Epigenetic drug screening for trophoblast syncytialization reveals a novel role for MLL1 in regulating fetoplacental growth.

BACKGROUND: Abnormal placental development is a significant factor contributing to perinatal morbidity and mortality, affecting approximately 5-7% of pregnant women. Trophoblast syncytialization plays a pivotal role in the establishment and maturation of the placenta, and its dysregulation is closely associated with several pregnancy-related disorders, including preeclampsia and intrauterine growth restriction. However, the underlying mechanisms and genetic determinants of syncytialization are largely unknown. METHODS: We conducted a systematic drug screen using an epigenetic compound library to systematically investigate the epigenetic mechanism essential for syncytialization, and identified mixed lineage leukemia 1 (MLL1), a histone 3 lysine 4 methyltransferase, as a crucial regulator of trophoblast syncytialization. BeWo cells were utilized to investigate the role of MLL1 during trophoblast syncytialization. RNA sequencing and CUT&Tag were further performed to search for potential target genes and the molecular pathways involved. Human placenta tissue was used to investigate the role of MLL1 in TEA domain transcription factor 4 (TEAD4) expression and the upstream signaling during syncytialization. A mouse model was used to examine whether inhibition of MLL1-mediated H3K4me3 regulated placental TEAD4 expression and fetoplacental growth. RESULTS: Genetic knockdown of MLL1 or pharmacological inhibition of the MLL1 methyltransferase complex (by MI-3454) markedly enhanced syncytialization, while overexpression of MLL1 inhibited forskolin (FSK)-induced syncytiotrophoblast formation. In human placental villous tissue, MLL1 was predominantly localized in the nuclei of cytotrophoblasts. Moreover, a notable upregulation in MLL1 expression was observed in the villus tissue of patients with preeclampsia compared with that in the control group. Based on RNA sequencing and CUT&Tag analyses, depletion of MLL1 inhibited the Hippo signaling pathway by suppressing TEAD4 expression by modulating H3K4me3 levels on the TEAD4 promoter region. TEAD4 overexpression significantly reversed the FSK-induced or MLL1 silencing-mediated trophoblast syncytialization. Additionally, decreased hypoxia-inducible factor 1A (HIF1A) enrichment at the MLL1 promoter was observed during syncytialization. Under hypoxic conditions, HIF1A could bind to and upregulate MLL1, leading to the activation of the MLL1/TEAD4 axis. In vivo studies demonstrated that the administration of MI-3454 significantly enhanced fetal vessel development and increased the thickness of the syncytial layer, thereby supporting fetoplacental growth. CONCLUSIONS: These results revealed a novel epigenetic mechanism underlying the progression of syncytialization with MLL1, and suggest potential avenues for identifying new therapeutic targets for pregnancy-related disorders.

Peroxiredoxin2 regulates trophoblast proliferation and migration through SPIB-HDAC2 pathway.

Adequate proliferation and migration of placental trophoblasts is the prerequisite of a successful pregnancy. Peroxiredoxin2 (Prdx2) is a multi-functional gene involved in various signal events to maintain essential biological functions and normal cellular homeostasis. In this study, substantially lower Prdx2 levels were found in the first trimester cytotrophoblasts of women who suffered from recurrent miscarriage (RM). Prdx2 downregulation inhibited trophoblast proliferation and migration. We demonstrated that histone deacetylase2 (HDAC2) acts downstream of Prdx2 in regulating trophoblast proliferation and migration. HDAC2 deacetylates histone-3-lysine-9 in E-cadherin (E-cad) promoter and reduces the transcription of E-cad epigenetically, whereas it promotes the expression of Slug and Snail genes. These molecular changes may contribute to the trophoblast epithelial-mesenchymal transition. We further verified whether Prdx2 modulated the expression of HDAC2 through SPIB. SPIB could bind to the HDAC2 promoter PU-box region and induce HDAC2 expression. In RM, down-regulated Prdx2 suppresses SPIB-HDAC2 pathway, leading to increased E-cad and decreased Slug and Snail, and eventually restrains trophoblast proliferation and migration. Our study unveils the role of Prdx2-regulated SPIB-HDAC2 pathway in the pathology of RM and provides diagnostic and therapeutic targets for RM as well as other "great obstetrical syndromes" including preeclampsia and intrauterine growth restriction.

