Impact of the Immunomodulatory Factor Soluble B7-H4 in the Progress of Preeclampsia by Inhibiting Essential Functions of Extravillous Trophoblast Cells.

A key aspect of preeclampsia pathophysiology is the reduced invasiveness of trophoblasts and the impairment of spiral artery remodelling. Understanding the causes of altered trophoblast function is critical to understand the development of preeclampsia. B7-H4, a checkpoint molecule, controls a wide range of processes, including T-cell activation, cytokine release, and tumour progression. Our previous findings indicated that B7-H4 levels are elevated in both maternal blood and placental villous tissue during the early stages of preeclampsia. Here, we investigated the function of B7-H4 in trophoblast physiology. Recombinant B7-H4 protein was used to treat human SGHPL-5 extravillous trophoblast cells. Biological functions were investigated using MTT, wound healing, and transwell assays. Signalling pathways were analysed by immunoblotting and immunofluorescence. The functionality of B7-H4 was further confirmed by immunoblotting and immunohistochemical analysis in placental tissues from control and preeclamptic patients following therapeutic plasma exchange (TPE) or standard of care treatment. This study showed that B7-H4 inhibited the proliferation, migration, and invasion capacities of SGHPL-5 extravillous cells while promoting apoptosis by downregulating the PI3K/Akt/STAT3 signalling pathway. These results were consistently confirmed in placental tissues from preterm controls compared to early-onset preeclamptic placental tissues from patients treated with standard of care or TPE treatment. B7-H4 may play a role in the development of preeclampsia by inhibiting essential functions of extravillous trophoblast cells during placental development. One possible mechanism by which TPE improves pregnancy outcomes in preeclampsia is through the elimination of B7-H4 amongst other factors.

Placental glycosylation senses the anti-angiogenic milieu induced by human sFLT1 during pregnancy.

Abnormal placental angiogenesis during gestation resulting from high levels of anti-angiogenic factors, soluble fms-like tyrosine kinase-1 (sFLT1) and soluble endoglin, has been implicated in the progression of preeclampsia (PE). This heterogeneous syndrome (defined by hypertension with or without proteinuria after 20 weeks of pregnancy) remains a major global health burden with long-term consequences for both mothers and child. Previously, we showed that in vivo systemic human (hsFLT1) overexpression led to reduced placental efficiency and PE-like syndrome in mice. Galectins (gal-1, -3 and -9) are critical determinants of vascular adaptation to pregnancy and dysregulation of the galectin-glycan circuits is associated with the development of this life-threatening disease. In this study, we assessed the galectin-glycan networks at the maternal-fetal interface associated with the hsFLT1-induced PE in mice. We observed an increase on the maternal gal-1 expression in the decidua and junctional zone layers of the placenta derived from hs FLT1high pregnancies. In contrast, placental gal-3 and gal-9 expression were not sensitive to the hsFLT1 overexpression. In addition, O- and N-linked glycan expression, poly-LacNAc sequences and terminal sialylation were down-regulated in hsFLT1 high placentas. Thus, the gal-1-glycan axis appear to play an important role counteracting the anti-angiogenic status caused by sFLT1, becoming critical for vascular adaptation at the maternal-fetal interface.

CCN1-Mediated Signaling in Placental Villous Tissues after SARS-CoV-2 Infection in Term Pregnant Women: Implications for Dysregulated Angiogenesis.

