The endothelial protein C receptor plays an essential role in the maintenance of pregnancy

Placenta-mediated pregnancy complications are a major challenge in the management of maternal-fetal health. Maternal thrombophilia is a suspected risk factor, but the role of thrombotic processes in these complications has remained unclear. Endothelial protein C receptor (EPCR) is an anticoagulant protein highly expressed in the placenta. EPCR autoantibodies and gene variants are associated with poor pregnancy outcomes. In mice, fetal EPCR deficiency results in placental failure and in utero death. We show that inhibition of molecules involved in thrombin generation or in the activation of maternal platelets allows placental development and embryonic survival. Nonetheless, placentae exhibit venous thrombosis in uteroplacental circulation associated with neonatal death. In contrast, maternal EPCR deficiency results in clinical and histological features of placental abruption and is ameliorated with concomitant Par4 deficiency. Our findings unveil a causal link between maternal thrombophilia, uterine hemorrhage, and placental abruption and identify Par4 as a potential target of therapeutic intervention.

Особенности морфофункционального состояния плаценты, инфицированной streptococcus agalactiae

Objective: To study morphofunctional peculiarities of S. agalactiae-infected placenta. 32 placentas from preterm birth were examined. Morphometric assessment of terminal villi was made with Carl Zeiss microscope using AxioVision software. DNA of intrauterine pathogens was analyzed by a PCR method using domestic test systems. S. agalactiae was found in 9,4 % of the placentas examined. S. agalactiae-infected placentas from preterm birth showed evidence of inflammation and infection processes in membranes, placenta and umbilical cord, signs of chronic placental failure. Morphometric study showed reduced cross-sectional area and perimeter as compared to the mean parameters of infected or non-infected placentas. The specific vascularization features of S.agalactiae-infected placenta are indicative of decreased gas exchange, emergent placental hypoxia and pathogen involvement in the pathogenesis of preterm birth.

232 Protecting pigs from viral infectivity with gene editing

Pig models were created that were fully resistant to Porcine Reproductive and Respiratory Syndrome (PRRS) and the alphacoronaviruses, transmissible gastroenteritis virus (TGEV) by DNA editing of the cluster of differentiation (CD163) gene and aminopeptidase N (ANPEP) gene via CRISPR/Cas, respectively. Litters of pigs were bred with a gene edit in exon 7 of CD163 that resulted no CD163 protein expression (CD163-/-). The pigs were either challenged with multiple PRRSV isolates at 3 weeks of age or bred at maturity for a challenge with pregnant sows. The challenges demonstrated that the pigs were completely resistant to infectivity to both Type 1 and 2 isolates as measured by clinical signs, viremia, antibody response and lung histopathology. In a follow-up study, pregnant CD163 edited pigs were challenged with PRRSV to determine if absence of CD163 in the dam should be sufficient to protect the fetuses that have functional CD163 protein. The CD163-/- sows carrying both the CD163-/- and CD163+/- fetuses were all negative for PRRSV nucleic acid and showed no sign of fetal or placental failure clearly demonstrating that the absence of CD163 in the sow is sufficient to protect a PRRSV-susceptible CD163+/- fetus. Gene editing of CD163 in pigs, via CRISPR/Cas9, successfully blocked PRRSV infectivity in young growing pigs and pregnant sows and their fetuses. Similar methods were used to determine if TGEV infectivity could be prevented by editing the ANPEP gene. Exon 2 of ANPEP was edited and resulted an ANPEP -/- genotype. ANPEP -/- pigs and age matched wild type pigs were challenged with TGEV. The presence of virus nucleic acid was determined by PCR in fecal samples. ANPEP -/- pigs were fully resistant to TGEV infectivity. These studies clearly show that precise gene editing can be successfully used in pigs to prevent viral infectivity of both PRRSV and TGEV.

