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ORIGINAL RESEARCH |
From the Research Division, NTD Laboratories, Huntington Station, New York; Centro Di Diagnosi Prenatale, Palermo, Italy; GeneCare Medical Genetics Center, Chapel Hill, North Carolina; and the Department of Obstetrics and Gynecology, The George Washington University Medical Center, Washington, DC.
Address reprint requests to: David A. Krantz NTD Laboratories 403 Oakwood Road Huntington Station, NY 11746
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Abstract |
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Methods: Risks were calculated for Down syndrome and trisomy 18 based on maternal age and biochemistry only (n = 10,251), nuchal translucency only (n = 5809), and the combination of nuchal translucency and biochemistry (n = 5809).
Results: The study population included 50 Down syndrome and 20 trisomy 18 cases. Nuchal translucency measurement was done on 33 Down syndrome and 13 trisomy 18 cases. Down syndrome screening using combined biochemistry and ultrasound resulted in a false-positive rate of 4.5% (95% confidence interval [CI] 3.9%, 5.2%) and detection rate of 87.5% (95% CI 47%, 100%) in patients under age 35 years. In older patients, the false-positive rate was 14.3% (95% CI 12.7%, 15.8%) and detection rate was 92% (95% CI 74%, 99%). For trisomy 18 screening, the false-positive rate was 0.4% (95% CI 0.24%, 0.69%) and detection rate was 100% (95% CI 40%, 100%) in younger patients, whereas in older patients the false-positive rate was 1.4% (95% CI 0.9%, 2.0%) and detection rate was 100% (95% CI 66%, 100%). Using modeling, at a fixed 5% false-positive rate, the Down syndrome detection rate was 91%. Conversely, at a fixed 70% Down syndrome detection rate, the false-positive rate was 1.4%.
Conclusion: First-trimester screening for Down syndrome and trisomy 18 is effective and offers substantial benefits to clinicians and patients.
Maternal serum Down syndrome and trisomy 18 screening is conducted in the United States during the second trimester of pregnancy with protocols that include two or more of the biochemical markers alpha-fetoprotein (AFP), hCG, free ß-hCG, and unconjugated estriol (E3). Those screening protocols have detection rates for Down syndrome in young, apparently healthy families in the range of 38%1 to 75% (Macri JN, Spencer K. Toward the optimal protocol for Down syndrome screening [letter]. Am J Obstet Gynecol 1996;174:1668–9). Previous studies2–4 have shown that free ß-hCG, a second-trimester marker, and pregnancy-associated plasma protein A, a biochemical marker not effective in the second trimester, are both productive screening markers for Down syndrome in the first trimester of pregnancy. More recently, ultrasound measurement of fetal nuchal translucency also has been found to be effective in screening for Down syndrome.5
Because these biochemical and biophysical screening approaches are relatively independent, first-trimester screening for Down syndrome can be optimized beyond the capabilities of either approach alone by combining the two. Several recent studies6–11 with small data sets or studies based on modeling have shown that such combined screening could detect 76–89% of Down syndrome cases in the first trimester. A recent opinion by the ACOG Committee on Genetics12 stated that first-trimester screening for chromosome abnormalities offers many advantages over second-trimester screening, and it suggested further studies to confirm the efficacy of nuchal translucency screening with or without serum markers. In light of this suggestion, we prospectively collected data from first-trimester pregnancies to evaluate the effectiveness of maternal serum biochemistry (free ß-hCG and pregnancy-associated plasma protein A), ultrasound measurement of fetal nuchal translucency, and their combination in screening for Down syndrome and trisomy 18.
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Materials and Methods |
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Dried blood specimens were analyzed for free ß-hCG and pregnancy-associated plasma protein A by using previously described enzyme-linked immunosorbent assay procedures.6 Of 10,251 specimens, 7801 were assayed at NTD Laboratories (Huntington Station, NY) and 2450 were assayed at Centro Di Diagnosi Prenatale (Palermo, Italy) using identical assay reagents and procedures.
Gestational ages ranged from 9 weeks 0 days to 13 weeks 6 days and were based on ultrasound or last menstrual period if ultrasound was not done. All pregnancies were apparently healthy, singleton, and not complicated by diabetes. All nuchal translucency measurements were conducted according to the protocol of the Fetal Medicine Foundation, London, United Kingdom. If gestational age was between 10 weeks 4 days and 13 weeks 6 days and an ultrasonographer who was trained by the Fetal Medicine Foundation was available, a nuchal translucency measurement was determined. Of the 10,251 women in the study, 5809 met this criterion and nuchal translucency measurement was successfully done in all in addition to biochemical analysis.
