Villus Tissue Research Articles

Dysregulation of Histone Deacetylases Inhibits Trophoblast Growth during Early Placental Development Partially through TFEB-Dependent Autophagy-Lysosomal Pathway.

Dysregulated biological behaviors of trophoblast cells can result in recurrent spontaneous abortion (RSA)-whose underlying etiology still remains insufficient. Autophagy, a conserved intracellular physiological process, is precisely monitored throughout whole pregnancy. Although the exact mechanism or role remains elusive, epigenetic modification has emerged as an important process. Herein, we found that a proportion of RSA patients exhibited higher levels of autophagy in villus tissues compared to controls, accompanied with impaired histone deacetylase (HDAC) expression. The purpose of this study is to explore the connection between HDACs and autophagy in the pathological course of RSA. Mechanistically, using human trophoblast cell models, treatment with HDAC inhibitor (HDACI)-trichostatin A (TSA) can induce autophagy by promoting nuclear translocation and transcriptional activity of the central autophagic regulator transcription factor EB (TFEB). Specifically, overactivated autophagy is involved in the TSA-driven growth inhibition of trophoblast, which can be partially reversed by the autophagy inhibitor chloroquine (CQ) or RNA interference of TFEB. In summary, our results reveal that abnormal acetylation and autophagy levels during early gestation may be associated with RSA and suggest the potential novel molecular target TFEB for RSA treatment.

Four Decades of Carrier Detection and Prenatal Diagnosis in Hemophilia A: Historical Overview, State of the Art and Future Directions.

Hemophilia A (HA), a rare recessive X-linked bleeding disorder, is caused by either deficiency or dysfunction of coagulation factor VIII (FVIII) resulting from deleterious mutations in the F8 gene encoding FVIII. Over the last 4 decades, the methods aimed at determining the HA carrier status in female relatives of HA patients have evolved from phenotypic studies based on coagulation tests providing merely probabilistic results, via genetic linkage studies based on polymorphic markers providing more accurate results, to next generation sequencing studies enabling highly precise identification of the causative F8 mutation. In parallel, the options for prenatal diagnosis of HA have progressed from examination of FVIII levels in fetal blood samples at weeks 20-22 of pregnancy to genetic analysis of fetal DNA extracted from chorionic villus tissue at weeks 11-14 of pregnancy. In some countries, in vitro fertilization (IVF) combined with preimplantation genetic diagnosis (PGD) has gradually become the procedure of choice for HA carriers who wish to prevent further transmission of HA without the need to undergo termination of pregnancies diagnosed with affected fetuses. In rare cases, genetic analysis of a HA carrier might be complicated by skewed X chromosome inactivation (XCI) of her non-hemophilic X chromosome, thus leading to the phenotypic manifestation of moderate to severe HA. Such skewed XCI may be associated with deleterious mutations in X-linked genes located on the non-hemophilic X chromosome, which should be considered in the process of genetic counseling and PGD planning for the symptomatic HA carrier. Therefore, whole exome sequencing, combined with X-chromosome targeted bioinformatic analysis, is highly recommended for symptomatic HA carriers diagnosed with skewed XCI in order to identify additional deleterious mutations potentially involved in XCI skewing. Identification of such mutations, which may profoundly impact the reproductive choices of HA carriers with skewed XCI, is extremely important.

Alpha-enolase 1 knockdown facilitates the proliferation and invasion of villous trophoblasts by upregulating COX-2.

Enolase 1 (ENO1) is a metabolic enzyme which participates in pyruvate synthesis and ATP production in cells. Previously, differential expression of ENO1 was discovered in villous tissues between recurrent miscarriage and induced abortion. This study was designed to explore whether ENO1 influences the proliferation and invasion of villous trophoblasts and the related molecular mechanisms. First, ENO1 expression in placental villus tissues collected from recurrent miscarriage (RM) patients and women for induced abortion as well as in trophoblast-derived cell lines was detected by RT-qPCR and western blotting. ENO1 localization and expression in villus tissues were further confirmed through immunohistochemistry staining. Then, the effects of ENO1 downregulation on trophoblast Bewo cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) process were evaluated by CCK-8 assay, transwell assay, and western blotting. As for the regulatory mechanism of ENO1, the expression of COX-2, c-Myc and cyclin D1 in Bewo cells after ENO1 knockdown was finally evaluated by RT-qPCR and western blotting. ENO1 was mainly localized in the cytoplasm, with very small amounts in the nucleus of trophoblast cells. ENO1 expression in the villi tissues of RM patients was significantly increased, when compared with the villous tissues of healthy controls. Furthermore, Bewo cells, a trophoblast cell line with relatively higher expression of ENO1, was used to downregulate the ENO1 expression by ENO1-siRNA transfection. ENO1 knockdown significantly facilitated Bewo cell growth, EMT process, migration, and invasion. ENO1 silencing markedly elevated COX-2, c-Myc, and cyclin D1 expression. ENO1 may participate in the development of RM via suppressing the growth and invasion of villous trophoblasts via reducing the expression of COX-2, c-Myc, and cyclin D1.

