Placental Vascular Bed Research Articles

Role of progesterone on dexamethasone-induced alterations in placental vascularization and progesterone receptors in rats†.

Intrauterine growth restriction (IUGR) is manifested by lower maternal progesterone levels, smaller placental size, and decreased placental vascularity indicated by lower expression of vascular endothelial growth factor (VEGF). Studies showed that progesterone increases angiogenesis and induces VEGF expression in different tissues. Therefore, the aim of the present study is to evaluate the effect of progesterone on placental vascular bed and VEGF expression and the modulation of nuclear and membranous progesterone receptors (PR) in dexamethasone-induced rat IUGR model. Pregnant Sprague-Dawley rats were allocated into four groups and given intraperitoneal injections of either saline, dexamethasone, dexamethasone, and progesterone or progesterone. Injections started on gestation day (DG) 15 and lasted until the days of euthanization (19 and 21 DG). Enzyme-linked immunosorbent assay was used to evaluate plasma progesterone levels. Real-time PCR and western blotting were used to evaluate gene and protein expressions of VEGF, and PR in labyrinth and basal placental zones. Immunohistochemistry was used to locate VEGF and different PRs in placental cells. Immunofluorescence was used to monitor the expression of blood vessel marker (αSMA). Dexamethasone decreased the vascular bed fraction and the expression of VEGF in both placental zones. Progesterone co-treatment with dexamethasone prevented this reduction. Nuclear and membrane PRs showed tissue-specific expression in different placental zones and responded differently to both dexamethasone and progesterone. Progesterone treatment improves the outcomes in IUGR pregnancy. Progesterone alleviated DEX-induced IUGR probably by promoting placental VEGF and angiogenesis.

ROLE OF ULTRASOUND AND COLOR FLOW DOPPLER IN DIAGNOSIS OF INTRAUTERINE GROWTH RESTRICTION

BACKGROUND: Multi-vessel Doppler ultrasonography is the principal surveillance tools in pregnancies complicated by fetal growth restriction. OBJECTIVE:To determine diagnostic accuracy of the fetal umbilical artery, middle cerebral artery pulsatility indices and their ratio in intrauterine growth restricted fetuses. PATIENTS and METHODS: Forty patients were studied that had been diagnosed clinically and ultrasonographically as intra-uterine growth restriction (IUGR). All patients in the study underwent uniform antenatal assessment protocol that includes umbilical artery (UA) and middle cerebral artery (MCA) Doppler ultrasound studies. These were conducted either twice weekly or daily according to the severity of the condition. RESULTS: Abnormal UA PI was found in 25 cases, 11cases due to maternal cause , 5 cases due to placental cause , 9 cases with idiopathic cause of the IUGR group . Abnormal MCA PI was found in 26 cases,11 cases due to maternal cause , 5 cases due to placental cause , 10 cases with idiopathic cause of the IUGR group. Abnormal C/U Ratio was found in 24 cases,11 cases due to maternal cause, 5 cases due to placental cause , 8 cases with idiopathic cause of the IUGR group . CONCLUSION: Among the Doppler indices, the C/U ratio is a better predictor of IUGR fetuses than either the UAPI or the MCA PI alone, with a high specificity and PPV. However, measurement of the UAPI (among all the Doppler indices) is enough to detect IUGR per se, probably because UAPI is a direct reflection of the resistance in the placental vascular bed.

An MRI approach to assess placental function in healthy humans and sheep.

