ENO1 promotes trophoblast invasion regulated by E2F8 in recurrent miscarriage.

Recurrent miscarriage (RM) is related to the dysfunction of extravillous trophoblast cells (EVTs), but the comprehensive mechanisms remain largely unexplored. We analyzed single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing and microarray datasets obtained from Gene Expression Omnibus (GEO) database to explore the hub genes in the mechanisms of RM. We identified 1724 differentially expressed genes (DEGs) in EVTs from the RM, and they were all expressed along the trajectory of EVTs. These DEGs were associated with hypoxia and glucose metabolism. Single-cell Regulatory Network Inference and Clustering (SCENIC) analysis revealed that E2F transcription factor (E2F) 8 (E2F8) was a key transcription factor for these DEGs. And the expression of ENO1 can be positively regulated by E2F8 via RNA sequencing analysis. Subsequently, we performed immunofluorescence assay (IF), plasmid transfection, western blotting, chromatin immunoprecipitation (ChIP), real-time quantitative polymerase chain reaction (qRT-PCR), and transwell assays for validation experiments. We found that the expression of alpha-Enolase 1 (ENO1) was lower in the placentas of RM. Importantly, E2F8 can transcriptionally regulate the expression of ENO1 to promote the invasion of trophoblast cells by inhibiting secreted frizzled-related protein 1/4 (SFRP1/4) to activate Wnt signaling pathway. Our results suggest that ENO1 can promote trophoblast invasion via an E2F8-dependent manner, highlighting a potential novel target for the physiological mechanisms of RM.

Progesterone Attenuates SIRT1-Deficiency-Mediated Pre-Eclampsia.

Pre-eclampsia is a severe hypertensive disorder of pregnancy (HDP), mainly characterized by new-onset hypertension with proteinuria after 20-week gestation. Sirtuin1 (SIRT1), a class III histone deacetylase, is associated with the regulation of various pathophysiological processes, including inflammation, immune response, metabolism, and autophagy. However, the effect of SIRT1 in the pathogenesis of pre-eclampsia remains to be elucidated. In this study, we found that the expression of SIRT1 was relatively lower in the placentas and serum samples of pre-eclampsia patients. Typical pre-eclampsia-like symptoms, such as hypertension, proteinuria, fetal growth restriction, kidney injury, and a narrow placental labyrinth layer, were observed in SIRT1 knockdown (SIRT1+/-) mice. Of note, these performances could be improved after the intraperitoneal injection of SIRT1 agonist SRT2104. More importantly, we found that the efficacy of progesterone on attenuating symptoms of PE was profoundly better than that of metformin in SIRT1+/- mice. In addition, our results suggested that progesterone can promote the invasion and inhibit the apoptosis of trophoblasts. These data suggest that SIRT1 plays an important role in pre-eclampsia and that progesterone alleviates pre-eclampsia-like symptoms mediated by SIRT1 deficiency.

Procyanidin B2 ameliorates endothelial dysfunction and impaired angiogenesis via the Nrf2/PPARγ/sFlt-1 axis in preeclampsia.

Preeclampsia is a severe complication of pregnancy characterised by variable degrees of placental malperfusion. A growing body of evidence indicates that soluble endoglin and soluble fms-like tyrosine kinase-1 (sFlt-1) play important pathophysiological roles in preeclampsia, causing endothelial dysfunction, hypertension, and multiorgan injury. A drug that is safe in pregnancy and inhibits placental sFlt-1 and soluble endoglin secretion would be an attractive treatment strategy for preeclampsia. Procyanidin B2, a bioactive food compound, has been reported to exert multiple beneficial functions. Placental explant cultures in vitro are useful for studying tissue functions including release of secretory components, pharmacology, toxicology, and disease processes. The reduced uterine perfusion pressure (RUPP) rat model has been widely used as a model of preeclampsia. We aimed to investigate the effect of procyanidin B2 on preeclampsia via using placental explant cultures and RUPP rat model. In this study, we demonstrated that procyanidin B2 reduced soluble endoglin and sFlt-1 secretion from human umbilical vein endothelial cells (HUVECs), primary trophoblasts, and placental explants from preeclamptic pregnancies. Moreover, procyanidin B2 alleviated endothelial dysfunction and impaired angiogenesis induced by sFlt-1, including increasing the migration, invasion and angiogenesis of endothelial cells and decreasing the expression of vascular cell adhesion molecule-1 (VCAM-1) and leukocyte adhesion on HUVECs. In addition, procyanidin B2 promoted nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear accumulation and induced peroxisome proliferator-activated receptor γ (PPARγ) expression in primary placental tissues and endothelial cells. Importantly, Nrf2 specifically binds to the PPARγ promoter region (-1227/-1217) and enhances its transcriptional activity. Procyanidin B2 inhibits sFlt-1 secretion via the Nrf2/PPARγ axis. In the RUPP rat model of preeclampsia, procyanidin B2 attenuated RUPP-induced maternal angiogenic imbalance, hypertension and improved placental and foetal weight. Taken together, our results demonstrate that procyanidin B2 inhibits sFlt-1 secretion and ameliorates endothelial dysfunction and impaired angiogenesis via the Nrf2/PPARγ axis in preeclampsia. Procyanidin B2 may be a novel therapeutic agent for treatment of preeclampsia.

