Consequences of imprecision in fetal fraction estimation on performance of cell-free DNA screening for Down syndrome.

BACKGROUND: There is a significant variability in reported fetal fraction (FF), a common cause for no-calls in cell-free (cf)DNA based non-invasive prenatal screening. We examine the effect of imprecision in FF measurement on the performance of cfDNA screening for Down syndrome, when low FF samples are classified as no-calls. METHODS: A model for the reported FF was constructed from the FF measurement precision and the underlying true FF. The model was used to predict singleton Down syndrome detection rates (DRs) for various FF cut-offs and underlying discriminatory powers of the test. RESULTS: Increasing the FF cut-off led to slightly increased apparent DR, when no-calls are excluded, and an associated larger decrease in effective DR, when no-calls are included. These effects were smaller for tests with higher discriminatory power and larger as maternal weight increased. CONCLUSIONS: Most no-calls due to a low reported FF have a true FF above the cut-off. The discriminatory power of a test limits its effective DR and FF precision determines the tradeoff between apparent and effective DR when low FF is used to discard samples. Tests with high discriminatory power do not benefit from current FF measurements.

Extrapolation of maternal weight in sequential aneuploidy screening.

OBJECTIVE: The aim of this study was to determine the impact on the risk calculation of various ways of handling maternal weight when these data are provided in the first part but not the second part of a sequential screening protocol. METHOD: A retrospective analysis of 38,986 sequential screens was carried out in which weight was provided in both the first and second trimesters. Three potential strategies for calculating multiples of the median values when the weight is not recorded at the time of second trimester risk evaluation were evaluated. First, perform no weight adjustment. Second, use the first trimester weight. Third, use the predicted second trimester weight on the basis of the first trimester weight. To predict the second trimester weight, we used a random-effects, multi-level model. RESULTS: The screen positive rate for Down syndrome was 3.0% (1151/38,986) and trisomy 18 alone 0.12% (47/38,986). The three strategies resulted in 196 (0.50%), 41 (0.11%), and 23 (0.06%) patients switching risk categories with the no adjustment, first trimester weight, and predicted weight strategies, respectively. CONCLUSION: Utilizing the first trimester weight or the predicted second trimester weight in sequential screening when second trimester weight is not provided offers an affordable alternative for laboratories to provide robust risk calculations and interpretations without requiring excessive use of resources.

Combined screening for open spina bifida at 11-13 weeks using fetal biparietal diameter and maternal serum markers.

OBJECTIVE: Screening at 11-13 weeks with ultrasound biparietal diameter (BPD) can detect half of open spina bifida cases. Maternal serum α-fetoprotein (AFP) levels at 15-19 weeks are increased 3- to 4-fold, in open spina bifida. We assessed whether combined screening using BPD, AFP, and other serum markers at 11-13 weeks would increase detection. STUDY DESIGN: Maternal AFP levels were measured on serum stored at 11-13 weeks in 44 open spina bifida and 182 unaffected pregnancies, and results were expressed in multiples of the median (MoM) for gestational age. All samples had been measured for free ß-human chorionic gonadotropin (ß-hCG) and pregnancy-associated plasma protein (PAPP)-A. A multivariate Gaussian model was used to predict screening performance from the serum data and BPD measurements on 80 cases, including 36 previously published. RESULTS: The median AFP level in cases was 1.201 MoM, significantly higher than in unaffected pregnancies (P < .01, 1 tail). The median free ß-hCG was significantly reduced to 0.820 MoM (P < .02), but the median PAPP-A was similar in cases and controls. Modeling predicted the following: BPD alone would detect 50% of cases for a 5% false-positive rate or 63% for 10%; adding AFP increases detection by 2%; and a combined test with BPD, AFP, and free ß-hCG detects 58% for 5% or 70% for 10%. CONCLUSION: Combining AFP and BPD with free ß-hCG as part of first-trimester aneuploidy screening would also allow early detection about two-thirds of cases with open spina bifida.

Screening for fetal spina bifida by ultrasound examination in the first trimester of pregnancy using fetal biparietal diameter.

