The stability of free-ß human chorionic gonadotrophin and pregnancy-associated plasma protein-A in first trimester dried blood spots.

OBJECTIVES: To determine the stability of first trimester free-ß human chorionic gonadotrophin (free-ß hCG) and pregnancy-associated plasma protein-A (PAPP-A) in dried blood spots (DBSs) under typical storage conditions. METHODS: First trimester maternal blood was spotted onto filter paper and left to dry. DBSs were analysed for PAPP-A and free-ß hCG using an AutoDELFIA dual assay at t = 0. Cards were stored at one of - 20 °C, refrigerator temperature, room temperature or 30 °C and reanalysed at set future time points. RESULTS: Free-ß hCG was stable (<10% change in concentration) under all temperatures tested for at least 35 days. PAPP-A was stable at - 20 °C and refrigerator temperature for at least 35 days. However, PAPP-A levels decreased by 10% at 4.1 days at room temperature and at 3.9 days at 30 °C. Longer-term storage at - 20 °C and refrigerator temperature showed that both PAPP-A and free-ß hCG levels were significantly decreased by 107 and 244 days. CONCLUSIONS: Free-ß hCG stability is greatly improved in DBS compared to serum storage; however PAPP-A stability is decreased in the DBS medium. Despite this DBS, screening may not necessitate such strict storage and transportation rules compared to serum screening programmes.

Maternal plasma levels of follistatin-related gene protein in the first trimester of pregnancies with Down syndrome.

OBJECTIVE: To determine maternal plasma levels of follistatin-related gene protein (FLRG) in the first trimester of pregnancy and assess its potential role as a marker for prenatal screening of Down syndrome. METHODS: Maternal plasma levels of FLRG were determined in 100 pregnant women with normal fetuses in their first trimester of pregnancy (i.e. 11th to 15th weeks). These results were compared with 20 cases with Down syndrome fetuses, taking into consideration clinical and demographic variables, such as maternal age, maternal weight, gestational age, smoking status and ethnicity. RESULTS: Maternal plasma median of FLRG in the normal population was 1.41 ng/mL with 95% confidence interval (CI) of 1.37-1.70 and interquartile range (IQR) of 0.88, during the 11th to 15th weeks of pregnancy. Maternal age and weight were the only variables significantly related to FLRG levels (p = 0.030 and 0.020, respectively). Only maternal and gestational ages were related to Down syndrome (p = 0.039 and 0.006, respectively). Maternal plasma levels of FLRG were not significantly different in the presence of Down syndrome fetuses compared to normal population (p = 0.63). CONCLUSION: FLRG can be successfully detected in maternal plasma in the first trimester of pregnancy. However, its levels are not significantly altered in the presence of Down syndrome fetuses.

PAPP-A and free ss-hCG stability in first trimester serum using PerkinElmer AutoDELFIA and DELFIA Xpress systems.

BACKGROUND: In this study we aim to investigate the stability of free-beta-hCG and PAPP-A over time in serum and whole blood in typical routine temperatures. METHODS: Serum pools were stored under the following temperatures: 30 degrees C, room temperature, refrigerator temperature and -20 degrees C, for up to 240 days. Stability of the markers in whole blood was examined in a shorter study and compared to serum. Samples were analysed using the AutoDELFIA and DELFIA Xpress analysers. RESULTS: On the AutoDELFIA, considering a 10% change acceptable, PAPP-A levels are stable in serum for 142 days at refrigerator temperature, 37 days at room temperature and 20 days at 30 degrees C. Free-beta hCG is stable in serum for 94 days at refrigerator temperature, 3 days at room temperature and 12 h at 30 degrees C. There was no significant change with either analyte after -20 degrees C storage for up to 240 days or after six repeated freeze-thaw cycles. In whole blood, free-beta hCG levels increased more rapidly compared to serum, especially at 30 degrees C. CONCLUSION: Normal handling of samples is only likely to minimally effect the risk assessment of chromosomal anomalies. However, careful attention should be paid to minimise the increase of free-beta hCG levels in samples shipped as whole blood.