Identification of MiR-223 Associated with Diagnosis in Ectopic Pregnancy.

In this study, we conducted an integrated study of the diagnostic value of MiR-223 in ectopic pregnancy (EP). We used GSE44731 downloaded from GEO and GEO2R to identify differentially expressed miRNA. The hub genes corresponding to the differential miRNA were then identified by using the Xiantao academic tool, GO (Gene Ontology), and KEGG (Kyoto Encyclopedia of Genes and Genomes). Afterward, we used the miEAA database to perform gene set enrichment analysis (GSEA) of differential miRNA, and used Xiantao academic tools again to conduct the ceRNA network based on the target genes. Protein-protein interaction (PPI) network construction and lncRNA of hub miRNA target genes were then predicted by the starbase database. For validation, the villus tissue from intrauterine pregnancy and tubal pregnancy was collected and assayed by qPCR. In total 19 differentially expressed miRNAs were screened out, of which MiR-223 had a relatively clear diagnostic significance. Hub genes were enriched and analyzed by GO, KEGG, and GSEA, and the results showed that regulation of NF-κB and other signaling pathways are primarily enriched in ectopic pregnancy. We also obtained 215 key genes from PPI analysis. Our ceRNA analysis indicated that LRRC75A-AS1 and PITPNA-AS1 were associated with MiR-223, and the expression of MiR-223 in qPCR was significantly high in tubal pregnancy group. We found that MiR-223 can be used in the diagnosis of EP. Our findings provide valuable information and direction for future research into novel targets for EP diagnosis.

Association between HIF-1α, BNIP3, and autophagy in the chorionic villi of missed abortion.

To investigate the expression of autophagy mediated by the hypoxia-inducible factor 1α (HIF-1α)/BNIP3 signaling pathway in villus tissues of missed abortion and HTR-8/SVneo cells and to elucidate the association of HIF-1α and BNIP3 in autophagy of missed abortion. Villus tissues from 30 healthy women with induced abortion and 35 patients with missed abortion were collected, and HTR-8/SVneo cells were cultured under hypoxia and transfected with HIF-1α-siRNA. Real-time polymerase chain reaction was utilized to measure the mRNA levels of HIF-1α and BNIP3; Western blotting was performed to determine the protein levels of HIF-1α, BNIP3, LC3 II/I, and Beclin 1 in villus tissues and HTR-8/SVneo cells. Cellular invasion activity was detected by transwell matrigel assay. The level of autophagy was confirmed by transmission electron microscopy of autophagosome formation. The mRNA levels of HIF-1α and BNIP3 were significantly lower in the missed abortion villi than in the induced abortion samples. The protein levels of HIF-1α, BNIP3, Beclin 1, and LC3II/I were significantly decreased in villus tissues from missed abortion, and autophagosomes were significantly decreased in villus tissues from missed abortion. Under hypoxia, the mRNA expression of HIF-1α and BNIP3 was inhibited after silencing HIF-1α by RNAi, while the protein expression of HIF-1α, BNIP3, Beclin1, and LC3II/I was significantly downregulated. The number of invading cells was significantly decreased, and autophagosomes were significantly decreased after silencing HIF-1α by RNAi in HTR-8/SVneo cells. Autophagy mediated by the HIF-1α/BNIP3 signaling pathway in villous trophoblast cells may be associated with the progression and development of missed abortion.