Conservative management versus cesarean hysterectomy in patients with placenta increta or percreta.

OBJECTIVE: To compare conservative management and cesarean hysterectomy in patients with placenta increta or percreta. MATERIALS AND METHODS: In this multicenter retrospective study, we recorded data on 2219 patients with placenta increta or percreta from 20 tertiary care centers in China from 1 January 2011 to 31 December 2015. Propensity score analysis was used to control for baseline characteristics. We divided patients into conservative management (C) and hysterectomy (H) groups. The primary outcome was operative/postoperative maternal morbidity; secondary outcomes were maternal-neonatal outcomes. RESULTS: In total, 17.9% (398/2219) of patients had placenta increta and percreta; 82.1% (1821/2219) of the patients were in group C. After propensity score matching, 140 pairs of patients from the two groups underwent one-to-one matching. Group C showed less average blood loss within 24 h of surgery (1518 ± 1275 vs. 4309 ± 2550 ml in group H, p<.001). There were more patients with blood loss >1000 ml in group H than in group C (93.6% [131/140] vs. 61.4% [86/140], p<.001). More patients received blood transfusions in group H than in group C (p=.014). There was no significant difference between the groups in terms of bladder injury, postoperative anemia, fever, and disseminated intravascular coagulation. Neonatal outcomes in the two groups were similar. CONCLUSION: Either conservative management or hysterectomy should be considered after thorough evaluation and detailed discussion of risks and benefits. A balance between bleeding control and fertility can be achieved.

Pro-inflammatory cytokine-induced microRNA-212-3p expression promotes myocyte contraction via methyl-CpG-binding protein 2: a novel mechanism for infection-related preterm parturition.

Preterm labour is a common pregnancy complication contributing to major maternal and fetal morbidity and mortality. We have found microRNA (miR)-212-3p, a potential infection-associated molecule, was significantly over-expressed during human preterm labour. However, the mechanism remains unknown. In this study, we have adopted a lipopolysaccharide (LPS)-induced Institute of Cancer Research murine preterm model to examine the role of miR-212-3p in the infection-induced preterm labour. Myometrial miR-212-3p expression was increased by nearly 4-fold in the term labour group (P = 0.10) and 12-fold (P = 0.03) in the LPS-induced preterm labour group compared with the non-labour group. In vitro cellular experiments confirmed that a series of pro-inflammatory cytokines, including interleukin (IL)1B (P = 0.02) and IL-6 (P = 0.01), rather than LPS (P = 0.08) itself could significantly upregulate miR-212-3p expression in human myometrial smooth muscle cells. Methyl-CpG-binding protein 2 (MeCP2), as a target gene of miR-212-3p confirmed by our dual luciferase assay, influenced myocyte contractility and connexin 43 expression which is an important contraction-associated protein. Therefore, we conclude that miR-212-3p may be involved in infection-induced preterm labour through MeCP2 and it is a promoting molecule and novel target for the diagnosis and treatment of preterm labour in the future.

Identification of key microRNAs and genes in preeclampsia by bioinformatics analysis.

