Utility of metabolic profiling of serum in the diagnosis of pregnancy complications.

INTRODUCTION: Currently there are no clinical screening tests available to identify pregnancies at risk of developing preeclampsia (PET) and/or intrauterine growth restriction (IUGR), both of which are associated with abnormal placentation. Metabolic profiling is now a stable analytical platform used in many laboratories and has successfully been used to identify biomarkers associated with various pathological states. METHODS: We used nuclear magnetic resonance spectroscopy (NMR) to metabolically profile serum samples collected from 143 pregnant women at 26-41 weeks gestation with pregnancy outcomes of PET, IUGR, PET IUGR or small for gestational age (SGA) that were age-matched to normal pre/term pregnancies. RESULTS: Spectral analysis found no difference in the measured metabolites from normal term, pre-term and SGA samples, and of 25 identified metabolites, only glutamate was marginally different between groups. Of the identified metabolites, 3-methylhistidine, creatinine, acetyl groups and acetate, were determined to be independent predictors of PET and produced area under the curves (AUC) = 0.938 and 0.936 for the discovery and validation sets. Only 3-hydroxybutyrate was determined to be an independent predictor of IUGR, however the model had low predictive power (AUC = 0.623 and 0.581 for the discovery and validation sets). CONCLUSIONS: A sub-panel of metabolites had strong predictive power for identifying PET samples in a validation dataset, however prediction of IUGR was more difficult using the identified metabolites. NMR based metabolomics can identify metabolites strongly associated with disease and has the potential to be useful in developing early clinical screening tests for at risk pregnancies.

Role for the thromboxane A2 receptor ß-isoform in the pathogenesis of intrauterine growth restriction.

Intrauterine growth restriction (IUGR) is a pathology of pregnancy that results in failure of the fetus to reach its genetically determined growth potential. In developed nations the most common cause of IUGR is impaired placentation resulting from poor trophoblast function, which reduces blood flow to the fetoplacental unit, promotes hypoxia and enhances production of bioactive lipids (TXA2 and isoprostanes) which act through the thromboxane receptor (TP). TP activation has been implicated as a pathogenic factor in pregnancy complications, including IUGR; however, the role of TP isoforms during pregnancy is poorly defined. We have determined that expression of the human-specific isoform of TP (TPß) is increased in placentae from IUGR pregnancies, compared to healthy pregnancies. Overexpression of TPα enhanced trophoblast proliferation and syncytialisation. Conversely, TPß attenuated these functions and inhibited migration. Expression of the TPß transgene in mice resulted in growth restricted pups and placentae with poor syncytialisation and diminished growth characteristics. Together our data indicate that expression of TPα mediates normal placentation; however, TPß impairs placentation, and promotes the development of IUGR, and represents an underappreciated pathogenic factor in humans.

High maternal serum ferritin in early pregnancy and risk of spontaneous preterm birth.

Previous studies have reported inconsistent associations between maternal serum ferritin concentrations and the risk of spontaneous preterm birth (sPTB). The aim of the present study was to examine the association between Fe biomarkers, including serum ferritin concentrations, and the risk of total ( 75th percentile ( ≥ 43 µg/l) (OR 1.49, 95% CI 1.06, 2.10) and >90th percentile ( ≥ 68 µg/l) (OR 1.92, 95% CI 1.25, 2.96). Increased odds of early and moderate-to-late sPTB were associated with ferritin levels >90th percentile (OR 2.50, 95% CI 1.32, 4.73) and >75th percentile (OR 1.56, 95% CI 1.03, 2.37), respectively. No association was found between the risk of sPTB and elevated sTfR levels or Fe deficiency. In conclusion, elevated maternal serum ferritin levels in early pregnancy are associated with an increased risk of sPTB from 34 weeks of gestation. The usefulness of early pregnancy ferritin levels in identifying women at risk of sPTB warrants further investigation.

Evaluation of first trimester serum soluble endothelial cell-specific tyrosine kinase receptor in normal and affected pregnancies.

AIMS: To assess soluble endothelial cell-specific tyrosine kinase receptor (sTie-2) levels in the first trimester of pregnancy and its association with adverse pregnancy outcomes; and examine the predictive accuracy. STUDY DESIGN: In this nested case-control study, serum sTie-2 levels were measured in 2616 women with singleton pregnancies attending first trimester screening in New South Wales, Australia. Multivariate logistic regression models were used to assess the association and predictive accuracy of serum sTie-2 with subsequent adverse pregnancy outcomes. RESULTS: Median (interquartile range) sTie-2 for the total population was 19.6 ng/ml (13.6-26.4). Maternal age, weight, and smoking status significantly affected sTie-2 levels. There was no difference in serum sTie-2 between unaffected and women with adverse pregnancy outcomes. After adjusting for maternal and clinical risk factors, low sTie-2 (<25th centile) was associated with preeclampsia (Adjusted odds ratio: 1.61; 95% CI: 1.01-2.57), however, the accuracy of sTie-2 in predicting preeclampsia was not different from chance (AUC = 0.54; p = 0.08) and does not add valuable predictive information to maternal and clinical risk factors. CONCLUSIONS: Our findings suggest that low sTie-2 levels are associated with preeclampsia, however, it does not add valuable information to clinical and maternal risk factor information in predicting preeclampsia or any other adverse pregnancy outcomes.