The effect of Rhesus status on first-trimester pregnancy screening markers free beta human chorionic gonadotropin, pregnancy-associated plasma protein-A and nuchal translucency.

OBJECTIVE: To examine whether maternal Rhesus status has any effect on the levels of first-trimester markers free beta-human chorionic gonadotropin (beta-hCG), pregnancy-associated plasma protein-A (PAPP-A) and nuchal translucency (NT). METHODS: First-trimester free-beta-hCG and PAPP-A levels from pregnant women attending three hospitals in Kent were converted into MoMs and corrected for maternal weight, ethnicity, and smoking status. Maternal Rhesus status data were merged with screening data and free-beta-hCG and PAPP-A medians multiples of medians (MoMs) and NT from the Rhesus positive and Rhesus negative group were compared. RESULTS: Totally, 15 045 normal, singleton pregnancies were retrieved with full records. Altogether, 16.0% of the population were Rhesus negative. There was no difference between the medians MoMs of both free-beta-hCG nor PAPP-A nor NT in the two Rhesus status groups (p > 0.05). Demographic analysis on the distribution of Rhesus status in different ethnic origins showed that Caucasians have lower percentages of RhD-positive antigen compared to Asians and Afro-Caribbeans. CONCLUSION: Maternal Rhesus status does not influence the levels of free-beta-hCG and PAPP-A in the first trimester of pregnancy in this almost exclusively Caucasian population studied; therefore correction for maternal Rhesus status is not suggested.

Is maternal renal disease a cause of elevated free beta-hCG in first trimester aneuploidy screening?

OBJECTIVE: To asses whether supra elevated levels of maternal serum free beta hCG in the first trimester are associated with impaired renal function. METHOD: A cohort of 553 women with maternal serum free beta-hCG greater than 5 multiple of median (MoM) with a single euploid fetus was matched with a control of the same maternal age (+/-1 year), ethnic origin and with a free beta-hCG within the range 0.50-1.50 MoM. Screening samples were analysed for serum creatinine and estimated glomerular filtration rate was calculated. Renal function in the two groups was compared. The database was examined to find outcomes registered as known renal disease amongst the high free beta-hCG group. RESULTS: In the group with a supra elevated free beta-hCG MoM there was a significant reduction in estimated glomerular filtration rate (eGFR) (122.85, 95% CI 120.43-124.74 vs 118.80, 95% CI 114.90-121.59; p = 0.009) suggesting a small but increased risk of renal disease. CONCLUSIONS: The presence of renal disease should also be considered as one potential cause of supra elevated levels of free beta-hCG in addition to the possibility of paternally derived triploidy and trisomy 21.

The impact of fetal gender on first trimester nuchal translucency and maternal serum free beta-hCG and PAPP-A MoM in normal and trisomy 21 pregnancies.

OBJECTIVE: To investigate if fetal sex has an impact on 1st trimester combined screening for aenuploidy. METHODS: We studied the first trimester PAPP-A, free beta-human chorionic gonadatropin (beta-hCG) and nuchal translucency levels in 56,024 normal, singleton pregnancies with known fetal sex at birth. We also examined the distributions in 722 pregnancies with trisomy 21 of known fetal sex. RESULTS: We have found a 14.74% increase in first trimester maternal serum (MS) median free beta-hCG MoM, 6.25% increase of PAPP-A and a 9.41% decrease in delta NT, when the fetus was female. Analysis of data has shown that women carrying a female fetus were 1.084 times more likely to be in the 'at risk' group than those carrying a male fetus. In examining data from 722 pregnancies in which the fetus was affected by trisomy 21, we observed a similar 20.8% increase in free beta-hCG MoM, 5.7% increase in PAPP-A and a 12% decrease in delta NT when the fetus was female. Amongst the trisomy 21 cases, 88.8% of male trisomy 21 cases were detected compared with 91.2% in female cases, this difference was not statistically significant. Correcting for fetal sex redressed the balance in screen-positive rate between the sexes and had a minimal impact on detection rate. CONCLUSION: Correcting for fetal sex may be a worthwhile consideration. A cost-benefit analysis would be required to determine if it is feasible to introduce fetal gender assignment into the routine first trimester scan for the purpose of marker correction and whether this would have any significant impact.

