Distinct First Trimester Cytokine Profiles for Gestational Hypertension and Preeclampsia.

OBJECTIVE: Gestational hypertension and preeclampsia involve dysregulated maternal inflammatory responses to pregnancy, but whether such responses differ between the disorders has not been determined. We aimed to investigate disease-specific early pregnancy serum cytokine profiles of women subsequently developing gestational hypertension or preeclampsia for new insight into the underlying pathogeneses and differences between the disorders. APPROACH AND RESULTS: The study cohort consisted of 548 pregnant Norwegian women who were either multiparous with previous gestational hypertension or preeclampsia or were nulliparous. Maternal sera at gestational weeks 11(0)-13(6) were assayed for 27 cytokines, C-reactive protein, total cholesterol, high-density lipoprotein, triglyceride, creatinine, calcium, uric acid, and placental growth factor. Compared with normotensive women, women with both hypertensive conditions presented an atherogenic lipid profile at early gestation, but only those later developing gestational hypertension had significantly higher serum levels of interleukin (IL)-5 and IL-12. Comparing the 2 hypertensive pregnancy disorders, women subsequently developing gestational hypertension had higher serum levels of IL-1ß, IL-5, IL-7, IL-8, IL-13, basic fibroblast growth factor, and vascular endothelial growth factor than the women subsequently developing preeclampsia. CONCLUSIONS: This study identifies early pregnancy differences in serum cytokine profiles for gestational hypertension and preeclampsia.

The Norwegian preeclampsia family cohort study: a new resource for investigating genetic aspects and heritability of preeclampsia and related phenotypes.

BACKGROUND: Preeclampsia is a major pregnancy complication without curative treatment available. A Norwegian Preeclampsia Family Cohort was established to provide a new resource for genetic and molecular studies aiming to improve the understanding of the complex pathophysiology of preeclampsia. METHODS: Participants were recruited from five Norwegian hospitals after diagnoses of preeclampsia registered in the Medical birth registry of Norway were verified according to the study's inclusion criteria. Detailed obstetric information and information on personal and family disease history focusing on cardiovascular health was collected. At attendance anthropometric measurements were registered and blood samples were drawn. The software package SPSS 19.0 for Windows was used to compute descriptive statistics such as mean and SD. P-values were computed based on t-test statistics for normally distributed variables. Nonparametrical methods (chi square) were used for categorical variables. RESULTS: A cohort consisting of 496 participants (355 females and 141 males) representing 137 families with increased occurrence of preeclampsia has been established, and blood samples are available for 477 participants. Descriptive analyses showed that about 60% of the index women's pregnancies with birth data registered were preeclamptic according to modern diagnosis criteria. We also found that about 41% of the index women experienced more than one preeclamptic pregnancy. In addition, the descriptive analyses confirmed that preeclamptic pregnancies are more often accompanied with delivery complications. CONCLUSION: The data and biological samples collected in this Norwegian Preeclampsia Family Cohort will provide an important basis for future research. Identification of preeclampsia susceptibility genes and new biomarkers may contribute to more efficient strategies to identify mothers "at risk" and contribute to development of novel preventative therapies.

First Trimester Urine and Serum Metabolomics for Prediction of Preeclampsia and Gestational Hypertension: A Prospective Screening Study.

