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ORIGINAL RESEARCH |
From the Division of Human Genetics, Department of Pediatrics, and Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, University of Connecticut Health Center, Farmington; Department of Statistics, University of Connecticut, Storrs; and the Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, St. Francis Hospital and Medical Center, Hartford, Connecticut.
Address reprint requests to: Peter A. Benn, PhD, University of Connecticut Health Center, Division of Human Genetics, Department of Pediatrics, 263 Farmington Avenue, Farmington, CT 06030-6140; E-mail: benn{at}nso1.uchc.edu.
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ABSTRACT |
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 ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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METHODS: Ultrasound measurements of nuchal fold, femur length, and humerus length were reviewed for 72 second-trimester Down syndrome and 7063 unaffected fetuses. Derived statistical variables for these parameters were entered into a multivariable Gaussian model together with the statistical variables used in the "quad" test (maternal serum alpha-fetoprotein, unconjugated estriol, human chorionic gonadotropin, and inhibin A). Maternal age-specific sensitivities, false-positive rates, and positive predictive values were generated together with receiver operating characteristic curves. Overall efficacy of ultrasound screening alone, the quad test, and the combination of the ultrasound and quad test were compared using a 1:270 second-trimester risk cutoff applied to 1999 US births.
RESULTS: Using ultrasound, a sensitivity of 79.9% and false-positive rate of 6.7% may be achieved (positive predictive value: 1 in 42). The quad test has a sensitivity of 81.5% and false-positive rate of 6.9% (positive predictive value: 1 in 42). The combination of the quad test with nuchal fold and long bone measurements may achieve 90% sensitivity and a 3.1% false-positive rate (positive predictive value: 1 in 18).
CONCLUSION: Combining second-trimester serum testing and fetal biometry is a feasible approach to Down syndrome screening, compatible with current obstetric practice. This modality is substantially more effective than either serum screening or ultrasound alone. Efficacy may be comparable to that reported for combined first- and second-trimester (integrated) screening.
In the United States, there has been a marked increase in the number of women who are delaying childbearing until their fourth decade of life.1 Because Down syndrome prevalence is strongly dependent on maternal age, this trend has resulted in a higher proportion of women at increased risk for an affected pregnancy. Although a number of effective first- and second-trimester screening protocols have been developed that help identify women at increased risk for a fetus affected by Down syndrome, each of these approaches has a relatively high false-positive rate, particularly for older women. Consequently, there remains a need for improved Down syndrome screening protocols that minimize the number of invasive diagnostic tests while retaining a high sensitivity. In addition to the requirement that the screening has a high positive predictive value, emerging first- and second-trimester protocols must have widespread practical applicability and acceptability if they are to benefit large numbers of women.
Second-trimester maternal serum screening is one approach to the identification of women at increased risk for fetal Down syndrome.2 For example, the "quad" screen modifies the maternal age-specific risk for fetal Down syndrome by a likelihood ratio that reflects the measured concentrations of maternal serum alpha-fetoprotein (MSAFP), human chorionic gonadotropin (hCG), unconjugated estriol (E3), and inhibin A.3,4 Estimates for the 1998 United States pregnancy population indicate that when all four analytes were measured (quad screening), this Down syndrome screening protocol has a sensitivity of 79.3% and a false-positive rate of 7.4%.5
Ultrasonography provides an alternative approach to second-trimester screening for fetal Down syndrome. The identification of specific anatomic markers associated with Down syndrome (ie, relatively short femur length [FL] or humerus length, and enlarged nuchal fold thickness) provide a basis for modifying the maternal age-specific risk for fetal Down syndrome.6–9 These approaches have treated these markers as "categoric" in nature (ie, the measurement is either abnormal or normal). Categorization diminishes the ability of the marker to modify patient-specific risk based on the observed extent of the variance from normal. Bahado-Singh et al10 have further noted that there is a lack of standardization in the criteria used to identify markers, and the definitions of "abnormality" vary. A further complication is the lack of data to indicate that the markers are independent of each other and independent of maternal serum screening tests. As a result of these difficulties, the usefulness of this second-trimester ultrasound screening has been questioned.11
The degree of interaction, or independence, for normally distributed markers can be determined and incorporated into a multivariable Gaussian model for screening. This approach has been routinely used for maternal serum screening and has also been applied to the "integrated test" that combines an ultrasound biometric variable, first-trimester nuchal translucency, with first- and second-trimester serum tests.12
Our objective was to determine if an effective Down syndrome screening protocol could be developed that combined second-trimester maternal serum analytes with the commonly measured second-trimester ultrasound continuous variables of nuchal thickness and proximal long bone measurements.
