Three-Dimensional Sonographic Assessment of Placental Volume and Vascularization in Pregnancies Complicated by Hypertensive Disorders

Objectives-The purpose of this study was to evaluate the association between placental volumes, placental vascularity, and hypertensive disorders in pregnancy.Methods A prospective case-control study was conducted between April 2011 and July 2012. Placental volumes and vascularity were evaluated by 3-dimensional sonographic, 3-dimensional power Doppler histographic, and 2-dimensional color Doppler studies. Pregnant women were classified as normotensive or hypertensive and stratified by the nature of their hypertensive disorders. The following variables were evaluated: observed-to-expected placental volume ratio, placental volume-to-estimated fetal weight ratio, placental vascular indices, and pulsatility indices of the right and left uterine and umbilical arteries.Results Sixty-six healthy pregnant women and 62 pregnant women with hypertensive disorders were evaluated (matched by maternal age, gestational age at sonography, and parity). Placental volumes were not reduced in pregnancy in women with hypertensive disorders (P > .05). Conversely, reduced placental vascularization indices (vascularization index and vascularization-flow index) were observed in pregnancies complicated by hypertensive disorders (P < .01; P < .01), especially in patients with superimposed preeclampsia (P = .04; P = .02). A weak correlation was observed between placental volumes, placental vascular indices, and Doppler studies of the uterine and umbilical arteries.Conclusions Pregnancies complicated by hypertensive disorders are associated with reduced placental vascularity but not with reduced placental volumes. These findings are independent of changes in uterine artery Doppler studies. Future studies of the prediction of preeclampsia may focus on placental vascularity in combination with results of Doppler studies of the uterine arteries.

Quantitative Analysis of Placental Vasculature by Three-Dimensional Power Doppler Ultrasonography in Normal Pregnancies From 12 to 40 Weeks of Gestation

Evaluate the distribution and variation of placental vascular indices according to gestational age and placental volume. From March to November 2007, three-dimensional (3D)-power Doppler ultrasound was performed in 295 normal pregnancies from 12 to 40 weeks of gestation. Using the same preestablished settings for all patients, power Doppler was applied to the placenta and placental Volume was obtained by the rotational technique (VOCAL(TM)). The 3D-power histogram was used to determine the placental vascular indices: vascularization index (VI), flow index (FI) and vascularization-flow index (VFI). The placental vascular indices were then plotted against gestational age and placental volume. All placental vascular indices showed constant distribution throughout gestation. A tendency for a reduction in placental vascular indices with increased placental volume was observed, but was only statistically significant when placental FI was considered (p < 0.05). All placental vascular indices estimated by 3D-power Doppler ultrasonography presented constant distribution throughout gestation, despite the increase in placental volume according to gestational age. (C) 2008 Elsevier Ltd. All rights reserved.

Does Aldosterone participate in placental angiogenesis via PLGF?

Introduction Angiogenic signals are a vital signal of placental integrity. Aldosterone has recently been shown to enhance placental growth factor (PlGF) expression in the peripheral vasculature [1] and to promote trophoblast growth [2]. The plgf gene possesses a functional mineralocorticoid receptor responsive element in the promoter region. Objectives Thus, we hypothesized that aldosterone adapts placental angiogenesis to trophoblast growth by secreting PlGF. Methods The human choriocarcinoma cell line BeWo and first and third trimester human primary trophoblasts cells were subjected to several syncytialization signals. Upon visual confirmation, the cultured cells were subjected to either control conditions, the known stimulator forskolin, and increasing amounts of aldosterone (10−9 to 10−6 M) with and without the competitive aldosterone receptor blocker spironolactone. After 6 and 24 h of incubation, RNA and protein were extracted. PlGF transcripts were quantified by Taqman PCR normalized to several housekeeping genes. Protein expression was quantified by ELISA. Results PlGF mRNA expression increased 3-fold with forskolin in BeWo cells. In this cell line, aldosterone could slightly stimulate PlGF production. In non-syncytialized primary human first trimester trophoblasts, aldosterone did not exert a specific effect. In contrast, the term primary human trophoblasts did respond with a 2.5-fold increase after incubation with aldosterone (10−7 M) in the presence of forskolin to allow forming a syncytial layer. PlGF protein was already slightly upregulated following 6 h of incubation with aldosterone. Conclusion We concluded that aldosterone does regulate PlGF expression in specified conditions during pregnancy. Inappropriately low aldosterone levels such as in preeclampsia might such not only compromise plasma volume and trophoblast growth but also placental vascularization and systemic PlGF availability. These observations merit further investigation.

