) in some STB nuclei. In this study, we confirm the presence of transcriptional activity in STB nuclei by demonstrating immunoreactivity for a transcription factor and an RNA polymerase I (RNA Pol I) co-factor, phospho-cAMP response element-binding protein and phospho-upstream binding factor, respectively. We also show, through immunoco-localisation studies, that a proportion of STB nuclei are both RNA Pol I and II transcriptionally active. Finally, we quantify the numerical densities of nuclei immunopositive and immunonegative for RNA Pol II in the STB of normal placentas of 11-39 weeks gestational age using an unbiased stereological counting tool, the physical disector. These data were combined with estimates of the volume of trophoblast to calculate total numbers of both types of nuclei at each gestational age. We found no correlation between gestational age and the numerical density of RNA Pol II-positive nuclei in the villous trophoblast (r = 0.39, P > 0.05). As the number of STB nuclei increases exponentially during gestation, we conclude that the number of transcriptionally active nuclei increases in proportion to trophoblast volume. The ratio of active to inactive nuclei remains constant at 3.9:1. These findings confirm that the majority of STB nuclei have intrinsic transcriptional activity, and that the STB is not dependent on CTB fusion for the provision of transcripts.

Adaptations in placental phenotype support fetal growth during undernutrition of pregnant mice.

Undernutrition during pregnancy reduces birth weight and programmes adult phenotype with consequences for life expectancy, but its effects on the phenotype of the placenta, responsible for supplying nutrients for fetal growth, remain largely unknown. Using molecular, morphological and functional analyses, placental phenotype was examined in mice during restriction of dietary intake to 80% of control from day 3 of pregnancy. At day 16, undernutrition reduced placental, but not fetal, weight in association with decreased junctional zone volume and placental expression of glucose transporter Slc2a1. At day 19, both placental and fetal weights were reduced in undernourished mice (91% and 87% of control, respectively, P < 0.01), as were the volume and surface area of the labyrinthine zone responsible for placental nutrient transfer (85% and 86%, respectively, P < 0.03). However, unidirectional materno-fetal clearance of tracer glucose was maintained and methyl-aminoisobutyric acid increased 166% (P < 0.005) per gram of undernourished placenta, relative to controls. This was associated with an 18% and 27% increased placental expression of glucose and system A amino acid transporters Slc2a1 and Slc38a2, respectively, at day 19 (P < 0.04). At both ages, undernutrition decreased expression of the placental specific transcript of the Igf2 gene by 35% (P < 0.01), although methylation of its promoter was unaffected. The placenta, therefore, adapts to help maintain fetal growth when its own growth is compromised by maternal undernutrition. Consequently, placental phenotype is responsive to environmental conditions and may help predict the risk of adult disease programmed in utero.

Placental efficiency and adaptation: endocrine regulation.

Size at birth is critical in determining life expectancy and is dependent primarily on the placental supply of nutrients. However, the fetus is not just a passive recipient of nutrients from the placenta. It exerts a significant acquisitive drive for nutrients, which acts through morphological and functional adaptations in the placenta, particularly when the genetically determined drive for fetal growth is compromised by adverse intrauterine conditions. These adaptations alter the efficiency with which the placenta supports fetal growth, which results in optimal growth for prevailing conditions in utero. This review examines placental efficiency as a means of altering fetal growth, the morphological and functional adaptations that influence placental efficiency and the endocrine regulation of these processes.

Placental endoplasmic reticulum stress and oxidative stress in the pathophysiology of unexplained intrauterine growth restriction and early onset preeclampsia.

The pregnancy complications of unexplained intrauterine growth restriction and early onset preeclampsia are thought to share a common aetiology in placental malperfusion secondary to deficient maternal spiral artery conversion. A key question is whether the contrasting clinical manifestations reflect different placental pathologies, or whether they are due to altered maternal responses to a common factor derived from the placenta. Recently, molecular evidence of protein synthesis inhibition secondary to endoplasmic reticulum stress has provided an explanation for the small placental phenotype in both conditions. However, other pathways activated by more severe endoplasmic reticulum stress are only observed in placentas from pregnancies associated with early onset preeclampsia. Here, we review the literature and conclude that there is evidence of greater maternal vascular compromise of the placenta in these cases. We speculate that in cases of normotensive intrauterine growth restriction the placental pathology is centred predominantly around endoplasmic reticulum stress, whereas in cases complicated by preeclampsia oxidative stress is further superimposed. This causes the release of a potent mix of pro-inflammatory cytokines, anti-angiogenic factors and trophoblastic aponecrotic debris into the maternal circulation that causes the peripheral syndrome. Maternal and fetal constitutional factors may modulate how the placenta responds to the maternal vascular insult, and how the mother is affected by the placental factors released. However, the principal conclusion is that the difference between these two conditions lies in the severity of the initiating deficit in spiral arterial conversion, and the relative degrees of endoplasmic reticulum stress and oxidative stress induced in the placenta as a result.

