Uteroplacental Unit Research Articles

Augmented dilation to nitric oxide in uterine arteries from rats with type 2 diabetes: implications for vascular adaptations to pregnancy

Pre-existing diabetes increases the risk of maternal and fetal complications during pregnancy, which may be due to underlying maternal vascular dysfunction and impaired blood supply to the uteroplacental unit. Endothelial dysfunction and reduced vascular smooth muscle responsiveness to nitric oxide (NO) are common vascular impairments in type 2 diabetes (T2D). We hypothesized that uterine arteries from diabetic rats would have reduced vascular smooth muscle sensitivity to NO compared with nondiabetic rats due to impairment in the NO/soluble guanylate cyclase (sGC)/cGMP signaling pathway. Uterine arteries from pregnant Goto-Kakizaki (GK; model of T2D) and Wistar (nondiabetic) rats were studied in a wire myograph. GK nonpregnant uterine arteries had reduced responses to ACh and sodium nitroprusside (SNP) but increased responses to propylamine propylamine NONOate and greater sensitivity to sildenafil compared with Wistar nonpregnant arteries. In late pregnancy, Wistar rats had reduced uterine vascular smooth muscle responsiveness to SNP, but GK rats failed to show this adaptation and had reduced expression of sGC compared with the nonpregnant state. GK rats had a smaller litter size (13.9 ± 0.48 vs. 9.8 ± 0.75; P < 0.05) and a greater number of resorptions compared with Wistar controls (0.8 ± 0.76% vs. 19.9 ± 6.06%; P < 0.05). These results suggest that uterine arteries from rats with T2D show reduced sensitivity of uterine vascular smooth muscle sGC to NO. During pregnancy, the GK uterine vascular smooth muscle fails to show relaxation responses similar to those of arteries from nondiabetic rats.

Gene Expression Analysis of the Microdissected Trophoblast Layer of Human Placenta after the Spontaneous Onset of Labor

BackgroundDespite increasing evidence that human parturition is associated with alteration in gene expression in the uteroplacental unit, the precise mechanisms that elicit spontaneous term labor in humans remain unknown. Our goal in this study was to compare the mRNA expression pattern of the trophoblast layer of normal term placenta between women who had given natural birth (labor group) and those who had undergone an elective cesarean section without labor (non-labor group).MethodsWe collected placental tissue samples from six pregnant women after term vaginal deliveries (labor group) and from six pregnant women after scheduled Cesarean sections (non-labor group). Frozen sections were made immediately after placental delivery. Because the placenta is a heterogeneous tissue composed of several cell types, we used laser capture microdissection to separate the trophoblast layer from the rest of the placental tissues.ResultsA number of genes were differentially expressed in the trophoblast layer when the labor and non-labor groups were compared. The expression of SIRT1, KAP1, and CRH was significantly lower in the trophoblast layer of the labor group than of the non-labor group. The expression of IL-1b, NF-kB1 and TLR 8 in the labor group was significantly higher than that in the non-labor group.ConclusionsHuman term labor may be closely associated with inflammatory response. We suggest that downregulation of SIRT1, KAP1, and CRH gene expression in the trophoblast may play a key role in parturition and initiation of labor in pregnant human females.

Macrophage-Derived IL-33 Is a Critical Factor for Placental Growth

IL-33, the most recently discovered member of the IL-1 superfamily and ligand for the transmembrane form of ST2 (ST2L), has been linked to several human pathologies including rheumatoid arthritis, asthma, and cardiovascular disease. Deregulated levels of soluble ST2, the natural IL-33 inhibitor, have been reported in sera of preeclamptic patients. However, the role of IL-33 during healthy pregnancy remains elusive. In the current study, IL-33 was detected in the culture supernatants of human placental and decidual macrophages, identifying them as a major source of secreted IL-33 in the uteroplacental unit. Because flow cytometry and immunofluorescence stainings revealed membranous ST2L expression on specific trophoblast populations, we hypothesized that IL-33 stimulates trophoblasts in a paracrine manner. Indeed, BrdU incorporation assays revealed that recombinant human IL-33 significantly increased proliferation of primary trophoblasts as well as of villous cytotrophoblasts and cell column trophoblasts in placental explant cultures. These effects were fully abolished upon addition of soluble ST2. Interestingly, Western blot and immunofluorescence analyses demonstrated that IL-33 activates AKT and ERK1/2 in primary trophoblasts and placental explants. Inhibitors against PI3K (LY294002) and MEK1/2 (UO126) efficiently blocked IL-33-induced proliferation in all model systems used. In summary, with IL-33, we define for the first time, to our knowledge, a macrophage-derived regulator of placental growth during early pregnancy.

