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.

Is Afamin a novel biomarker for gestational diabetes mellitus? A pilot study.

BACKGROUND: In search of potential early biomarkers for timely prediction of gestational diabetes mellitus (GDM), we focused on afamin, a vitamin E-binding protein in human plasma.. Afamin plays a role in anti-apoptotic cellular processes related to oxidative stress and is associated with insulin resistance and other features of metabolic syndrome. During uncomplicated pregnancy its serum concentrations increase linearly. The aim of this study was to investigate the suitability of afamin as early marker for predicting GDM. METHODS: In a first-trimester cohort from a prospective observational study of adverse pregnancy outcomes we secondarily analyzed afamin concentrations in 59 patients diagnosed with GDM and 51 controls. Additionally, afamin concentrations were cross-sectionally examined in a mid-trimester cohort of 105 women and compared with results from a simultaneously performed oral glucose tolerance test (OGTT). Subgroup analysis comparing patients treated with either insulin (iGDM) or dietary intervention (dGDM) was performed in both cohorts. Patients were recruited at the University Hospital Essen, Germany, between 2003 and 2016. RESULTS: Results were adjusted for body-mass-index (BMI) and gestational age. First and mid-trimester cohorts yielded significantly elevated afamin concentrations in patients with pathological OGTT compared to patients without GDM (first trimester cohort: mean, 113.4 mg/l; 95% CI, 106.4-120.5 mg/l and 87.2 mg/l; 95% CI, 79.7-94.7 mg/l; mid-trimester cohort: mean, 182.9 mg/l; 95% CI, 169.6-196.2 mg/l and 157.3 mg/l; 95% CI, 149.1-165.4 mg/l, respectively). In the first-trimester cohort, patients developing iGDM later in pregnancy presented with significantly higher afamin concentrations compared to patients developing dGDM and compared to patients without GDM. In the mid-trimester cohort, mean concentrations of afamin differed significantly between patients with dGDM compared to controls and between patients with iGDM and controls. Patients with iGDM showed only slightly higher afamin levels compared to patients with dGDM. CONCLUSION: Afamin may serve as a new early biomarker for pathological glucose metabolism during pregnancy. Further research is needed to determine afamin's concentrations during pregnancy, its predictive value for early detection of pregnancies at high risk to develop GDM and its diagnostic role during the second trimester.

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.

Afamin: an early predictor of preeclampsia.

PURPOSE: Oxidative stress is involved in the pathogenesis of hypertensive disorders such as preeclampsia (PE) and associated with the human vitamin E-binding protein afamin. The aim of this study was, therefore, to analyse afamin in the first trimester of patients developing PE later in pregnancy and in control subjects without pregnancy complications. METHODS: In this retrospective study, 137 serum samples from the first trimester of pregnancy were analysed in a case-control study design. 39 patients developed PE (10 patients with early-onset and 29 patients with late onset disease) and 98 women had an uncomplicated pregnancy. Mann-Whitney U test, t test, logistic regression and ROC analyses were performed for statistical evaluation. RESULTS: Pregnant women developing PE presented with higher afamin concentrations in the first trimester [median 101.81 mg/L; interquartile range (IQR) 88.94-113.26] compared to subjects with uncomplicated pregnancy (median 86.40; IQR 75.26-96.92; p < 0.001). After adjusting for confounders, the odds ratio per afamin standard deviation was 1.60 (95% CI: 1.04-2.58; p = 0.04). An afamin threshold concentration of 87.8 mg/L exhibited the best sensitivity (79.5%) and specificity (57.1%) in predicting PE. Subgroup analysis of early- and late-onset disease resulted in substantially higher afamin concentrations in women with developing late-onset PE compared to controls (p < 0.001) with an odds ratio per afamin standard deviation of 1.62 (95% CI: 0.98-2.70; p = 0.06). CONCLUSIONS: Serum afamin concentrations are elevated in the first trimester among patients developing PE compared to controls. Substantial differences were observed mainly among patients with late-onset PE.

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.

Increased Rat Placental Fatty Acid, but Decreased Amino Acid and Glucose Transporters Potentially Modify Intrauterine Programming.

