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Maternal serum ADAM12 (A disintegrin and metalloprotease) in chromosomally abnormal pregnancy at 11-13 weeks
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Maternal serum ADAM12 (A disintegrin and metalloprotease) in chromosomally abnormal pregnancy at 11-13 weeks Leona C. Y. Poon, MD; Teodora Chelemen, MD; Ryoko Minekawa, MD; Veronika Frisova, MD; Kypros H. Nicolaides, MD OBJECTIVE: The objective of this study was to investigate the potential
value of ADAM12 (A disintegrin and metalloprotease) in first-trimester screening for trisomy 21 and other major chromosomal abnormalities. STUDY DESIGN: The concentration of ADAM12 was measured at
11-13 weeks in cases of trisomy 21 (n ⫽ 49), trisomy 18 (n ⫽ 28), trisomy 13 (n ⫽ 20), Turner syndrome (n ⫽ 29), triploidy (n ⫽ 10), and euploid pregnancies (n ⫽ 272). The levels of ADAM12, expressed as multiples of median (MoM), were compared in cases and controls and were assessed for association with free -human chorionic gonadotropin (hCG) and pregnancy-associated plasma protein A (PAPP-A). RESULTS: The median ADAM12 value in trisomy 21 (0.961 MoM) was
not significantly different from the euploid fetuses (1.013 MoM), but in
trisomy 18 (0.697 MoM), trisomy 13 (0.577 MoM), triploidy (0.426 MoM), and Turner syndrome (0.747 MoM), the levels were significantly lower. In both the euploid and aneuploid pregnancies, there was a significant association between ADAM12 and free -hCG and PAPP-A. CONCLUSION: Maternal serum ADAM12 concentration at 11-13 weeks
of gestation is unlikely to be useful in first-trimester screening for chromosomal abnormalities because in trisomy 21 the levels are not significantly different from normal, and in the other chromosomal defects, there is a significant association between ADAM12 and the traditional biochemical markers of free -hCG and PAPP-A. Key words: ADAM12 (A disintegrin and metalloprotease), aneuploidies, Down syndrome, first trimester, screening
Cite this article as: Poon LCY, Chelemen T, Minekawa R, et al. Maternal serum ADAM12 (A disintegrin and metalloprotease) in chromosomally abnormal pregnancy at 11-13 weeks. Am J Obstet Gynecol 2009;200:508.e1-508.e6.
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here is some evidence that in pregnancies with fetal trisomy 21, the maternal serum concentration of ADAM12 (A disintegrin and metalloprotease) is reduced during the first trimester of pregnancy, but there are contradictory results as to the potential value of this metabolite in early screening for trisomy 21. Several studies have examined cases of trisomy 21 and reported their values as From the Harris Birthright Research Centre for Fetal Medicine, King’s College Hospital, London, UK. Received July 25, 2008; revised Oct. 7, 2008; accepted Dec. 22, 2008. Reprints: K. H. Nicolaides, MD, Harris Birthright Research Centre for Fetal Medicine, King’s College Hospital, Denmark Hill, London SE5 9RS, United Kingdom. kypros@fetalmedicine. com. This study was supported by a Grant from the Fetal Medicine Foundation (UK Charity #1037116). The assays were sponsored by PerkinElmer Life and Analytical Sciences, Wallac Oy, Turku, Finland. 0002-9378/$36.00 © 2009 Mosby, Inc. All rights reserved. doi: 10.1016/j.ajog.2008.12.029
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multiple of the median (MoM) of normal controls corrected for gestation and maternal weight.1-6 The median MoM of ADAM12 in trisomy 21 pregnancies at 6-9 weeks was reported to be extremely low by Laigaard et al1,2 (0.13 MoM; n ⫽ 14) but only modestly reduced by the group of Spencer et al3,4 (0.60 MoM; n ⫽ 35). Similarly, at the gestational age of 11-13 weeks, when successful screening is achieved by combined fetal nuchal translucency (NT) thickness and maternal serum-free -human chorionic gonadotropin (hCG) and pregnancy-associated plasma protein A (PAPP-A), Spencer et al5 reported that the median MoM of ADAM12 in 46 cases of trisomy 21 was 0.98. In contrast, Laigaard et al6 examined 212 cases of trisomy 21 at 11-13 weeks and reported that the median MoM was low at 11 and 12 weeks (0.49 and 0.74, respectively) and high at 13 weeks (1.38). They estimated that addition of ADAM12 to combined screening with fetal NT and serum-free -hCG and PAPP-A would improve the detection rate of trisomy 21 at both 10-11 weeks and 12-13 weeks, from about 85% to 90% at a false-positive rate of 5%.6
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In chromosomal abnormalities other than trisomy 21, there is evidence that serum ADAM12 at 11-13 weeks is also reduced to 0.79 MoM for trisomy 18, 0.66 MoM for trisomy 13, 0.68 MoM for Turner syndrome, and 0.55 MoM for triploidy.7,8 The aim of this study was to investigate further the potential value of ADAM12 in first-trimester screening for trisomy 21 and other major chromosomal abnormalities.
M ATERIALS AND M ETHODS Study population This was a case-control study. In our center, we performed screening for chromosomal abnormalities by a combination of maternal age, fetal NT thickness, and maternal serum-free -hCG and PAPP-A at 11-13 weeks of gestation.9,10 Written informed consent was obtained from the women agreeing to participate in a research study to identify potential biomarkers of pregnancy complications, which was approved by the King’s College Hospital Ethics Committee. They agreed for aliquots of their serum used for the measurement of free -hCG and
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TABLE 1
Maternal characteristics in each outcome group Maternal characteristic
Control (n ⴝ 272)
Maternal age, y, median (range)
32.6 (17.9-42.3)
Maternal weight, kg, median (range)
66.5 (45-124)
CRL, mm, median (range)
64.0 (47-84)
Trisomy 21 (n ⴝ 49)
Trisomy 18 (n ⴝ 28)
Trisomy 13 (n ⴝ 20)
Turner syndrome (n ⴝ 29)
Triploidy (n ⴝ 10)
38.1 (19.1-46.5)a
37.9 (25.3-42.6)a
34.8 (29.6-44.6)b
30.0 (18.1-37.9)c
31.9 (20.8-37.6)
................................................................................................................................................................................................................................................................................................................................................................................