Hypertensive disorders of pregnancy, including preeclampsia, are major contributors to maternal morbidity. The goal of this study was to evaluate the potential of metabolomics to predict preeclampsia and gestational hypertension from urine and serum samples in early pregnancy, and elucidate the metabolic changes related to the diseases. Metabolic profiles were obtained by nuclear magnetic resonance spectroscopy of serum and urine samples from 599 women at medium to high risk of preeclampsia (nulliparous or previous preeclampsia/gestational hypertension). Preeclampsia developed in 26 (4.3%) and gestational hypertension in 21 (3.5%) women. Multivariate analyses of the metabolic profiles were performed to establish prediction models for the hypertensive disorders individually and combined. Urinary metabolomic profiles predicted preeclampsia and gestational hypertension at 51.3% and 40% sensitivity, respectively, at 10% false positive rate, with hippurate as the most important metabolite for the prediction. Serum metabolomic profiles predicted preeclampsia and gestational hypertension at 15% and 33% sensitivity, respectively, with increased lipid levels and an atherogenic lipid profile as most important for the prediction. Combining maternal characteristics with the urinary hippurate/creatinine level improved the prediction rates of preeclampsia in a logistic regression model. The study indicates a potential future role of clinical importance for metabolomic analysis of urine in prediction of preeclampsia.

Genetic dissection of the pre-eclampsia susceptibility locus on chromosome 2q22 reveals shared novel risk factors for cardiovascular disease.

Pre-eclampsia is an idiopathic pregnancy disorder promoting morbidity and mortality to both mother and child. Delivery of the fetus is the only means to resolve severe symptoms. Women with pre-eclamptic pregnancies demonstrate increased risk for later life cardiovascular disease (CVD) and good evidence suggests these two syndromes share several risk factors and pathophysiological mechanisms. To elucidate the genetic architecture of pre-eclampsia we have dissected our chromosome 2q22 susceptibility locus in an extended Australian and New Zealand familial cohort. Positional candidate genes were prioritized for exon-centric sequencing using bioinformatics, SNPing, transcriptional profiling and QTL-walking. In total, we interrogated 1598 variants from 52 genes. Four independent SNP associations satisfied our gene-centric multiple testing correction criteria: a missense LCT SNP (rs2322659, P = 0.0027), a synonymous LRP1B SNP (rs35821928, P = 0.0001), an UTR-3 RND3 SNP (rs115015150, P = 0.0024) and a missense GCA SNP (rs17783344, P = 0.0020). We replicated the LCT SNP association (P = 0.02) and observed a borderline association for the GCA SNP (P = 0.07) in an independent Australian case-control population. The LRP1B and RND3 SNP associations were not replicated in this same Australian singleton cohort. Moreover, these four SNP associations could not be replicated in two additional case-control populations from Norway and Finland. These four SNPs, however, exhibit pleiotropic effects with several quantitative CVD-related traits. Our results underscore the genetic complexity of pre-eclampsia and present novel empirical evidence of possible shared genetic mechanisms underlying both pre-eclampsia and other CVD-related risk factors.

Validity of the diagnosis of pre-eclampsia in the Medical Birth Registry of Norway.

OBJECTIVE: Evaluating the validity of pre-eclampsia registration in the Medical Birth Registry of Norway (MBRN) according to both broader and restricted disease definitions. DESIGN: Retrospective nested cohort study. SETTING: Multicenter study. POPULATION: In this study, two cohorts of women with pre-eclamptic pregnancies registered in the MBRN were selected. Study group 1 contained 966 pregnancies from 1967 to 2002. Concomitant participation in the Nord-Trøndelag Health Study 2 was required. Study group 2 comprised 1138 pregnancies recorded in 1967-2005, examined as a pre-eclampsia biobank was established. METHODS: Diagnostic criteria vary. The broader criteria for pre-eclampsia, used by the MBRN, are one measurement of hypertension and proteinuria (Criterion A). Criteria used internationally today require two measurements of hypertension and proteinuria (Criterion B). The diagnostic validities in Study groups 1 and 2 were judged against medical records according to Criterion A and B, respectively. MAIN OUTCOME MEASURES: Positive predictive value (PPV) and trend analyses. RESULTS: The diagnosis was confirmed in 88.3% of pregnancies in Study group 1, and in 63.6% in Study group 2. PPV was high for Study group 1 throughout the period. For Study group 2, results improved significantly after 1986. CONCLUSIONS: This study ascertains high PPV of pre-eclampsia in the MBRN using broader traditional criteria, although the PPV decreases through assessment using restricted modern criteria. This illustrates how inclusion of direct measurements may improve registration of complex disorders defined by changing diagnostic criteria.

