K. Orendi

Factors involved in regulating trophoblast fusion: potential role in the development of preeclampsia.

In the human placenta, turnover of villous trophoblast involves proliferation, differentiation and fusion of mononucleated cytotrophoblasts with the overlying syncytiotrophoblast. In this way the syncytiotrophoblast is continuously supplied with compounds derived from the fusing cytotrophoblasts. Acquisition of fresh cellular components is balanced by a concomitant release of apoptotic material as syncytial knots from the syncytiotrophoblast to the maternal circulation. In the turnover of villous trophoblast, fusion is an essential step and has been shown to be regulated by multiple factors, such as cytokines, hormones, protein kinases, transcription factors, proteases and membrane proteins. Dysregulation of one or more of these fusion factors entails aberrant fusion of the cytotrophoblast with the syncytiotrophoblast, which adversely affects the maintenance and integrity of the placental barrier. Unbalanced trophoblast fusion and release of apoptotic material into the intervillous space may provoke a massive systemic inflammatory response by the mother and thus lead to preeclampsia.

The choriocarcinoma cell line BeWo: syncytial fusion and expression of syncytium-specific proteins

Fusion of the trophoblast-derived choriocarcinoma cell line BeWo can be triggered by forskolin. BeWo cells are regularly used as a cell culture model to mimic in vivo syncytialisation of placental villous trophoblast. The β subunit of human chorionic gonadotropin (CGB), placental alkaline phosphatase as well as placental protein 13 (PP13, LGALS13) are exclusively expressed in the syncytiotrophoblast of the human placenta, and CGB is commonly used as a marker of syncytial differentiation. Here we tested the hypothesis that syncytial fusion precedes CGB and LGALS13 expression in trophoblast-derived BeWo cells. BeWo cells were cultured for 48 h in the presence or absence of forskolin and varying concentrations of H-89, a protein kinase A inhibitor that interferes with the forskolin-mediated pathway of syncytial fusion. LGALS13 and CGB expression were quantified by DELFIA and real-time PCR. Cell fusion was determined by morphological analysis and cell counting after immunofluorescence staining. In forskolin-stimulated BeWo cells that were hindered to fuse by treatment with H-89, levels of CGB protein expression were not altered, while LGALS13 protein and mRNA expression decreased significantly to control levels without forskolin. The LGALS13 protein expression data coincided with a significant decrease in syncytial fusion, while CGB protein expression was unaffected by rates of cell fusion and proliferation. We postulate that CGB protein expression is not necessarily linked to syncytial fusion, and thus CGB should be used with great caution as a marker of BeWo cell fusion.

Placental and trophoblastic in vitro models to study preventive and therapeutic agents for preeclampsia

In the field of preeclampsia, enormous efforts are ongoing to identify biomarkers predicting the syndrome already in the first trimester of pregnancy. At the same time, there is the need for in vitro models to test such biomarkers prior to their use in clinical trials. In addition, in vitro models may accelerate the development and evaluation of the benefit of any putative therapeutics. Therefore, in vitro systems have been established to evaluate the release of biomarkers and measure the effect of putative therapeutics using placental villous explants as well as the choriocarcinoma cell line BeWo. For explants, a cryogenic method to freeze, transport and thaw villous explants was developed to use such tissues for a multi-site tissue culture evaluation. Here we focus on three out of many in vitro models that have been established for human placental trophoblast. (1) Choriocarcinoma cell lines such as BeWo, Jeg-3 and Jar cells (2) isolated primary trophoblast cells, and (2) villous explants from normal placentas delivered at term. Cell lines were used to assess the effect of differentiation and fusion on the expression and release of a preeclampsia marker (placental protein 13; PP13) and beta-hCG. Moreover, cell lines were used to study the effect of putative preeclampsia therapeutics such as vitamins C and E, heparin and aspirin on marker release and viability. Cryopreservation of villous explants enabled shipment to a remote laboratory and testing of parameters in different countries using explants from one and the same placenta. Recently published data make it tempting to speculate that the choriocarcinoma cell line BeWo as well as fresh and cryogenically stored placental villous explants may well serve as in vitro models to study preventive and therapeutic agents in the field of preeclampsia.