Effects of the SPI/lncRNA NEAT1 Axis on Functions of Trophoblast and Decidual Cells in Patients with Recurrent Miscarriage

Recurrent miscarriage (RM) is a frustrating and complex pregnancy disorder and long noncoding RNAs (lncRNAs) modulate susceptibility to RM. This study expounded on the role of specificity protein 1 (SP1) in functions of chorionic trophoblast and decidual cells via regulating lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1). Chorionic villus tissues and decidual tissues of RM patients and normal pregnant women were collected. Real-time quantitative polymerase chain reaction and Western blotting revealed that SP1 and NEAT1 were downregulated in trophoblast and decidual tissues of RM patients, and the Pearson correlation analysis detected that they were positively correlated in expression level. Chorionic trophoblast and decidual cells of RM patients were isolated and intervened by vectors over-expressing SP1 or NEAT1 siRNAs. Thereafter, the cell counting kit-8, Transwell, flow cytometry assays detected that SP1 overexpression accelerated trophoblast cell proliferation, invasion, and migration, meanwhile, enhancing decidual cell proliferation while repressed apoptosis. Next, the dual-luciferase and Chromatin immunoprecipitation assays showed that SP1 bound to the NEAT1 promoter region and further activated NEAT1 transcription. Silencing NEAT1 reversed the efforts of SP1 overexpression on the functions of trophoblast and decidual cells. Overall, SP1 activated NEAT1 transcription, accelerating trophoblast cell proliferation, invasion, and migration and mitigating decidual cell apoptosis.

RGS2 promotes estradiol biosynthesis by trophoblasts during human pregnancy

Production of estradiol (E2) by the placenta during human pregnancy ensures successful maintenance of placental development and fetal growth by stimulating trophoblast proliferation and the differentiation of cytotrophoblasts into syncytiotrophoblasts. Decreased levels of E2 are closely associated with obstetrical diseases such as preeclampsia (PE) in the clinic. However, the mechanisms underlying the inhibition of placental E2 biosynthesis remain poorly understood. Here, we report that regulator of G-protein signaling 2 (RGS2) affects E2 levels by regulating aromatase, a rate-limiting enzyme for E2 biosynthesis, by using human trophoblast-derived JEG-3 cells and human placental villus tissues. RGS2 enhanced the protein degradation of the transcription factor heart and neural crest derivatives expressed 1 (HAND1) by suppressing ubiquitin-specific protease 14 (USP14)-mediated deubiquitination of HAND1, resulting in the restoration of HAND1-induced trans-inactivation of the aromatase gene and subsequent increases in E2 levels. However, aromatase bound to RGS2 and repressed RGS2 GTPase activating protein (GAP) activity. Moreover, we observed a positive correlation between RGS2 and aromatase expression in clinical normal and preeclamptic placental tissues. Our results uncover a hitherto uncharacterized role of the RGS2-aromatase axis in the regulation of E2 production by human placental trophoblasts, which may pinpoint the molecular pathogenesis and highlight potential biomarkers for related obstetrical diseases.

Comparison of trace element concentrations in paired formalin-fixed paraffin-embedded and frozen human placentae

IntroductionThere is increasing interest in measuring metals concentrations in human placentas to better understand physiology, disease, and toxic and diagnostic exposures. For these purposes, formalin-fixed paraffin embedded (FFPE) tissues obtained at clinical pathology examination represent a valuable potential store of well-characterized tissues for analysis. However, the limited data that exist comparing metal concentrations in FFPE tissue to recently collected frozen tissues paints a confusing picture, and there is no published data directly comparing frozen and FFPE placental villus tissues. MethodsPaired samples of fresh frozen and FFPE tissue from 22 rapidly processed human singleton placentae were weighed and digested using standard clean laboratory procedures and subsequently analyzed for a suite of 13 metals using a PerkinElmer DRC II ICP-MS. The analytical results were compared using either a paired t-test or a sign test depending on data normality. ResultsConcentrations of metals (aluminum (Al), arsenic (As), barium (Ba), cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), gadolinium (Gd), mercury (Hg), manganese (Mn), lead (Pb), strontium (Sr), and zinc (Zn)) measured in both types of tissue preparations (frozen and FFPE) displayed a consistent range with other studies and did not display significantly different values from each of the paired specimens for any of the 13 specific metals analyzed. DiscussionWithin placentae, metals concentrations of measured trace, toxic and diagnostic elements (Al, As, Ba, Cd, Cr, Cu, Fe, Gd, Hg, Mn, Pb, Sr, and Zn) are consistent between FFPE and fresh placental villus tissue, without indications of systematic element loss or bias. FFPE from archived pathology specimens may offer an important and convenient alternative for measuring trace metals in human frozen placental tissues.