Elevated histone demethylase KDM5C increases recurrent miscarriage risk by preventing trophoblast proliferation and invasion.

KDM5C is a histone H3K4-specific demethylase, which has been shown to play a key role in biological disease and development. However, the role of KDM5C in trophoblasts at early pregnancy is currently unknown. Here, we showed that KDM5C was upregulated in placental trophoblasts from recurrent miscarriage (RM) patients compared with healthy controls (HCs). Trophoblast proliferation and invasion was inhibited by KDM5C overexpression and was promoted by KDM5C knockdown. Transcriptome sequencing revealed that elevated KDM5C exerted anti-proliferation and anti-invasion effects by repressing the expression of essential regulatory genes. The combination analysis of RNA-seq, ChIP-seq and CUT&Tag assay showed that KDM5C overexpression leads to the reduction of H3K4me3 on the promoters and the corresponding downregulation of expression of several regulatory genes in trophoblasts. Among these genes, TGFß2 and RAGE are essential for the proliferation and invasion of trophoblasts. Importantly, overexpression of KDM5C by a systemically delivered KDM5C adenovirus vector (Ad-KDM5C) promoted embryo resorption rate in mouse. Our results support that KDM5C is an important regulator of the trophoblast function during early pregnancy, and suggesting that KDM5C activity could be responsible for epigenetic alterations seen RM disease.

Retraction Notice to: Elevated Tristetraprolin Impairs Trophoblast Invasion in Women with Recurrent Miscarriage by Destabilization of HOTAIR.

[This retracts the article DOI: 10.1016/j.omtn.2018.07.001.].

TCF3 regulates human endometrial stromal cell proliferation and migration in RPL.

Recurrent pregnancy loss (RPL) is a multifactorial condition with no explanation of miscarriage in approximately half of the RPL patients, consequently leaving deep physical and emotional sequels. Transcription factor 3 (TCF3 or E2A), is a unique member of the LEF/TCF family and plays an important role in embryogenesis. However, its function in RPL is poorly understood. Using real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry, we demonstrated that TCF3 was downregulated in decidual tissues from RPL patients compared with healthy control (HC). Further, TCF3 knockdown inhibited proliferation, induced G0/G1 phase arrest, and promoted migration in human endometrial stromal cells (HESCs), while overexpression of TCF3 exhibited the opposite effects. RNA-sequencing analysis combined with gene-set enrichment analysis results showed that the mitogen-activated protein kinase pathway is potentially downstream of TCF3. Knockdown of TCF3 confirmed increased p38 phosphorylation, while overexpression of TCF3 inhibited p38 phosphorylation. Furthermore, we found that TCF3 protein level was decreased in HESCs under hypoxic incubation, while hypoxia-inducible factor-1α (HIF1A) knockdown increased the expression of TCF3. TCF3 overexpression recovered the proliferation ability of HESCs inhibited by hypoxia and reversed hypoxia-induced migration. Consistently, we found that RPL patients had a significantly higher level of HIF1A in the decidual tissue than HC. Overall, this study clarifies that increased HIF1A in the decidua contributes to the occurrence of RPL through the TCF3/p38 signaling pathway.

The role of extravillous trophoblasts and uterine NK cells in vascular remodeling during pregnancy.

Successful embryo implantation requires both a receptive endometrium and competent blastocysts. After implantation, the maternal decidua undergoes a series of changes, including uterine spiral artery (SA) remodeling to accommodate the fetus and provide nutrients and oxygen for the fetus to survive. Uterine spiral arteries transform from small-diameter, high-resistance arteries to large-diameter and low-resistance arteries during pregnancy. This transformation includes many changes, such as increased permeability and dilation of vessels, phenotypic switching and migration of vascular smooth muscle cells (VSMCs), transient loss of endothelial cells (ECs), endovascular invasion of extravillous trophoblasts (EVTs), and presence of intramural EVT, which are regulated by uterine NK (uNK) cells and EVTs. In this review, we mainly focus on the separate and combined roles of uNK cells and EVTs in uterine SA remodeling in establishing and maintaining pregnancy. New insight into related mechanisms will help us better understand the pathogenesis of pregnancy complications such as recurrent pregnancy loss (RPL) and preeclampsia (PE).

CLDN1 regulates trophoblast apoptosis and proliferation in preeclampsia.