Essential role for p38α mitogen-activated protein kinase in placental angiogenesis

The p38 family of mitogen-activated protein kinases (MAPKs) mediates signaling in response to environmental stresses and inflammatory cytokines, but the requirements for the p38 MAPK pathway in normal mammalian development have not been elucidated. Here, we show that targeted disruption of the p38α MAPK gene results in homozygous embryonic lethality because of severe defects in placental development. Although chorioallantoic placentation is initiated appropriately in p38α null homozygotes, placental defects are manifest at 10.5 days postcoitum as nearly complete loss of the labyrinth layer and significant reduction of the spongiotrophoblast. In particular, p38α mutant placentas display lack of vascularization of the labyrinth layer as well as increased rates of apoptosis, consistent with a defect in placental angiogenesis. Furthermore, p38α mutants display abnormal angiogenesis in the embryo proper as well as in the visceral yolk sac. Thus, our results indicate a requirement for p38α MAPK in diploid trophoblast development and placental vascularization and suggest a more general role for p38 MAPK signaling in embryonic angiogenesis.

Upregulation of urotensin II receptor in preeclampsia causes in vitro placental release of soluble vascular endothelial growth factor receptor 1 in hypoxia

Preeclampsia is a hypertensive disorder of pregnancy caused by abnormal placental function, partly because of chronic hypoxia at the utero-placental junction. The increase in levels of soluble vascular endothelial growth factor receptor 1, an antiangiogenic agent known to inhibit placental vascularization, is an important cellular factor implicated in the onset of preeclampsia. We investigated the ligand urotensin II (U-II), a potent endogenous vasoconstrictor and proangiogenic agent, for which levels have been reported to increase in patients with preeclampsia. We hypothesized that an increased sensitivity to U-II in preeclampsia might be achieved by upregulation of placental U-II receptors. We further investigated the role of U-II receptor stimulation on soluble vascular endothelial growth factor receptor 1 release in placental explants from diseased and normal patients. Immunohistochemistry, real-time PCR, and Western blotting analysis revealed that U-II receptor expression was significantly upregulated in preeclampsia placentas compared with controls (P<0.01). Cellular models of syncytiotrophoblast and vascular endothelial cells subjected to hypoxic conditions revealed an increase in U-II receptor levels in the syncytiotrophoblast model. This induction is regulated by the transcriptional activator hypoxia-inducible factor 1a. U-II treatment is associated with increased secretion of soluble vascular endothelial growth factor receptor 1 only in preeclamptic placental explants under hypoxia but not in control conditions. Interestingly, normal placental explants did not respond to U-II stimulation.

Avaliação do volume e índices vasculares placentários pela ultrassonografia tridimensional em gestações com restrição grave de crescimento fetal