Human placental function is evaluated using non-invasive Doppler ultrasound of umbilical and uterine artery pulsatility indices as measures of resistance in placental vascular beds, while measurement of placental oxygen consumption ( ) is only possible during Caesarean delivery. This study shows the feasibility of using magnetic resonance imaging (MRI) in utero to measure blood flow and oxygen content in uterine and umbilical vessels to calculate oxygen delivery to and by the gravid uterus, uteroplacenta and fetus. Normal late gestational human uteroplacental by MRI was ∼4mlmin-1 kg-1 fetal weight, which was similar to our MRI measurements in sheep and to those previously measured using invasive techniques. Our MRI approach can quantify uteroplacental , which involves the quantification of maternal- and fetal-placental blood flows, fetal oxygen delivery and , and the oxygen gradient between uterine- and umbilical-venous blood, providing a comprehensive assessment of placental function with clinical potential. It has not been feasible to perform routine clinical measurement of human placental oxygen consumption ( ) and in vitro studies do not reflect true metabolism in utero. Here we propose an MRI method to non-invasively quantify in utero placental and fetal oxygen delivery ( ) and in healthy humans and sheep. Women (n=20) and Merino sheep (n=10; 23sets of measurements) with singleton pregnancies underwent an MRI in late gestation (36±2weeks and 128±9days, respectively; mean±SD). Blood flow (phase-contrast) and oxygen content (T1 and T2 relaxometry) were measured in the major uterine- and umbilical-placental vessels, allowing calculation of uteroplacental and fetal and . Maternal (mlmin-1 kg-1 fetus) to the gravid uterus was similar in humans and sheep (human=54±15, sheep=53±21, P=0.854), while fetal (human=25±4, sheep=22 ± 5, P=0.049) was slightly lower in sheep. Uteroplacental and fetal (mlmin-1 kg-1 fetus; uteroplacental: human=4.1±1.5, sheep=3.5±1.9, P=0.281; fetus: human=6.8±1.3, sheep=7.2±1.7, P=0.426) were similar between species. Late gestational uteroplacental:fetal ratio did not change with age (human, P=0.256; sheep, P=0.121). Human umbilical blood flow (mlmin-1 kg-1 fetus) decreased with advancing age (P=0.008), while fetal was preserved through an increase in oxygen extraction (P=0.046). By contrast, sheep fetal was preserved through stable umbilical flow (mlmin-1 kg-1 ; P=0.443) and oxygen extraction (P=0.582). MRI derived measurements of uteroplacental and fetal between humans and sheep were similar and in keeping with prior data obtained using invasive techniques. Taken together, these data confirm the reliability of our approach, which offers a novel clinical 'placental function test'.

Comparative morphofunctional analysis of the state of fetoplacental complex in diabetes mellitus (literature review)

This article reviews the literature on placental morphofunctional changes in placenta of patients with type 1 and type 2 diabetes mellitus and gestational diabetes mellitus. The detailed analysis of features of pathogenesis of various abnormalities of the fetoplacental complex depending on the type of diabetes, its influence on the formation of the placental vascular bed. The analysis of mechanisms of development of placenta formation disorders, pathologies of placental vascular bed, the role of hyperglycemia and hyperinsulinemia in villous maturation, placental weight gain, perinatal outcomes. The discussed anomalies have a significant impact on the fetoplacental complex, acting as epigenetic factors, forming the environment for the fetus, which may later affect the health of the unborn child. They lead to adverse perinatal outcomes, including high infant morbidity and mortality. Literature search was performed in Russian (eLibrary, CyberLeninka.ru) and international (PubMed, Cochrane Library) databases in Russian and English languages. The free access to the full text of the articles was in priority. The selection of sources was prioritized for the period from 2016 to 2020. However, due to the lack of knowledge of the chosen topic, the selection of sources was dated from 2001.

Near to One's Heart: The Intimate Relationship Between the Placenta and Fetal Heart.

The development of the fetal heart is exquisitely controlled by a multitude of factors, ranging from humoral to mechanical forces. The gatekeeper regulating many of these factors is the placenta, an external fetal organ. As such, resistance within the placental vascular bed has a direct influence on the fetal circulation and therefore, the developing heart. In addition, the placenta serves as the interface between the mother and fetus, controlling substrate exchange and release of hormones into both circulations. The intricate relationship between the placenta and fetal heart is appreciated in instances of clinical placental pathology. Abnormal umbilical cord insertion is associated with congenital heart defects. Likewise, twin-to-twin transfusion syndrome, where monochorionic twins have unequal sharing of their placenta due to inter-twin vascular anastomoses, can result in cardiac remodeling and dysfunction in both fetuses. Moreover, epidemiological studies have suggested a link between placental phenotypic traits and increased risk of cardiovascular disease in adult life. To date, the mechanistic basis of the relationships between the placenta, fetal heart development and later risk of cardiac dysfunction have not been fully elucidated. However, studies using environmental exposures and gene manipulations in experimental animals are providing insights into the pathways involved. Likewise, surgical instrumentation of the maternal and fetal circulations in large animal species has enabled the manipulation of specific humoral and mechanical factors to investigate their roles in fetal cardiac development. This review will focus on such studies and what is known to date about the link between the placenta and heart development.