68.0 (45-95)
71.4 (52-90)
72.0 (52-85)
67.0 (39-114)
65.7 (50-89)
................................................................................................................................................................................................................................................................................................................................................................................ a c c
63.4 (47-84)
57.7 (47-71)
60.5 (51-73)
64.2 (50-79)
58.4 (45-74)
................................................................................................................................................................................................................................................................................................................................................................................
Ethnicity
....................................................................................................................................................................................................................................................................................................................................................................... c a
White, n (%)
195 (71.7)
43 (87.8)
19 (67.9)
15 (75.0)
27 (93.1)
8 (80.0)
African American, n (%)
52 (19.1)
2 (4.1)
4 (14.3)
3 (15.0)
2 (6.9)
2 (20.0)
Indian or Pakistani, n (%)
12 (4.4)
....................................................................................................................................................................................................................................................................................................................................................................... b
.......................................................................................................................................................................................................................................................................................................................................................................
3 (6.1)
4 (14.3)
1 (5.0)
0
0
.......................................................................................................................................................................................................................................................................................................................................................................
Chinese or Japanese, n (%)
4 (1.5)
Mixed, n (%)
9 (3.3)
1 (2.0)
0
0
0
0
.......................................................................................................................................................................................................................................................................................................................................................................
0
1 (3.6)
1 (5.0)
0
0
................................................................................................................................................................................................................................................................................................................................................................................ a
Nulliparous, n (%)
118 (43.4)
19 (38.8)
12 (42.9)
4 (20.0)
14 (48.3)
7 (70.0)
Cigarette smoker, n (%)
14 (5.1)
5 (10.2)
1 (3.6)
1 (5.0)
2 (6.9)
1 (10.0)
................................................................................................................................................................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................................................................................................................................
Conception
....................................................................................................................................................................................................................................................................................................................................................................... a a b a c
Spontaneous, n (%)
269 (98.9)
23 (46.9)
12 (42.9)
16 (80.0)
19 (65.5)
8 (80.0)
....................................................................................................................................................................................................................................................................................................................................................................... a a b a c
Ovulation drugs, n (%)
3 (1.1)
In vitro fertilization, n (%)
0
25 (51.0)
16 (57.1)
4 (20.0)
10 (34.5)
2 (20)
....................................................................................................................................................................................................................................................................................................................................................................... a
1 (2.0)
0
0
0
0
................................................................................................................................................................................................................................................................................................................................................................................
CRL, crown-rump length. Comparison with euploid group (2 or Fisher exact test for categorical variables and Student t test for continuous variables). P ⬍ .0001; b P ⬍ .01; c P ⬍ .05. Poon. ADAM12 and chromosomal abnormalities. Am J Obstet Gynecol 2009.
a
PAPP-A to be stored at -80oC for future studies. Transabdominal ultrasound examination was performed to diagnose any major fetal defects and for measurement of fetal crown-rump length (CRL) and NT thickness.9,10 Gestational age was based on the CRL at the time of the screening and was calculated using the formula obtained from Robinson and Fleming.11 Automated machines that provide reproducible results within 30 minutes were used to measure PAPP-A and free -hCG (DELFIA Xpress system; PerkinElmer Life and Analytical Sciences, Waltham, MA). Maternal demographic characteristics, ultrasononographic measurements, and biochemical results were recorded in a computer da-
tabase. Karyotype results and details on pregnancy outcomes were added into the database as soon as they became available.
Base cohort population The base cohort study population, wherein the present case-control study was nested, was examined between March 2006 and March 2007. During this period, first-trimester combined screening was carried out in 10,641 singleton pregnancies. There were 734 cases (6.9%) in which it was not possible to ascertain the fetal karyotype either because the pregnancies were lost to follow-up (n ⫽ 492) or they resulted in fetal death or miscarriage without pathologic examination (n ⫽ 242). The base cohort
population included 9792 pregnancies with a normal karyotype or delivery of a phenotypically normal baby, 85 cases with fetal trisomy 21, and 102 cases with chromosomal abnormalities other than trisomy 21.
Case-control population In this study, we measured ADAM12 in samples from 49 cases with fetal trisomy 21, 28 with trisomy 18, 20 with trisomy 13, 29 with Turner syndrome, and 10 with triploidy. All 10 cases of triploidy had the phenotype of digynic triploidy characterized by a thin but normal-looking placenta with severe asymmetrical fetal growth restriction. Each case with chromosomal abnormalities was matched with 2 control subjects who were examined on
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the same day from pregnancies with no complications and resulting in the live birth of phenotypically normal neonates (euploid group, n ⫽ 272). The median storage time of the samples was 499 days (range, 198-591).
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FIGURE 1
Relationship between ADAM12 MoM and PAPP-A MoM in euploid and aneuploid pregnancies
Sample analysis A single serum sample of 25 L was used to measure ADAM12 concentration by a heterogeneous time-resolved fluorescent immunoassay in which the ADAM12 concentration was directly proportional to the fluorescence measured on a time-resolved fluorometer at 615 nm (DELFIA/ AutoDELFIA ADAM12 research kit; PerkinElmer Life and Analytical Sciences, Turku, Finland). Fresh aliquots of ADAM12 quality-control samples of 77.6, 294.4, and 736.2 pg/mL concentrations were measured in duplicate at the beginning and at the end of each run. The mean coefficients of variation were 5.4%, 2.8%, and 3.2%, respectively. Statistical analysis The measured concentration of ADAM12 was log transformed to make the distribution Gaussian. Multiple regression analysis was used to determine which of the factors among maternal age, ethnicity, weight, parity, smoking status, method of conception, and fetal CRL were significant predictors of log ADAM12 in the control group and from the regression model, the value in each case and control was expressed as a MoM of the control group. Box-whisker plot of ADAM12 MoM for each of the outcome groups was created. A Mann-Whitney test was used to determine the significance of differences in the median MoM in each chromosomal abnormality to that in the euploid group. The measured free -hCG and PAPP-A were converted into a MoM for gestational age adjusted for maternal weight, ethnicity, smoking status, method of conception, and parity.12 Regression analysis was then used to determine the significance of association between ADAM12 MoM with free -hCG MoM and PAPP-A MoM. Similarly, the measured NT was expressed as a difference from the expected normal mean for gestation (delta value) and regression analysis was then used to determine the 508.e3
Relationship between log ADAM12 MoM and log PAPP-A MoM in euploid (open circles and dashed regression line) and aneuploid pregnancies (solid dots and solid regression line). Poon. ADAM12 and chromosomal abnormalities. Am J Obstet Gynecol 2009.
significance of association between ADAM12 MoM and delta NT. The statistical software package SPSS 15.0 (SPSS Inc, Chicago, IL) was used for all data analyses.
