Exosomal small RNA profiling in first-trimester maternal blood explores early molecular pathways of preterm preeclampsia.

Introduction: Preeclampsia (PE) is a severe obstetrical syndrome characterized by new-onset hypertension and proteinuria and it is often associated with fetal intrauterine growth restriction (IUGR). PE leads to long-term health complications, so early diagnosis would be crucial for timely prevention. There are multiple etiologies and subtypes of PE, and this heterogeneity has hindered accurate identification in the presymptomatic phase. Recent investigations have pointed to the potential role of small regulatory RNAs in PE, and these species, which travel in extracellular vesicles (EVs) in the circulation, have raised the possibility of non-invasive diagnostics. The aim of this study was to investigate the behavior of exosomal regulatory small RNAs in the most severe subtype of PE with IUGR. Methods: We isolated exosomal EVs from first-trimester peripheral blood plasma samples of women who later developed preterm PE with IUGR (n=6) and gestational age-matched healthy controls (n=14). The small RNA content of EVs and their differential expression were determined by next-generation sequencing and further validated by quantitative real-time PCR. We also applied the rigorous exceRpt bioinformatics pipeline for small RNA identification, followed by target verification and Gene Ontology analysis. Results: Overall, >2700 small RNAs were identified in all samples and, of interest, the majority belonged to the RNA interference (RNAi) pathways. Among the RNAi species, 16 differentially expressed microRNAs were up-regulated in PE, whereas up-regulated and down-regulated members were equally found among the six identified Piwi-associated RNAs. Gene ontology analysis of the predicted small RNA targets showed enrichment of genes in pathways related to immune processes involved in decidualization, placentation and embryonic development, indicating that dysregulation of the induced small RNAs is connected to the impairment of immune pathways in preeclampsia development. Finally, the subsequent validation experiments revealed that the hsa_piR_016658 piRNA is a promising biomarker candidate for preterm PE associated with IUGR. Discussion: Our rigorously designed study in a homogeneous group of patients unraveled small RNAs in circulating maternal exosomes that act on physiological pathways dysregulated in preterm PE with IUGR. Therefore, our small RNA hits are not only suitable biomarker candidates, but the revealed biological pathways may further inform us about the complex pathology of this severe PE subtype.

Decreased Expression of Placental Proteins in Recurrent Pregnancy Loss: Functional Relevance and Diagnostic Value.

Miscarriages affect 50-70% of all conceptions and 15-20% of clinically recognized pregnancies. Recurrent pregnancy loss (RPL, ≥2 miscarriages) affects 1-5% of recognized pregnancies. Nevertheless, our knowledge about the etiologies and pathophysiology of RPL is incomplete, and thus, reliable diagnostic/preventive tools are not yet available. Here, we aimed to define the diagnostic value of three placental proteins for RPL: human chorionic gonadotropin free beta-subunit (free-ß-hCG), pregnancy-associated plasma protein-A (PAPP-A), and placental growth factor (PlGF). Blood samples were collected from women with RPL (n = 14) and controls undergoing elective termination of pregnancy (n = 30) at the time of surgery. Maternal serum protein concentrations were measured by BRAHMS KRYPTOR Analyzer. Daily multiple of median (dMoM) values were calculated for gestational age-specific normalization. To obtain classifiers, logistic regression analysis was performed, and ROC curves were calculated. There were differences in changes of maternal serum protein concentrations with advancing healthy gestation. Between 6 and 13 weeks, women with RPL had lower concentrations and dMoMs of free ß-hCG, PAPP-A, and PlGF than controls. PAPP-A dMoM had the best discriminative properties (AUC = 0.880). Between 9 and 13 weeks, discriminative properties of all protein dMoMs were excellent (free ß-hCG: AUC = 0.975; PAPP-A: AUC = 0.998; PlGF: AUC = 0.924). In conclusion, free-ß-hCG and PAPP-A are valuable biomarkers for RPL, especially between 9 and 13 weeks. Their decreased concentrations indicate the deterioration of placental functions, while lower PlGF levels indicate problems with placental angiogenesis after 9 weeks.

Classification of preeclampsia according to molecular clusters with the goal of achieving personalized prevention.

The prevention of pre-eclampsia is difficult due to the syndromic nature and multiple underlying mechanisms of this severe complication of pregnancy. The current clinical distinction between early- and late-onset disease, although clinically useful, does not reflect the true nature and complexity of the pathologic processes leading to pre-eclampsia. The current gaps in knowledge on the heterogeneous molecular pathways of this syndrome and the lack of adequate, specific diagnostic methods are major obstacles to early screening and tailored preventive strategies. The development of novel diagnostic tools for detecting the activation of the identified disease pathways would enable early, accurate screening and personalized preventive therapies. We implemented a holistic approach that includes the utilization of different proteomic profiling methods of maternal plasma samples collected from various ethnic populations and the application of systems biology analysis to plasma proteomic, maternal demographic, clinical characteristic, and placental histopathologic data. This approach enabled the identification of four molecular subclasses of pre-eclampsia in which distinct and shared disease mechanisms are activated. The current review summarizes the results and conclusions from these studies and the research and clinical implications of our findings.