Maternal body weight and first trimester screening for chromosomal anomalies.

Prenatal risk ratios for Down syndrome adjust for maternal weight because maternal serum biomarker levels decrease with increasing maternal weight. This is accomplished by converting serum biomarker values into a multiple of the expected median (MoM) for women of the same gestational age. Weight is frequently not recorded, and the impact of using MoMs not adjusted for weight for calculating risk ratios is unknown. The aim of this study is to examine the effect of missing weight on first trimester Down syndrome risk ratios by comparing risk ratios calculated using weight-unadjusted-and-adjusted MoMs. Findings at the population level indicate that the impact of not adjusting for maternal weight on first trimester screening results for chromosomal anomalies would lead to under-identification of 84 per 10,000 pregnancies.

Effects of maternal serum 25-hydroxyvitamin D concentrations in the first trimester on subsequent pregnancy outcomes in an Australian population.

BACKGROUND: Low serum 25-hydroxyvitamin D [25(OH)D] concentrations during pregnancy have been associated with adverse pregnancy outcomes in a few studies but not in other studies. OBJECTIVES: We assessed the serum 25(OH)D concentration at 10-14 wk of pregnancy and its association with adverse pregnancy outcomes and examined the predictive accuracy. DESIGN: In this nested case-control study, we measured serum 25(OH)D in 5109 women with singleton pregnancies who were attending first-trimester screening in New South Wales, Australia. Multivariate logistic regression was conducted to examine the association between low 25(OH)D concentrations and adverse pregnancy outcomes (small for gestational age, preterm birth, preeclampsia, gestational diabetes, miscarriage, and stillbirth). The predictive accuracy of models was assessed. RESULTS: The median (IQR) 25(OH)D concentration for the total population was 56.4 nmol/L (43.3-69.8 nmol/L). Serum 25(OH)D concentrations showed significant variation by parity, smoking, weight, season of sampling, country of birth, and socioeconomic status. After adjustment for maternal and clinical risk factors, low 25(OH)D concentrations were not associated with most adverse pregnancy outcomes. The area under the receiver operating characteristic curve (AUC) and likelihood ratio for a composite of severe adverse pregnancy outcomes of 25(OH)D concentrations <25 nmol/L were 0.51 and 1.44, respectively, and, for risk factors alone, were 0.64 and 2.87, respectively. The addition of 25(OH)D information to maternal and clinical risk factors did not improve the ability to predict severe adverse pregnancy outcomes (AUC: 0.64; likelihood ratio: 2.32; P = 0.39). CONCLUSION: Low 25(OH)D serum concentrations in the first trimester of pregnancy are not associated with adverse pregnancy outcomes and do not predict complications any better than routinely assessed clinical and maternal risk-factor information.

Angiopoietin 1 and 2 serum concentrations in first trimester of pregnancy as biomarkers of adverse pregnancy outcomes.

OBJECTIVE: To assess angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), and the Ang-1/Ang-2 ratio levels in the first trimester of pregnancy, their association with adverse pregnancy outcomes, and their predictive accuracy. STUDY DESIGN: This cohort study measured serum Ang-1 and Ang-2 levels in 4785 women with singleton pregnancies attending first trimester screening in New South Wales, Australia. Multivariate logistic regression models were used to assess the association and predictive accuracy of serum biomarkers with subsequent adverse pregnancy outcomes (small for gestational age, preterm birth, preeclampsia, miscarriage >10 weeks, and stillbirth). RESULTS: Median (interquartile range) levels for Ang-1, Ang-2, and the Ang-1/Ang-2 ratio for the total population were 19.6 ng/mL (13.6-26.4), 15.5 ng/mL (10.3-22.7), and 1.21 (0.83-1.73), respectively. Maternal age, weight, country of birth, and socioeconomic status significantly affected Ang-1, Ang-2, and the Ang-1/Ang-2 ratio levels. After adjusting for maternal and clinical risk factors, women with low Ang-2 levels (<10th percentile) and high Ang-1/Ang-2 ratio (>90th percentile) had increased risk of developing most adverse pregnancy outcomes. Compared with the Ang-1/Ang-2 ratio alone, maternal and clinical risk factors had better predictive accuracy for most adverse pregnancy outcomes. The exception was miscarriage (Ang-1/Ang-2 ratio area under receiver operating characteristic curve = 0.70; maternal risk factors = 0.58). Overall, adding the Ang-1/Ang-2 ratio to maternal risk factors did not improve the ability of the models to predict adverse pregnancy outcomes. CONCLUSION: Our findings suggest that the Ang-1/Ang-2 ratio in first trimester is associated with most adverse pregnancy outcomes, but do not predict outcomes any better than clinical and maternal risk factor information.