The global spread of SARS-CoV-2 has increased infections among pregnant women. This study aimed to explore placental pathology alterations and angiogenic factor levels in term pregnant women after SARS-CoV-2 infection in a retrospective single-center study. Additionally, we investigated the role and underlying mechanism of the vascular inflammation-promoting, cysteine-rich protein 61 (CYR61/CCN1) in this context. All analyses were performed in term pregnant women infected with or without SARS-CoV-2. The sFlt-1, PlGF, and sEng serum levels were quantified using ELISA. Placental protein expressions were examined by immunoblot and immunostaining. Additionally, the effect of CCN1 protein on SGHPL-5 trophoblast cells was examined. We found that SARS-CoV-2 activated the inflammatory response in pregnant women, leading to pronounced vascular alterations in placental villous tissues. Elevated serum anti-angiogenic factors (sFlt-1, sEng) upon SARS-CoV-2 infection may directly contribute to these pathological changes. Upregulated CCN1 and pNF-κB in placental villous tissues of infected patients are identified as crucial factors in placental alterations. As a conclusion, CCN1 was significantly elevated in the placentas of term pregnant women infected with SARS-CoV-2. By activating a cascade of inflammatory responses, CCN1 induced the production of the anti-angiogenic factors sFlt-1 and sEng, which may lead to abnormal placental vascular architecture.

Overexpression of Human sFLT1 in the Spongiotrophoblast Is Sufficient to Induce Placental Dysfunction and Fetal Growth Restriction in Transgenic Mice.

Preeclampsia (PE) is characterized by maternal hypertension and placental dysfunction, often leading to fetal growth restriction (FGR). It is associated with an overexpression of the anti-angiogenic sFLT1 protein, which originates from the placenta and serves as a clinical biomarker to predict PE. To analyze the impact of sFLT1 on placental function and fetal growth, we generated transgenic mice with placenta-specific human sFLT1 (hsFLT1) overexpression. Immunohistochemical, morphometrical, and molecular analyses of the placentas on 14.5 dpc and 18.5 dpc were performed with a focus on angiogenesis, nutrient transport, and inflammation. Additionally, fetal development upon placental hsFLT1 overexpression was investigated. Dams exhibited a mild increase in serum hsFLT1 levels upon placental hsFLT1 expression and revealed growth restriction of the fetuses in a sex-specific manner. Male FGR fetuses expressed higher amounts of placental hsFLT1 mRNA compared to females. FGR placentas displayed an altered morphology, hallmarked by an increase in the spongiotrophoblast layer and changes in labyrinthine vascularization. Further, FGR placentas showed a significant reduction in placental glycogen storage and nutrient transporter expression. Moreover, signs of hypoxia and inflammation were observed in FGR placentas. The transgenic spongiotrophoblast-specific hsFLT1 mouse line demonstrates that low hsFLT1 serum levels are sufficient to induce significant alterations in fetal and placental development in a sex-specific manner.

Growth-Restricted Fetuses and Offspring Reveal Adverse Sex-Specific Metabolic Responses in Preeclamptic Mice Expressing Human sFLT1.

Fetal adaptations to harmful intrauterine environments due to pregnancy disorders such as preeclampsia (PE) can negatively program the offspring's metabolism, resulting in long-term metabolic changes. PE is characterized by increased circulating levels of sFLT1, placental dysfunction and fetal growth restriction (FGR). Here we examine the consequences of systemic human sFLT1 overexpression in transgenic PE/FGR mice on the offspring's metabolic phenotype. Histological and molecular analyses of fetal and offspring livers as well as examinations of offspring serum hormones were performed. At 18.5 dpc, sFLT1 overexpression resulted in growth-restricted fetuses with a reduced liver weight, combined with reduced hepatic glycogen storage and histological signs of hemorrhages and hepatocyte apoptosis. This was further associated with altered gene expression of the molecules involved in fatty acid and glucose/glycogen metabolism. In most analyzed features males were more affected than females. The postnatal follow-up revealed an increased weight gain of male PE offspring, and increased serum levels of Insulin and Leptin. This was associated with changes in hepatic gene expression regulating fatty acid and glucose metabolism in male PE offspring. To conclude, our results indicate that sFLT1-related PE/FGR in mice leads to altered fetal liver development, which might result in an adverse metabolic pre-programming of the offspring, specifically targeting males. This could be linked to the known sex differences seen in PE pregnancies in human.

The immunological role of B7-H4 in pregnant women with Sars-Cov2 infection.