Proteomic profile of extracellular vesicles in maternal plasma of women with fetal death.

OBJECTIVES: Fetal death is a complication of pregnancy caused by multiple etiologies rather than being the end-result of a single disease process. Many soluble analytes in the maternal circulation, such as hormones and cytokines, have been implicated in its pathophysiology. However, changes in the protein content of extracellular vesicles (EVs), which could provide additional insight into the disease pathways of this obstetrical syndrome, have not been examined. This study aimed to characterize the proteomic profile of EVs in the plasma of pregnant women who experienced fetal death and to evaluate whether such a profile reflected the pathophysiological mechanisms of this obstetrical complication. Moreover, the proteomic results were compared to and integrated with those obtained from the soluble fraction of maternal plasma. METHODS: This retrospective case-control study included 47 women who experienced fetal death and 94 matched, healthy, pregnant controls. Proteomic analysis of 82 proteins in the EVs and the soluble fractions of maternal plasma samples was conducted by using a bead-based, multiplexed immunoassay platform. Quantile regression analysis and random forest models were implemented to assess differences in the concentration of proteins in the EV and soluble fractions and to evaluate their combined discriminatory power between clinical groups. Hierarchical cluster analysis was applied to identify subgroups of fetal death cases with similar proteomic profiles. A p-value of <.05 was used to infer significance, unless multiple testing was involved, with the false discovery rate controlled at the 10% level (q < 0.1). All statistical analyses were performed by using the R statistical language and environment-and specialized packages. RESULTS: Nineteen proteins (placental growth factor, macrophage migration inhibitory factor, endoglin, regulated upon activation normal T cell expressed and presumably secreted (RANTES), interleukin (IL)-6, macrophage inflammatory protein 1-alpha, urokinase plasminogen activator surface receptor, tissue factor pathway inhibitor, IL-8, E-Selectin, vascular endothelial growth factor receptor 2, pentraxin 3, IL-16, galectin-1, monocyte chemotactic protein 1, disintegrin and metalloproteinase domain-containing protein 12, insulin-like growth factor-binding protein 1, matrix metalloproteinase-1(MMP1), and CD163) were found to have different plasma concentrations (of an EV or a soluble fraction) in women with fetal death compared to controls. There was a similar pattern of change for the dysregulated proteins in the EV and soluble fractions and a positive correlation between the log2-fold changes of proteins significant in either the EV or the soluble fraction (ρ = 0.89, p < .001). The combination of EV and soluble fraction proteins resulted in a good discriminatory model (area under the ROC curve, 82%; sensitivity, 57.5% at a 10% false-positive rate). Unsupervised clustering based on the proteins differentially expressed in either the EV or the soluble fraction of patients with fetal death relative to controls revealed three major clusters of patients. CONCLUSION: Pregnant women with fetal death have different concentrations of 19 proteins in the EV and soluble fractions compared to controls, and the direction of changes in concentration was similar between fractions. The combination of EV and soluble protein concentrations revealed three different clusters of fetal death cases with distinct clinical and placental histopathological characteristics.

Molecular subclasses of preeclampsia characterized by a longitudinal maternal proteomics study: distinct biomarkers, disease pathways and options for prevention.

OBJECTIVES: The heterogeneous nature of preeclampsia is a major obstacle to early screening and prevention, and a molecular taxonomy of disease is needed. We have previously identified four subclasses of preeclampsia based on first-trimester plasma proteomic profiles. Herein, we expanded this approach by using a more comprehensive panel of proteins profiled in longitudinal samples. METHODS: Proteomic data collected longitudinally from plasma samples of women who developed preeclampsia (n=109) and of controls (n=90) were available from our previous report on 1,125 proteins. Consensus clustering was performed to identify subgroups of patients with preeclampsia based on data from five gestational-age intervals by using select interval-specific features. Demographic, clinical, and proteomic differences among clusters were determined. Differentially abundant proteins were used to identify cluster-specific perturbed KEGG pathways. RESULTS: Four molecular clusters with different clinical phenotypes were discovered by longitudinal proteomic profiling. Cluster 1 involves metabolic and prothrombotic changes with high rates of early-onset preeclampsia and small-for-gestational-age neonates; Cluster 2 includes maternal anti-fetal rejection mechanisms and recurrent preeclampsia cases; Cluster 3 is associated with extracellular matrix regulation and comprises cases of mostly mild, late-onset preeclampsia; and Cluster 4 is characterized by angiogenic imbalance and a high prevalence of early-onset disease. CONCLUSIONS: This study is an independent validation and further refining of molecular subclasses of preeclampsia identified by a different proteomic platform and study population. The results lay the groundwork for novel diagnostic and personalized tools of prevention.

