Soluble endoglin in urine as an early-pregnancy preeclampsia marker: antenatal longitudinal feasibility study.

This study aimed to evaluate soluble endoglin (sEng) in urine as a preeclampsia predictor. Ninety-three pregnant women at risk for preeclampsia were followed. Spot urine sample ELISA analysis before 20 weeks of gestation was done to assess protein levels. Logistic regression analysis evaluated associations between preeclampsia with sEng/creatinine ratio, pg/mg, adjusted for risk factors. Preeclampsia incidence was 22.8% (20/92). Urinary sEng/creatinine (pg/mg) 0.001 (95% CI 0.001-0.136) was associated, adjusted for body mass index > 28 kg/m2 OR 6.44 (95% CI 1.11-37.47) and mean arterial pressure OR 1.20 (1.07-1.35). During the first half of gestation sEng urinary excretion was lower in pregnant women developing preeclampsia.Impact statementWhat is already known on this subject? The angiogenesis factors present in the plasma of pregnant women have shown good preclinical predictors of preeclampsia. Studies on urinary markers in pregnancy are infrequent, despite the ease of obtaining urine specimens.What do the results of this study add? Values of the sEng/creatinine ratio during the first half of pregnancy were related to a higher chance of preeclampsia occurring when it was evaluated alone or adjusted by body mass index and mean arterial pressure values.What are the implications of these findings for clinical practice and/or further research? The potential benefits of a urinary test compared to one of the blood levels include its non-invasive nature and ease of performing the test, even during prenatal care. Future research is expected to evaluate the sEng/creatinine ratio relevance to improve clinical scores of preeclampsia prediction for the identification of women at risk for this disease.

Prenatal arsenic exposure alters the placental expression of multiple epigenetic regulators in a sex-dependent manner.

BACKGROUND: Prenatal exposure to arsenic has been linked to a range of adverse health conditions in later life. Such fetal origins of disease are frequently the result of environmental effects on the epigenome, leading to long-term alterations in gene expression. Several studies have demonstrated effects of prenatal arsenic exposure on DNA methylation; however the impact of arsenic on the generation and decoding of post-translational histone modifications (PTHMs) is less well characterized, and has not been studied in the context of prenatal human exposures. METHODS: In the current study, we examined the effect of exposure to low-to-moderate levels of arsenic in a US birth cohort, on the expression of 138 genes encoding key epigenetic regulators in the fetal portion of the placenta. Our candidate genes included readers, writers and erasers of PTHMs, and chromatin remodelers. RESULTS: Arsenic exposure was associated with the expression of 27 of the 138 epigenetic genes analyzed. When the cohort was stratified by fetal sex, arsenic exposure was associated with the expression of 40 genes in male fetal placenta, and only 3 non-overlapping genes in female fetal placenta. In particular, we identified an inverse relationship between arsenic exposure and expression of the gene encoding the histone methyltransferase, PRDM6 (p < 0.001). Mutation of PRDM6 has been linked to the congenital heart defect, patent ductus arteriosus. CONCLUSIONS: Our findings suggest that prenatal arsenic exposure may have sex-specific effects on the fetal epigenome, which could plausibly contribute to its subsequent health impacts.

Reference Intervals for Urine Sediment Analysis of Healthy Pregnant Women.

BACKGROUND: Urine sediment parameters of pregnant women are different from those of non-pregnant women, and it is necessary to establish reference intervals for pregnant women. The aim of this study was to establish reference intervals of white blood cell (WBC), red blood cell (RBC), bacteria (BACT), squamous epithelial cell (EC), small round epithelial cell (SRC), and mucous strands (MUS) for urine sediment test of pregnant women using a UF-1000i analyzer as the detection device. The differences between pregnant women and non-pregnant women in terms of the aforementioned parameters as well as the differences of such parameters in different trimesters of pregnancy were clarified. METHODS: The experimental subjects were divided into two groups: the experiment group (612 healthy pregnant women) and the control group (582 healthy non-pregnant women). Subjects of both groups are women between the age of 22 and 46. The urine specimens were analyzed using the Sysmex UF-1000i analyzer, followed by manual correction. A statistical analysis was performed by SPSS 22.0. Results were considered significant at p < 0.01. RESULTS: The pregnancy reference intervals of WBC, RBC, BACT, EC, SRC, and MUS were 0 ~ 30/µL, 0 ~ 23/µL, 0 ~ 698/µL, 0 ~ 28/µL, 0 ~ 8/µL, and 0 ~ 3/µL, respectively. In the experiment group, the concentrations of WBC, BACT, EC, and SRC were significantly higher than those of the control group (p < 0.01), while the concentrations of RBC and MUS were significantly lower than those of the control group (p < 0.01). The inter-trimester differences in terms of the concentrations of WBC, BACT, EC, and SRC were statistically indistinguishable (p > 0.05). However, the concentration of RBC was significantly lower with the increase of trimester of pregnancy (the comparison between the first trimester with the second trimester: p = 0.000 < 0.01; the comparison between the second trimester and the third trimester: p = 0.004 < 0.01). The WBC, BACT, EC, and SRC had moderate intercorrelations (0.569 ~ 0.681, p < 0.01). CONCLUSIONS: There were significant differences in the aforementioned parameters between the two groups. The intervals of WBC, RBC, BACT, EC, SRC, and MUS for urine sediment analysis of healthy pregnant women using a UF-1000i should be established.

