Epigenetic Dysregulation of Trophoblastic Gene Expression in Gestational Trophoblastic Disease

Gestational trophoblastic diseases (GTDs) have not been investigated for their epigenetic marks and consequent transcriptomic changes. Here, we analyzed genome-wide DNA methylation and transcriptome data to reveal the epigenetic basis of disease pathways that may lead to benign or malignant GTDs. RNA-Seq, mRNA microarray, and Human Methylation 450 BeadChip data from complete moles and choriocarcinoma cells were bioinformatically analyzed. Paraffin-embedded tissues from complete moles and control placentas were used for tissue microarray construction, DNMT3B immunostaining and immunoscoring. We found that DNA methylation increases with disease severity in GTDs. Differentially expressed genes are mainly upregulated in moles while predominantly downregulated in choriocarcinoma. DNA methylation principally influences the gene expression of villous trophoblast differentiation-related or predominantly placenta-expressed genes in moles and choriocarcinoma cells. Affected genes in these subsets shared focal adhesion and actin cytoskeleton pathways in moles and choriocarcinoma. In moles, cell cycle and differentiation regulatory pathways, essential for trophoblast/placental development, were enriched. In choriocarcinoma cells, hormone biosynthetic, extracellular matrix-related, hypoxic gene regulatory, and differentiation-related signaling pathways were enriched. In moles, we found slight upregulation of DNMT3B protein, a developmentally important de novo DNA methylase, which is strongly overexpressed in choriocarcinoma cells that may partly be responsible for the large DNA methylation differences. Our findings provide new insights into the shared and disparate molecular pathways of disease in GTDs and may help in designing new diagnostic and therapeutic tools.

Regulation of Epigenetic Modifications in the Placenta During Preeclampsia: PPARγ Influences H3K4me3 and H3K9ac in Extravillous Trophoblast Cells

The aim of this study was to analyze the expression of peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RxRα), a binding heterodimer playing a pivotal role in the successful trophoblast invasion, in the placental tissue of preeclamptic patients. Furthermore, we aimed to characterize a possible interaction between PPARγ and H3K4me3 (trimethylated lysine 4 of the histone H3), respectively H3K9ac (acetylated lysine 9 of the histone H3), to illuminate the role of histone modifications in a defective trophoblast invasion in preeclampsia (PE). Therefore, the expression of PPARγ and RxRα was analyzed in 26 PE and 25 control placentas by immunohistochemical peroxidase staining, as well as the co-expression with H3K4me3 and H3K9ac by double immunofluorescence staining. Further, the effect of a specific PPARγ-agonist (Ciglitazone) and PPARγ-antagonist (T0070907) on the histone modifications H3K9ac and H3K4me3 was analyzed in vitro. In PE placentas, we found a reduced expression of PPARγ and RxRα and a reduced co-expression with H3K4me3 and H3K9ac in the extravillous trophoblast (EVT). Furthermore, with the PPARγ-antagonist treated human villous trophoblast (HVT) cells and primary isolated EVT cells showed higher levels of the histone modification proteins whereas treatment with the PPARγ-agonist reduced respective histone modifications. Our results show that the stimulation of PPARγ-activity leads to a reduction of H3K4me3 and H3K9ac in trophoblast cells, but paradoxically decreases the nuclear PPARγ expression. As the importance of PPARγ, being involved in a successful trophoblast invasion has already been investigated, our results reveal a pathophysiologic connection between PPARγ and the epigenetic modulation via H3K4me3 and H3K9ac in PE.

AN UNUSUAL CASE OF POSTPARTUM CHORIOCARCINOMA WITH VAGINAL METASTASIS AND UTERINE PERFORATION

Gestational trophoblastic disease is a heterogenous group of interrelated lesions that arise from abnormal proliferation of placental 1 trophoblasts . Choriocarcinoma differs from any type of villous trophoblast and is the most aggressive GTN and is highly chemosensitive. When a patient reports with irregular bleeding during her postpartum or postabortion, pregnancy related causes should be ruled out. We describe a patient who presented to us after a term pregnancy and was diagnosed to have stage III choriocarcinoma.

Comparison of matrix metalloproteinase-9 and E-cadherin expression in early- and late-onset preeclampsia

BACKGROUNDPreeclampsia (PE) is one of the most common pregnancy complications worldwide. Turnover of villous trophoblast is affected by impaired placental perfusion in preeclampsia. Among the various factors that influence pro and antiangiogenic factors in trophoblast invasion of PE are E-cadherin and matrix metalloproteinase-9 (MMP-9). The current classification scheme differentiates PE into two variants early-onset (EO) and late-onset (LO) PE. The aim of this study was to compare MMP-9 and E-cadherin expression between early- (EO) and late-onset (LO) PE. METHODSThis study used a cross-sectional design involving 26 women with gestational age <34 weeks (EO) and 38 women with gestational age ≥34 weeks (LO) from PE patients. Placentas born to preeclamptic mothers were taken in the form of small pieces of the maternal side to measure the levels of MMP-9 and E-cadherin by the ELISA method. Statistical analysis was assessed using the Mann Whitney and independent t-test with a significant p value <0.05. RESULTSSemiquantitative proteinuria levels were significantly higher in EO-PE group compared to LO-PE group (p=0.000). Mean E-cadherin levels were significant lower in the EO-PE group (125.94 ± 54.22 pg/mg) compared to LO-PE group (157.95 ± 54.12 pg/mg) (p=0.024). However, there was no significance difference in median MMP-9 levels between EO-PE group and LO-PE group (p=0.376). CONCLUSIONThis study demonstrate that E-cadherin had lower levels in preeclampsia patients who gave birth <34 weeks. This study indicated that lower levels of e-cadherin can lead to early delivery in preeclampsia patients.