Oxidative stress and endothelial function in normal pregnancy versus pre-eclampsia, a combined longitudinal and case control study

BackgroundPre-eclampsia (PE) is related to an impaired endothelial function. Endothelial dysfunction accounts for altered vascular reactivity, activation of the coagulation cascade and loss of vascular integrity. Impaired endothelial function originates from production of inflammatory and cytotoxic factors by the ischemic placenta and results in systemic oxidative stress (OS) and an altered bioavailability of nitric oxide (•NO). The free radical •NO, is an endogenous endothelium-derived relaxing factor influencing endothelial function. In placental circulation, endothelial release of •NO dilates the fetal placental vascular bed, ensuring feto-maternal exchange. The Endopreg study was designed to evaluate in vivo endothelial function and to quantify in vitro OS in normal and pre-eclamptic pregnancies.Methods/designThe study is divided into two arms, a prospective longitudinal study and a matched case control study. In the longitudinal study, pregnant patients ≥18 years old with a singleton pregnancy will be followed throughout pregnancy and until 6 months post-partum. In the case control study, cases with PE will be compared to matched normotensive pregnant women. Maternal blood concentration of superoxide (O2•) and placental concentration of •NO will be determined using EPR (electron paramagnetic resonance). Endothelial function and arterial stiffness will be evaluated using respectively Peripheral Arterial Tonometry (PAT), Flow-Mediated Dilatation (FMD) and applanation tonometry. Placental expression of eNOS (endothelial NOS) will be determined using immune-histochemical staining. Target recruitment will be 110 patients for the longitudinal study and 90 patients in the case-control study.DiscussionThe results of Endopreg will provide longitudinal information on in vivo endothelial function and in vitro OS during normal pregnancy and PE. Adoption of these vascular tests in clinical practice potentially predicts patients at risk to develop cardiovascular events later in life after PE pregnancies. •NO, O2•− and eNOS measurements provide further inside in the pathophysiology of PE.Trial registrationThis trial has been registered on clinicaltrials.gov. ClinicalTrials.gov Identifier: NCT02603913. Registered October 2015.

Expression of von Willebrand factor and caldesmon in the placental tissues of pregnancies complicated with intrauterine growth restriction

Purpose: The decreased placental perfusion is the underlying reason for intrauterine growth restriction that in turn leads to reduced placental perfusion and ischemia. However, there are several issues to be understood in the pathophysiology of intrauterine growth restriction. We aimed to study whether any compensatory response in placental vascular bed occur in pregnancies complicated with intrauterine growth restriction by the immunohistochemical staining of von Willebrand factor and caldesmon in placental tissues.Materials and methods: A total of 103 pregnant women was enrolled in the study including 50 patients who were complicated with IUGR and 50 uncomplicated control patients. The study was designed in a prospective manner. All placentas were also stained with von Willebrand factor and caldesmon monoclonal kits.Results: The immunohistochemical staining of von Willebrand factor and caldesmon expressions in placental tissues were different between normal and intrauterine growth restriction group. The percentages of 2+ and 3+ von Willebrand factor expression were higher in the intrauterine growth restriction group comparing with the normal group, although the difference was not statistically significant. The intensity of caldesmon expression was significantly lower in the intrauterine growth restriction group in comparison with the normal group (p < .001).Conclusion: Angiogenesis occurs as a placental response to intrauterine growth restriction which is a hypoxic condition. But newly formed vessels are immature and not strong enough. Our study is important to clarify the pathophysiology and placental compensatory responses in intrauterine growth restriction.

Evaluation of the relation between placental weight and placental weight to foetal weight ratio and the causes of stillbirth: a retrospective comparative study