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MATERIALS AND METHODS |
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Normal values for proximal long bone measurements and biparietal diameter (BPD) were established from a database of 7063 referrals from January 1992 through March 2001. All cases of Down syndrome or other significant aneuploidy were excluded. Remaining cases either had normal karyotypes or were presumed to be normal on the basis of absence of clinical evidence for chromosome abnormality in the newborn. Median maternal age at delivery for the group was 31.3 years. The BPD/FL and BPD/humerus length ratios were calculated for each fetus.
The normal nuchal fold values were derived from a subset of 699 of these pregnancies. Figure 1
demonstrates the axial view of the fetal head through a plane including the cavum septum pellucidum and the bicerebellar diameter with measurement of nuchal fold. An appropriate axial view of the fetal head with measurement of nuchal fold was not made, or recorded, in many cases. However, presence of a nuchal fold measurement was not based on reason for referral, presence of other abnormal ultrasound findings, or gestational age, and the subset was therefore considered to represent an unbiased sample of all normal cases.
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A cohort of pregnancies complicated by Down syndrome was identified through a follow-up database for maternal serum screening. On the basis that the observed number of Down syndrome–affected pregnancies approximated that expected in the population of women screened, ascertainment for affected pregnancies was considered to be substantially complete.13 Fetal biometry data were available for 72 of these pregnancies and included women referred for ultrasonography between January 1991 and March 2000. The median maternal age at delivery for these cases was 34.4 years, which can be compared with an expected median of 33.9 for reported Down syndrome cases in the United States for 1999.14 For the 72 Down syndrome pregnancies, the median values of maternal serum analytes were MSAFP 0.80 multiples of the median (MoM) (n = 72), hCG 2.10 MoM (n = 71), E3 0.65 MoM (n = 71), and inhibin A 2.99 MoM (n = 30). These values were comparable to those seen in a random sample of affected pregnancies,4 further indicating that the cases could be considered to be broadly representative. Biparietal diameter/femur length, BPD/humerus length, and nuchal fold values for these Down syndrome–affected fetuses were expressed as MoM using the gestational age-specific median values for normal pregnancies.
The distributions of log10 transformed BPD/FL, BPD/humerus length, and nuchal fold values, expressed as MoM, were evaluated for departure from normal distributions. Normality was evaluated by determining skewness and kurtosis, plotting histograms, reviewing normal probability (Q-Q) plots, and performing the Shapiro-Wilks and Lilliefors tests.
For both affected and unaffected pregnancies the ultrasound biometric parameter means, standard deviations, and Pearson correlation coefficients were calculated. To model the performance of screening protocols that included maternal serum analytes, we used the statistical parameters derived by Wald et al 4 for pregnancies dated by ultrasound because they were derived from a larger data set of unselected affected pregnancies. The correlation coefficients between serum analytes and the biometric parameters were derived from the affected and unaffected pregnancies identified in our study.
The methods used to determine sensitivity and false-positive rates have been described elsewhere.5 Briefly, the means, standard deviations, and correlation coefficients were assumed to be applicable to large populations of affected and unaffected pregnancies. For any particular screening test (or test combination), screening test results for 100,000 normal pregnancies and 100,000 Down syndrome–affected pregnancies were computer generated using these statistical factors. For each set of results, the ratio of the probabilities that the results were from the affected and unaffected distributions (likelihood ratio) was calculated. At each maternal age, these likelihood ratios were multiplied with the age-specific risk for Down syndrome15 to derive a theoretic distribution of risks that would arise if the screening test were used. The proportion of affected cases with these risks exceeding 1:270 defined the sensitivity. Similarly, the proportion of unaffected cases with risks exceeding 1:270 defined the false-positive rate.
Positive predictive values (true positives divided by true plus false positives) were calculated using the second-trimester maternal age-specific sensitivities, false-positive rates, and prevalence. Total amniocentesis procedure-related losses were calculated using a second-trimester loss rate of 1:200.16 The overall evaluation of efficacy was carried out using the maternal age distribution for 1999 United States births from the National Center for Health Statistics.14 This distribution was used to calculate the average sensitivity and false-positive rate, weighted for the number of women at each maternal age.