R ESULTS The maternal characteristics of each of the outcome groups are compared in Table 1.
Euploid group Multiple regression analysis in the control group demonstrated that for log ADAM12 significant independent contributions were provided by fetal CRL, maternal weight, and ethnic origin: log expected ADAM12 ⫽ 2.759 ⫹ 0.006 ⫻ CRL (in mm) – 0.005 ⫻ weight (in kg) ⫹ (0.058 [if black], 0 [if other ethnic origins]; r2 ⫽ 0.263; P ⬍ .0001). In each patient, we used this formula to derive
American Journal of Obstetrics & Gynecology MAY 2009
the expected log ADAM12 and then expressed the observed value as a MoM of the expected. In the univariate analysis, cigarette smokers had significantly lower ADAM12 than nonsmokers (r ⫽ 0.141; P ⫽ .020) but in the multivariate analysis, smoking did not have a significant contribution in predicting ADAM12 (P ⫽ .098). In the euploid group, the mean log ADAM12 MoM was 0 with a standard deviation (SD) of 0.134. There was a significant association between log ADAM12 MoM and log PAPP-A MoM (r ⫽ 0.470; P ⬍ .0001) and log-free -hCG MoM (r ⫽ 0.219; P ⬍ .0001) (Figures 1 and 2), but not with delta NT (P ⫽ .775).
Aneuploid group The median ADAM12 MoM in each chromosomally abnormal group, except
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FIGURE 2
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FIGURE 3
Relationship between ADAM12 MoM and free -hCG MoM in euploid and aneuploid pregnancies
ADAM12 in fetuses with specific chromosome abnormalities as compared with those that were euploid
Box-whisker plot of ADAM12 in the outcome groups: euploid, trisomy 21, trisomy 18, trisomy 13, Turner syndrome, and triploidy. Poon. ADAM12 and chromosomal abnormalities. Am J Obstet Gynecol 2009.
Relationship between log ADAM12 MoM and log-free -hCG MoM in euploid (open circles and dashed regression line) and aneuploid pregnancies (solid dots and solid regression line). Poon. ADAM12 and chromosomal abnormalities. Am J Obstet Gynecol 2009.
in trisomy 21, was significantly lower than in the euploid group (Figure 3 and Table 2). For the observed difference in ADAM12 between euploid and trisomy 21 pregnancies to have been significant at the 5% level with a power of 90%, it would have been necessary to study 98 cases of trisomy 21. In the aneuploid group, the mean log ADAM12 MoM was -0.121 with an SD of 0.158. There was a significant association between log ADAM12 MoM and log PAPP-A MoM (r ⫽ 0.336; P ⬍ .0001) and log-free -hCG MoM (r ⫽ 0.414; P ⬍ .0001; Figures 1 and 2) but not with delta NT (P ⫽ .117). In trisomy 21, log ADAM12 MoM increased with fetal CRL (log ADAM12 MoM ⫽ – 0.478 ⫹ 0.007 ⫻ CRL (in mm); r2 ⫽ 0.181, P ⫽ .002; Figure 4). The median (interquartile range [IQR]) ADAM12 MoM was 0.805 (IQR, 0.686-
1.009) at 11 weeks of gestation (n ⫽ 8), 0.951 (IQR, 0.733-1.140) at 12 weeks (n ⫽ 30), and 1.068 (IQR, 0.977-1.235) at 13 weeks (n ⫽ 11). Compared with the euploid group the median free -hCG MoM was higher in trisomy 21 and lower in trisomies 18 and 13; PAPP-A MoM was lower in all chromosomal abnormalities; and delta NT was higher in trisomies 21, 18, and 13 and Turner syndrome (Table 2).
C OMMENT The findings of this study demonstrate that at 11-13 weeks of gestation, the maternal serum ADAM12 concentration in trisomy 21 pregnancies is not significantly different from euploid pregnancies, but in trisomy 18, trisomy 13, triploidy, and Turner syndrome, the level is reduced.
In this study, the cases and controls were matched for storage time, and ADAM12 was measured by a time-resolved fluorescent immunoassay using a monoclonal tracer antibody that recognizes a stable epitope on the molecule. The results were highly reproducible with low coefficients of variation and SDs of 0.13 and 0.16 for the euploid and chromosomally abnormal groups, respectively. In previous publications, either there was no statement on the method of matching cases and controls or this was merely based on gestational age, and the research assays used were based on an antibody pair (6E6 and 8F8), which is not fully optimized. The reported SD was wide (euploid group, 0.28-0.43; aneuploid group, 0.26-0.78), suggesting that the binding site for the antibodies used may be either degrading or altering its conformation. Consequently, the very low levels of ADAM12 found in cases of trisomy 21 may at least in part reflect the longer storage time for the cases than the controls. In euploid pregnancies, the maternal serum ADAM12 concentration is dependent on fetal CRL, maternal weight, and ethnic origin, being higher in black than in white women. We used multiple re-
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TABLE 2
Median (interquartile range) of maternal serum ADAM12 MoM, free -hCG MoM, PAPP-A MoM, and delta NT in fetuses with specific chromosome abnormalities as compared with those that were euploid Karyotype
n
ADAM12 MoM
Free -hCG MoM
PAPP-A MoM
Delta NT (mm)
Euploid
272
1.01 (0.82-1.24)
0.96 (0.66-1.41)
0.99 (0.68-1.45)
0.020 (-0.17 to 0.24)
Trisomy 21
49
0.96 (0.76-1.12)
2.26 (1.42-3.50)
0.47 (0.34-0.70)
2.17 (1.13-4.28)
Trisomy 18
28
0.70 (0.55-1.0)
0.19 (0.14-0.30)
0.17 (0.14-0.25)
4.13 (0.97-5.95)
Trisomy 13
20
0.58 (0.44-0.84)
0.39 (0.29-0.47)
0.27 (0.21-0.37)
2.21 (0.20-4.63)
Turner syndrome
29
0.75 (0.61-0.87)
0.97 (0.61-1.75)
0.53 (0.42-0.81)
7.81 (6.70-10.51)
Triploidy
10
0.43 (0.26-0.84)
0.13 (0.04-0.34)
0.06 (0.04-0.08)
0.09 (-0.01 to 0.65)
................................................................................................................................................................................................................................................................................................................................................................................ a a a ................................................................................................................................................................................................................................................................................................................................................................................ a a a a ................................................................................................................................................................................................................................................................................................................................................................................ a a a b ................................................................................................................................................................................................................................................................................................................................................................................ a a a ................................................................................................................................................................................................................................................................................................................................................................................ c a a ................................................................................................................................................................................................................................................................................................................................................................................
