Serine protease HtrA1 is developmentally regulated in trophoblast and uterine decidual cells during placental formation in the mouse.

Development of a hemochorial placenta involves trophoblast proliferation, differentiation, and invasion into the uterus to promote blood flow to the embryo. Trophoblast invasion is tightly controlled by expression of specific proteases in the trophoblast and highly coordinated activities in the uterus. One uterine event essential for placentation is the developmentally regulated formation and regression of the decidua. In mice, decidual regression takes place in a temporal- and spatial-specific manner that is coordinated with placental development. In this study, we identified that the serine protease HtrA1 (high temperature requirement factor A1) was specifically expressed in differentiated trophoblast cells, especially the giant cells, during the early stages of placental development. A high level of HtrA1 expression was also detected in decidua capsularis specifically at the decidual-trophoblast interface where active involution occurs. Thus, we have identified a previously unknown role for HtrA1 as a protease potentially important for trophoblast differentiation/invasion and uterine decidual regression during placental development.     

Molecular and immunological developments in placentas

Cytotrophoblasts differentiate in two directions during early placentation: syncytiotrophoblasts (STBs) and extravillous trophoblasts (EVTs). STBs face maternal immune cells in placentas, and EVTs, which invade the decidua and uterine myometrium, face the cells in the uterus. This situation, in which trophoblasts come into contact with maternal immune cells, is known as the maternal-fetal interface. Despite fetuses and fetus-derived trophoblast cells being of the semi-allogeneic conceptus, fetuses and placentas are not rejected by the maternal immune system because of maternal-fetal tolerance. The acquired tolerance develops during normal placentation, resulting in normal fetal development in humans. In this review, we introduce placental development from the viewpoint of molecular biology. In addition, we discuss how the disruption of placental development could lead to complications in pregnancy, such as hypertensive disorder of pregnancy, fetal growth restriction, or miscarriage.     

The role of platelets during reproduction

The availability of mice with defined defects within the hemostatic system enabled researchers to identify a role the coagulation system for embryonic and placental development. However, the role of platelets during development has only recently been experimentally addressed, giving some insight into potential functions of platelets during development. Thus, a quantitative embryonic platelet defect (severe thrombopenia secondary to NF-E2 deficiency) is associated with an embryonic growth retardation and reduced vascularisation of the placenta. Maternal platelet deficiency is associated with placental hemorrhage, which, however, does not impair embryonic or maternal survival. In vitro studies established that platelets or platelet conditioned medium regulate the invasive properties of human extravillous trophoblast cells and induce a phenotypical switch of trophoblast cells. These data imply that platelets are of relevance during placentation. Conversely, platelets and the formation of platelet-fibrin aggregates are dispensable for the development of the embryo proper, establishing that the lethal phenotypes observed in some embryo slacking coagulation regulators does not result from an inability to form platelet-fibrin aggregates, but likely reflects altered protease dependent signaling during vascular development.     

Pre-eclampsia: a mistake of trophoblastic cells for tumour cells?

Pre-eclampsia is a severe form of hypertension induced by pregnancy. In pre-eclampsia, there is deficient trophoblast invasion of the spiral arteries terminating in the placental bed. Perhaps some abnormality occurs in the immunosuppressive process as the maternal immune system encounters paternal antigens expressed by immunosuppressive decidual cells. This immunosuppressive abnormality might cause the deficient trophoblast invasion. Abnormal placentation might then lead to maternal endothelial cell damage by an ongoing process. There might be a recurring sequence of 4 steps: (1) The placenta releases trophoblastic cells with potentially cytotoxic characteristics. These circulating trophoblastic cells have an abnormal pattern of expression of integrins and perhaps other glycoproteins or proteins. (2) The circulating trophoblastic cells loosely bind to maternal endothelial cells, targeting them for anti-tumourigenesis. (3) The maternal immune system reacts against targeted maternal endothelial cells through anti-tumourigenic mechanisms. (4) Widespread maternal endothelial damage causes the characteristic kidney lesion called glomerula endotheliosis.     

Localization of heparanase in normal and pathological human placenta

Degradation of extracellular matrix (ECM) components is critical for invasion. Heparan sulphate proteoglycans are abundant in the ECM of the placenta and the decidua, hence their degradation may disassemble the matrix and facilitate placentation and trophoblast invasion. This study investigates the expression of heparanase in normal and pathological placentation using RT-PCR, in-situ hybridization and immunohistochemistry analysis to detect heparanase in specific cells of the placenta and at the fetal-maternal interface throughout pregnancy. Heparanase was observed in villous cytotrophoblasts (CT), syncytial trophoblasts (ST) and in intermediate trophoblast cell columns in normal first trimester, molar and ectopic pregnancies. The heparanase protein was preferentially expressed in the endothelium of fetal capillaries, and to a much lesser extent in larger fetal vessels. Extravillous trophoblasts (EVT) invading the decidua and the maternal vessels were also heparanase positive. In the second and third trimesters, villous CT remained heparanase positive whereas ST showed variable heparanase expression. EVT invading the placental implantation site were also positively stained. A similar pattern was observed in samples obtained from pre-eclamptic placentae and from placenta accreta. Our results indicate consistent expression of heparanase in normal and abnormal placenta, in small fetal vessels and in a variety of trophoblast subpopulations with different invasive potentials.     