First trimester screening of serum soluble fms-like tyrosine kinase-1 and placental growth factor predicting hypertensive disorders of pregnancy.

OBJECTIVE: To assess the accuracy of first trimester soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) in predicting pregnancy hypertension and pre-eclampsia; and compare with the accuracy of routinely collected maternal and clinical risk factors. STUDY DESIGN: In this population-based cohort study, serum sFlt-1 and PlGF levels were measured in first trimester in 2,681 women with singleton pregnancies in New South Wales, Australia. MAIN OUTCOME MEASURES: Prediction of pregnancy hypertension and pre-eclampsia. RESULTS: There were 213 (7.9%) women with pregnancy hypertension, including 68 (2.5%) with pre-eclampsia. The area under the curve (AUC) for both sFlt-1 and PlGF was not different from chance, but combined was 0.55 (P=0.005). Parity and previous diagnosed hypertension had better predictive accuracy than serum biomarkers (AUC=0.64, P<0.001) and the predictive accuracy for all maternal and clinical information was fair (AUC=0.70, P<0.001 for pregnancy hypertension and AUC=0.74, P<0.001 for pre-eclampsia). Adding sFlt-1 and PlGF to maternal risk factors did not improve the ability of the models to predict pregnancy hypertension or pre-eclampsia. CONCLUSIONS: Maternal first trimester serum concentrations of sFlt-1 and PlGF do not predict hypertensive disorders in pregnancy any better than routinely collected clinical and maternal risk factor information. Screening for sFlt-1 and PlGF levels in early pregnancy would not identify those pregnancies at-risk.

Association and predictive accuracy of high TSH serum levels in first trimester and adverse pregnancy outcomes.

CONTEXT: High serum levels of TSH have been associated with adverse pregnancy outcomes by some studies, and not by others. OBJECTIVE: The aim of the study was to assess the association between high levels of TSH in the first trimester of pregnancy and adverse pregnancy outcomes; and to examine the predictive accuracy as a screening test. SETTING AND PARTICIPANTS: Serum levels of TSH were measured in a cohort of 2801 women with a singleton pregnancy attending first trimester Down syndrome screening. Information on maternal and infant outcomes was obtained through record linkage to population-based birth and hospital data. Association between high TSH (>95th and >97.5th centiles) multiple of the median levels, and risk of adverse pregnancy outcomes was evaluated using multivariable logistic regression, and the predictive accuracy of models was assessed. MAIN OUTCOMES: Rates of infants being small for gestational age (SGA), preterm birth, preeclampsia, miscarriage, and stillbirth were investigated. RESULTS: High TSH multiple of the median levels were associated with SGA (<10th centile) [adjusted odds ratio (aOR), 1.71; 95% confidence interval (CI), 0.99-2.94]; preterm birth at less than 37 wk gestation (aOR, 2.59; 95% CI, 1.21-5.53); miscarriage (aOR, 3.66; 95% CI, 1.59-8.44); and a composite measure of any study outcome (aOR, 2.10; 95% CI, 1.23-3.59). The area under the receiver operator characteristic curves were 0.69 (95% CI, 0.65-0.73) for SGA; 0.56 (95% CI, 0.51-0.61) for preterm birth; 0.70 (95% CI, 0.61-0.79) for miscarriage; and 0.63 (95% CI, 0.60-0.65) for any adverse pregnancy outcome. CONCLUSIONS: High TSH serum levels during the first trimester of pregnancy were associated with adverse pregnancy outcomes; however, the predictive accuracy was poor. Screening for high TSH levels in the first trimester would be of no benefit to identify women at risk.

Record linkage to obtain birth outcomes for the evaluation of screening biomarkers in pregnancy: a feasibility study.