Partial correlation network analyses to detect altered gene interactions in human disease: using preeclampsia as a model.

Differences in gene expression between cases and controls have been identified for a number of human diseases. However, the underlying mechanisms of transcriptional regulation remain largely unknown. Beyond comparisons of absolute or relative expression levels, disease states may be associated with alterations in the observed correlational patterns among sets of genes. Here we use partial correlation networks aiming to compare the transcriptional co-regulation for 222 genes that are differentially expressed in decidual tissues between preeclampsia (PE) cases and non-PE controls. Partial correlation coefficients (PCCs) have been calculated in cases (N = 37) and controls (N = 58) separately. For all PCCs, we tested if they were significant non-zero in the cases and controls separately. In addition, to examine if a given PCC is different between the cases and controls, we tested if the difference between two PCCs were significant non-zero. In the group with PE cases, only five PCCs were significant (FDR p value ≤ 0.05), of which none were significantly different from the PCCs in the controls. However, in the controls we identified a total of 56 statistically significant PCCs (FDR p value ≤ 0.05), of which 31 were also significantly different (FDR p value ≤ 0.05) from the PCCs in the PE cases. The identified partial correlation networks included genes that are potentially relevant for developing PE, including both known susceptibility genes (EGFL7, HES1) and novel candidate genes (CFH, NADSYN1, DBP, FIGLA). Our results might suggest that disturbed interactions, or higher order relationships between these genes play an important role in developing the disease.

A transcriptional profile of the decidua in preeclampsia.

OBJECTIVE: We sought to obtain insight into possible mechanisms underlying preeclampsia using genomewide transcriptional profiling in decidua basalis. STUDY DESIGN: Genomewide transcriptional profiling was performed on decidua basalis tissue from preeclamptic (n = 37) and normal (n = 58) pregnancies. Differentially expressed genes were identified and merged into canonical pathways and networks. RESULTS: Of the 26,504 expressed transcripts detected, 455 were differentially expressed (P < .05; false discovery rate, P < .1). Both novel (ARL5B, SLITRK4) and previously reported preeclampsia-associated (PLA2G7, HMOX1) genes were identified. Pathway analysis revealed that tryptophan metabolism, endoplasmic reticulum stress, linoleic acid metabolism, notch signaling, fatty acid metabolism, arachidonic acid metabolism, and NRF2-mediated oxidative stress response were overrepresented canonical pathways. CONCLUSION: In the present study single genes, canonical pathways, and gene-gene networks that are likely to play an important role in the pathogenesis of preeclampsia have been identified. Future functional studies are needed to accomplish a greater understanding of the mechanisms involved.

The ERAP2 gene is associated with preeclampsia in Australian and Norwegian populations.

Preeclampsia is a heritable pregnancy disorder that presents new onset hypertension and proteinuria. We have previously reported genetic linkage to preeclampsia on chromosomes 2q, 5q and 13q in an Australian/New Zealand (Aust/NZ) familial cohort. This current study centered on identifying the susceptibility gene(s) at the 5q locus. We first prioritized candidate genes using a bioinformatic tool designed for this purpose. We then selected a panel of known SNPs within ten prioritized genes and genotyped them in an extended set of the Aust/NZ families and in a very large, independent Norwegian case/control cohort (1,139 cases, 2,269 controls). In the Aust/NZ cohort we identified evidence of a genetic association for the endoplasmic reticulum aminopeptidase 1 (ERAP1) gene (rs3734016, P (uncorr) = 0.009) and for the endoplasmic reticulum aminopeptidase 2 (ERAP2) gene (rs2549782, P (uncorr) = 0.004). In the Norwegian cohort we identified evidence of a genetic association for ERAP1 (rs34750, P (uncorr) = 0.011) and for ERAP2 (rs17408150, P (uncorr) = 0.009). The ERAP2 SNPs in both cohorts remained statistically significant (rs2549782, P (corr) = 0.018; rs17408150, P (corr) = 0.039) after corrections at an experiment-wide level. The ERAP1 and ERAP2 genes encode enzymes that are reported to play a role in blood pressure regulation and essential hypertension in addition to innate immune and inflammatory responses. Perturbations within vascular, immunological and inflammatory pathways constitute important physiological mechanisms in preeclampsia pathogenesis. We herein report a novel preeclampsia risk locus, ERAP2, in a region of known genetic linkage to this pregnancy-specific disorder.