The aim of the present study was to evaluate the clinical importance of placental weight (PW) and placental weight to foetal weight (PW/FW) ratio according to maternal characteristics, pathological conditions in obstetrics and the causes of foetal death by category in stillbirths. The results of autopsies and placental histopathological examinations for 145 singleton stillbirths were reviewed retrospectively. Pathological features of the placenta were significantly associated with lower PW compared to the group with no pathological placental parameters (230 grams versus 295 grams, p = .045). Foetal growth restriction (FGR) with pre-eclampsia (PE) was accompanied by significantly lower FW, PW and PW/FW compared to FGR cases without PE (1045 grams versus 1405 grams, p = .026, 200 grams versus 390 grams, p = .006 and .19 versus .24, p = .037, respectively), whereas a similar trend was not observed in the non-FGR pregnancies complicated by PE. Oligohydramnios was accompanied by lower foetal weight compared to those who had normal amount of amniotic fluid (650 grams versus 1400 grams, p = .006). Among the clinical factors, only PE and oligohydramnios contributed to disproportionate fetoplacental growth in stillbirth, while none of the categories of stillbirth was related to unequal fetoplacental growth.Impact statementWhat is already known on this subject: In 27% of stillbirths, pathological features of the placenta or placental vascular bed are recorded. Underlying placental pathology contributes to foetal growth restriction (FGR) in approximately 50%. Although placental weight relative to foetal weight (PW/FW ratio) is an indicator of foetal as well as placental growth, data on PW/FW in stillbirth has not yet been published.What the results of this study add: Causes of death do not show any correlation with PW/FW ratio. Placentas derived from pregnancies complicated by pre-eclampsia (PE) and concomitant FGR are smaller and PW/FW is also diminished. Oligohydramnios is associated with an enhanced risk of restricted placental growth. FGR is not correlated with any categories of causes of death.What the implications are of these findings for clinical practice and/or further research: Sonographic follow-up of placental volume and FW can predict the stillbirth in PE complicated by FGR and oligohydramnios.

Insulin Induces Relaxation and Decreases Hydrogen Peroxide-Induced Vasoconstriction in Human Placental Vascular Bed in a Mechanism Mediated by Calcium-Activated Potassium Channels and L-Arginine/Nitric Oxide Pathways.

HIGHLIGHTS Short-term incubation with insulin increases the L-arginine transport in HUVECs.Short-term incubation with insulin increases the NO synthesis in HUVECs.Insulin induces relaxation in human placental vascular bed.Insulin attenuates the constriction induced by hydrogen peroxide in human placenta.The relaxation induced by insulin is dependent on BKCa channels activity in human placenta. Insulin induces relaxation in umbilical veins, increasing the expression of human amino acid transporter 1 (hCAT-1) and nitric oxide synthesis (NO) in human umbilical vein endothelial cells (HUVECs). Short-term effects of insulin on vasculature have been reported in healthy subjects and cell cultures; however, its mechanisms remain unknown. The aim of this study was to characterize the effect of acute incubation with insulin on the regulation of vascular tone of placental vasculature. HUVECs and chorionic vein rings were isolated from normal pregnancies. The effect of insulin on NO synthesis, L-arginine transport, and hCAT-1 abundance was measured in HUVECs. Isometric tension induced by U46619 (thromboxane A2 analog) or hydrogen peroxide (H2O2) were measured in vessels previously incubated 30 min with insulin and/or the following pharmacological inhibitors: tetraethylammonium (KCa channels), iberiotoxin (BKCa channels), genistein (tyrosine kinases), and wortmannin (phosphatidylinositol 3-kinase). Insulin increases L-arginine transport and NO synthesis in HUVECs. In the placenta, this hormone caused relaxation of the chorionic vein, and reduced perfusion pressure in placental cotyledons. In vessels pre-incubated with insulin, the constriction evoked by H2O2 and U46619 was attenuated and the effect on H2O2-induced constriction was blocked with tetraethylammonium and iberiotoxin, but not with genistein, or wortmannin. Insulin rapidly dilates the placental vasculature through a mechanism involving activity of BKCa channels and L-arginine/NO pathway in endothelial cells. This phenomenon is related to quick increases of hCAT-1 abundance and higher capacity of endothelial cells to take up L-arginine and generate NO.