Decreased B7-H3 promotes unexplained recurrent miscarriage via RhoA/ROCK2 signaling pathway and regulates the secretion of decidual NK cells†.

The cause for at least 50% of recurrent miscarriages is unclear, which is defined as unexplained recurrent miscarriages. The B7-H1 (PD-L1), a molecule of the B7 family, promotes tumor development by modulating immune evasion, and recent researchers have also attached importance to the role of B7-H3, another molecule of B7 family, in tumor. Based on the similarity between growth and immune response in tumors and pregnancy, we first explored the role of B7-H3 in unexplained recurrent miscarriages. We found reduced levels of B7-H3 in the villus tissue of unexplained recurrent miscarriage patients, and it was mainly expressed on the cell membrane of extravillous trophoblasts. Further, the HTR-8/SVneo and JEG-3 cells were selected to explore the role of B7-H3 in proliferation, apoptosis, tube formation, migration, and invasion. We found that B7-H3 regulated trophoblast migration and invasion via RhoA/ROCK2 signaling pathway. Inflammatory cytokines were detected through enzyme-linked immunosorbent assay after co-culturing with decidual natural killer cells and B7-H3-knockout JEG-3. Results showed that B7-H3 inhibited IL-8 and IP-10 secretion from the decidual natural killer cells. In a CBA/J × DBA/2 abortion-prone mice model, treatment with B7-H3-Fc protein successfully reduced the rate of embryo resorption. In conclusion, our results revealed a possible mechanism by which decreased B7-H3 on trophoblasts of unexplained recurrent miscarriages inhibited trophoblast migration and invasion and increased IL-8 and IP-10 secretion from the decidual natural killer cells. Furthermore, B7-H3 may be a promising new therapeutic target in unexplained recurrent miscarriage patients.

Decreased expression and DNA hypermethylation of syncytin-1 in human villus tissues with unexplained recurrent spontaneous abortion

Recurrent spontaneous abortion (RSA) affects approximately 5 % of women of reproductive age worldwide. The etiology and pathogenesis of approximately 50 % of RSA cases currently remain unclear, which known as unexplained RSA (URSA). Syncytin-1, an envelope protein encoded by HERV-W gene, is essential for human embryonic development. The purpose of this study was to explore the correlation between syncytin-1 expression and URSA occurrence. The villi tissues of URSA patients and patients with voluntary termination of pregnancy for non-medical reasons in early pregnancy (Control group) were collected. Compared with the Control group, syncytin-1 was abnormally low expressed in URSA villus tissues, and the HERV-W gene promoter was hypermethylated. Compared with the control group, the global DNA methylation level and the expression level of DNA methylases in the villus tissues of the URSA group had no significant difference. In addition, compared with the Control group, URSA villus tissue showed obviously abnormal apoptosis. Overexpression of syncytin-1 promoted the proliferation of HTR-8 cells and inhibited their apoptosis; while knockdown of syncytin-1 inhibited cell proliferation and promoted cell apoptosis. URSA villus tissue exhibited hypermethylation of the HERV-W gene and down-regulation of syncytin-1 expression. Syncytin-1 has the potential to be a predictive and diagnostic biomarker for URSA.

Trophoblastic microcirculation occlusion as an adjunct to electric vacuum aspiration for the treatment of endogenous cesarean scar pregnancy

