FOXM1 Participates in Trophoblast Migration and Early Trophoblast Invasion: Potential Role in Blastocyst Implantation.

Successful implantation requires coordinated migration and invasion of trophoblast cells into a receptive endometrium. Reduced forkhead box M1 (FOXM1) expression limits trophoblast migration and angiogenesis in choriocarcinoma cell lines, and in a rat model, placental FOXM1 protein expression was significantly upregulated in the early stages of pregnancy compared to term pregnancy. However, the precise role of FOXM1 in implantation events remains unknown. By analyzing mice blastocysts at embryonic day (E3.5), we have demonstrated that FOXM1 is expressed as early as the blastocyst stage, and it is expressed in the trophectoderm of the blastocyst. Since controlled oxygen tension is determinant for achieving normal implantation and placentation and a chronic hypoxic environment leads to shallow trophoblast invasion, we evaluated if FOXM1 expression changes in response to different oxygen tensions in the HTR-8/SVneo first trimester human trophoblast cell line and observed that FOXM1 expression was significantly higher when trophoblast cells were cultured at 3% O2, which coincides with oxygen concentrations in the uteroplacental interface at the time of implantation. Conversely, FOXM1 expression diminished in response to 1% O2 that resembles a hypoxic environment in utero. Migration and angiogenesis were assessed following FOXM1 knockdown and overexpression at 3% O2 and 1% O2, respectively, in HTR-8/SVneo cells. FOXM1 overexpression increased transmigration ability and tubule formation. Using a 3D trophoblast invasion model with trophospheres from HTR-8/SVneo cells cultured on a layer of MATRIGEL and of mesenchymal stem cells isolated from menstrual fluid, we observed that trophospheres obtained from 3D trophoblast invasion displayed higher FOXM1 expression compared with pre-invasion trophospheres. Moreover, we have also observed that FOXM1-overexpressing trophospheres increased trophoblast invasion compared with controls. HTR-8/SVneo-FOXM1-depleted cells led to a downregulation of PLK4, VEGF, and MMP2 mRNA expression. Our current findings suggest that FOXM1 participates in embryo implantation by contributing to trophoblast migration and early trophoblast invasion, by inducing transcription activation of genes involved in these processes. Maternal-fetal communication is crucial for trophoblast invasion, and maternal stromal cells may induce higher levels of FOXM1 in trophoblast cells.

Mesenchymal Stem Cells-Induced Trophoblast Invasion Is Reduced in Patients with a Previous History of Preeclampsia.

Endometrial stromal cells play an important role in reproductive success, especially in implantation and placentation. Although Mesenchymal stem cells (MSCs) have been studied to assess decidualization disorders in preeclampsia (PE), their role during trophoblast invasion remains unclear. This study aims to determine: (i) whether MSCs isolated from menstrual fluid (MenSCs) from nulliparous, multiparous, and women with a previous history of preeclampsia exhibited different patterns of proliferation and migration and (ii) whether reproductive history (i.e., prior pregnancy or prior history of PE) was able to produce changes in MenSCs, thus altering trophoblast invasion capacity. MenSCs were collected from nulliparous and multiparous women without a history of PE and from non-pregnant women with a history of PE. Proliferation and migration assays were performed on MenSCs with sulforhodamine B and transwell assays, respectively. Trophoblast invasion was analyzed by culturing HTR-8/SVneo trophospheres on a matrigel overlying MenSCs for 72 h at 5% O2, simulating a 3D implantation model. A previous history of pregnancy or PE did not impact the proliferative capacity or migratory behavior of MenSCs. Following exposure to physiological endometrial conditions, MenSCs demonstrated upregulated expression of IGFBP-1 and LIF mRNA, decidualization and window of implantation markers, respectively. The mRNA expression of VIM, NANOG, and SOX2 was upregulated upon trophosphere formation. Relative to co-culture with multiparous MenSCs, co-culture with PE-MenSCs was associated with reduced trophoblast invasion. The findings of this study suggest a potential role for communication between maternal MenSCs and invading trophoblast cells during the implantation process that could be implicated in the etiology of PE.

Identification of LOC101927355 as a Novel Biomarker for Preeclampsia.