Maternal extracellular vesicles and platelets promote preeclampsia via inflammasome activation in trophoblasts

AbstractPreeclampsia (PE) is a placenta-induced inflammatory disease associated with maternal and fetal morbidity and mortality. The mechanisms underlying PE remain enigmatic and delivery of the placenta is the only known remedy. PE is associated with coagulation and platelet activation and increased extracellular vesicle (EV) formation. However, thrombotic occlusion of the placental vascular bed is rarely observed and the mechanistic relevance of EV and platelet activation remains unknown. Here we show that EVs induce a thromboinflammatory response specifically in the placenta. Following EV injection, activated platelets accumulate particularly within the placental vascular bed. EVs cause adenosine triphosphate (ATP) release from platelets and inflammasome activation within trophoblast cells through purinergic signaling. Inflammasome activation in trophoblast cells triggers a PE-like phenotype, characterized by pregnancy failure, elevated blood pressure, increased plasma soluble fms-like tyrosine kinase 1, and renal dysfunction. Intriguingly, genetic inhibition of inflammasome activation specifically in the placenta, pharmacological inhibition of inflammasome or purinergic signaling, or genetic inhibition of maternal platelet activation abolishes the PE-like phenotype. Inflammasome activation in trophoblast cells of women with preeclampsia corroborates the translational relevance of these findings. These results strongly suggest that EVs cause placental sterile inflammation and PE through activation of maternal platelets and purinergic inflammasome activation in trophoblast cells, uncovering a novel thromboinflammatory mechanism at the maternal-embryonic interface.

A novel tryptophan-based mechanism regulating the tonus of the placental vascular bed is impaired in intrauterine growth restriction and preeclampsia

A novel tryptophan-based mechanism regulating the tonus of the placental vascular bed is impaired in intrauterine growth restriction and preeclampsia

Decidualisation of human endometrial stromal cells is associated with increased expression and secretion of prorenin.

BackgroundIn pregnancy, the decidualised endometrium expresses high levels of prorenin and other genes of the renin-angiotensin system (RAS) pathway. In this study we aimed to determined if the RAS was present in endometrial stromal cells and if decidualisation upregulated the expression of prorenin, the prorenin receptor ((P)RR) and associated RAS pathways. Immortalised human endometrial stromal cells (HESCs) can be stimulated to decidualise by combined treatment with medroxyprogesterone acetate (MPA), 17β-estradiol (E2) and cAMP (MPA-mix) or with 5-aza-2’-deoxycytidine (AZA), a global demethylating agent.MethodsHESCs were incubated for 10 days with one of the following treatments: vehicle, MPA-mix, a combination of medroxyprogesterone acetate (MPA) and estradiol-17β alone, or AZA. Messenger RNA abundance and protein levels of prorenin (REN), the (P)RR (ATP6AP2), angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AGTR1), vascular endothelial growth factor (VEGF), and plasminogen activator inhibitor-1 (PAI-1) were measured by real-time PCR and ELISA’s, respectively. Promyelocytic zinc finger (PLZF) and phospho-inositol-3 kinase (PIK3R1) mRNA abundances were also measured.ResultsHESCs expressed the prorenin receptor (ATP6AP2), REN, AGT, ACE and low levels of AGTR1. MPA-mix and AZA stimulated expression of REN. Prorenin protein secretion was increased in MPA-mix treated HESCs. E2 + MPA had no effect on any RAS genes. MPA-mix treatment was associated with increased VEGF (VEGFA) and PAI-1 (SERPINE1) mRNA and VEGF protein.ConclusionsAn endometrial prorenin receptor/renin angiotensin system is activated by decidualisation. Since (P)RR is abundant, the increase in prorenin secretion could have stimulated VEGF A and SERPINE1 expression via Ang II, as both ACE and AGTR1 are present, or by Ang II independent pathways. Activation of the RAS in human endometrium with decidualisation, through stimulation of VEGF expression and secretion, could be critical in establishing an adequate blood supply to the developing maternal placental vascular bed.

Porcine placental immunoexpression of vascular endothelial growth factor, placenta growth factor, Flt-1 and Flk-1

Porcine embryo mortalities cause economic losses. Development of the placental vascular bed is required for successful gestation and postnatal survival. We studied the temporal and spatial distributions of vascular endothelial growth factor (VEGF), placenta growth factor (PlGF) and their receptors, Flt-1 and Flk-1. We used crossbred swine placental tissues from 30, 60, 80, 90 and 114 (term) days of gestation. Both VEGF and PlGF were present during gestation. At early pregnancy and at term, VEGF probably acts through Flt-1; during intermediate periods, its function is mediated by Flk-1. By day 90, factors other than members of VEGF family appear to be involved.