Log/linear regression was carried out using an Excel (Microsoft Corp., Redmond, WA) spreadsheet. Descriptive statistics, t tests, Pearson correlation coefficients, and other statistical analyses were performed using JMP (SAS Institute Inc., Cary, NC) and SPSS (SPSS Inc., Chicago, IL). Simulation to generate sensitivity and false-positive rates was performed using S-Plus (Math-Soft Inc., Seattle, WA). For tests of significance a P value of less than .05 was considered to be significant.
The collection of the clinical data used in this study was approved by the investigational review boards of the University of Connecticut Health Center and St. Francis Hospital and Medical Center.
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RESULTS |
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Expressed in MoM, log10(BPD/FL), log10(BPD/humerus length), and log10(nuchal fold) were considered to show Gaussian distributions. Skewness and kurtosis values were low, the Shapiro-Wilks and Lilliefors tests were compatible with the normal approximation, histograms showed bell-shaped distributions, and normal probability (Q-Q) plots showed points falling close to a straight line. In addition, these variables were also essentially independent of maternal age. Log10(BPD/FL), log10(BPD/humerus length), and log10(nuchal fold) were therefore all considered to be acceptable parameters for the screening algorithm.
For Down syndrome fetuses, the median values for BPD/FL (1.09 MoM) and BPD/humerus length (1.10 MoM) were only modestly higher than that in unaffected pregnancies (1.00 MoM). However, the difference between the means in affected and unaffected pregnancies was statistically significant (P < .001), indicating that these parameters had some utility in screening.
In the Down syndrome pregnancies, the median nuchal fold was 1.50 MoM (compared with 1.00 MoM in unaffected fetuses), indicating a high potential for this marker in screening. The log10 (nuchal fold) values in affected pregnancies were significantly larger than those of the controls (P < .001).
Table 1 summarizes the means, standard deviations, and correlation coefficients of the fetal biometric parameters entered into the multivariable screening model. Significant correlations between BPD/humerus length and BPD/FL ratios were found. Additionally, other significant associations between fetal biometric markers and maternal serum analytes were noted.
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shows the receiver operating characteristic curves (plots of sensitivity versus false-positive rates) for age and the quad screen; age and ultrasound screening using nuchal fold, humerus length, and FL; and age with the seven-parameter combination of quad plus ultrasound. The area under the ultrasound curve was 0.91, the quad screen 0.91, and the combined seven-parameter screen 0.97. The area under the curve is a measure of how often a randomly selected woman with an affected pregnancy will have a risk greater than that found for a randomly selected woman with an unaffected pregnancy (without weighting for differences in the numbers of women screened at each maternal age). At every maternal age from 14 to 49, the combined screen had a lower false-positive rate, whereas it maintained or exceeded the detection rate achievable with ultrasound alone or with the quad screen alone.
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summarizes estimated sensitivity for various protocols when the false-positive rate was held at 5%. Table 3 presents the overall expected performance of these protocols applied to the 1999 United States pregnancy population.16 Results are based on a 1:270 second-trimester Down syndrome risk cutoff. Using ultrasound, a sensitivity of 79.9% and false-positive rate of 6.7% could be achieved (positive predictive value: 1 in 42). The quad test has a sensitivity of 81.5% and false-positive rate of 6.9% (positive predictive value: 1 in 42). The most effective screening was achieved using all parameters. The combination of the quad test with nuchal fold and long bone measurements had an expected overall 90% sensitivity and 3.1% false-positive rate (positive predictive value: 1 in 18). For women 35 or older, the combined screening had 95% sensitivity and a 9.1% false-positive rate. Because of the strong correlation between humerus length and FL measurements, the incremental benefit of including both of these was small and was primarily seen in the reduction of the false-positive rates.
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summarizes the Down syndrome cases detected, cases missed, and the amniocentesis-related fetal losses for five different screening protocols. We estimated that, relative to advanced maternal age alone, the combined seven-marker screening protocol could reduce the annual number of amniocentesis procedure-related fetal losses from 2653 to 658; a four-fold reduction.