Unhealthy Levels of Phthalates and Bisphenol A in Mexican Pregnant Women with Gestational Diabetes and Its Association to Altered Expression of miRNAs Involved with Metabolic Disease.

Several studies indicate that bisphenol A (BPA) and phthalates may have a role in the development of metabolic diseases using different molecular pathways, including epigenetic regulatory mechanisms. However, it is unclear whether exposure to these chemicals modifies serum levels of miRNAs associated with gestational diabetes mellitus (GDM) risk. In the present study, we evaluated the serum levels of miRNAs associated with GDM (miR-9-5p, miR-16-5p, miR-29a-3p and miR-330-3p) and urinary levels of phthalate metabolites (mono-n-butyl phthalate (MBP), mono-isobutyl phthalate (MiBP), mono-benzyl phthalate (MBzP) and mono(2-ethyl hexyl) phthalate (MEHP)) and bisphenol A in GDM patients and women without GDM during the second trimester of gestation. We observed higher levels of miR-9-5p, miR-29a-3p and miR-330-3p in sera of patients with GDM compared to non-diabetic subjects. Phthalates were detected in 97-100% of urine samples, while BPA only in 40%. Urinary MEHP and BPA concentrations were remarkably higher in both study groups compared to previously reported data. Unadjusted MEHP levels and adjusted BPA levels were higher in non-diabetics than in GDM patients (p = 0.03, p = 0.02). We found positive correlations between adjusted urinary MBzP levels and miR-16-5p expression levels (p < 0.05), adjusted MEHP concentrations and miR-29a-3p expression levels (p < 0.05). We also found negative correlations between unadjusted and adjusted MBP concentrations and miR-29a-3p expression levels (p < 0.0001, p < 0.05), unadjusted MiBP concentrations and miR-29a-3p expression levels (p < 0.01). Urinary MEHP levels reflect a striking exposure to di(2-ethylhexyl) phthalate (DEHP) in pregnant Mexican women. This study highlights the need for a regulatory strategy in the manufacture of several items containing endocrine disruptors in order to avoid involuntary ingestion of these compounds in the Mexican population.

Prenatal exposure to phthalates and autism spectrum disorder in the MARBLES study.

BACKGROUND: Evidence from experimental and observational studies suggests that prenatal phthalate exposures may be associated with autism spectrum disorder (ASD). We examined whether prenatal phthalate exposures were associated with an increased risk of ASD. METHODS: We quantified 14 metabolites of eight phthalates in 636 multiple maternal urine samples collected during 2nd and 3rd trimesters of pregnancy from 201 mother-child pairs in MARBLES (Markers of Autism Risk in Babies - Learning Early Signs), a high-risk ASD longitudinal cohort. At 3 years old, children were clinically assessed for ASD and classified into three diagnostic categories: ASD (n = 46), non-typical development (Non-TD, n = 55), and typical development (TD, n = 100). We used multinomial logistic regression to evaluate the association of phthalate metabolite concentrations with ASD and Non-TD. RESULTS: Most associations of phthalate biomarkers with both ASD and Non-TD were null, with the exception that monoethyl phthalate (MEP) was significantly associated with an increased risk of Non-TD (per 2.72-fold relative increase in concentration: Relative risk ratio (RRR) = 1.38; 95% confidence interval (CI): 1.01, 1.90). When stratified by prenatal vitamin use during the first month of pregnancy, among mothers who took vitamins, ASD risk was inversely associated with mono-isobutyl phthalate (MiBP, RRR = 0.44; 95% CI: 0.21, 0.88), mono(3-carboxypropyl) phthalate (MCPP, RRR = 0.41; 95% CI: 0.20, 0.83) and mono-carboxyisooctyl phthalate (MCOP, RRR = 0.49; 95% CI: 0.27, 0.88), but among mothers who did not take prenatal vitamins, Non-TD risk was positively associated with MCPP (RRR = 5.09; 95% CI: 2.05, 12.6), MCOP (RRR = 1.86; 95% CI: 1.01, 3.39), and mono-carboxyisononyl phthalate (MCNP, RRR = 3.67; 95% CI: 1.80, 7.48). When stratified by sex, among boys, MEP, monobenzyl phthalate, MCPP, MCNP, and sum of di(2-ethylhexyl) phthalate metabolites (ΣDEHP) were positively associated with Non-TD risk, but associations with ASD were null. Among girls, associations with both ASD and Non-TD were null. CONCLUSIONS: Our study showed that phthalate exposures in mid- to late pregnancy were not associated with ASD in children from this high-risk ASD cohort. Further studies should be conducted in the general population without high-risk genes to confirm our findings.