The HLA-G 14bp gene polymorphism and decidual HLA-G 14bp gene expression in pre-eclamptic and normal pregnancies.

Trophoblast expression of the non-classical MHC, HLA-G, is considered essential for feto-maternal immune tolerance and successful placentation in pregnancy. The HLA-G 14bp polymorphism in the 3'-untranslated region (UTR) of the HLA-G gene has been reported to be associated with development of pre-eclampsia (PE). In this study, maternal (peripheral blood, n=54) and fetal (cord blood, n=57) HLA-G 14bp genotypes have been determined by PCR in pre-eclamptic and normal pregnancies. In addition, HLA-G 14bp gene expression in decidua basalis (n=59) was analyzed by RT-PCR. The pre-eclamptic syndrome was neither associated with the HLA-G 14bp genotype (maternal or fetal), nor with altered decidual HLA-G 14bp gene expression. Furthermore, the HLA-G 14bp mRNA expressed in decidua basalis was of fetal origin and all potential transcripts, as predicted from the fetal HLA-G 14bp genotype, were expressed. In contrast to previous findings, we found no correlation between the HLA-G 14bp polymorphism and fetal growth. In conclusion, the fetal HLA-G 14bp genotype is reflected in the decidual HLA-G mRNA splice form profile, but does not appear to be associated with increased risk for development of PE.

Genetic association of preeclampsia to the inflammatory response gene SEPS1.

OBJECTIVE: The objective of the study was to test for a genetic association between the G-105A promoter polymorphism of the inflammatory mediator Selenoprotein S (SEPS1) and preeclampsia. STUDY DESIGN: A retrospective study in a large Norwegian case-control cohort compared maternal genotype and allele frequencies of the SEPS1 g.-105G>A polymorphism genotyped by SNPlex assay in preeclamptic (n = 1139) and control (n = 2269) women. Statistical significance was determined by chi2 and multivariate regression analyses. RESULTS: Women with preeclampsia were 1.34 times more likely to have the GA or AA genotype (P = .0039; 95% confidence interval [CI] 1.09 to 1.64) and 1.22 times more likely to carry the A allele (P = .023; odds ratio, 1.22; 95% CI, 1.02 to 1.46). CONCLUSION: The A allele of the SEPS1-105G>A polymorphism is a significant risk factor for preeclampsia in this population.

Decidual expression and maternal serum levels of heme oxygenase 1 are increased in pre-eclampsia.

BACKGROUND: Pre-eclampsia (PE) is associated with increased oxidative stress and excessive maternal inflammatory response. Heme oxygenase 1 (HMOX1) is an important stress response enzyme and a mediator of cytoprotection against a wide variety of tissue injuries. METHODS: In the present study, microarray technology was used to compare the expression of HMOX1 and other genes involved in stress and inflammatory responses in decidua basalis from 16 pregnancies complicated by PE and 17 healthy controls. In addition, the presence of HMOX1 protein in decidua basalis was examined by means of immunohistochemistry, and ELISA was used to measure the maternal serum concentration of HMOX1. RESULTS: Fifteen transcripts involved in stress response including HMOX1 were up-regulated in cases, using a cut-off value at p=0.01. HMOX1 protein expression in decidua basalis was significantly increased in cases compared to controls reflected by more pronounced intensity of HMOX1 positive decidual cells (1.8+/-0.3 versus 1.5+/-0.4, p=0.02) and an increased proportion of HMOX1 positive decidual leukocytes (31+/-29 versus 9+/-6%, p=0.001). Finally, serum HMOX1 levels were significantly higher among cases compared to controls (3.1+/-1.3 versus 1.9+/-0.5 ng/ml, p=0.008). CONCLUSIONS: Increased decidual and serum HMOX1 levels, together with altered decidual expression of some stress-related genes in cases, support the role of oxidative stress and excessive maternal inflammatory response in the pathogenesis of PE.

