Antenatal screening for Down's syndrome: Revised nuchal translucency upper truncation limit due to improved precision of measurement.

OBJECTIVE: To determine whether the improved precision of nuchal translucency (NT) measurement used in antenatal screening for Down's syndrome observed over time as evidenced by a decrease in the multiple of the median (MoM) standard deviation requires a modification to the NT MoM truncation limits to maintain accurate risk estimation. METHODS: Probability plots were derived from the measurements of NT MoM values used in a 2018 audit of 22,362 unaffected pregnancies. The plots were used to determine whether the NT MoM upper truncation limit should be lowered. Validation plots were used to assess the screening accuracy of Down's syndrome risk estimates calculated from observed NT MoM values in the 22,362 unaffected pregnancies and 69 Down's syndrome pregnancies for original and revised NT MoM truncation limits. RESULTS: Probability plots indicated that with improved precision of NT measurements, there was deviation from a Gaussian distribution at less high MoM values than with less precise measurements. Validation plots showed that using the current NT MoM upper truncation limit of 2.5 MoM with improved precision NT measurements overestimates the Down's syndrome risk (median risk in highest risk category expressed as an odds was 53.3:1 and observed prevalence was 1:1.1). The large discrepancy was corrected by changing the NT upper truncation limit to 2.0 MoM (median risk in highest risk category expressed as an odds was 1:1.78 and observed prevalence 1:2.7). CONCLUSION: The NT MoM upper truncation limit should be reduced from 2.5 to 2.0 MoM.

A simple method to allow for guanine-cytosine amplification error in prenatal DNA screening for trisomy 18.

BACKGROUND: A source of error in prenatal screening for trisomies is PCR amplification error associated with guanine-cytosine (GC) content of DNA fragments in maternal plasma. We describe a simple method of allowing for this. METHODS: Data from a Reflex DNA screening programme (67 trisomy 18 and 83 unaffected pregnancies) were used to compare the ratio of chromosome 18 DNA fragment counts to chromosome 8 DNA fragment counts (because chromosome 8 has a similar GC content to chromosome 18) with the percentage of chromosome 18 DNA counts using counts from all autosomes in the denominator, with and without an all autosome correction for the GC content of the DNA fragments. RESULTS: A chromosome 18 to 8 ratio of DNA fragment counts was more discriminatory than the percentage of all autosome counts arising from chromosome 18 without, or with an all autosome correction for GC content bias. It achieves a high screening performance, eg. for a 0.25% false-positive rate, a 97% detection rate instead of 49% without a correction for GC content, and 91% with an all autosome correction for GC content. CONCLUSION: Consideration can be given to using the ratio of chromosome 18 DNA fragment counts to chromosome 8 DNA fragment counts in cell-free DNA prenatal screening for trisomy 18, avoiding the need for more complex methods of making a correction for the GC content currently used.

Prenatal screening for Down syndrome in twin pregnancies: Estimates of screening performance based on 61 affected and 7302 unaffected twin pregnancies.

The aims of this study were to determine whether assumptions used in prenatal screening for Down syndrome in twin pregnancies are valid and derive estimates of risk and screening performance in twin pregnancies using observed data. Data were collected on nuchal translucency, chorionicity, pregnancy associated plasma protein-A (PAPP-A), and free ß human chorionic gonadotrophin (free ß-hCG) from 61 twin pregnancies with Down syndrome and 7302 unaffected twin pregnancies. Distribution parameters were determined and used to estimate screening performance. The assumption that proportional differences in serum marker levels in affected and unaffected singleton pregnancies apply to twin pregnancies was not confirmed. Median free ß-hCG value in monochorionic affected twin pregnancies (2.63 multiples of the median [MoM]; 95% CI, 1.79-3.22 MoM) was lower than that assuming proportionality (3.76 MoM), and the median PAPP-A value in dichorionic affected twin pregnancies (1.88 MoM; 95% CI, 1.60-2.17 MoM) was higher than that based on proportionality (1.33 MoM). The detection rate was 87% for a 3% false-positive rate in monochorionic twin pregnancies and 74% in dichorionic twin pregnancies compared with 86% in singleton pregnancies. Estimates of screening performance in Down syndrome twin pregnancies do not need to rely on assumptions and can take account of chorionicity and gestational age.

Serum marker truncation limits in first trimester antenatal screening for trisomy 18.