Natural killer cells direct hemochorial placentation by regulating hypoxia-inducible factor dependent trophoblast lineage decisions

Natural killer (NK) cells are recruited into the uterine stroma during establishment of the hemochorial placenta and are proposed regulators of uterine spiral artery remodeling. Failures in uterine spiral artery remodeling are linked to diseases of pregnancy. This prompted an investigation of the involvement of NK cells in placentation. NK cell depletion decreased the delivery of proangiogenic factors and delayed uterine spiral artery development, leading to decreased oxygen tension at the placentation site, stabilized hypoxia-inducible factor 1A protein, and redirected trophoblast differentiation to an invasive phenotype. Trophoblast cells replaced the endothelium of uterine spiral arteries extending the depth of the placental vascular bed and accelerating vessel remodeling. Hypoxia-regulated trophoblast lineage decisions, including expansion of invasive trophoblast, could be reproduced in vitro by using rat trophoblast stem cells and were dependent on hypoxia-inducible factor signaling. We conclude that NK cells guide hemochorial placentation through controlling a hypoxia-sensitive adaptive reflex regulating trophoblast lineage decisions.

Role of nitric oxide in placental vascular development and function

Nitric oxide (NO) is one of the most pleiotropic signaling molecules at systemic and cellular levels, participating in vascular tone regulation, cellular respiration, proliferation, apoptosis and gene expression. Indeed NO actively participates in trophoblast invasion, placental development and represents the main vasodilator in this tissue. Despite the large number of studies addressing the role of NO in the placenta, its participation in placental vascular development and the effect of altered levels of NO on placental function remains to be clarified. This review draws a time-line of the participation of NO throughout placental vascular development, from the differentiation of vascular precursors to the consolidation of vascular function are considered. The influence of NO on cell types involved in the origin of the placental vasculature and the expression and function of the nitric oxide synthases (NOS) throughout pregnancy are described. The developmental processes involved in the placental vascular bed are considered, such as the participation of NO in placental vasculogenesis and angiogenesis through VEGF and Angiopoietin signaling molecules. The role of NO in vascular function once the placental vascular tree has developed, in normal pregnancy as well as in pregnancy-related diseases, is then discussed.

MRNA and protein expression of FGF-1, FGF-2 and their receptors in the porcine umbilical cord during pregnancy.

The fibroblast growth factors (FGFs) are multifunctional proteins that, among other roles, regulate structural reorganization of uterine and placental vascular bed during pregnancy. Thus, we analyzed mRNA and protein expression and immunohistochemical localization of FGF-1 and FGF-2, and their receptors (FGFR-1 and FGFR-2) in the developing umbilical cord (UC) on days 40, 60, 75 and 90 of pregnancy and after the physiological delivery in the pig (day 114). qPCR analysis demonstrated an increase in FGF-1 and FGF-2 mRNA levels beginning on day 75 and on day 114 of pregnancy, respectively. In addition, significantly increased FGFR-1IIIc mRNA expression was also found on day 114. On the other hand, no significant changes in FGFR-2IIIb mRNA expression were observed. Western Blot analysis revealed a decrease in FGF-1 and FGFR-2 protein expression after day 40. Beside an increased protein expression of FGF-2 on day 60, no significant changes in FGFR-1 protein expression were detected. Immunohistochemical staining enabled detection of FGF-FGFR system, with different intensity of immunoreaction in endothelial and tunica media cells of the umbilical vessels and in allantoic duct and amniotic epithelium as well as in myofibroblasts. In conclusion, our results show that members of FGF-FGFR system are expressed specifically in UC structures. Furthermore their day of pregnancy-related expression suggest that they may be an important players during UC formation and development.

The Effect of a Mast Cell Degranulating Agent on Vascular Resistance in the Human Placental Vascular Bed and on the Tone of Isolated Placental Vessels

The objective of this study is to investigate the effect of a mast cell degranulating agent, compound 48/80, on vascular resistance in the perfused human placenta and on the tone of isolated human chorionic vessels. Human placenta was obtained from term nonlaboring women undergoing cesarean delivery. Placental vascular bed perfusion pressure and isometric tension for segments of chorionic plate artery and vein were obtained in response to potassium chloride, compound 48/80, a mast cell stabilizer (cromolyn), and thromboxane A2 mimetic (U46619). Compound 48/80 significantly increased perfusion pressure in isolated human placental cotyledons. This effect was significantly potentiated further after induction of active vascular tone by thromboxane A2 mimetic U46619. Cromolyn significantly attenuated responses to compound 48/80 in these preparations. Compound 48/80 also significantly increased tone in isolated human chorionic artery and vein rings, and responses were abolished by cromolyn. In conclusion, degranulation of placental and intravascular mast cells by compound 48/80 leads to the release of vasoconstrictive substances. This could impair placental blood flow and result in growth restriction in fetuses of women with type l hypersensitivity reactions.