INTRODUÇÃO: A restrição de crescimento fetal (RCF) representa uma das principais complicações da gravidez e está associada a elevadas taxas de morbimortalidade perinatal. A frequência de desfechos desfavoráveis neonatais está diretamente relacionada à gravidade da RCF, sendo que os casos de pior evolução estão relacionados com peso abaixo do percentil 3. O mecanismo do crescimento fetal não está totalmente esclarecido, mas resulta da interação entre potencial genético de crescimento e fatores placentários, maternos e ambientais. Dentre os fatores etiológicos, o desenvolvimento anormal da placenta e a diminuição da perfusão uteroplacentária são as principais causas de RCF. Este estudo teve por objetivo avaliar volume e índices de vascularização placentários, por meio da ultrassonografia tridimensional (US3D), em gestações com RCF grave, e as correlações dos parâmetros placentários com valores de normalidade e dopplervelocimetria materno-fetal. MÉTODOS: Foram avaliadas 27 gestantes cujos fetos apresentavam peso estimado abaixo do percentil 3 para a idade gestacional. Por meio da US3D, utilizando-se a técnica VOCAL, foram mensurados o volume placentário (VP) e os índices vasculares: índice de vascularização (IV), índice de fluxo (IF) e índice de vascularização e fluxo (IVF). Os dados foram comparados com a curva de normalidade para a idade gestacional e peso fetal descrita por De Paula e cols. (2008, 2009). Desde que os volumes placentários variam durante a gravidez, os valores observados foram comparados com os valores esperados para a idade gestacional e peso fetal. Foram criados os índices volume observado/ esperado para a idade gestacional (Vo/e IG) e volume placentário observado/ esperado para o peso fetal (Vo/e PF). Os parâmetros placentários foram correlacionados com índice de pulsatilidade (IP) médio de (AUt) e IP de artéria umbilical (AU), e avaliados segundo a presença de incisura protodiastólica bilateral em AUt. RESULTADOS: Quando comparadas à curva de normalidade, as placentas de gestação com RCF grave apresentaram VP, IV, IF e IVF significativamente menores (p < 0,0001 para todos os parâmetros). Houve correlação inversa estatisticamente significante da média do PI de AUt com o Vo/e IG (r= -0,461, p= 0,018), IV (r= -0,401, p= 0,042) e IVF (r= -0,421, p= 0,048). No grupo de gestantes que apresentavam incisura protodiastólica bilateral de artérias uterinas, Vo/e IG (p= 0,014), Vo/e PF (p= 0,02) e IV (p= 0,044) foram significativamente mais baixos. Nenhum dos parâmetros placentários apresentou correlação significativa com IP de AU. CONCLUSÕES: Observou-se que o volume e os índices de vascularização placentários apresentam-se diminuídos nos fetos com RCF grave. IP médio de AUT apresenta correlação negativa com Vo/e IG, IV e IVF, e Vo/e IG, Vo/e PF e IV apresentaram-se reduzidos nos casos de incisura bilateral. Não houve correlação significativa dos parâmetros placentários com IP de AU

Hydrogen sulphide rescues preeclampsia-like phenotype aggravated by high sFlt-1 in Placenta Growth Factor (PlGF) deficiency

INTRODUCTION: Low circulating levels of placenta growth factor (PlGF) is strongly associated with the onset of preeclampsia, a maternal hypertensive disorder characterized by high blood pressure and proteinuria after 20 weeks of gestation. Although, PlGF-deficient mice are born healthy and fertile at a Mendelian ratio, the physiological importance of PlGF in the pathogenesis of preeclampsia is unclear. We hypothesised that decreased levels of PlGF in pregnancy exacerbates the fetal growth restriction associated with preeclampsia in the presence of high sFlt-1. METHODS: Pregnant PlGF-/- mice were injected with adenovirus encoding sFlt-1 (Ad-sFlt-1) at high (i) 1.5x109 pfu/ml and low (ii) 0.5x109 pfu/ml doses. Mean arterial blood pressure (MBP), biochemical and histological assessments of maternal kidney, placenta and embryos were performed. RESULTS: Ad-sFlt-1 significantly increased MBP and induced severe glomerular endotheliosis in PlGF-/- mice at E10.5 gestation compared to wild-type animals. High sFlt-1 also significantly elevated albumincreatinine ratio and increased levels of urinary kidney injury molecule-1, a marker for proximal tubule injury.At a high dose of sFlt-1, there was complete fetal resorption in the pregnant PlGF-/- mice, and even the lower dose of sFlt-1 induced severe fetal resorption and abnormal placental vascularization. Hydrogen sulphide-releasing agent, GYY4137, significantly reduced resorption, hypertension and proteinuria in Ad-sFlt-1 treated pregnant PlGF-/- mice. To determine if placental PlGF is critical for preventing fetal growth restriction associated with preeclampsia, we generated haploinsufficient PlGF+/- placentas and embryos were generated in wild-time dams and exposed to high sFlt-1 environment. This resulted in reduced fetal resorption, gestational hypertension and proteinuria when compared to pregnant PlGF-/- mice. CONCLUSIONS: Placental PlGF is a critical protective factor against the damaging effects of high sFlt-1 in preeclampsia and the hydrogen sulphide pathway may rescue preeclampsia phenotypes.