West Nile virus infection of the placenta

Intrauterine infection of fetuses with West Nile virus (WNV) has been implicated in cases of women infected during pregnancy. Infection of timed-pregnant mice on 5.5, 7.5, and 9.5 days post-coitus (dpc) resulted in fetal infection. Infection of dams on 11.5 and 14.5 dpc resulted in little and no fetal infection, respectively. Pre-implantation embryos in culture were also infected with WNV after the blastocyst stage and the formation of trophectoderm. Green fluorescent protein (GFP) expression was observed in a trophoblast stem (TS) cell line after infection with a GFP-expressing WNV construct. However, no fluorescence was observed in differentiated trophoblast giant cell (TGC) cultures. GFP fluorescence was present in TGC cultures if infected TS cells were induced to differentiate. These results suggest that embryos are susceptible to WNV infection after the formation of the trophectoderm around 3.5 dpc through the formation of the functional placenta around 10.5 dpc.     

Blocking of the placental immune-modulatory ferritin activates Th1 type cytokines and affects placenta development, fetal growth and the pregnancy outcome

BACKGROUND: Placenta immunomodulatory ferritin (PLIF) cDNA was recently cloned from the human placenta, where it is expressed in syncytiotrophoblast and decidual mononuclear cells. PLIF and its subcloned bioactive domain (C48), expressed in Escherichia coli, are immunosuppressive proteins and induce pronounced IL-10 production in vitro and in vivo. METHODS AND RESULTS: PLIF serum level, measured by enzyme-linked immunosorbent assay, was elevated in pregnant mice throughout gestation and declined towards delivery. Blocking of PLIF activity by vaccination of mice with C48 prior to mating inhibited pregnancy development. Passive transfer of anti-C48 immunoglobulin (Ig) starting at 3.5-12.5 days post coitum (dpc) resulted in high rate of embryo resorption. Furthermore, treatment with anti-C48 Ig resulted in placental and embryonal growth restriction. At gestation day 13.5, growth retardation was especially notable in the placentae, while at 16.5 dpc it was pronounced in the embryos. Histopathological examination revealed that experimental placentae were globally hypoplastic and the labyrinth was strikingly pale and contained less maternal blood compared with control. Immune-activated spleen cells harvested at 13.5 dpc from anti-C48 Ig-treated pregnant mice secreted in vitro increased level of Th1 cytokines (IL-2, TNF-alpha, IL-12) and decreased level of Th2 cytokines (IL-10, IL-4, IL-5, IL-6) as compared with the level of the respective cytokines secreted by spleen cells from control pregnant mice. CONCLUSION: This study provides the first in vivo evidence that PLIF plays a major role in placentation and embryonic growth.     

Placental vasculogenesis is regulated by keratin-mediated hyperoxia in murine decidual tissues

The mammalian placenta represents the interface between maternal and embryonic tissues and provides nutrients and gas exchange during embryo growth. Recently, keratin intermediate filament proteins were found to regulate embryo growth upstream of the mammalian target of rapamycin pathway through glucose transporter relocalization and to contribute to yolk sac vasculogenesis through altered bone morphogenetic protein 4 signaling. Whether keratins have vital functions in extraembryonic tissues is not well understood. Here, we report that keratins are essential for placental function. In the absence of keratins, we find hyperoxia in the decidual tissue directly adjacent to the placenta, because of an increased maternal vasculature. Hyperoxia causes impaired vasculogenesis through defective hypoxia-inducible factor 1α and vascular endothelial growth factor signaling, resulting in invagination defects of fetal blood vessels into the chorion. In turn, the reduced labyrinth, together with impaired gas exchange between maternal and embryonic blood, led to increased hypoxia in keratin-deficient embryos. We provide evidence that keratin-positive trophoblast secretion of prolactin-like protein a (Prlpa) and placental growth factor (PlGF) during decidualization are altered in the absence of keratins, leading to increased infiltration of uterine natural killer cells into placental vicinity and increased vascularization of the maternal decidua. Our findings suggest that keratin mutations might mediate conditions leading to early pregnancy loss due to hyperoxia in the decidua.     