OBJECTIVE: Prenatal screening for aneuploidies is best achieved in the first trimester when there is no reliable screening test for spina bifida. Early ultrasound features may be too complex for routine screening. We assessed screening potential of simple and reproducible fetal biometric measurements at 11-14 weeks of gestation. STUDY DESIGN: A total of 34,951 unselected consecutive pregnancies included 18 with spina bifida. Another 28 cases were referred for assessment. Biometric measurements were expressed in multiples of the median for crown-rump length. RESULTS: Biparietal diameter (BPD) was smaller in spina bifida (P < .0001). In all, 22 of 44 (50%) cases with spina bifida aperta had a BPD <5th centile. BPD was independent of maternal adiposity and smoking status. CONCLUSION: Simple and reproducible BPD at 11-14 weeks of gestation could detect half the cases of open fetal spina bifida by identifying 5% of pregnancies for expert scanning in first- and second-trimester examinations of the fetal spine and cranium.

Digital PCR for noninvasive detection of aneuploidy: power analysis equations for feasibility.

OBJECTIVE: To determine the feasibility of digital PCR analysis for noninvasive prenatal diagnosis of trisomy 21. METHODS: Through power equations, we modeled the number of wells necessary to determine the feasibility of digital PCR as a practical method for trisomy 21 risk assessment. RESULTS: The number of wells needed is a direct correlate of the ability to isolate free fetal DNA. If a 20% fetal DNA enhancement can be achieved, then 2,609 counts would be sufficient to achieve a 99% detection rate for a 1% false-positive rate and potentially feasible with readily available plates. However, if only a 2% increase is seen, then 220,816 counts will be necessary, and over 110,000 would be needed just to achieve 95% for a 5% false-positive rate - both far beyond current commercially available technology. CONCLUSION: There are several noninvasive prenatal diagnostic methods which may reach commercialization; all have differing potential advantages and disadvantages. Digital PCR is potentially a cheaper methodology for trisomy 21, but it is too early to determine the optimal method.

Performance adjusted risks: a method to improve the quality of algorithm performance while allowing all to play.

OBJECTIVE: Differences in quality among ultrasound nuchal translucency providers or laboratories can profoundly affect Down syndrome screening results. A new method, performance adjusted risks (PAR), is developed to allow for such differences and improve performance. METHODS: Individual provider and laboratory marker distribution parameters are compared with national expectations. The maximum absolute deviation over the operating range is used to derive a handicap and weighting factors. Down syndrome risks obtained from commercial software for individual women can be corrected using the weights. Those with the biggest handicap will have the greatest correction. Five theoretical examples are used to illustrate handicap calculation and prospective combined test results on 32 Down syndrome and 7205 unaffected pregnancies are used to indicate the influence of weighting on performance. RESULTS: In the theoretical examples, a 10% systematic change (inaccuracy) in one or more markers or a similar change in the standard deviation (imprecision) yielded a handicap ranging from 4 to 11. Over the operating range, the individual risk reduced 40% or increased 250%. When the prospective combined test results were artificially adjusted to create 10% inaccurracy in all three markers, the detection rate was only 59% but after PAR weighting this increased to 75%. CONCLUSIONS: PAR recognizes that not all providers are equal, and perfection is unrealistic. Using this approach all 'can play' while patients are protected from poor performance.

Down's Syndrome Screening in the First Trimester with Additional Serum Markers: Indian Parameters.

OBJECTIVE: To derive a risk calculation algorithm suitable for use in India when screening for Down's syndrome using four first-trimester maternal serum markers either alone or with ultrasound nuchal translucency (NT). METHODS: Stored maternal serum samples (- 20 °C) from 411 singleton unaffected pregnancies were retrieved and measured for pregnancy-associated plasma protein (PAPP-A), free ß-human chorionic gonadotropin (hCG), placental growth factor and α-fetoprotein. Samples were taken at 10-13 weeks' gestation. Equations were derived to express marker levels in multiples of the gestation-specific normal median, adjusted for maternal weight. Gaussian model parameters were derived and compared with six published non-Indian studies; NT parameters were derived from 27,647 women screened in India. On the basis of the maternal age distribution in 64,473 Indian women screened in 2016-2017, the model was used to predict test performance. RESULTS: The model predicted a detection rate for a serum-only protocol of 80% for a 5% false-positive rate. Using a 1 in 250 at term Down's syndrome risk cut-off, the predicted detection rate was 78% and the false-positive rate was 4.1%. When NT was also included, the rates were 95% for 5% and 90% for 1.4%, respectively. CONCLUSION: First-trimester screening using four serum markers only can be carried out in India. Performance is expected to be similar to the second-trimester Quad test and will also facilitate early screening for preeclampsia and open spina bifida. A protocol of NT plus the four serum markers enhances the performance compared with NT, PAPP-A and free ß-hCG.

Prediction of patient-specific risk for fetal loss using maternal characteristics and first- and second-trimester maternal serum Down syndrome markers.