MORPHOLOGICAL ASSESSMENT OF GESTATION REBUILDING OF THE VASCULAR BED IN THE UNTERINE-PLACENTAL AREA IN CASE OF IRON DEFICIENCY ANEMIA OF PREGNANCY IN THE ASPECT OF CHRONIC PLACENTAL FAILURE

According to the analysis of the results of own studies, the authors found that in the uterine-placental area during pregnancy with iron deficiency anemia of II-III degrees, there are no adequate gestational changes in 60±0.1 % of the spiral arteries, and insufficiency of venous flow is shown by the decrease of the number of vessels to 4.4±0.22 per 1 mm2 of histologic section area and signs of venous stagnation, mainly in the projection of the peripheral areas of the placenta. For the first time, in order to evaluate gestational immaturity of the uterine-placental region depending on the degree of iron deficiency anemia in the aspect of uterineplacental form of lacuna insufficiency, the technique of immunohistochemical imaging of vimentin in the walls of vessels in the projection of the attachment was applied. The revealed features of immune expression of vimentin in the walls of the spiral arteries in the observations with anemia indicated the preservation of leiomyocytes with incomplete gestational transformation of the arteries. The peculiarities of the concentration of vimentin in the walls of the vessels of the microcirculatory bed reflected the disturbances of the processes of angiogenesis and vasculogenesis in the uterine-placental region with iron deficiency anemia of II - III degrees.

Genetic Absence of Thrombin Receptor Par4 Overcomes the Obligate Requirement of EPCR in Mouse Placenta

Introduction: Murine models suggest that the Thrombomodulin-Protein C system plays a critical role in placentation and the maintenance of pregnancy. Severe Protein C deficiency in the mother results in pregnancy failure in early gestation. Thrombomodulin (Thbd) or the Endothelial Protein C Receptor (EPCR/ProcR) gene deletions result in embryonic death, secondary to developmental and functional abnormalities of the placenta. These molecules play multiple roles in coagulation and inflammation. The mechanisms governing their role in placental development and maintenance of placental function remain to be fully understood. The objective of this work is to identify the critical functions of EPCR and Thbd that are required for placental development. Both Thbd and EPCR augment activated protein C generation, albeit to different extents. We have examined if reduced activated Protein C generation mediates placental abnormalities of EPCR- and Thbd-null mice. Activation of thrombin receptors expressed on platelets and trophoblast cells can also contribute to placental failure. We examined the role of thrombin receptor Par4 in placental failure of EPCR-null mice. Methods: To assess the role of a PC generation in placental phenotype of Thbd- and EPCR-null mice, we used a transgene to express a hyperactivatable form of murine protein C (hMPC) under the control of transthyretin promoter. Thrombin cleaves this mutant form of Protein C 30-fold more efficiently than wild type protein C, without requiring the cofactor function of thrombomodulin. Wild type mice expressing hMPC show 2-fold increase in PC and 3-fold increase in aPC levels. hMPC expression in PC-null mice restores their ability to carry pregnancies. Breeding strategies were used to generate hMPCtg ProcR+/- or hMPCtg Thbd+/- female mice. These were mated to ProcR+/- or Thbd+/- males, respectively, and survival of ProcR-/- and Thbd-/- embryos was analyzed. Similar genetic strategy was used to analyze the role of thrombin receptor Par4 in the demise of EPCR-null embryos. Placental phenotypes and embryonic survival was compared with experiments in which the mother was continuously infused with LMWH using a subcutaneous osmotic pump. Results: As previously reported, EPCR-null mice die before 10.5 days post coitum (dpc) (ProcR+/- intercrosses, out of 41 live embryos none were ProcR-/-, 10 were expected, 21 aborted not genotyped, 7 pregnancies analyzed at 11.5 dpc) and none are found at wean (out of 30 live pups none were ProcR-/-, 8 were expected, 5 litters analyzed). Transgenic expression of hMPC in the mother resulted in some live ProcR-/- embryos at 11.5 dpc (4 ProcR-/- out of 41 live embryos, 10 were expected, 15 aborted not genotyped, 7 pregnancies at 11.5 dpc) and pups at wean (2 ProcR-/- out of 28 live, 7 litters analyzed). Despite transgenic hMPC expression ProcR-/- embryos and pups were underrepresented (P=0.007, chi square GOF test). Surviving ProcR-/- embryos showed normal placental histology grossly comparable to littermate controls. Expression of hMPC in the mother did not ameliorate fetal death of Thbd-null mice (out of 38 live embryos none were Thbd-/-, 10 expected, 16 aborted not genotyped, 7 pregnancies at 9.5 dpc). Continuous infusion of LMWH also resulted in some live ProcR-/- embryos at 11.5 dpc (3 ProcR-/- out of 19 live embryos, 5 expected, 11 aborted not genotyped, 3 pregnancies analyzed), but two were growth retarded and all 3 placentae showed markedly reduced placental labyrinth formation. In contrast to transgenic expression of hMPC and treatment with LMWH, when Par4-/- ProcR+/- animals were intercrossed, ProcR-/- animals were born at an expected Mendelian frequency (7 ProcR-/- out of 35 live pups, 9 expected, 7 litters analyzed). Conclusions: Our results show that transgenic expression of hMPC allows normal placental development and rescues a fraction of EPCR-null embryos. Thus, placental defect of EPCR-null mice is in part mediated by reduced generation of aPC on placental cells. In contrast to the transgenic expression of hMPC and LMWH treatment, genetic absence of Par4 completely overcame the placental defect and allowed development of EPCR-null embryos. Further studies will clarify contributions of maternal versus fetal Par4 in this phenomenon. Disclosures No relevant conflicts of interest to declare.