Regulation of placental nutrient transport significantly affects fetal development and may modify intrauterine growth restriction (IUGR) and fetal programming. We hypothesized that placental nutrient transporters are differentially affected both by utero-placental insufficiency and prenatal surgical stress. Pregnant rats underwent bilateral uterine artery and vein ligation (LIG), sham operation (SOP) or no operation (controls, C) on gestational day E19. Placentas were obtained by caesarean section 4 h (LIG, n=20 placentas; SOP, n=24; C, n=12), 24 h (LIG, n=28; SOP, n=20; C, n=12) and 72 h (LIG, n=20; SOP, n=20; C, n=24) after surgery. Gene and protein expression of placental nutrient transporters for fatty acids (h-FABP, CD36), amino acids (SNAT1, SNAT2) and glucose (GLUT-1, Connexin 26) were examined by qRT-PCR, western blot and immunohistochemistry. Interestingly, the mean protein expression of h-FABP was doubled in placentas of LIG and SOP animals 4, 24 (SOP significant) and 72 h (SOP significant) after surgery. CD36 protein was significantly increased in LIG after 72 h. SNAT1 and SNAT2 protein and gene expressions were significantly reduced in LIG and SOP after 24 h. Further significantly reduced proteins were GLUT-1 in LIG (4 h, 72 h) and SOP (24 h), and Connexin 26 in LIG (72 h). In conclusion, placental nutrient transporters are differentially affected both by reduced blood flow and stress, probably modifying the already disturbed intrauterine milieu and contributing to IUGR and fetal programming. Increased fatty acid transport capacity may affect energy metabolism and could be a compensatory reaction with positive effects on brain development. J. Cell. Biochem. 117: 1594-1603, 2016. © 2015 Wiley Periodicals, Inc.

Physiological roles of connexins and pannexins in reproductive organs.

Reproductive organs are complex and well-structured tissues essential to perpetuate the species. In mammals, the male and female reproductive organs vary on their organization, morphology and function. Connectivity between cells in such tissues plays pivotal roles in organogenesis and tissue functions through the regulation of cellular proliferation, migration, differentiation and apoptosis. Connexins and pannexins can be seen as major regulators of these physiological processes. In the present review, we assembled several lines of evidence demonstrating that these two families of proteins are essential for male and female reproduction.

Oxygen Sensitivity of Placental Trophoblast Connexins 43 and 46: A Role in Preeclampsia?

Several gap junction connexins have been shown to be essential for appropriate placental development and function. It is known that the expression and distribution of connexins change in response to environmental oxygen levels. The placenta develops under various oxygen levels, beginning at a low oxygen tension of approximately 2% and increasing to a tension of 8% after the onset of the uteroplacental circulation. Moreover, it has been shown that during preeclampsia (PE) placentas are subjected to chronic hypoxia. Therefore, we investigated oxygen sensitivity of placental connexins 43 and 46. Using the trophoblast cell line Jar, we demonstrated that the expression of connexin43 increased during acute hypoxia but decreased during chronic hypoxia. Chronic hypoxia resulted in the translocation of connexin43 from the membrane to the cytoplasm and in a reduction in its communication properties. In contrast, the expression of connexin46 was down-regulated during chronic hypoxia and was translocated from perinuclear areas to the cell membrane. Hypoxia-inducible factor (HIF) knockdown showed that the translocation of connexin43 but not that of connexin46 was HIF-2α dependent and was mediated by phosphoinositide 3-kinase. The up-regulation of connexin43 in combination with the down-regulation of connexin46 was confirmed in placental explants cultivated under low oxygen and in placentas with early-onset PE. Taken together, in Jar cells, placental connexins 43 and 46 are regulated during periods of low oxygen in opposite manners. The oxygen sensing of connexins in the trophoblast may play a role in physiological and pathophysiological oxygen conditions and thus may contribute to PE.

Reduced Gene Dosage of Tfap2c Impairs Trophoblast Lineage Differentiation and Alters Maternal Blood Spaces in the Mouse Placenta.