Trimester-specific phthalate concentrations and glucose levels among women from a fertility clinic.

BACKGROUND: Subfertile women are at increased risk of glucose intolerance in pregnancy. Based on epidemiologic studies, exposure to certain phthalates is associated with diabetes, elevated glucose, and increased insulin resistance. OBJECTIVES: To evaluate the association between urinary phthalate metabolites and pregnancy glucose levels in women seeking medically assisted reproduction. METHODS: We evaluated 245 women participating in a prospective cohort study based at a large fertility clinic who delivered live births and had data on pregnancy urinary phthalate metabolite concentrations and blood glucose levels. Urinary phthalate metabolite concentrations were from single spot urine samples collected in 1st and 2nd trimesters. Blood glucose data was abstracted from medical records for non-fasting 50-g glucose challenge tests at 24-28 weeks gestation. Multivariable linear regression models were used to evaluate associations between 7 urinary phthalate metabolites in quartiles and mean glucose adjusted for potential confounders. RESULTS: Eighteen percent of women had glucose levels ≥ 140 mg/dL. Second trimester monoethyl phthalate (MEP) concentrations were positively associated with glucose levels, with adjusted mean (95%CI) glucose levels of 121 mg/dl (114, 128) vs. 109 mg/dL (103, 116) for women in highest and lowest quartiles, respectively. Women in the highest quartile of second trimester mono-isobutyl phthalate (MiBP) concentrations had a mean glucose level 14 mg/dL lower compared to women in the lowest quartile. No other urinary phthalate metabolites were associated with glucose levels. CONCLUSIONS: MEP and MiBP-metabolites of diethyl phthalate and dibutyl phthalate, respectively-were associated with higher pregnancy glucose in subfertile women-a population at high risk of glucose intolerance in pregnancy.

First and second trimester urinary metabolic profiles and fetal growth restriction: an exploratory nested case-control study within the infant development and environment study.

BACKGROUND: Routine prenatal care fails to identify a large proportion of women at risk of fetal growth restriction (FGR). Metabolomics, the comprehensive analysis of low molecular weight molecules (metabolites) in biological samples, can provide new and earlier biomarkers of prenatal health. Recent research has suggested possible predictive first trimester urine metabolites correlating to fetal growth restriction in the third trimester. Our objective in this current study was to examine urinary metabolic profiles in the first and second trimester of pregnancy in relation to third trimester FGR in a US population from a large, multi-center cohort study of healthy pregnant women. METHODS: We conducted a nested case-control study within The Infant Development and the Environment Study (TIDES), a population-based multi-center pregnancy cohort study. We identified 53 cases of FGR based on the AUDIPOG [Neonatal growth - AUDIPOG [Internet]. [cited 29 Nov 2016]. Available from: http://www.audipog.net/courbes_morpho.php?langue=en ] formula for birthweight percentile considering maternal height, age, and prenatal weight, as well as infant sex, gestational age, and birth rank. Cases were matched to 106 controls based on study site, maternal age (± 2 years), parity, and infant sex. NMR spectroscopy was used to assess concentrations of four urinary metabolites that have been previously associated with FGR (tyrosine, acetate, formate, and trimethylamine) in first and second trimester urine samples. We fit multivariate conditional logistic regression models to estimate the odds of FGR in relation to urinary concentrations of these individual metabolites in the first and second trimesters. Exploratory analyses of custom binned spectroscopy results were run to consider other potentially related metabolites. RESULTS: We found no significant association between the relative concentrations of each of the four metabolites and odds of FGR. Exploratory analyses did not reveal any significant differences in urinary metabolic profiles. Compared with controls, cases delivered earlier (38.6 vs 39.8, p < 0.001), and had lower birthweights (2527 g vs 3471 g, p < 0.001). Maternal BMI was similar between cases and controls. CONCLUSIONS: First and second trimester concentrations of urinary metabolites (acetate, formate, trimethylamine and tyrosine) did not predict FGR. This inconsistency with previous studies highlights the need for more rigorous investigation and data collection in this area before metabolomics can be clinically applied to obstetrics.

Identification of urinary biomarkers for the prediction of gestational diabetes mellitus in early second trimester of young gravidae based on iTRAQ quantitative proteomics.