Preeclampsia, a disorder with a heterogeneous physiopathology, can be attributed to maternal, fetal, and/or placental factors. Long non-coding RNAs (lncRNAs) refer to a class of non-coding RNAs, the essential regulators of biological processes; their differential expression has been associated with the pathogenesis of multiple diseases. The study aimed to identify lncRNAs, expressed in the placentas and plasma of patients who presented with preeclampsia, as potential putative biomarkers of the disease. In silico analysis was performed to determine lncRNAs differentially expressed in the placentas of patients with preeclampsia, using a previously published RNA-Seq dataset. Seven placentas and maternal plasma samples collected at delivery from preterm preeclamptic patients (≤37 gestational weeks of gestation), and controls were used to validate the expression of lncRNAs by qRT-PCR. Six lncRNAs were validated and differentially expressed (p < 0.05) in the preeclampsia and control placentas: UCA1 and HCG4 were found upregulated, and LOC101927355, LINC00551, PART1, and NRAD1 downregulated. Two of these lncRNAs, HCG4 and LOC101927355, were also detected in maternal plasma, the latter showing a significant decrease (p = 0.03) in preeclamptic patients compared to the control group. In silico analyses showed the cytoplasmic location of LOC101927355, which suggests a role in post-transcriptional gene regulation. The detection of LOC101927355 in the placenta and plasma opens new possibilities for understanding the pathogenesis of preeclampsia and for its potential use as a biomarker.

The Role of Long Non-Coding RNAs in Trophoblast Regulation in Preeclampsia and Intrauterine Growth Restriction.

Preeclampsia (PE) and Intrauterine Growth Restriction (IUGR) are two pregnancy-specific placental disorders with high maternal, fetal, and neonatal morbidity and mortality rates worldwide. The identification biomarkers involved in the dysregulation of PE and IUGR are fundamental for developing new strategies for early detection and management of these pregnancy pathologies. Several studies have demonstrated the importance of long non-coding RNAs (lncRNAs) as essential regulators of many biological processes in cells and tissues, and the placenta is not an exception. In this review, we summarize the importance of lncRNAs in the regulation of trophoblasts during the development of PE and IUGR, and other placental disorders.

Diagnostic potential of peri-implant crevicular fluid microRNA-21-3p and microRNA-150-5p and extracellular vesicles in peri-implant diseases.

BACKGROUND: To explore the diagnostic usefulness of extracellular vesicles (EVs), and their subpopulations (micro-vesicles and exosomes), and microRNAs (miRNA-21-3p, miRNA-150-5p, and miRNA-26a-5p) in peri-implant crevicular fluid (PICF) of subjects with healthy, peri-implant mucositis and peri-implantitis implants. METHODS: A total of 54 patients were enrolled into healthy, peri-implant mucositis, and peri-implantitis groups. PICF samples were collected, EVs subpopulations (MVs and Exo) were isolated and characterized by nanoparticle tracking analysis and transmission electron microscopy. The expression of miRNA-21-3p, miRNA-150-5p and miRNA-26a-5p was quantified by qRT-PCR. Logistic regression models and accuracy performance tests were estimated. RESULTS: PICF samples show the presence of EVs delimited by a bi-layered membrane, in accordance with the morphology and size (< 200 nm). The concentration of PICF-EVs, micro-vesicles and exosomes was significantly increased in peri-implantitis implants compared to healthy implants (P = 0.023, P = 0.002, P = 0.036, respectively). miRNA-21-3p and miRNA-150-5p expression were significantly downregulated in patients with peri-implantitis in comparison with peri-implant mucositis sites (P = 0.011, P = 0.020, respectively). The reduced expression of miRNA-21-3p and miRNA-150-5p was associated with peri-implantitis diagnosis (OR:0.23, CI 0.08-0.66, P = 0.007 and OR:0.07, CI 0.01-0.78, P = 0.031, respectively). The model which included the miRNA-21-3p and miRNA-150-5p expression had a sensitivity of 93.3%, a specificity of 76.5%, a positive predictive value of 77.8%, and a negative predictive value of 92.9%. The positive and negative likelihood ratios were 3.97 and 0.09, respectively. The area under the receiver operating characteristics curve for the model was 0.84. CONCLUSIONS: An increase concentration of EVs with a downregulation expression of miRNA-21-3p and miRNA-150-5p could be related with the peri-implantitis development.

Molecular signatures of extracellular vesicles in oral fluids of periodontitis patients.