An Experimental Model of Platelet Activation at the Feto-Maternal Interface

Background and Objectives: A developing fetus relies on the placenta for nutrition and other essential functions. To perform these functions human and rodent placenta forms unusual vascular spaces filled with maternal blood and directly accessible to specialized placental cells, called trophoblast cells. Such vascular spaces are not found anywhere else in the body. Trophoblast cells are of zygotic origin and they express a repertoire of molecules with anticoagulant and anti-platelet activities (PMID 16380449). In addition, they constitutively express Tissue Factor. Given these peculiarities, it is unclear how blood flow is maintained in these vascular spaces. The mechanisms by which activated coagulation causes placental pathology also remain undefined. We have previously developed and characterized a murine model of placental pathology precipitated by combining maternal and fetal prothrombotic mutations at the feto-maternal interface (PMID 17438064). We have shown that placental failure in this model can be prevented by depletion of maternal platelets or genetic ablation of thrombin receptor, Par4. Treatment of the mother with heparin significantly improves pregnancy outcome, but equivalent anticoagulation with fondaparinux, hirudin or direct Xa inhibitors is ineffective (PMID 23325830). We combine genetic tools with platelet transfusion experiments to present new data showing a critical role of Par4 and factor V carried by maternal platelets in placental failure and fetal death. We further show that the mechanism of placental failure, although mediated by maternal platelets, does not require integrin αIIb-mediated aggregation. Methods and Results: Factor V Leiden and thrombomodulin Pro mice have been previously described (PMID 11110695, 9576763). Both strains are viable in homozygous state for the mutated allele on C57Bl6 genetic background. We combined factor V Leiden mutation in the mother (homozygous FVQ/Q) with a fetal prothrombotic Glu387Pro mutation in thrombomodulin (homozygous ThbdPro/Pro) by crossing FVQ/QThbdPro/+ females with ThbdPro/Pro males. Almost all ThbdPro/Pro embryos conceived from these pregnancies die in utero due to placental failure (Table 1, row 1). The near complete loss of ThbdPro/Pro embryos is maternal genotype specific and is not observed in FV+/+ThbdPro/+ mothers. While >95% antibody-mediated platelet depletion in the mother rescues ThbdPro/Pro embryos (Table 1, row 2), we show that partial platelet depletion (~75%) is ineffective (Table 1, row 3). Genetic absence of Par4 in the mother completed rescues pregnancies (Table 1, row 4). We show that transfusion of Par4+/+FVQ/Q platelets to 25% of host platelets re-precipitates placental failure and death of ThbdPro/Pro embryos (table 1, row 5). In contrast, transfusion of wild type (Par4+/+FV+/+) platelets did not precipitate placental failure and death of ThbdPro/Pro embryos (Table 1, row 6). Genetic absence of integrin αIIb in the mother did not protect ThbdPro/Proembryos from intrauterine death. Conclusions: We explore pathogenic mechanisms in a murine model of platelet-mediated placental development