Gestational Diabetes Mellitus (GDM) has brought great harm to maternal and fetus. Up to now, only a few plasma biomarkers for its early diagnosis have been reported; nevertheless, there is no report about identification of urinary biomarkers for prediction of GDM. Thus, it is necessary to correct this deficiency. In our study, urine samples were collected from 889 healthy young gravidae at the early second trimester (15 to 20 weeks), 69 of whom were subsequently diagnosed with GDM at 24 to 28 weeks. iTRAQ (the isobaric tags for relative and absolute quantification) quantitative proteomics was conducted on sixteen GDM (trial group) and an equal number of matched healthy young gravidae (control group). Validation was performed in 40 cases of each group by ELISA. A total of 1,901 proteins were identified in this study, including 119 significantly differential proteins (fold change ≧1.2 or ≦0.83 and p < 0.05). Compared with control group, 83 differential proteins were increased and 36 proteins were decreased in GDM group. The validation for expression of CD59 and IL1RA showed significant difference and the area under the receiver operating characteristic curve was 0.729 and 0.899, respectively (p < 0.05). The two candidate protein biomarkers (CD59 and IL1RA) in urine could be an early, noninvasive diagnostic predictors of young pravidae with GDM, and IL1RA is stronger diagnostic power than CD59.

Perinatal Biochemical Confirmation of Smoking Status by Trimester.

INTRODUCTION: Tobacco use during pregnancy is the most modifiable risk factor associated with poor pregnancy outcomes. Self-reported tobacco use has been demonstrated to have high misclassification rates. The aims were to examine misclassification rates of perinatal tobacco use during each trimester of pregnancy and 8 weeks postpartum, and to evaluate characteristics associated with misclassification of tobacco use status. METHODS: This is secondary analysis of a prospective, multicenter trial of pregnant women, and it includes participants who were biochemically identified as tobacco users during their first trimester (N = 103). Each trimester and once postpartum, tobacco use was assessed via self-report and validated using a cutoff of 100 ng/mL for urine cotinine via NicAlert test strips to indicate current use. Those who self-reported as nonusers but were identified as users via urine cotinine were considered misclassified; misclassification rates were determined for each time period. Logistic regression assessed maternal factors associated with misclassification status. RESULTS: Misclassification rates declined from 35.0% at first trimester to 31.9% and 26.6% at the second and third; the postpartum rate was 30.4%. These rates did not differ significantly from each other at the 0.05 level. Race/ethnicity was associated with misclassification status; white/non-Hispanic women were 87% less likely to be misclassified (p < .001). CONCLUSION: Misclassification of prenatal smoking status decreases as pregnancy progresses, though the observed rate change was not significant. Minority women may be at particular risk for non-disclosure of tobacco use. Biochemical validation should be considered when assessing perinatal tobacco use via self-report, given high misclassification rates throughout the perinatal period. IMPLICATIONS: These results demonstrate that regardless of trimester, more than one-quarter of tobacco-using pregnant women may not disclose tobacco use throughout pregnancy and early postpartum. Although the rate of misclassification decreased from first to third trimester and then increased in the immediate postpartum, these changes in misclassification rates were not significant. Minority groups may be at particular risk of misclassification compared with white/non-Hispanic women. Biochemical validation is warranted throughout pregnancy to encourage cessation as tobacco use is one of the most easily-modified risk factors for poor birth outcomes.

Correlation between markers of DNA and lipid oxidative damage in maternal and fetoplacental compartment in the mid-trimester of pregnancy.

OBJECTIVE: To determine the levels of 8-isoprostane (8-IP) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in urine and in amniotic fluid (AF) of pregnant women and to assess the correlation between oxidative status in the maternal and fetal compartment in the second trimester of pregnancy. METHODS: One hundred and forty-six women with singleton pregnancies, undergoing amniocentesis at the Department of Obstetrics and Gynaecology at the University Medical Centre Ljubljana, were prospectively enrolled. AF and maternal urine were collected in the second trimester of pregnancy. Paired urinary and AF 8-IP and 8-OHdG were measured and evaluated cross-sectionally. RESULTS: 8-IP and 8-OHdG concentrations were higher in maternal urine compared to AF and the ratios were 47:1 and 50:1, respectively. AF 8-OHdG was very low and in 74% was below the limit of detection (LOD). We found a positive correlation between 8-IP in maternal and fetal compartment (ρ=0.217, P=0.008), which stayed unchanged also after adjustment for possible confounding factors. CONCLUSIONS: Oxidative damage to lipids and DNA is also a part of physiologic processes during healthy pregnancy. 8-IP and 8-OHdG are constantly present in urine and AF. A weak positive correlation between maternal and fetal unit suggests a weak reflection of fetal oxidative status in maternal urine in the mid-trimester.

sFlt-1, PlGF, sFlt-1/PlGF ratio and uterine artery Doppler for preeclampsia diagnostics.