Risk calculations were determined for biochemical markers alone (n = 10,251), nuchal translucency alone (n = 5809), and for both (n = 5809). For statistical purposes only, risks were also calculated based on nuchal translucency alone and biochemistry alone. Risks were calculated based on biochemistry only for women on whom nuchal translucency measurement was not performed. As part of risk assessment, free ß-hCG, pregnancy-associated plasma protein A, and nuchal translucency values were divided by their respective day-specific median level to determine the multiples of the median (MoM) for each marker. Each laboratory developed separate analyte medians to account for interlaboratory assay variation. Likelihood ratios were calculated from multivariate log-gaussian distributions of MoM values in unaffected, Down syndrome, and trisomy 18 cases. Risks were determined by multiplying the likelihood ratio by the women’s risk for Down syndrome and trisomy 18 before screening, which was based on maternal age14 and gestational age, using the formula of Snidjers et al.15 If blood collection and nuchal translucency measurement were not done on the same day, the later of the two gestational ages was used to determine prior risk. Women with risks greater than that of a 35-year-old at the same gestational age were considered to be at increased risk for Down syndrome. For trisomy 18 screening, a risk cut-off of one in 150 was used.
Exact confidence intervals (CI) based on binomial distribution were determined for false-positive and detection rates. The yield was determined by dividing the sum of the Down syndrome and trisomy 18 cases that were at increased risk by the total number of women who were at increased risk after excluding women with other outcomes. False-positive and detection rates were modeled using observed likelihood ratios and the maternal age distribution of live births. For each maternal age 14–49 years, age-specific false-positive and detection rates were determined on the basis of the observed likelihood ratios, the prior risk at that maternal age, and a cut-off risk. The overall false-positive rate was then determined by taking a weighted average of the age-specific false-positive rates, where the weights were equal to the number of unaffected pregnancies in the United States at each maternal age divided by the total number of unaffected pregnancies in the United States. Similarly, the overall detection rate was determined by taking a weighted average of the age-specific detection rates, where the weights were equal to the number of Down syndrome pregnancies at each maternal age divided by the total number of Down syndrome pregnancies in the United States. The number of Down syndrome pregnancies was estimated by multiplying the number of live births at each maternal age by the incidence rate of Down syndrome. The cut-off risk was varied until it reached a 5% false-positive rate and the detection rate at a 5% false-positive rate was determined. Similarly, a separate analysis was done to determine the false-positive rate at a 70% detection rate.
We also present distribution parameters for free ß-hCG, pregnancy-associated plasma protein A, and nuchal translucency based on the samples in the study plus 58 Down syndrome and eight trisomy 18 cases analyzed previously. A total of 108 Down syndrome and 28 trisomy 18 cases were used to determine these parameters.
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shows the distribution of gestational and maternal ages.
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shows results of prospective screening using the combined screening protocol for the 5809 women who had both nuchal translucency and biochemical analysis done. In women under 35 years old, the observed false-positive rate for Down syndrome screening was 4.5% (95% CI 3.9%, 5.2%) with a detection rate of 87.5% (95% CI 47%, 100%). The false-negative rate was 12.5% (95% CI 0%, 53%). In older women the observed false-positive rate was 14.3% (95% CI 12.7%, 15.8%) and detection rate was 92% (95% CI 74%, 99%). The false-negative rate was 8% (95% CI (1%, 26%). For trisomy 18 analysis in the younger women, the observed false-positive rate was 0.4% (95% CI 0.24%, 0.69%) and observed detection rate was 100% (95% CI 40%, 100%). The false-negative rate was 0% (95% CI 0%, 60%). In the older women the observed false-positive rate was 1.4% (95% CI 0.9%, 2.0%) and observed detection rate was 100% (95% CI 66%, 100%). The false-negative rate was 0% (95% CI 0%, 34%). The yield of either Down syndrome or trisomy 18 for every increased-risk result was 11 of 195 (one in 18) in younger women and 32 of 343 (one in 11) in older patients. Including other adverse outcomes, the yield would be 19 of 203 (one in 11) in younger patients and 45 of 356 (one in eight) in older women. For results that were within normative range, the negative predictive value was 99.97% for younger women and 99.88% in older women after excluding other outcomes. Both cases of triploidy, all five cases of Turner syndrome, and two of three cases of trisomy 13 were detected with combined screening. Table 3 lists the median MoM values for the other adverse outcomes in the study.