Placental abruption : Studies on incidence, risk factors and potential predictive biomarkers

Placental abruption, one of the most significant causes of perinatal mortality and maternal morbidity, occurs in 0.5-1% of pregnancies. Its etiology is unknown, but defective trophoblastic invasion of the spiral arteries and consequent poor vascularization may play a role. The aim of this study was to define the prepregnancy risk factors of placental abruption, to define the risk factors during the index pregnancy, and to describe the clinical presentation of placental abruption. We also wanted to find a biochemical marker for predicting placental abruption early in pregnancy. Among women delivering at the University Hospital of Helsinki in 1997-2001 (n=46,742), 198 women with placental abruption and 396 control women were identified. The overall incidence of placental abruption was 0.42%. The prepregnancy risk factors were smoking (OR 1.7; 95% CI 1.1, 2.7), uterine malformation (OR 8.1; 1.7, 40), previous cesarean section (OR 1.7; 1.1, 2.8), and history of placental abruption (OR 4.5; 1.1, 18). The risk factors during the index pregnancy were maternal (adjusted OR 1.8; 95% CI 1.1, 2.9) and paternal smoking (2.2; 1.3, 3.6), use of alcohol (2.2; 1.1, 4.4), placenta previa (5.7; 1.4, 23.1), preeclampsia (2.7; 1.3, 5.6) and chorioamnionitis (3.3; 1.0, 10.0). Vaginal bleeding (70%), abdominal pain (51%), bloody amniotic fluid (50%) and fetal heart rate abnormalities (69%) were the most common clinical manifestations of placental abruption. Retroplacental blood clot was seen by ultrasound in 15% of the cases. Neither bleeding nor pain was present in 19% of the cases. Overall, 59% went into preterm labor (OR 12.9; 95% CI 8.3, 19.8), and 91% were delivered by cesarean section (34.7; 20.0, 60.1). Of the newborns, 25% were growth restricted. The perinatal mortality rate was 9.2% (OR 10.1; 95% CI 3.4, 30.1). We then tested selected biochemical markers for prediction of placental abruption. The median of the maternal serum alpha-fetoprotein (MSAFP) multiples of median (MoM) (1.21) was significantly higher in the abruption group (n=57) than in the control group (n=108) (1.07) (p=0.004) at 15-16 gestational weeks. In multivariate analysis, elevated MSAFP remained as an independent risk factor for placental abruption, adjusting for parity ≥ 3, smoking, previous placental abruption, preeclampsia, bleeding in II or III trimester, and placenta previa. MSAFP ≥ 1.5 MoM had a sensitivity of 29% and a false positive rate of 10%. The levels of the maternal serum free beta human chorionic gonadotrophin MoM did not differ between the cases and the controls. None of the angiogenic factors (soluble endoglin, soluble fms-like tyrosine kinase 1, or placental growth factor) showed any difference between the cases (n=42) and the controls (n=50) in the second trimester. The levels of C-reactive protein (CRP) showed no difference between the cases (n=181) and the controls (n=261) (median 2.35 mg/l [interquartile range {IQR} 1.09-5.93] versus 2.28 mg/l [IQR 0.92-5.01], not significant) when tested in the first trimester (mean 10.4 gestational weeks). Chlamydia pneumoniae specific immunoglobulin G (IgG) and immunoglobulin A (IgA) as well as C. trachomatis specific IgG, IgA and chlamydial heat-shock protein 60 antibody rates were similar between the groups. In conclusion, although univariate analysis identified many prepregnancy risk factors for placental abruption, only smoking, uterine malformation, previous cesarean section and history of placental abruption remained significant by multivariate analysis. During the index pregnancy maternal alcohol consumption and smoking and smoking by the partner turned out to be the major independent risk factors for placental abruption. Smoking by both partners multiplied the risk. The liberal use of ultrasound examination contributed little to the management of women with placental abruption. Although second-trimester MSAFP levels were higher in women with subsequent placental abruption, clinical usefulness of this test is limited due to low sensitivity and high false positive rate. Similarly, angiogenic factors in early second trimester, or CRP levels, or chlamydial antibodies in the first trimester failed to predict placental abruption.