Bioaugmentation of thiabendazole-contaminated soils from a wastewater disposal site: Factors driving the efficacy of this strategy and the diversity of the indigenous soil bacterial community.

The application of the fungicide thiabendazole (TBZ) in fruit packaging plants (FPP) results in the production of effluents which are often disposed in adjacent field sites. These require remediation to prevent further environmental dispersal of TBZ. We assessed the bioaugmentation potential of a newly isolated TBZ-degrading bacterial consortium in a naturally contaminated soil (NCS) exhibiting a natural gradient of TBZ levels (12000, 400, 250 and 12 mg kg-1). The effect of aging on bioaugmentation efficacy was comparatively tested in a soil with similar physicochemical properties and soil microbiota, which was artificially, contaminated with the same TBZ levels (ACS). The impact of bioaugmentation and TBZ on the bacterial diversity in the NCS was explored via amplicon sequencing. Bioaugmentation effectively removed TBZ from both soils at levels up to 400 mg kg-1 but failed at the highest contamination level (12000 mg kg-1). Dissipation of TBZ in bioaugmented samples showed a concentration-dependent pattern, while aging of TBZ had a slight effect on bioaugmentation efficiency. Bioaugmentation had no impact on the soil bacterial diversity, in contrast to TBZ contamination. Soils from the hotspots of TBZ contamination (12000 mg kg-1) showed a drastically lower α-diversity driven by the dominance of ß- and γ-proteobacteria at the expense of all other bacterial phyla, especially Actinobacteria. Overall, bioaugmentation with specialized microbial inocula could be an effective solution for the recovery of disposal sites contaminated with persistent chemicals like TBZ.

Placental protein-13 and pregnancy-associated plasma protein-A as first trimester screening markers for hypertensive disorders and small for gestational age outcomes.

OBJECTIVE: To investigate the role of placenta protein 13 (PP13) and pregnancy-associated plasma protein-A (PAPP-A) in hypertensive disorders and small for gestational age (SGA) during first trimester of pregnancy. METHODS: In this case-control study, first trimester serum samples (11(+0) to 13(+6) weeks) were retrieved from frozen storage of which 452 were from normal pregnancies and 47 samples were identified to have pregnancies with at least one of the following adverse outcomes: SGA, preeclampsia (PE), hemolysis, elevated liver enzymes, and low platelets (HELLP), or gestational hypertension (GH). PP13 concentrations were measured by a new AutoDELFIA method. Levels of PAPP-A were measured for a first trimester screening program using Kryptor analyzer. RESULTS: First trimester levels of PAPP-A are significantly lower in cases of SGA, PE, and most subgroups including HELLP. Levels of PP13 were not found to differ between control and affected pregnancies. CONCLUSION: PP13 needs to be studied further as our results contrast the majority of previous studies.

ADAM12s in maternal serum as a potential marker of pre-eclampsia.