OBJECTIVE: To develop and evaluate a method of estimating patient-specific risk for fetal loss by combining maternal characteristics with serum markers. STUDY DESIGN: Data were obtained on 36,014 women from the FaSTER trial. Separate likelihood ratios were estimated for significant maternal characteristics and serum markers. Patient-specific risk was calculated by multiplying the incidence of fetal loss by the likelihood ratios for each maternal characteristic and for different serum marker combinations. RESULTS: Three hundred eighteen women had fetal loss < 24 weeks (early) and 103 > 24 weeks (late). Clinical characteristics evaluated included maternal age, body mass index, race, parity, threatened abortion, previous preterm delivery, and previous early loss. Serum markers studied as possible predictors of early loss included first-trimester pregnancy-associated plasma protein A and second-trimester alpha-fetoprotein, and unconjugated estriol. A risk assessment for early loss based on all of these factors yielded a 46% detection rate, for a fixed 10% false-positive rate, 39% for 5% and 28% for 1%. The only significant marker for late loss was inhibin A. The detection rate was 27% for a fixed 10% false-positive rate and only increased slightly when clinical characteristics were added to the model. CONCLUSION: Patient-specific risk assessment for early fetal loss using serum markers, with or without maternal characteristics, has a moderately high detection. Patient-specific risk assessment for late fetal loss has low detection rates.

First-trimester maternal serum PP13 in the risk assessment for preeclampsia.

OBJECTIVE: The objective of the study was to determine whether first-trimester maternal serum placental protein 13 (PP13) concentrations can be used in the risk assessment for preeclampsia. STUDY DESIGN: This case-control study included 50 patients with preeclampsia and 250 patients with normal pregnancies. Samples were collected between 8 and 13 weeks of gestation. Serum PP13 concentrations were measured by immunoassay and expressed as medians and multiples of the median (MoM) for gestational age. Sensitivity and specificity were derived from receiver-operating characteristic curve analysis. RESULTS: (1) Serum PP13 concentration in the first trimester was significantly lower in patients who developed preterm and early-onset preeclampsia than in those with normal pregnancies; and (2) at 80% specificity, a cutoff of 0.39 MoM had a sensitivity of 100% for early-onset preeclampsia and 85% for preterm preeclampsia. CONCLUSION: Maternal serum first-trimester PP13 appears to be a reasonable marker for risk assessment for preterm preeclampsia but a weak marker for severe preeclampsia at term, and ineffective for identifying mild preeclampsia at term.

Decisions about testing and termination of pregnancy for different fetal conditions: a qualitative study of European White and Pakistani mothers of affected children.

The aim of this study is to explore reasons for and against prenatal testing and termination for a range of conditions in women from two different ethnic backgrounds. A total of 19 Pakistani and European women in West Yorkshire, UK, who either had a child with a genetic condition or had terminated a pregnancy for one, completed a questionnaire about their attitudes regarding prenatal testing and termination for 30 different fetal conditions and were interviewed about their reasons for their responses. There were more similarities than differences between the Pakistani and European white women. The most important factor in most women's decisions about termination of pregnancy was their perception of the quality of the life of a child with the genetic condition, in particular, whether the child would be "suffering." This was described as either physical suffering, as a result of medical treatment, or as emotional suffering, as a result of psychological and/or social factors. These findings highlight the need for detailed information about the potential quality of life for the child and the child's family to enable parents to make informed choices, particularly the extent to which the child is likely to suffer, the nature of such potential "suffering" and the extent to which the child could lead a "normal" life. The findings also challenge stereotypes about cultural differences in attitudes about termination of pregnancy.

First- and second-trimester screening: detection of aneuploidies other than Down syndrome.