P ⬍ .0001 (comparison with euploid [Mann-Whitney test]); b P ⬍ .01 (comparison with euploid [Mann-Whitney test]); c P ⬍ .05 (comparison with euploid [Mann-Whitney test]). Poon. ADAM12 and chromosomal abnormalities. Am J Obstet Gynecol 2009.
a
gression analysis to define the contribution of maternal variables that influence the measured concentration of ADAM12 and the interaction between these covariates because the alternative method of sequential adjustment for each individual parameter fails to take into account the interaction between the covariates.12 In previous publications on ADAM12, adjustments were made only for gestational age.1-6 The results demonstrate that, contrary to the expectations raised by a FIGURE 4
Relationship between ADAM12 MoM and fetal CRL in trisomy 21 pregnancies
Relationship between log ADAM12 MoM and fetal crown-rump length (CRL) in trisomy 21 pregnancies. Poon. ADAM12 and chromosomal abnormalities. Am J Obstet Gynecol 2009.
508.e5
previous publication,6 measurement of maternal serum ADAM12 at 11-13 weeks is not useful in screening for trisomy 21. The finding that in trisomy 21 pregnancies the median ADAM12 MoM increases with gestation is compatible with the report of Christiansen et al13 that at 14-19 weeks the levels in affected pregnancies are significantly higher than in euploid pregnancies and that this metabolite could potentially improve second-trimester serum biochemical screening. Similarly, before 10 weeks serum ADAM12 in trisomy 21 pregnancies is likely to be significantly lower than in euploid pregnancies, but the magnitude of this difference remains uncertain.1-4 In addition, we found that in both euploid and aneuploid pregnancies, there is a strong association between the levels of ADAM12 and both PAPP-A and free -hCG, and therefore, the potential performance of biochemical screening by a combination of ADAM12, PAPP-A, and free -hCG at 8-9 weeks is likely to be substantially lower than that suggested by Laigaard et al6 who predicted a detection rate of 92% at a false-positive rate of 5%. In chromosomal abnormalities other than trisomy 21, the level of reduction in serum ADAM12 at 11-13 weeks is similar to that reported by Spencer et al.7,8 However, in these chromosomal abnormalities, the magnitude of the reduction in ADAM12 is substantially smaller than the reduction in PAPP-A and free -hCG. Furthermore, there is
American Journal of Obstetrics & Gynecology MAY 2009
a strong association between the levels of ADAM12 and both PAPP-A and free -hCG. Consequently, measurement of ADAM12 is unlikely to improve the performance of first-trimester screening for these abnormalities achieved by the combination of maternal age, fetal NT, fetal heart rate, and maternal serumfree -hCG and PAPP-A.14 f ACKNOWLEDGMENTS The assays were performed by Keith Burling and Fiona Tulloch (Department of Clinical Biochemistry, Addenbrookes National Health Service Trust, Cambridge, UK).
REFERENCES 1. Laigaard J, Sørensen T, Fröhlich C, et al. ADAM12: a novel first-trimester maternal serum marker for Down syndrome. Prenat Diagn 2003;23:1086-91. 2. Laigaard J, Cuckle H, Wewer UM, Christiansen M. Maternal serum ADAM12 levels in Down and Edwards’ syndrome pregnancies at 9-12 weeks’ gestation. Prenat Diagn 2006; 26:689-91. 3. Spencer K, Vereecken A, Cowans NJ. Maternal serum ADAM12s as a potential marker of trisomy 21 prior to 10 weeks of gestation. Prenat Diagn 2008;28:209-11. 4. Spencer K, Cowans NJ, Uldbjerg N, Tørring N. First-trimester ADAM12s as early markers of trisomy 21: a promise still unfulfilled? Prenat Diagn 2008;28:338-42. 5. Spencer K, Cowans NJ, Stamatopoulou A. Maternal serum ADAM12s in the late first trimester of pregnancies with Trisomy 21. Prenat Diagn 2008;28:422-4. 6. Laigaard J, Spencer K, Christiansen M, et al. ADAM12 as a first-trimester maternal serum marker in screening for Down syndrome. Prenat Diagn 2006;26:973-9.
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www.AJOG.org 7. Spencer K, Cowans NJ. ADAM12 as a marker of trisomy 18 in the first and second trimester of pregnancy. J Matern Fetal Neonatal Med 2007;20:645-50. 8. Spencer K, Cowans NJ, Stamatopoulou A. Maternal serum ADAM12s as a marker of rare aneuploidies in the first or second trimester of pregnancy. Prenat Diagn 2007;27:1233-7. 9. Snijders RMJ, Noble P, Sebire N, Souka A, Nicolaides KH. UK 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;351:343-6.
10. Nicolaides KH, Spencer K, Avgidou K, Faiola S, Falcon O. Multicenter study of firsttrimester screening for trisomy 21 in 75 821 pregnancies: results and estimation of the potential impact of individual risk-orientated twostage first-trimester screening. Ultrasound Obstet Gynecol 2005;25:221-6. 11. Robinson HP, Fleming JE. A critical evaluation of sonar crown-rump length measurements. BJOG 1975;82:702-10. 12. Kagan KO, Wright D, Spencer K, Molina FS, Nicolaides KH. First-trimester screening for trisomy 21 by free -hCG and PAPP-A:
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impact of maternal and pregnancy characteristics. Ultrasound Obstet Gynecol 2008;31: 493-502. 13. Christiansen M, Spencer K, Laigaard J, Cowans NJ, Larsen SO, Wewer UM. ADAM 12 as a second-trimester maternal serum marker in screening for Down syndrome. Prenat Diagn 2007;27:611-5. 14. Kagan KO, Wright D, Valencia C, Maiz N, Nicolaides KH. Screening for trisomies 21, 18 and 13 by maternal age, fetal NT, fetal heart rate, free  hCG and PAPP-A. Hum Reprod 2008;23:1968-75.