Preeclampsia is a gestational hypertensive disease; however, preeclampsia remains poorly understood. Bioinformatics analysis was applied to find novel genes involved in the pathogenesis of preeclampsia and identified CLDN1 as one of the most differentially expressed genes when comparing patients with preeclampsia and healthy controls. The results of the qRT-PCR, Western blotting and immunohistochemistry experiments demonstrated that CLDN1 was significantly downregulated in the chorionic villi in samples from patients with preeclampsia. Furthermore, knockdown of CLDN1 in HTR-8/SVneo cells resulted in the inhibition of proliferation and induction of apoptosis, and overexpression of CLDN1 reversed these effects. In addition, RNA-seq assays demonstrated that the gene BIRC3 is potentially downstream of CLDN1 and is involved in the regulation of apoptosis. Knockdown of CLDN1 confirmed that the expression level of BIRC3 was obviously decreased and was associated with a significant increase in cleaved PARP. Interestingly, the apoptotic effect in CLDN1 knockdown cells was rescued after BIRC3 overexpression. Overall, these results indicate that a decrease in CLDN1 inhibits BIRC3 expression and increases cleaved PARP levels thus participating in the pathogenesis of preeclampsia.

Proteomic analysis of decidua in patients with recurrent pregnancy loss (RPL) reveals mitochondrial oxidative stress dysfunction.

BACKGROUND: Pregnancy is a complicated physiological process. The multifaceted regulation of maternal-fetal interface is of great importance for maintaining normal pregnancy and avoiding fetal rejection and secondary abortion. Previous studies have focused on the clinical features or pathological biomarkers of fetal rejection and abortion. However, no significant breakthrough has been made. Therefore, it is important to understand the molecular mechanisms of recurrent pregnancy loss (RPL) to identify potential therapeutic strategies. The aim of this study was to investigate the pathogenesis of RPL. METHODS: In this study, Relative and absolute quantitation (iTRAQ) technology integrated with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to identify differentially expressed proteins in decidual from RPL patients and matched normal controls. Further, Molecules NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3 (ndufb3) and cyclooxygenase-2 (COX-2) were validated by immunohistochemistry (IHC), Western blotting, CCK8 and mitochondrial red fluorescent probe (Mito-Tracker Red CMXRos). RESULTS: A total of 456 proteins reached the threshold of a 1.5-fold change were identified for further bioinformatics analysis. Upon mapping the differentially expressed proteins using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways database, iTRAQ results were confirmed by assessing NDUFB3 and COX-2 protein levels in specimens of decidual tissue by Western blotting. Our study indicates that the level of COX-2 and NDUFB3 were significantly increased in decidual cell from RPL patients. Overexpression of NDUFB3 inhibited cell vitality and oxidative stress of decimal cell. Further, our found that overexpression NDUFBD3 in decidual cell decreased the mitochondrial membrane potential expression levels. These results suggest that NDUFB3 might play an important role in promote the pathological process of RPL. CONCLUSIONS: This comprehensive analysis of RPL proteomics reveals novel candidate: NDUFB3, which could be further investigated for explanation of the pathological mechanism of RPL.

PDIA3 regulates trophoblast apoptosis and proliferation in preeclampsia via the MDM2/p53 pathway.

Protein disulfide isomerase 3 (PDIA3) is a chaperone protein that modulates the folding of newly synthesized glycoproteins, has isomerase and redox activity, and has been implicated in the pathogenesis of many diseases. However, the role of PDIA3 in pregnancy-associated diseases remains largely unknown. Our present study reveals a key role for PDIA3 in the biology of placental trophoblasts from women with preeclampsia (PE). Immunohistochemistry and Western blot analysis revealed that PDIA3 expression was decreased in villous trophoblasts from women with PE compared to normotensive pregnancies. Further, using a Cell Counting Kit-8 assay, flow cytometry, and 5-ethynyl-2'-deoxyuridine (EdU) staining, we found that siRNA-mediated PDIA3 knockdown significantly promoted apoptosis and inhibited proliferation in the HTR8/SVneo cell line, while overexpression of PDIA3 reversed these effects. Furthermore, RNA sequencing and Western blot analysis demonstrated that knockdown of PDIA3 inhibited MDM2 protein expression in HTR8 cells, concurrent with marked elevation of p53 and p21 expression. Conversely, overexpression of PDIA3 had the opposite effects. Immunohistochemistry and Western blot further revealed that MDM2 protein expression was downregulated and p21 was increased in trophoblasts of women with PE compared to women with normotensive pregnancies. Our findings indicate that PDIA3 expression is decreased in the trophoblasts of women with PE, and decreased PDIA3 induces trophoblast apoptosis and represses trophoblast proliferation through regulating the MDM2/p53/p21 pathway.

