Objective To examine the expression pattern of biomarker proteins in extravillous trophoblast (EVT) cells obtained noninvasively by trophoblast retrieval and isolation from the cervix (TRIC) in patients with early pregnancy loss compared with control patients with uncomplicated term delivery. Design Case-control study. Setting Academic medical center. Patient(s) Women with either early pregnancy loss (EPL, n = 10) or an uncomplicated term delivery (N = 10). Intervention(s) Endocervical specimens obtained from ongoing pregnancies at gestational ages of 5–10 weeks to generate an archive of EVT cells isolated by TRIC, with medical records examined to select specimens matched for gestational age at the time of endocervical sampling. Main Outcome Measure(s) Known serum biomarkers for adverse pregnancy outcome that are expressed by EVT cells were evaluated by semiquantitative immunocytochemistry, using antibodies against endoglin (ENG), FMS-like tyrosine kinase-1 (FLT-1), α-fetoprotein (AFP), pregnancy-associated plasma protein-A (PAPP-A), galectin-13 (LGALS13), galectin-14 (LGALS14), and placental growth factor (PGF). Result(s) The EVT purity was over 95% in all specimens, based on chorionic gonadotropin expression; however, the number of EVT cells obtained was significantly lower in women with EPL than the control group. There was a statistically significant elevation of AFP, ENG, and FLT-1, and statistically significant reduction of PAPP-A, LGALS14, and PGF in the EPL group compared with controls. Conclusion(s) In this pilot study, EVT cells isolated by TRIC early in gestation exhibited altered protein expression patterns before an EPL compared with uncomplicated term pregnancies.
Abstract Despite the high incidence of trophoblast-related diseases, the molecular mechanism of inadequate early trophoblast development is still unclear due to the lack of an appropriate cellular model in vitro. In the present study, we reprogrammed the amniotic cells to be induced pluripotent stem cells (iPSCs) via a non-virus and non-integrated method and subsequently differentiated them into trophoblast-like cells by a modified BMP4 strategy in E6 medium. Compared with the previously studied trophoblast-like cells from ESCs, the iPSCs derived trophoblast-like cells behave similarly in terms of gene expression profiles and biofunctions. Also we confirmed the differentiating tendency from iPSCs to be syncytiotrophoblasts-like cells might be caused by inappropriate differentiating oxygen condition. Additionally, we preliminarily indicated in vitro “artificial” differentiation of iPSCs also undergoing a possible trophoblastic stem cell stage, as witnessed in vivo. In conclusion, we provided an in vitro cellular model to study early trophoblast development for specific individual, by using the feasible amnion.
Our understanding of how cells communicate has undergone a paradigm shift since the recent recognition of the role of exosomes in intercellular signaling. In this study, we investigated whether oxygen tension alters the exosome release and miRNA profile from extravillous trophoblast (EVT) cells, modifying their bioactivity on endothelial cells (EC). Furthermore, we have established the exosomal miRNA profile at early gestation in women who develop pre-eclampsia (PE) and spontaneous preterm birth (SPTB). HTR-8/SVneo cells were used as an EVT model. The effect of oxygen tension (i.e. 8% and 1% oxygen) on exosome release was quantified using nanocrystals (Qdot (R)) coupled to CD63 by fluorescence NTA. A real-time, live-cell imaging system (Incucyte (TM)) was used to establish the effect of exosomes on EC. Plasma samples were obtained at early gestation (< 18 weeks) and classified according to pregnancy outcomes. An Illumina TrueSeq Small RNA kit was used to construct a small RNA library from exosomal RNA obtained from EVT and plasma samples. The number of exosomes was significantly higher in EVT cultured under 1% compared to 8% oxygen. In total, 741 miRNA were identified in exosomes from EVT. Bioinformatic analysis revealed that these miRNA were associated with cell migration and cytokine production. Interestingly, exosomes isolated from EVT cultured at 8% oxygen increased EC migration, whilst exosomes cultured at 1% oxygen decreased EC migration. These changes were inversely proportional to TNF-alpha released from EC. Finally, we have identified a set of unique miRNAs in exosomes from EVT cultured at 1% oxygen and exosomes isolated from the circulation of mothers at early gestation, who later developed PE and SPTB. We suggest that aberrant exosomal signalling by placental cells is a common aetiological factor in pregnancy complications characterised by incomplete SpA remodeling and is therefore a clinically relevant biomarker of pregnancy complications.