Caspases rather than calpains mediate remodelling of the fodrin skeleton during human placental trophoblast fusion

Fusion of cytotrophoblasts with the overlying syncytiotrophoblast is an integral step in differentiation of the human placental villous trophoblast. Multiple factors, such as growth factors, hormones, cytokines, protein kinases, transcription factors and structural membrane proteins, were described to modulate trophoblast fusion. However, the knowledge on remodelling of the membrane-associated cytoskeleton during trophoblast fusion is very limited. This study describes the link between remodelling of spectrin-like α-fodrin and intercellular trophoblast fusion. Experiments with primary trophoblasts isolated from term placentas and the choriocarcinoma cell line BeWo revealed a biphasic strategy of the cells to achieve reorganization of α-fodrin. Syncytialization of trophoblasts was accompanied by down-regulation of α-fodrin mRNA, whereas the full-length α-fodrin protein was cleaved into 120 and 150 kDa fragments. Application of calpeptin and calpain inhibitor III did not affect α-fodrin fragmentation in primary term trophoblasts and forskolin-treated BeWo cells, but decreased secretion of β human chorionic gonadotropin. In contrast, inhibitors of caspases 3, 8 and 9 attenuated generation of the 120 kDa fragment and a general caspase inhibitor completely blocked fragmentation, suggesting an exclusive function of caspases in α-fodrin remodelling. Immunofluorescence double staining of human placenta revealed co-localization of active caspase 8 with α-fodrin positive vesicles in fusing villous cytotrophoblasts. These results suggest that caspase-dependent fragmentation of α-fodrin may be important for reorganization of the sub-membranous cytoskeleton during trophoblast fusion.

Oxygen as modulator of trophoblast invasion

At the time of blastocyst implantation the uterine spiral arteries have already undergone morphological changes in the absence of any extravillous trophoblast invasion. Only 2 weeks after implantation, extravillous trophoblast cells develop and come into first contact with decidual tissues. Invading through the decidual interstitium, extravillous trophoblasts potentially reach and transform spiral arteries into uteroplacental arteries. Spiral arterial erosion starts at about mid‐first trimester, whereas flow of maternal blood into the intervillous space is continuously established only at the beginning of the second trimester. One key regulator of the number of extravillous trophoblasts is oxygen. The steep gradient in oxygen concentration within the first trimester placenta is diminished with the onset of maternal blood flow. This gradient is used by the trophoblast to generate a large number of invasive cells to adapt the arterial vasculature in the placental bed to the growing needs of the fetus. Changes in oxygen concentrations or other factors leading to alterations in the rates of proliferation and/or apoptosis of extravillous trophoblast clearly impact on the remodelling of the vessels. The respective consequences of a failure in trophoblast invasion are growth restrictions of the baby and perhaps other pregnancy complications.

The art of identification of extravillous trophoblast

Immunohistochemical staining with specific markers for the respective cell type facilitates tracking and identification of cells such as extravillous trophoblast in the uterine wall. Cytokeratin has been recommended as a marker for all kinds of trophoblasts and is commonly used as a marker to identify interstitial as well as endovascular trophoblast. With immunohistochemical double staining of specimens of first trimester placental bed we show that staining with anti-cytokeratin alone is not sufficient to track all routes of trophoblast invasion. Endovascular trophoblasts can be easily mixed up with endoglandular trophoblasts. Thus, additional application of specific markers for extravillous trophoblast such as anti-HLA-G is strongly recommended, ideally in combination with other markers in immunohistochemical or immunofluorescence double staining.

Effects of vitamins C and E, acetylsalicylic acid and heparin on fusion, beta-hCG and PP13 expression in BeWo cells.

Preeclampsia is one of the leading causes for maternal and fetal morbidity. Placental protein 13 (PP13) is a placenta specific protein and with its decreased maternal serum levels in the first trimester it is one of the most promising markers to predict the syndrome in early pregnancy. In clinical trials attempts to prevent preeclampsia have already been made using low-dose aspirin, low-molecular-weight heparin, and antioxidants such as vitamins C and E. Here we investigated the effect of these agents on PP13 and beta-hCG levels using choriocarcinoma cell lines as surrogates for primary villous trophoblast. Five different cell lines were triggered with forskolin and cultured for 48 h. Amongst the five tested cell lines BeWo cells showed the strongest increase in PP13 mRNA after forskolin treatment compared to controls. Hence these cells were used to investigate the effect of varying concentrations of vitamin C, acetylsalicylic acid (ASA), Trolox) and heparin on cell fusion and PP13 and beta-hCG levels. The response to vitamin C was a dose-dependent increase in protein expression, while the other drugs showed only modest effects. Since first trimester PP13 has been shown to be significantly decreased in women subsequently developing preeclampsia, this data might point to a beneficial effect of very early vitamin C treatment of such women already in the early first trimester of pregnancy.