PROBLEM: T-cells are key players in fighting the coronavirus disease 2019 (COVID-19). The checkpoint molecule B7-H4, a member of the B7 family, can inhibit T-cell activation and proliferation by inhibiting NF-kb expression. We aimed to elucidate the immunological role of soluble B7-H4 (sB7-H4) and B7-H4 in pregnant women suffered from an acute Sars-Cov2 infection. METHODS: Expression levels of sB7-H4 and cytokines were detected by enzyme linked immunosorbent assay. B7-H4 and cytokines mRNA expression was analyzed by qPCR, and B7-H4 and NF-κb (p65) protein levels were investigated by western blot and immunofluorescence staining in placenta chorionic villous and decidual basalis tissues of COVID-19 affected women and healthy controls. RESULTS: Fibrinoid necrosis in the periphery of placental villi was increased in the COVID-19-affected patients. sB7-H4 protein in maternal and cord blood serum and IL-6/IL-10 were increased while leukocytes were decreased during SARS-CoV-2 infection. Serum sB7-H4 level was increased according to the severity of SARS-Cov-2 infection. Cytokines (IL-6, IL-18, IL-1ß, TNF-α), B7-H4 mRNA and protein in the decidual basalis tissues of COVID-19-infected pregnant women were significantly increased compared to healthy controls. IL-18 and IL-1ß were significantly increased in the placenta chorionic villous samples of COVID-19 affected patients, while NF-κb (p65) expression was decreased. CONCLUSIONS: The expression of the immunological marker sB7-H4 correlated with the severity of COVID-19 disease in pregnant women. sB7-H4 and B7-H4 can be used to monitor the progression of COVID-19 infection during pregnancy, and for evaluating of the maternal immune status.

The Impact of Increased Maternal sFlt-1/PlGF Ratio on Motor Outcome of Preterm Infants.

Background: The sFlt-1 (soluble fms-like tyrosine kinase-1)/PlGF (placental growth factor) ratio serves as a clinical biomarker to predict the hypertensive, placenta-derived pregnancy disorder pre-eclampsia which is often associated with placental dysfunction and fetal growth restriction. Additionally elevated levels also indicate an increased risk for prematurity. However, its predictive value for subsequent neonatal neurological outcome has not been studied. Objective: This study aimed to evaluate the correlation of maternal sFlt-1/PlGF ratio with early motor outcome of preterm infants. Design/Methods: 88 preterm infants (gestational age ≤ 34 + 0) born between February 2017 and August 2020 at the Department of Obstetrics and Gynecology, University Hospital Essen in Germany, were included, when the following variables were available: maternal sFlt-1/PlGF levels at parturition and general movement assessment of the infant at the corrected age of 3 to 5 months. The infants were stratified into high and low ratio groups according to maternal sFlt-1/PlGF cut-off values of 85. To investigate the early motor repertoire and quality of spontaneous movements of the infant, the Motor Optimality Score (MOS-R) based on antigravity movements and posture patterns, was applied. In the given age, special attention was paid to the presence of fidgety movements. Linear regressions were run to test differences in infants motor repertoire according to the maternal sFlt-1/PIGF ratio. Results: Linear regression analysis showed that the sFlt-1/PlGF ratio does not predict the MOS-R score (ß=≤0.001; p=0.282). However, children with birth weight below the 10th percentile scored significantly lower (mean 20.7 vs 22.7; p=0.035). These children were 91% in the group with an increased ratio, which in turn is a known predictor of low birth weight (ß= -0.315; p <0.001). In the group with a high sFlt-1/PLGF ratio above 85 the mothers of female infants had a lower average sFlt-1/PlGF ratio compared to a male infant (median: 438 in female vs. 603 in male infant, p=0.145). Conclusions: In our cohort, especially low birth weight, which correlated with an elevated sFlt-1/PlGF ratio, had a negative effect on the outcome in the MOS-R. A direct correlation between an increased ratio and a worse motor outcome was not demonstrated.