Evidence for transcriptional activity in the syncytiotrophoblast of the human placenta.

The aim was to test for evidence of transcriptional activity within the nuclei of the syncytiotrophoblast of the human placenta. The syncytiotrophoblast forms the epithelial covering of the villous tree, and is a multinucleated, terminally-differentiated syncytium generated through fusion of the underlying progenitor cytotrophoblast cells. Its nuclei are heterogeneous with respect to chromatin condensation, and previous functional studies of 3H-uridine uptake in vitro have indicated that they are transcriptionally inactive. This observation is surprising given the key roles this tissue plays in active transport, hormone synthesis and metabolic regulation, and has widespread implications for trophoblast physiology and pathophysiology. We used three different approaches to look for evidence of transcriptional activity. First, immunofluorescence staining was performed on paraffin-embedded early pregnancy and term placental villi, using an antibody directed specifically against the actively transcribing form of RNA polymerase II. Second, a nucleoside incorporation assay was applied to placental villi maintained in short-term culture, with and without the transcription blocker alpha-amanitin. Third, histone modifications associated with active chromatin were identified by immunohistochemistry and immunofluorescence. Each of these methods showed transcription to be occurring in a proportion of syncytiotrophoblast nuclei, with qualitative evidence for transcription being more abundant in the first trimester than at term. These findings correlated with electron microscopical observations of prominent nucleoli within the nuclei, particularly during early pregnancy, signifying transcription of ribosomal RNA. Contrary to previous findings, these results confirm that a proportion of syncytiotrophoblast nuclei actively produce mRNA transcripts.

IFPA meeting 2008 workshops report.

Workshops are an important part of the IFPA annual meeting. At the IFPA meeting 2008 diverse topics were discussed in 12 themed workshops. Topics covered included: immunology of placentation; galectins and trophoblast invasion; signaling in implantation and invasion; markers to identify trophoblast subpopulations; placental pathology; placental toxicology; stereology; placental transport of fatty acids; placental mesenchymal stem cells; comparative placentation; trophoblast and neoplasia; trophoblast differentiation. This report is a summary of the various topics covered.

Regulation of vascular growth and function in the human placenta.

During the course of 9 months, the human placenta develops into a highly vascular organ. Vasculogenesis starts during the third week post-conception. Hemangioblastic cell cords differentiate in situ from mesenchymal cells in the villous cores, most probably under the influence of vascular endothelial growth factor (VEGFA) secreted by the overlying trophoblast. The cords elongate through proliferation and cell recruitment, and connect with the vasculature of the developing fetus. A feto-placental circulation starts around 8 weeks of gestation. Elongation of the capillaries outstrips that of the containing villi, leading to looping of the vessels. The obtrusion of both capillary loops and new sprouts results in the formation of terminal villi. Branching and non-branching angiogenesis therefore play key roles in villous morphogenesis throughout pregnancy. Maternal circulating levels of VEGFA and placental growth factor vary across normal pregnancy, and in complicated pregnancies. Determining the impact of these changes on placental angiogenesis is difficult, as the relationship between levels of factors in the maternal circulation and their effects on fetal vessels within the placenta remains unclear. Furthermore, the trophoblast secretes large quantities of soluble receptors capable of binding both growth factors, influencing their bioavailability. Villous endothelial cells are prone to oxidative stress, which activates the apoptotic cascade. Oxidative stress associated with onset of the maternal circulation, and with incomplete conversion of the spiral arteries in pathological pregnancies, plays an important role in sculpting the villous tree. Suppression of placental angiogenesis results in impoverished development of the placenta, leading ultimately to fetal growth restriction.

Disproportional effects of Igf2 knockout on placental morphology and diffusional exchange characteristics in the mouse.