SRY-specific cell free fetal DNA in maternal plasma in twin pregnancies throughout gestation

OBJECTIVES Levels of SRY-specific cell free fetal DNA (SRY-cffDNA) in maternal plasma were investigated in twin pregnancies with two male fetuses versus one male and one female fetus and singleton male pregnancies during second and third trimester. The aim was to evaluate at which gestational age the amount of SRY-cffDNA reflects the number of fetuses and placentas respectively. METHODS 251 venous blood samples were analyzed from a total of 178 women with male or mixed-gender twin pregnancies and male singleton pregnancies in the second and the third trimester. The concentration of SRY-cffDNA was determined by quantitative real time PCR using the Y-chromosome specific SRY assay. For statistical analysis these three groups were divided into four subgroups according to their gestational age. RESULTS During second trimester levels of SRY-cffDNA showed no differences between twin and singleton pregnancies. After 28 weeks SRY-cffDNA of male twin pregnancies was significantly increased compared to singleton male pregnancies and mixed-gender twin pregnancies with no differences between the latter two. CONCLUSION The level of SRY-cffDNA in maternal serum of twin pregnancies reflects the number of fetuses only during the third trimester. Hence its use as a diagnostic tool for complications related to altered SRY-cffDNA levels in twin pregnancies should be evaluated at different weeks of gestation, especially during the second trimester.

Fibulin-5 expression in the human placenta

Fibulin-5 is a secreted extracellular matrix glycoprotein and displays a diverse panel of biological functions, which can be segregated into elastogenic as well as extra-elastogenic functions. While elastogenic functions of fibulin-5 include essential roles in early steps of elastic fibre assembly, extra-elastogenic functions are widespread. Depending on the cell type used, fibulin-5 mediates cell adherence via a subset of integrins, antagonizes angiogenesis and inhibits migration as well as proliferation of endothelial and smooth muscle cells. In this study, we focused on the spatiotemporal expression of fibulin-5 in the human placenta. With progressing gestation, placental fibulin-5 expression increased from first trimester towards term. At term, placental fibulin-5 mRNA expression is lower when compared with other well-vascularized organs such as lung, kidney, heart, uterus and testis. In first trimester, placenta immunohistochemistry localized fibulin-5 in villous cytotrophoblasts and extravillous cytotrophoblasts of the proximal cell column. In term placenta, fibulin-5 was detected in the endothelial basement membrane and adventitia-like regions of vessels in the chorionic plate and stem villi. Cell culture experiments with the villous trophoblast-derived cell line BeWo showed that fibulin-5 expression was downregulated during functional differentiation and intercellular fusion. Moreover, cultivation of BeWo cells under low oxygen conditions impaired intercellular fusion and upregulated fibulin-5 expression. The spatiotemporal shift from the trophoblast compartment in first trimester to the villous vasculature at term suggests a dual role of fibulin-5 in human placental development.

Complete and pure trisomy 18p due to a complex chromosomal rearrangement in a male adult with mild intellectual disability.

Since its first description in 1968 [Jacobsen and Mikkelsen, 1968], complete and pure trisomy of the short arm of chromosome 18 has remained a rare finding with only 23 reported patients [Jacobsen and Mikkelsen, 1968; Taylor et al., 1975; Rosano et al., 1977; Meinecke and Koske-Westphal, 1981; Habedank and Trost-Brinkhues, 1983; Johansson et al., 1988; Takeda et al., 1989; Wolff et al., 1991; Moog et al., 1994; Li et al., 1998; Grosso et al., 2005; Mabboux et al., 2007; Marical et al., 2007; Rodriguez et al., 2007] (Table I) and another three entries in the DECIPHER and ISCA databases (Table II). Furthermore,manyof these reportswere based on traditional cytogenetic banding methods instead of high-resolution technologies such as array-CGH. Due to these technical limitations and its rare occurrence, phenotypic consequences of trisomy 18p are poorly defined. Here we describe a patient with a complex chromosomal rearrangement which has resulted in complete and pure trisomy 18p and provide a review of the literature. The patient was seen by our genetic counseling service at 30 years of age. He was born to a 27-year-old mother at term after an uneventful pregnancy. He was the first child of unrelated, healthy Caucasian parents. At birth, he had aweight of 2,800 g (9th centile), a length of 47 cm (9th centile) and anOFC of 34 cm (25th centile). Initial feeding difficulties and frequent vomiting were reported. Milestones in psychomotor development were slightly delayed: he could sit at the ageof 12months, started towalk and talk at the ageof 18months. Between 4 and 8 years of age, absence epilepsy occurred. The EEG showed a focal hyperexcitability with monomorphic delta-theta waves intermingled with high potentials and spikewave complexes in the occipital areas. Therapeutic intervention with several antiepileptic drugs helped to reduce absence epilepsy and he is currently being successfully treated with lamotrigine. During childhood the patient attended an integration school and learned to read and write, although difficulties with calculating still remain. Puberty and growth were delayed. He later attended and successfully completed a vocational school and today lives mainly independently with parental support due to a reported mild intellectual disability. At present, he has a weight of 78 kg (92nd centile), a height of 168 cm(8th centile) and anOFCof 56 cm(38th centile). Our physical examination showed a healthy young man with a short neck, deep-set eyes, short palpebral fissures, a short philtrum, micrognathia, a high-arched palate and the presence of