BACKGROUND AND OBJECTIVE: Angiogenic factors such as soluble fms-like tyrosine kinase 1 (sFlt-1) and placental growth factor (PlGF) play a key role in the pathogenesis of preeclampsia. Uterine artery (UA) blood flow is important for preeclamptic pregnancy outcome, but small amount of evidence suggests UA dopplerometry for preeclampsia diagnostics and management. The aim of our study was to compare the value of angiogenic factors and UA dopplerometry in preeclampsia diagnosis and determine cut-off values to obtain the highest sensitivity and specificity of the parameter. MATERIALS AND METHODS: We performed a case controlled study of 72 pregnant women with preeclampsia and 72 healthy matched controls. SFlt-1 and PlGF were measured in serum samples, the sFlt-1/PlGF ratio was calculated and UA pulsatility (PI) and resistance (RI) indexes were registered. RESULTS: Significantly higher levels of sFlt-1, sFlt-1/PlGF ratio and mean UAPI and UARI and lower levels of PlGF were found in preeclampsia group when compared to controls. The highest sensitivity and specificity for preeclampsia had SFlt-1/PlGF and PlGF with the cut-off values of ≥35 (sensitivity of 95.8% and specificity of 96.2%, respectively) and ≤138.6pg/mL (sensitivity of 95.8% and specificity of 93.7%, respectively). For diagnostics of early-onset preeclampsia, all factors sFlt-1, PlGF and sFlt-1/PlGF had equal significance with the cut-off values of ≥7572pg/mL (specificity of 97.5%, sensitivity 92.3%), ≤100.5pg/mL (specificity 96.2%, sensitivity of 100%) and ≥54.6 (specificity 97.5%, sensitivity 97.5%) respectively. CONCLUSIONS: The sFlt-1/PlGF ratio and PlGF are superior to sFlt-1, UAPI and UARI for preeclampsia diagnosis. For early-onset preeclampsia diagnostics either sFlt-1 or PlGF is sufficient.

Urine tests for Down's syndrome screening.

BACKGROUND: Down's syndrome occurs when a person has three copies of chromosome 21, or the specific area of chromosome 21 implicated in causing Down's syndrome, rather than two. It is the commonest congenital cause of mental disability and also leads to numerous metabolic and structural problems. It can be life-threatening, or lead to considerable ill health, although some individuals have only mild problems and can lead relatively normal lives. Having a baby with Down's syndrome is likely to have a significant impact on family life. The risk of a Down's syndrome affected pregnancy increases with advancing maternal age.Noninvasive screening based on biochemical analysis of maternal serum or urine, or fetal ultrasound measurements, allows estimates of the risk of a pregnancy being affected and provides information to guide decisions about definitive testing. Before agreeing to screening tests, parents need to be fully informed about the risks, benefits and possible consequences of such a test. This includes subsequent choices for further tests they may face, and the implications of both false positive and false negative screening tests (i.e. invasive diagnostic testing, and the possibility that a miscarried fetus may be chromosomally normal). The decisions that may be faced by expectant parents inevitably engender a high level of anxiety at all stages of the screening process, and the outcomes of screening can be associated with considerable physical and psychological morbidity. No screening test can predict the severity of problems a person with Down's syndrome will have. OBJECTIVES: To estimate and compare the accuracy of first and second trimester urine markers for the detection of Down's syndrome. SEARCH METHODS: We carried out a sensitive and comprehensive literature search of MEDLINE (1980 to 25 August 2011), EMBASE (1980 to 25 August 2011), BIOSIS via EDINA (1985 to 25 August 2011), CINAHL via OVID (1982 to 25 August 2011), The Database of Abstracts of Reviews of Effectiveness (The Cochrane Library 2011, Issue 7), MEDION (25 August 2011), The Database of Systematic Reviews and Meta-Analyses in Laboratory Medicine (25 August 2011), The National Research Register (archived 2007), Health Services Research Projects in Progress database (25 August 2011). We studied reference lists and published review articles. SELECTION CRITERIA: Studies evaluating tests of maternal urine in women up to 24 weeks of gestation for Down's syndrome, compared with a reference standard, either chromosomal verification or macroscopic postnatal inspection. DATA COLLECTION AND ANALYSIS: We extracted data as test positive or test negative results for Down's and non-Down's pregnancies allowing estimation of detection rates (sensitivity) and false positive rates (1-specificity). We performed quality assessment according to QUADAS (Quality Assessment of Diagnostic Accuracy Studies) criteria. We used hierarchical summary ROC (receiver operating characteristic) meta-analytical methods to analyse test performance and compare test accuracy. We performed analysis of studies allowing direct comparison between tests. We investigated the impact of maternal age on test performance in subgroup analyses. MAIN RESULTS: We included 19 studies involving 18,013 pregnancies (including 527 with Down's syndrome). Studies were generally of high quality, although differential verification was common with invasive testing of only high-risk pregnancies. Twenty-four test combinations were evaluated formed from combinations of the following seven different markers with and without maternal age: AFP (alpha-fetoprotein), ITA (invasive trophoblast antigen), ß-core fragment, free ßhCG (beta human chorionic gonadotrophin), total hCG, oestriol, gonadotropin peptide and various marker ratios. The strategies evaluated included three double tests and seven single tests in combination with maternal age, and one triple test, two double tests and 11 single tests without maternal age. Twelve of the 19 studies only evaluated the performance of a single test strategy while the remaining seven evaluated at least two test strategies. Two marker combinations were evaluated in more than four studies; second trimester ß-core fragment (six studies), and second trimester ß-core fragment with maternal age (five studies).In direct test comparisons, for a 5% false positive rate (FPR), the diagnostic accuracy of the double marker second trimester ß-core fragment and oestriol with maternal age test combination was significantly better (ratio of diagnostic odds ratio (RDOR): 2.2 (95% confidence interval (CI) 1.1 to 4.5), P = 0.02) (summary sensitivity of 73% (CI 57 to 85) at a cut-point of 5% FPR) than that of the single marker test strategy of second trimester ß-core fragment and maternal age (summary sensitivity of 56% (CI 45 to 66) at a cut-point of 5% FPR), but was not significantly better (RDOR: 1.5 (0.8 to 2.8), P = 0.21) than that of the second trimester ß-core fragment to oestriol ratio and maternal age test strategy (summary sensitivity of 71% (CI 51 to 86) at a cut-point of 5% FPR). AUTHORS' CONCLUSIONS: Tests involving second trimester ß-core fragment and oestriol with maternal age are significantly more sensitive than the single marker second trimester ß-core fragment and maternal age, however, there were few studies. There is a paucity of evidence available to support the use of urine testing for Down's syndrome screening in clinical practice where alternatives are available.