OBJECTIVE: To examine whether maternal serum ADAM12s, a potential first- and second-trimester marker of fetal aneuploidy and fetal growth, had altered concentrations in the first or second trimester of pregnancies subsequently developing pre-eclampsia. METHODS: ADAM12s was measured by a time-resolved fluoroimmunoassay developed by PerkinElmer Life Science. Maternal serum samples from women taking part in early first-trimester aneuploidy screening in whom the pregnancy resulted in pre-eclampsia (64) were identified from a cohort of 4,390 singleton pregnancies in which uterine artery Doppler mean Pulsatility Index (PI) had been measured at 22-24 weeks. From amongst those cases delivering a normal term infant with birth weight greater than the 10th centile for gestational age 240 cases were selected as gestational age-matched controls. A second study group consisting of maternal serum taken at 22-24 weeks at the time of uterine artery Doppler in a group of 12 women developing pre-eclampsia were compared with 86 matched controls from a previously studied cohort of 24 cases and 144 controls. Serum ADAM12s concentrations were converted to multiple of the median (MoM) to take account of gestational age variation. RESULTS: First-trimester maternal serum ADAM12s levels in women who developed pre-eclampsia were reduced with a median MoM of 0.71 which was further reduced in those delivering prior to 35 weeks (0.50). At the 5th centile of normal (0.48 MoM) ADAM12s identified 27% of cases with pre-eclampsia and 47% of those with early pre-eclampsia. Combining ADAM12s with PAPP-A from a previous study only resulted in a further 1% increase in detection of all women developing pre-eclampsia. However combining ADAM12s with mean PI increased the detection rate to 66%. In the second trimester at 22-24 weeks the maternal serum ADAM12s levels were increased in those women developing pre-eclampsia compared to controls (709 vs 486 ug/L, p = 0.045). CONCLUSION: ADAM12s in addition to being a potential marker of aneuploidy may also be a marker of pre-eclampsia. Further studies are required to see if this can improve on the clinical discrimination already provided by PAPP-A in the early first trimester.

Maternal serum ADAM12s in the late first trimester of pregnancies with Trisomy 21.

BACKGROUND: ADAM12s is a placenta-derived glycoprotein that is involved in growth and differentiation, and has been shown to be a potential first-trimester and second-trimester marker of Trisomy 21 and other aneuploides. Maternal ADAM12s concentrations show a considerable temporal variation with gestational age and here we study the levels at 11-13 weeks of gestation to establish the effectiveness or otherwise at a time when other established markers are used. MATERIALS AND METHODS: Samples collected as part of routine first-trimester screening were retrieved from storage. In total, 46 samples from pregnancies with Trisomy 21 were identified and collected between 11 and 13 weeks of gestation-of these 83% had been identified by combined first-trimester screening. A series of 414 gestational age-matched samples collected during the same period formed the control group. ADAM12s was measured by a new DELFIA assay incorporating two monoclonals (6E6 and 8F8). Results were expressed as weight-corrected multiples of the median (MoM). RESULTS: The median MoM ADAM12s rose from 0.914 at 11 weeks to 1.032 at 13 weeks. CONCLUSIONS: Combining the data from this study and other published studies suggests that ADAM12s is unlikely to be of much additional value when screening for Trisomy 21 in the period 11-13 weeks. More studies are required looking at the potential of ADAM12s prior to 10 weeks.

Maternal serum ADAM12s as a marker of rare aneuploidies in the first or second trimester of pregnancy.

OBJECTIVE: To assess whether the maternal serum ADAM12s concentrations are altered in the first and second trimester of pregnancies complicated by rare aneuploides. METHODS: ADAM12s was measured by a semi-automated time-resolved immunofluorometric assay in a series of 60 first-trimester cases with trisomy 13, 78 first-trimester cases with Turner's syndrome, 38 first-trimester cases with triploidy and 24 first-trimester cases with sex aneuploidy-the cases were compared with the data from 389 first-trimester controls. In the second trimester, a smaller number of 6, 7, 2 and 13 cases, respectively, were compared with the data from 341 controls. All data were expressed as multiple of the median (MoM) and corrected for maternal weight. Correlation with previously analysed markers (PAPP-A, free beta-hCG and delta NT) was performed. RESULTS: The first-trimester median MoM ADAM12s was significantly lower than 1.0 in all types of rare aneuploidy with the possible exception of triploidy type II. A significant positive correlation with gestational age was reported for trisomy 13 and Turner's syndrome. ADAM12s was not significantly correlated with any other first-trimester marker. In the second trimester, ADAM12s values were marginally increased in these rare aneuploidies. CONCLUSIONS: ADAM12s has already been shown to be a possible early first- and second-trimester marker of trisomies 21 and 18. Our data also show the possibility of levels of this marker being altered in the first and second trimester of pregnancies with rare aneuploidies. This may be a useful addition to screening strategies in the future.