MicroRNA-3935 promotes human trophoblast cell epithelial-mesenchymal transition through tumor necrosis factor receptor-associated factor 6/regulator of G protein signaling 2 axis

Background Insufficient migration and invasion during trophoblast epithelial-mesenchymal transition (EMT) results in the occurrence and development of preeclampsia (PE), and our previous study has screened 52 miRNAs, whose expression levels are altered in the placental samples from PE patients, compared with the normal group. Among those, miR-3935 is one of the miRNAs being most significantly down-regulated, indicating its involvement in PE. However, the exact effect and molecular mechanisms remain unknown. Methods In the present study, we investigate the roles and underlying mechanisms of miR-3935 in trophoblast EMT by use of the human extra-villous trophoblast cell line HTR-8/SVneo as well as human placental tissues and maternal blood samples obtained from 15 women with normal pregnancies and 15 women with PE. Experimental methods include transfection, quantitative reverse transcription-PCR (qRT-PCR), western blot, immunofluorescence staining, dual-luciferase assays, in vitro invasion and migration assays, RNA-Seq analysis, bisulfite sequencing and immunohistochemistry staining. Results MiR-3935 expression is significantly decreased in both placentas and peripheral blood specimens of PE, and functionally, miR-3935 promotes EMT of trophoblast cells. Mechanistically, TRAF6 is identified to be a direct target of miR-3935 and TRAF6 exerts its negative effect on EMT of trophoblast cells by inhibition of RGS2, which down-regulates the methylation status of promoter of CDH1 gene that encodes E-Cadherin protein through induction of ALKBH1, resulting in increase of E-Cadherin and subsequently insufficient trophoblast EMT. Conclusions Together these results uncover a hitherto uncharacterized role of miR-3935/TRAF6/RGS2 axis in the function of human trophoblasts, which may pinpoint the molecular pathogenesis of PE and may be a prognostic biomarker and therapeutic target for such obstetrical diseases as PE.

Does LIN28B gene dysregulation make women more likely to abort?

Background: LIN28B plays an important role in early embryonic development, but its role in villous trophoblast implantation and differentiation remains unknown. To verify the role of LIN28B in trophoblastic villous tissue and cells from women with URSA(unexplained recurrent spontaneous abortion)and artificial termination of pregnancy (negative control, NC). Methods:The Lin28b gene and its protein expression level were detected with real-time quantitative PCR, Western immunoblotting analysis, and immunocytochemistry. The gene was also overexpressed in chorionic villous cell lines (HTR-8/SVneo and BeWo) to examine its effect on trophoblast function. Results: The expression of LIN28B mRNA and protein of URSA villi was lower than that in the NC group. At the cellular level, overexpression of LIN28B enhanced cellular migration, and invasion, and inhibited apoptosis. LIN28B may inhibit apoptosis by promoting Akt phosphorylation and by inhibiting Bad phosphorylation and Bcl-2 expression. In addition, LIN28B inhibited cell fusion and reduced cellular syncytia. Conclusions: LIN28B can inhibit cell invasion and migration in vitro, and promote apoptosis and fusion. The low expression of LIN28B in URSA villous trophoblast cells may be one of the causes of abortion. The role of LIN28B in villous trophoblasts needs further study.

Reduced Placental CD24 in Preterm Preeclampsia Is an Indicator for a Failure of Immune Tolerance

Introduction: CD24 is a mucin-like glycoprotein expressed at the surface of hematopoietic and tumor cells and was recently shown to be expressed in the first trimester placenta. As it was postulated as an immune suppressor, CD24 may contribute to maternal immune tolerance to the growing fetus. Preeclampsia (PE), a major pregnancy complication, is linked to reduced immune tolerance. Here, we explored the expression of CD24 in PE placenta in preterm and term cases. Methods: Placentas were derived from first and early second trimester social terminations (N = 43), and third trimester normal term delivery (N = 67), preterm PE (N = 18), and preterm delivery (PTD) (N = 6). CD24 expression was determined by quantitative polymerase chain reaction (qPCR) and Western blotting. A smaller cohort included 3–5 subjects each of term and early PE, and term and preterm delivery controls analyzed by immunohistochemistry. Results: A higher expression (2.27-fold) of CD24 mRNA was determined in the normal term delivery compared to first and early second trimester cases. The mRNA of preterm PE cases was only higher by 1.31-fold compared to first and early second trimester, while in the age-matched PTD group had a fold increase of 5.72, four times higher compared to preterm PE. The delta cycle threshold (ΔCt) of CD24 mRNA expression in the preterm PE group was inversely correlated with gestational age (r = 0.737) and fetal size (r = 0.623), while correlation of any other group with these parameters was negligible. Western blot analysis revealed that the presence of CD24 protein in placental lysate of preterm PE was significantly reduced compared to term delivery controls (p = 0.026). In immunohistochemistry, there was a reduction of CD24 staining in villous trophoblast in preterm PE cases compared to gestational age-matched PTD cases (p = 0.042). Staining of PE cases at term was approximately twice higher compared to preterm PE cases (p = 0.025) but not different from normal term delivery controls. Conclusion: While higher CD24 mRNA expression levels were determined for normal term delivery compared to earlier pregnancy stages, this expression level was found to be lower in preterm PE cases, and could be said to be linked to reduced immune tolerance in preeclampsia.