Second trimester maternal urine for the diagnosis of trisomy 21 and prediction of poor pregnancy outcomes.

Given the recognized lack of prenatal clinical methods for the early diagnosis of preterm delivery, intrauterine growth restriction, preeclampsia and gestational diabetes mellitus, and the continuing need for optimized diagnosis methods for specific chromosomal disorders (e.g., trisomy 21) and fetal malformations, this work sought specific metabolic signatures of these conditions in second trimester maternal urine, using (1)H Nuclear Magnetic Resonance ((1)H NMR) metabolomics. Several variable importance to the projection (VIP)- and b-coefficient-based variable selection methods were tested, both individually and through their intersection, and the resulting data sets were analyzed by partial least-squares discriminant analysis (PLS-DA) and submitted to Monte Carlo cross validation (MCCV) and permutation tests to evaluate model predictive power. The NMR data subsets produced significantly improved PLS-DA models for all conditions except for pre-premature rupture of membranes. Specific urinary metabolic signatures were unveiled for central nervous system malformations, trisomy 21, preterm delivery, gestational diabetes, intrauterine growth restriction and preeclampsia, and biochemical interpretations were proposed. This work demonstrated, for the first time, the value of maternal urine profiling as a complementary means of prenatal diagnostics and early prediction of several poor pregnancy outcomes.

Metabolic biomarkers of prenatal disorders: an exploratory NMR metabonomics study of second trimester maternal urine and blood plasma.

This work describes an exploratory NMR metabonomic study of second trimester maternal urine and plasma, in an attempt to characterize the metabolic changes underlying prenatal disorders and identify possible early biomarkers. Fetal malformations have the strongest metabolic impact in both biofluids, suggesting effects due to hypoxia (leading to hypoxanthine increased excretion) and a need for enhanced gluconeogenesis, with higher ketone bodies (acetone and 3-hydroxybutyric acid) production and TCA cycle demand (suggested by glucogenic amino acids and cis-aconitate overproduction). Choline and nucleotide metabolisms also seem affected and a distinct plasma lipids profile is observed for mothers with fetuses affected by central nervous system malformations. Urine from women who subsequently develop gestational diabetes mellitus exhibits higher 3-hydroxyisovalerate and 2-hydroxyisobutyrate levels, probably due to altered biotin status and amino acid and/or gut metabolisms (the latter possibly related to higher BMI values). Other urinary changes suggest choline and nucleotide metabolic alterations, whereas lower plasma betaine and TMAO levels are found. Chromosomal disorders and pre-preterm delivery groups show urinary changes in choline and, in the latter case, in 2-hydroxyisobutyrate. These results show that NMR metabonomics of maternal biofluids enables the noninvasive detection of metabolic changes associated to prenatal disorders, thus unveiling potential disorder biomarkers.

Prenatal screening of sialic acid storage disease and confirmation in cultured fibroblasts by LC-MS/MS.