Both complete knockout of the Igf2 gene (Igf2null(+/-)) and knockout of its placental specific transcript alone (Igf2P0(+/-)) lead to fetal growth restriction in mice. However, in the Igf2null(+/-) this growth restriction occurs concurrently in gestation with placental growth restriction, whereas, placental growth restriction precedes fetal growth restriction in the Igf2P0(+/-) mouse. Previous studies have shown that the Igf2P0(+/-) placenta has proportionate reductions in its cellular compartments and its diffusional exchange characteristics. Yet, nothing is known about the structural development or diffusional exchange characteristics of the Igf2null(+/-) mouse. Hence, this study compares the structural properties (using stereology) and diffusional exchange characteristics (using measurement of permeability-surface area product, P.S, of three inert hydrophilic tracers) of the Igf2null(+/-) and the Igf2P0(+/-) placenta to identify the role of Igf2 in the development of the labyrinthine exchange membrane and its functional consequences. Our data show disproportionate effects of complete Igf2 ablation on the compartments of the placenta, not seen when the placental-specific transcript alone is deleted. Furthermore, although the theoretical diffusing capacity (calculated from the stereological data) of the Igf2null(+/-) placenta was reduced relative to control, there was no effect of the complete knockout on permeability surface area available for small hydrophilic tracers. This is in contrast to the Igf2P0(+/-) placenta, where theoretical diffusion capacity and P.S values were reduced similarly. Total ablation of the Igf2 gene from the fetoplacental unit in the mouse therefore results in a disproportionate growth of placental compartments whereas, deleting the placental specific transcript of Igf2 alone results in proportional placental growth restriction. Thus, placental phenotype depends on the degree of Igf2 gene ablation and the interplay between placental and fetal Igf2 in the mouse.

Adaptations in placental nutrient transfer capacity to meet fetal growth demands depend on placental size in mice.

Experimental reduction in placental growth often leads to increased placental efficiency measured as grams of fetus produced per gram of placenta, although little is known about the mechanisms involved. This study tested the hypothesis that the smallest placenta within a litter is the most efficient at supporting fetal growth by examining the natural intra-litter variation in placental nutrient transfer capacity in normal pregnant mice. The morphology, nutrient transfer and expression of key growth and nutrient supply genes (Igf2P0, Grb10, Slc2a1, Slc2a3, Slc38a1, Slc38a2 and Slc38a4) were compared in the lightest and heaviest placentas of a litter at days 16 and 19 of pregnancy, when mouse fetuses are growing most rapidly in absolute terms. The data show that there are morphological and functional adaptations in the lightest placenta within a litter, which increase active transport of amino acids per gram of placenta and maintain normal fetal growth close to term, despite the reduced placental mass. The specific placental adaptations differ with age. At E16, they are primarily morphological with an increase in the volume fraction of the labyrinthine zone responsible for nutrient exchange, whereas at E19 they are more functional with up-regulated placental expression of the glucose transporter gene, Slc2a1/GLUT1 and one isoform the System A family of amino acid transporters, Slc38a2/SNAT2. Thus, this adaptability in placental phenotype provides a functional reserve capacity for maximizing fetal growth during late gestation when placental growth is compromised.

Gestational diseases -- a workshop report.

Between 11% and 20% of all clinically recognised pregnancies are lost before the 20th week of gestation, with huge financial and personal implications. Immune mechanisms have been proposed to play a role in unexplained recurrent miscarriage. Considerable attention has focused on endometrial leucocyte populations in recurrent miscarriage, although the underlying pathogenesis remains largely unexplained. The mechanisms underlying sporadic miscarriage are even less well understood, although aneuploidy is the commonest attributable cause of early (

New imaging markers for preconceptional and first-trimester utero-placental vascularization.

INTRODUCTION: The availability of imaging makers of early placental circulation development is limited. This study aims to develop a feasible and reliable method to assess preconceptional and early first-trimester utero-placental vascular volumes using three-dimensional power Doppler (3D PD) ultrasound on two different Virtual Reality (VR) systems. METHODS: 3D PD ultrasound images of the uterine and placental vasculature were obtained in 35 women, either preconceptionally (n = 5), or during pregnancy at 7 (n = 10), 9 (n = 10) or 11 (n = 10) weeks of gestation. Preconceptional uterine vascular volume (UVV), first-trimester placental vascular volume (PVV) and embryonic vascular volume (EVV) were measured by two observers on two VR systems, i.e., a Barco I-Space and VR desktop. Intra- and inter-observer agreement and intersystem agreement were assessed by intra-class correlation coefficients (ICC) and absolute and relative differences. RESULTS: Uterine-, embryonic- and placental vascular volume measurements showed good to excellent intra- and inter-observer agreement and inter-system reproducibility with most ICC above 0.80 and relative differences of less than 20% preconceptionally and almost throughout the entire gestational age range. Inter-observer agreement of PVV at 11 weeks gestation was suboptimal (ICC 0.69, relative difference 50.1%). DISCUSSION: Preconceptional and first-trimester 3D PD ultrasound utero-placental and embryonic vascular volume measurements using VR are feasible and reliable. Longitudinal cohort studies with repeated measurements are needed to further validate this and assess their value as new imaging markers for placental vascular development and ultimately for the prediction of placenta-related pregnancy complications.