Systemic Maternal Human sFLT1 Overexpression Leads to an Impaired Foetal Brain Development of Growth-Restricted Foetuses upon Experimental Preeclampsia.

The pregnancy disorder preeclampsia (PE) is characterized by maternal hypertension, increased level of circulating antiangiogenic soluble fms-like tyrosine kinase-1 (sFLT1), and reduced placental perfusion, leading to foetal growth restriction (FGR) and preterm birth. All these adverse effects are associated with neurocognitive disorders in the offspring. However, the direct interplay between increased antiangiogenesis during PE and disturbed foetal brain development independent of prematurity has not been investigated yet. To examine foetal brain development in sFLT1-related PE, hsFLT1/rtTA-transgenic mice with systemic (maternal or maternal/fetoplacental) human sFLT1 (hsFLT1) overexpression since 10.5 days postconception (dpc) were used, and histological and molecular analyses of foetal brains were performed at 18.5 dpc. Consequences of elevated hsFLT1 on placental/foetal vascularization and hypoxia of placentas and foetal brains were analysed using the hypoxia markers pimonidazole and hemeoxygenase-1 (HO-1). Immunohistochemical analysis revealed increased hypoxia in placentas of PE-affected pregnancies. Moreover, an increase in HO-1 expression was observed upon elevated hsFLT1 in placentas and foetal brains. PE foetuses revealed asymmetrical FGR by increased brain/liver weight ratio. The brain volume was reduced combined with a reduction in the cortical/hippocampal area and an increase of the caudate putamen and its neuroepithelium, which was associated with a reduced cell density in the cortex and increased cell density in the caudate putamen upon hsFLT1 overexpression. Mild influences were observed on brain vasculature shown by free iron deposits and mRNA changes in Vegf signalling. Of note, both types of systemic hsFLT1 overexpression (indirect: maternal or direct: maternal/fetoplacental) revealed similar changes with increasing severity of impaired foetal brain development. Overall, circulating hsFLT1 in PE pregnancies impaired uteroplacental perfusion leading to disturbed foetal oxygenation and brain injury. This might be associated with a disturbed cell migration from the caudate putamen neuroepithelium to the cortex which could be due to disturbed cerebrovascular adaption.

Circulating Maternal sFLT1 (Soluble fms-Like Tyrosine Kinase-1) Is Sufficient to Impair Spiral Arterial Remodeling in a Preeclampsia Mouse Model.

[Figure: see text].

Abnormal expression of the costimulatory molecule B7-H4 in placental chorionic villous and decidual basalis tissues of patients with preeclampsia and HELLP syndrome.

BACKGROUND: B7-H4, a checkpoint molecule of the B7 family, regulates a broad spectrum such as T-cell activation, cytokine secretion, tumour progression, and invasion capacities. Our previous data revealed that soluble B7-H4 (sB7-H4) blood serum levels are elevated in women at high risk for the hypertensive pregnancy disorder preeclampsia (PE) in the first trimester, as well as in patients with confirmed early/late-onset PE. AIM: We here aim to investigate the expression pattern of B7-H4 in placental tissues of PE and HELLP Syndrome versus control group. METHODS: B7-H4 protein expression and localization were investigated by immunoblotting and co-immunohistochemistry in placental chorionic villous and decidual basalis tissues. RESULTS: B7-H4 protein was prominently expressed at the cell membrane, in the cytoplasm of the syncytiotrophoblast (STB) and interstitial extravillous trophoblast (EVT). B7-H4 protein levels in placental chorionic villous tissue were significantly higher in women with early-onset/late-onset PE and HELLP, while it was decreased in decidual basalis tissues of early-onset PE and HELLP compared with controls. CONCLUSION: B7-H4 was inversely expressed in placental chorionic villous and decidual basalis tissues of PE and HELLP patients. The increase in B7-H4 in the STB in PE and HELLP may lead to excessive apical expression and release of soluble B7-H4 in the maternal circulation. In contrast, the decrease in B7-H4 in decidual basalis tissues could be related to the decrease in invasion ability of the EVT in PE. Thus, the current results strongly suggest that B7-H4 is involved in the pathogenesis of PE and HELLP.