OBJECTIVE: To evaluate the performance of first- and second-trimester screening methods for the detection of aneuploidies other than Down syndrome. METHODS: Patients with singleton pregnancies at 10 weeks 3 days through 13 weeks 6 days of gestation were recruited at 15 U.S. centers. All patients had a first-trimester nuchal translucency scan, and those without cystic hygroma had a combined test (nuchal translucency, pregnancy-associated plasma protein A, and free beta-hCG) and returned at 15-18 weeks for a second-trimester quadruple screen (serum alpha-fetoprotein, total hCG, unconjugated estriol, and inhibin-A). Risk cutoff levels of 1:300 for Down syndrome and 1:100 for trisomy 18 were selected. RESULTS: Thirty-six thousand one hundred seventy-one patients completed first-trimester screening, and 35,236 completed second-trimester screening. There were 77 cases of non-Down syndrome aneuploidies identified in this population; 41 were positive for a cystic hygroma in the first trimester, and a further 36 had a combined test, of whom 29 proceeded to quadruple screening. First-trimester screening, by cystic hygroma determination or combined screening had a 78% detection rate for all non-Down syndrome aneuploidies, with an overall false-positive rate of 6.0%. Sixty-nine percent of non-Down syndrome aneuploidies were identified as screen-positive by the second-trimester quadruple screen, at a false-positive rate of 8.9%. In the combined test, the use of trisomy 18 risks did not detect any additional non-Down syndrome aneuploidies compared with the Down syndrome risk alone. In second-trimester quadruple screening, a trisomy 18-specific algorithm detected an additional 41% non-Down syndrome aneuploidies not detected using the Down syndrome algorithm. CONCLUSION: First-trimester Down syndrome screening protocols can detect the majority of cases of non-Down aneuploidies. Addition of a trisomy 18-specific risk algorithm in the second trimester achieves high detection rates for aneuploidies other than Down syndrome. LEVEL OF EVIDENCE: II.

Primary prevention of Down's syndrome.

BACKGROUND: Antenatal screening has the capacity to detect more than 90% of Down's syndrome pregnancies leading to therapeutic abortion. Successes in recent years with such so-called 'secondary' prevention have not been matched with progress in primary prevention. Despite considerable research over many decades the principle cause of the disorder is unknown. METHODS: This paper considers three potential primary prevention strategies, (1) avoiding reproduction at advanced maternal age, (2) pre-implantation genetic diagnosis for couples who are at high risk of Down's syndrome, and (3) folic acid supplementation. The principle aetiological hypotheses are also reviewed. INTERPRETATION: A strategy of completing the family before a maternal age of 30 could more than halve the birth prevalence of this disorder. Women with a high a priori risk should have access to pre-implantation genetic diagnosis, which can lead to a reasonably high pregnancy rate with an extremely low risk of a Down's syndrome. The evidence suggesting an aetiological role for defective folate and methyl metabolism is not sufficient to justify an active preventative strategy of folic acid supplementation without performing a large clinical trial. Current supplementation policies designed to prevent neural tube defects may incidentally prevent Down's syndrome, provided a sufficiently high dose of folic acid is used. Further progress in primary prevention is hampered by limited aetiological knowledge and there is an urgent need to refocus research in that direction.

Role of second-trimester genetic sonography after Down syndrome screening.

OBJECTIVE: To estimate the effectiveness of second-trimester genetic sonography in modifying Down syndrome screening test results. METHODS: The First and Second Trimester Evaluation of Risk (FASTER) aneuploidy screening trial participants were studied from 13 centers where a 15- to 23-week genetic sonogram was performed in the same center. Midtrimester Down syndrome risks were estimated for five screening test policies: first-trimester combined, second-trimester quadruple, and testing sequentially by integrated, stepwise, or contingent protocols. The maternal age-specific risk and the screening test risk were modified using likelihood ratios derived from the ultrasound findings. Separate likelihood ratios were obtained for the presence or absence of at least one major fetal structural malformation and for each "soft" sonographic marker statistically significant at the P<.005 level. Detection and false-positive rate were calculated for the genetic sonogram alone and for each test before and after risk modification. RESULTS: A total of 7,842 pregnancies were studied, including 59 with Down syndrome. Major malformations and 8 of the 18 soft markers evaluated were highly significant. The detection rate for a 5% false-positive rate for the genetic sonogram alone was 69%; the detection rate increased from 81% to 90% with the combined test, from 81% to 90% with the quadruple test, from 93% to 98% with the integrated test, from 97% to 98% with the stepwise test, and from 95% to 97% with the contingent test. The stepwise and contingent use of the genetic sonogram after first-trimester screening both yielded a 90% detection rate. CONCLUSION: Genetic sonography can increase detection rates substantially for combined and quadruple tests and more modestly for sequential protocols. Substituting sonography for quadruple markers in sequential screening was not useful. LEVEL OF EVIDENCE: II.

Contingent screening for Down syndrome--results from the FaSTER trial.