Clinical, ultrasonographic, and endocrinological studies on donkey pregnancy

Although donkey breeding has gained new interest in the past two decades, knowledge about donkey reproduction is still scarce, particularly on jenny pregnancy. The aim of this study was to describe the ultrasonographic and endocrine profiles of the physiological pregnancy in the jenny. The study was performed on 12 pregnancies of 7 Amiata donkeys from Day 10 after ovulation to delivery. Because three pregnancies, respectively at weeks 42, 44, and 45, were considered pathologic and treated pharmacologically, data collected from 2 weeks before diagnosis to the end of pregnancy were removed from the analysis. Average length of the normal pregnancies was 353.4 ± 13.0 days (range, 339–370 days). Timing, dimensions, and development during the first phases of embryonic growth, evaluated using transrectal ultrasound, were similar to that previously described in jennies and mares: first detection of embryonic vesicle was at 11.8 ± 1.3 days of gestation and diameter was 6.5 ± 1.9 mm, loss of spherical shape occurred at 18.5 ± 1.4 days, and embryo and heart beat were first seen at 22.0 ± 1.1 and 25 ± 1.1 days, respectively. The intrauterine growth in the second half of pregnancy, evaluated using the transrectal and transabdominal approach, also showed strong positive correlations, similar to that reported for the mare. The trends of the combined thickness of the utero-placental unit and the echogenicity of the amniotic and allantoic fluids are examples. The diameters (mm) of fetal chest, eye orbit, and aorta increased throughout pregnancy and were 40.6 ± 2.9, 8.7 ± 1.5, and 3.5 ± 0.7, respectively, at week 13, and 190.9 ± 12.0, 21.4 ± 1.5, and 30.6 ± 1.8 at the last evaluation before parturition. In contrast, heart rate decreased as pregnancy progressed. Regression analyses between these parameters and day of gestation were statistically significant (P < 0.001). All fetuses consistently showed some intrauterine activity. Maternal plasma progestagens and estrone sulfate concentrations followed a pattern similar to that seen in mares, although the prepartal progestagen peak was lower in jennies. This study provides a range of ultrasonographic and endocrine values for normal pregnancy in jennies.

Abstract 281: HO-1 Induction Increases ßENaC in Ischemic Placentas and Cultured Cytotrophoblasts

Preeclampsia is characterized by abnormal placentation that is thought to arise from inadequate cytotrophoblast migration and invasion of the spiral arteries. The resulting reduced perfusion of the utero-placental unit promotes the development of hypertension in preeclampsia. Beta Epithelial Na+ Channel (βENaC) protein, a mediator of cytotrophoblast migration in vitro, is decreased in placentas of preeclamptic patients. We have recently reported that hemeoxygenase-1 (HO-1) and its by-products reduce blood pressure in placental ischemic rats. Since HO and its byproducts can regulate ENaC expression, including βENaC, are important mediators of migration, the goal of this study was to test the hypothesis that HO-1 induction 1) stimulates βENaC expression and migration in cultured cytotrophoblasts and 2) restores βENaC expression in ischemic placentas. Cobalt protoporhyrin (CoPP, 1 - 10 μM, 48 hrs) stimulated HO-1 induction in cultured cytotrophoblasts (BEWO cells), upregulated βENaC expression, and increased spontaneous migration by 39.2 ± 6.0% (n=7, p&lt;0.001). To determine if HO-1 induction increased placental βENaC expression and prevented hypertension, CoPP was injected subcutaneously (5mg/kg) on gestational day 14 in timed pregnant Sprague-Dawley rats with placental ischemia. MAP was increased on gestational day 19 in placental ischemic rats (141 ± 4 vs.101 ± 1 mmHg, p&lt;0.001; n=4 per group). While HO-1 induction did not affect MAP in normal pregnant rats (101 ± 5 mmHg), MAP was significantly attenuated (p&lt;0.01) in placental ischemic rats treated with CoPP (117 ± 1 mmHg). Western blot analysis revealed a 62 ± 22% decrease in βENaC expression in ischemic placentas, which was restored by HO-1 induction (n= 4, p&lt;0.05). These findings suggest that HO-1 induction stimulates βENaC expression in cultured cytotrophoblasts and ischemic placentas. Future studies will determine if the beneficial effects of HO-1 occur through βENaC-mediated cytotrophoblast migration and spiral artery remodeling in ischemic placentas. Targeting the HO-1 pathway presents a potential therapeutic approach for improving placentation in preeclamptic women.