Early pathways, biomarkers, and four distinct molecular subclasses of preeclampsia: The intersection of clinical, pathological, and high-dimensional biology studies.

Preeclampsia is a syndromic disease of the mother, fetus, and placenta. The main limitation in early and accurate diagnosis of preeclampsia is rooted in the heterogeneity of this syndrome as reflected by diverse molecular pathways, symptoms, and clinical outcomes. Gaps in our knowledge preclude successful early diagnosis, personalized treatment, and prevention. The advent of "omics" technologies and systems biology approaches addresses this problem by identifying the molecular pathways associated with the underlying mechanisms and clinical phenotypes of preeclampsia. Here, we provide a brief overview on how the field has progressed, focusing on studies utilizing state-of-the-art transcriptomics and proteomics methods. Moreover, we summarize our systems biology studies involving maternal blood proteomics and placental transcriptomics, which identified early maternal and placental disease pathways and showed that their interaction influences the clinical presentation of preeclampsia. We also present an analysis of maternal blood proteomics data which revealed distinct molecular subclasses of preeclampsia and their molecular mechanisms. Maternal and placental disease pathways behind these subclasses are similar to those recently reported in studies on the placental transcriptome. These findings may promote the development of novel diagnostic tools for the distinct subtypes of preeclampsia syndrome, enabling early detection and personalized follow-up and tailored care of patients.

Prediction of preeclampsia throughout gestation with maternal characteristics and biophysical and biochemical markers: a longitudinal study.

BACKGROUND: The current approach to predict preeclampsia combines maternal risk factors and evidence from biophysical markers (mean arterial pressure, Doppler velocimetry of the uterine arteries) and maternal blood proteins (placental growth factor, soluble vascular endothelial growth factor receptor-1, pregnancy-associated plasma protein A). Such models require the transformation of biomarker data into multiples of the mean values by using population- and site-specific models. Previous studies have focused on a narrow window in gestation and have not included the maternal blood concentration of soluble endoglin, an important antiangiogenic factor up-regulated in preeclampsia. OBJECTIVE: This study aimed (1) to develop models for the calculation of multiples of the mean values for mean arterial pressure and biochemical markers; (2) to build and assess the predictive models for preeclampsia based on maternal risk factors, the biophysical (mean arterial pressure) and biochemical (placental growth factor, soluble vascular endothelial growth factor receptor-1, and soluble endoglin) markers collected throughout pregnancy; and (3) to evaluate how prediction accuracy is affected by the presence of chronic hypertension and gestational age. STUDY DESIGN: This longitudinal case-cohort study included 1150 pregnant women: women without preeclampsia with (n=49) and without chronic hypertension (n=871) and those who developed preeclampsia (n=166) or superimposed preeclampsia (n=64). Mean arterial pressure and immunoassay-based maternal plasma placental growth factor, soluble vascular endothelial growth factor receptor-1, and soluble endoglin concentrations were available throughout pregnancy (median of 5 observations per patient). A prior-risk model for preeclampsia was established by using Poisson regression based on maternal characteristics and obstetrical history. Next, multiple regression was used to fit biophysical and biochemical marker data as a function of maternal characteristics by using data collected at 8 to 15+6, 16 to 19+6, 20 to 23+6, 24 to 27+6, 28 to 31+6, and 32 to 36+6 week intervals, and observed values were converted into multiples of the mean values. Then, multivariable prediction models for preeclampsia were fit based on the biomarker multiples of the mean data and prior-risk estimates. Separate models were derived for overall, preterm, and term preeclampsia, which were evaluated by receiver operating characteristic curves and sensitivity at fixed false-positive rates. RESULTS: (1) The inclusion of soluble endoglin in prediction models for all preeclampsia, together with the prior-risk estimates, mean arterial pressure, placental growth factor, and soluble vascular endothelial growth factor receptor-1, increased the sensitivity (at a fixed false-positive rate of 10%) for early prediction of superimposed preeclampsia, with the largest increase (from 44% to 54%) noted at 20 to 23+6 weeks (McNemar test, P<.05); (2) combined evidence from prior-risk estimates and biomarkers predicted preterm preeclampsia with a sensitivity (false-positive rate, 10%) of 55%, 48%, 62%, 72%, and 84% at 8 to 15+6, 16 to 19+6, 20 to 23+6, 24 to 27+6, and 28 to 31+6 week intervals, respectively; (3) the sensitivity for term preeclampsia (false-positive rate, 10%) was 36%, 36%, 41%, 43%, 39%, and 51% at 8 to 15+6, 16 to 19+6, 20 to 23+6, 24 to 27+6, 28 to 31+6, and 32 to 36+6 week intervals, respectively; (4) the detection rate for superimposed preeclampsia among women with chronic hypertension was similar to that in women without chronic hypertension, especially earlier in pregnancy, reaching at most 54% at 20 to 23+6 weeks (false-positive rate, 10%); and (5) prediction models performed comparably to the Fetal Medicine Foundation calculators when the same maternal risk factors and biomarkers (mean arterial pressure, placental growth factor, and soluble vascular endothelial growth factor receptor-1 multiples of the mean values) were used as input. CONCLUSION: We introduced prediction models for preeclampsia throughout pregnancy. These models can be useful to identify women at risk during the first trimester who could benefit from aspirin treatment or later in pregnancy to inform patient management. Relative to prediction performance at 8 to 15+6 weeks, there was a substantial improvement in the detection rate for preterm and term preeclampsia by using data collected after 20 and 32 weeks' gestation, respectively. The inclusion of plasma soluble endoglin improves the early prediction of superimposed preeclampsia, which may be valuable when Doppler velocimetry of the uterine arteries is not available.

