An assessment of serum-dependent impacts on intracellular accumulation and genomic response of per- and polyfluoroalkyl substances in a placental trophoblast model.

Per- and polyfluoroalkyl substances (PFAS), a class of environmental contaminants, have been detected in human placenta and cord blood. The mechanisms driving PFAS-induced effects on the placenta and adverse pregnancy outcomes are not well understood. This study investigated the impact of perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), and a replacement PFAS known as hexafluoropropylene oxide dimer acid (HFPO-DA, tradename GenX) on placental trophoblasts in vitro. Several key factors were addressed. First, PFAS levels in cell culture reagents at baseline were quantified. Second, the role of supplemental media serum in intracellular accumulation of PFAS in a human trophoblast (JEG3) cell line was established. Finally, the impact of PFAS on the expression of 96 genes involved in proper placental function in JEG3 cells was evaluated. The results revealed that serum-free media (SFM) contained no detectable PFAS. In contrast, fetal bovine serum-supplemented media (SSM) contained PFNA, PFUdA, PFTrDA, and 6:2 FTS, but these PFAS were not detected internally in cells. Intracellular accumulation following 24 hr treatments was significantly higher when cultured in SFM compared to SSM for PFOS and PFOA, but not HFPO-DA. Treatment with PFAS was associated with gene expression changes (n = 32) in pathways vital to placental function, including viability, syncytialization, inflammation, transport, and invasion/mesenchymal transition. Among the most robust PFAS-associated changes were those observed in the known apoptosis-related genes, BAD and BAX. These results suggest a complex relationship between PFAS, in vitro culture conditions, and altered expression of key genes necessary for proper placentation.

Real-time microscopic assessment of fatty acid uptake kinetics in the human term placenta.

INTRODUCTION: The placenta employs an efficient and selective fatty acid transport system to supply lipids for fetal development. Disruptions in placental fatty acid transport lead to restricted fetal growth along with cardiovascular and neurologic deficits. Nevertheless, little is known about the molecular mechanisms involved in human placental fatty acid trafficking during the initial steps of uptake, or the importance of fatty acid chain length in determining uptake rates. METHODS: We employed BODIPY fluorophore conjugated fatty acid analogues of three chain lengths, medium (BODIPY-C5), long (BODIPY-C12), and very-long (BODIPY-C16), to study fatty acid uptake in isolated human trophoblast and explants using confocal microscopy. The three BODIPY-labeled fatty acids were added to freshly isolated explants and tracked for up to 30 min. Fatty acid uptake kinetics were quantified in trophoblast (cytotrophoblast and syncytiotrophoblast together) and the fetal capillary lumen. RESULTS: Long- (BODIPY-C12) and Very long-chain (BODIPY-C16) fatty acids accumulated more rapidly in the trophoblast layer than did medium-chain (BODIPY-C5) whereas BODIPY-C5 accumulated more rapidly in the fetal capillary than did the longer chain length fatty acids. The long-chain fatty acids, BODIPY-C12 and BODIPY-C16, are esterified and stored in lipid droplets in the cytotrophoblast layer, but medium-chain fatty acid, BODIPY-C5, is not. DISCUSSION: Fatty acids accumulate in trophoblast and fetal capillaries inversely according to their chain length. BODIPY-C5 accumulates in the fetal capillary in concentrations far greater than in the trophoblast, suggesting that medium-chain length BODIPY-labeled fatty acids are capable of being transported against a concentration gradient.

Assessment of the cellular localisation of the annexin A2/S100A10 complex in human placenta.

The AnxA2/S100A10 complex has been implicated in various placental functions but although the localisation of these proteins individually has been studied, there is no information about the localisation of their complex in situ at the cellular level. Using the proximity ligation technique, we have investigated the in situ localisation of AnxA2/S100A10 complex in the placenta and have compared this with the location patterns of the individual proteins. High levels of expression of AnxA2/S100A10 complexes were observed in the amniotic membrane and in blood vessel endothelial cells. Lower levels were detected in the brush border area of the syncytium and in the trophoblasts. Immunohistochemical analysis of AnxA2 and S100A10 individually revealed broadly similar patterns of localisation. The brush border staining pattern suggests that in this location at least some of the AnxA2 is not in complex with S100A10. The formal location of the AnxA2/S100A10 complex is compatible with a role in cell-cell interaction, intracellular transport and secretory processes and regulation of cell surface proteases, implying contributions to membrane integrity, nutrient exchange, placentation and vascular remodelling in different parts of the placenta. Future applications will allow specific assessment of the association of the complex with pathophysiological disorders.

Toxicity assessment on trophoblast cells for some environment polluting chemicals and 17ß-estradiol.