Macrophage-derived interleukin-33 is a critical factor for placental growth

s / Placenta 34 (2013) A1–A99 A11 including SynA and Gcm1. Similar treatment of Sirt1-null mTSC showed this effect to be Sirt1-dependent. Conclusion: Sirt1 regulates mTSC proliferation and differentiation. Lack of Sirt1 hinders mTSC differentiation. Sirt1 activation, in contrast, specifically induces the labyrinthine lineage. Further characterization of Sirt1-null mTSC and placentas may help elucidate mechanisms important for placental development and disease. http://dx.doi.org/10.1016/j.placenta.2013.06.036 NI.13. MACROPHAGE-DERIVED INTERLEUKIN-33 IS A CRITICAL FACTOR FOR PLACENTAL GROWTH Valerie Fock, Harald Zeisler, Martin Knofler, Jurgen Pollheimer Medical University of Vienna, Vienna, Austria Interleukin (IL)-33, a novel IL-1 superfamily member and ligand for the Thelper type-2-associated receptor ST2L, has been linked to several human pathologies including rheumatoid arthritis, asthma and cardiovascular disease. Recently, elevated levels of the natural IL-33 decoy receptor soluble ST2 have been reported in sera of pre-eclamptic women. Objectives: Our aim was to characterize the expression pattern of IL-33 and its receptor in the human placenta and to investigate the yet unknown role of the IL-33/ST2L pathway during healthy pregnancy. Methods: Placental and decidual specimens were obtained from elective terminations offirst trimester pregnancies. Expression and localization of IL33 and ST2L were analyzed by flow cytometry, Western blot and immunofluorescence stainings. IL-33 levels in culture supernatants of macrophages were determined by ELISA. Bromodeoxyuridine incorporation assays and ki67 stainingswere performed to assess proliferation inprimary trophoblast model systems. The phosphorylation status of proliferation-associated kinases was evaluated by Western blot and immunofluorescence stainings. Results: In this studywe show that CD45+CD14+macrophages represent the major source of cytoplasmic IL-33 in the uteroplacental unit. Furthermore, the presence of IL-33 was confirmed in culture supernatants of primary villous and decidual macrophages. Membranous expression of ST2L was detected on different trophoblast subtypes, identifying them as potential target cells for secreted IL-33. Indeed, recombinanthuman IL-33 significantly increased proliferation of primary trophoblasts as well as of villous cytotrophoblasts (CTBs) and cell column trophoblasts (CCTs) in placental explant cultures. These effects were fully abolished upon addition of soluble ST2. IL33-dependent AKT and ERK1/2 activation was observed in primary trophoblasts, villous CTBs and CCTs. In agreement with this observation, chemical inhibitors against PI3K (LY294002) andMEK1/2 (UO126) efficiently blocked IL-33-induced trophoblast proliferation in all model systems used. Conclusion: With IL-33 we define for the first time a macrophage-derived regulator of placental growth during early pregnancy. This studywas funded by the Austrian National Bank (grant 13955) and the Austrian Science Fund (P-22687-B13, P-25187-B13). http://dx.doi.org/10.1016/j.placenta.2013.06.037 P1.1. DECIDUAL ACUTE ATHEROSISUPDATING AND TESTING DIAGNOSTIC CRITERIA Patji Alnaes-Katjavivi , Fiona Lyall , Borghild Roald , Christopher Redman , Annetine Staff 1 Women and Children's Division, Oslo University Hospital (OUS), Oslo, Norway; 2 Institute of Medical Genetics, University of Glasgow, Glasgow, UK; Nuffield Department of ObstetricsG Department of Pathology, Oslo University Hospital (OUS), Oslo, Norway Background: Acute atherosis (AA) of the uteroplacental spiral arteries is an understudied lesion, associatedwith infarction of the placental parenchyma and preeclampsia, both contributing to deleterious pregnancy outcome. The lesion resembles early stage atherosclerosis, and we hypothesize that it has a spectrum of expression and may also identify a subset of women at elevated risk for atherosclerotic arterial disease later in life. AA is not specific to pregnancies affected by preeclampsia, but can affect uncomplicated, normotensive pregnancies. Acute atherosis, originally described by Hertig (1945), is defined by presence of subendothelial lipidfilled foam cells, fibrinoid necrosis and perivascular leukocyte infiltration (Hanssens M et al. Eur J Obstet Gynecol 1998). Previous definitions lack quantitative criteria and are subject to observer bias. The aim of the study was to update the diagnostic criteria of decidual acute atherosis, and to develop methods of quantification of lesions. Methods: We have developed revised criteria to diagnose and quantify decidual uterospiral acute atherosis. We evaluate serial sections of formalin fixated and paraffin embedded decidual basalis tissue samples, collected by vacuum suction from 250 caesarean deliveries. Results: Our revised definition expands on the traditional CD68+ marker for the macrophage-type of foam cells, recording residual smooth muscle (desmin antibody), fibrinoid (PAS), quantity and localization of trophoblast (cytokeratin antibody), and perivascular mononuclear cells. The findings for each patient yield a stratified diagnosis of decidual acute atherosis, depending on the sum of characteristics present for the visualized decidual arteries. Reliability of diagnosis for acute atherosis, using these predefined criteria, will be presented as Kappa values between the first three abstract authors. Conclusions:We present a new, quantitativemethod for defining decidual acute atherosis. Quantification may prove useful when exploring the association between acute atherosis and pregnancy phenotype, and whether presence of lesion relates to excessive risk of future atherosclerotic CVD. http://dx.doi.org/10.1016/j.placenta.2013.06.038 P1.2. ABNORMAL PERICYTE RECRUITMENT IN FETAL PLACENTAL VASCULATURE: CAUSE OF IUGR AND FETAL THROMBOTIC VASCULOPATHY IN ADAMS-OLIVER SYNDROME?