Epigenetic Dysregulation of Trophoblastic Gene Expression in Gestational Trophoblastic Disease.

Gestational trophoblastic diseases (GTDs) have not been investigated for their epigenetic marks and consequent transcriptomic changes. Here, we analyzed genome-wide DNA methylation and transcriptome data to reveal the epigenetic basis of disease pathways that may lead to benign or malignant GTDs. RNA-Seq, mRNA microarray, and Human Methylation 450 BeadChip data from complete moles and choriocarcinoma cells were bioinformatically analyzed. Paraffin-embedded tissues from complete moles and control placentas were used for tissue microarray construction, DNMT3B immunostaining and immunoscoring. We found that DNA methylation increases with disease severity in GTDs. Differentially expressed genes are mainly upregulated in moles while predominantly downregulated in choriocarcinoma. DNA methylation principally influences the gene expression of villous trophoblast differentiation-related or predominantly placenta-expressed genes in moles and choriocarcinoma cells. Affected genes in these subsets shared focal adhesion and actin cytoskeleton pathways in moles and choriocarcinoma. In moles, cell cycle and differentiation regulatory pathways, essential for trophoblast/placental development, were enriched. In choriocarcinoma cells, hormone biosynthetic, extracellular matrix-related, hypoxic gene regulatory, and differentiation-related signaling pathways were enriched. In moles, we found slight upregulation of DNMT3B protein, a developmentally important de novo DNA methylase, which is strongly overexpressed in choriocarcinoma cells that may partly be responsible for the large DNA methylation differences. Our findings provide new insights into the shared and disparate molecular pathways of disease in GTDs and may help in designing new diagnostic and therapeutic tools.

Decreased Expression of ZNF554 in Gliomas is Associated with the Activation of Tumor Pathways and Shorter Patient Survival.

Zinc finger protein 554 (ZNF554), a member of the Krüppel-associated box domain zinc finger protein subfamily, is predominantly expressed in the brain and placenta in humans. Recently, we unveiled that ZNF554 regulates trophoblast invasion during placentation and its decreased expression leads to the early pathogenesis of preeclampsia. Since ZNF proteins are immensely implicated in the development of several tumors including malignant tumors of the brain, here we explored the pathological role of ZNF554 in gliomas. We examined the expression of ZNF554 at mRNA and protein levels in normal brain and gliomas, and then we searched for genome-wide transcriptomic changes in U87 glioblastoma cells transiently overexpressing ZNF554. Immunohistochemistry of brain tissues in our cohort (n = 62) and analysis of large TCGA RNA-Seq data (n = 687) of control, oligodendroglioma, and astrocytoma tissues both revealed decreased expression of ZNF554 towards higher glioma grades. Furthermore, low ZNF554 expression was associated with shorter survival of grade III and IV astrocytoma patients. Overexpression of ZNF554 in U87 cells resulted in differential expression, mostly downregulation of 899 genes. The "PI3K-Akt signaling pathway", known to be activated during glioma development, was the most impacted among 116 dysregulated pathways. Most affected pathways were cancer-related and/or immune-related. Congruently, cell proliferation was decreased and cell cycle was arrested in ZNF554-transfected glioma cells. These data collectively suggest that ZNF554 is a potential tumor suppressor and its decreased expression may lead to the loss of oncogene suppression, activation of tumor pathways, and shorter survival of patients with malignant glioma.