Tfap2c is required for placental development and trophoblast stem cell maintenance. Deletion of Tfap2c results in early embryonic loss because of failure in placental development. We evaluated the effect of reduced Tfap2c expression on fetal outcome and placental development. Sixty percent of the heterozygous mice were lost directly after birth. Labyrinthine differentiation was impaired, as indicated by enhanced proliferation and inclusions of cobblestone-shaped cell clusters characterized by expression of Tfap2c and glycogen stores. Moreover, expression of marker genes such as Cdx2, Eomes, Gata3, and Ascl2 are decreased in the spongiotrophoblast and indicate a lowered stem cell potential. On Day 18.5 postcoitum, the labyrinth layer of Tfap2c(+/-) placentas exhibited massive hemorrhages in the maternal blood spaces; these hemorrhages might have contributed to the significantly reduced number of live-born pups. These morphological alterations were accompanied by a shift toward sinusoidal trophoblast giant cells as the cell subpopulation lining the maternal sinusoids and toward reduction in expression of the prolactin gene family member Prl2c2, a finding characteristic of the spiral arteries lining trophoblast cells. The trophoblast stem cells heterozygous for Tfap2c exhibited a reduction in the expression level of stem cell markers and in their proliferation and differentiation capacity but did not exhibit changes in marker genes of the trophoblast giant cell lineage. Taken together, these findings indicate that a reduction in the gene dosage of placental Tfap2c leads to morphological changes in the labyrinth at midgestation and in the maternal blood spaces during late pregnancy.

The role of the CCN family of proteins in female reproduction.

The CCN family of proteins consists of six high homologous matricellular proteins which act predominantly by binding to heparin sulphate proteoglycan and a variety of integrins. Interestingly, CCN proteins are regulated by ovarian steroid hormones and are able to adapt to changes in oxygen concentration, which is a necessary condition for successful implantation. CCN1 is involved in processes of angiogenesis within reproductive systems, thereby potentially contributing to diseases such as endometriosis and disturbed angiogenesis in the placenta and fetus. In the ovary, CCN2 is the key factor for follicular development, ovulation and corpora luteal luteolysis, and its deletion leads to fertility defects. CCN1, CCN2 and CCN3 seem to be regulators for human trophoblast proliferation and migration, but with CCN2 acting as a counterweight. Alterations in the expression of these three proteins could contribute to the shallow invasion properties observed in preeclampsia. Little is known about the role of CCN4-6 in the reproductive organs. The ability of CCN1, CCN2 and CCN3 to interact with numerous receptors enables them to adapt their biological function rapidly to the continuous remodelling of the reproductive organs and in the development of the placenta. The CCN proteins mediate their specific cell physiological function through the receptor type of their binding partner followed by a defined signalling cascade. Because of their partly overlapping expression patterns, they could act in a concert synergistically or in an opposite way within the reproductive organs. Imbalances in their expression levels are correlated to different human reproductive diseases, such as endometriosis and preeclampsia.

Maternal serum copeptin, MR-proANP and procalcitonin levels at 11-13 weeks gestation in the prediction of preeclampsia.

PURPOSE: We investigated the potential value of maternal serum copeptin, midregional proatrial natriuretic peptide (MR-proANP) and Procalcitonin (PCT) levels at 11-13 weeks' gestation in the prediction of preeclampsia (PE) in a case-control study. MATERIALS AND METHODS: Maternal serum concentration of copeptin, MR-proANP and PCT were measured at 11-13 weeks' gestation in cases of PE (n = 35) and controls (n = 100). The PE group was divided into early-onset PE (EO-PE) and late-onset PE (LO-PE). From the regression model, the value in each case and control was expressed as a multiple of the expected median (MoM). The Mann-Whitney test was used to determine the significance of differences in the median MoM in each outcome group from that in the controls. RESULTS: In the PE group, compared to controls, maternal serum concentrations of copeptin, MR-proANP and PCT were not significantly different. CONCLUSION: The maternal serum copeptin, MR-proANP and PCT levels are higher in EO-PE and LO-PE patients, but the difference is not significant. Thus, their levels in first trimester are not proven to be effective markers to screen for PE.

Impact of 2 years of COVID-19 pandemic on preterm birth: Experience from a tertiary center of obstetrics in western Germany.