BACKGROUND: Linking population health data to pathology data is a new approach for the evaluation of predictive tests that is potentially more efficient, feasible and efficacious than current methods. Studies evaluating the use of first trimester maternal serum levels as predictors of complications in pregnancy have mostly relied on resource intensive methods such as prospective data collection or retrospective chart review. The aim of this pilot study is to demonstrate that record-linkage between a pathology database and routinely collected population health data sets provides follow-up on patient outcomes that is as effective as more traditional and resource-intensive methods. As a specific example, we evaluate maternal serum levels of PAPP-A and free beta-hCG as predictors of adverse pregnancy outcomes, and compare our results with those of prospective studies. METHODS: Maternal serum levels of PAPP-A and free beta-hCG for 1882 women randomly selected from a pathology database in New South Wales (NSW) were linked to routinely collected birth and hospital databases. Crude relative risks were calculated to investigate the association between low levels (multiples of the median < or = 5th percentile) of PAPP-A or free beta-hCG and the outcomes of preterm delivery (<37 weeks), small for gestational age (<10th percentile), fetal loss and stillbirth. RESULTS: Using only full name, sex and date of birth for record linkage, pregnancy outcomes were available for 1681 (89.3%) of women included in the study. Low levels of PAPP-A had a stronger association with adverse pregnancy outcomes than a low level of free beta-hCG which is consistent with results in published studies. The relative risk of having a preterm birth with a low maternal serum PAPP-A level was 3.44 (95% CI 1.96-6.10) and a low free beta-hCG level was 1.31 (95% CI 0.55-6.16). CONCLUSION: This study provides data to support the use of record linkage for outcome ascertainment in studies evaluating predictive tests. Linkage proportions are likely to increase if more personal identifiers are available. This method of follow-up is a cost-efficient technique and can now be applied to a larger cohort of women.

PAX8-peroxisome proliferator-activated receptor gamma (PPARgamma) disrupts normal PAX8 or PPARgamma transcriptional function and stimulates follicular thyroid cell growth.

Follicular thyroid carcinomas are associated with a chromosomal translocation that fuses the thyroid-specific transcription factor paired box gene 8 (PAX8) with the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma). This study investigated the transcriptional mechanisms by which PAX8-PPARgamma regulates follicular thyroid cells. In HeLa cells, rat follicular thyroid (FRTL-5) cells, or immortalized human thyroid cells, PAX8-PPARgamma stimulated transcription from PAX8-responsive thyroperoxidase and sodium-iodide symporter promoters in a manner at least comparable with wild-type PAX8. In contrast, PAX8-PPARgamma failed to stimulate transcription from the thyroglobulin promoter and blocked the synergistic stimulation of this promoter by wild-type PAX8 and thyroid transcription factor-1. Unexpectedly, PAX8-PPARgamma transcriptional function on a PPARgamma-responsive promoter was cell-type dependent; in HeLa cells, PAX8-PPARgamma dominantly inhibited expression of the PPARgamma-responsive promoter, whereas in FRTL-5 and immortalized human thyroid cells PAX8-PPARgamma stimulated this promoter. In gel shift analyses, PAX8-PPARgamma bound a PPARgamma-response element suggesting that its transcriptional function is mediated via direct DNA contact. A biological model of PAX8-PPARgamma function in follicular thyroid cells was generated via constitutive expression of the fusion protein in FRTL-5 cells. In this model, PAX8-PPARgamma expression was associated with enhanced growth as assessed by soft agar assays and thymidine uptake. Therefore, PAX8-PPARgamma disrupts normal transcriptional regulation by stimulating some genes and inhibiting others, the net effect of which may mediate follicular thyroid cell growth and loss of differentiation that ultimately leads to carcinogenesis.

Bioactivity of PTH/PTHrP analogs lacking the 1-14 N-terminal domain.

The N-terminal regions of 1-34 parathyroid hormone (PTH) and 1-34 parathyroid hormone related protein (PTHrP) are thought to be required for full agonist activity of these molecules and for signal transduction by cyclic AMP (cAMP). The C-terminal regions are thought to be involved in receptor binding and protein kinase C activation. In this study, two analogs of PTH/PTHrP lacking the segment 1-14 exhibited agonist activity in opossum kidney (OK) 3B2 cells. Analogs cPTHrP(15-34) and ANA NPY(13-36), an analog of neuropeptide Y, which both have amphipathic alpha helices, inhibited phosphate uptake and stimulated cAMP production in a dose-dependent manner, with half maximal activity in the microM range, compared to the nM range for hPTHrP(1-34) and hPTH(1-34). They also exhibited proportionately lower receptor binding affinities. cAMP production by these analogs was suppressed by the antagonist hPTHrP(7-34). Inhibition of phosphate uptake in response to the analogs was partially suppressed by H-89, but not by bisindolylmaleimide. The analogs also inhibited phosphate uptake and stimulated cAMP in parent OK cells and stimulated cAMP production in UMR-106 cells. These studies present the novel finding that in these cell types, a C-terminal region encompassing PTH/PTHrP(24-31), with the alpha-helical structure maintained, is sufficient for full activity at reduced potency.