Proteomic identification of Placental Protein 1 (PP1), PP8, and PP22 and characterization of their placental expression in healthy pregnancies and in preeclampsia.

INTRODUCTION: Placental Protein 1 (PP1), PP8, and PP22 were isolated from the placenta. Herein, we aimed to identify PP1, PP8, and PP22 proteins and their placental and trophoblastic expression patterns to reveal potential involvement in pregnancy complications. METHODS: We analyzed PP1, PP8, and PP22 proteins with LC-MS. We compared the placental behaviors of PP1, PP8, and PP22 to the predominantly placenta-expressed PP5/TFPI-2. Placenta-specificity scores were generated from microarray data. Trophoblasts were isolated from healthy placentas and differentiated; total RNA was isolated and subjected to microarray analysis. We assigned the placentas to the following groups: preterm controls, early-onset preeclampsia, early-onset preeclampsia with HELLP syndrome, term controls, and late-onset preeclampsia. After histopathologic examination, placentas were used for tissue microarray construction, immunostaining with anti-PP1, anti-PP5, anti-PP8, or anti-PP22 antibodies, and immunoscoring. RESULTS: PP1, PP8, and PP22 were identified as 'nicotinate-nucleotide pyrophosphorylase', 'serpin B6', and 'protein disulfide-isomerase', respectively. Genes encoding PP1, PP8, and PP22 are not predominantly placenta-expressed, in contrast with PP5. PP1, PP8, and PP22 mRNA expression levels did not increase during trophoblast differentiation, in contrast with PP5. PP1, PP8, and PP22 immunostaining were detected primarily in trophoblasts, while PP5 expression was restricted to the syncytiotrophoblast. The PP1 immunoscore was higher in late-onset preeclampsia, while the PP5 immunoscore was higher in early-onset preeclampsia. DISCUSSION: PP1, PP8, and PP22 are expressed primarily in trophoblasts but do not have trophoblast-specific regulation or functions. The distinct dysregulation of PP1 and PP5 expression in either late-onset or early-onset preeclampsia reflects different pathophysiological pathways in these preeclampsia subsets.

Placental Protein 13 (Galectin-13) Polarizes Neutrophils Toward an Immune Regulatory Phenotype.

Termed as galectin-13, placental protein 13 (PP13) is exclusively expressed in the placenta of anthropoid primates. Research on PP13 in normal and pathologic pregnancies show alteration of PP13 concentrations in pregnancy affected by preeclampsia or gestational diabetes. Galectins are also described as potent immunomodulators, and PP13 regulates T cell function in the placenta. Therefore, this study aims to investigate the effects of PP13 on neutrophils; a cell type often ignored in pregnancy, but present in the uterus and placenta from the early stages of pregnancy. Since neutrophil function is dysregulated during pathologic pregnancies, a link between PP13 and neutrophil activity is possible. We determined that PP13 reduces the apoptosis rate in neutrophils. Also, PP13 increases the expression of PD-L1 and production of HGF, TNF-α, reactive oxygen species (ROS), and MMP-9 in these cells. This phenotype resembles one observed in permissive tumor neutrophils; able to sustain tissue and vessel growth, and inhibit T cell activation. At the same time, PP13 does not alter all neutrophil functions, i.e., extrusion of neutrophil extracellular traps, degranulation, phagocytosis, and ROS production following bacterial exposure. PP13 seems to play an essential role in regulating the activity of neutrophils in the placenta by polarizing them toward a placental-growth-permissive phenotype.

Dysregulation of Placental Functions and Immune Pathways in Complete Hydatidiform Moles.

Gene expression studies of molar pregnancy have been limited to a small number of candidate loci. We analyzed high-dimensional RNA and protein data to characterize molecular features of complete hydatidiform moles (CHMs) and corresponding pathologic pathways. CHMs and first trimester placentas were collected, histopathologically examined, then flash-frozen or paraffin-embedded. Frozen CHMs and control placentas were subjected to RNA-Seq, with resulting data and published placental RNA-Seq data subjected to bioinformatics analyses. Paraffin-embedded tissues from CHMs and control placentas were used for tissue microarray (TMA) construction, immunohistochemistry, and immunoscoring for galectin-14. Of the 14,022 protein-coding genes expressed in all samples, 3,729 were differentially expressed (DE) in CHMs, of which 72% were up-regulated. DE genes were enriched in placenta-specific genes (OR = 1.88, p = 0.0001), of which 79% were down-regulated, imprinted genes (OR = 2.38, p = 1.54 × 10-6), and immune genes (OR = 1.82, p = 7.34 × 10-18), of which 73% were up-regulated. DNA methylation-related enzymes and histone demethylases were dysregulated. "Cytokine-cytokine receptor interaction" was the most impacted of 38 dysregulated pathways, among which 17 were immune-related pathways. TMA-based immunoscoring validated the lower expression of galectin-14 in CHM. In conclusion, placental functions were down-regulated, imprinted gene expression was altered, and immune pathways were activated, indicating complex dysregulation of placental developmental and immune processes in CHMs.