OBJECTIVE: To assess whether the accuracy of risk estimation in antenatal screening for trisomy 18 using the Combined test can be improved by revising the truncation limits of two serum markers. METHODS: In an audit of data from 420 trisomy 18 and 573,754 unaffected singleton pregnancies screened at the Wolfson Institute of Preventive Medicine, London (March 2003 to June 2017), the accuracy of risk estimation was assessed by inspection of a validation plot (the median predicted late first trimester Combined test risk plotted against observed prevalence within categories of predicted risk estimates). Using validation and probability plots, we assessed whether the revised pregnancy-associated plasma protein A (PAPP-A) and free ß-human chorionic gonadotrophin (free ß-hCG) truncation limits led to more accurate risk estimation and improved screening performance. RESULTS: With the lower truncation limits currently used for PAPP-A and free ß-hCG (0.15 and 0.30 multiples of the median [MoM], respectively), risk was underestimated. Revised lower truncation limits of 0.05 MoM for both PAPP-A and free ß-hCG led to greater accuracy, with an increase in the number of trisomy 18 pregnancies detected (from 85.4% to 90.2%) for a small increase in the false-positive rate (from 0.20% to 0.29%) at a 1 in 100 late first trimester risk cut-off. CONCLUSION: The revised truncation limits for PAPP-A and free ß-hCG increase the accuracy of trisomy 18 risk estimation and improve screening performance using the Combined test. Validation and probability plots are useful in setting screening marker truncation limits.

Specifying a Gold Standard for the Validation of Fetal Fraction Estimation in Prenatal Screening.

BACKGROUND: An estimate of fetal fraction (FF) is needed for DNA-based screening for trisomy 21 and other aneuploidies, but there is no gold standard to validate FF measurement methods. We specify a gold standard and use it to validate a method of measuring FF (SeqFF) in singleton pregnancies. METHODS: The gold standard was a formula derived from 2 elements: (a) an estimate of the percentage of DNA fragments in maternal plasma from chromosome 21 (%Ch21) in pregnancies without trisomy 21, 18, or 13 (PU) and (b) calculation of %Ch21 with increasing FF in trisomy 21 pregnancies (P21). The SeqFF method was evaluated by plotting regression lines of %Ch21 and SeqFF estimates of FF in 31 singleton male and 31 female trisomy 21 pregnancies and comparing the regressions with the reference line derived from the gold standard formula. RESULTS: The gold standard formula was P21 = (1/2)PUFF + PU, with FF expressed as a proportion, or converting %Ch21 to multiples of the median (MoM), P21(MoM) = (1/2)FF + 1. Based on 3865 pregnancies, the PU was 1.2935%. The regression lines for trisomy 21 pregnancies with male and female fetuses were almost identical to the gold standard reference line (regression slopes in MoMs 0.52 and 0.50, respectively, compared with 0.50 for the gold standard reference line). CONCLUSIONS: The proposed gold standard can be used to validate different methods of estimating FF in singleton pregnancies. SeqFF is an accurate method of estimating FF.

Prenatal reflex DNA screening for trisomies 21, 18, and 13.

PURPOSE: The purpose of the study was to determine the screening performance of prenatal reflex DNA screening for trisomies 21 (T21), 18 (T18), and 13 (T13) as part of a routine service at five hospitals. METHODS: Women who accepted screening had a first-trimester combined test (pregnancy-associated plasma protein A, free ß-human chorionic gonadotropin, nuchal translucency interpreted with maternal age). Those with a risk of having an affected pregnancy ≥1 in 800 were reflexed to a DNA sequencing test using stored plasma from the original blood sample, thereby avoiding the need to recall them. RESULTS: Of 22,812 women screened (including 106 with affected pregnancies), 2,480 (10.9%) were reflexed to DNA testing; 101/106 were detected (69/73 T21, 24/25 T18, and 8/8 T13), a 95% detection rate (95% confidence interval 89-98%) with four false positives (0.02%, 95% confidence interval 0.00-0.05%). The odds of being affected given a positive result were 25:1. Of the 105 screen-positive pregnancies, 91 (87%) had an invasive diagnostic test. Reflex DNA screening avoided up to 530 invasive diagnostic tests compared with using the combined test. CONCLUSION: Reflex DNA screening was successfully implemented in routine care, achieving a high detection rate, low false-positive rate, and, consequently, greater safety with fewer invasive diagnostic tests than other methods of screening.

The estimation of median nuchal translucency values between 10 and 14 weeks of pregnancy.