To describe a simple adjunct treatment option, trophoblastic microcirculation occlusion (TMO), which could change subsequent high-risk electric vacuum aspiration in patients with an endogenous cesarean scar pregnancy (CSP) into a routine conventional surgical procedure. Electric vacuum aspiration for the treatment of CSP may be associated with several significant complications, including hemorrhage and tissue remnants. Consequently, a second intervention, such as uterine artery embolization, resectoscopy, or methotrexate, may be required, thus complicating the surgical treatment of CSP. We demonstrate the principle and detailed technique of TMO treatment using animations, video clips, and slides. The study was approved by the institutional review board of Hangzhou Women's Hospital. Tertiary maternity hospital. The video shows a 30-year-old woman with a 5-week endogenous CSP. The procedural steps were repeated in another 6 patients suffering from endogenous CSP (Table1). Written informed consent was obtained from each patient. In patients with a CSP, the syncytiotrophoblastic cells erode the helicine arteries in the inner uterine myometrium. The cytotrophoblastic cells extend as villi tissue into the syncytiotrophoblast. Maternal blood enters the intervillous space between the cytotrophoblast and syncytiotrophoblast. The syncytiotrophoblast, cytotrophoblast, and intervillous space constitute the trophoblastic microcirculation, which subsequently becomes the main blood supply to the gestational sac (Fig.1). During TMO treatment, the trophoblastic microcirculation is identified by contrast-enhanced ultrasound with the cubital vein injected with sulfur hexafluoride microbubbles (Bracco, Switzerland). A 21-gauge needle (200 mm long) was punctured vaginally into the uterine myometrium surrounding the syncytiotrophoblast at 3 different points, and a total of 8 mL sclerosant was injected directly to seal the trophoblastic microcirculation. Complete compression of the helicine arteries for trophoblastic blood supply is the key to TMO treatment (Fig.2, see video interpretation for detailed information). A repeat contrast-enhanced ultrasound was performed after injection to confirm a significant reduction of blood flow in the trophoblast. An electric vacuum aspiration was performed 24 hours after TMO treatment to remove the products of conception completely. Surgical safety was enhanced by deep sedation and concurrent transabdominal ultrasound guidance. The levels of β-human chorionic gonadotropin (β-hCG) were measured weekly until a nonpregnant level was achieved. A follow-up ultrasound then was performed to confirm that the treatment was complete. The clinical value and feasibility of TMO treatment as an adjunct before high-risk electric vacuum aspiration for the management of endogenous CSP. The TMO treatment was performed successfully in all 7 patients; the clinical outcomes are presented in Table2. The TMO treatment significantly reduced the risk of hemorrhage during electric vacuum aspiration. No complications were detected perioperatively. Patients were discharged on day 1 postoperatively. Follow-up ultrasound, performed when the levels of β-hCG had fallen to normal, failed to identify any remnants of the uterine corpus. One patient achieved an intrauterine pregnancy 7 months postoperatively and gave birth to a live baby at 39 weeks of gestation via repeat cesarean section. No cesarean scar dehiscence was observed. Surgical abortion of an endogenous CSP, with a high risk of intraoperative hemorrhage, can be performed with minimal blood loss in a single electric vacuum aspiration when assisted by TMO treatment. This technique provides good results in terms of bleeding reduction that are comparable to those in conventional patients. The TMO treatment can be accomplished via ultrasound-guided needle puncture, a technique that is available widely in the practice of reproductive medicine with a reduced need for consumable resources. The use of a simple adjunct treatment option, TMO, expands the indications of electric vacuum aspiration. This sequential treatment allows for completion of clinical management without the need for uterine artery embolization, resectoscopy, or methotrexate, thus simplifying the originally complicated form of surgical treatment for CSP.

Clinical efficacy of laparoscopic surgery used for the excision of villus tissue and the repair of uterine scar in patients with cesarean scar pregnancy-II.

The present study described the clinical presentation of patients with cesarean scar pregnancy type II (CSP)-II diagnosed by ultrasound or magnetic resonance imaging, who underwent laparoscopic surgery resection or hysteroscopic treatment. The aim of the present study was to evaluate the outcomes of different treatments for CSP. The patients with CSP-II were assigned to the laparoscopy and hysteroscopy groups according to the type of surgery performed. The general indicators and post-operative outcomes were compared between the two groups. Statistically significant differences (P<0.05) were noted in the treatment efficacy indices between the two groups. The laparoscopic group exhibited a lower value of β-human chorionic gonadotropin (β-HCG) following surgery (P=0.017), a shorter time required for β-HCG to return to normal levels (P=0.001), a reduced post-operative thickness of the uterine scar (P<0.001) and a reduced menstruation recovery time (P<0.001). However, no significant differences were noted in blood loss during operation (P>0.05). On the whole, the data indicated that the laparoscopic resection of the scar with gestational tissue and wound repair may be used to preserve the uterus and may thus be an effective method for CSP-II. The appropriate treatment needs to be selected according to the condition of each patient. Based on the latter and on the available technology, priority may perhaps be given to laparoscopic surgery.