The identification of reproductive toxicants is a major scientific challenge for human health. We investigated the effects of a selected group of environmental polluting chemicals mostly provided with estrogenic activity on the human trophoblast cell lines BeWo and HTR-8/SVneo. Cells were exposed for 24h to various concentrations (from 0.1 pM to 1 mM) of atrazine (ATR), diethylstilbestrol (DES), para-nonylphenol (p-NP), resveratrol (RES) and 17 ß-estradiol (E2) and assayed for cell viability and human beta-Chorionic Gonadotropin (ß-hCG) secretion. Decrease of cell viability as respect to control, vehicle-treated, cultures was obtained for all chemicals in the concentration range of 1 µM-1 mM in both cell types. A parallel decrease of ß-hCG secretion was observed in BeWo cells, at 1 µM-1 mM concentrations, with the only exception of ATR which caused an increase at concentrations up to 1mM. ß-hCG release was also unexpectedly inhibited by ATR, DES, p-NP and RES at non-toxic (pM-nM) concentrations. These findings raise concern about the negative, potential effects of various environmental polluting chemicals on pregnancy success and fetal health.

Exaggerated placental site reaction detected during caesarean delivery: a case report.

Exaggerated placental site (EPS) reaction is an exuberant physiologic process in which intermediate trophoblasts infiltrate the underlying endometrium and myometrium at the implantation site. During a caesarean section, we noted a polypoid well shaped smooth lesion, about 3 cm in diameter on the anterior wall of the uterus apart from the placenta. The histopathologic examination revealed an exuberant proliferation of trophoblastic cells in the placental site, a low Ki-67 labelling index and the absence of mitotic activity. Distinguishing EPS reaction from the other intermediate trophoblastic tumours is critical, as the latter may likely involve surgical intervention and/or chemotherapy, although no specific treatment and follow-up is required for EPS reaction. It is necessary to be aware of this pathology and take biopsies from suspicious lesions in the placental site for pathologic examination.

Pregenesys pre-eclampsia markers consensus meeting: What do we require from markers, risk assessment and model systems to tailor preventive strategies?

The Pregenesys Consensus Meeting held in Cambridge on 13 July 2009 was organized by the Pregenesys Consortium to review and critically discuss current knowledge regarding early markers of preeclampsia, to identify priorities and opportunities for future research, to consider issues that may need to be addressed in future recommendations and to highlight key issues in cost effectiveness and national policies concerning prediction and early screening for the risk of developing preeclampsia. This report summarizes the outcome of the Consensus Meeting and draws attention to issues for further investigation with specific regard to single versus multiple markers, early versus late risk identification, and the long-term effects on both maternal and perinatal health and the need to include these in any future cost-benefit assessment.

Trophoblast invasion: assessment of cellular models using gene expression signatures.

Invasive, extravillous trophoblasts (EVT) of the human placenta are critically involved in successful pregnancy outcome since they remodel the uterine spiral arteries to increase blood flow and oxygen delivery to the placenta and the developing fetus. To gain more insights into their biological role different primary cell culture models are commonly utilised. However, access to early placental tissue may be limited and primary trophoblasts rapidly cease proliferation in vitro impairing genetic manipulation. Hence, trophoblastic cell lines have been widely used as surrogates to study EVT function. Although the cell lines share some molecular markers with their primary counterpart, it is unknown to what extent they recapitulate the invasive phenotype of EVT. Therefore, we here report the first thorough GeneChip analyses of SGHPL-5, HTR-8/SVneo, BeWo, JEG-3 and the novel ACH-3P trophoblast cells in comparison to previously analysed primary villous cytotrophoblasts (CTBs) and extravillous trophoblasts (EVTs). Analyses of approximately 14,000 commonly expressed genes revealed that EVTs most closely resemble CTBs with considerable differences to the group of choriocarcinoma cells (JEG-3, BeWo, ACH-3P) and the group of SV40 Large T Antigen-selected cell types (SGHPL-5, HTR-8/SVneo). Similarly, analyses of 912 genes discriminating EVT from CTB, or 370 EVT-specific genes did not unravel a particular cell line with close similarity to any of the primary cell types, although molecular signatures common to EVT and each group of cell lines could be identified. Considering the diversity of mRNA expression patterns it is suggested that molecular studies in trophoblast cell lines require verification of the critical steps in an appropriate primary model system.

E-cadherin in the assessment of aberrant placental cytotrophoblast turnover in pregnancies complicated by pre-eclampsia.