Placental Apoptosis in Health and Disease

Citation Sharp AN, Heazell AEP, Crocker IP, Mor G. Placental apoptosis in health and disease. Am J Reprod Immunol 2010; 64: 159–169 Apoptosis, programmed cell death, is an essential feature of normal placental development but is exaggerated in association with placental disease. Placental development relies upon effective implantation and invasion of the maternal decidua by the placental trophoblast. In normal pregnancy, trophoblast apoptosis increases with placental growth and advancing gestation. However, apoptosis is notably exaggerated in the pregnancy complications, hydatidiform mole, pre‐eclampsia, and intrauterine growth restriction (IUGR). Placental apoptosis may be initiated by a variety of stimuli, including hypoxia and oxidative stress. In common with other cell‐types, trophoblast apoptosis follows the extrinsic or intrinsic pathways culminating in the activation of caspases. In contrast, the formation of apoptotic bodies is less clearly identified, but postulated by some to involve the clustering of apoptotic nuclei and liberation of this material into the maternal circulation. In addition to promoting a favorable maternal immune response, the release of this placental‐derived material is thought to provoke the endothelial dysfunction of pre‐eclampsia. Widespread apoptosis of the syncytiotrophoblast may also impair trophoblast function leading to the reduction in nutrient transport seen in IUGR. A clearer understanding of placental apoptosis and its regulation may provide new insights into placental pathologies, potentially suggesting therapeutic targets.     

Global analysis of differentially expressed genes in early gestational decidua and chorionic villi using a 9600 human cDNA microarray

The global gene expression profiles of the decidua and chorionic villi of early human pregnancies were analysed by using cDNA microarray technology. Decidual and villous placental tissues were obtained from first trimester abortus and mRNA was extracted for cDNA microarray analysis. The human cDNA microarray [9600 clones, including known regulatory genes and expressed sequence tags (EST)] with colorimetric detection was used to identify differentially expressed genes between early gestational decidua and villi. According to cDNA microarray analysis, we have identified 641 genes with highly expressed mRNA in both decidua and villi, 49 genes with higher expressions in decidua, and 75 genes with higher expression in chorionic villi. These differentially expressed genes were further grouped into categories by their putative functions, including: cell growth-related factors, hormones/cytokines, cell adhesion molecules, signal transduction molecules, apoptosis-related factors, cytoskeleton/extracellular matrix proteins, and EST. Immunohistochemical stainings of cathepsin L, leukaemia inhibitory factor-receptor, and proliferative cell nuclear antigen showed results consistent with the microarray data. Identification of the differentially expressed genes between decidua and villi by microarray provide a global profiling of the gene expression pattern. This work adds to our understanding of placentation by reporting the gene expression profiles during first trimester human pregnancies using cDNA microarray.     

Comparative immunohistochemical study of M-CSF and G-CSF in feto-maternal interface in a multiparity mouse model

PROBLEM: Multiparity status has been found to bring beneficial effects both to the maintenance of pregnancy and to the offspring; however, these effects have not been fully explained. We have previously reported that placentae obtained from multiparous females belonging to a syngeneic mouse crossbreeding showed an important increase in the number of placental macrophages, suggesting that they might constitute a protective subpopulation. Taking into account that macrophage-colony stimulating factor (M-CSF) and granulocyte-colony stimulating factor (G-CSF) have proved to modulate macrophage activity and that both factors and/or their receptors have been found at feto-maternal interface, in this paper we analyzed the presence of M-CSF and G-CSF in placental tissue employing the same multiparity mouse model in order to investigate the influence of parity status on local immunoregulation factors of macrophage activity. METHOD OF STUDY: Three groups of mice (CBA/J x CBA/J) were analyzed: Primiparous Young, 3.0 +/- 0.5 months old (PY); Primiparous Old, 8.5 +/- 0.5 months old (PO) and Multiparous Old, 8.5 +/- 0.5 months old, with three to four previous pregnancies (MO). The presence of M-CSF and G-CSF in placental tissue was analyzed by immunohistochemistry. Cytokeratin (CK) and vimentin (VIM) expression and PAS staining were also studied. RESULTS: The three groups showed a similar immunostaining pattern for M-CSF in the whole placental trophoblast, while the expression of G-CSF was significantly higher only in the spongy zone in the MO group. Furthermore, all the MO placentae showed 5-11 layers of cells adjacent to the decidua, where G-CSF and M-CSF were highly detected. Conversely, they constituted a thin layer in PY and PO placentae. These cells were proved to be CK(+) and VIM(-) thus demonstrating their trophoblast origin. In addition, the layers closer to the decidua were also PAS+ suggesting that they could be interstitial cells, a type of invading trophoblast. CONCLUSIONS: In our mouse model, we observed an increase in the expression of G-CSF in placental spongiotrophoblast cells in multiparous females, which have been previously proposed as progenitors of the interstitial cells. Furthermore, this is the first report that indicates that parity status increases trophoblast invasion inducing a proliferative effect of the invading cells on the maternal tissue. We suggest that M-CSF and G-CSF secreted by these invading cells could favor the recruitment of macrophages to the trophoblast and might modulate their activity inducing a switch to a protective, non-inflammatory population.     