Nuchal translucency (NT) is a useful marker in antenatal screening for Down's syndrome in the late first trimester of pregnancy. NT measurements increase with increasing crown rump length (CRL) so multiple of the median (MoM) values are used to allow for this. Log-linear and log-quadratic regressions of NT in relation to CRL have previously been proposed to calculate MoM values. Using data on 288,079 women, these models were compared with a log-sigmoid regression. The log-linear regression overestimated the median NT above a CRL of 75 mm; for example, 1.9 mm versus 1.8 mm observed at 75-79 mm, and 2.0 mm versus 1.8 mm at 80-84 mm. The log-quadratic regression underestimated the median NT below a CRL of 45 mm at 1.03 mm versus 1.2 mm observed. The sigmoid regression provided the best overall fit to the data across the range of CRL values (40-84 mm) corresponding to gestational ages of 76 to 99 days. The differences between the three models are small. If a log-linear regression appears to be a poor fit using local data, a log-sigmoid regression could be considered.

Prevention of neural tube defects: a cross-sectional study of the uptake of folic acid supplementation in nearly half a million women.

BACKGROUND: Taking folic acid supplements before pregnancy to reduce the risk of a neural tube defect (NTD) is especially important in countries without universal folic acid fortification. The extent of folic acid supplementation among women who had antenatal screening for Down's syndrome and NTDs at the Wolfson Institute of Preventive Medicine, London between 1999 and 2012 was assessed. METHODS AND FINDINGS: 466,860 women screened provided details on folic acid supplementation. The proportion of women who took folic acid supplements before pregnancy was determined according to year and characteristics of the women. The proportion of women taking folic acid supplements before pregnancy declined from 35% (95% CI 34%-35%) in 1999-2001 to 31% (30%-31%) in 2011-2012. 6% (5%-6%) of women aged under 20 took folic acid supplements before pregnancy compared with 40% of women aged between 35 and 39. Non-Caucasian women were less likely to take folic acid supplements before pregnancy than Caucasian women; Afro-Caribbean 17% (16%-17%), Oriental 25% (24%-25%) and South Asian 20% (20%-21%) compared with 35% (35%-35%) for Caucasian women. 51% (48%-55%) of women who previously had an NTD pregnancy took folic acid supplements before the current pregnancy. CONCLUSIONS: The policy of folic acid supplementation is failing and has led to health inequalities. This study demonstrates the need to fortify flour and other cereal grain with folic acid in all countries of the world.

Detection of trisomy 18 and trisomy 13 using first and second trimester Down's syndrome screening markers.

OBJECTIVE: To estimate the detection rates (DRs) and false-positive rates (FPRs) in the incidental identification of trisomy 18 (T18) and trisomy 13 (T13) as part of antenatal screening for Down's syndrome (DS) using the Combined, Quadruple and Integrated test markers. METHODS: Screening marker levels on 224 T18 and 67 T13 pregnancies screened for DS were evaluated. Estimated means, standard deviations and correlation coefficients were used with published estimates for unaffected pregnancies to derive detection algorithms for the two disorders. DRs and FPRs of the algorithms were estimated using Monte Carlo simulation. RESULTS: In T18 and T13 pregnancies first trimester nuchal translucency was raised, free ß-human chorionic gonadotrophin (hCG) and pregnancy associated plasma protein-A reduced. In T18 pregnancies second trimester alphafetoprotein, unconjugated oestriol and free ß-hCG were reduced. In T13 pregnancies second trimester inhibin-A was raised. These markers specified T18 and T13 algorithms. The DS Combined test algorithm detected 42% of T18 and 59% of T13 (2.00% FPR); 88% and 74% by adding the T18 Combined test algorithm (2.17% FPR) and 89% and 75% by further adding the T13 Combined test algorithm (2.19% FPR). The corresponding detection rates for the Quadruple test were: 5% and 21% (2.00% FPR), 59% and 21% (2.16% FPR) and 59% and 24% (2.28% FPR), and for the Integrated test were: 40% and 60% (2.00% FPR), 92% and 68% (2.12% FPR) and 92% and 74% (2.18% FPR).[Corrected] CONCLUSIONS: Antenatal screening for DS detects about 40% of T18 and about 60% of T13 pregnancies. The addition of a T18 algorithm substantially increases the detection of both trisomies with a small increase in the FPR. The further addition of a T13 algorithm results in a small increase in the detection of T13.

Antenatal screening for Down syndrome using serum placental growth factor with the combined, quadruple, serum integrated and integrated tests.