Evaluation of carcinoembryonic antigen-related cell adhesion molecule 1 blood serum levels in women at high risk for preeclampsia.

PROBLEM: The aim of this study was to evaluate the sCEACAM1 concentrations in serum from patients in the first trimester who have a high risk for developing PE during pregnancy. METHOD OF THE STUDY: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) levels were determined with ELISA. The patients (n = 109) were divided into two groups: patients who have a high risk of developing PE early-onset and a control group. Patients who have a high risk of developing PE were then divided into two subgroups depending on PE development in third trimester of pregnancy: PE in third trimester versus no PE in third trimester. RESULTS: sCEACAM1 concentrations in patients who were screened as having a high risk for developing PE were significantly higher than in healthy pregnant women in the first trimester (p = .03). The highest sCEACAM1 concentration was found in the high-risk group with PE development compared to the control group (p = .004). CONCLUSION: Elevated sCEACAM1 blood serum levels in women with PE suggest that there is immune dysregulation in early pregnancy, which may be helpful in PE prediction and therapy.

CCN3 Signaling Is Differently Regulated in Placental Diseases Preeclampsia and Abnormally Invasive Placenta.

Objectives: An adequate development of the placenta includes trophoblast differentiation with the processes of trophoblast migration, invasion, cellular senescence and apoptosis which are all crucial to establishing a successful pregnancy. Altered placental development and function lead to placental diseases such as preeclampsia (PE) which is mainly characterized by insufficient trophoblast invasion and abnormally invasive placenta (AIP) disorders (Placenta accreta, increta, or percreta) which are characterized by excessive trophoblast invasion. Both of them will cause maternal and fetal morbidity/mortality. However, the etiology of these diseases is still unclear. Our previous study has shown that the matricellular protein nephroblastoma overexpressed (NOV, CCN3) induces G0/G1 cell cycle arrest, drives trophoblast cells into senescence and activates FAK and Akt kinases resulting in reduced cell proliferation and enhanced migration capability of the human trophoblast cell line SGHPL-5. The present study focuses on whether CCN3 can alter cell cycle-regulated pathways associated with trophoblast senescence and invasion activity in pathological versus gestational age-matched control placentas. Methods: Cell cycle regulator proteins were investigated by immunoblotting and qPCR. For localization of CCN3, p16, p21, and Cyclin D1 proteins, co-immunohistochemistry was performed. Results: In early-onset PE placentas, CCN3 was expressed at a significantly lower level compared to gestational age-matched controls. The decrease of CCN3 level is associated with an increase in p53, Cyclin E1 and pRb protein expression, whereas the level of cleaved Notch-1, p21, Cyclin D1, pFAK, pAKT, and pmTOR protein decreased. In term AIP placentas, the expression of CCN3 was significantly increased compared to matched term controls. This increase was correlated to an increase in p53, p16, p21, Cyclin D1, cleaved Notch-1, pFAK, pAkt, and pmTOR whereas pRb was significantly decreased. However, in late PE and early AIP placentas, no significant differences in CCN3, p16, p21, Cyclin D1, p53, and cleaved Notch-1 expression were found when matched to appropriate controls. Conclusions: CCN3 expression levels are correlated to markers of cell cycle arrest oppositely in PE and AIP by activating the FAK/AKT pathway in AIP or down-regulating in PE. This may be one mechanism to explain the different pathological features of placental diseases, PE and AIP.

A double-hit pre-eclampsia model results in sex-specific growth restriction patterns.