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shows projected Down syndrome detection rates based on a general United States pregnancy population (ages 14–49 years) for each marker at a fixed 5% false-positive rate and the false-positive rate at a fixed 70% detection rate. At a fixed 5% false-positive rate, the detection rates of biochemistry, ultrasound, and the combination were 63%, 74%, and 91%, respectively. Conversely, to detect 70% of Down syndrome cases, the false-positive rates of biochemistry, ultrasound, and both were 6.8%, 3.4%, and 1.4%, respectively. A receiver operating characteristic (ROC) curve (Figure 1) shows the relationship between detection and false-positive rates for the combined protocol. Table 5 shows the incremental detection rate of all three screening markers in achieving a 91% detection rate for Down syndrome. Table 6 shows false-positive and detection rates increasing with maternal age. For trisomy 18, a combined screening approach can achieve a detection rate of 96% at a 1.1% false-positive rate using a one in 100 cut-off or a 97% detection rate at a 1.2% false-positive rate using a one in 150 cut-off.
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shows distribution parameters based on 108 cases of Down syndrome and 28 cases of trisomy 18. Among Down syndrome pregnancies, the Spearman rank correlation coefficients of MoM values compared with gestational age were 0.10 (P = .302), 0.26 (P = .007), and 0.27 (P = .134) for free ß-hCG, pregnancy-associated plasma protein A, and nuchal translucency, respectively.
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Discussion |
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). In addition to increased detection, first-trimester screening offers inherent advantages of early detection. Most women are found to be at low risk of chromosomal abnormalities and thus can be reassured much earlier in pregnancy. For patients found to be at increased risk, more time is available to decide on diagnostic options. Finally, for patients who choose to terminate an affected pregnancy, safer and earlier procedures are available than those used at or around 20 weeks’ gestation. Our experience with second-trimester screening indicates that there is significant maternal anxiety after an increased-risk result. It is essential, therefore, that diagnostic procedures be available and offered as quickly as possible after screening results have been reported. Chorionic villus sampling (CVS) and early amniocentesis are the primary diagnostic procedures used between 11 and 14 weeks’ gestation. After the initial introduction of CVS, reports indicated an increased incidence of fetal complications, including specific limb defects.16,17 However, more recent studies showed lower rates of limbreduction defects if CVS is done at 10 weeks or later.18 Similarly, there are reports concerning the safety of early amniocentesis.19,20 On balance, it is likely that both CVS and early amniocentesis will result in similar low but acceptable risks for patients who have been given appropriate preprocedure counseling. Randomized studies are currently under way to assess this issue further.
Our detection rate of 91% at a 5% false-positive rate based on combined ultrasound and biochemical screening in the first trimester is similar to or slightly greater than the detection rate of 76–89% reported by other studies6–11 using smaller data sets or modeled data using the same protocol, and it is unlikely that bias could have artificially enhanced screening results. One potential bias could result from not accounting for the fetal loss rate for Down syndrome, which could overestimate the detection rate because of cases of Down syndrome that are undetected and spontaneously abort before term. This overestimation affects both first- and second-trimester screening studies, although the overestimation will tend to be greater in the first trimester where the loss rate is greater. Two recent studies by Snijders et al15 and Morris et al21 found that the fetal loss rate between late first trimester (when this screening is done) and term was approximately 31%. Using the model of Dunstan and Nix,22 which addresses the issue of fetal loss, true detection rate in our study might be adjusted to 88% (based on an average gestational age of 12 weeks and the fetal loss data of Snidjers et al).15 This detection capability still surpasses commonly used second-trimester protocols that yield detection rates of 38–75%. Further, the first-trimester detection of affected cases destined to miscarry would mean that treatment can be offered in a medically controlled setting, and the diagnosis can alert patients to increased risks in future pregnancies. Another bias could result from failure to ascertain live-born Down syndrome cases that were not identified by the screening process, causing an overestimation of detection efficiency. In the current study, however, the number of Down syndrome and trisomy 18 cases observed in the population was similar to that expected, based on maternal age and gestational age. Therefore it is unlikely that there was a significant underreporting of undetected affected cases.