Sex hormone-binding globulin provides a novel entry pathway for estradiol and influences subsequent signaling in lymphocytes via membrane receptor.

The complex effects of estradiol on non-reproductive tissues/cells, including lymphoid tissues and immunocytes, have increasingly been explored. However, the role of sex hormone binding globulin (SHBG) in the regulation of these genomic and non-genomic actions of estradiol is controversial. Moreover, the expression of SHBG and its internalization by potential receptors, as well as the influence of SHBG on estradiol uptake and signaling in lymphocytes has remained unexplored. Here, we found that human and mouse T cells expressed SHBG intrinsically. In addition, B lymphoid cell lines as well as both primary B and T lymphocytes bound and internalized external SHBG, and the amount of plasma membrane-bound SHBG decreased in B cells of pregnant compared to non-pregnant women. As potential mediators of this process, SHBG receptor candidates expressed by lymphocytes were identified in silico, including estrogen receptor (ER) alpha. Furthermore, cell surface-bound SHBG was detected in close proximity to membrane ERs while highly colocalizing with lipid rafts. The SHBG-membrane ER interaction was found functional since SHBG promoted estradiol uptake by lymphocytes and subsequently influenced Erk1/2 phosphorylation. In conclusion, the SHBG-SHBG receptor-membrane ER complex participates in the rapid estradiol signaling in lymphocytes, and this pathway may be altered in B cells in pregnant women.

Integrated Systems Biology Approach Identifies Novel Maternal and Placental Pathways of Preeclampsia.

Preeclampsia is a disease of the mother, fetus, and placenta, and the gaps in our understanding of the complex interactions among their respective disease pathways preclude successful treatment and prevention. The placenta has a key role in the pathogenesis of the terminal pathway characterized by exaggerated maternal systemic inflammation, generalized endothelial damage, hypertension, and proteinuria. This sine qua non of preeclampsia may be triggered by distinct underlying mechanisms that occur at early stages of pregnancy and induce different phenotypes. To gain insights into these molecular pathways, we employed a systems biology approach and integrated different "omics," clinical, placental, and functional data from patients with distinct phenotypes of preeclampsia. First trimester maternal blood proteomics uncovered an altered abundance of proteins of the renin-angiotensin and immune systems, complement, and coagulation cascades in patients with term or preterm preeclampsia. Moreover, first trimester maternal blood from preterm preeclamptic patients in vitro dysregulated trophoblastic gene expression. Placental transcriptomics of women with preterm preeclampsia identified distinct gene modules associated with maternal or fetal disease. Placental "virtual" liquid biopsy showed that the dysregulation of these disease gene modules originates during the first trimester. In vitro experiments on hub transcription factors of these gene modules demonstrated that DNA hypermethylation in the regulatory region of ZNF554 leads to gene down-regulation and impaired trophoblast invasion, while BCL6 and ARNT2 up-regulation sensitizes the trophoblast to ischemia, hallmarks of preterm preeclampsia. In summary, our data suggest that there are distinct maternal and placental disease pathways, and their interaction influences the clinical presentation of preeclampsia. The activation of maternal disease pathways can be detected in all phenotypes of preeclampsia earlier and upstream of placental dysfunction, not only downstream as described before, and distinct placental disease pathways are superimposed on these maternal pathways. This is a paradigm shift, which, in agreement with epidemiological studies, warrants for the central pathologic role of preexisting maternal diseases or perturbed maternal-fetal-placental immune interactions in preeclampsia. The description of these novel pathways in the "molecular phase" of preeclampsia and the identification of their hub molecules may enable timely molecular characterization of patients with distinct preeclampsia phenotypes.

Predicting the Risk to Develop Preeclampsia in the First Trimester Combining Promoter Variant -98A/C of LGALS13 (Placental Protein 13), Black Ethnicity, Previous Preeclampsia, Obesity, and Maternal Age.