OBJECTIVE: To estimate the value of first or second trimester placental growth factor (PlGF) as an additional antenatal screening marker for Down syndrome. DESIGN: Nested case-control study. SETTING: Antenatal screening service. POPULATION OR SAMPLE: 532 Down syndrome pregnancies and 1,155 matched unaffected pregnancies. METHODS: Stored maternal serum samples (-40°C) were assayed for PlGF. Monte Carlo simulation was used to estimate the screening performance of PlGF with the Combined, Quadruple, serum Integrated and Integrated tests. MAIN OUTCOME MEASURES: Median PlGF levels in affected and unaffected pregnancies and screening performance (detection rates [DR] for specified false-positive rates [FPR] and vice versa). RESULTS: First trimester median PlGF was 15%, 28% and 39% lower in Down syndrome than unaffected pregnancies at 11, 12 and 13 completed weeks' gestation respectively (all p<0.001). Second trimester median PlGF was 31% lower at 14 weeks (p<0.001), and the difference decreased (6% lower at 17 weeks). At a 90% DR with first trimester markers measured at 13 weeks, adding PlGF decreased the FPR from 11.1 to 5.1% using the Combined test, 9.3% to 4.5% using the serum Integrated test, and 3.4% to 1.5% using the Integrated test (or 1.5 to 1.4% with first trimester markers measured at 11 weeks). Adding PlGF to the Quadruple test (measured at 15 weeks) decreased the FPR from 10.0% to 9.6% at a 90% DR. CONCLUSIONS: First trimester PlGF measurements improve the performance of antenatal screening for Down syndrome using the Combined, serum Integrated and Integrated tests. Second trimester PlGF measurements are of limited value.

Effect of interrupting prenatal Down syndrome screening due to a large nuchal translucency.

OBJECTIVE: The aim of this study is to determine the effect of interrupting prenatal Down syndrome screening for women with large nuchal translucency (NT) measurements on the performance of combined and integrated tests. DESIGN: Distribution of large NT at 11-13 weeks of gestation was determined from a screening programme (204,982 pregnancies, 509 with Down syndrome). Monte Carlo simulation was used to estimate the effects of interrupting screening on the detection rate (DR) and false-positive rate (FPR) in (i) the remaining women and (ii) all women. RESULTS: At 12 weeks of gestation, 28% of affected (76/275) and 0.4% of unaffected (389/107,386) pregnancies had an NT of ≥ 3.5 mm. Among the remaining women, for an 85% DR, combined test FPR was 7.2% with interpretation of screening but 4.1% without interruption (1.8% and 0.9% respectively for integrated test). Among all women (women who interrupted screening with an NT of ≥ 3.5 mm and a 1/150 risk cut-off for others), the FPR was 0.1 and 0.2 percentage points higher for the same DR at 12-13 weeks compared with no interruption using the combined and integrated tests, respectively, and 0.3 and 0.7 percentage points higher at 11 weeks. CONCLUSION: Interrupting combined or integrated screening is acceptable using an NT of ≥ 3.5 mm, but screening performance in screened women will seem less than expected. This should be taken into account when performing audits.

First trimester Down's syndrome screening marker values and cigarette smoking: new data and a meta-analysis on free beta human chorionic gonadotophin, pregnancy-associated plasma protein-A and nuchal translucency.

OBJECTIVES: To examine the effect of smoking on three first trimester screening markers for Down's syndrome that constitute the Combined test, namely nuchal translucency (NT), pregnancy-associated plasma protein-A (PAPP-A) and free beta human chorionic gonadotophin (free beta-hCG) and to use the results to determine which of these markers need to be adjusted for smoking and by how much. METHODS: The difference in the median multiple of the median (MoM) values in smokers compared to non-smokers was determined for NT, PAPP-A and free beta-hCG in 12,517 unaffected pregnancies that had routine first trimester Combined test screening. These results were then included in a meta-analysis of published studies and the effect of adjusting for smoking on screening performance of the Combined test was estimated. RESULTS: The results using the routine screening data were similar to the summary estimates from the meta-analysis of all studies. The results from the meta-analysis were; median MoM in smokers compared to non-smokers: 1.06 NT (95% confidence interval 1.03 to 1.10), 0.81 PAPP-A (0.80 to 0.83) and 0.94 free beta-hCG (0.89 to 0.99). The effect of adjusting for smoking on the Combined test is small, with an estimated less than half percentage point increase in the detection rate (the proportion of affected pregnancies with a positive result) for a 3% false-positive rate (the proportion of unaffected pregnancies with a positive result) and less than 0.2 percentage point decrease in the false-positive rate for an 85% detection rate. CONCLUSION: Adjusting first trimester screening markers for smoking has a minimal favourable effect on screening performance, but it is simple to implement and this paper provides the adjustment factors needed if a decision is made to make such an adjustment.