The data indicate that the pregnancy-associated plasma protein A median MoM in Down syndrome pregnancies varies by gestational age, so separate distribution parameters might be needed at different gestational ages. We observed a similar effect for free ß-hCG and nuchal translucency values. However, the variation for these markers was not significant.
A recent study11 suggested that an alternative to first-trimester screening could include a protocol combining results from first-trimester and second-trimester screenings. Unfortunately, this protocol negates the substantial advantages of early screening and diagnosis and could unnecessarily increase anxiety among women who must wait to receive second-trimester screening results. Further, the improved screening performance of an integrated test relies mostly on the high detection capability of first-trimester markers, with small improvements obtained by second-trimester AFP, inhibin, and unconjugated E3, none of which are effective individual markers for Down syndrome. It is likely that further investigation will identify other first-trimester markers (either ultrasound or biochemical) that can make similar small improvements in screening performance while maintaining the substantial advantages of the first-trimester screen.
Results could be reported in terms of either first-trimester or term Down syndrome risk. Either method is valid as long as women are counseled properly about the meaning of the risks. In our Down syndrome screening protocol we used a first-trimester risk cut-off equal to the risk of a 35-year-old at the corresponding gestational age and obtained an acceptable false-positive rate (Table 2). With trisomy 18 screening, a logical age-related cut-off would have resulted in a false-positive rate that was too high. Therefore we chose a first-trimester cut-off risk of one in 150, which, based on the maternal age distribution of live births, resulted in a false-positive rate of approximately 1%.
Previous studies6,23 have shown that nuchal translucency measurements are effective in screening for other chromosomal abnormalities, such as trisomy 13, Turner syndrome, and triploidy. The data in this study confirm those results. Undoubtedly, ultrasound examinations done during the first trimester of pregnancy will lead to the detection of other fetal abnormalities as well.24 Although concerns have been raised about the cost of ultrasound, it has been argued that ultrasound examinations should be done on all gravidas before second-trimester triple screening to reduce false-positive rates.25
We believe that first-trimester screening using a combination of biochemistry and nuchal translucency measurement is feasible, results in improved Down syndrome detection compared with currently used second-trimester protocols, and provides substantial advantages to clinicians and patients. Further studies will refine risk algorithms for Down syndrome and trisomy 18, reduce CIs for false-positive and detection rates, provide more information on detection of autosomal trisomies, determine the impact of first-trimester screening on second-trimester screening, and assess the correlation of free ß-hCG, pregnancy-associated plasma protein A, and nuchal translucency with other congenital anomalies and perinatal complications.
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Footnotes |
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Authors Krantz and Hallahan are employees and author Macri is the owner of NTD Laboratories. Author Macri owns several patents related to the use of free beta hCG in Down syndrome screening.
Received November 23, 1999. Received in revised form February 2, 2000. Accepted February 10, 2000.
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2. Macri JN, Spencer K, Aitken D, Garver K, Buchanan PD, Muller F, et al. First-trimester free beta (hCG) screening for Down syndrome. Prenat Diagn 1993;13:557–62.[Medline]
3. Krantz DA, Larsen JW, Buchanan PD, Macri JN. First-trimester Down syndrome screening: Free beta human chorionic gonadotropin and pregnancy-associated plasma protein A. Am J Obstet Gynecol 1996;174:612–6.[Medline]
4. Haddow JE, Palomaki GE, Knight GJ, Williams J, Miller WA, Johnson A. Screening of maternal serum for fetal Down’s syndrome in the first trimester. N Engl J Med 1998;338:955–61.