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Maternal serum ADAM12 (A disintegrin and metalloprotease) in chromosomally abnormal pregnancy at 11-13 weeks Leona C. Y. Poon, MD; Teodora Chelemen, MD; Ryoko Minekawa, MD; Veronika Frisova, MD; Kypros H. Nicolaides, MD OBJECTIVE: The objective of this study was to investigate the potential
value of ADAM12 (A disintegrin and metalloprotease) in first-trimester screening for trisomy 21 and other major chromosomal abnormalities. STUDY DESIGN: The concentration of ADAM12 was measured at
11-13 weeks in cases of trisomy 21 (n ⫽ 49), trisomy 18 (n ⫽ 28), trisomy 13 (n ⫽ 20), Turner syndrome (n ⫽ 29), triploidy (n ⫽ 10), and euploid pregnancies (n ⫽ 272). The levels of ADAM12, expressed as multiples of median (MoM), were compared in cases and controls and were assessed for association with free -human chorionic gonadotropin (hCG) and pregnancy-associated plasma protein A (PAPP-A). RESULTS: The median ADAM12 value in trisomy 21 (0.961 MoM) was
not significantly different from the euploid fetuses (1.013 MoM), but in
trisomy 18 (0.697 MoM), trisomy 13 (0.577 MoM), triploidy (0.426 MoM), and Turner syndrome (0.747 MoM), the levels were significantly lower. In both the euploid and aneuploid pregnancies, there was a significant association between ADAM12 and free -hCG and PAPP-A. CONCLUSION: Maternal serum ADAM12 concentration at 11-13 weeks
of gestation is unlikely to be useful in first-trimester screening for chromosomal abnormalities because in trisomy 21 the levels are not significantly different from normal, and in the other chromosomal defects, there is a significant association between ADAM12 and the traditional biochemical markers of free -hCG and PAPP-A. Key words: ADAM12 (A disintegrin and metalloprotease), aneuploidies, Down syndrome, first trimester, screening
Cite this article as: Poon LCY, Chelemen T, Minekawa R, et al. Maternal serum ADAM12 (A disintegrin and metalloprotease) in chromosomally abnormal pregnancy at 11-13 weeks. Am J Obstet Gynecol 2009;200:508.e1-508.e6.
T
here is some evidence that in pregnancies with fetal trisomy 21, the maternal serum concentration of ADAM12 (A disintegrin and metalloprotease) is reduced during the first trimester of pregnancy, but there are contradictory results as to the potential value of this metabolite in early screening for trisomy 21. Several studies have examined cases of trisomy 21 and reported their values as From the Harris Birthright Research Centre for Fetal Medicine, King’s College Hospital, London, UK. Received July 25, 2008; revised Oct. 7, 2008; accepted Dec. 22, 2008. Reprints: K. H. Nicolaides, MD, Harris Birthright Research Centre for Fetal Medicine, King’s College Hospital, Denmark Hill, London SE5 9RS, United Kingdom. kypros@fetalmedicine. com. This study was supported by a Grant from the Fetal Medicine Foundation (UK Charity #1037116). The assays were sponsored by PerkinElmer Life and Analytical Sciences, Wallac Oy, Turku, Finland. 0002-9378/$36.00 © 2009 Mosby, Inc. All rights reserved. doi: 10.1016/j.ajog.2008.12.029
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multiple of the median (MoM) of normal controls corrected for gestation and maternal weight.1-6 The median MoM of ADAM12 in trisomy 21 pregnancies at 6-9 weeks was reported to be extremely low by Laigaard et al1,2 (0.13 MoM; n ⫽ 14) but only modestly reduced by the group of Spencer et al3,4 (0.60 MoM; n ⫽ 35). Similarly, at the gestational age of 11-13 weeks, when successful screening is achieved by combined fetal nuchal translucency (NT) thickness and maternal serum-free -human chorionic gonadotropin (hCG) and pregnancy-associated plasma protein A (PAPP-A), Spencer et al5 reported that the median MoM of ADAM12 in 46 cases of trisomy 21 was 0.98. In contrast, Laigaard et al6 examined 212 cases of trisomy 21 at 11-13 weeks and reported that the median MoM was low at 11 and 12 weeks (0.49 and 0.74, respectively) and high at 13 weeks (1.38). They estimated that addition of ADAM12 to combined screening with fetal NT and serum-free -hCG and PAPP-A would improve the detection rate of trisomy 21 at both 10-11 weeks and 12-13 weeks, from about 85% to 90% at a false-positive rate of 5%.6
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In chromosomal abnormalities other than trisomy 21, there is evidence that serum ADAM12 at 11-13 weeks is also reduced to 0.79 MoM for trisomy 18, 0.66 MoM for trisomy 13, 0.68 MoM for Turner syndrome, and 0.55 MoM for triploidy.7,8 The aim of this study was to investigate further the potential value of ADAM12 in first-trimester screening for trisomy 21 and other major chromosomal abnormalities.
M ATERIALS AND M ETHODS Study population This was a case-control study. In our center, we performed screening for chromosomal abnormalities by a combination of maternal age, fetal NT thickness, and maternal serum-free -hCG and PAPP-A at 11-13 weeks of gestation.9,10 Written informed consent was obtained from the women agreeing to participate in a research study to identify potential biomarkers of pregnancy complications, which was approved by the King’s College Hospital Ethics Committee. They agreed for aliquots of their serum used for the measurement of free -hCG and
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Research
TABLE 1
Maternal characteristics in each outcome group Maternal characteristic
Control (n ⴝ 272)
Maternal age, y, median (range)
32.6 (17.9-42.3)
Maternal weight, kg, median (range)
66.5 (45-124)
CRL, mm, median (range)
64.0 (47-84)
Trisomy 21 (n ⴝ 49)
Trisomy 18 (n ⴝ 28)
Trisomy 13 (n ⴝ 20)
Turner syndrome (n ⴝ 29)
Triploidy (n ⴝ 10)
38.1 (19.1-46.5)a
37.9 (25.3-42.6)a
34.8 (29.6-44.6)b
30.0 (18.1-37.9)c
31.9 (20.8-37.6)
................................................................................................................................................................................................................................................................................................................................................................................