Background Preeclampsia has become the world's major maternal health problem putting a huge burden on mothers, newborns and also on the health systems. The pathogenesis of preeclampsia seems to include events in very early pregnancy affecting differentiation of placental villous trophoblast. The arising changes of the cell death spectrum from apoptosis via increased autophagy and aponecrosis to necrosis in turn induce systemic inflammation of the mother. Methods Placental tissue samples and maternal serum samples from 40 pregnant women were collected from normal pregnancy, IUGR, early-onset and late-onset preeclampsia. Immunohistochemistry for LC3B and Beclin-1 was quantified using systematic random sampling techniques. Serum levels of LDH and other markers were assessed in serum. Results Expression of the autophagy markers LC3B and Beclin-1 was significantly different between groups as was the LC3B/Beclin-1 ratio. Early-onset preeclampsia and IUGR had the highest autophagy protein expression levels, while normal pregnancy and late-onset preeclampsia had the highest LC3B/Beclin-1 ratio. Early-onset preeclampsia had the highest negative correlation with free LDH as cell defect marker. Conclusions Autophagy plays a critical role in the cell death spectrum and cellular survival capacity of villous trophoblast. Alterations in autophagic protein expression are involved in pathological pregnancies such as preeclampsia.
Abstract Problem Reproductive failure including RM has been suggested to correlate with antibodies that cross react with HLA-negative syncytiotrophoblasts and we have reported that 17% of women with 2 or more miscarriages and 34% of women with 3 or more miscarriages express anti-trophoblast antibodies (ATAB). Until now, the mechanism, how ATAB interfere with pregnancy success is not known. HCG and progesterone both play fundamental roles in supporting human pregnancy. Therefore we investigated the effects of sera of RM patients containing ATAB on the hCG and progesterone production of cells of the choriocarcinoma cell line JEG-3. Method of study In vitro study to investigate effects of patient sera with and without ATAB on hCG and progesterone secretion of JEG-3 cells. The presence of ATAB was detected as described earlier. Effects of sera from ATAB positive and ATAB negative RM patients on hCG and progesterone secretion by JEG-3 cells were analysed 12 and 24 h after plating. Sera of women without pregnancy pathologies served as controls. Results Sera of ATAB-positive RM patients significantly inhibit hCG secretion of JEG-3 cells for 12 h after plating compared to sera of healthy controls (p = 0.019) and significantly reduce progesterone production for 12 h (p = 0.046) and 24 h (p = 0.027) of co-culture. Sera of ATAB-negative RM patient show no significant effect on progesterone secretion. Conclusions Inhibition of hCG and progesterone production might point to a mechanism, how ATAB interfere with early pregnancies.
Preimplantation factor (PIF) is a peptide secreted by viable mammalian embryos. Moreover, it can be detected in the circulation of pregnant women. Recently, it was shown that PIF promotes invasion in trophoblast cell lines in vitro. Successful human embryo implantation depends on a deep and highly controlled invasion of extravillous trophoblast (EVT) in the maternal endometrium. Trophoblast invasion is regulated in part by matrix metalloproteinase (MMP) activity and integrin expression. The present study demonstrates the presence of PIF in early pregnancy and characterizes its effects on primary human trophoblast invasion. At the fetomaternal interface, intense PIF labeling by immunohistochemistry was present during early gestation in villous trophoblasts and EVTs. A decrease of labeling was observed at term. Furthermore, PIF significantly promoted invasion of human EVT isolated from first-trimester placenta. The proinvasive regulatory effect of PIF in EVT was associated with 1) increased MMP9 activity and 2) reduced tissue inhibitor of metalloproteinase-1 (TIMP1) mRNA expression. PIF also regulated alpha v and alpha 1 integrin mRNA expressions. Last, the proinvasive effect of PIF appeared to be mediated by the mitogen-activated protein kinase (MAPK), phosphoinositide-3-kinase (PI3K), and Janus-kinase signal transducer and activator of transcription (JAK-STAT) signaling pathways. In summary, this work describes the direct, positive effect of PIF on the control of human trophoblastic cell invasion by modulation of MMP/TIMP balance and integrin expression. Moreover, these results suggest that PIF is involved in pathological pregnancies characterized by insufficient or excessive trophoblast invasion.