5. Snidjers RJM, Noble P, Sebire N, Souka A, Nicolaides KH. United Kingdom multicentre project on assessment of risk of trisomy 21 by maternal age and fetal nuchal-translucency thickness at 10–14 weeks of gestation. Lancet 1998;352:343–6.[Medline]
6. Orlandi F, Damiani G, Hallahan TW, Krantz DA, Macri JN. First-trimester screening for fetal aneuploidy: Biochemistry and nuchal translucency. Ultrasound Obstet Gynecol 1997;10:381–6.[Medline]
7. Biagotti R, Brizzi L, Periti E, d’Agata A, Vanzi E, Cariati E. First trimester screening for Down’s syndrome using maternal serum PAPP-A and free beta-hCG in combination with fetal nuchal translucency thickness. Br J Obstet Gynaecol 1998;105:917–20.[Medline]
8. De Biasio P, Siccardi M, Volpe G, Famularo L, Santi F, Canini S. First trimester screening for Down syndrome using nuchal translucency measurement with free beta-hCG and PAPP-A between 10 and 13 weeks of pregnancy—the combined test. Prenat Diagn 1999;19:360–3.[Medline]
9. De Graaf IM, Pajkrt E, Bilardo CM, Leschot NJ, Cuckle HS, van Lith JM. Early pregnancy screening for fetal aneuploidy with serum markers and nuchal translucency. Prenat Diagn 1999;19: 458–62.[Medline]
10. Spencer K, Souter V, Tul N, Snijders R, Nicolaides KH. A screening program for trisomy 21 at 10–14 weeks using fetal nuchal translucency, maternal serum free beta-human chorionic gonadotropin, and pregnancy-associated plasma protein A. Ultrasound Obstet Gynecol 1999;13:231–7.[Medline]
11. Wald NJ, Watt HC, Hackshaw AK. Integrated screening for Down’s syndrome based on tests performed during the first and second trimesters. N Engl J Med 1999;341:461–7.
12. American College of Obstetricians and Gynecologists. First-trimester screening for fetal anomalies with nuchal translucency. Committee on Genetics opinion no. 223. Washington, DC: American College of Obstetricians and Gynecologists, 1999.
13. Macri JN, Anderson RW, Krantz DA, Larsen JW, Buchanan PD.Prenatal maternal dried blood screening with alpha-fetoprotein and free beta-human chorionic gonadotropin for open neural tube defect and Down syndrome. Am J Obstet Gynecol 1996;174:566–72.[Medline]
14. Hecht CA, Hook EB. The imprecision in rates of Down syndrome by one-year maternal age intervals. A critical analysis of rates used in biochemical screening. Prenat Diagn 1994;14:729–38.[Medline]
15. Snidjers RJM, Sundberg K, Holzgreve W, Henry G, Nicolaides K.Maternal age and gestation specific risk for trisomy 21. Ultrasound Obstet Gynecol 1999;13:167–70.[Medline]
16. Firth HV, Boyd PA, Chamberlain P, MacKenzie IZ, Lindenbaum RH, Huson SM. Severe limb abnormalities after chorion villus sampling at 56–66 days’ gestation. Lancet 1991;337:762–3.[Medline]
17. Burton BK, Schulz CJ, Burd L. Limb abnormalities associated with chorionic villus sampling. Obstet Gynecol 1992;79:726–30.
18. American College of Obstetricians and Gynecologists. Chorionic villus sampling. Committee on Genetics opinion no. 160. Washington, DC: American College of Obstetricians and Gynecologists, 1995.
19. Nicolaides KH, de Lourdes Brizot M, Patel F, Snidjers R. Comparison of chorionic villus sampling and amniocentesis for fetal karyotyping at 10–13 weeks gestation. Lancet 1994;344:435–9.[Medline]
20. Canadian Early and Mid-Trimester Amniocentesis Trial (CEMAT) Group. Randomised trial to assess safety and fetal outcome of early and midtrimester amniocentesis. Lancet 1998;351:242–7.[Medline]
21. Morris JK, Wald NJ, Watt HC. Fetal loss in Down syndrome pregnancies. Prenat Diagn 1999;19:142–5.[Medline]
22. Dunstan FDJ, Nix ABJ. Screening for Down’s syndrome: The effect of test date on the detection rate. Ann Clin Biochem 1998;35:57–61.
23. Pandya PP, Snidjers RJM, Johnson SP, Brizot MDL, Nicolaides KH. Screening for fetal trisomies by maternal age and fetal nuchal translucency thickness at 10 to 14 weeks of gestation. Br J Obstet Gynaecol 1995;102:957–62.[Medline]
24. Souka AP, Snidjers RJM, Novakov A, Soares W, Nicolaides KH.Defects and syndromes in chromosomally normal fetuses with increased nuchal translucency thickness at 10–14 weeks of gestation. Ultrasound Obstet Gynecol 1998;11:391–400.[Medline]
25. Haddow JE, Palomaki GE, Knight GJ, Williams J, Pulkkinen A, Canick JA, et al. Prenatal screening for Down’s syndrome with use of maternal serum markers. N Engl J Med 1992;327:588–93.[Abstract]
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