Whole-genome microarray and targeted analysis of angiogenesis-regulating gene expression (ENG, FLT1, VEGF, PlGF) in placentas from pre-eclamptic and small-for-gestational-age pregnancies.

OBJECTIVE: To compare the placental pathology associated with pre-eclampsia (PE) and/or fetal growth restriction, the transcriptomes of placental tissues from PE and small-for-gestational-age (SGA) pregnancies were explored. In addition, a targeted analysis of angiogenesis-regulating gene expression was performed. METHODS: Whole-genome microarray analysis was performed on placental tissue from gestational age-matched PE (n = 10), SGA (n = 8) and PE + SGA (n = 10) pregnancies. The expression of genes regulating angiogenesis (endoglin (ENG), fms-related tyrosine kinase 1 (FLT1), vascular endothelial growth factor (VEGF) and placental growth factor (PlGF)) was analyzed by quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR). RESULTS: Microarray analysis did not reveal any significant differences between groups. However, an increased expression of ENG and FLT1 was detected by qRT-PCR in the PE + SGA group. CONCLUSIONS: The placental transcriptome did not differ between groups, although an increased anti-angiogenic gene expression in PE + SGA was observed with qRT-PCR analysis. Based on this, we conclude that although microarray technology may represent a powerful tool in generating new hypothesis in complex fields, it may not be sensitive enough to detect subtle changes in gene expression.

Fetal growth restriction is associated with reduced FasL expression by decidual cells.

The Fas-Fas ligand (FasL) system contributes to immune tolerance at the feto-maternal site and has been ascribed a role in implantation and placental development by regulating trophoblast invasion and spiral artery remodelling. In the present study, we have examined FasL expression in decidual tissue from pregnancies with impaired placental development. Women with pre-eclampsia (PE) and/or fetal growth restriction (FGR) were enrolled as cases (n=33), and women with normal pregnancies were used as controls (n=27). Decidua basalis tissue was obtained by vacuum suction of the placental bed after delivery. FasL expression by extravillous trophoblasts (EVTs) and decidual cells (DeCs), together with EVT apoptosis, were assessed by immunohistochemistry. Levels of soluble FasL in maternal serum and apoptosis-related gene expression in decidual tissue were determined. The proportion of FasL-expressing DeCs was high in controls (72.0+/-10.2%), with a significant reduction among cases (58.1+/-19.7%; p=0.002), especially in those with FGR (54.3+/-19.9%; p<0.001). EVTs had a lower proportion of FasL expression than DeCs, with a less pronounced reduction in cases compared to controls (10.9+/-3.9 and 8.3+/-4.0%, respectively; p=0.02). Decidual FasL expression correlated with placental growth. The EVT apoptosis rate did not differ between cases and controls (1.1+/-1.9 and 1.1+/-1.3%, respectively). These findings indicate a reduction of immune privilege in decidua of PE/FGR pregnancies by reduced FasL expression and that DeCs may have a central role in the Fas-FasL-based feto-maternal immune balance.

Incident Coronary Heart Disease After Preeclampsia: Role of Reduced Fetal Growth, Preterm Delivery, and Parity.

