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.

ChatGPT outscored human candidates in a virtual objective structured clinical examination in obstetrics and gynecology.

BACKGROUND: Natural language processing is a form of artificial intelligence that allows human users to interface with a machine without using complex codes. The ability of natural language processing systems, such as ChatGPT, to successfully engage with healthcare systems requiring fluid reasoning, specialist data interpretation, and empathetic communication in an unfamiliar and evolving environment is poorly studied. This study investigated whether the ChatGPT interface could engage with and complete a mock objective structured clinical examination simulating assessment for membership of the Royal College of Obstetricians and Gynaecologists. OBJECTIVE: This study aimed to determine whether ChatGPT, without additional training, would achieve a score at least equivalent to that achieved by human candidates who sat for virtual objective structured clinical examinations in Singapore. STUDY DESIGN: This study was conducted in 2 phases. In the first phase, a total of 7 structured discussion questions were selected from 2 historical cohorts (cohorts A and B) of objective structured clinical examination questions. ChatGPT was examined using these questions and responses recorded in a script. Of note, 2 human candidates (acting as anonymizers) were examined on the same questions using videoconferencing, and their responses were transcribed verbatim into written scripts. The 3 sets of response scripts were mixed, and each set was allocated to 1 of 3 human actors. In the second phase, actors were used to presenting these scripts to examiners in response to the same examination questions. These responses were blind scored by 14 qualified examiners. ChatGPT scores were unblinded and compared with historical human candidate performance scores. RESULTS: The average score given to ChatGPT by 14 examiners was 77.2%. The average historical human score (n=26 candidates) was 73.7 %. ChatGPT demonstrated sizable performance improvements over the average human candidate in several subject domains. The median time taken for ChatGPT to complete each station was 2.54 minutes, well before the 10 minutes allowed. CONCLUSION: ChatGPT generated factually accurate and contextually relevant structured discussion answers to complex and evolving clinical questions based on unfamiliar settings within a very short period. ChatGPT outperformed human candidates in several knowledge areas. Not all examiners were able to discern between human and ChatGPT responses. Our data highlight the emergent ability of natural language processing models to demonstrate fluid reasoning in unfamiliar environments and successfully compete with human candidates that have undergone extensive specialist training.

Development of machine learning models to predict gestational diabetes risk in the first half of pregnancy.

BACKGROUND: Early prediction of Gestational Diabetes Mellitus (GDM) risk is of particular importance as it may enable more efficacious interventions and reduce cumulative injury to mother and fetus. The aim of this study is to develop machine learning (ML) models, for the early prediction of GDM using widely available variables, facilitating early intervention, and making possible to apply the prediction models in places where there is no access to more complex examinations. METHODS: The dataset used in this study includes registries from 1,611 pregnancies. Twelve different ML models and their hyperparameters were optimized to achieve early and high prediction performance of GDM. A data augmentation method was used in training to improve prediction results. Three methods were used to select the most relevant variables for GDM prediction. After training, the models ranked with the highest Area under the Receiver Operating Characteristic Curve (AUCROC), were assessed on the validation set. Models with the best results were assessed in the test set as a measure of generalization performance. RESULTS: Our method allows identifying many possible models for various levels of sensitivity and specificity. Four models achieved a high sensitivity of 0.82, a specificity in the range 0.72-0.74, accuracy between 0.73-0.75, and AUCROC of 0.81. These models required between 7 and 12 input variables. Another possible choice could be a model with sensitivity of 0.89 that requires just 5 variables reaching an accuracy of 0.65, a specificity of 0.62, and AUCROC of 0.82. CONCLUSIONS: The principal findings of our study are: Early prediction of GDM within early stages of pregnancy using regular examinations/exams; the development and optimization of twelve different ML models and their hyperparameters to achieve the highest prediction performance; a novel data augmentation method is proposed to allow reaching excellent GDM prediction results with various models.

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.

Periodontitis and Gestational Diabetes Mellitus: A Potential Inflammatory Vicious Cycle.