Preeclampsia is a leading cause of perinatal maternal-foetal mortality and morbidity. The aim of this study is to identify the key microRNAs and genes in preeclampsia and uncover their potential functions. We downloaded the miRNA expression profile of GSE84260 and the gene expression profile of GSE73374 from the Gene Expression Omnibus database. Differentially expressed miRNAs and genes were identified and compared to miRNA-target information from MiRWalk 2.0, and a total of 65 differentially expressed miRNAs (DEMIs), including 32 up-regulated miRNAs and 33 down-regulated miRNAs, and 91 differentially expressed genes (DEGs), including 83 up-regulated genes and 8 down-regulated genes, were identified. The pathway enrichment analyses of the DEMIs showed that the up-regulated DEMIs were enriched in the Hippo signalling pathway and MAPK signalling pathway, and the down-regulated DEMIs were enriched in HTLV-I infection and miRNAs in cancers. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses of the DEGs were performed using Multifaceted Analysis Tool for Human Transcriptome. The up-regulated DEGs were enriched in biological processes (BPs), including the response to cAMP, response to hydrogen peroxide and cell-cell adhesion mediated by integrin; no enrichment of down-regulated DEGs was identified. KEGG analysis showed that the up-regulated DEGs were enriched in the Hippo signalling pathway and pathways in cancer. A PPI network of the DEGs was constructed by using Cytoscape software, and FOS, STAT1, MMP14, ITGB1, VCAN, DUSP1, LDHA, MCL1, MET, and ZFP36 were identified as the hub genes. The current study illustrates a characteristic microRNA profile and gene profile in preeclampsia, which may contribute to the interpretation of the progression of preeclampsia and provide novel biomarkers and therapeutic targets for preeclampsia.

Identification and functional analysis of microRNA in myometrium tissue from spontaneous preterm labor.

Spontaneous preterm labor is an important complication in perinatology characterized by early onset myometrium contractions leading to labor at preterm. However, the exact mechanism that maintain uterine quiescence and promote increased uterine contractility during labor were incompletely defined. MicroRNAs is a class of short non-coding RNAs that regulate gene expression at the post-transcriptional level by binding the 3' untranslated region of target mRNAs and play an important role in biological process and cellular functions. We hypothesized we could find differentially expressed microRNAs in the myometrium of women in spontaneous preterm labor. Thus, a microarray analysis of miRNAs of preterm myometrium was performed. 18 out of the 2006 detected microRNAs were found to be significantly dysregulated in myometrium in labor verse not in labor at preterm. Biological validation by quantitative real-time polymerase chain reaction confirms us a consistence rate of 83.3% (5 out of 6) with microarray analysis. The target genes for validated microRNAs were predicted by three algorithms (PicTar, TargetScan, and miRanda). Most of the potential targets of the miRNAs were relevant to positive regulation of cardiac muscle hypertrophy, reduction of cytosolic calcium ion concentration and relaxation of cardiac muscle as well as prostate cancer, adherents junction, regulation of actin cytoskeleton and regulation and other factor-regulated calcium reabsorption. Our result illustrates a characteristic microRNA profile in myometrium tissues and provides a new understanding of the process involved in spontaneous preterm labor.

Natural history and pregnancy outcome in patients with placental chorioangioma.

PURPOSE: To evaluate the natural history and outcome of pregnancies in patients with placental chorioangioma. METHODS: A total of 16 placentas with a histologic diagnosis of chorioangioma were identified, and the natural history and outcome of pregnancy were evaluated. This study was approved by the Institutional Ethics Committees of our unit, and written informed consent was obtained from all study participants. RESULTS: Thirteen of the 16 cases were associated with a wide variety of fetal complications. Two-thirds of the cases developed complications that either required elective delivery because of fetal distress (n = 4), fetal heart failure (n = 1), oligohydramnion (n = 1), and premature labor of dichorionic twins (n = 1) or resulted in intrauterine fetal death and termination of pregnancy (n = 2). CONCLUSIONS: Placental chorioangioma was associated with the development of polyhydramnios, fetal growth restriction, and fetal distress in a significant number of cases. The size, vascularity, and location of the chorioangioma may be three independent factors of maternal and fetal complications. Any of these three factors can influence the outcome of pregnancy. Close antenatal examination should be routinely practiced to allow the timely diagnosis of early fetal heart failure.