Placenta growth factor: potential role in pregnancy

PROBLEM: In spite of the known requirement for adequate vascularity during placentation, little is known regarding the regulation of angiogenic growth factor production by trophoblast. Placenta growth factor (PIGF) is a recently discovered angiogenic growth factor whose expression is relatively limited to trophoblast. METHOD OF STUDY: Current literature of PIGF was reviewed, with emphasis on its expression, regulation, role in angiogenesis, and potential function(s) at the maternal-fetal interface. RESULTS: PIGF is abundantly expressed by trophoblast, which implies that it could act in a paracrine manner to modulate vascular development, stability, and/or function within the decidua and placental villi. In addition, expression of the PIGF receptor, fms-like tyrosine kinase (flt-1) receptor, on trophoblast raises the potential for an autocrine role of PIGF in regulating trophoblast growth and/or function. CONCLUSIONS: The potential for PIGF to influence both vascular endothelial cells and trophoblast suggests that aberrant trophoblast production of PIGF could compromise cellular function during gestation and contribute to the vascular and placental pathologies noted in many obstetric complications.     

Cells of the fetomaternal interface: Their role in the maintenance of viviparous pregnancy

An immune system capable of discriminating between self and nonself evolved in nature long before the appearance of the viviparous mode of pregnancy, which brings maternal cells into a direct physical contact with genetically disparate cells of fetal origin. In the hemochorial type of placentation, the former include cells of the maternal immune system. This article briefly reviews the possible mechanisms that may protect the semiallogeneic conceptus in nature, with special reference to the role of the cells at the fetomaternal interface. We also present some new data on the antigenicity of pre- and postimplantation trophoblast cells and the immunobiology of decidual cells. Systemic changes in the maternal immune system appear to represent homeostatic responses to the presence of a semiallogeneic conceptus, unrelated to its protection; mechanisms for this protection must reside locally at the fetomaternal interface. We find that the lack of immunogenicity of the outer (trophoblast) cells of the preimplantation blastocyst can be explained by a transient disappearance of the major histocompatibility (MHC) antigens on their cell surface. However, following implantation and the formation of the placenta, class 1 MHC antigens reappear on certain classes of trophoblast cells, i.e., labyrinthine and spongiotrophoblast cells of the murine placenta. Similarly, cytotrophoblast cells of the early human placenta exhibit the presence of class 1 MHC antigens. An absence of class 2 MHC antigens despite the presence of class 1 antigens cannot entirely explain the lack of trophoblast immunogenicity. A local immunosuppression mediated by trophoblast cells themselves as well as maternal cells of hemopoietic origin in the decidua remain as a strong possibility. Typical decidual cells appear to play a central role in the maintenance of pregnancy because of their numerous functions: nutritive, endocrine, and immunoregulatory. Our studies reveal that they are descendants of bone-marrow-derived precursors, have unique surface markers recognizable with monoclonal antibodies nonreactive with other hemopoietic cell lineages, and have the ability to abrogate mixed lymphocyte reactions in vitro in a genetically unrestricted manner. Further studies directed at the cells of the fetomaternal interface should provide a better insight into the mode of survival of the nature's most commonplace allograft.     

Cardiovascular Development and Survival During Gestation in the Ts65Dn Mouse Model for Down Syndrome

The Ts65Dn mouse model for Down syndrome (DS) exhibits many phenotypes seen in human DS. Previous research has revealed a reduced rate of transmission of the T65Dn marker chromosome in neonates. To analyze potential fetal loss, litters from trisomic females at 10.5dpc through 14.5dpc were genotyped. No significant differences from the expected Mendelian ratio were found in transmission of T65Dn at any stage. Cardiovascular defects found in trisomic neonates are associated with formation of pharyngeal arch arteries. Vessel tracing was used to identify anomalies in 10.5dpc, 11.5dpc, and 13.5dpc embryos. Comparison of trisomic versus euploid embryos injected with India ink revealed delay and abnormality in cardiovascular development in trisomic embryos at each stage. Through the analysis of transmission rate and cardiovascular development in embryonic mice, we learn more about prenatal mortality and the origins of cardiac abnormality in the Ts65Dn mice to assist in understanding cardiovascular malformation associated with DS. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.