BACKGROUND: Preeclampsia is a severe pregnancy disorder often complicated by reduced fetal growth or preterm delivery and is associated with long-term maternal morbidity and mortality. We aimed to assess the association between preeclampsia phenotypes and risk of subsequent coronary heart disease and maternal cardiovascular mortality. METHODS AND RESULTS: Women aged 16 to 49 years who gave birth during 1980-2002 and registered in the Medical Birth Registry of Norway were followed prospectively (1-29 years) for an incident major coronary event and mortality through linkage with the Cardiovascular Disease in Norway 1994-2009 (CVDNOR) project and the Norwegian Cause of Death Registry. Preeclampsia was subdivided based on the presence of a child born small for gestational age or preterm delivery. Among 506 350 women with 1 to 5 singleton births, there were 1275 (0.3%) occurrences of major coronary event, 468 (0.1%) cardiovascular deaths, and 5411 (1.1%) deaths overall. Compared with women without preeclampsia, the hazard ratio (95% CI) for major coronary event was 2.1 (1.73-2.65) after preeclampsia alone, 3.3 (2.37-4.57) after preeclampsia in combination with small for gestational age, and 5.4 (3.74-7.74) after preeclampsia in combination with preterm delivery. Analyses distinguishing women with 1 (n=61 352) or >1 (n=281 069) lifetime pregnancy and analyses with cardiovascular mortality as outcome followed the same pattern. CONCLUSIONS: The occurrence of major coronary events was increased among women with preeclampsia and highest for preeclampsia combined with a child born small for gestational age and/or preterm delivery.

The antihypertensive MTHFR gene polymorphism rs17367504-G is a possible novel protective locus for preeclampsia.

OBJECTIVE: Preeclampsia is a complex heterogeneous disease commonly defined by new-onset hypertension and proteinuria in pregnancy. Women experiencing preeclampsia have increased risk for cardiovascular diseases (CVD) later in life. Preeclampsia and CVD share risk factors and pathophysiologic mechanisms, including dysregulated inflammation and raised blood pressure. Despite commonalities, little is known about the contribution of shared genes (pleiotropy) to these diseases. This study aimed to investigate whether genetic risk factors for hypertension or inflammation are pleiotropic by also being associated with preeclampsia. METHODS: We genotyped 122 single nucleotide polymorphisms (SNPs) in women with preeclampsia (n = 1006) and nonpreeclamptic controls (n = 816) from the Norwegian HUNT Study. SNPs were chosen on the basis of previously reported associations with either nongestational hypertension or inflammation in genome-wide association studies. The SNPs were tested for association with preeclampsia in a multiple logistic regression model. RESULTS: The minor (G) allele of the intronic SNP rs17367504 in the gene methylenetetrahydrofolate reductase (MTHFR) was associated with a protective effect on preeclampsia (odds ratio 0.65, 95% confidence interval 0.53-0.80) in the Norwegian cohort. This association did not replicate in an Australian preeclampsia case-control cohort (P = 0.68, odds ratio 1.05, 95% confidence interval 0.83-1.32, minor allele frequency = 0.15). CONCLUSION: MTHFR is important for regulating transmethylation processes and is involved in regulation of folate metabolism. The G allele of rs17367504 has previously been shown to protect against nongestational hypertension. Our study suggests a novel association between this allele and reduced risk for preeclampsia. This is the first study associating the minor (G) allele of a SNP within the MTHFR gene with a protective effect on preeclampsia, and in doing so identifying a possible pleiotropic protective effect on preeclampsia and hypertension.

Serious foetal growth restriction is associated with reduced proportions of natural killer cells in decidua basalis.