Upregulation of RND3 Affects Trophoblast Proliferation, Apoptosis, and Migration at the Maternal-Fetal Interface.

Trophoblasts as the particular cells of the placenta play an important role in implantation and formation of the maternal-fetal interface. RND3 (also known as RhoE) is a unique member of the Rnd subfamily of small GTP-binding proteins. However, its function in cytotrophoblasts (CTBs) at the maternal-fetal interface is poorly understood. In the present study, we found that RND3 expression was significantly increased in trophoblasts from the villous tissues of patients with recurrent miscarriage (RM). RND3 inhibited proliferation and migration and promoted apoptosis in HTR-8/SVneo cells. Using dual-luciferase reporter and chromatin immunoprecipitation assays, we found that forkhead box D3 (FOXD3) is a key transcription factor that binds to the RND3 core promoter region and regulates RND3 expression. Here, the level of FOXD3 was upregulated in the first-trimester CTBs of patients with RM, which in turn mediated RND3 function, including inhibition of cell proliferation and migration and promotion of apoptosis. Further, we found that RND3 regulates trophoblast migration and proliferation via the RhoA-ROCK1 signaling pathway and inhibits apoptosis via ERK1/2 signaling. Taken together, our findings suggest that RND3 and FOXD3 may be involved in pathogenesis of RM and may serve as potential therapeutic targets.

SPRY4 regulates trophoblast proliferation and apoptosis via regulating IFN-γ-induced STAT1 expression and activation in recurrent miscarriage.

PROBLEM: The dysregulation of trophoblast functions is one of the leading causes of recurrent miscarriage (RM), which frustrates 1%-5% of couples of childbearing ages. Sprouty 4 (SPRY4) is considered as a tumour suppressor and exerts a negative role in cell viability. However, its role in regulating trophoblast behaviors at the maternal-fetal interface remains largely unknown. METHOD OF STUDY: First-trimester villous samples were collected from RM patients and healthy controls (HCs) to determine the SPRY4 expression in human placenta during early pregnancy. The HTR8/SVneo cell line was introduced to clarify trophoblast cell functions via transfecting with specific short interfering RNA against SPRY4 or SPRY4-overexpressing lentivirus in vitro. In addition, gene expression microarray analysis was performed to explore the downstream molecules and pathways. RESULTS: Our results revealed that SPRY4 expression was significantly increased in the first-trimester cytotrophoblasts of RM patients compared with HCs. Furthermore, SPRY4 overexpression inhibited trophoblast proliferation and accelerated apoptosis in vitro, while SPRY4 knockdown reversed these effects. Mechanistically, IFN-γ -induced STAT1 expression and activation were involved in the regulation of trophoblast proliferation and apoptosis by SPRY4, and IFN-γ promoted SPRY4 expression and STAT1 phosphorylation through PI3K/AKT pathway. Additionally, both STAT1 and phosphorylated STAT (p-STAT) levels were also upregulated in trophoblasts from RM patients and positively correlated with SPRY4 expression. CONCLUSION: Our findings indicate that SPRY4 may act as a negative regulator of trophoblast functions through upregulating IFN-γ/PI3K/AKT-induced STAT1 activation. High levels of SPRY4 and STAT1 may contribute to RM development and progression, and blocking of either target could be a novel therapeutic strategy for RM patients.

PDL1 blockage increases fetal resorption and Tfr cells but does not affect Tfh/Tfr ratio and B-cell maturation during allogeneic pregnancy.