Temporo-spatial expression of adrenomedullin and its receptors in the bovine placenta

BackgroundAdrenomedullin (AM) is a potent vasodilator peptide and is also involved in various physiological activities. In humans and rodents, AM is found in the uteroplacental unit and may be responsible for fetal development and maintenance of placental function. This study investigated 1) the mRNA expression patterns of AM and its receptor components (calcitonin receptor-like receptor (CRLR), receptor activity modifying protein (RAMP) 2 and RAMP3) during pregnancy and 2) mRNA and protein localization of AM, CRLR and RAMPs in the bovine placentome.MethodsFor real-time quantitative RT-PCR, bovine uteroplacental tissues were collected from Day 25, 60, 100, 150, 200 and 250 of gestation and separated into uterine caruncle (CAR), intercaruncular endometrium (ICAR), extra-embryonic membranes on Day 25 and cotyledonary villous after Day 60 (EEM-COT) and intercotyledonary chorion (ICOT). In situ hybridization and immunohistochemistry was performed to investigate the cellular localization of mRNA and protein of AM, CRLR, RAMP2 and RAMP3 in the placentome on Day 56, 150 and 230 of gestation and interplacentomal tissues on Day 56 of gestation.ResultsAM mRNA was highly expressed on Day 200 in EEM-COT, CAR and ICAR. CRLR mRNA was highly expressed on Day 60 in all portions. RAMP2 mRNA was also highly expressed on Day 60 in ICOT and ICAR. In EEM-COT, mRNA expression of CRLR and RAMP2 decreased from Day 150 to 250. RAMP3 mRNA was highly expressed on Day 150 in EEM-COT, ICOT and ICAR. A distinct AM mRNA and protein signal were only found in trophoblast binucleate cells (BNCs), whereas those of CRLR, RAMP2 and RAMP3 were detected in cotyledonary villous and caruncular epithelial cells. In interplacentomal tissues, AM was detected in BNCs of fetal membrane and a small part of luminal epithelium, endothelial lineage of blood vessels and glandular epithelium of the endometrium. Distinct signals of CRLR, RAMP2 and RAMP3 were found in trophoblast cells, luminal epithelium, stroma under the epithelium, endothelial lineage of blood vessels and glandular epithelium.ConclusionsOur results indicate that the AM system in the bovine uteroplacental unit may be activated at placentation and transition from the mid to late gestation period. Locally produced AM in the BNCs may play a crucial role in regulation of placental vascular and cellular functions during pregnancy.

Screening for overt thyroid disease in early pregnancy may be preferable to searching for small aberrations in thyroid function tests

Thyroid hormones are important regulators of foetal development, and in recent years, there has been much focus on the screening and treatment of pregnant women for even small aberrations in thyroid function tests. We searched PubMed for publications on thyroid function and pregnancy outcomes including child cognition, and included references from the retrieved articles. Both small aberrations in thyroid function tests in early pregnancy and an increase in risk of pregnancy complications may be caused by a functional change in the uteroplacental unit. Thus, the association found in several studies between small thyroid test abnormalities and pregnancy complications may be due to confounding, and thyroid hormone therapy will have no effect. On the other hand, screening of thyroid function in early pregnancy may identify 200-300 women with undiagnosed overt hypothyroidism per 100,000 pregnancies, which is at least five times more than the number of hypothyroid newborns identified by screening. A number of studies indicate that untreated overt thyroid disease in pregnancy may lead to complications. The potential benefit of screening and early therapy is supported by evidence, indicating that even severe maternal hypothyroidism does not lead to neurocognitive deficiencies in the child, if the condition is detected and treated during the first half of pregnancy. Screening and therapy for overt thyroid dysfunction in early pregnancy may be indicated, rather than focusing on identifying and treating small aberrations in thyroid function tests.

AT1, AT2, and AT1-7 receptor expression in the uteroplacental unit of normotensive and hypertensive rats during early and late pregnancy