Extravillous trophoblasts are major participants in placental development and remodelling of spiral arteries. Trophoblast invasion is regulated by maternal immune cells, and abnormal leucocyte subpopulation composition has been reported in implantation failure. In pre-eclampsia (PE), with or without foetal growth restriction (FGR), superficial trophoblast invasion and insufficient remodelling of spiral arteries are common findings. In the present study, we have compared spiral artery remodelling and leucocyte composition in decidual tissue from 30 cases (PE=8, FGR=5, PE + FGR=17) and 31 controls. Six histological remodelling criteria were established, and each pregnancy obtained a remodelling score. Numbers of natural killer (NK) cells (CD56+), T cells (CD3+) and activated (CD25+ or CD69+) leucocytes were determined and related to total leucocyte (CD45+) numbers in serial sections. Cases demonstrated significantly impaired spiral artery remodelling, inappropriate placental growth and reduced NK cell proportions, as compared to controls (P=0.02, P<0.001 and P=0.01, respectively). Reduced NK cell proportion was primarily found in pregnancies complicated by FGR, with or without PE, and a significant positive correlation was observed between NK cell proportion, trophoblast infiltration and placental growth. Our in vivo observations support the hypothesized association between NK cells, impaired placental development and pathogenesis of PE/FGR.

A comparative study of immunomagnetic methods used for separation of human natural killer cells from peripheral blood.

Immunomagnetic sorting of natural killer (NK) cells from the peripheral blood of healthy donors has been evaluated in a comparative study of composition, yield and activation of target cells obtained by positive (Dynabeads, Microbeads) and negative (Microbeads) sorting procedures. Positively sorted target cells were selected by expression of the NK cell marker CD56, whereas NK cells obtained by negative sorting were those remaining after steps to remove all non-NK cell leukocyte populations were accomplished. In positive sorting, both CD56+CD3- NK cells and CD56+CD3+ natural killer T (NKT) cells were included. The NKT cell fraction differed between individuals, but not between the positive sorting methods. Whereas 20-30% of positively sorted target cells were NKT cells, only approximately 3% of negatively sorted cells were CD3+. Contamination with monocytes and B cells was low (1-3%) in all methods studied. Sorting with Microbeads (both positive and negative) gave higher cell yields than those obtained with Dynabeads (14% vs. 5% of total leukocyte numbers). A higher CD56 fluorescence intensity of NK cells and a better discrimination between the CD56bright and CD56dim NK cell subpopulations was obtained after negative sorting. Dynabeads-separated cells had, shortly after separation, a significantly higher expression (approximately 30%) of the early activation marker CD69 than cells either positively or negatively separated by Microbeads (approximately 8%). CD56+ cells positively sorted by Microbeads demonstrated a significantly higher production of TNF-alpha and IFN-gamma after IL-2 stimulation than Dynabeads-sorted cells. However, the cytotoxicity of cells obtained by the two positive sorting procedures did not differ. In conclusion, positive selection of CD56+ cells by Microbeads is better than Dynabeads, as determined from cell yield and procedure-associated cell activation and should be chosen for in vitro studies of NK/NKT cells. However, when pure NK cells and phenotypic subtypes are to be studied, negative sorting seems most appropriate.

Metabolic profiles of placenta in preeclampsia using HR-MAS MRS metabolomics.

INTRODUCTION: Preeclampsia is a heterogeneous gestational disease characterized by maternal hypertension and proteinuria, affecting 2-7% of pregnancies. The disorder is initiated by insufficient placental development, but studies characterizing the placental disease components are lacking. METHODS: Our aim was to phenotype the preeclamptic placenta using high-resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS MRS). Placental samples collected after delivery from women with preeclampsia (n = 19) and normotensive pregnancies (n = 15) were analyzed for metabolic biomarkers including amino acids, osmolytes, and components of the energy and phospholipid metabolism. The metabolic biomarkers were correlated to clinical characteristics and inflammatory biomarkers in the maternal sera. RESULTS: Principal component analysis showed inherent differences in placental metabolic profiles between preeclamptic and normotensive pregnancies. Significant differences in metabolic profiles were found between placentas from severe and non-severe preeclampsia, but not between preeclamptic pregnancies with fetal growth restricted versus normal weight neonates. The placental metabolites correlated with the placental stress marker sFlt-1 and triglycerides in maternal serum, suggesting variation in placental stress signaling between different placental phenotypes. DISCUSSION: HR-MAS MRS is a sensitive method for defining the placental disease component of preeclampsia, identifying several altered metabolic pathways. Placental HR-MAS MRS analysis may improve insight into processes affected in the preeclamptic placenta, and represents a novel long-required tool for a sensitive placental phenotyping of this heterogeneous disease.