OBJECTIVE: To characterize extracellular vesicles (EVs) in gingival crevicular fluid (GCF) and saliva samples from healthy/gingivitis and periodontitis patients and correlate them with clinical inflammatory periodontal parameters. MATERIAL AND METHOD: An exploratory study, including 86 subjects, was conducted. Clinical and periodontal data were recorded, and oral fluid samples were obtained. EVs were precipitated by ExoQuick-TC™ and characterized by nanoparticle tracking (NanoSight™), Western blot (WB), transmission electron microscopy (TEM), and ELISA analysis. RESULTS: TEM showed nanoparticles morphologically compatible with EVs, and WB analysis revealed bands of specific EV markers (CD9, TSG101, and Alix) in both oral fluids of periodontitis and healthy/gingivitis subjects. The total concentration of EVs in GCF was increased in periodontitis patients compared to healthy/gingivitis subjects (p = .017). However, we did not observe differences in the EV concentration of saliva samples (p = .190). The size of GCF-EVs was 144.2 nm in periodontitis and 160.35 nm in healthy/gingivitis patients (p = .038). The CD63 exosome marker was increased in GCF of periodontitis patients (p = .00001). The total concentration of EVs in GCF was correlated with bleeding on probing (rho = 0.63, p = .002), periodontal probing depth (rho = 0.56, p = .009), and clinical attachment level (rho = 0.48, p = .030). CONCLUSION: Periodontitis patients have an increased concentration of EVs in GCF, and their role in periodontitis should be clarified.

Increased Circulating Levels of Tissue-Type Plasminogen Activator Are Associated with the Risk of Spontaneous Abortion During the First Trimester of Pregnancy.

Spontaneous abortion is a common complication in early pregnancy, with an incidence of around 20%. Ultrasound scan and measurement of human chorionic gonadotropin are used to identify patients at risk of spontaneous abortion; however, there is a clinical need to find new biomarkers to prospectively identify patients before the onset of clinical symptoms. Here, we aim to investigate potential biomarkers of spontaneous abortion taken in the first clinical appointment of pregnancy. A case-control study was conducted based on a prospectively collected cohort in which cases and controls were retrospectively stratified based on pregnancy outcome: normal healthy pregnancies (controls = 33) and pregnancies that ended in spontaneous abortion (cases = 10). We evaluated extracellular vesicles isolated by precipitation with ExoQuick™ and protein concentrations of tissue plasminogen activator, leptin, and adiponectin measured by ELISA. The extracellular vesicles showed the typical morphology and membrane proteins: CD63, Alix, and Flotilin-1. The size distributions of the isolated extracellular vesicles were 112 ± 27 and 118 ± 28 nm in diameter for controls and spontaneous abortion, respectively, and the total amount of extracellular vesicles did not show any difference between controls and the spontaneous abortion group. The tissue plasminogen activator showed a significant difference (p = 0.0004) between both groups, although neither adiponectin nor leptin revealed significant changes, indicating that women who had spontaneous abortions have significantly higher levels of tissue plasminogen activator than women who had normal pregnancies.

Vesículas extracelulares como predictores tempranos de diabetes gestacional / Extracellular vesicle concentration in maternal plasma as an early marker of gestational diabetes

Background During pregnancy, there is an increase in the amount of extracellular vesicles, especially placental exosomes, in maternal plasma. Aim To isolate and characterize extracellular vesicles from blood during the three trimesters of pregnancy and to evaluate their capacity to identify patients at risk of developing gestational diabetes. Material and Methods A case-control study was conducted in a cohort of 50 pregnant women with plasma samples taken in each trimester. Six women who developed gestational diabetes were paired with three healthy controls per case (a total of 19). Clinical characteristics were recorded at first prenatal appointment, and blood samples were obtained during the first, second and third trimesters. Extracellular vesicles were isolated from plasma by the commercial kit, ExoQuick™. Nanoparticle tracking analysis, was used to characterize the obtained extracellular vesicles. Results The total concentration of extracellular particles isolated from maternal plasma increased along with gestational age. The size of the extracellular vesicles obtained in the first trimester of pregnancy was very similar between groups (144 ± 37 nm for controls and 143 ± 34 nm for patients with gestational diabetes mellitus). Moreover, the concentration of extracellular vesicles collected in the first trimester, was significantly higher in patients who developed gestational diabetes mellitus later in pregnancy compared to normoglycemic pregnant women (7.94 x 10 8 and 5.15 x 10 8 , p = 0.03). Conclusions Our results provide an insight into the potential capacity of first trimester plasma extracellular vesicles as early biomarkers for the prediction of gestational diabetes mellitus.

Small Extracellular Vesicles Released from Ovarian Cancer Spheroids in Response to Cisplatin Promote the Pro-Tumorigenic Activity of Mesenchymal Stem Cells.