Sialic acid storage disease (SASD) is an inborn error resulting from defects in the lysosomal membrane protein sialin. The SASD phenotypical spectrum ranges from a severe presentation, infantile sialic acid storage disease (ISSD) which may present as hydrops fetalis, to a relatively mild form, Salla disease. Screening for SASD is performed by determination of free sialic acid (FSA) in urine or amniotic fluid supernatant (AFS). Subsequent diagnosis of SASD is performed by quantification of FSA in cultured fibroblasts and by mutation analysis of the sialin gene, SLC17A5. We describe simple quantitative procedures to determine FSA as well as conjugated sialic acid in AFS, and FSA in cultured fibroblasts, using isotope dilution ((13)C(3)-sialic acid) and multiple reaction monitoring LC-ESI-MS/MS. The whole procedure can be performed in 2-4 h. Reference values in AFS were 0-8.2 µmol/L for 15-25 weeks of gestation and 3.2-12.0 µmol/L for 26-38 weeks of gestation. In AFS samples from five fetuses affected with ISSD FSA was 23.9-58.9 µmol/L demonstrating that this method is able to discriminate ISSD pregnancies from normal ones. The method was also validated for determination of FSA in fibroblast homogenates. FSA in SASD fibroblasts (ISSD; 20-154 nmol/mg protein, intermediate SASD; 12.9-15.1 nmol/mg, Salla disease; 5.9-7.4 nmol/mg) was clearly elevated compared to normal controls (0.3-2.2 nmol/mg). In conclusion, we report simple quantitative procedures to determine FSA in AFS and cultured fibroblasts improving both prenatal diagnostic efficacy for ISSD as well as confirmatory testing in cultured fibroblasts following initial screening in urine or AFS.

First-trimester increase in oxidative stress and risk of small-for-gestational-age fetus.

OBJECTIVE: Investigation of increased oxidative stress in early pregnancy and association with an increased risk of small-for-gestational-age (SGA) fetus. DESIGN: Longitudinal case-control study. SETTING: University Hospitals of Leicester NHS Trust, Leicester, UK. POPULATION: Low-risk pregnant women with no current or pre-existing medical illness were recruited at a large teaching hospital from 2004 to 2006. METHODS: Recruitment performed at the time of the dating ultrasound scan (12+/-2 weeks of gestation). Spot urine samples collected at 12+/-2 and 28+/-2 weeks of gestation were analysed for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) by liquid chromatography with tandem mass spectrometry). SGA was defined as birthweight <10th centile based on customised centile calculator (www.gestation.net). This identified the cases (n=55), whereas controls (n=55) were mothers whose babies were appropriate for gestational age (AGA, birthweight 10th-90th centile). Statistical analysis was performed using GraphPad Prism v.5. The relationship between maternal urinary 8-oxodG at different gestations and customised SGA was investigated by nonparametric tests. MAIN OUTCOME MEASURES: Customised SGA and AGA pregnancies. RESULTS: Urinary 8-oxodG concentrations were significantly increased in pregnancies with subsequent SGA compared with concentrations in normal pregnancies; 12 weeks: 2.8 (interquartile range [IQR] 1.96-3.67) versus 2.2 (IQR 1.26-3.28) pmol 8-oxodG/micromol creatinine (P=0.0007); 28 weeks: 2.21 (IQR 1.67-3.14) versus 1.68 (IQR 1.16-2.82) pmol 8-oxodG/micromol creatinine (P<0.0002). Concentrations decreased significantly between week 12 and 28 (P=0.04 and P=0.02 for controls and cases). CONCLUSIONS: In this study, urinary 8-oxodG at 12 and 28 weeks were elevated in SGA compared with AGA pregnancies. This may reflect early placental changes predating clinical features of SGA.

Maternal urine and serum steroid measurements to identify steroid sulfatase deficiency (STSD) in second trimester pregnancies.

OBJECTIVE: To document the performance of second trimester maternal urine and serum steroid measurements for detecting fetal steroid sulfatase deficiency (STSD). METHODS: We studied detection rate and false positive rate (DR, FPR) of analytes in maternal urine [combinations of 16alpha-OH-dehydroepiandrosterone sulfate (16alpha-OH-DHEAS), 11beta-hydroxyandrosterone, total estriol] and serum [combinations of 16alpha-OH-DHEAS, 11beta-hydroxyandrosterone, total estriol, unconjugated estriol (uE3)]. Samples were obtained from pregnancies which were screen positive for Smith-Lemli-Opitz syndrome (SLOS). RESULTS: Among 1 079 301 pregnancies, 3083 (0.29%) were screen positive for SLOS. Urine and/or serum samples were available from 917 viable pregnancies with known gender. We assigned likelihood ratios (LRs) to steroid measurements from male fetuses with known STSD and unaffected female fetuses. An LR > or = 100 was present in urine from 84 of 86 STSD pregnancies (98% DR, 95% CI 92-99), along with 0 of 198 pregnancies with normal female fetuses (0.0% FPR, CI 0-1.9). LRs were > or = 100 in 4 of 129 female fetuses with major abnormalities (3% FPR). In maternal serum, steroid measurements performed less effectively, achieving a 71% DR for STSD at a 1.6% FPR. CONCLUSION: Maternal urine steroid measurements are effective for detecting STSD, including those with point mutations and those with full deletions.

Study on the Potential Biomarkers of Maternal Urine Metabolomics for Fetus with Congenital Heart Diseases Based on Modified Gas Chromatograph-Mass Spectrometer.