Pre-eclampsia is a multifactorial pregnancy-associated disorder characterized by angiogenic dysbalance and systemic inflammation; however, animal models that combine these two pathophysiological conditions are missing. Here, we introduce a novel double-hit pre-eclampsia mouse model that mimics the complex multifactorial conditions present during pre-eclampsia and allows for the investigation of early consequences for the fetus. Adenoviral overexpression of soluble fms-like tyrosine kinase (sFlt-1) and lipopolysaccharide (LPS) administration at mid-gestation in pregnant mice resulted in hypertension and albuminuria comparable to that of the manifestation in humans. A metabolomics analysis revealed that pre-eclamptic dams have increased plasma concentrations of phosphadytilcholines. The fetuses of both sexes were growth restricted; however, in males a brain-sparing effect was seen as compensation for this growth restriction. According to the plasma metabolomics, male fetuses showed changes in amino acid metabolism, while female fetuses showed pronounced alterations in lipid metabolism. Our results show that combined exposure to sFlt-1 and LPS mimics the clinical symptoms of pre-eclampsia and affects fetal growth in a sex-specific manner, with accompanying metabolome changes.

Trends in anti-Müllerian hormone concentrations across different stages of pregnancy in women with polycystic ovary syndrome.

RESEARCH QUESTION: What are the trends in anti-Müllerian hormone (AMH) concentrations from pre-conception to the third trimester of pregnancy in women with polycystic ovary syndrome (PCOS)? DESIGN: Observational study including cross-sectional and longitudinal data analysis. The Beckman Coulter AMH Gen II Assay was used to determine AMH levels longitudinally before pregnancy from 52 women with PCOS and 51 controls during all trimesters. Differences in AMH levels across successive stages of pregnancy were examined with the Wilcoxon signed-rank test for paired values. Linear regression models, adjusted for body-mass index (BMI), gestational and maternal age were used to compare AMH levels of PCOS and controls. RESULTS: AMH levels decreased significantly (all P < 0.05) from pre-pregnancy level throughout each trimester in women with PCOS and healthy controls. After adjusting for maternal age, gestational age and maternal BMI, AMH levels before pregnancy were 1.89 (95% CI 1.46 to 2.44; P < 0.0001) times higher among women with PCOS compared with controls (median 7.66 versus 2.67 ng/ml). During the first trimester, AMH levels were 1.61 (95% CI 1.22 to 2.13; P = 0.001) times higher among women with PCOS compared with controls (median 5.33 versus 2.48 ng/ml). Differences in AMH levels between women with PCOS and controls in the second trimester (1.68 times higher; 95% CI 0.94 to 3.01; median: 5.50 versus 2.20 ng/ml) and the third trimester (1.45 times higher; 95% CI 1.01 to 2.07; median: 1.36 versus 1.06 ng/ml) were not statistically significant. CONCLUSION: These findings indicate a pregnancy-associated AMH-decline independent of pre-pregnancy elevated AMH levels.

Tight interplay in early pregnancy between follistatin and anti-mullerian hormone in women with polycystic ovarian syndrome (PCOS).

PURPOSE: Follistatin levels increase during the course of pregnancy and may play a role in ovarian arrest, reflected by the simultaneous decrease of anti-mullerian-hormone (AMH) levels. The aim of the study was to investigate AMH and follistatin levels during the hormonal window at the beginning of pregnancy. Since both parameters are described as deregulated in polycystic ovarian syndrome (PCOS), subgroup analysis of PCOS patients may additionally elucidate their interplay and effects on ovarian activity. METHODS: Serum samples were retrospectively analyzed using the AMH Gen II ELISA and the Human Follistatin Quantikine ELISA Kit. Samples were collected longitudinally from 57 patients (32 with PCOS and 25 controls) before conception and during the first trimester. In 18 patients, measurements from the early and the late first trimester were available. Potential associations of AMH and follistatin levels with PCOS-related parameters were compared between the subgroups as well as longitudinally before and in the first trimester of pregnancy. For statistical analysis, the Spearman's correlation, Wilcoxon test, t test, Friedman test and multiple linear regression analysis was performed. RESULTS: In contrast to AMH, follistatin levels differed not between controls and PCOS patients before and in pregnancy. In both subgroups, AMH levels significantly decreased and follistatin levels significantly increased in longitudinally performed measurements before conceiving and in the first trimester of pregnancy. CONCLUSION: Follistatin levels are not suited as a biomarker for PCOS, but could be involved in suppressing ovarian activity, as reflected by AMH levels at the beginning of pregnancy.