CircFBXW4 regulates human trophoblast cell proliferation and invasion via targeting miR-324–3p/TJP1 axis in recurrent spontaneous abortion

IntroductionIncreasing evidence has shown that circular RNAs (circRNAs) play vital roles in embryonic development. However, the function of circRNAs in recurrent spontaneous abortion (RSA) is largely unknown. This study aimed to investigate the expression profile of human circRNAs and their functional mechanisms in regulating RSA. MethodsThe profiles of circRNAs in placental villus tissues from women with RSA and healthy pregnancy with induced abortion were investigated using RNA-sequencing and bioinformatics. Nine circRNAs were verified in the 50 placental villus samples. RNase R digestion, actinomycin D treatment, and fluorescence in situ hybridization were performed to characterize circFBXW4. Furthermore, direct binding of circFBXW4 to miR-324–3p was confirmed by dual-luciferase reporter assay. The roles of circFBXW4 were determined by loss- and gain-of-function assays including cell proliferation, invasion, and apoptosis using CCK8 kit, transwell migration assay, and TUNEL kit in vitro, respectively. ResultsA total of 417 aberrantly expressed circRNAs was detected. circFBXW4, a circRNA significantly up-regulated in the RSA group, was further evaluated. circFBXW4 showed higher stability than FBXW4 mRNA and was localized in the cytoplasm and nucleus in HTR-8/SVneo cells. MiR-324–3p was lowly expressed in the RSA group, and directly regulated circFBXW4 and TJP1 expression in a targeted manner. Overexpression and knockdown of circFBXW4 and miR-324–3p mimic/inhibitor could increase or decrease HTR-8/SVneo cell proliferation and invasion. circFBXW4 regulated TJP1 expression, cell proliferation, and invasion by sponging miR-324–3p. DiscussionThe circFBXW4/miR-324–3p/TJP1 axis is involved in the occurrence and progression of RSA and may be a promising therapeutic target in RSA.

MiR-135a-5p suppresses trophoblast proliferative, migratory, invasive, and angiogenic activity in the context of unexplained spontaneous abortion

BackgroundSpontaneous abortions (SA) is amongst the most common complications associated with pregnancy in humans, and the underlying causes cannot be identified in roughly half of SA cases. We found miR-135a-5p to be significantly upregulated in SA-associated villus tissues, yet the function it plays in this context has yet to be clarified. This study explored the function of miR-135a-5p and its potential as a biomarker for unexplained SA.MethodRT-qPCR was employed for appraising miR-135a-5p expression within villus tissues with its clinical diagnostic values being assessed using ROC curves. The effects of miR-135a-5p in HTR-8/SVneo cells were analyzed via wound healing, Transwell, flow cytometry, EdU, CCK-8, and tube formation assays. Moreover, protein expression was examined via Western blotting, and interactions between miR-135a-5p and PTPN1 were explored through RIP-PCR, bioinformatics analyses and luciferase reporter assays.ResultsRelative to normal pregnancy (NP), villus tissue samples from pregnancies that ended in unexplained sporadic miscarriage (USM) or unexplained recurrent SA (URSA) exhibited miR-135a-5p upregulation. When this miRNA was overexpressed in HTR-8/SVneo cells, their migration, proliferation, and cell cycle progression were suppressed, as were their tube forming and invasive activities. miR-135a-5p over-expression also downregulated the protein level of cyclins, PTPN1, MMP2 and MMP9. In RIP-PCR assays, the Ago2 protein exhibited significant miR-135a-5p and PTPN1 mRNA enrichment, and dual-luciferase reporter assays indicated PTPN1 to be a bona fide miR-135a-5p target gene within HTR-8/SVneo cells.ConclusionmiR-135a-5p may suppress trophoblast migratory, invasive, proliferative, and angiogenic activity via targeting PTPN1, and it may thus offer value as a biomarker for unexplained SA.

Whole-transcriptome sequencing identifies key mRNAs, miRNAs, lncRNAs, and circRNAs associated with unexplained recurrent pregnancy loss.