68.0 (45-95)
71.4 (52-90)
72.0 (52-85)
67.0 (39-114)
65.7 (50-89)
................................................................................................................................................................................................................................................................................................................................................................................ a c c
63.4 (47-84)
57.7 (47-71)
60.5 (51-73)
64.2 (50-79)
58.4 (45-74)
................................................................................................................................................................................................................................................................................................................................................................................
Ethnicity
....................................................................................................................................................................................................................................................................................................................................................................... c a
White, n (%)
195 (71.7)
43 (87.8)
19 (67.9)
15 (75.0)
27 (93.1)
8 (80.0)
African American, n (%)
52 (19.1)
2 (4.1)
4 (14.3)
3 (15.0)
2 (6.9)
2 (20.0)
Indian or Pakistani, n (%)
12 (4.4)
....................................................................................................................................................................................................................................................................................................................................................................... b
.......................................................................................................................................................................................................................................................................................................................................................................
3 (6.1)
4 (14.3)
1 (5.0)
0
0
.......................................................................................................................................................................................................................................................................................................................................................................
Chinese or Japanese, n (%)
4 (1.5)
Mixed, n (%)
9 (3.3)
1 (2.0)
0
0
0
0
.......................................................................................................................................................................................................................................................................................................................................................................
0
1 (3.6)
1 (5.0)
0
0
................................................................................................................................................................................................................................................................................................................................................................................ a
Nulliparous, n (%)
118 (43.4)
19 (38.8)
12 (42.9)
4 (20.0)
14 (48.3)
7 (70.0)
Cigarette smoker, n (%)
14 (5.1)
5 (10.2)
1 (3.6)
1 (5.0)
2 (6.9)
1 (10.0)
................................................................................................................................................................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................................................................................................................................
Conception
....................................................................................................................................................................................................................................................................................................................................................................... a a b a c
Spontaneous, n (%)
269 (98.9)
23 (46.9)
12 (42.9)
16 (80.0)
19 (65.5)
8 (80.0)
....................................................................................................................................................................................................................................................................................................................................................................... a a b a c
Ovulation drugs, n (%)
3 (1.1)
In vitro fertilization, n (%)
0
25 (51.0)
16 (57.1)
4 (20.0)
10 (34.5)
2 (20)
....................................................................................................................................................................................................................................................................................................................................................................... a
1 (2.0)
0
0
0
0
................................................................................................................................................................................................................................................................................................................................................................................
CRL, crown-rump length. Comparison with euploid group (2 or Fisher exact test for categorical variables and Student t test for continuous variables). P ⬍ .0001; b P ⬍ .01; c P ⬍ .05. Poon. ADAM12 and chromosomal abnormalities. Am J Obstet Gynecol 2009.
a
PAPP-A to be stored at -80oC for future studies. Transabdominal ultrasound examination was performed to diagnose any major fetal defects and for measurement of fetal crown-rump length (CRL) and NT thickness.9,10 Gestational age was based on the CRL at the time of the screening and was calculated using the formula obtained from Robinson and Fleming.11 Automated machines that provide reproducible results within 30 minutes were used to measure PAPP-A and free -hCG (DELFIA Xpress system; PerkinElmer Life and Analytical Sciences, Waltham, MA). Maternal demographic characteristics, ultrasononographic measurements, and biochemical results were recorded in a computer da-
tabase. Karyotype results and details on pregnancy outcomes were added into the database as soon as they became available.
Base cohort population The base cohort study population, wherein the present case-control study was nested, was examined between March 2006 and March 2007. During this period, first-trimester combined screening was carried out in 10,641 singleton pregnancies. There were 734 cases (6.9%) in which it was not possible to ascertain the fetal karyotype either because the pregnancies were lost to follow-up (n ⫽ 492) or they resulted in fetal death or miscarriage without pathologic examination (n ⫽ 242). The base cohort
population included 9792 pregnancies with a normal karyotype or delivery of a phenotypically normal baby, 85 cases with fetal trisomy 21, and 102 cases with chromosomal abnormalities other than trisomy 21.
Case-control population In this study, we measured ADAM12 in samples from 49 cases with fetal trisomy 21, 28 with trisomy 18, 20 with trisomy 13, 29 with Turner syndrome, and 10 with triploidy. All 10 cases of triploidy had the phenotype of digynic triploidy characterized by a thin but normal-looking placenta with severe asymmetrical fetal growth restriction. Each case with chromosomal abnormalities was matched with 2 control subjects who were examined on
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the same day from pregnancies with no complications and resulting in the live birth of phenotypically normal neonates (euploid group, n ⫽ 272). The median storage time of the samples was 499 days (range, 198-591).
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FIGURE 1
Relationship between ADAM12 MoM and PAPP-A MoM in euploid and aneuploid pregnancies
Sample analysis A single serum sample of 25 L was used to measure ADAM12 concentration by a heterogeneous time-resolved fluorescent immunoassay in which the ADAM12 concentration was directly proportional to the fluorescence measured on a time-resolved fluorometer at 615 nm (DELFIA/ AutoDELFIA ADAM12 research kit; PerkinElmer Life and Analytical Sciences, Turku, Finland). Fresh aliquots of ADAM12 quality-control samples of 77.6, 294.4, and 736.2 pg/mL concentrations were measured in duplicate at the beginning and at the end of each run. The mean coefficients of variation were 5.4%, 2.8%, and 3.2%, respectively. Statistical analysis The measured concentration of ADAM12 was log transformed to make the distribution Gaussian. Multiple regression analysis was used to determine which of the factors among maternal age, ethnicity, weight, parity, smoking status, method of conception, and fetal CRL were significant predictors of log ADAM12 in the control group and from the regression model, the value in each case and control was expressed as a MoM of the control group. Box-whisker plot of ADAM12 MoM for each of the outcome groups was created. A Mann-Whitney test was used to determine the significance of differences in the median MoM in each chromosomal abnormality to that in the euploid group. The measured free -hCG and PAPP-A were converted into a MoM for gestational age adjusted for maternal weight, ethnicity, smoking status, method of conception, and parity.12 Regression analysis was then used to determine the significance of association between ADAM12 MoM with free -hCG MoM and PAPP-A MoM. Similarly, the measured NT was expressed as a difference from the expected normal mean for gestation (delta value) and regression analysis was then used to determine the 508.e3
Relationship between log ADAM12 MoM and log PAPP-A MoM in euploid (open circles and dashed regression line) and aneuploid pregnancies (solid dots and solid regression line). Poon. ADAM12 and chromosomal abnormalities. Am J Obstet Gynecol 2009.
significance of association between ADAM12 MoM and delta NT. The statistical software package SPSS 15.0 (SPSS Inc, Chicago, IL) was used for all data analyses.
