Fetal cardiac dysfunction in intrahepatic cholestasis of pregnancy is associated with elevated serum bile acid concentrations.

BACKGROUND & AIMS: Intrahepatic cholestasis of pregnancy (ICP) is associated with an increased risk of stillbirth. This study aimed to assess the relationship between bile acid concentrations and fetal cardiac dysfunction in patients with ICP who were or were not treated with ursodeoxycholic acid (UDCA). METHODS: Bile acid profiles and NT-proBNP, a marker of ventricular dysfunction, were assayed in umbilical venous serum from 15 controls and 76 ICP cases (36 untreated, 40 UDCA-treated). Fetal electrocardiogram traces were obtained from 43 controls and 48 ICP cases (26 untreated, 22 UDCA-treated). PR interval length and heart rate variability (HRV) parameters were measured in 2 behavioral states (quiet and active sleep). RESULTS: In untreated ICP, fetal total serum bile acid (TSBA) concentrations (r = 0.49, p = 0.019), hydrophobicity index (r = 0.20, p = 0.039), glycocholate concentrations (r = 0.56, p = 0.007) and taurocholate concentrations (r = 0.44, p = 0.039) positively correlated with fetal NT-proBNP. Maternal TSBA (r = 0.40, p = 0.026) and alanine aminotransferase (r = 0.40, p = 0.046) also positively correlated with fetal NT-proBNP. There were no significant correlations between maternal or fetal serum bile acid concentrations and fetal HRV parameters or NT-proBNP concentrations in the UDCA-treated cohort. Fetal PR interval length positively correlated with maternal TSBA in untreated (r = 0.46, p = 0.027) and UDCA-treated ICP (r = 0.54, p = 0.026). Measures of HRV in active sleep and quiet sleep were significantly higher in untreated ICP cases than controls. HRV values in UDCA-treated cases did not differ from controls. CONCLUSIONS: Elevated fetal and maternal serum bile acid concentrations in untreated ICP are associated with an abnormal fetal cardiac phenotype characterized by increased NT-proBNP concentration, PR interval length and HRV. UDCA treatment partially attenuates this phenotype. LAY SUMMARY: The risk of stillbirth in intrahepatic cholestasis of pregnancy (ICP) is linked to the level of bile acids in the mother which are thought to disrupt the baby's heart rhythm. We found that babies of women with untreated ICP have abnormally functioning hearts compared to those without ICP, and the degree of abnormality is closely linked to the level of harmful bile acids in the mother and baby's blood. Babies of women with ICP who received treatment with the drug UDCA do not have the same level of abnormality in their hearts, suggesting that UDCA could be a beneficial treatment in some ICP cases, although further clinical trials are needed to confirm this.

Measurement of the total angiotensinogen and its reduced and oxidised forms in human plasma using targeted LC-MS/MS.

Angiotensinogen (AGT) is a critical protein in the renin-angiotensin-aldosterone system and may have an important role in the pathogenesis of pre-eclampsia. The disulphide linkage between cysteines 18 and 138 has a key role in the redox switch of AGT which modulates the release of angiotensin I with consequential effects on blood pressure. In this paper, we report a quantitative targeted LC-MS/MS method for the reliable measurement of the total AGT and its reduced and oxidised forms in human plasma. AGT was selectively enriched from human plasma using two-dimensional chromatography employing concanavalin A lectin affinity and reversed phase steps and then deglycosylated using PNGase F. A differential alkylation approach was coupled with targeted LC-MS/MS method to identify the two AGT forms in the plasma chymotryptic digest. An additional AGT proteolytic marker peptide was identified and used to measure total AGT levels. The developed MS workflow enabled the reproducible detection of total AGT and its two distinct forms in human plasma with analytical precision of ≤ 15%. The LC-MS/MS assay for total AGT in plasma showed a linear response (R2 = 0.992) with a limit of quantification in the low nanomolar range. The method gave suitable validation characteristics for biomedical application to the quantification of the oxidation level and the total level of AGT in plasma samples collected from normal and pre-eclamptic patients.

Regulation of the human placental (pro)renin receptor-prorenin-angiotensin system by microRNAs.