Recurrent pregnancy loss is a common obstetric complication affecting approximately 1-2% of reproductive population worldwide, but the precise causes for approximately a half of such patients remain unexplained. In this study, we compared the expression profiles of messenger RNA (mRNA), long non-coding RNA (lncRNA), microRNA (miRNA), and circular RNA (circRNA) in villi tissues from patients with unexplained recurrent pregnancy loss (URPL) and elective termination of pregnancy (ETP) using whole-transcriptome sequencing. A number of differentially expressed RNAs were confirmed by real-time PCR analysis. As a result, we identified a total of 1,703 mRNAs, 798 lncRNAs, 199 miRNAs, and 163 circRNAs that were significantly differentially expressed between villi tissues from URPL and ETP. The data of real-time PCR were consistent with those of the sequencing results. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the majority of differentially expressed mRNAs and target genes of ncRNAs were associated with focal adhesion, extracellular matrix-receptor interaction, and the PI3K-Akt signaling pathway. Additionally, two co-expression networks (lncRNA-miRNA-mRNA and lncRNA-circRNA-miRNA-mRNA) were constructed based on the correlation analysis between the differentially expressed RNAs. Taken together, this study provides a large number of valuable candidates for elucidating regulatory mechanisms of ncRNAs, which may ultimately assist in understanding the pathogenesis of URPL.

Placental Transcription Profiling in 6-23 Weeks' Gestation Reveals Differential Transcript Usage in Early Development.

The human placenta is a rapidly developing transient organ that is key to pregnancy success. Early development of the conceptus occurs in a low oxygen environment before oxygenated maternal blood begins to flow into the placenta at ~10–12 weeks’ gestation. This process is likely to substantially affect overall placental gene expression. Transcript variability underlying gene expression has yet to be profiled. In this study, accurate transcript expression profiles were identified for 84 human placental chorionic villus tissue samples collected across 6–23 weeks’ gestation. Differential gene expression (DGE), differential transcript expression (DTE) and differential transcript usage (DTU) between 6–10 weeks’ and 11–23 weeks’ gestation groups were assessed. In total, 229 genes had significant DTE yet no significant DGE. Integration of DGE and DTE analyses found that differential expression patterns of individual transcripts were commonly masked upon aggregation to the gene-level. Of the 611 genes that exhibited DTU, 534 had no significant DGE or DTE. The four most significant DTU genes ADAM10, VMP1, GPR126, and ASAH1, were associated with hypoxia-responsive pathways. Transcript usage is a likely regulatory mechanism in early placentation. Identification of functional roles will facilitate new insight in understanding the origins of pregnancy complications.

Overexpression of long non-coding RNA NEAT1 enhances cell viability and inhibits apoptosis in recurrent spontaneous abortion by targeting the miR-125b/BCL-2 axis.

The current study aimed to investigate the function of the long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) in the pathogenesis of recurrent spontaneous abortion (RSA) and to examine its potential mechanism. The expression of NEAT1, microRNA (miR)-125b and Bcl-2 in the villi of patients with RSAs and women with normal pregnancies was measured by reverse transcription-quantitative PCR. Cell viability was detected by the MTT assay and cell apoptosis was evaluated by flow cytometry. A dual-luciferase reporter assay was performed to verify the associations between NEAT1 and miR-125b. The protein expression of Bcl-2 was detected by western blot analysis. In the present study, the expression of NEAT1 and Bcl-2 was reduced and that of miR-125b was increased in clinical samples of villus tissues from patients with RSAs. In vitro, overexpression of NEAT1 enhanced the viability and suppressed the apoptosis of JEG-3 cells. It was demonstrated that miR-125b acts as a molecular sponge of NEAT1 and its expression was negatively regulated by NEAT1. miR-125b overexpression reduced the viability and promoted the apoptosis of JEG-3 cells. The expression of BCL-2, a target gene of miR-125b, was inversely correlated with that of miR-125b. Overexpression of miR-125b and inhibition of BCL-2 partially reversed the effect of NEAT1 overexpression on the viability and apoptosis of JEG-3 cells. Collectively, it was demonstrated that the NEAT1/miR-125b/BCL-2 axis plays a pivotal role in regulating the viability and apoptosis of JEG-3 cells. The findings of the present study offer new insights into the pathogenesis of RSA and may provide information on RSA treatment.

Expression and Relationship of Netrin-1, DCC, UNC5B, and VEGF in Villous Tissues of Patients with Delayed Abortion.