Periodontitis is a chronic inflammatory immune disease associated with a dysbiotic state, influenced by keystone bacterial species responsible for disrupting the periodontal tissue homeostasis. Furthermore, the severity of periodontitis is determined by the interaction between the immune cell response in front of periodontitis-associated species, which leads to the destruction of supporting periodontal tissues and tooth loss in a susceptible host. The persistent bacterial challenge induces modifications in the permeability and ulceration of the sulcular epithelium, which facilitates the systemic translocation of periodontitis-associated bacteria into distant tissues and organs. This stimulates the secretion of pro-inflammatory molecules and a chronic activation of immune cells, contributing to a systemic pro-inflammatory status that has been linked with a higher risk of several systemic diseases, such as type 2 diabetes mellitus (T2DM) and gestational diabetes mellitus (GDM). Although periodontitis and GDM share the common feature of systemic inflammation, the molecular mechanistic link of this association has not been completely clarified. This review aims to examine the potential biological mechanisms involved in the association between periodontitis and GDM, highlighting the contribution of both diseases to systemic inflammation and the role of new molecular participants, such as extracellular vesicles and non-coding RNAs, which could act as novel molecular intercellular linkers between periodontal and placental tissues.

Care of the pregnant woman with coronavirus disease 2019 in labor and delivery: anesthesia, emergency cesarean delivery, differential diagnosis in the acutely ill parturient, care of the newborn, and protection of the healthcare personnel.

Coronavirus disease 2019, caused by the severe acute respiratory syndrome coronavirus 2, has been declared a pandemic by the World Health Organization. As the pandemic evolves rapidly, there are data emerging to suggest that pregnant women diagnosed as having coronavirus disease 2019 can have severe morbidities (up to 9%). This is in contrast to earlier data that showed good maternal and neonatal outcomes. Clinical manifestations of coronavirus disease 2019 include features of acute respiratory illnesses. Typical radiologic findings consists of patchy infiltrates on chest radiograph and ground glass opacities on computed tomography scan of the chest. Patients who are pregnant may present with atypical features such as the absence of fever as well as leukocytosis. Confirmation of coronavirus disease 2019 is by reverse transcriptase-polymerized chain reaction from upper airway swabs. When the reverse transcriptase-polymerized chain reaction test result is negative in suspect cases, chest imaging should be considered. A pregnant woman with coronavirus disease 2019 is at the greatest risk when she is in labor, especially if she is acutely ill. We present an algorithm of care for the acutely ill parturient and guidelines for the protection of the healthcare team who is caring for the patient. Key decisions are made based on the presence of maternal and/or fetal compromise, adequacy of maternal oxygenation (SpO2 >93%) and stability of maternal blood pressure. Although vertical transmission is unlikely, there must be measures in place to prevent neonatal infections. Routine birth processes such as delayed cord clamping and skin-to-skin bonding between mother and newborn need to be revised. Considerations can be made to allow the use of screened donated breast milk from mothers who are free of coronavirus disease 2019. We present management strategies derived from best available evidence to provide guidance in caring for the high-risk and acutely ill parturient. These include protection of the healthcare workers caring for the coronavirus disease 2019 gravida, establishing a diagnosis in symptomatic cases, deciding between reverse transcriptase-polymerized chain reaction and chest imaging, and management of the unwell parturient.

Mechanisms and evidence of vertical transmission of infections in pregnancy including SARS-CoV-2s.