1A.1 Animal model of fetal loss in factor V Leiden carriers: pathogenic mechanism and therapeutic targets

Recurrent pregnancy loss (RPL), defined as two or more spontaneous abortions affects approximately 5% of all women of reproductive age. Epidemiological studies suggest that inherited and acquired thrombophilia of the mother, such as that caused by the Leiden polymorphism in blood coagulation factor V, contributes to the pathogenesis of fetal loss, as well as other adverse pregnancy outcomes [1 3]. These studies also demonstrate a high study-to-study variability of association strength, suggesting the existence of as yet uncharacterized risk modifiers. The pathogenesis of thrombophilia-associated fetal loss remains to be established. Evaluation of placental pathologies has not revealed a clear correlation between thrombophilia status of the mother and the presence of villous infarcts, blood clots or fibrin deposits in the placenta [4,5], raising the question whether thrombotic processes are indeed causative in thrombophilia-associated fetal loss or constitute an epiphenomenon. On the other hand, clinical trials suggest that heparin anticoagulation may indeed mitigate pregnancy failure in prothrombotic women [6 9]. In view of the uncertain etiology of thrombophilia-associated pregnancy failure, and the lack of established criteria for a more precise risk stratification enabling identification of truly atrisk pregnancies, the prophylactic anticoagulation of asymptomatic women and the risk-to-benefit balance of anticoagulation therapy during pregnancy are subjects of intense debate [10 12]. Pregnancy failure in carriers of the factor V Leiden (FVL) polymorphism, which renders coagulation factor V refractory to inactivating proteolysis by activated protein C, has not been replicated in experimental animal models. Homozygous status for the FVL polymorphism elicits pronounced thrombophilia in mice, but is not associated with intrauterine fetal loss or impaired fecundity [13]. In contrast, observations in animals lacking the receptor components of the protein C anticoagulant pathway, thrombomodulin (TM) and the endothelial protein C receptor (EPCR), demonstrate that the integrity of this pathway within the placenta is essential for the maintenance of pregnancy [14 16]. TMor EPCR knockout mice die in utero due to a placental malfunction that results from a lack of these receptors on trophoblast cells constituting fetal lining of the placental vascular bed. Placental malfunction is triggered by the initiator of blood coagulation, tissue factor (TF), and at least in the case of TM is not associated with thrombosis, nor prevented by the absence of fibrinogen [15]. These observations led us to propose a “coagulotoxic” mechanism distinct from thrombosis, in which localized, tissue factor-dependent engagement of coagulation factor receptors (i.e. PAR 1, PAR2, and PAR4) expressed by trophoblast cells would cause deleterious effects on placental growth and morphogenesis. Importantly, fetal loss in these animal models occurs in the absence of maternal thrombophilia. The relevance of these knockout models for fetal loss in thrombophilic mothers, and the pathogenesis of fetal loss remain unclear. Here, we experimentally test the role of coagulation receptors PAR1, 2, and 4 in the fetal loss of TM / embryos; and develop a mouse model of pregnancy loss in FVL carriers, in which fetal loss is triggered by the interaction of maternal FVL carrier status and reduced ability of fetal trophoblast cells to sustain the protein C anticoagulant pathway. Interaction of the same risk factors across the blood-endothelial interface of the systemic vasculature does not precipitate thrombosis. Fetal loss in this animal model is caused by defective placental morphogenesis in the absence of placental thrombosis, infarction or perfusion defects, and can be prevented by treatment with low molecular weight heparin, by platelet depletion, or by elimination of the thrombin receptor PAR4 from maternal platelets. Therapeutic intervention studies suggest that the efficacy of low molecular weight heparin in preventing fetal loss does not correlate with its anti-coagulant activity. These findings validate a cause effect relation between FVL carrier status and pregnancy failure, show that fetal