BACKGROUND: LGALS13 (placental protein 13 [PP13]) promoter DNA polymorphisms was evaluated in predicting preeclampsia (PE), given PP13's effects on hypotension, angiogenesis, and immune tolerance. METHODS: First-trimester plasma samples (49 term and 18 intermediate) of PE cases matched with 196 controls were collected from King's College Hospital, London, repository. Cell-free DNA was extracted and the LGALS13 exons were sequenced after PCR amplification. Expression of LGALS13 promoter reporter constructs was determined in BeWo trophoblast-like cells with luciferase assays. Adjusted odds ratio (OR) was calculated for the A/A genotype combined with maternal risk factors. RESULTS: The A/A, A/C, and C/C genotypes in the -98 promoter position were in Hardy-Weinberg equilibrium in the control but not in the PE group (p < 0.036). The dominant A/A genotype had higher frequency in the PE group (p < 0.001). The A/C and C/C genotypes protected from PE (p < 0.032). The ORs to develop term and all PE, calculated for the A/A genotype, previous PE, body mass index (BMI) >35, black ethnicity, and maternal age >40 were 15.6 and 11.0, respectively (p < 0.001). In luciferase assays, the "-98A" promoter variant had lower expression than the "-98C" variant in non-differentiated (-13%, p = 0.04) and differentiated (-26%, p < 0.001) BeWo cells. Forskolin-induced differentiation led to a larger expression increase in the "-98C" variant than in the "-98A" variant (4.55-fold vs. 3.85-fold, p < 0.001). CONCLUSION: Lower LGALS13 (PP13) expression with the "A" nucleotide in the -98 promoter region position (compared to "C") and high OR calculated for the A/A genotype in the -98A/C promoter region position, history of previous PE, BMI >35, advanced maternal age >40, and black ethnicity could serve to aid in PE prediction in the first trimester.

Cerebral Palsy-Trends in Epidemiology and Recent Development in Prenatal Mechanisms of Disease, Treatment, and Prevention.

Cerebral palsy (CP) is the most common motor disability in childhood. This syndrome is the manifestation of intrauterine pathologies, intrapartum complications, and the postnatal sequel, especially among preterm neonates. A double hit model theory is proposed suggesting that an intrauterine condition along with intrapartum or postnatal insult lead to the development of CP. Recent reports demonstrated that treatment during the process of preterm birth such as magnesium sulfate and postnatal modalities such as cooling may prevent or reduce the prevalence of this syndrome. Moreover, animal models demonstrated that postnatal treatment with anti-inflammatory drugs coupled with nanoparticles may affect the course of the disease in pups with neuroinflammation. This review will describe the changes in the epidemiology of this disease, the underlying prenatal mechanisms, and possible treatments that may reduce the prevalence of CP and alter the course of the disease.

Characteristic Changes in Decidual Gene Expression Signature in Spontaneous Term Parturition.

BACKGROUND: The decidua has been implicated in the "terminal pathway" of human term parturition, which is characterized by the activation of pro-inflammatory pathways in gestational tissues. However, the transcriptomic changes in the decidua leading to terminal pathway activation have not been systematically explored. This study aimed to compare the decidual expression of developmental signaling and inflammation-related genes before and after spontaneous term labor in order to reveal their involvement in this process. METHODS: Chorioamniotic membranes were obtained from normal pregnant women who delivered at term with spontaneous labor (TIL, n = 14) or without labor (TNL, n = 15). Decidual cells were isolated from snap-frozen chorioamniotic membranes with laser microdissection. The expression of 46 genes involved in decidual development, sex steroid and prostaglandin signaling, as well as pro- and anti-inflammatory pathways, was analyzed using high-throughput quantitative real-time polymerase chain reaction (qRT-PCR). Chorioamniotic membrane sections were immunostained and then semi-quantified for five proteins, and immunoassays for three chemokines were performed on maternal plasma samples. RESULTS: The genes with the highest expression in the decidua at term gestation included insulin-like growth factor-binding protein 1 (IGFBP1), galectin-1 (LGALS1), and progestogen-associated endometrial protein (PAEP); the expression of estrogen receptor 1 (ESR1), homeobox A11 (HOXA11), interleukin 1ß (IL1B), IL8, progesterone receptor membrane component 2 (PGRMC2), and prostaglandin E synthase (PTGES) was higher in TIL than in TNL cases; the expression of chemokine C-C motif ligand 2 (CCL2), CCL5, LGALS1, LGALS3, and PAEP was lower in TIL than in TNL cases; immunostaining confirmed qRT-PCR data for IL-8, CCL2, galectin-1, galectin-3, and PAEP; and no correlations between the decidual gene expression and the maternal plasma protein concentrations of CCL2, CCL5, and IL-8 were found. CONCLUSIONS: Our data suggests that with the initiation of parturition, the decidual expression of anti-inflammatory mediators decreases, while the expression of pro-inflammatory mediators and steroid receptors increases. This shift may affect downstream signaling pathways that can lead to parturition.

A Role for the Inflammasome in Spontaneous Labor at Term with Acute Histologic Chorioamnionitis.