There remain unanswered questions concerning mother-to-child-transmission of SARS-CoV-2. Despite reports of neonatal COVID-19, SARS-CoV-2 has not been consistently isolated in perinatal samples, thus definitive proof of transplacental infection is still lacking. To address these questions, we assessed investigative tools used to confirm maternal-fetal infection and known protective mechanisms of the placental barrier that prevent transplacental pathogen migration. Forty studies of COVID-19 pregnancies reviewed suggest a lack of consensus on diagnostic strategy for congenital infection. Although real-time polymerase chain reaction of neonatal swabs was universally performed, a wide range of clinical samples was screened including vaginal secretions (22.5%), amniotic fluid (35%), breast milk (22.5%) and umbilical cord blood. Neonatal COVID-19 was reported in eight studies, two of which were based on the detection of SARS-CoV-2 IgM in neonatal blood. Histological examination demonstrated sparse viral particles, vascular malperfusion and inflammation in the placenta from pregnant women with COVID-19. The paucity of placental co-expression of ACE-2 and TMPRSS2, two receptors involved in cytoplasmic entry of SARS-CoV-2, may explain its relative insensitivity to transplacental infection. Viral interactions may utilise membrane receptors other than ACE-2 thus, tissue susceptibility may be broader than currently known. Further spatial-temporal studies are needed to determine the true potential for transplacental migration.

Development and analytical validation of real-time PCR for the detection of Streptococcus agalactiae in pregnant women.

BACKGROUND: Group B Streptococcus (GBS) is the leading cause of invasive neonatal infection. In this study, we aimed to evaluate the analytical validation of qualitative real-time polymerase chain reaction (qPCR) as a means to detect GBS. METHODS: Genomic DNA (gDNA) was purified from 12 ATCC bacterial strains, two belonging to GBS and the remainder acting as negative controls. Additionally, gDNA was isolated from 21 strains of GBS from various serotypes (Ia, Ib and II-VIII). All gDNA was used to evaluate the analytical validation of the qPCR method employing a specific Taqman probe. Inclusivity, exclusivity, anticipated reportable range, the limit of detection and robustness were evaluated. The methods used are described in international guidelines and other existing reports. The performance of this qPCR method for detecting GBS was compared to other microbiological methods used with vaginal-rectal samples from pregnant women. RESULTS: Our qPCR method for detecting GBS was analytically validated. It has a limit of detection of 0.7 GE/µL and 100% analytical specificity. It detects all strains of GBS with the same level of performance as microbiological methods. CONCLUSION: Data suggest that this qPCR method performs adequately as a means to detect GBS in vaginal-rectal swabs from pregnant women.

Ultrasound-Guided Extraction of Intrauterine Devices With Nonvisible Threads: 254 Consecutive Cases: An Effective, Noninvasive Technique.

Intrauterine devices (IUDs) are among the most efficient and widely used contraceptive methods available. Removal is recommended after a certain time or in some cases when adverse effects are observed. A considerable number of patients have nonvisible guides or "lost threads" on speculum examinations, hindering the extraction. In this article, we report a consecutive series of 254 nonpregnant patients referred to our center after 1 or more failed attempts at IUD removal. We describe a novel ultrasound-guided approach, using a laparoscopic forceps to safely and effectively remove IUDs in nonpregnant patients.

First trimester prediction of gestational diabetes mellitus using plasma biomarkers: a case-control study.

Objectives To evaluate the first trimester maternal biomarkers for early pregnancy prediction of gestational diabetes mellitus (GDM). Methods The study was a case-control study of healthy women with singleton pregnancies at the first trimester carried out at the Obstetrics and Gynecology Unit, Clinica Davila, Santiago, Chile. After obtaining informed consent, peripheral blood samples of pregnant women under 14 weeks of gestation were collected. At 24-28 weeks of pregnancy, women were classified as GDM (n=16) or controls (n=80) based on the results of a 75-g oral glucose tolerance test (OGTT). In all women, we measured concentrations of fasting blood glucose, insulin, glycated hemoglobin, uric acid, cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), very low density lipoprotein (VLDL), triglycerides, aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (GGT), alkaline phosphatase (AP), sex hormone-binding globulin (SHBG), adiponectin, tissue plasminogen activator (t-PA), leptin and placental growth factor (PGF). Results The GDM group displayed an increased median concentration of cholesterol (P=0.04), triglycerides (P=0.003), insulin (P=0.003), t-PA (P=0.0088) and homeostatic model assessment (HOMA) (P=0.003) and an increased mean concentration of LDL (P=0.009) when compared to the control group. The receiver operating characteristic (ROC) curve for significant variables achieved an area under the curve (AUC) of 0.870, a sensitivity of 81.4% and a specificity of 80.0%. The OGTT was positive for GDM according to the IADPSG (International Diabetes in Pregnancy Study Group) criteria. Conclusion Women who subsequently developed GDM showed higher levels of blood-borne biomarkers during the first trimester, compared to women who did not develop GDM. These data warrant validation in a larger cohort.