R ESULTS The maternal characteristics of each of the outcome groups are compared in Table 1.
Euploid group Multiple regression analysis in the control group demonstrated that for log ADAM12 significant independent contributions were provided by fetal CRL, maternal weight, and ethnic origin: log expected ADAM12 ⫽ 2.759 ⫹ 0.006 ⫻ CRL (in mm) – 0.005 ⫻ weight (in kg) ⫹ (0.058 [if black], 0 [if other ethnic origins]; r2 ⫽ 0.263; P ⬍ .0001). In each patient, we used this formula to derive
American Journal of Obstetrics & Gynecology MAY 2009
the expected log ADAM12 and then expressed the observed value as a MoM of the expected. In the univariate analysis, cigarette smokers had significantly lower ADAM12 than nonsmokers (r ⫽ 0.141; P ⫽ .020) but in the multivariate analysis, smoking did not have a significant contribution in predicting ADAM12 (P ⫽ .098). In the euploid group, the mean log ADAM12 MoM was 0 with a standard deviation (SD) of 0.134. There was a significant association between log ADAM12 MoM and log PAPP-A MoM (r ⫽ 0.470; P ⬍ .0001) and log-free -hCG MoM (r ⫽ 0.219; P ⬍ .0001) (Figures 1 and 2), but not with delta NT (P ⫽ .775).
Aneuploid group The median ADAM12 MoM in each chromosomally abnormal group, except
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FIGURE 2
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FIGURE 3
Relationship between ADAM12 MoM and free -hCG MoM in euploid and aneuploid pregnancies
ADAM12 in fetuses with specific chromosome abnormalities as compared with those that were euploid
Box-whisker plot of ADAM12 in the outcome groups: euploid, trisomy 21, trisomy 18, trisomy 13, Turner syndrome, and triploidy. Poon. ADAM12 and chromosomal abnormalities. Am J Obstet Gynecol 2009.
Relationship between log ADAM12 MoM and log-free -hCG MoM in euploid (open circles and dashed regression line) and aneuploid pregnancies (solid dots and solid regression line). Poon. ADAM12 and chromosomal abnormalities. Am J Obstet Gynecol 2009.
in trisomy 21, was significantly lower than in the euploid group (Figure 3 and Table 2). For the observed difference in ADAM12 between euploid and trisomy 21 pregnancies to have been significant at the 5% level with a power of 90%, it would have been necessary to study 98 cases of trisomy 21. In the aneuploid group, the mean log ADAM12 MoM was -0.121 with an SD of 0.158. There was a significant association between log ADAM12 MoM and log PAPP-A MoM (r ⫽ 0.336; P ⬍ .0001) and log-free -hCG MoM (r ⫽ 0.414; P ⬍ .0001; Figures 1 and 2) but not with delta NT (P ⫽ .117). In trisomy 21, log ADAM12 MoM increased with fetal CRL (log ADAM12 MoM ⫽ – 0.478 ⫹ 0.007 ⫻ CRL (in mm); r2 ⫽ 0.181, P ⫽ .002; Figure 4). The median (interquartile range [IQR]) ADAM12 MoM was 0.805 (IQR, 0.686-
1.009) at 11 weeks of gestation (n ⫽ 8), 0.951 (IQR, 0.733-1.140) at 12 weeks (n ⫽ 30), and 1.068 (IQR, 0.977-1.235) at 13 weeks (n ⫽ 11). Compared with the euploid group the median free -hCG MoM was higher in trisomy 21 and lower in trisomies 18 and 13; PAPP-A MoM was lower in all chromosomal abnormalities; and delta NT was higher in trisomies 21, 18, and 13 and Turner syndrome (Table 2).
C OMMENT The findings of this study demonstrate that at 11-13 weeks of gestation, the maternal serum ADAM12 concentration in trisomy 21 pregnancies is not significantly different from euploid pregnancies, but in trisomy 18, trisomy 13, triploidy, and Turner syndrome, the level is reduced.
In this study, the cases and controls were matched for storage time, and ADAM12 was measured by a time-resolved fluorescent immunoassay using a monoclonal tracer antibody that recognizes a stable epitope on the molecule. The results were highly reproducible with low coefficients of variation and SDs of 0.13 and 0.16 for the euploid and chromosomally abnormal groups, respectively. In previous publications, either there was no statement on the method of matching cases and controls or this was merely based on gestational age, and the research assays used were based on an antibody pair (6E6 and 8F8), which is not fully optimized. The reported SD was wide (euploid group, 0.28-0.43; aneuploid group, 0.26-0.78), suggesting that the binding site for the antibodies used may be either degrading or altering its conformation. Consequently, the very low levels of ADAM12 found in cases of trisomy 21 may at least in part reflect the longer storage time for the cases than the controls. In euploid pregnancies, the maternal serum ADAM12 concentration is dependent on fetal CRL, maternal weight, and ethnic origin, being higher in black than in white women. We used multiple re-
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TABLE 2
Median (interquartile range) of maternal serum ADAM12 MoM, free -hCG MoM, PAPP-A MoM, and delta NT in fetuses with specific chromosome abnormalities as compared with those that were euploid Karyotype
n
ADAM12 MoM
Free -hCG MoM
PAPP-A MoM
Delta NT (mm)
Euploid
272
1.01 (0.82-1.24)
0.96 (0.66-1.41)
0.99 (0.68-1.45)
0.020 (-0.17 to 0.24)
Trisomy 21
49
0.96 (0.76-1.12)
2.26 (1.42-3.50)
0.47 (0.34-0.70)
2.17 (1.13-4.28)
Trisomy 18
28
0.70 (0.55-1.0)
0.19 (0.14-0.30)
0.17 (0.14-0.25)
4.13 (0.97-5.95)
Trisomy 13
20
0.58 (0.44-0.84)
0.39 (0.29-0.47)
0.27 (0.21-0.37)
2.21 (0.20-4.63)
Turner syndrome
29
0.75 (0.61-0.87)
0.97 (0.61-1.75)
0.53 (0.42-0.81)
7.81 (6.70-10.51)
Triploidy
10
0.43 (0.26-0.84)
0.13 (0.04-0.34)
0.06 (0.04-0.08)
0.09 (-0.01 to 0.65)
................................................................................................................................................................................................................................................................................................................................................................................ a a a ................................................................................................................................................................................................................................................................................................................................................................................ a a a a ................................................................................................................................................................................................................................................................................................................................................................................ a a a b ................................................................................................................................................................................................................................................................................................................................................................................ a a a ................................................................................................................................................................................................................................................................................................................................................................................ c a a ................................................................................................................................................................................................................................................................................................................................................................................