Objective Placenta accreta is clinically associated with maternal uterine scar. Our objective was to investigate the biochemical contribution of maternal scarring to hyperinvasive trophoblast. We hypothesised that trophoblast over‐invasion in placenta accreta is associated with aberrant invasion‐site signalling of growth and angiogenic factors known to be involved in wound healing and promotion of cell invasion through the epithelial to mesenchymal cellular programme. Design Cross‐sectional series. Setting Yale–New Haven Hospital. Population Women with histologically confirmed normal and abnormal placentation. Methods Placental invasion site tissue sections were immunostained for endoglin and other angiogenic regulators, and transforming growth factor β (TGFβ) proteins. Maternal serum endoglin, and the vascular endothelial growth factor (VEGF) mediators hypoxia‐inducible factor‐1α (HIF1α) and endostatin, were assessed using immunoassay. Main outcome measures Differences in median H‐score by immunostaining and in mean serum level by immunoassay. Results By immunostaining, placenta accreta samples demonstrated intervillous endoglin shedding and increased trophoblast expression of its cleavage protein matrix metalloproteinase‐14. Absent decidual HIF1α and endostatin were observed in areas of VEGF upregulation. TGFβ1 was present in myocytes but not in collagen bundles into which accreta trophoblast invaded. Maternal serum endoglin decreased in praevia and accreta when corrected for gestational age. Conclusion Angiogenic and growth factors at the placental invasion site are altered in accreta, both by decidual absence and within myometrial scar. We postulate this promotes the invasive phenotype of placenta accreta by activating hyperinvasive trophoblast and by dysregulating placental vascular remodelling. Funding Yale Department of Obstetrics, Gynecology and Reproductive Sciences funds. Tweetable Placenta accreta histology shows dysregulation of angiogenic and growth factors. Tweetable Placenta accreta histology shows dysregulation of angiogenic and growth factors. This article includes Author Insights, a video available at https://vimeo.com/rcog/authorinsights15176
Background The maternal signs of preeclampsia, which include the new onset of high blood pressure, can occur because of faulty placentation. We theorized that transcriptomic analyses of trophoblast subpopulations in situ would lend new insights into the role of these cells in preeclampsia pathogenesis. Objective Our goal was to enrich syncytiotrophoblasts, invasive cytotrophoblasts, or endovascular cytotrophoblasts from the placentas of severe preeclampsia cases. Total RNA was subjected to global transcriptional profiling to identify RNAs that were misexpressed compared with controls. Study Design This was a cross-sectional analysis of placentas from women who had been diagnosed with severe preeclampsia. Gestational age-matched controls were placentas from women who had a preterm birth with no signs of infection. Laser microdissection enabled enrichment of syncytiotrophoblasts, invasive cytotrophoblasts, or endovascular cytotrophoblasts. After RNA isolation, a microarray approach was used for global transcriptional profiling. Immunolocalization identified changes in messenger RNA expression that carried over to the protein level. Differential expression of non–protein-coding RNAs was confirmed by in situ hybridization. A 2-way analysis of variance of non-coding RNA expression identified particular classes that distinguished trophoblasts in cases vs controls. Cajal body foci were visualized by coilin immunolocalization. Results Comparison of the trophoblast subtype data within each group (severe preeclampsia or noninfected preterm birth) identified many highly differentially expressed genes. They included molecules that are known to be expressed by each subpopulation, which is evidence that the method worked. Genes that were expressed differentially between the 2 groups, in a cell-type–specific manner, encoded a combination of molecules that previous studies associated with severe preeclampsia and those that were not known to be dysregulated in this pregnancy complication. Gene ontology analysis of the syncytiotrophoblast data highlighted the dysregulation of immune functions, morphogenesis, transport, and responses to vascular endothelial growth factor and progesterone. The invasive cytotrophoblast data provided evidence of alterations in cellular movement, which is consistent with the shallow invasion