E-cadherin is a cell-cell adhesion protein expressed in cytotrophoblasts, which is lost as they differentiate and syncytialise. We have exploited E-cadherin as a marker of cytotrophoblasts to investigate villous tissue composition in first and third trimester placentae, both in normal pregnancy and pregnancies complicated by pre-eclampsia. We have achieved this by measuring expression levels of E-cadherin at the mRNA level, using Q-PCR, and at the protein level using semi-quantitative Western blotting. We have also combined E-cadherin immunohistochemistry with morphometric analysis of area measurements to define cytotrophoblast and syncytiotrophoblast compartments. This novel use of E-cadherin has revealed a decrease in the proportion of cytotrophoblasts in villous tissue as pregnancy progresses, in the absence of changes in syncytiotrophoblast cover. Moreover, in pre-eclampsia, placental E-cadherin was raised compared to syncytiotrophoblast, suggesting either exaggerated cytotrophoblast proliferation or impaired cytotrophoblast differentiation, both alterations of potential pathogenic importance.

Ectopic pregnancy: histopathology and assessment of cell proliferation with and without methotrexate treatment.

OBJECTIVE: To evaluate tubal morphology, trophoblast proliferation, and inflammatory reaction in response to methotrexate (MTX) treatment of ectopic pregnancy (EP). DESIGN: Nonrandomized controlled study. SETTING: Academic hospital. PATIENTS: Archival specimens from 10 EP unsuccessfully treated with MTX and 10 cases primarily treated by surgery. INTERVENTIONS: Ki67/hCG and Ki67/human placental lactogen double immunohistochemical methods were used to examine trophoblastic spread, placentation, hormone production, decidualization, vascular invasion, hemorrhage, rupture, and proliferative index of the cytotrophoblast. B and T-lymphocyte responses were evaluated by CD3 and CD20. RESULTS: Trophoblastic spread and placentation were confined to the tubal mucosa after MTX treatment, whereas invasion of the muscularis and subserosa was common in the controls. The proliferative index was reduced (19 percent versus 93 percent), although a high proliferative index was found in two of three cases complicated by rupture. Polar proliferation of Ki67-positive cytotrophoblast toward the implantation site was abolished in MTX-treated cases. Decidual reaction was not observed. No correlation was observed between the above-mentioned findings and gestational age, level of beta-hCG, dose of MTX, or interval to surgery. CONCLUSION: Trophoblastic spread, differentiation, and invasion were compromised by MTX treatment. Methotrexate seems to decrease cytotrophoblast proliferation. Whether a missing decrease in proliferation index reflects treatment failure awaits a larger population-based study.

Assessment of expression of the receptor for colony-stimulating factor-1 (fms) in bovine trophoblast.

Bovine trophoblast was employed to assess the questions of whether the receptor for CSF-1 is expressed by noninvasive trophoblast and whether expression changes with differentiation within placentomes. Bovine placental poly(A) mRNA contained sequences cross-reactive with cDNA probes to c-fms and v-fms. Using a monoclonal antibody to v-fms, immunohistochemistry of postattachment bovine trophoblast showed expression of an fms-like protein between Day 29 and term. Expression occurred in both the intercotyledonary and cotyledonary trophoblast. Reactivity that was fms-like was also demonstrated on preattachment conceptuses flushed at Days 14 and 7 of gestation and on Day 7 embryos derived from in vitro oocyte maturation and fertilization. Unexpectedly, in the second half of pregnancy some cells, including binucleate cells, showed nuclear rather than cytoplasmic reactivity to the antibody. These data indicate expression of an fms-like protein in bovine placenta that does not correlate with properties of trophoblast cell invasiveness or major morphological differentiation. The data do support a universal role for this protein during mammalian placental development.

[Determination of HCG excretion for diagnosis of trophoblast vitality and assessment of age of early pregnancy (author's transl)]. / Zur Diagnostik der Vitalität des Trophoblasten und zur Tragzeitbestimmung in der Frühschwangerschaft durch Bestimmung der HCG-Ausscheidung.

A convenient technique for quantitative determination of human chorionic gonadotrophin in urine is described in this paper, Gravimun test kit reagents are used to establish inhibition of haemagglutination, using in glass tubes one tenth (Gravimun) or one fifteenth (Gravimun II) of the amounts of reagents which would be required for the original test. Erythrocytes undergo sedimentation as two longitudinal stripes in parallel on the bottom of the tubes. The spacing between them is taken as the measure of the HCG quantity added to the system and related to a specially prepared standard curve. The Second International Standard of HCG for Bioassay is used for comparison. - Relation c = t3 (c = HCG concentration, IU/L; t = days from nidation, 20 days from last menstruation) was established for this period of time from values which had been recorded from 500 normal early pregnancies up to the tenth week. That relation can be used as a rule of thumb by which to determine age of pregnancy or to calculate expected average HCG excretion up to the tenth week of pregnancy.