OBJECTIVE: Comparison of contingent, step-wise and integrated screening policies. METHODS: Mid-trimester Down syndrome risks were retrospectively calculated from FaSTER trial data. For contingent screening, initial risk was calculated from ultrasound measurement of nuchal translucency (NT), maternal serum pregnancy-associated plasma protein (PAPP)-A and free beta-human chorionic gonadotrophin (hCG) at 11-13 weeks, and classified positive (>1 in 30), borderline (1 in 30-1500) or negative. Borderline risks were recalculated using alpha-fetoprotein, hCG, unconjugated estriol (uE3) and inhibin at 15-18 weeks, and reclassified as positive (>1 in 270) or negative. For step-wise screening, initial negative risks were also recalculated. For integrated screening, a single risk was calculated from NT, PAPP-A and the second trimester markers. RESULTS: There were 86 Down syndrome and 32,269 unaffected pregancies. The detection rate for contingent screening was 91% and false-positive rate was 4.5%; initial detection rate was 60%, initial false-positive rate was 1.2% and borderline risk was 23%. Step-wise screening had 92% detection rate and 5.1% false-positive rate; integrated screening had 88% and 4.9% respectively. CONCLUSION: As predicted by modelling, the contingent screening detection rate for a fixed false-positive rate is comparable with step-wise and integrated screening, but substantially reduces the number needing to return for second trimester testing.

Serum müllerian-inhibiting substance in Down's syndrome pregnancies.

BACKGROUND: To examine whether maternal serum levels of müllerian-inhibiting substance (MIS) differ in Down's syndrome and unaffected pregnancies. METHODS: Case-control study was conducted using stored serum from an antenatal screening programme. Sera from 25 Down's syndrome pregnancies were retrieved from -20 degrees C storage together with 125 unaffected controls individually matched for maternal age, weeks of gestation and duration of storage. Results were expressed in multiples of the gestation-specific median value (MoM) in controls. RESULTS: The median value in Down's syndrome pregnancies was 0.83 MoM (P = 0.77, two-tail Wilcoxon rank sum test). Among unaffected pregnancies, there was a statistically significant correlation between MIS and pregnancy-associated plasma protein-A (P < 0.05). MIS levels were elevated in pregnancies where assisted reproduction techniques had been used. CONCLUSION: There is no evidence for a substantial reduction in maternal serum MIS levels in Down's syndrome pregnancies. This study provides useful information regarding serum MIS levels in pregnancy.

Attitudes to prenatal testing and termination of pregnancy for fetal abnormality: a comparison of white and Pakistani women in the UK.

OBJECTIVES: To compare the attitudes of women from two different ethnic backgrounds to prenatal testing for a range of conditions, and to see if "clusters" of attitudes to different conditions could be identified, for which prenatal testing might be offered as a package. METHODS: Four hundred and twenty white and Pakistani women living in the UK were surveyed about their attitudes to prenatal testing and termination for 30 different fetal conditions. All participants had recently had a baby. RESULTS: Pakistani women held more favourable attitudes to prenatal testing, but less favourable attitudes to termination than their white counterparts. Both groups were most in favour of termination for the same four conditions: anencephaly, trisomy 13 or 18, quadriplegia, Duchenne muscular dystrophy. The rank ordering of conditions was also similar. Only 4% of Pakistani and 2% of white women wanted no prenatal testing at all. Fewer than a quarter of participants would consider a termination of pregnancy for 85% of the conditions, but only 25% of Pakistani women and 6% of white women would consider termination for none of the conditions. More advanced statistical analyses were used to measure how closely associated the 30 conditions were in respondents' answers. These analyses identified a principal dimension, reflecting the overall perceived seriousness of the conditions, and a cluster of severely disabling conditions-anencephaly, trisomy 13 or 18, quadriplegia, Duchenne muscular dystrophy, and severe learning difficulties-which stood out from the rest. Even within this cluster there remained a considerable amount of individual variation. CONCLUSION: Women in the study wanted to make up their own minds about the conditions that to them merited testing or termination. These findings have implications for obtaining informed consent.

Aneuploidy screening: what test should I use?

Screening for the risk of fetal aneuploidy can be effectively performed in either the first or second trimester. When obtained independently, the interpretation of those data is straightforward. However, the effectiveness of screening can be enhanced by combining studies performed in each trimester in a variety of ways. In this commentary we will define and discuss both the advantages and disadvantages of using integrated, stepwise, sequential or contingency screening for risk assessment of fetal aneuploidy.

Down syndrome screening marker levels in women with a previous aneuploidy pregnancy.