Esx1 is an X-chromosome-imprinted regulator of placental formation and fetal growth

Placental formation and genomic imprinting are two important features of embryonic development in placental mammals. Genetic studies have demonstrated that imprinted genes play a prominent role in regulating placental formation. In marsupials, mice and humans, the paternally derived X chromosome is preferentially inactivated in the placental tissues of female embryos. This special form of genomic imprinting may have evolved under the same selective forces as autosomal imprinted genes. This chromosomal imprinting phenomenon predicts the existence of maternally expressed X-linked genes that regulate placental development.^ In this study, an X-linked homeobox gene, designated Esx1 has been isolated. During embryogenesis, Esx1 was expressed in a subset of placental tissues and regulates formation of the chorioallantoic placenta. Esx1 acted as an imprinted gene. Heterozygous female mice that inherit an Esx1-null allele from their father developed normally. However, heterozygous females that inherit the Esx1 mutation from their mother were born 20% smaller than normal and had an identical phenotype to hemizygous mutant males and homozygous mutant females. Surprisingly, although Esx1 mutant embryos were initially comparable in size to wild-type controls at 13.5 days post coitum (E13.5) their placentas were significantly larger (51% heavier than controls). Defects in the morphogenesis of the labyrinthine layer were observed as early as E11.5. Subsequently, vascularization abnormalities developed at the maternal-fetal interface, causing fetal growth retardation. These results identify Esx1 as the first essential X-chromosome-imprinted regulator of placental development that influences fetal growth and may have important implications in understanding human placental insufficiency syndromes such as intrauterine growth retardation (IUGR). ^

Provirus integration into a gene encoding a ubiquitin-conjugating enzyme results in a placental defect and embryonic lethality.

Ubiquitin-conjugating enzymes (E2 or Ubc) constitute a family of conserved proteins that play a key role in ubiquitin-dependent degradation of proteins in eukaryotes. We describe here a transgenic mouse strain where retrovirus integration into an Ubc gene, designated UbcM4, results in a recessive-lethal mutation. UbcM4 is the mouse homologue of the previously described human UbcH7 that is involved in the in vitro ubiquitination of several proteins including the tumor suppressor protein p53. The provirus is located in the first intron of the gene. When both alleles are mutated the level of steady-state mRNA is reduced by about 70%. About a third of homozygous mutant embryos die around day 11.5 of gestation. Embryos that survive that stage are growth retarded and die perinatally. The lethal phenotype is most likely caused by impairment of placenta development as this is the only organ that consistently showed pathological defects. The placental labyrinth is drastically reduced in size and vascularization is disturbed. The UbcM4 mouse mutant represents the first example in mammals of a mutation in a gene involved in ubiquitin conjugation. Its recessive-lethal phenotype demonstrates that the ubiquitin system plays an essential role during mouse development.

Dysregulation of hydrogen sulfide producing enzyme cystathionine γ-lyase contributes to maternal hypertension and placental abnormalities in preeclampsia