Despite the different strategies used to treat ovarian cancer, around 70% of women/patients eventually fail to respond to the therapy. Cancer stem cells (CSCs) play a role in the treatment failure due to their chemoresistant properties. This capacity to resist chemotherapy allows CSCs to interact with different components of the tumor microenvironment, such as mesenchymal stem cells (MSCs), and thus contribute to tumorigenic processes. Although the participation of MSCs in tumor progression is well understood, it remains unclear how CSCs induce the pro-tumorigenic activity of MSCs in response to chemotherapy. Small extracellular vesicles, including exosomes, represent one possible way to modulate any type of cell. Therefore, in this study, we evaluate if small extracellular vesicle (sEV) derived from ovarian cancer spheroids (OCS), which are enriched in CSCs, can modify the activity of MSCs to a pro-tumorigenic phenotype. We show that sEV released by OCS in response to cisplatin induce an increase in the migration pattern of bone marrow MSCs (BM-MSCs) and the secretion interleukin-6 (IL-6), interleukin-8 (IL-8), and vascular endothelial growth factor A (VEGFA). Moreover, the factors secreted by BM-MSCs induce angiogenesis in endothelial cells and the migration of low-invasive ovarian cancer cells. These findings suggest that cisplatin could modulate the cargo of sEV released by CSCs, and these exosomes can further induce the pro-tumorigenic activity of MSCs.

Angiogenic Properties of Menstrual Stem Cells Are Impaired in Women with a History of Preeclampsia.

Preeclampsia is a pregnancy-specific disorder defined by the new onset of hypertension and proteinuria after 20 weeks of gestation. Although its precise etiology is still unknown, there is evidence suggesting that it may be a consequence of impaired decidual and stromal cell function. Recently, a stem cell population derived from endometrial tissue and isolated from menstrual effluent called menstrual stem cells (MenSCs) has been identified. MenSCs exhibit important angiogenic and inflammatory properties that may contribute to both normal and pathological complications of implantation and placentation, including preeclampsia. We hypothesized that the angiogenic and inflammatory activity of MenSCs is altered in women who have a past history of preeclampsia and that this phenotype persists postpartum. The primary outcome measures were stromal progenitor cell formation, in vitro induction of endothelial tube formation, and release of proinflammatory cytokines. MenSCs obtained from women with a previous normal or preeclamptic pregnancy displayed similar phenotypic characteristics, tri-differentiation capacity, and proliferation. MenSCs derived from women who had preeclampsia on their previous pregnancy had reduced angiogenic capacity (~30% fewer junctions and nodes, p < 0.05) and stromal progenitor cell formation (<50% measured at a serial dilution of 1 : 10.000, p < 0.05) when compared to controls. In vitro, MenSCs obtained from patients with a history of preeclampsia expressed less endoglin and secreted less VEGF but more IL-6 than controls did. These data are consistent with the hypothesis that the angiogenic and inflammatory properties of MenSCs of women with a previous pregnancy complicated by preeclampsia have reduced angiogenic capacity and are more proinflammatory than those of MenSCs of women with a previous normal pregnancy. This altered phenotype of MenSCs observed following preeclampsia could either be present before the development of the pathology, predisposing the endometrial milieu to and consequently leading to limited vascular remodeling, or be a consequence of preeclampsia itself. The former may afford opportunity for targeted therapeutic intervention; the latter, a putative biomarker for future risk of pregnancy complications.

Reduced FOXM1 Expression Limits Trophoblast Migration and Angiogenesis and Is Associated With Preeclampsia.

Trophoblast cells are often compared to highly invasive carcinoma cells due to their capacity to proliferate in hypoxic conditions and to exhibit analogous vascular, proliferative, migratory, and invasive capacities. Thus, genes that are important for tumorigenesis, such as forkhead box M1 ( FOXM1) may also be involved in processes of trophoblast invasion. Indeed, we found Foxm1 protein and messenger RNA (mRNA) levels decreased as gestational age increased in rat's placentae. Accordingly, when mimicking early placental events in vitro, protein and mRNA expression of FOXM1 increased from 21% to 8% O2, reaching its highest expression at 3% oxygen tension, which reflects early implantation environment, and dropping to very low levels at 1% O2. Remarkably, FOXM1 silencing in JEG-3 cells was able to significantly decrease migration by 27.9%, in comparison with those cells transfected with control siRNA. Moreover, angiogenesis was compromised when conditioned media (CM) from FOXM1-siRNA -JEG-3 (3% O2) was added to human umbilical vein endothelial cells (HUVEC) cells; however, when CM of JEG-3 cells overexpressing FOXM1 at 1% O2 was added, the ability of HUVEC to form tubule networks was restored. Additionally, quantitative real-time polymerase chain reaction (PCR) assays of FOXM1 knockdown and overexpression experiments in JEG-3 cells revealed that the depletion of FOXM1 at 3% O2 and overexpression of FOXM1 at 1% O2 led to downregulation and upregulation of vascular endothelial growth factor transcriptional (VEGF) levels, respectively. Conversely, we also observed deregulation of FOXM1 in placentae derived from pregnancies complicated by preeclampsia (PE). Therefore, we demonstrate that FOXM1 may be a new regulatory protein of early placentation processes and that under chronic hypoxic conditions (1% O2) and in patients with severe PE, its levels decrease.