STUDY QUESTION: Are any microRNAs (miRNAs) that target the placental renin-angiotensin system (RAS) in the human placenta suppressed in early gestation? SUMMARY ANSWER: Overall, 21 miRNAs with predicted RAS mRNA targets were less abundant in early versus term placentae and nine were more highly expressed. WHAT IS KNOWN ALREADY: Regulation of human placental RAS expression could alter placental development and therefore normal pregnancy outcome. The expression of genes encoding prorenin (REN), angiotensinogen, (pro)renin receptor, angiotensin converting enzyme 2, and the angiotensin II type 1 receptor are highest in early gestation, at a time when oxygen tension is at its lowest. Studies have shown that the human placental RAS is sensitive to oxygen, as are some miRNAs that regulate RAS mRNAs. We propose that in early pregnancy, the prevailing low O2 tension, by suppression of levels of miRNAs that target RAS mRNAs, results in increased expression of RAS mRNAs and encoded proteins. As gestation proceeds and the prevailing oxygen tension rises, abundance of these miRNAs increases, and placental RAS mRNA expression is suppressed. STUDY DESIGN, SIZE, DURATION: The expression of miRNAs was compared in human placentae collected in early (10-11 weeks; n = 7) and mid-gestation (14-18 weeks; n = 8) with placenta collected at term (38-40 weeks; n = 8). Expression of placental miRNAs in women with early (29-35.1 weeks; n = 8) or late-onset pre-eclampsia (PE) (>34-weeks gestation; n = 8) and gestational age matched preterm (31.6-35.1 weeks; n = 8) and term normotensive controls were also compared. PARTICIPANTS/MATERIALS, SETTING, METHODS: Agilent Human miRNA microarray v19 was used to detect up to 2006 miRNAs in four placentae from each group. Statistically different levels of expression were determined and refined using predictive modelling. Placental miRNAs predicted to target RAS mRNAs were identified in three databases. Differences detected on the array were confirmed for some miRNAs by semi-quantitative RT-PCR (qPCR, n = 7-8 for all groups). Two differentially expressed miRNAs that were known to target human renal REN mRNA (miR-181a-5p and miR-663) were transfected into human HTR-8/SVneo trophoblast cells to examine their effect on placental REN expression and prorenin levels. MAIN RESULTS AND THE ROLE OF CHANCE: In early gestation placentae, 186 miRNAs were differentially expressed compared with term placentae (109 increased, 77 decreased). Thirty of the differentially expressed miRNAs were predicted to target RAS components. In mid-gestation placentae, 117 miRNAs were differentially expressed compared with term placentae (69 increased, 48 decreased). Of these, 19 had RAS mRNAs as predicted targets. Eight miRNAs that were lower in early gestation and predicted to target RAS mRNAs were confirmed by qPCR. All showed an increase during gestation and could influence the transgestational profile of the human placental RAS. Additionally, on the array, three miRNAs predicted to target RAS mRNAs (miR-892c-3p, miR-378c and miR-514b-3p) were overexpressed in placentae from women with late-onset PE (P = 3.6E-10, P = 1.8E-05, P = 5.3E-06; respectively). miR-663, which suppresses renal REN mRNA expression, was overexpressed in early-onset PE placentae as determined by qRT-PCR analysis (P = 0.014). Transfection of miR-181a-5p and miR-663 into HTR-8/SVneo trophoblast cells suppressed REN mRNA expression (P = 0.05) and prorenin protein production (P = 0.001). LARGE SCALE DATA: Data can be found via GEO accession number GSE109832. LIMITATIONS, REASONS FOR CAUTION: Further validation that the differentially expressed miRNAs do indeed directly target RAS mRNAs and affect placental development and function is required. This study is limited by the small sample size. Therefore independent validation in a larger cohort is required. WIDER IMPLICATIONS OF THE FINDINGS: We propose that suppression of miRNAs that target the placental RAS in early gestation is partly responsible for the increase in RAS expression at this time, in order to promote placental development. Later in pregnancy, we have detected overexpression of several miRNAs in placentae from women with PE. These may prove to be biomarkers for early detection of women at risk of developing PE. Since the placenta produces at least two miRNAs that were found in the kidney to target REN mRNA, and that also target placental REN mRNA, the escape of these miRNAs into the maternal circulation in excess amounts could affect maternal renal REN mRNA production and thereby disturb maternal fluid and electrolyte homoeostasis. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Health and Medical Research Council, Australia (APP1043537). K.G.P. is supported by an Australian Research Council Future Fellowship (FT150100179). C.T.R. is supported by a Lloyd Cox Professorial Research Fellowship from the University of Adelaide. F.Z.M. is supported by a National Heart Foundation Future Leader Fellowship and Baker Heart and Diabetes Institute Fellowship. The authors declare that they have no competing interests.

Maternal selenium, copper and zinc concentrations in pregnancy associated with small-for-gestational-age infants.