IntroductionWe investigated the expression of angiotensin receptors in early pregnancy and established whether normal pregnancy or preeclampsia alters the expression and distribution of the uteroplacental AT1R, AT2R and mas/AT1-7R at late gestation. MethodsThe percentage of each receptor subtype present in tissues from virgin rats and from normotensive and RUPP hypertensive pregnant rats was established by in vitro receptor autoradiography. Receptor mRNA levels were determined by quantitative PCR at early and late pregnancy. ResultsAT1R mRNA levels were up-regulated in the interimplantation (IIS) site at day 7 of gestation. AT2R mRNA levels were decreased at day 5 and 7 in the IIS but increased in the implantation site (IS) at day 5 and 7 as compared to the IIS at day 5. Mas/AT1-7R mRNA was increased in early pregnancy. In normal pregnancy and RUPP the mRNA for all angiotensin receptors was reduced in the uterus at late gestation. The AT1R accounted for the majority of binding in the uterus of virgin and the placenta of pregnant and RUPP. In RUPP pregnancy there was a significant competition with d-Ala in the placenta labyrinth. Discussion and conclusionThe expression of angiotensin receptors suggests their involvement in the maintenance of early stages of pregnancy. During late gestation down-regulation of Ang receptors in the uterus may arise from feedback down-regulation by Ang II. In the placenta the levels of AT1Rs are equivalent in the RUPP model. The increased binding of mas/AT1-7R at late gestation in RUPP may represent a compensatory mechanism to reduce uteroplacental vascular resistance.

Interleukin-15 is required for maximal lipopolysaccharide-induced abortion

The maternal immune response during pregnancy is critical for the survival of the fetus yet can be detrimental during infection and inflammation. Previously, IL-15 has been observed to mediate inflammation during LPS-induced sepsis. Therefore, we sought to determine whether IL-15 mediates the inflammatory process during LPS-induced abortion through the use of IL-15(-/-) and WT mice. Administration of 2.5 μg LPS i.p. on gd 7.5 drastically reduced fetal viability in WT mice, whereas it had a minimal effect on fetal survival in IL-15(-/-) mice. The uteroplacental sites of LPS-treated WT mice were characterized by vast structural degradation and inflammation compared with treated IL-15(-/-) and untreated controls. This suggests that IL-15 may mediate the inflammation responsible for LPS-induced resorption. As IL-15(-/-) mice are deficient in NK cells and resistant to LPS-induced abortion, these effects suggest that IL-15 may mediate abortion through their homeostatic and/or activation effects on NK cells. WT uteroplacental units exposed to LPS had an increase in the overall number and effector number of NK cells compared with their control counterparts. Furthermore, NK cell depletion before administration of LPS in WT mice partially restored fetal viability. Overall, this paper suggests that IL-15 mediates the inflammatory environment during LPS-induced fetal resorption, primarily through its effects on NK cells.

Current Perspectives in Uterine Research: Reviews From the Society for Gynecologic Investigation Myometrium and Parturition Satellite Symposia

It is difficult to imagine examples of greater physiological adaptations than those of the uteroplacental unit during pregnancy. The sheer scale of molecular, cellular and tissue remodeling, organogenesis, and organism development in a relatively short period of time seems the epitome of ‘‘systems biology.’’ The processes are challenging to comprehend, and investigate experimentally, but undoubtedly fascinating. Efforts to understand the physiology and pathology of these phenomena are of prime importance due to the considerable health and socioeconomic pressures brought to bear by the high incidences of compromised pregnancies and, especially, preterm birth (PTB). The World Health Organization-sponsored studies equate the worldwide incidence of PTB to be approximately 13 million births per year with approximately 1 million neonatal deaths attributable to PTB. If improved diagnosis and therapeutic treatment of PTB are to be achieved, an integrated understanding of the physiological basis of pregnancy and parturition is required. With this in mind, each year the Myometrium and Parturition Satellite Symposium of the Society for Gynecologic Investigation meeting gathers international experts in this field to share their experimental findings. We are delighted that several plenary speakers from the 2011 and 2012 symposia have provided reviews on their topics for this issue of Reproductive Sciences. The articles provide an up-to-date compendium that integrates many of the important facets of uteroplacental remodeling and function leading to term and preterm labor. Jerome Strauss (Virginia) introduces the importance of dynamic remodeling of the extracellular matrix (ECM) and its molecular components in uteroplacental tissues. With a focus on fetal membrane interfaces, he summarizes the ECMs’ multiplicity of roles including receptor signaling, cytokine/ growth factor sequestration, and tensile strength. Among the important molecular components are fetal fibronectin, hyaluronan, and up to 28 collagens, regulated by different metallomatrix proteinases. Mutations in many genes encoding such ECM proteins are associated with increased incidence of preterm premature rupture of membranes and the roles of environmental and/or epigenetic factors are being increasingly elucidated. In a similar vein, Shynlova et al (Toronto, Canada) describe pregnancy in terms of progressive changes of tissue remodeling under the control of localized inflammatory events (physiological inflammation). The authors posit a carefully structured theory whereby spatially restricted inflammatory events are under the influence of ever-changing endocrine (and a focus is given to the role of progesterone) and mechanical inputs activating a rich milieu of chemokine/cytokine signaling. These, in turn, serve to coordinate diverse processes such as decidualization, myometrial cell proliferation and growth, leukocyte migration, cervical remodeling, and postparturient involution. Olcese et al (Tallahassee, USA) review an oft-neglected consideration of the mechanisms responsible for the timing of parturition. They too give credence to the notion that pregnancy and parturition involve dynamic physiological adaptations—in this case that uterine contractions of labor may be initiated by activity of the central circadian oscillator of the maternal hypothalamic suprachiasmatic nuclei via the feedback ‘‘gating’’ actions of neuroendocrine outputs. Of key importance may well be circadian secretion of melatonin given recent interest in the uterotonic actions of this hormone and uterine expression of melatonin receptors. Regulation of contractile activity of the uterus is also the focus of reviews by Europe-Finner et al (Newcastle, UK) and Wim Lammers (Al Ain, UAE). The former concentrates on intracellular signaling and describes the possibility that posttranslational modification by acetylation may be a mechanism of wider significance to the uterus than first realized when considering ‘‘‘only’’ nuclear events of histone modification, chromatin remodeling, and gene transcription. They suggest