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DISCUSSION |
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). Our model is based on a relatively small number of affected pregnancies. These cases were ascertained in a population of women referred for ultrasound examination and who may have a higher a priori risk for a pregnancy complicated by fetal Down syndrome. Inclusion of Down syndrome cases with the most abnormal findings could inflate the apparent difference between normal and affected pregnancies and thus overestimate the efficacy of the combined screening. Based on the maternal serum screening results, the 72 cases of Down syndrome included in this study appear to be broadly typical for a random sample of affected pregnancies. However, independent confirmation of the statistical variables derived from a large sample of affected and unaffected pregnancies is required. In addition to the issue of the reproducibility of statistics obtainable at various centers, it may also be necessary to consider national standards and the training of ultrasonographers before widespread use of this screening protocol.17
Previous studies that have approached a combined second-trimester fetal ultrasound and maternal serum screen have not treated nuchal fold, FL, and humerus length as continuous variables11 or have not considered the potential contributions of all seven parameters used in our model.18 Combining fetal biometry with maternal serum screening requires that the correlation between parameters be fully considered. The significant association between hCG concentration and nuchal fold is consistent with the idea that enlarged nuchal translucency or thickness is a manifestation of fluid accumulation19,20 and that elevated hCG is associated with fetal and placental edema.21 A significant correlation between long bone measurements and E3 could reflect this marker’s ability to strongly correlate with fetal size as well as gestational age.13 We also observed a significant association between MSAFP and nuchal fold thickness in normal pregnancies. Each of these associations would indicate that serum screening test results and ultrasound measurements are not totally independent risk factors for Down syndrome. After maternal serum screening, the patient-specific risk for Down syndrome should not be modified using fetal biometric data unless the correlations between the various ultrasonographic and biochemical markers are taken into account.
The results of the modeling indicate that the Combined II-7 protocol has an efficacy that exceeds that of first-trimester screening22 and could approach that expected for the "integrated test" in which first- and second-trimester serum tests are combined with first-trimester nuchal translucency measurement.12 The integrated test can, theoretically, achieve approximately 94% sensitivity with a 5% false-positive rate when applied to the United Kingdom pregnancy population. The Combined II-7 protocol should detect 93% of affected pregnancies with a 5% false-positive rate in the United States (where the median maternal age may be somewhat higher). Integrated testing requires early referral of patients to specialized ultrasound centers, and there are ethical concerns regarding the management of pregnancies that show an indication of a high risk after only the first-trimester phase of screening has been completed.23 Given these practical limitations, a combined screening approach, limited to the second trimester, may be preferred.
Our estimates of the efficacy of the Combined II-7 protocol are based on all patients receiving both maternal serum screening and ultrasound. In practice, laboratory testing and ultrasound examinations are likely to be sequential, and those patients with very low risks after the first component of the testing may not proceed with additional testing. Additional analysis is needed to determine if there is an optimal approach that has a high sensitivity, low false-positive rate, and low procedure-related fetal loss rate while maintaining financial costs within reasonable limits. The current widely adopted practice of referring only those women with second-trimester risks greater than 1:270 by serum testing for an ultrasound examination is not necessarily optimal.
An advantage of the proposed model is that the measurement of nuchal fold, humerus length, and FL is relatively straightforward and is compatible with current standards and practice. Guidelines from the American Institute of Ultrasound in Medicine require that fetal biometry, including BPD and FL, be obtained with every second- and third-trimester ultrasound.24 They also suggest that a view of the posterior fossa (including cerebellar hemispheres and cisterna magna) be documented. This is the same view used to obtain the nuchal fold measurement (Figure 1). Humerus length, though not part of the American Institute of Ultrasound in Medicine standards, is readily obtained when long bones are assessed. Alternatively, screening risks could be based on biometry without the humerus length, although this would be associated with a somewhat higher false-positive rate.
Obstetric ultrasound may be performed in the second trimester for dating or an anatomic survey. Optimal maternal serum screening is achieved when gestational age is based on ultrasound measurements,25,26 and most screen-positive patients already receive a second-trimester ultrasound examination, separately or as a component of the amniocentesis procedure. A second-trimester ultrasound examination may also identify the presence of other fetal biometric or structural markers for Down syndrome such as major structural abnormalities, cardiac defects, echogenic bowel, pyelectasis, and ventriculomegaly.7 These additional markers have not been included in our analyses. Generation of appropriate likelihood ratios for the presence or absence of these anatomic features should provide a basis for further enhancement to this second-trimester approach to Down syndrome screening.
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Footnotes |
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Received March 25, 2002. Received in revised form June 4, 2002. Accepted June 27, 2002.