Patients with acute cervical insufficiency without intra-amniotic infection/inflammation treated with cerclage have a good prognosis.

Background The frequency of intra-amniotic infection/inflammation (IAI/I) in patients with midtrimester cervical insufficiency is up to 50%. Our purpose was to determine the perinatal outcomes of cervical cerclage in patients with acute cervical insufficiency with bulging membranes, and to compare the admission-to-delivery interval and pregnancy outcomes according to the results of amniotic fluid (AF) analysis and cerclage placement. Methods This was a retrospective cohort study including singleton pregnancies with cervical insufficiency between 15 and 26.9 weeks in two tertiary health centers. IAI/I was defined when at least one of the following criteria was present in AF: (a) a white blood cell (WBC) count >50 cells/mm3; (b) glucose concentration <14 mg/dL; and/or (c) a Gram stain positive for bacteria. Three different groups were compared: (1) absence of IAI/I with placement of a cerclage; (2) amniocentesis not performed with placement of a cerclage; and (3) IAI/I with or without a cerclage. Results Seventy patients underwent an amniocentesis to rule out IAI/I. The prevalence of IAI/I was 19%. Forty-seven patients underwent a cerclage. Patients with a cerclage had a longer median admission-to-delivery interval (33 vs. 2 days; P < 0.001) and delivered at a higher median gestational age (27.4 vs. 22.6 weeks; P = 0.001) than those without a cerclage. The neonatal survival rate in the cerclage group was 62% vs. 23% in those without a cerclage (P = 0.01). Patients without IAI/I who underwent a cerclage had a longer median admission-to-delivery interval (43 vs. 1 day; P < 0.001), delivered at a higher median gestational age (28 vs. 22.1 weeks; P = 0.001) and had a higher neonatal survival rate (67% vs. 8%; P < 0.001) than those with IAI/I. Conclusion The pregnancy outcomes of patients with midtrimester cervical insufficiency and bulging membranes are poor as they have a high prevalence of IAI/I. Therefore, a pre-operative amniocentesis is key to identify the best candidates for the subsequent placement of a cerclage.

Normality Ranges of Menstrual Fluid Volume During Reproductive Life Using Direct Quantification of Menses with Vaginal Cups.

OBJECTIVE: To determine the normal limits of menstrual fluid volume during reproductive life, quantified by direct measurement. METHODS: This was an observational, prospective clinical trial of healthy women aged 20-49 years old, with normal menstrual periods, recruited in a Natural Family Planning Unit. Women collected their menstrual fluid for at least 3 menstrual periods using a vaginal cup. Menstrual volume and different covariables were evaluated using a multilevel mixed-effects linear regression. RESULTS: Ninety-six cycles from 28 patients between 24 and 49 years old were analyzed. The average menstrual volume was 86.7 mL with a range from 15 to 271 mL. The 50th percentile of all samples was 81 mL and the 95th percentile was 162 mL. For multiparous patients the 50th percentile was 93 mL and the 95th was 169 mL. Menstrual fluid volume was higher in multigravida (99.1 mL) than in nulliparous women (45.9 Ml; p < 0.02). No statistically significant associations were identified between different variables and menstrual volume. CONCLUSION: A menstrual volume over 169 mL should be considered abnormal on multiparous patients. Age was not associated with changes on menstrual fluid volume.

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.

[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.