A successful pregnancy requires sophisticated regulation of uterine microenvironment to guarantee the existence of semi-allogeneic conceptus without immune rejection. T follicular regulatory (Tfr) cells exert a suppressive effect on Tfh-cell expansion, B-cell response, and antibody production. Although accumulating evidence has demonstrated that dysregulations of Tfr cells can bring on various immunological diseases, their immunomodulatory roles during pregnancy still remain unheeded. Herein, we introduced an allogeneic normal-pregnant mouse model and found that CD4+CXCR5hiPD-1hiFoxp3+ Tfr cells were preferentially accumulated in the uterus at mid-gestation and displayed a distinct phenotype. In addition, the absence of PDL1 resulted in increased fetal resorption by favoring Tfr cells accumulation and upregulating PD-1 expression on these cells. However, PDL1 blockade affected neither the ratio of Tfh/Tfr cells nor the maturation and differentiation of B cells. Overall, our results are the first to present a correlation of Tfr cells accumulation with healthy allogeneic pregnancy and PDL1 blockade-induced miscarriage, and to indicate that appropriate assembly of Tfr cells is important for pregnancy maintenance. Since blockade of PD-1-PDL1 pathway leads to more Tfr cells and fetal losses, the reproductive safety must be taken into consideration when PD-1/PD-L1 checkpoint blockade immunotherapy is used in pregnancy.

Upregulation of PUM1 Expression in Preeclampsia Impairs Trophoblast Invasion by Negatively Regulating the Expression of the lncRNA HOTAIR.

Pumilio (PUM) proteins are members of a highly conserved RNA-binding protein family that posttranscriptionally regulate gene expression in many organisms. However, their roles in the placenta are unclear. In the present study, we report the requirement for the PUM homolog 1 (PUM1) gene in preeclampsia (PE). Immunofluorescence and immunohistochemical data showed that PUM1 was highly expressed in human placental villi from women with PE compared to healthy controls (HCs). Further, PUM1 overexpression repressed, and knockdown enhanced, the invasion and proliferation of trophoblasts. Interestingly, PUM1 knockdown promoted trophoblast invasion in a villous explant culture model, while PUM1 overexpression repressed these effects. Furthermore, lncRNA transcriptome sequencing coupled with RNA immunoprecipitation (RIP) revealed that PUM1 inhibits trophoblast invasion in PE by downregulating the expression of lncRNA HOTAIR. Moreover, PUM1 regulates HOTAIR expression via a posttranscriptional mechanism. Using RNA-protein pull-down and mRNA stability assays, we identified PUM1 as a specific binding partner that decreased the half-life of HOTAIR and lowered the steady-state level of HOTAIR expression, suggesting a novel posttranscriptional regulatory mechanism. Collectively, these findings identified a novel RNA regulatory mechanism, revealing a new pathway governing the regulation of PUM1/HOTAIR in trophoblast invasion in the pathogenesis of PE.

Urothelial carcinoma associated 1 promotes trophoblast invasion by regulating MMP9.

BACKGROUND: The long non-coding RNA UCA1 is reportedly increased in several human tumors and critical for the cell migration, invasion, or proliferation of several cancer cells. However, the potential roles of UCA1 in trophoblasts at early pregnancy still poorly understood. Here, we sought to unravel the roles of UCA1 in the occurrence of the recurrent miscarriage (RM) disorders. RESULTS: The knockdown of UCA1 in human HTR-8 trophoblast cell line reduced their cell proliferative and invasive ability. Conversely, the UCA1 overexpression promoted the cell proliferation and invasion of HTR-8 cells. Quantitative RT-PCR screening revealed that UCA1 overexpression significantly enhanced MMP9, but not MMP2, mRNA expression in trophoblast cells. The overexpression of UCA1 also promoted trophoblast invasion by upregulating MMP9 expression and activity both in vitro and ex vivo. Consistently, UCA1 and MMP9 mRNA expression level was notably reduced in placental villi derived from patients with RM diseases. CONCLUSION: This study revealed that UCA1 is critical for the regulation of invasive ability in trophoblasts. The abnormal UCA1/MMP9 pathway might result in the impaired trophoblast activities and lead to the development of RM. Our data may also provide a novel angle for the treatment in RM patients.

ANXA7 regulates trophoblast proliferation and apoptosis in preeclampsia.