BACKGROUND: There has been significant research on the genetic and environmental factors of congenital heart defects (CHDs), but few causes of teratogenicity, especially teratogenic mechanisms, can be clearly identified. Metabolomics has a potential advantage in researching the relationship between external factors and CHD. OBJECTIVE: To find and identify the urinary potential biomarkers of pregnancy (including in the second and third trimesters) for fetuses with CHD based on modified gas chromatograph-mass spectrometer (GC-MS), which could reveal the possibility of high-risk factors for CHD and lay the foundation for early intervention, treatment, and prevention. METHODS: Using a case-control design, we measured the urinary potential biomarkers of maternal urine metabolomics based on GC-MS in a population-based sample of women whose infants were diagnosed with CHD (70 case subjects) or were healthy (70 control subjects). SIMCA-P 13.0 software, principal component analysis (PCA), orthogonal partial least squares-discriminant analysis (OPLS-DA), Wilcoxon-Mann-Whitney test, and logistics regression were used to find significant potential biomarkers. RESULT: The 3D score graph of the OPLS-DA showed that the CHD and control groups were fully separated. The fitting parameters were R2x=0.78 and R2y=0.69, and the forecast rate was Q2=0.61, indicating a high forecast ability. According to the ranking of VIPs from the OPLS-DA models, we found 34 potential metabolic markers with a VIP > 1, and after two pairwise rank sum tests, we found 20 significant potential biomarkers, which were further used in multifactor logistic regressions. Significant substances, including 4-hydroxybenzeneacetic acid (OR=4.74, 95% CI: 1.06-21.06), 5-trimethylsilyloxy-n-valeric acid (OR=15.78, 95% CI: 2.33-106.67), propanedioic acid (OR=5.37, 95% CI: 1.87-15.45), hydracrylic acid (OR=6.23, 95% CI: 1.07-36.21), and uric acid (OR=5.23, 95% CI: 1.23-22.32), were associated with CHD. CONCLUSION: The major potential biomarkers in maternal urine associated with CHD were 4-hydroxybenzeneacetic acid, 5-trimethylsilyloxy-n-valeric acid, propanedioic acid, hydracrylic acid, and uric acid, respectively. These results indicated that the short chain fatty acids (SCFAs) and aromatic amino acid metabolism may be relevant with CHD.

Urine albumin concentration and albumin-to-creatinine ratio at 11(+0) to 13(+6) weeks in the prediction of pre-eclampsia.

OBJECTIVE: To determine the performance of screening for pre-eclampsia by maternal characteristics, urine albumin concentration and albumin-to-creatinine ratio (ACR) at 11(+0) to 13(+6) weeks. DESIGN: Prospective cross-sectional observational study. SETTING: Routine antenatal visit. POPULATION: A total of 2679 pregnant women at 11(+0) to 13(+6) weeks of gestation. METHODS: Maternal variables, urine albumin concentrations and ACR of 51 women who developed pre-eclampsia were compared with 2364 women who were unaffected by hypertensive disorders. Regression analysis was used first to determine which of the factors among the maternal characteristics were significant predictors of urine albumin concentration and ACR in the unaffected group and second to determine the contribution of urine albumin concentration and ACR in the prediction of pre-eclampsia. MAIN OUTCOME MEASURES: Development of pre-eclampsia. RESULTS: In the unaffected group, log urine albumin concentration and log ACR were influenced by ethnic origin, age, body mass index (BMI), parity and smoking. In the prediction of pre-eclampsia, significant contributions were provided by log urine albumin concentration, log ACR, ethnic origin, BMI, age, family and history of pre-eclampsia. The median urine albumin concentration and the median ACR in the pre-eclampsia group were significantly higher than those in the unaffected group. However, in screening for pre-eclampsia, the area under the receiver operating characteristic curve was not significantly improved by the combined models than with maternal variables alone. The value of urine albumin concentration was not improved by correcting for the creatinine concentration. CONCLUSION: In the prediction of pre-eclampsia, urine albumin concentration at 11(+0) to 13(+6) weeks does not provide additional value to maternal variables.

Second-trimester urinary neutrophil gelatinase-associated lipocalin levels in gestational diabetes: preliminary results.

OBJECTIVE: The objective of this study is to investigate the urinary neutrophil gelatinase-associated lipocalin (uNGAL) levels in the second trimester of pregnant patients at the time of gestational diabetes mellitus (GDM) screening. MATERIALS AND METHODS: Urinary samples from 88 pregnant women who underwent gestational diabetes screening test were collected in late second trimester (24-28 weeks) prospectively. After an overnight fasting, 75 g GTT was performed. The blood samples were drawn for measurement of glucose, insulin, and HbA1c. The urinary and blood parameters were compared for pregnant women with or without gestational diabetes. RESULTS: uNGAL levels were significantly elevated in pregnant women with gesting compared with the control groups (p < .014). There was a positive correlation between uNGAL and HbA1c levels (p = .001). CONCLUSIONS: In the second trimester, at the time of GDM screening, high levels of uNGAL indicate tubular injury in GDM cases which seems to be a result of hyperglycemia. uNGAL may correlate with an inflammatory renal involvement in GDM.