OBJECTIVE: To compare preterm birth rates and reasons before and during the COVID-19-pandemic using a monocentric, retrospective study. METHODS: Univariate analysis identified differences in rates and reasons for preterm birth and neonatal outcomes between the pre-pandemic period (January 1, 2018 to December 31, 2019) and during the pandemic (January 1, 2020 to December 31, 2021) among all births at our tertiary obstetrical center, the University Hospital of Essen. RESULTS: The cohort consisted of 6086 deliveries with 593 liveborn preterm singletons. During the pandemic, the incidence of preterm birth decreased (10.7% vs. 8.6%; odds ratio [OR] 0.79; 95% confidence interval [CI] 0.66-0.93). Spontaneous preterm birth (43.2% vs. 52.3%; OR 1.47; 95% CI 1.05-2.03), and placenta accreta spectrum disorder (3.7% vs. 8.2%; OR 2.36; 95% CI 1.15-4.84) were more common reasons for preterm birth. Placental dysfunction was a less common reason (34.1% vs. 24.3%; OR 0.62; 95% CI 0.43-0.90). Incidences of preterm premature rupture of membranes (28.13% vs. 40.25%; OR 1.72; 95% CI 1.12-2.43) and oligo-/anhydramnios (3.98% vs. 7.88%; OR 2.06; 95% CI 1.02-4.21) increased. Iatrogenic preterm birth decreased (54.5% vs. 49.5%; OR 0.81; 95% CI 0.58-1.13). Stillbirth rates did not change significantly. Among term births, there were fewer spontaneous deliveries (71.0% vs. 65.8%; OR 0.78; 95% CI 0.69-0.88), and more elective (12.3% vs. 15.1%; OR 1.26; 95% CI 1.07-1.50) and unplanned (9.3% vs. 10.9%; OR 1.19; 95% CI 0.98-1.45) cesarean sections. During the pandemic, more term newborns were admitted to neonatal intensive care (1.4% vs. 2.5%; OR 1.86; 95% CI 1.20-2.88). CONCLUSION: Our results, in line with data from other high-income countries, suggest that the likely reason for the decreased preterm birth rates is the underdiagnosis of pregnancy complications.

The Importance of Vaccination, Variants and Time Point of SARS-CoV-2 Infection in Pregnancy for Stillbirth and Preterm Birth Risk: An Analysis of the CRONOS Register Study.

Background: The risk of preterm birth (PTB) and stillbirth increases after a SARS-CoV-2 infection during gestation. We aimed to estimate the risk depending on gestational age at infection (early <28 + 0 and late ≥28 weeks of gestation, WoG), virus variants, severity of infection, and vaccination. Methods: PTB was divided into early PTB (<32 + 0) and late PTB (32 + 0-36 + 6 WoG). The prospective register COVID-19 Related Obstetrics and Neonatal Outcome Study (CRONOS) included 8032 pregnant women with a confirmed SARS-CoV-2 infection from 3 April 2020 to 31 December 2022, in Germany and Austria. Results: Stillbirth and early preterm births rates were higher during the Alpha (1.56% and 3.13%) and Delta (1.56% and 3.44%) waves than during the Omicron wave (0.53% and 1.39%). Early SARS-CoV-2 infection increased the risk for stillbirth (aRR 5.76, 95% CI 3.07-10.83) and early PTB before 32 + 0 (aRR, 6.07, 95% CI 3.65-10.09). Hospital admission increased the risks further, especially in the case of ICU admission. Vaccination against SARS-CoV-2 significantly reduced the risk of stillbirth (aRR 0.32, 95% CI 0.16-0.83). Conclusions: This multicentric prospective study shows an increased risk of stillbirth and preterm birth after infection early in pregnancy and therefore the importance of obstetrical surveillance thereafter. Vaccination offers effective protection.

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.

Peripheral lymphangiogenesis in mice depends on ectodermal connexin-26 (Gjb2).