The role of the renin–angiotensin–aldosterone system in preeclampsia: genetic polymorphisms and microRNA

The compensatory alterations in the rennin-angiotensin-aldosterone system (RAAS) contribute to the salt-water balance and sufficient placental perfusion for the subsequent well-being of the mother and fetus during normal pregnancy and is characterized by an increase in almost all the components of RAAS. Preeclampsia, however, breaks homeostasis and leads to a disturbance of this delicate equilibrium in RAAS both for circulation and the uteroplacental unit. Despite being a major cause for maternal and neonatal morbidity and mortality, the pathogenesis of preeclampsia remains elusive, where RAAS has been long considered to be involved. Epidemiological studies have indicated that preeclampsia is a multifactorial disease with a strong familial predisposition regardless of variations in ethnic, socioeconomic, and geographic features. The heritable allelic variations, especially the genetic polymorphisms in RAAS, could be the foundation for the genetics of preeclampsia and hence are related to the development of preeclampsia. Furthermore, at a posttranscriptional level, miRNA can interact with the targeted site within the 3'-UTR of the RAAS gene and thereby might participate in the regulation of RAAS and the pathology of preeclampsia. In this review, we discuss the recent achievements of genetic polymorphisms, as well as the interactions between maternal and fetal genotypes, and miRNA posttranscriptional regulation associated with RAAS in preeclampsia. The results are controversial but utterly inspiring and attractive in terms of potential prognostic significance. Although many studies suggest positive associations with genetic mutations and increased risk for preeclampsia, more meticulously designed large-scale investigations are needed to avoid the interference from different variations.

Utero-placental expression of angiotensin-(1–7) and ACE2 in the pregnant guinea-pig

BackgroundIn humans, trophoblast invasion, vascular remodeling and placental development are critical to determine the fate of pregnancy. Since guinea-pigs (GP) and humans share common pregnancy features including extensive trophoblast invasion, transformation of the uterine spiral arteries and a haemomonochorial placenta, the GP animal model was deemed suitable to extend our knowledge on the spatio-temporal immunoreactive expression of the vasodilator arpeptide of the renin-angiotensin system, angiotensin-(1–7) [Ang-(1–7)] and its main generating enzyme, angiotensin converting enzyme 2 (ACE2).MethodsUtero-placental units were collected in days 15, 20, 40 and 60 of a 64–67 day long pregnancy in 25 Pirbright GP. Ang-(1–7) and ACE2 expression in utero-placental units were evaluated by immunohistochemistry.ResultsAng-(1–7) and ACE2 were detected in the endothelium and syncytiotrophoblast of the labyrinthine placenta, interlobium, subplacenta, giant cells, syncytial sprouts, syncytial streamers, and myometrium throughout pregnancy. In late pregnancy, perivascular or intramural trophoblasts in spiral and mesometrial arteries expressed both factors. Immunoreactive Ang-(1–7) and ACE2 were present in decidua and in the vascular smooth muscle of spiral, myometrial and mesometrial arteries, which also express kallikrein (Kal), the bradykinin receptor 2 (B2R), vascular endothelial growth factor (VEGF) and its type 2 receptor (KDR), but no endothelial nitric oxide synthase (eNOS). In addition, the signal of Ang-(1–7) and ACE2 was especially remarkable in giant cells, which also show Kal, B2R. eNOS, VEGF and KDR.ConclusionsThe spatio-temporal expression of Ang-(1–7) and ACE2 in GP, similar to that of humans, supports a relevant evolutionary conserved function of Ang-(1–7) and ACE2 in decidualization, trophoblast invasion, vascular remodeling and placental flow regulation, as well as the validity of the GP model to understand the local adaptations of pregnancy. It also integrates Ang-(1–7) to the utero-placental vasodilatory network. However, its antiangiogenic effect may counterbalance the proangiogenic activity of some of the other vasodilator components.

OC-06 Effects of low-molecular-weight heparin within the Uteroplacental Unit

OC-06 Effects of low-molecular-weight heparin within the Uteroplacental Unit

Renin-angiotensin system in pre-eclampsia: everything old is new again.