often associated with severe preeclampsia. Other dysregulated pathways included immune, lipid, oxygen, and transforming growth factor-beta responses. The data for endovascular cytotrophoblasts showed disordered metabolism, signaling, and vascular development. Additionally, the transcriptional data revealed the differential expression in severe preeclampsia of 2 classes of non-coding RNAs: long non-coding RNAs and small nucleolar RNAs. The long non-coding RNA, urothelial cancer associated 1, was the most highly up-regulated in this class. In situ hybridization confirmed severe preeclampsia-associated expression in syncytiotrophoblasts. The small nucleolar RNAs, which chemically modify RNA structure, also correlated with severe preeclampsia. Thus, we enumerated Cajal body foci, sites of small nucleolar RNA activity, in primary cytotrophoblasts that were isolated from control and severe preeclampsia placentas. In severe preeclampsia, cytotrophoblasts had approximately double the number of these foci as the control samples. Conclusion A laser microdissection approach enabled the identification of novel messenger RNAs and non-coding RNAs that were misexpressed by various trophoblast subpopulations in severe preeclampsia. The results suggested new avenues of investigation, in particular, the roles of PRG2, Kell blood group determinants, and urothelial cancer associated 1 in syncytiotrophoblast diseases. Additionally, many of the newly identified dysregulated molecules might have clinical utility as biomarkers of severe preeclampsia.
Clinical pregnancies increasingly end in recurrent miscarriage (RM) during the first trimester, with genetic factors shouldering the main responsibility. MicroRNAs (miRNAs) regulate gene expression in a wide array of important biological processes. We examined the potential role of dysregulated miRNAs in RM pathogenesis and trophoblast development as an approach to elucidate the molecular mechanism behind RM. miRNA profiles from clinical specimens of RM and induced abortion (IA) were compared, and several miRNAs were found to be aberrantly expressed in RM samples. Among the miRNAs, miR‐365 was significantly differentially expressed in RM decidual tissues. Furthermore, our results demonstrate that miR‐365 functions as an upstream regulator of MDM2/p53 expression, cell cycle progression and apoptosis in trophoblasts. Bioinformatic prediction and experimental validation assays identified SGK1 as a direct target of miR‐365; consistently, its protein levels were low in decidual tissues. Additionally, functional studies revealed that SGK1 silencing elicits cell cycle arrest and apoptosis in trophoblasts and that SGK1 overexpression attenuates the effects of miR‐365 on apoptosis and MDM2/p53 expression. Collectively, our data provide evidence that the up‐regulation of miR‐365 may contribute to RM by decreasing SGK1 expression, which suggests its potential utility as a prognostic biomarker and therapeutic target for RM.
The maternal syndrome of pre-eclampsia is thought to result from endothelial cell damage caused by a circulating factor derived from the placenta. This study investigates the hypothesis that trophoblast deportation may be part of the process by which this factor enters the maternal circulation. The nature and incidence of trophoblast deportation was studied in uterine vein and peripheral blood taken from normal and pre-eclamptic women at caesarean section. Trophoblasts were enriched using immunomagnetic beads to deplete leucocytes and labelled with trophoblast-specific monoclonal antibodies. Syncytiotrophoblast, cytotrophoblast, cytotrophoblast clumps and anucleate trophoblast cells were found in uterine vein blood. Cytotrophoblast cells were found to be shed less frequently than syncytiotrophoblast and the majority were probably villous in origin. Trophoblasts were found in the uterine vein blood of normal pregnant women with higher levels in pre-eclampsia. However, trophoblasts were rarely found in the peripheral circulation. There was no correlation between trophoblast numbers and either the severity of the disease, the extent of placental pathology or the inhibitory effect of uterine and peripheral vein plasma on endothelial growth in vitro. Thus, it is speculated that increased trophoblast deportation in pre-eclampsia is secondary to the structural and functional changes occurring in the placenta, rather than directly linked with the circulating endothelial cell damaging factor in pre-eclampsia. (C) 1999 Harcourt Publishers Ltd.