Soluble CEACAM1 and CEACAM6 are differently expressed in blood serum of pregnant women during normal pregnancy.

PROBLEM: CEACAM1 and CEACAM6 belong to the carcinoembryonic antigen (CEA) family and may play an immune-modulatory role during pregnancy. The aim of the study was to determine the blood serum levels of soluble CEACAM1 and CEACAM6 over the course of pregnancy and postpartum. METHOD OF STUDY: CEACAM1 and CEACAM6 levels were determined with customized in-house Sandwich-enzyme-linked immunosorbent assay (ELISA) systems. The study population (n=125) was divided into four groups according to the pregnancy trimester and postpartum. Additionally, samples of non-pregnant women (n=14) were analyzed. RESULTS: Serum levels of CEACAM1 in healthy pregnant women were much lower than in non-pregnant women, a difference not seen for CEACAM6. Comparison between the trimesters and postpartum revealed a significant difference in CEACAM1 serum levels. The highest CEACAM1 levels were detected in third trimester. These levels were statistically significantly different from the CEACAM1 levels in first trimester and second trimester. The lowest levels were observed in the second trimester. Postpartum CEACAM1 serum concentrations were slightly lower than in the third trimester, but higher than in the first trimester and significantly higher compared to levels in the second trimester. CONCLUSION: Decreased concentration of CEACAM1 during the pregnancy suggests its regulatory role in the immune tolerance during the course of pregnancy.

Impact of maternal serum levels of Visfatin, AFP, PAPP-A, sFlt-1 and PlGF at 11-13 weeks gestation on small for gestational age births.

OBJECTIVE: Investigating potential value of maternal serum Visfatin, sFlt-1, PlGF, AFP, PAPP-A levels at first trimester for prediction of small for gestational age (SGA) at birth. METHODS: Measurements were performed in 20 SGA and 65 control cases. Logistic regression analysis adjusted for age and weeks of pregnancy at data collection was performed to estimate odds ratios (OR), 95% confidence intervals (95% CI) and p values separately for each potential predictor. A multiple regression model was used to assess the impact of all the promising predictors adjusted for each other. Receiver operating characteristic (ROC) analysis was used to indicate the ability to discriminate between SGA cases and controls. RESULTS: There was an association of serum PlGF levels (OR 0.53 per interquartile range [IQR] increase in PlGF; 95% CI 0.24-1.16), sFlt-1/PlGF ratio (OR 1.42 per IQR increase in sFlt-1/PlGF; 95% CI 1.03-1.96), serum Visfatin levels (OR 0.31 per IQR increase in Visfatin; 95% CI 0.10-0.95) and smoking (OR 4.24; 95% CI 1.10-16.37) with SGA at birth. CONCLUSIONS: Associations between SGA and lower PlGF, Visfatin levels as well as increased sFlt-1/PlGF ratio and smoking status were detected which may contribute to predict SGA.

Placental-Specific Overexpression of sFlt-1 Alters Trophoblast Differentiation and Nutrient Transporter Expression in an IUGR Mouse Model.