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REFERENCES |
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2. Wald NJ, Cuckle HS, Densem JW, Nanchahal K, Royston P, Chard T, et al. Maternal serum screening for Down’s syndrome in early pregnancy. BMJ 1988;297:883–7.
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6. Nyberg DA, Souter VL. Sonographic markers for fetal trisomies: Second trimester. J Ultrasound Med 2001;20: 655–74.
7. Bennaceraf BR, Barss VA, Laboda LA. A sonographic sign for the detection in the second trimester of the fetus with Down’s syndrome. Am J Obstet Gynecol 1985;151: 1078–81.[Medline]
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10. Bahado-Singh R, Oz AU, Hsu CD, Deren O, Copel J, Mahoney M. Ratio of nuchal thickness to humerus length for Down syndrome detection. Am J Obstet Gynecol 2001;184:1284–8.[Medline]
11. Smith-Bindman R, Hosmer W, Feldstein VA, Deeks JJ, Goldberg JD. Second-trimester ultrasound to detect fetuses with Down syndrome: A meta-analysis. JAMA 2001;285: 1044–55.
12. Wald NJ, Watt HC, Hackshaw AK. Integrated screening for Down’s syndrome on the basis of tests performed during the first and second trimesters. N Engl J Med 1999;341:461–7.
13. Benn PA. Preliminary evidence for associations between second trimester human chorionic gonadotropin and unconjugated estriol levels with pregnancy outcome in Down syndrome pregnancies. Prenat Diagn 1998;18: 319–24.[Medline]
14. 1999 natality data set. CD-ROM series 21, no. 12. Hyattsville, Maryland: National Center for Health Statistics, 2001.
15. Bray I, Wright DE, Davies C, Hook EB. Joint estimation of Down syndrome risk and ascertainment rates: A meta-analysis of nine published data sets. Prenat Diagn 1998;18: 9–20.[Medline]
16. Elias S, Simpson JL. Amniocentesis. In: Milunsky A, ed. Genetic disorders and the fetus. 3rd ed. Baltimore: Johns Hopkins University Press, 1992:33–57.
17. Snijders RJM, Thom EA, Zachary JM, Platt LD, Greene N, Jackson LG, et al. First-trimester trisomy screening: Nuchal translucency measurement training and quality assurance to correct and unify technique. Ultrasound Obstet Gynecol 2002;19:353–9.[Medline]
18. Bahado-Singh R, Oz AU, Kovanci E, Deren O, Copel J, Baumgarten A, et al. New Down syndrome screening algorithm: Ultrasonographic biometry and multiple serum markers combined with maternal age. Am J Obstet Gynecol 1998;179:1627–31.[Medline]
19. Hyett J, Moscoso G, Nicolaides K. Abnormalities of the heart and great arteries in first trimester chromosomally abnormal fetuses. Am J Med Genet 1997;69:207–16.[Medline]
20. Souter VL, Nyberg DA, El-Bastawissi A, Zebelman A, Luthhardt F, Luthy DA. Correlation of ultrasound findings and biochemical markers in the second trimester of pregnancy in fetuses with trisomy 21. Prenat Diagn 2002; 22:175–82.[Medline]
21. Benn PA, Gainey A, Ingardia CJ, Rodis JF, Egan JFX. Second trimester maternal serum analytes in triploid pregnancies: Correlation with phenotype and sex chromosome complement. Prenat Diagn 2001;21:680–6.[Medline]
22. Cuckle HS, van Lith JM. Appropriate biochemical parameters in first-trimester screening for Down syndrome. Prenat Diagn 1999;19:505–12.[Medline]
23. Copel JA, Bahado-Singh RO. Prenatal screening for Down syndrome—a search for the family values. N Engl J Med 1999;341:521–2.
24. American Institute of Ultrasound in Medicine. Standards for performance of the antepartum obstetrical ultrasound examination. Available at: http://www.aium.org/consumer/standards/obstetrical.pdf. Accessed 2002 Aug 12.
25. Wald NJ, Cuckle HS, Densem JW, Kennard A, Smith D. Maternal serum screening for Down’s syndrome: The effect of routine ultrasound scan determination of gestational age and adjustment for maternal weight. Br J Obstet Gynaecol 1992:99:144–9.[Medline]
26. Benn PA, Borgida A, Horne D, Briganti S, Collins R, Rodis JF. Down syndrome and neural tube defect screening: The value of using gestational age by ultrasonography:Am J Obstet Gynecol 1997;176;1056–61.[Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