Cyclosporin A enhances the ability of trophoblasts to displace the activated human umbilical vein endothelial cell monolayers.

Transformation of the spiral arteries including the displacement of vascular endothelial cells by extravillous trophoblasts is an essential prerequisite to normal placentation. However, the activated endothelial cells resist the invasion of trophoblasts, which contributes to the pathologies of some pregnant disorders. Our previous studies have demonstrated that Cyclosporin A (CsA) promotes the migration and invasion of human first-trimester trophoblasts. In the present study, we further investigated whether CsA could promote the ability of trophoblasts to displace the activated human umbilical vein endothelial cell (HUVEC) monolayers and the possible molecular mechanisms. Human choriocarcinoma Jar cells were used as a model of invasive trophoblasts. CsA pretreated JAR cells (red) were added to HUVEC monolayers (green) activated with either necrotic JAR cells or tumor necrosis factor alpha (TNFα). The ability of JAR cells to displace HUVECs from the monolayers was examined by confocal microscopy. The effects of CsA on Titin and E-cadherin expression, matrix metalloproteinases (MMPs) activity and CXCL12 secretion of JAR cells were evaluated by western blot, gelatin zymography and enzyme-linked immunosorbent assay (ELISA), respectively. We found that CsA pretreatment increased the ability of JAR cells to displace activated HUVECs from the monolayers. However, the displacement was reduced by untreated JAR cells. Moreover, CsA pretreatment up-regulated Titin expression, down-regulated E-cadherin expression, improved MMP2 and MMP9 activity, and increased the CXCL12 secretion in JAR cells. These results indicate that CsA may improve the trophoblast invasion to activated HUVEC monolayers through different downstream targets, and ultimately, improve the transformation and remodeling of spiral arteries.

[Effects on human leukocyte antigen-G on proliferation and invasion of JEG-3 cell line].

OBJECTIVE: To investigate the effect of human leukocyte antigen-G (HLA-G) on the growth and invasion of JEG-3 cell line and the role of HLA-G in the onset and development of pre-eclampsia. METHODS: The experiment was composed of three groups: groups of transfection, negative control and blank control, which corresponded to groups of HLA-G siRNA transfection, negative siRNA transfection and no transfection. HLA-G overexpressed choriocarcinoma cell line JEG-3 was used. The role of HLA-G in JEG-3 cell monolayer was examined by RNA interference technology using HLA-G specific small interfering RNA (siRNA). Expression of HLA-G was detected by reverse transcriptase-polymerase chain reaction and western blot analysis. Changes of cell cycle, apoptosis, proliferation and invasion were respectively detected by methyl thiazolyl tetrazolium (MTT), flow cytometry assay and transwell test. RESULTS: (1) The mRNA and protein levels of HLA-G control group and blank control group were 0.0013 +/- 0.0014, 0.0163 +/- 0.0007 and 0.1923 +/- 0.0384, 0.2184 +/- 0.0153, respectively, which were both significantly different (P<0.05); the number of negative transfection group was 0.1606 +/- 0.0133 and 0.2020 +/- 0.0132, which had no significant difference compared with blank control group (P>0.05). (2) The integral absorbance (IA) values of the HLA-G transfection group and blank control group were 0.44 +/- 0.04 and 0.75 +/- 0.13 respectively, which was significantly different (P<0.01); the IA value of negative control group was 0.69 +/- 0.10, which was not significantly different compared with blank group (P>0.05). (3) The ratios of G2/M and S phase cells in transfection group were (10.9 +/- 2.2)% and (58.6 +/- 0.8)% respectively, significantly different compared with the blank control group [(15.4 +/- 1.9)% and (52.9 +/- 2.3)% respectively; P<0.01]. (4) The ratio of early apoptosis cells in transfection group [(14.5 +/- 2.7)%] was significantly increased compared with negative [(5.3 +/- 1.1)%] and blank control group [(4.7 +/- 0.6)% ; P<0.01]. (5) The invasion number of transfection group and blank control group were 121+/- 12 and 452 +/- 17 respectively, with a significant difference between them (P<0.01). CONCLUSION: HLA-G is probably involved in the onset of preeclampsia by regulating proliferation and invasion of trophoblast.