Since it is known that placental overexpression of the human anti-angiogenic molecule sFlt-1, the main candidate in the progression of preeclampsia, lead to intrauterine growth restriction (IUGR) in mice by lentiviral transduction of mouse blastocysts, we hypothesize that sFlt-1 influence placental morphology and physiology resulting in fetal IUGR. We therefore examined the effect of sFlt-1 on placental morphology and physiology at embryonic day 18.5 with histologic and morphometric analyses, transcript analyses, immunoblotting, and methylation studies. Interestingly, placental overexpression of sFlt-1 leads to IUGR in the fetus and results in lower placental weights. Moreover, we observed altered trophoblast differentiation with reduced expression of IGF2, resulting in a smaller placenta, a smaller labyrinth, and the loss of glycogen cells in the junctional zone. Changes in IGF2 are accompanied by small changes in its DNA methylation, whereas overall DNA methylation is unaffected. In addition, the expression of placental nutrient transporters, such as the glucose diffusion channel Cx26, is decreased. In contrast, the expression of the fatty acid transporter CD36 and the cholesterol transporter ABCA1 is significantly increased. In conclusion, placental sFlt-1 overexpression resulted in a reduction in the differentiation of the spongiotrophoblast into glycogen cells. These findings of a reduced exchange area of the labyrinth and glycogen stores, as well as decreased expression of glucose transporter, could contribute to the intrauterine growth restriction phenotype. All of these factors change the intrauterine availability of nutrients. Thus, we speculate that the alterations triggered by increased anti-angiogenesis strongly affect fetal outcome and programming. J. Cell. Biochem. 118: 1316-1329, 2017. © 2016 Wiley Periodicals, Inc.

Genome-wide methylation analysis of retrocopy-associated CpG islands and their genomic environment.

Gene duplication by retrotransposition, i.e., the reverse transcription of an mRNA and integration of the cDNA into the genome, is an important mechanism in evolution. Based on whole-genome bisulfite sequencing of monocyte DNA, we have investigated the methylation state of all CpG islands (CGIs) associated with a retrocopy (n = 1,319), their genomic environment, as well as the CGIs associated with the ancestral genes. Approximately 10% of retrocopies are associated with a CGI. Whereas almost all CGIs of the human genome are unmethylated, 68% of the CGIs associated with a retrocopy are methylated. In retrocopies resulting from multiple retrotranspositions of the same ancestral gene, the methylation state of the CGI often differs. There is a strong positive correlation between the methylation state of the CGI/retrocopy and their genomic environment, suggesting that the methylation state of the integration site determined the methylation state of the CGI/retrocopy, or that methylation of the retrocopy by a host defense mechanism has spread into the adjacent regions. Only a minor fraction of CGI/retrocopies (n = 195) has intermediate methylation levels. Among these, the previously reported CGI/retrocopy in intron 2 of the RB1 gene (PPP1R26P1) as well as the CGI associated with the retrocopy RPS2P32 identified in this study carry a maternal methylation imprint. In conclusion, these findings shed light on the evolutionary dynamics and constraints of DNA methylation.

CCN1 (CYR61) and CCN3 (NOV) signaling drives human trophoblast cells into senescence and stimulates migration properties.

During placental development, continuous invasion of trophoblasts into the maternal compartment depends on the support of proliferating extravillous trophoblasts (EVTs). Unlike tumor cells, EVTs escape from the cell cycle before invasion into the decidua and spiral arteries. This study focused on the regulation properties of glycosylated and non-glycosylated matricellular CCN1 and CCN3, primarily for proliferation control in the benign SGHPL-5 trophoblast cell line, which originates from the first-trimester placenta. Treating SGHPL-5 trophoblast cells with the glycosylated forms of recombinant CCN1 and CCN3 decreased cell proliferation by bringing about G0/G1 cell cycle arrest, which was accompanied by the upregulation of activated Notch-1 and its target gene p21. Interestingly, both CCN proteins increased senescence-associated ß-galactosidase activity and the expression of the senescence marker p16. The migration capability of SGHPL-5 cells was mostly enhanced in response to CCN1 and CCN3, by the activation of FAK and Akt kinase but not by the activation of ERK1/2. In summary, both CCN proteins play a key role in regulating trophoblast cell differentiation by inducing senescence and enhancing migration properties. Reduced levels of CCN1 and CCN3, as found in early-onset preeclampsia, could contribute to a shift from invasive to proliferative EVTs and may explain their shallow invasion properties in this disease.