Background—The exact etiology of preeclampsia is unknown, but there is growing evidence of an imbalance in angiogenic growth factors and abnormal placentation. Hydrogen sulfide (H2S), a gaseous messenger produced mainly by cystathionine ?-lyase (CSE), is a proangiogenic vasodilator. We hypothesized that a reduction in CSE activity may alter the angiogenic balance in pregnancy and induce abnormal placentation and maternal hypertension. Methods and Results—Plasma levels of H2S were significantly decreased in women with preeclampsia (P<0.01), which was associated with reduced placental CSE expression as determined by real-time polymerase chain reaction and immunohistochemistry. Inhibition of CSE activity by DL-propargylglycine reduced placental growth factorproduction from first-trimester (8–12 weeks gestation) human placental explants and inhibited trophoblast invasion in vitro. Knockdown of CSE in human umbilical vein endothelial cells by small-interfering RNA increased the release of soluble fms-like tyrosine kinase-1 and soluble endoglin, as assessed by enzyme-linked immunosorbent assay, whereas adenoviral-mediated CSE overexpression in human umbilical vein endothelial cells inhibited their release. Administration of DL-propargylglycine to pregnant mice induced hypertension and liver damage, promoted abnormal labyrinth vascularization in the placenta, and decreased fetal growth. Finally, a slow-releasing H2S-generating compound, GYY4137, inhibited circulating soluble fms-like tyrosine kinase-1 and soluble endoglin levels and restored fetal growth in mice that was compromised by DL-propargylglycine treatment, demonstrating that the effect of CSE inhibitor was attributable to inhibition of H2S production. Conclusions—These results imply that endogenous H2S is required for healthy placental vasculature and that a decrease in CSE/H2S activity may contribute to the pathogenesis of preeclampsia. (Circulation. 2013;127:2514-2522.)

Dysregulation of hydrogen sulfide producing enzyme cystathionine γ-lyase contributes to maternal hypertension and placental abnormalities in preeclampsia

Background-The exact etiology of preeclampsia is unknown, but there is growing evidence of an imbalance in angiogenic growth factors and abnormal placentation. Hydrogen sulfide (H2S), a gaseous messenger produced mainly by cystathionine γ-lyase (CSE), is a proangiogenic vasodilator. We hypothesized that a reduction in CSE activity may alter the angiogenic balance in pregnancy and induce abnormal placentation and maternal hypertension. Methods and Results-Plasma levels of H2S were significantly decreased in women with preeclampsia (P<0.01), which was associated with reduced placental CSE expression as determined by real-time polymerase chain reaction and immunohistochemistry. Inhibition of CSE activity by DL-propargylglycine reduced placental growth factorproduction from first-trimester (8-12 weeks gestation) human placental explants and inhibited trophoblast invasion in vitro. Knockdown of CSE in human umbilical vein endothelial cells by small-interfering RNA increased the release of soluble fms-like tyrosine kinase-1 and soluble endoglin, as assessed by enzyme-linked immunosorbent assay, whereas adenoviral-mediated CSE overexpression in human umbilical vein endothelial cells inhibited their release. Administration of DL-propargylglycine to pregnant mice induced hypertension and liver damage, promoted abnormal labyrinth vascularization in the placenta, and decreased fetal growth. Finally, a slow-releasing H2S-generating compound, GYY4137, inhibited circulating soluble fms-like tyrosine kinase-1 and soluble endoglin levels and restored fetal growth in mice that was compromised by DL-propargylglycine treatment, demonstrating that the effect of CSE inhibitor was attributable to inhibition of H2S production. Conclusions-These results imply that endogenous H2S is required for healthy placental vasculature and that a decrease in CSE/H2S activity may contribute to the pathogenesis of preeclampsia. © 2013 American Heart Association, Inc.

Closed system isolation and scalable expansion of human placental mesenchymal stem cells

Mesenchymal stem cells (MSC) are emerging as a leading cellular therapy for a number of diseases. However, for such treatments to become available as a routine therapeutic option, efficient and cost-effective means for industrial manufacture of MSC are required. At present, clinical grade MSC are manufactured through a process of manual cell culture in specialized cGMP facilities. This process is open, extremely labor intensive, costly, and impractical for anything more than a small number of patients. While it has been shown that MSC can be cultivated in stirred bioreactor systems using microcarriers, providing a route to process scale-up, the degree of numerical expansion achieved has generally been limited. Furthermore, little attention has been given to the issue of primary cell isolation from complex tissues such as placenta. In this article we describe the initial development of a closed process for bulk isolation of MSC from human placenta, and subsequent cultivation on microcarriers in scalable single-use bioreactor systems. Based on our initial data, we estimate that a single placenta may be sufficient to produce over 7,000 doses of therapeutic MSC using a large-scale process.