Inflammasomes are cytosolic signaling platforms that regulate the activation of caspase (CASP)-1, which induces the maturation of interleukin (IL)-1ß and IL-18. Herein, we determined whether the chorioamniotic membranes from women in spontaneous labor at term with acute histologic chorioamnionitis express major inflammasome components and whether these changes are associated with the activation of CASP-1 and CASP-4 and the release of mature IL-1ß and IL-18. When comparing the chorioamniotic membranes from women in spontaneous labor at term with acute histologic chorioamnionitis to those without this placental lesion, we found that (1) the messenger RNA (mRNA) abundance of NLR family pyrin domain containing 3 ( NLRP3), NLR family CARD domain containing 4 ( NLRC4), absent in melanoma 2 ( AIM2), and nucleotide binding oligomerization domain 2 ( NOD2) was higher; (2) the NLRP3 and NLRC4 protein quantities were increased; (3) the mRNA and protein expressions of CASP-1 and its active forms were greater; (4) CASP-4 was increased at the mRNA level only; (5) the mRNA and protein expressions of IL-1ß and its mature form were higher; and (6) a modest increase in the total protein concentration and abundance of the mature form of IL-18 was observed. In vitro incubation of the chorioamniotic membranes with the CASP-1 inhibitor, VX765, decreased the release of endotoxin-induced IL-1ß and IL-18 (2-fold) but not IL-6 or tumor necrosis factor α. In conclusion, spontaneous labor at term with acute histologic chorioamnionitis is characterized by an upregulation of inflammasome components which, in turn, may participate in the activation of CASP-1 and lead to the release of mature IL-1ß by the chorioamniotic membranes. These results support a role for the inflammasome in the mechanisms responsible for spontaneous labor at term with acute histologic chorioamnionitis.

A longitudinal study of placental perfusion using dynamic contrast enhanced magnetic resonance imaging in murine pregnancy.

INTRODUCTION: To evaluate changes in placental perfusion with advancing gestation in normal murine pregnancy using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI). METHODS: Seven timed-pregnant CD-1 mice underwent DCE-MRI scanning longitudinally on gestational days (GD) 13, 15 and 17. Placentas were segmented into high (HPZ) and low perfusion zones (LPZ) using tissue similarity mapping. Blood perfusion of the respective regions and the whole placenta was quantified using the steepest slope method. The diameter of the maternal central canal (CC) was also measured. RESULTS: An increase in perfusion was observed between GD13 and GD17 in the overall placenta (p = 0.04) and in the HPZ (p = 0.02). Although perfusion in the LPZ showed a slight increasing trend, it was not significant (p = 0.07). Perfusion, in units of ml/min/100 ml, in the overall placenta and the HPZ was respectively 61.2 ± 31.2 and 106.2 ± 56.3 at GD13 (n = 19 placentas); 90.3 ± 43.7 and 139 ± 55.4 at GD15 (n = 20); and 104.9 ± 76.1 and 172.2 ± 85.6 at GD17 (n = 14). The size of the CC increased with advancing gestation (p < 0.05). DISCUSSION: Using longitudinal DCE-MRI, the gestational age-dependent perfusion change in the normal murine placenta and in its regional compartments was quantified. In mid and late gestations, placental constituent regions differ significantly in their perfusion rates. The CC diameter also showed increase with advancing gestation, which may be playing an important role toward the gestational age-dependent increase in placental perfusion.

Galectins: Double-edged Swords in the Cross-roads of Pregnancy Complications and Female Reproductive Tract Inflammation and Neoplasia.

Galectins are an evolutionarily ancient and widely expressed family of lectins that have unique glycan-binding characteristics. They are pleiotropic regulators of key biological processes, such as cell growth, proliferation, differentiation, apoptosis, signal transduction, and pre-mRNA splicing, as well as homo- and heterotypic cell-cell and cell-extracellular matrix interactions. Galectins are also pivotal in immune responses since they regulate host-pathogen interactions, innate and adaptive immune responses, acute and chronic inflammation, and immune tolerance. Some galectins are also central to the regulation of angiogenesis, cell migration and invasion. Expression and functional data provide convincing evidence that, due to these functions, galectins play key roles in shared and unique pathways of normal embryonic and placental development as well as oncodevelopmental processes in tumorigenesis. Therefore, galectins may sometimes act as double-edged swords since they have beneficial but also harmful effects for the organism. Recent advances facilitate the use of galectins as biomarkers in obstetrical syndromes and in various malignancies, and their therapeutic applications are also under investigation. This review provides a general overview of galectins and a focused review of this lectin subfamily in the context of inflammation, infection and tumors of the female reproductive tract as well as in normal pregnancies and those complicated by the great obstetrical syndromes.