Pregnancy during adolescence increases the risk of adverse pregnancy outcome, especially small-for-gestational-age (SGA) birth, which has been linked to micronutrient deficiencies. Smoking has been shown to be related to lower micronutrient concentrations. Different ethnicities have not been examined. We used a subset from a prospective observational study, the About Teenage Eating study consisting of 126 pregnant adolescents (14-18-year-olds) between 28 and 32 weeks gestation. Micronutrient status was assessed by inductively coupled mass spectrometry. Smoking was assessed by self-report and plasma cotinine, and SGA was defined as infants born <10th corrected birthweight centile. The main outcome measures were as follows: (1) maternal plasma selenium, copper and zinc concentrations in adolescent mothers giving birth to SGA vs. appropriate-for-gestational-age (AGA) infants; and (2) comparison of micronutrient concentrations between women of different ethnicities and smoking habits. The plasma selenium {mean ± standard deviation (SD) [95% confidence interval (CI)]} concentration was lower in the SGA [n = 19: 49.4 ± 7.3 (CI: 45.9, 52.9) µg L(-1)] compared with the AGA [n = 107: 65.1 ± 12.5 (CI: 62.7, 67.5) µg L(-1); P < 0.0001] group. Smoking mothers had a lower selenium concentration compared with non-smokers (P = 0.01) and Afro-Caribbean women had higher selenium concentrations compared with White Europeans (P = 0.02). Neither copper nor zinc concentrations varied between groups. Low plasma selenium concentration in adolescent mothers could contribute to the risk of delivering an SGA infant, possibly through lowering placental antioxidant defence, thus directly affecting fetal growth. Differences in plasma selenium between ethnicities may relate to variation in nutritional intake, requiring further investigation.

A pilot study of alterations in oxidized angiotensinogen and antioxidants in pre-eclamptic pregnancy.

The oxidation status of angiotensinogen (AGT) may have a critical role in pre-eclampsia. We used a validated, quantitative, mass spectrometry-based method to measure the oxidized and total AGT levels in plasma of pre-eclamptic women (n = 17), normotensive-matched controls (n = 17), and healthy non-pregnant women (n = 10). Measurements of plasma glutathione peroxidase (GPx) activity and serum selenium concentrations were performed as markers of circulating antioxidant capacity. Higher proportions of oxidized AGT in plasma from pre-eclamptic women compared to matched normotensive pregnant controls (P = 0.006), whilst maintaining a similar total plasma AGT concentration were found. In the pre-eclamptic group, blood pressure were correlated with the proportion of oxidized AGT; no such correlation was seen in the normotensive pregnant women. Plasma GPx was inversely correlated with oxidized AGT, and there was an inverse association between serum selenium concentration and the proportion of oxidized AGT. This is the first time that oxidized AGT in human plasma has been linked directly to antioxidant status, providing a mechanism for the enhanced oxidative stress in pre-eclampsia. We now provide pathophysiological evidence that the conversion of the reduced form of AGT to its more active oxidized form is associated with inadequate antioxidant status and could indeed contribute to the hypertension of pre-eclampsia.

Genetic predisposition to hypertension is associated with preeclampsia in European and Central Asian women.

Preeclampsia is a serious complication of pregnancy, affecting both maternal and fetal health. In genome-wide association meta-analysis of European and Central Asian mothers, we identify sequence variants that associate with preeclampsia in the maternal genome at ZNF831/20q13 and FTO/16q12. These are previously established variants for blood pressure (BP) and the FTO variant has also been associated with body mass index (BMI). Further analysis of BP variants establishes that variants at MECOM/3q26, FGF5/4q21 and SH2B3/12q24 also associate with preeclampsia through the maternal genome. We further show that a polygenic risk score for hypertension associates with preeclampsia. However, comparison with gestational hypertension indicates that additional factors modify the risk of preeclampsia.

Variants in the fetal genome near FLT1 are associated with risk of preeclampsia.

Preeclampsia, which affects approximately 5% of pregnancies, is a leading cause of maternal and perinatal death. The causes of preeclampsia remain unclear, but there is evidence for inherited susceptibility. Genome-wide association studies (GWAS) have not identified maternal sequence variants of genome-wide significance that replicate in independent data sets. We report the first GWAS of offspring from preeclamptic pregnancies and discovery of the first genome-wide significant susceptibility locus (rs4769613; P = 5.4 × 10-11) in 4,380 cases and 310,238 controls. This locus is near the FLT1 gene encoding Fms-like tyrosine kinase 1, providing biological support, as a placental isoform of this protein (sFlt-1) is implicated in the pathology of preeclampsia. The association was strongest in offspring from pregnancies in which preeclampsia developed during late gestation and offspring birth weights exceeded the tenth centile. An additional nearby variant, rs12050029, associated with preeclampsia independently of rs4769613. The newly discovered locus may enhance understanding of the pathophysiology of preeclampsia and its subtypes.