failure. Our results demonstrate that placental failure in this model is caused by Par4-mediated activation of maternal platelets. The mechanism, however, does not involve αIIb-mediated platelet aggregation. Our results suggest that platelet factor V makes an important contribution to the development of placental pathology. These observations demonstrate the existence of vascular bed specific pathogenic mechanisms. Involvement of platelet secretion and/or recruitment of immune cells is being explored. Table 1: Results from experimental cross, FVQ/QThbdPro/+ females mated to ThbdPro/Pro males, are shown. Pregnancies were analyzed on or after gestation day 12.5. * P <0.05, c2 goodness of fit. ** P <0.05, c2 test of independence. Experimental manipulation Genotype of live embryos Number (%age) of aborted embryos Total number of embryos analyzed c2 test of independence (Compared to Par4-/- mother) ThbdPro/+(50%) ThbdPro/Pro(50%) None 22 0* 51 (70%) 73 (8) ** >95% Platelet depletion 13 18 (58%) 5 (14%) 36 (4) NS Partial platelet depletion 11 2* (15%) 19 (59%) 32 (4) ** Par4-/- mother 18 15 (46%) 3 (8%) 36 (4) - QQ platelet transfusion 9 1* (10%) 21 (68%) 31 (3) ** WT platelet infusion 18 13 (42%) 10 (26%) 41 (5) NS αIIb-/-mother 36 2* (5%) 41 (52%) 79 (9) ** Disclosures No relevant conflicts of interest to declare.

Ultrasound Features of the Formation of the Maternal-Placental Complex in Cases of Hyperandrogenism as Observed during the First and Second Trimesters of Pregnancy

ABSTRACT An excess of maternal androgen adversely affects the implantation process and contributes to uterine circulatory disturbances and myometrium vessel sclerosis. Placental insufficiency is typically diagnosed at the end of the second trimester of pregnancy or at the beginning of the third trimester, when the treatment is not effective Many researchers have attempted to use early sonographic diagnostics to identify placental failure in various pathological states. Nevertheless, the peculiarities of the formation of the fetal-placental complex during the first and second trimesters are still undefined. In this study, we traced the process of placental formation under conditions of excess maternal androgen using ultrasound and Dopplerometry. Morphological examinations of the ñhorion and placental tissue were undertaken to ñompare any changes. Unfavorable chorion locations, heterogeneous chorion tissue structures, and marginal sinus dilatations were revealed by ultrasound. According to a Dopplerometric investigation, a statistically significant increase in peripheral vessel resistance in cases of hyperandrogenism was identified compared with healthy pregnancies. The negative influence of hyperandrogenism on placental formation was confirmed by morphological data (including the presence of immature decidual cells, defective villi that covered the epithelium, and the dissociative development of cotyledons). How to cite this article Olga M, Elena T, Luybov A, Irina B, Sergey L. Ultrasound Features of the Formation of the Maternalplacental Complex in Cases of Hyperandrogenism as Observed during the First and Second Trimesters of Pregnancy. Donald School J Ultrasound Obstet Gynecol 2014;8(2):149-154.