The immunosuppressive signature of menstrual blood mesenchymal stem cells entails opposite effects on experimental arthritis and graft versus host diseases.

Recently, a noninvasive and highly proliferative stem cell population from menstrual blood called MenSCs has been identified. Despite their use in clinical studies, their immunomodulatory properties have not yet been investigated. In this context, we studied the immunosuppressive properties of MenSCs in comparison with the well-characterized bone marrow derived-MSCs (BM-MSCs). Using an in vitro proliferation assays, we showed that MenSCs displayed a lower suppressive effect on peripheral blood mononuclear cells and in particular on the proinflammatory CD4(+) IFN-γ(+) and CD8(+) IFNγ(+) cells than BM-MSCs. Moreover, compared to BM-MSCs, MenSCs activated with IFN-γ and IL-1ß produced lower amounts of immunosuppressive factors such as IDO, PDL-1, PGE2, and Activin A and exhibited a substantial lower expression level of IFN-γ receptor subunits. In the collagen induced arthritis model, while BM-MSCs administration resulted in a potent therapeutic effect associated with a significant decrease of proinflammatory T cell frequency in the lymph nodes, MenSCs injection did not. In contrast, in the xeno-GVHD model, only MenSCs administration significantly increased the survival of mice. This beneficial effect mediated by MenSCs was associated with a higher capacity to migrate into the intestine and liver and not to their anti-inflammatory capacities. All together our results demonstrate for the first time that the therapeutic potential of MSC in the experimental xeno-GVHD model is independent of their immunosuppressive properties. These findings should be taken into consideration for the development of safe and effective cell therapies.

Oxidative damage and nitric oxide synthase induction by surgical uteroplacental circulation restriction in the rabbit fetal heart.

OBJECTIVE: This study investigated the role of oxidative damage and nitric oxide (NO) synthases in the fetal heart using a model of intrauterine growth restriction induced by uteroplacental circulation restriction (UCR). METHODS: New Zealand white rabbits kept under 12-h light cycles, with food and water provided ad libitum, were subjected at day 25 of pregnancy to 40-50% uteroplacental artery ligation. We analyzed the gene expression of enzymes linked to nitric oxide synthesis (iNOS, eNOS, HO-1, and ARG-2), hypoxia inducible factor 1 alpha (HIF-1α), and the state of oxidative stress (protein carbonyl levels) in fetal heart homogenates. Additionally, we studied the histological morphology of the fetal heart. RESULTS: We found that fetal growth restriction was associated with a significant reduction in heart weight but a normal heart/body weight ratio in UCR animals. Hematoxylin and eosin staining showed normal left and right ventricular thickness but increased vessel dilatation with hyperemia in the hearts of the UCR group. We observed HIF-1α, eNOS, p-eNOS, and iNOS induction concomitant with intensified protein carbonyl levels but observed no changes in HO-1 or ARG-2 expression, suggesting increased NO and oxidative stress in the hearts of UCR animals. CONCLUSION: Uteroplacental circulation restriction increased NO-linked enzymes, oxidative damage, and dilated coronary vessels in fetal hearts. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Jumonji Domain Containing Protein 6 Is Decreased in Human Preeclamptic Placentas and Regulates sFLT-1 Splice Variant Production.