PROBLEM: Preeclampsia (PE) is a unique gestational disorder leading to maternal and neonatal morbidity and mortality. AnnexinA7 (ANXA7) is a calcium-dependent phospholipid-binding protein that promotes membrane fusion during exocytosis. However, the function of ANXA7 in placental trophoblast is poorly understood. The present study aimed to investigate a possible association between ANXA7 and human trophoblast apoptosis. METHODS: We collected human placental tissues from patients with PE and normal pregnant women to elucidate the expression level of ANXA7. The ANXA7-knockdown and ANXA7-overexpressing HTR8/SVneo cells were utilized for studying the function of ANXA7 in trophoblast. The proliferation and apoptosis levels of trophoblast were examined with Western blot assay, flow cytometry, Cell Counting Kit-8 assay, and immunohistochemistry. RESULTS: ANXA7 expression was significantly lower in placentas from patients with PE patients compared with that in from normal pregnant controls. Knockdown of ANXA7 induced cell apoptosis and inhibited cell proliferation in HTR-8 via by downregulating BCL2 protein levels. Overexpression of ANXA7 reduced apoptosis and promoted HTR8 proliferation. Further analyses showed that ANXA7 knockdown inhibited the activation of the JAK1/STAT3 pathway in HTR-8 cells. CONCLUSION: Our findings revealed a new regulatory pathway of ANXA7/JAK1/STAT3 in trophoblast apoptosis in preeclampsia, suggesting that ANXA7 is a potential therapeutic target for preeclampsia.

The m6A demethylase ALKBH5 controls trophoblast invasion at the maternal-fetal interface by regulating the stability of CYR61 mRNA.

N6-Methyladenosine (m6A) is the most prevalent internal modification in mammalian mRNAs. Although m6A is important in many biological processes, its roles in the placenta are unclear. Methods: Levels of global mRNA m6A methylation and ALKBH5 expression in recurrent miscarriage (RM) patients were determined using quantitative reverse transcription-PCR (qRT-PCR), m6A RNA methylation quantification, and immunohistochemical methods. Using ALKBH5 overexpression and knockdown methods, we determined the role of ALKBH5 in trophoblast invasion at the maternal interface through trophoblasts and an extravillous explant culture experiments. Furthermore, the regulation of CYR61 by ALKBH5 was explored by RNA-sequencing coupled with methylated RNA immunoprecipitation. Results: We found that the level of global mRNA m6A methylation was significantly decreased in placental villous tissue from RM patients, while ALKBH5 expression was specifically unregulated. Furthermore, we demonstrated that ALKBH5 knockdown in human trophoblast promoted trophoblast invasion. Conversely, overexpression of ALKBH5 inhibited cell invasion. ALKBH5 knockdown promoted trophoblast invasion in villous explant culture experiments, while overexpression of ALKBH5 repressed these effects. Furthermore, we clarified that ALKBH5 inhibited trophoblast invasion by regulating CYR61 mRNA stability, and this RNA regulation is m6A dependent. Mechanistic analyses showed that decreased ALKBH5 in trophoblast increased the half-life of CYR61 mRNA and promoted steady-state CYR61 mRNA expression levels. Conclusions: We elucidated the functional roles of ALKBH5 and mRNA m6A methylation in trophoblast and identified a novel RNA regulatory mechanism, providing a basis for further exploration of broad RNA epigenetic regulatory patterns in RM diseases.

The orphan nuclear receptor NUR77 promotes trophoblast invasion at early pregnancy through paracrine placental growth factor.

NR4A1 (NUR77) is an orphan nuclear receptor that has been implicated in both cell survival and apoptosis. However, the role of NUR77 in trophoblast function during early placenta development has not been fully elucidated. In this study, we showed that NUR77 expression was significantly lower in the villi of the recurrent miscarriage (RM) group compared to that in the healthy controls (HCs) group. We used immunohistochemistry and found that NUR77 was highly expressed in human placental villi during early pregnancy, especially in syncytiotrophoblast (STB), and was expressed at a much lower level in STB from the RM group than in those from HC group. Western blotting data further confirmed that NUR77 was highly expressed in primary human term placental STB and the FSK-induced BeWo cell line. Moreover, antibody array screening and ELISA revealed that NUR77 promoted significant placental growth factor (PGF) expression during trophoblast fusion. Ectopic overexpression and knockdown experiments demonstrated that PGF was a novel downstream target of NUR77, and serum PGF expression correlated positively with trophoblast NUR77 mRNA levels in HCs and RM patients. Importantly, bioinformatics analysis identified two NUR77 binding sites in the PGF promoter region, and chromatin immunoprecipitation (ChIP) coupled with Western blotting analysis further verified that NUR77 bound directly to the PGF promoter region and promoted PGF expression. Furthermore, in a BeWo/HTR-8 co-culture system, FSK-induced BeWo-secreted PGF promoted HTR-8 cell migration and invasion, and an anti-PGF antibody reversed this effect. Collectively, these results indicated that NUR77 may play a key role in regulating trophoblast invasion at early pregnancy. KEY MESSAGES: NUR77 expression was significantly decreased in the syncytiotrophoblast of the recurrent miscarriage group compared to that in the healthy control group. NUR77 promoted PGF expression during trophoblast fusion. ChIP and western blotting experiments verified that NUR77 bound directly to the PGF promoter region and activated PGF expression in trophoblast. Trophoblast-derived PGF promoted HTR-8 cell migration and invasion in a cell co-culture system.