In order to study the specific function of connexin-26 (Cx26, also known as gap junction beta-2 protein; Gjb2), we generated knockin mice that expressed either a floxed lacZ reporter or, after Cre-mediated deletion, connexin-32 (Cx32)-coding DNA, both driven by the endogenous Cx26 promoter. Heterozygous Cx26knock-inCx32 (Cx26KICx32) embryos developed normally until embryonic day 14.5 but died before birth with severe lymphedemas. Although the jugular lymph sacs were normally developed, these embryos had a strongly reduced dermal lymphatic capillary network. By analyses of ß-galactosidase reporter protein expression and lymphatic or blood endothelial-specific marker proteins, we demonstrated that Cx26 expression is temporally closely linked to lymphangiogenesis. No obvious phenotypic abnormalities were observed in Cx26KICx32 mice when Cre-mediated recombination was directed to mesenchyme or blood endothelium using the Prx1-Cre or Tie2-Cre mouse strains, respectively. By contrast, keratin-5-Cre-mediated replacement of Cx26 with Cx32 or deletion of both Cx26 alleles revealed severe lymphedemas similar to the general Cx26KICx32 phenotype. Thus, conditional ablation of Cx26 (loss of function) in ectoderm leads to partial disruption of lymphatic capillaries and embryonic death. We conclude that appropriate development of dermal lymphatic vessels in mice is dependent on the expression of Cx26 in the ectoderm.

Decidual angiogenesis and placental orientation are altered in mice heterozygous for a dominant loss-of-function Gja1 (connexin43) mutation.

Connexin43 (CX43), encoded by Gja1 in the mouse, is highly expressed in decidual cells and is known to be important for the transformation of stromal cells into the compact decidua and for neoangiogenesis. Here we investigated if the dominant Gja1(Jrt) mutation encoding CX43(G60S) in mice, which results in a phenotype resembling oculodentodigital dysplasia in humans, has an impact on decidualization, angiogenesis, and implantation. We found a reduced mean weight of fetuses at Gestational Day 17.5 in dams carrying this mutation, with the growth deficiency being independent of fetal genotype. Although the mutant implantation sites exhibited a reduction in CX43 protein, with most immunoreactivity being cytoplasmic, the decidua was morphologically intact at Embryonic Days 5.5 to 7.5. However, the mutation resulted in enhanced and irregular angiogenesis and an increased level of expression of the angiogenic factor-encoding genes Vegfa, Flt1, Kdr, and Fgf2 as well as the prolactin-related gene Prl6a. Moreover, immunolocalization of VEGFA, FLT1, and KDR revealed a homogeneous distribution pattern in the mesometrial as well as antimesometrial decidua of the mutants. Most obviously, uterine NK cells are drastically diminished in the mesometrial decidua of the mutant mice. Invasion of ectoplacental cone cells was disoriented, and placentation was established more laterally in the implantation chambers. It was concluded that the CX43(G60S) mutant impairs control of decidual angiogenesis, leading to dysmorphic placentation and fetal growth restriction. This phenomenon could contribute to the reduced fetal weights and viability of pups born of Gja1(Jrt)/+ dams.

CCN3 regulates proliferation and migration properties in Jeg3 trophoblast cells via ERK1/2, Akt and Notch signalling.

Previous studies showed that CCN3 is deregulated in early-onset pre-eclampsia (PE), a pregnancy disease associated with impaired trophoblast invasion, which leads to reduced fetal oxygen and nutrition support. Recently, we identified the glycosylated (g-CCN3) and the non-glycosylated (ng-CCN3) form of matricellular CCN3 as key factors in regulation of trophoblast proliferation and invasion. While Jeg3 cells revealed a decreased proliferation upon stimulation with both forms of CCN3, enhanced migration and invasion properties were only found for ng-CCN3. Here, we focused on the signalling cascades mitogen-activated protein kinase (MAPK), PI3 kinase/Akt and Notch/p21 for mediating the dual function of CCN3 on trophoblast proliferation versus migration in Jeg3 cells upon stimulation with g- and ng-recombinant CCN3 (g/ng-rCCN3). Analysis of the CCN3-mediated signalling pathways showed that ng-rCCN3 stimulated migration properties by activating the Akt as well as the MAPK pathway. Moreover, cell migration stimulated by ng-rCCN3 was mediated via Akt and integrin α5ß1 but not the antiproliferative effect of CCN3. There was evidence that the Notch pathway might contribute to the antiproliferative properties of both forms of CCN3 by an increase in Notch1 expression and its target gene, the cell cycle inhibitor p21. Our data showed that the presence of both forms of CCN3 is accompanied by a balance of trophoblast proliferation and migration/invasion properties, which are triggered by different signalling pathways. Thus, a deregulated expression of g/ng-CCN3 could lead to an imbalance in proliferation versus invasion, and might contribute to the shallow trophoblast invasion observed in PE.