The anti-angiogenic protein, soluble fms-like tyrosine kinase-1 (sFLT-1), plays a central role in preeclamptic pathophysiology. A splice variant of FLT-1 (VEGF receptor 1), sFLT-1 is released in excessive amounts from the preeclamptic placenta into the maternal circulation, where it causes endothelial dysfunction manifesting as end-organ disease. However, the mechanisms regulating its production within the placenta remain poorly understood. Recently it was shown in endothelial cells that Jumonji domain containing protein 6 (JMJD6) hydroxylates U2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65, a component of the splicesome). The hydroxylation by JMJD6 is oxygen dependent. Under hypoxia, JMJD6 is less able to hydroxylate U2AF65, and this unhydroxylated form of U2AF65 biases splicing of FLT-1 to sFLT-1. We assessed whether oxygen-sensing JMJD6 is differentially expressed in preeclamptic placenta and regulates sFLT-1 splicing in placenta via U2AF65. JMJD6 protein expression was significantly reduced in preterm preeclamptic placenta (P < 0.0001; n = 21) relative to preterm controls (n = 10). Exposing both placental and endothelial cells to hypoxia significantly reduced JMJD6 mRNA and increased sFLT-1 mRNA and protein expression. Silencing JMJD6 in primary endothelial and trophoblast cells significantly increased sFLT-1 secretion. Next, we examined whether these molecules may be directly interacting. We demonstrated that placental U2AF65 colocalized with JMJD6. In turn, we found JMJD6 directly interacts with U2AF65, which in turn produces sFLT-1 mRNA transcripts. Taken together, our findings provide evidence that JMJD6 may play a role in regulating the production of sFLT-1 in the preeclamptic placenta. Decreased placental JMJD6 expression may be an important component to the pathophysiology of preeclampsia.

Metformin as a prevention and treatment for preeclampsia: effects on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion and endothelial dysfunction.

BACKGROUND: Preeclampsia is associated with placental ischemia/hypoxia and secretion of soluble fms-like tyrosine kinase 1 and soluble endoglin into the maternal circulation. This causes widespread endothelial dysfunction that manifests clinically as hypertension and multisystem organ injury. Recently, small molecule inhibitors of hypoxic inducible factor 1α have been found to reduce soluble fms-like tyrosine kinase 1 and soluble endoglin secretion. However, their safety profile in pregnancy is unknown. Metformin is safe in pregnancy and is also reported to inhibit hypoxic inducible factor 1α by reducing mitochondrial electron transport chain activity. OBJECTIVE: The purposes of this study were to determine (1) the effects of metformin on placental soluble fms-like tyrosine kinase 1 and soluble endoglin secretion, (2) to investigate whether the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion are regulated through the mitochondrial electron transport chain, and (3) to examine its effects on endothelial dysfunction, maternal blood vessel vasodilation, and angiogenesis. STUDY DESIGN: We performed functional (in vitro and ex vivo) experiments using primary human tissues to examine the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from placenta, endothelial cells, and placental villous explants. We used succinate, mitochondrial complex II substrate, to examine whether the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion were mediated through the mitochondria. We also isolated mitochondria from preterm preeclamptic placentas and gestationally matched control subjects and measured mitochondrial electron transport chain activity using kinetic spectrophotometric assays. Endothelial cells or whole maternal vessels were incubated with metformin to determine whether it rescued endothelial dysfunction induced by either tumor necrosis factor-α (to endothelial cells) or placenta villous explant-conditioned media (to whole vessels). Finally, we examined the effects of metformin on angiogenesis on maternal omental vessel explants. RESULTS: Metformin reduced soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from primary endothelial cells, villous cytotrophoblast cells, and preterm preeclamptic placental villous explants. The reduction in soluble fms-like tyrosine kinase 1 and soluble endoglin secretion was rescued by coadministration of succinate, which suggests that the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin were likely to be regulated at the level of the mitochondria. In addition, the mitochondrial electron transport chain inhibitors rotenone and antimycin reduced soluble fms-like tyrosine kinase 1 secretion, which further suggests that soluble fms-like tyrosine kinase 1 secretion is regulated through the mitochondria. Mitochondrial electron transport chain activity in preterm preeclamptic placentas was increased compared with gestation-matched control subjects. Metformin improved features of endothelial dysfunction relevant to preeclampsia. It reduced endothelial cell messenger RNA expression of vascular cell adhesion molecule 1 that was induced by tumor necrosis factor-α (vascular cell adhesion molecule 1 is an inflammatory adhesion molecule up-regulated with endothelial dysfunction and is increased in preeclampsia). Placental conditioned media impaired bradykinin-induced vasodilation; this effect was reversed by metformin. Metformin also improved whole blood vessel angiogenesis impaired by fms-like tyrosine kinase 1. CONCLUSION: Metformin reduced soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from primary human tissues, possibly by inhibiting the mitochondrial electron transport chain. The activity of the mitochondrial electron transport chain was increased in preterm preeclamptic placenta. Metformin reduced endothelial dysfunction, enhanced vasodilation in omental arteries, and induced angiogenesis. Metformin has potential to prevent or treat preeclampsia.

