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This study investigated the roles of Rho protein in epidermal growth factor (EGF)-induced trophoblast cell migration and its mechanism. Using choriocarcinoma cell lines JEG-3 and JAR and first-trimester human chorionic villus explant cultures on matrigel, we examined EGF-mediated stimulation of trophoblast migration. EGF is shown to have a dose-dependent effect on trophoblast migration. A low concentration of EGF (1 ng/ml) has a stimulatory effect on cell migration, whereas high concentrations of EGF (100 ng/ml) shows an inhibitory effect. EGF (1 ng/ml) activates RhoA and RhoC, but not RhoB, through elevated protein levels and activity. EGF-induced migration was shown to be inhibited by either cell-permeable C3 exoenzyme transferase or selective RhoA or RhoC small interfering RNAs. The inhibition was not mitigated by the addition of EGF, suggesting that RhoA and RhoC play an important role in trophoblast migration and are obligatory for EGF action. Treatment of JEG-3 and JAR cells with RhoA small interfering RNA induced F-actin cytoskeleton disruption and cell shrinkage, which is consistent with the effect of C3 exoenzyme transferase, and this action was not mitigated by EGF treatment. RhoC small interfering RNA had no apparent effect on the F-actin arrangement, suggesting that RhoA but not RhoC takes part in the EGF-induced migration through F-actin rearrangement. These results indicate that RhoA and RhoC play more important roles than RhoB in EGF-mediated migration of trophoblast cells, and RhoA but not RhoC regulates this migration through F-actin cytoskeleton reorganization.

Analysis of fetal DNA in maternal plasma has recently been introduced for non-invasive prenatal diagnosis. We have now investigated the feasibility of polymerase chain reaction (PCR)/ligase detection reaction (LDR)/capillary electrophoresis for the detection of fetal point mutations, such as the beta-thalassemia mutation, IVS2 654(C --T), in maternal plasma DNA.The sensitivity of LDR/capillary electrophoresis was examined by quantifying the mutant PCR products in the presence of a vast excess of non-mutant competitor template, a situation that mimics the detection of rare fetal mutations in the presence of excess maternal DNA. PCR/LDR/capillary electrophoresis was applied to detect the mutation, IVS2 654(C --T), in an experimental model at different sensitivity levels and from 10 maternal plasma samples.Our results demonstrated that this approach to detect a low abundance IVS2 654(C --T) mutation achieved a sensitivity of approximately 1:10,000. The approach was applied to maternal plasma DNA to detect the paternally inherited fetal IVS2 654(C --T) mutation, and the results were equivalent to those obtained by PCR/reverse dot blot of amniotic fluid cell DNA.PCR/LDR/capillary electrophoresis has a very high sensitivity that can distinguish low abundance single nucleotide differences and can detect paternally inherited fetal point mutations in maternal plasma.

Objectives To characterize the H19 gene imprinting in the placental tissues of normal pregnancy and pre-eclampsia. Methods A total of 188 subjects at 5–9 weeks, 54 at 10–12 weeks, 50 at the second trimester, 65 at term of gestation, and 30 with pre-eclampsia at the third trimester were recruited. Their DNA and RNA were extracted from the placental tissues. The H19 genotypes for individuals were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the H19 gene imprinting was identified by reverse transcription-PCR (RT-PCR) and the Rsa I-based RFLP. The levels of H19 RNA transcripts in the pre-eclampsia patients with the H19 heterozygote were measured by quantitative RT-PCR. The frequency of the H19 heterozygote, the H19 imprinting status among different trimesters of pregnancy and the clinical symptoms in the pre-eclampsia patients were analyzed. Results (1) About 40–46% of subjects at varying stages of normal pregnancy showed the H19 heterozygote, and 24 out of 87 (27.59%) heterozygous cases were at 5–9 weeks of gestation, but no single one in other stages displayed biallelic expression of the H19 gene. (2) Thirteen out of 30 pre-eclampsia patients were H19 heterozygous and six of these showed the biallelic expression of the H19 gene (loss of imprinting). (3) The pre-eclampsia patients with the biallelic expression of H19 tended to have severer hypertension although their H19 RNA transcription level was similar to that in those with monoallelic expression. Conclusions (1) The biallelic expression of the H19 gene exists in some cases at the early stage of normal pregnancy and changes into monoallelic expression near 10 weeks of gestation. The dynamic alternations in the patterns of the H19 gene imprinting may regulate the maintenance of normal pregnancy. (2) The loss of the H19 gene imprinting in the placental tissues of pre-eclampsia patients may be associated with severe hypertension, contributing to the pathogenic process of pre-eclampsia.

Increased circulating syncytiotrophoblast microparticles (STBMs) are often associated with preeclampsia (PE) but the molecular mechanisms regulating STBM shedding remain elusive. Experimental evidence has shown that actin plays a key role in STBM shedding and that Rho/ROCK is important in regulating actin rearrangement. To investigate the role of RhoB/ROCK-regulated actin arrangement in STBM shedding in PE, chorionic villous explants were prepared from placenta of patients with normotensive or PE pregnancies and BeWo cells were fused to imitate syncytiotrophoblasts. The oxygen–glucose deprivation (OGD) conditions were applied to imitate the pathophysiology of PE in vitro. The results showed that RhoB and ROCK were activated in the preeclamptic placenta, accompanied by increased actin polymerization and decreased outgrowing microvilli. In villous tissue cultures or BeWo cells, OGD activated RhoB, ROCK1 and ROCK2 and promoted STBM shedding and actin stress fibers formation. In BeWo cells, RhoB overexpression activated ROCK1 and ROCK2, leading to F-actin redistribution and STBM shedding and the OGD-induced actin polymerization and STBM shedding could be reversed by RhoB or ROCK knockdown. These results reveal that RhoB and ROCK play a key role in PE by targeting STBM shedding through actin rearrangement and that RhoB/ROCK intervention may be a potential therapeutic strategy for PE.

Objectives: It was the aim of this study to investigate the feasibility of polymerase chain reaction (PCR)/ligase detection reaction (LDR)/capillary electrophoresis for the detection of paternally inherited fetal CD17 (A→T) mutation of β-thalassemia in maternal plasma. Methods: The target DNA in maternal plasma was amplified by PCR and the mutant signal was detected by LDR. Unique LDR products were produced and separated by capillary electrophoresis. PCR/LDR/capillary electrophoresis was applied to detect CD17 (A→T) mutation in an experimental model at different sensitivity levels and from 3 maternal plasma samples. Results: The sensitivity of PCR/LDR/capillary electrophoresis for detecting low-abundance CD17 (A→T) mutation was 1:5,000 at least. The technique was applied in maternal plasma DNA for detecting paternally inherited fetal CD17 (A→T) mutation, and the results were concordant with that of PCR/reverse dot blot of amniotic fluid cell DNA. Conclusions: PCR/LDR/capillary electrophoresis has a very high sensitivity to distinguish low-abundance single nucleotide differences and probably detects paternally inherited fetal point mutations in maternal plasma.