BACKGROUND: In Down syndrome screening programmes, women with a previous affected pregnancy are assumed to have the same marker distribution as those without a family history. This assumption needs to be tested. METHODS: Information on previous aneuploidy pregnancies was routinely sought on the test request forms in three centres, Leeds, Romford and the Fetal Medicine Centre, London. For each woman with a previous aneuploidy (case), five unaffected pregnancies to women without a history were selected as controls. The markers tested included maternal serum free beta-human chorionic gonadotrophin (hCG), pregnancy-associated plasma protein A (PAPP-A), alpha-fetoprotein, unconjugated estriol and ultrasound nuchal translucency thickness. RESULTS: There were 375 cases: 303 with previous Down syndrome, 63 with Edwards syndrome and 9 with Patau's syndrome. There was a statistically significant difference between cases and controls, in the distribution of free beta-hCG and PAPP-A levels, adjusted for gestation. On average, free beta-hCG was increased by 10% in a subsequent pregnancy after aneuploidy (p < 0.005, Wilcoxon rank sum test) and for PAPP-A the increase was 15% (p < 0.0001). No other marker was significantly different. CONCLUSION: Risk calculation algorithms need to be modified to take account of the increased marker levels. Until data from sufficient affected pregnancies are available for study, it would be prudent to assume that the same increase as in unaffected pregnancies applies.

Assessment of risk for the development of pre-eclampsia by maternal characteristics and uterine artery Doppler.

OBJECTIVE: To develop a method for the estimation of patient-specific risk for the development of pre-eclampsia by combining maternal history and uterine artery Doppler. DESIGN: Prospective multicentre observational study. SETTING: Antenatal clinics in seven hospitals in the UK and three overseas centres. POPULATION: Unselected women with singleton pregnancies attending for routine antenatal care. METHODS: Doppler studies of the uterine arteries were performed using colour flow mapping and pulsed wave Doppler at 23 weeks of gestation. The mean pulsatility index (PI) of the two uterine arteries was calculated. Doppler and maternal history variables were combined to develop a model for risk assessment. The incidence of pre-eclampsia was used to derive the prior risk for this complication. The posterior risk was derived by multiplying the prior odds with likelihood ratios (LRs) derived from independent risk factors identified from the maternal history, and the LR estimated from the heights of the frequency distributions of mean PI in affected and unaffected pregnancies. MAIN OUTCOME MEASURE: Pre-eclampsia. RESULTS: There were 17,480 women recruited to the study, in which 17,319 (99.1%) of these Doppler examination of both uterine arteries were completed, and outcome data were available in 16,806 (97.0%). Pre-eclampsia occurred in 369 (2.20%) cases. Significant independent prediction of pre-eclampsia was provided by mean PI, ethnic origin, body mass index (BMI), parity, cigarette smoking, history of hypertension and family or personal history of pre-eclampsia. Models were derived allowing calculation of patient-specific risk for development of pre-eclampsia. For a false-positive rate of 25%, the detection rate of pre-eclampsia by screening using maternal history was 45.3%, with uterine artery Doppler it was 63.1% and with combined assessment it was 67.5%. CONCLUSIONS: Combining risk factors in the mother's history with Doppler of the uterine arteries allows calculation of patient-specific risk for the development of pre-eclampsia.

Screening for chromosomal anomalies in the first trimester: does repeat maternal serum screening improve detection rates?

OBJECTIVE: To assess the within person biological variability of first trimester maternal serum biochemical markers of trisomy 21 across the 10-14 week gestational period. To evaluate whether repeat sampling and testing of free beta-hCG and PAPP-A during this period would result in an improved detection rate. METHODS: Women presenting at the first trimester OSCAR clinic have blood collected prior to ultrasound dating and nuchal translucency measurement. All samples are analysed for free beta-hCG and PAPP-A before an accurate estimate of gestation is available. In 10% of cases the gestation is prior to the minimum time for NT measurement (11 weeks) and these women are rebooked for a repeat visit to the clinic at the appropriate time. Our fetal database was interrogated to obtain cases in which two maternal blood samples had been collected and analysed in the 10-14 week period. Using data from the marker correlations and statistical modelling, the impact of repeat testing on detection rate for trisomy 21 at a fixed 5% false positive rate, was assessed. RESULTS: 261 pairs of data were available for analysis collected over a 3 year period. The correlation between free beta-hCG in sample 1 and sample 2 was 0.890 and that for PAPP-A was 0.827. The average within person biological variation for free beta-hCG was 21% and 32% for PAPP-A. The increase in detection rate when using both sets of marker data was 3.5% when using serum biochemistry and maternal age, and 1.3% when using nuchal translucency, serum biochemistry and maternal age. CONCLUSION: Repeat sampling and testing of maternal serum biochemical markers is unlikely to substantially improve first trimester screening performance.