414 COLOR DOPPLER EVALUATION OF UMBILICAL BLOOD FLOW DURING PREGNANCY IN COWS

There are few reports describing the use of Doppler for reproductive imaging in large animals; only one study (in mares) assessed umbilical blood flow (Bollwein et al. 2004 Theriogenology 61, 499–509). Conditions that constrict the placental vascular bed (i.e. hemorrhage, thrombosis, abnormal development, etc.) increase resistance to incoming blood. Thus, blood flow in umbilical arteries can be used to monitor placental development and function (Giles et al. 1985 Brit. J. Obstet. Gynaecol. 92, 31–38). The objective of the present study was to characterize the Doppler flow velocity waveform in umbilical arteries of cows with apparently normal pregnancies. Twenty-three multiparous, nonlactating Aberdeen Angus cows with pregnancies achieved after transfer of embryos derived by IVF (n = 10) or multiple ovulation and embryo transfer (MOET) (n = 13) were examined weekly from 5 to 38 weeks of gestation. Ultrasonography (Toshiba Nemio 20, Tokyo, Japan) was done using a 5–10 MHz intraoperative finger transducer (transrectal) from 5 to 17 weeks of pregnancy and thereafter using a 3–6 MHz linear-array transducer (transabdominal). The interrogation angle (between the ultrasound beam and the artery) ranged from 45° to 60°. Three resistance indices were calculated: A/B ratio, Resistance Index (RI) = (A − B)/A, and Pulsatility Index (PI) = (A − B)/M. [A = systole, B = diastole, and M = mean maximum Doppler-Shift frequency over the cardiac cycle.] The mean ± SEM duration of pregnancy was 285 ± 1.6 days (range: 269 to 291 days). A repeated measure ANOVA was used to detect differences between groups for every week using the Wald-Wolfowitz test (InfoStat V1.5, FCA, Universidad de Córdoba, Córdoba, Argentina). All 3 resistance indices decreased (by &amp;gt;50%) until 26 weeks, with no substantial change thereafter (Table 1). From 5 to 18 weeks of pregnancy, blood flow was characterized by a systolic pattern (i.e. high resistance with absence of diastolic flow); at 20 weeks, all fetuses had a diastolic flow, consistent with low resistance. There were differences (P &amp;lt; 0.05) between IVF- and MOET-derived pregnancies for RI and PI at 38 and 26 weeks of gestation, respectively, indicating differences in the placental vascular development at these weeks of gestation. In conclusion, umbilical cord blood flow in cattle was characterized by high resistance (5 weeks of gestation), but resistance decreased until 26 weeks; consequently, blood flow was initially systolic but became diastolic. Doppler sonography was useful for assessment of umbilical blood flow from 5 to 38 weeks of pregnancy, and may be useful for assessing placental function in pregnancies under risk, e.g. clone-derived pregnancies (Bertolini et al. 2002 Theriogenology 57, 181–187). Table 1. RI and PI at 19, 22, 26, 30, and 38 weeks of pregnancy in IVF- and MOET-derived pregnancies (mean ± sd)

Fetomaternal cross talk in the placental vascular bed: control of coagulation by trophoblast cells

AbstractHumans and rodents exhibit a peculiar type of placentation in which zygote-derived trophoblast cells, rather than endothelial cells, line the terminal maternal vascular space. This peculiar aspect of the placental vasculature raises important questions about the relative contribution of fetal and maternal factors in the local control of hemostasis in the placenta and how these might determine the phenotypic expression of thrombophilia-associated complications of pregnancy. Using genomewide expression analysis, we identify a panel of genes that determine the ability of fetal trophoblast cells to regulate hemostasis at the fetomaternal interface. We show that spontaneous differentiation of trophoblast stem cells is associated with the acquisition of an endothelial cell–like thromboregulatory gene expression program. This program is developmentally regulated and conserved between mice and humans. We further show that trophoblast cells sense, via the expression of protease activated receptors, the presence of activated coagulation factors. Engagement of these receptors results in cell-type specific changes in gene expression. Our observations define candidate fetal genes that are potential risk modifiers of maternal thrombophilia-associated pregnancy complications and provide evidence that coagulation activation at the fetomaternal interface can affect trophoblast physiology altering placental function in the absence of frank thrombosis.