Downregulation of CCNA2 disturbs trophoblast migration, proliferation, and apoptosis during the pathogenesis of recurrent miscarriage.

PROBLEM: Recurrent miscarriage (RM) is defined as two or more pregnancy losses until 24 weeks of gestation, which distresses up to 1-5% of couples worldwide. Cyclin A2 (CCNA2) regulates the cell cycle by promoting transition through G1/S and G2/M. Little is known about CCNA2's functions in trophoblast, although it is highly expressed in the placenta. We therefore sought to explore the role of CCNA2 in RM and early pregnancy. METHOD OF STUDY: First-trimester human placental tissues were collected from patients with RM and normal pregnant women to clarify the expression level of CCNA2. First-trimester human villi explants culture was performed as an ex vivo model to study the functions of CCNA2. The HTR8/SVneo cells were studied for in vitro experiments. The migration, proliferation, and apoptosis levels of trophoblast were examined in the CCNA2-knockdown and CCNA2-overexpressing HTR8 cells. Results Our study revealed that CCNA2 was downregulated in trophoblast of RM first-trimester villi. Results showed that CCNA2 promotes migration of HTR8 cells via the RhoA-ROCK signaling and that CCNA2 increased HTR8 proliferation and inhibited their apoptosis via p53 pathway. In addition, the relationship between ROCK signaling and p53 pathway was found. Further, mechanistic research attributed the regulatory function of CCNA2 on p53 pathway to the involvement of DNA damage. Conclusion Our study revealed the downregulation of CCNA2 in first-trimester chorionic villi of patients with RM and showed that this protein regulates migration, proliferation, and apoptosis in HTR8 cells, suggesting that CCNA2 may be the potential therapeutic target for RM.

Myeloid-derived suppressor cells depletion may cause pregnancy loss via upregulating the cytotoxicity of decidual natural killer cells.

PROBLEM: Maternal immune system tolerance to the semiallogeneic fetus is critical for a successful pregnancy. Studies have shown that myeloid-derived suppressor cells (MDSCs) play an important role in maintaining feto-maternal tolerance. However, the mechanisms remain poorly understood. METHODS: Flow cytometry was used to evaluate the percentage of MDSCs in an allogeneic-normal-pregnant mouse model during different periods of gestation. We further assessed the percentage of MDSCs and their subtypes (granulocytic MDSCs [GR-MDSCs] and monocytic MDSCs [MO-MDSCs]) in a spontaneous abortion mouse model. The levels of the immunosuppressive molecules ARG-1, iNOS, IL-10, and TGF-ß in MDSCs were also evaluated. MDSCs were depleted by anti-Gr-1 injection, and the resorption rate was calculated. The cytotoxicity of decidual natural killer (NK) cells was evaluated, and the percentage of regulatory NK (NKreg) cells and regulatory T lymphocytes (Tregs) were evaluated. RESULTS: Myeloid-derived suppressor cells was accumulated in a time-dependent manner during pregnancy. However, the percentage of MDSCs was decreased in the spontaneous abortion mice compared with that in the control mice. In addition, the levels of ARG-1, iNOS, IL-10, and TGF-ß in MDSCs decreased differentially. Finally, depletion of MDSCs was associated with increased rates of resorption and the proportion of NKreg and Treg cells in uterine tissues; meanwhile, the cytotoxicity of decidual NK cells was upregulated by increasing the level of perforin, granzyme B, and natural killer group protein 2 D-activating NK receptor (NKG2D). CONCLUSION: Depletion of MDSCs may cause pregnancy loss, while upregulating the cytotoxicity of decidual NK cells and increasing NKreg and Treg cell numbers.