This review presents an update of the role of the renin-angiotensin system in normal pregnancy and pre-eclampsia. We have known for years that the circulatory renin-angiotensin system in pre-eclampsia is suppressed. We now know that the circulating renin-angiotensin system does not only have a vasoconstrictor arm, but also a vasodilator arm, which is upregulated in normal pregnancy; this balance is probably disturbed in pre-eclampsia. Recent studies show the importance of the local renin-angiotensin system in the uteroplacental unit for early placentation and regulation of placental blood flow. We discuss the possible role of autoantibodies against the AT1-receptor in pre-eclampsia and the suggestion that activation of the AT1-receptor in the placenta may lead to placental dysfunction and the clinical syndrome of pre-eclampsia.

Placental expression and localization of endothelin-1 system and nitric oxide synthases during bovine pregnancy

This study aimed to investigate mRNA expression of the endothelin-1 (EDN1) system (preproEDN1; precursor, ECE-1; converting enzyme, EDNRA and EDNRB; receptor subtypes A and B) and endothelial and inducible nitric oxide synthases (eNOS and iNOS) in the bovine utero-placental unit during pregnancy. We also investigated the cellular localization of mRNA and protein of components of the EDN1 system in the placentome. The bovine utero-placental unit on Day 60, 100, 150, 200 and 250 of gestation was separated into carunclar areas (CAR), intercaruncular areas (ICAR), cotyledonary villi (COT) and intercotyledonary areas (ICOT). PreproEDN1, ECE1, EDNRA, EDNRB, eNOS and iNOS mRNA expression was determined by real-time quantitative RT-PCR. In situ hybridization and immunohistochemistry were performed using placentomes on Day 94 or Day 250 of gestation. PreproEDN1 and ECE1 mRNA expression was higher on Day 100 than on other gestation days. The mRNA expression for EDNRA in COT and ICOT and eNOS in COT, CAR and ICAR were higher on Day 150 than on other gestation days. EDNRB mRNA expression increased from Day 60 to Day 150 then decreased. iNOS mRNA expression in COT and CAR was higher on Day 250 than on other gestation days. PreproEDN1, ECE1 and EDNRA mRNA was localized in the caruncular epithelial cells (CEs) and the COT. EDNRB mRNA was found in the CEs and the trophoblast binucleate giant cells (BNCs). PreproEDN1, EDNRA and EDNRB proteins were detected in COT and CEs, whereas ECE-1 was found in the BNCs. Our results demonstrate that differential cell-specific and spatiotemporal expression of the EDN1 system and NOS in the bovine utero-placental unit may be associated with regulation of vascular and cellular functions during pregnancy.

Quantification of utero-placental vascularization in a rabbit model of IUGR with three-dimensional power Doppler angiography

ObjectivesOur objective was to evaluate the 3D power Doppler angiography (PDA) in terms of feasibility and ability to detect placental hypo-perfusion in an experimental rabbit model of intrauterine growth restriction (IUGR). Study design14 pregnant females were treated with NG-nitro-L-arginine methylester (L-NAME), a nitric oxide synthase inhibitor, from day 24 to day 28 of gestation, to induce an IUGR. Concomitantly, 8 pregnant rabbits were used as controls. On day 28, 3D power Doppler indices were quantified in each utero-placental unit. Morphological examination of the placentas for the control group (n = 4) and the L-NAME group (500 mg/day, n = 4) were performed with immunohistochemical staining to discriminate the fetal capillaries in the labyrinthine area. ResultsA total of 180 live fetuses were obtained, 108 from the L-NAME group and 72 from the control group. G28 fetal weight was significantly lower in the L-NAME group than in the control group (27.40 ± 0.55 g vs 33.14 ± 0.62 g, p < 0.0001). In the L-NAME group the vascularization index (VI), flow index (FI) and vascularization flow index (VFI) were significantly lower than in the control group (2.6 [1.4; 6.0] vs 7.6 [3.5; 12.6], p < 0.05; 28.7 [26.5; 31.3] vs 32.9 [28.3; 38.1], p < 0.05; 0.8 [0.4; 1.8] vs 2.5 [1.1; 4.1], p < 0.05, for VI, FI and VFI, respectively). Morphological examinations revealed a substantial disorganization of the placental vascular architecture in the L-NAME group. ConclusionThis experimental study demonstrates that quantitative 3D PDA indices are sensitive enough to detect placental vascular insufficiency in an experimental rabbit model of IUGR.