P ⬍ .0001 (comparison with euploid [Mann-Whitney test]); b P ⬍ .01 (comparison with euploid [Mann-Whitney test]); c P ⬍ .05 (comparison with euploid [Mann-Whitney test]). Poon. ADAM12 and chromosomal abnormalities. Am J Obstet Gynecol 2009.
a
gression analysis to define the contribution of maternal variables that influence the measured concentration of ADAM12 and the interaction between these covariates because the alternative method of sequential adjustment for each individual parameter fails to take into account the interaction between the covariates.12 In previous publications on ADAM12, adjustments were made only for gestational age.1-6 The results demonstrate that, contrary to the expectations raised by a FIGURE 4
Relationship between ADAM12 MoM and fetal CRL in trisomy 21 pregnancies
Relationship between log ADAM12 MoM and fetal crown-rump length (CRL) in trisomy 21 pregnancies. Poon. ADAM12 and chromosomal abnormalities. Am J Obstet Gynecol 2009.
508.e5
previous publication,6 measurement of maternal serum ADAM12 at 11-13 weeks is not useful in screening for trisomy 21. The finding that in trisomy 21 pregnancies the median ADAM12 MoM increases with gestation is compatible with the report of Christiansen et al13 that at 14-19 weeks the levels in affected pregnancies are significantly higher than in euploid pregnancies and that this metabolite could potentially improve second-trimester serum biochemical screening. Similarly, before 10 weeks serum ADAM12 in trisomy 21 pregnancies is likely to be significantly lower than in euploid pregnancies, but the magnitude of this difference remains uncertain.1-4 In addition, we found that in both euploid and aneuploid pregnancies, there is a strong association between the levels of ADAM12 and both PAPP-A and free -hCG, and therefore, the potential performance of biochemical screening by a combination of ADAM12, PAPP-A, and free -hCG at 8-9 weeks is likely to be substantially lower than that suggested by Laigaard et al6 who predicted a detection rate of 92% at a false-positive rate of 5%. In chromosomal abnormalities other than trisomy 21, the level of reduction in serum ADAM12 at 11-13 weeks is similar to that reported by Spencer et al.7,8 However, in these chromosomal abnormalities, the magnitude of the reduction in ADAM12 is substantially smaller than the reduction in PAPP-A and free -hCG. Furthermore, there is
American Journal of Obstetrics & Gynecology MAY 2009
a strong association between the levels of ADAM12 and both PAPP-A and free -hCG. Consequently, measurement of ADAM12 is unlikely to improve the performance of first-trimester screening for these abnormalities achieved by the combination of maternal age, fetal NT, fetal heart rate, and maternal serumfree -hCG and PAPP-A.14 f ACKNOWLEDGMENTS The assays were performed by Keith Burling and Fiona Tulloch (Department of Clinical Biochemistry, Addenbrookes National Health Service Trust, Cambridge, UK).
REFERENCES 1. Laigaard J, Sørensen T, Fröhlich C, et al. ADAM12: a novel first-trimester maternal serum marker for Down syndrome. Prenat Diagn 2003;23:1086-91. 2. Laigaard J, Cuckle H, Wewer UM, Christiansen M. Maternal serum ADAM12 levels in Down and Edwards’ syndrome pregnancies at 9-12 weeks’ gestation. Prenat Diagn 2006; 26:689-91. 3. Spencer K, Vereecken A, Cowans NJ. Maternal serum ADAM12s as a potential marker of trisomy 21 prior to 10 weeks of gestation. Prenat Diagn 2008;28:209-11. 4. Spencer K, Cowans NJ, Uldbjerg N, Tørring N. First-trimester ADAM12s as early markers of trisomy 21: a promise still unfulfilled? Prenat Diagn 2008;28:338-42. 5. Spencer K, Cowans NJ, Stamatopoulou A. Maternal serum ADAM12s in the late first trimester of pregnancies with Trisomy 21. Prenat Diagn 2008;28:422-4. 6. Laigaard J, Spencer K, Christiansen M, et al. ADAM12 as a first-trimester maternal serum marker in screening for Down syndrome. Prenat Diagn 2006;26:973-9.
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www.AJOG.org 7. Spencer K, Cowans NJ. ADAM12 as a marker of trisomy 18 in the first and second trimester of pregnancy. J Matern Fetal Neonatal Med 2007;20:645-50. 8. Spencer K, Cowans NJ, Stamatopoulou A. Maternal serum ADAM12s as a marker of rare aneuploidies in the first or second trimester of pregnancy. Prenat Diagn 2007;27:1233-7. 9. Snijders RMJ, Noble P, Sebire N, Souka A, Nicolaides KH. UK 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;351:343-6.
10. Nicolaides KH, Spencer K, Avgidou K, Faiola S, Falcon O. Multicenter study of firsttrimester screening for trisomy 21 in 75 821 pregnancies: results and estimation of the potential impact of individual risk-orientated twostage first-trimester screening. Ultrasound Obstet Gynecol 2005;25:221-6. 11. Robinson HP, Fleming JE. A critical evaluation of sonar crown-rump length measurements. BJOG 1975;82:702-10. 12. Kagan KO, Wright D, Spencer K, Molina FS, Nicolaides KH. First-trimester screening for trisomy 21 by free -hCG and PAPP-A:
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impact of maternal and pregnancy characteristics. Ultrasound Obstet Gynecol 2008;31: 493-502. 13. Christiansen M, Spencer K, Laigaard J, Cowans NJ, Larsen SO, Wewer UM. ADAM 12 as a second-trimester maternal serum marker in screening for Down syndrome. Prenat Diagn 2007;27:611-5. 14. Kagan KO, Wright D, Valencia C, Maiz N, Nicolaides KH. Screening for trisomies 21, 18 and 13 by maternal age, fetal NT, fetal heart rate, free  hCG and PAPP-A. Hum Reprod 2008;23:1968-75.
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