In order to investigate the correlation between neuroaxon-guiding factor (Netrin-1), deleted in colorectal cancer (DCC), uncoordinated 5B (UNC5B), and vascular endothelial growth factor (VEGF) expression machine in villus tissues of delayed abortion in colorectal cancer, a total of 120 pregnant women are selected from February 2019 to August 2021. The two groups of subjects improve the relevant examinations before surgery and the underwent negative pressure induces abortion hysterectomy guided by abdominal ultrasound under intravenous anesthesia. The mRNA and protein expressions of netrin-1, DCC, UNC5B, and VEGF in the villi of the two groups are detected by real-time fluorescence quantitative polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC), and the correlation of the four indicators is analyzed by Pearson's test.

Crypt and Villus Transcriptomic Responses in Mouse Small Intestine Following Oral Exposure to Hexavalent Chromium.

Oral exposure to hexavalent chromium (Cr(VI)) induces tumors in the mouse duodenum. Previous microarray-based transcriptomic analyses of homogenized mouse duodenal tissue have demonstrated Cr(VI)-induced alterations in various cellular pathways and processes. However, X-ray fluorescence microscopy indicates that chromium localizes primarily to the duodenal villi following exposure to Cr(VI), suggesting that previous transcriptomic analyses of homogenized tissue provide an incomplete picture of transcriptomic responses in the duodenum. Herein, transcriptomic analyses were conducted separately on crypt and villus tissue from formalin-fixed paraffin-embedded transverse duodenal sections from the same study in which microarray-based analyses were previously conducted. A total of 28 groups (7 doses × 2 timepoints × 2 tissue compartments) were analyzed for differential gene expression, dose-response, and gene set enrichment. Tissue compartment isolation was confirmed by differences in expression of typical markers of crypt and villus compartments. Fewer than 21 genes were altered in the crypt compartment of mice exposed to 0.1-5 ppm Cr(VI) for 7 or 90 days, which increased to hundreds or thousands of genes at ≥20 ppm Cr(VI). Consistent with histological evidence for crypt proliferation, a significant, dose-dependent increase in genes that regulate mitotic cell cycle was prominent in the crypt, while subtle in the villus, when compared with samples from time-matched controls. Minimal transcriptomic evidence of DNA damage response in either the crypts or the villi is consistent with published in vivo genotoxicity data. These results are also discussed in the context of modes of action that have been proposed for Cr(VI)-induced small intestine tumors in mice.

Methylation Status of the miR-141-3p Promoter Regulates miR-141-3p Expression, Inflammasome Formation, and the Invasiveness of HTR-8/SVneo Cells

MicroRNA-141 (miR-141-3p) is upregulated in preeclampsia. This study investigated the effect of methylation of the miR-141-3p promoter on cell viability, invasion capability, and inflammasomes in vitro. The expression of miR-141-3p and methylation status of the miR-141-3p promoter were examined by RT-qPCR and pyrosequencing in villus tissues of women with spontaneous delivery (VTsd), villus tissues of women with preeclampsia (VTpe), and also in HTR-8/SVneo cells treated with a miR-141-3p inhibitor and 20 μmol/L 5-aza-2′-deoxycytidine (5-Aza), a DNA methyltransferase inhibitor. Cell viability and invasion were evaluated by CCK-8 and transwell assays. In addition, the levels of CXCL12, CXCR4, CXCR2, MMPs, NLRP3, and ASC expression were assessed by western blotting, and IL-1β and IL-18 concentrations were assayed by ELISA. miR-141-3p expression was upregulated, and the levels of miR-141-3p promoter methylation and CXCL12, CXCR4, and CXCR2 expression were decreased in VTpe relative to VTsd. In HTR-8/SVneo cells, hypomethylation caused by 5-Aza treatment increased miR-141-3p expression, while DNA methyltransferase 3 (DNMT3) transfection decreased miR-141-3p expression. miRNA-141-3p induced NLRP3, IL-1β, and IL-18 production, decreased CXCR4, MMP, and MMP2 production, and suppressed cell growth and invasion. Furthermore, we observed that NLRP3 plays an important mediatory role in the effects of miR-141-3p described above. Decreased methylation of the miR-141-3p promoter increases miR-141-3p expression, which in turn increases NLRP3 expression, resulting in higher IL-1β and IL-18 levels and lower levels of MMP2/9 and CXCR4. We conclude that modification of the miR-141-3p promoter might be a curial mediator in preeclampsia.