PP085. Angiotensin II type 1 receptor antibodies increase angiotensin II sensitivity in pregnant rats

Pregnant women who subsequently develop preeclampsia are highly sensitive to infused angiotensin (Ang) II; the sensitivity persists postpartum. Activating autoantibodies against the Ang II type 1 (AT1) receptor are present in preeclampsia. In vitro and in vivo data suggest that they could be involved in the disease process. The aim of the study was to show if AT1-AB generated by immunisation alters Ang II sensitivity in pregnant rats. We generated and purified activating antibodies against the AT1 receptor (AT1-AB) by immunizing rabbits against the AFHYESQ epitope of the second extracellular loop, which is the binding epitope of endogenous activating autoantibodies against AT1 from patients with preeclampsia. We then purified AT1-AB using affinity chromatography with the AFHYESQ peptide. We were able to detect AT1-AB both by ELISA and a functional bioassay. We then passively transferred AT1-AB into pregnant rats, alone or combined with Ang II. AT1-AB activated protein kinase C-alpha and extracellular-related kinase 1/2. Passive transfer of AT1-AB alone or Ang II (435ng/kg per minute) infused alone did not induce a preeclampsia-like syndrome in pregnant rats. However, the combination (AT1-AB plus Ang II) induced hypertension, proteinuria, intrauterine growth retardation, and arteriolosclerosis in the uteroplacental unit. We next performed gene-array profiling of the uteroplacental unit and found that hypoxia-inducible factor 1alpha was upregulated by Ang II plus AT1-AB, which we then confirmed by Western blotting in villous explants. Furthermore, endothelin 1 was upregulated in endothelial cells by Ang II plus AT1-AB. We show that AT1-AB induces Ang II sensitivity. Our mechanistic study supports the existence of an "autoimmune-activating receptor" that could contribute to Ang II sensitivity and possibly to preeclampsia.

Complement C5a Regulates Prolabor Mediators in Human Placenta1

Human preterm and term parturition is associated with inflammatory cascades in the uteroplacental unit. Activation of the complement cascade releases potent proinflammatory mediators, including the anaphylatoxin C5a, which exerts its biological effects through its receptors, C5AR (also known as CD88) and C5L2, official symbol GPR77. To date, there are few data available on the role of C5a and CD88 in human pregnancy, so the aim of this study was to determine the effect of C5a and CD88 on some key inflammatory pathways involved in human parturition. Placental tissue samples were obtained from normal pregnancies at the time of Cesarean section. Human placental and fetal membranes were incubated in the absence (basal control) or presence of 0.5 μg/ml (~60 nM) human recombinant C5a for 24 h. Concentrations of proinflammatory cytokines, prostaglandins, and 8-isoprostane (a marker of oxidative stress) were quantified by ELISA and secretory matrix metalloproteinases (MMPs) activity by zymography. NFKB DNA binding activity and NFKBIA (IkappaB-alpha; inhibitor of NFKB) protein degradation were analyzed by ELISA and Western blotting, respectively. In the presence of C5a, proinflammatory cytokines (IL6 and IL8), cyclooxygenase (COX)-2; official symbol PTGS2) expression, and subsequent prostaglandin (PGE(2) and PGF(2alpha)), MMP9 enzyme production, and NFKB DNA activation were all significantly increased. The C5a-induced prolabor responses were significantly reduced by treatment with the selective CD88 antagonist PMX53 and the NFKB inhibitor BAY 11-7082. We conclude that C5a upregulates prolabor mediators in human gestational tissues via CD88-mediated NFKB activation.

Cardiovascular Biomarker Midregional Proatrial Natriuretic Peptide During and After Preeclamptic Pregnancies

Preeclampsia is associated with increased risk of cardiovascular disease. Midregional proatrial natriuretic peptide (MR-proANP), a precursor of the atrial natriuretic peptide, is a biomarker for cardiovascular disease. We obtained plasma from 184 pregnant women in gestational weeks 24 to 42 (normotensive pregnancies: n=77, preeclampsia: n=107), from 25 of these women at 5 to 8 years after index pregnancy (normotensive pregnancies: n=11, preeclampsia: n=14), and from 49 normotensive, nonpregnant women and analyzed them by immunoassay for MR-proANP. To investigate potential sources, placental and decidual atrial natriuretic peptide mRNA expression levels were analyzed by quantitative real-time PCR in 21 normotensive and 23 preeclamptic pregnancies, as well as in human heart and kidney samples. For further confirmation, we measured circulating MR-proANP and performed expression studies in a transgenic rat model for preeclampsia. MR-proANP was significantly elevated in maternal plasma in preeclampsia compared with normotensive pregnancies (135 versus 56 pmol/L; P<0.001). However, 5 to 8 years after pregnancy, there was no difference (formerly preeclamptic women versus formerly normotensive in pregnancy: 53 versus 49 pmol/L; P=0.5). Our preeclamptic rat model confirmed the acute MR-proANP differences between preeclamptic and normotensive pregnancies (10.9±1.9 versus 4.3±0.3 pmol/L; P=0.05). Atrial natriuretic peptide expression was high in the heart but negligible in the uteroplacental unit in both normotensive humans and rats, whereas expression in maternal and fetal hearts in the preeclamptic rats was significantly increased, compared with controls. MR-proANP is a serviceable biomarker in preeclampsia, both in humans and a rat model, probably reflecting cardiovascular hemodynamic stress.