Refined phenotyping identifies links between preeclampsia and related diseases in a Norwegian preeclampsia family cohort.

OBJECTIVE: Preeclampsia is a complex genetic disease of pregnancy with a heterogenous presentation, unknown cause and potential severe outcomes for both mother and child. Preeclamptic women have increased risk for atherothrombotic cardiovascular disease. We aimed to identify heritabilities and phenotypic correlations of preeclampsia and related conditions in the Norwegian Preeclampsia Family Biobank. METHODS: By applying a variance components model, a total of 493 individuals (from 138 families with increased occurrence of preeclampsia) were classified according to 30 disease-related phenotypes. RESULTS: Of parous women, 75.7% (263/338) had experienced preeclampsia and 35.7% of women with and 22.4% without preeclampsia delivered children small for gestational age (SGA). We identified 11 phenotypes as heritable. The increased occurrence of preeclampsia was reflected by the presence [heritability (H2r) = 0.60)] and severity (H2r = 0.15) of preeclampsia and being born in a preeclamptic pregnancy (H2r = 0.25). Other heritable phenotypes identified included SGA (H2r = 0.40), chronic hypertension (H2r = 0.57), severity of atherothrombotic cardiovascular disease (H2r = 0.31), BMI (H2r = 0.60) and pulmonary disease (H2r = 0.91). The heritable phenotype preeclampsia overlapped with SGA (P = 0.03), whereas pulmonary disease was phenotypically correlated with atherothrombotic cardiovascular disease (P < 0.01), SGA (P = 0.02) and BMI (P = 0.02). CONCLUSION: This is the first study identifying the H2r of a range of health-related conditions in preeclamptic families. Our study demonstrates how refinement of phenotypes leads to better H2r estimation and the identification of a biological relationship between preeclampsia and related traits.

Pre-eclampsia: associated with increased syncytial apoptosis when the infant is small-for-gestational-age.

The present investigation was undertaken to study the association between placental apoptosis and pre-eclampsia, discriminating between pre-eclamptic pregnancies with appropriate-, and small-for-gestational-age (SGA), infants. Twenty pregnancies with pre-eclampsia and SGA (birth weight at or below -2 standard deviations) infants were selected in a retrospective study. Subsequently, corresponding numbers of gestational age-matched pre-eclampsia cases with appropriate-gestational-age (AGA) (birth weight at or above the 50% centile) infants and AGA controls without pre-eclampsia were selected. Formalin-fixed placental tissue was obtained from all groups. Apoptosis was assessed by a monoclonal antibody (M30), detecting a neoepitope of cytokeratin that is generated early in the apoptotic cascade. M30-positive cells were counted in villous and decidual/ basal plate tissue fields, and results were given as numbers of M30-positive cells per field. The study was performed blinded. Increased apoptosis was found in the syncytiotrophoblast layer in pre-eclampsia with SGA infants (0.14 apototic incidents per field of villous tissue, with 0.04-0.23 as the corresponding 25-75% inter quartile range (IQR) (P=0.05)). Syncytial apoptosis in the syncytial layer in the pre-eclampsia group with AGA infants was lower (0.09, IQR 0.03-0.15) and corresponded to the level detected among controls (0.06, IQR 0.03-0.17). Apoptosis in other placental cellular compartments did not differ between groups. The increased syncytial apoptosis found in placentas from pregnancies with SGA infants may either be due to specific mechanisms associated with pre-eclampsia complicated with growth restriction, or may simply reflect the presence of syncytiotrophoblast layer damage, regardless of underlying pathological condition.