[Extracellular vesicle concentration in maternal plasma as an early marker of gestational diabetes]. / Vesículas extracelulares como predictores tempranos de diabetes gestacional.

Background During pregnancy, there is an increase in the amount of extracellular vesicles, especially placental exosomes, in maternal plasma. Aim To isolate and characterize extracellular vesicles from blood during the three trimesters of pregnancy and to evaluate their capacity to identify patients at risk of developing gestational diabetes. Material and Methods A case-control study was conducted in a cohort of 50 pregnant women with plasma samples taken in each trimester. Six women who developed gestational diabetes were paired with three healthy controls per case (a total of 19). Clinical characteristics were recorded at first prenatal appointment, and blood samples were obtained during the first, second and third trimesters. Extracellular vesicles were isolated from plasma by the commercial kit, ExoQuick™. Nanoparticle tracking analysis, was used to characterize the obtained extracellular vesicles. Results The total concentration of extracellular particles isolated from maternal plasma increased along with gestational age. The size of the extracellular vesicles obtained in the first trimester of pregnancy was very similar between groups (144 ± 37 nm for controls and 143 ± 34 nm for patients with gestational diabetes mellitus). Moreover, the concentration of extracellular vesicles collected in the first trimester, was significantly higher in patients who developed gestational diabetes mellitus later in pregnancy compared to normoglycemic pregnant women (7.94 x 10 8 and 5.15 x 10 8 , p = 0.03). Conclusions Our results provide an insight into the potential capacity of first trimester plasma extracellular vesicles as early biomarkers for the prediction of gestational diabetes mellitus.

Periodontitis and placental growth factor in oral fluids are early pregnancy predictors of gestational diabetes mellitus.

BACKGROUND: Gestational diabetes mellitus (GDM) affects around 7% to 10% of all pregnancies. Early detection of predisposition to GDM is the first step in developing efficacious preventive treatment. The objective of the present study was to establish the utility of placental proteins presents in oral fluids (gingival crevicular fluid [GCF] and saliva), and periodontal disease status as early pregnancy predictors of GDM. METHODS: A nested case control within a prospective cohort was conducted. Pregnant systemically healthy women, aged between 18 and 40 years at 11 to 14 weeks gestation were included. Samples of oral fluids were collected and a complete maternal/obstetric and periodontal history was obtained. The concentration of placental growth factor (PlGF) and soluble Fms-like tyrosine kinase 1 (sFlt-1) were measured by enzyme-linked immunosorbent assay in a nested case control sample of the prospective cohort. Multiple logistic regression models assessed the association. The evaluation of the diagnostic accuracy of the biomarkers was performed through receiver operating characteristic (ROC) curves by calculating the area under the curve (AUC). RESULTS: There were recruited 212 pregnant women at 11 to 14 weeks of pregnancy, of these, 14 women (i.e., 6.6%) developed GDM, and displayed significant greater bleeding on probing (BOP) [P = 0.0003]; periodontal probing depth (PD) [P = 0.0028]; clinical attachment level (AL) [P = 0.0008] and periodontal inflamed surface area (PISA) [P = 0.0001]. Similarly, initial glycemia and GCF-PlGF concentrations were significantly greater in women with GDM [P = 0.0012, and P = 0.0019, respectively]. When data were subjected to ROC curve analysis, the combination of initial glycemia and GCF-PlGF concentration delivered an area under the ROC curve of 0.897. Multiple logistic regression analyses demonstrate an association between glycemia (OR 1.21, 95% confidence interval [CI] 1.06 to 1.38; P = 0.005) and GCF-PlGF concentrations in women who developed GDM (OR 1.68, CI 1.05 to 2.68 P = 0.03). CONCLUSIONS: Within the limitations of the present study, the results support that first trimester maternal glycemia combined with GCF-PlGF concentrations could be a surrogate biomarker for the future development of GDM in pre-symptomatic women.