Nitric oxide synthase and changes in oxidative stress levels in embryonic kidney observed in a rabbit model of intrauterine growth restriction.

OBJECTIVE: This work aimed to study the effect of uteroplacental circulation restriction on endothelial kidney damage in a fetal rabbit model. METHODS: New Zealand rabbits were subjected to 40% to 50% of uteroplacental artery ligation at day 25 of pregnancy. After 5 days, surviving fetuses were harvested by cesarean section. The gene and protein expressions of selected enzymes associated with nitric oxide production and oxidative stress were analyzed in fetal kidney homogenates. RESULTS: The placenta weight (6.06 ± 0.27, p < 0.0319) and fetal body (19.90 ± 1.03, p < 0.0001) were significantly reduced in the uteroplacental circulation restriction group. The kidneys from restricted fetuses presented a mild vascular congestion and glomerular capillary congestion, without inflammation or hypertrophy. We found endothelial nitric oxide synthase phosphorylation inhibition (0.23 ± 0.13, p < 0.012) and arginase-2 (0.29 ± 0.14, p < 0.023) protein induction in fetal kidneys of the circulation restriction group. Finally, the kidneys from circulation-restricted fetuses showed increased inducible nitric oxide synthase messenger RNA (mRNA) (2.68 ± 0.24, p < 0.01) and reduced heme oxygenase-1 mRNA (23 ± 1.3, p < 0.003), with increased reactive oxygen species (1.69 ± 0.09, p < 0.001) and nitrotyrosine protein (1.74 ± 0.28, p < 0.003) levels, without changes in Nox mRNA. CONCLUSION: We describe significant deregulation of vascular activity and oxidative damage in kidneys of fetal rabbits that have been exposed to restriction of the uterine circulation. © 2016 John Wiley & Sons, Ltd.

Applying SWATH Mass Spectrometry to Investigate Human Cervicovaginal Fluid During the Menstrual Cycle.

Inherent interindividual and intraindividual variation in the length of the menstrual cycle limits the accuracy of predicting days of peak fertility. To improve detection of days of peak fertility, a more detailed understanding of longitudinal changes in cervicovaginal fluid (CVF) biomarkers during the normal menstrual cycle is needed. The aim of this study, therefore, was to characterize longitudinal changes in CVF proteins during the menstrual cycle using a quantitative, data-independent acquisition mass spectrometry approach. Six serial samples were collected from women (n = 10) during the menstrual cycle. Samples were obtained at two time points for each phase of the cycle: early and late preovulatory, ovulatory, and postovulatory. Information-dependent acquisition (IDA) of mass spectra from all individual CVF samples was initially performed and identified 278 total proteins. Samples were then pooled by time of collection (n = 6 pools) and analyzed using IDA and information-independent acquisition (Sequential Windowed Acquisition of All Theoretical Mass Spectra [SWATH]). The IDA library generated contained 176 statistically significant protein identifications (P < 0.000158). The variation in the relative abundance of CVF proteins across the menstrual cycle was established by comparison with the SWATH profile against the IDA library. Using time-series, pooled samples obtained from 10 women, quantitative data were obtained by SWATH analysis for 43 CVF proteins. Of these proteins, 28 displayed significant variation in relative abundance during the menstrual cycle (assessed by ANOVA). Statistical significant changes in the relative expression of CVF proteins during preovulatory, ovulatory, and postovulatory phases of menstrual cycle were identified. The data obtained may be of utility not only in elucidating underlying physiological mechanisms but also as clinically useful biomarkers of fertility status.