PRENATAL DIAGNOSISPrenat Diagn 2008; 28: 209–211.Published online 8 February 2008 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/pd.1939

Maternal serum ADAM12s as a potential marker of trisomy21 prior to 10 weeks of gestation

Kevin Spencer1,2*, Annie Vereecken3 and Nicholas J. Cowans1

1Prenatal Screening Unit, Clinical Biochemistry Department, King George Hospital, Goodmayes, Essex IG3 8YB, UK2Harris Birthright Research Unit for Fetal Medicine, Kings College Hospital, London, UK3Algemeen Medisch Laboratorium, Antwerp, Belgium

Background ADAM12s (a disintegrin and metalloprotease) is a placenta-derived glycoprotein that is involvedin growth and differentiation and has been shown to be a potential first-trimester and second-trimester marker oftrisomy 21 and other aneuploides. Maternal ADAM12s concentrations show a considerable temporal variationwith gestational age and in the initial study levels were found to be significantly reduced in the early firsttrimester. Here we study the levels prior to 10 weeks of gestation to establish further the effectiveness orotherwise of ADAM12s as an early screening marker.

Materials and Methods Samples collected as part of routine first-trimester screening were retrieved fromstorage. In total, ten samples from singleton pregnancies with trisomy 21 were identified and were collectedbetween the 8th and 9th weeks of gestation—of these 80% had been identified by combined first-trimesterscreening. A series of 62 gestational age-matched samples from singleton pregnancies collected during thesame period formed the control group. ADAM12s was measured by a new DELFIA assay incorporating twomonoclonals (6E6 and 8F8). Results were expressed as multiples of the median (MoM).

Results The median MoM ADAM12s at a median gestation of 9.3 weeks was 0.61 which was significantlylower than in the controls (p = 0.011) when compared by the Mann–Whitney test. The corresponding medianpregnancy associated plasma protein (PAPP-A) was 0.30 and free beta-human chorionic gonadotropin (β-hCG)2.02.

Conclusions Combining the data from this study and from the only other published study with data prior to10 weeks suggests that ADAM12s may have the potential as an early screening marker for trisomy 21, butmay not be as reduced as first thought. Copyright 2008 John Wiley & Sons, Ltd.

KEY WORDS: aneuploidy; screening; growth factors; PAPP-A; free β-hCG

INTRODUCTION

ADAM12s (a disintegrin and metalloprotease) (Gilpinet al., 1998) has been shown to have proteolytic activityagainst insulin-like growth factor binding proteins 3and 5 (Loechel et al., 2000; Shi et al., 2000) and isthought to be part of a mechanism controlling foetalgrowth during pregnancy (Cowans and Spencer, 2007).In pregnancies, with trisomy 21, reduced levels ofADAM12s have been found in the early first trimester(Laigaard et al., 2003, 2006a,b) with levels increasingabove normal in the second trimester (Christiansen et al.,2007). Similar patterns have been observed for trisomy18 (Laigaard et al., 2005a, 2006a; Spencer and Cowans,2007) and also with other rare aneuploidies (Spenceret al., 2007). Laigaard et al., 2006a and Spencer et al.(2007) have both concluded that it is unlikely thatADAM12s will be of value in screening during the 11to the 13 week window—a time commonly used forscreening using the combined test of nuchal translucency(NT), pregnancy associated plasma protein (PAPP-A)and free beta-human chorionic gonadotropin (β-hCG).

*Correspondence to: Kevin Spencer, Clinical BiochemistryDepartment, King George Hospital, Barley Lane, Goodmayes,Essex IG3 8YB, UK. E-mail: KevinSpencer1@aol.com

The early work of Laigaard et al. (2003) with samplescollected predominantly prior to 10 weeks suggestedvery low levels of ADAM12s in cases with trisomy 21.In this study, we seek to understand better the likelyclinical performance of ADAM12s prior to 10 completedweeks of pregnancy.

MATERIALS AND METHODS

First-trimester screening for Down Syndrome was per-formed in a sequential (2-step) method as reported else-where (Gyselaers et al., 2005). Briefly, blood was sam-pled between 8–10 weeks of pregnancy and analysedfor PAPP-A and free-β HCG using the Kryptor anal-yser (Brahms AG, Berlin, Germany). The performanceof this system has been previously described (Spenceret al., 1999) NT was measured between 11–13 weeks ofpregnancy (CRL 45–85 mm) and a combined risk wascalculated. The risk cut-off level was 1 : 300.

In total from this routine screening program 72 sam-ples from singleton pregnancies with known outcome,collected as part of routine prospective screening andwhich had not previously had ADAM12s measured wereselected for further analysis. Of the trisomy 21, three

Copyright 2008 John Wiley & Sons, Ltd. Received: 1 October 2007Revised: 19 November 2007

Accepted: 29 November 2007Published online: 8 February 2008

210 K. SPENCER ET AL.

cases out of ten resulted in live-born infants with tri-somy 21 and of the ten cases, two were missed by thescreening program. In one live-born case, the motherrefused further diagnosis.

As a control group, we used gestational age-matcheddata from 62 first-trimester samples collected duringthe same time period and stored under the same con-ditions. In all first-trimester cases, gestational age atsample collection (median controls = 65.1 days; mediancases = 65.8 days) was determined by CRL measure-ment. Samples had on average been frozen and thawedas aliquots once (range 0–2). The control group had amedian maternal age of 29.1 years, while that for thegroup of cases was 35.04 years. The maternal weight ofthe two groups was similar (median controls = 62.0 Kg;median cases was 61.8 Kg). The group was exclusivelyof Caucasian origin. An informed consent for use of left-over material is not obligatory in Belgium, as screeningfor Down Syndrome is the standard procedure in thefollow-up of pregnancies.

Serum ADAM12s was measured blindly to clinicaloutcome, using a newly developed manual DELFIAassay (PerkinElmer Life & Analytical Sciences, Turku,Finland) incorporating the monoclonal antibodies 6E6and 8F8, which was based on previously describedenzyme-linked immunosorbent assay (ELISA) andAutoDELFIA assays (Laigaard et al., 2003, 2005b)as previously described (Cowans and Spencer, 2007).PAPP-A and free β-hCG results were available fromroutine analysis in which a Kryptor was used.

Regression analysis was used to establish the medianADAM12s marker concentrations across the narrowwindow for 8 to 10 weeks and the concentrations wereconverted into multiples of the median (MoM) bydividing each result by the expected median marker levelfrom the control pregnancies at the same gestational ageas derived from this equation.

RESULTS

Although the cases had a maternal age greater thanthe controls, previous studies have shown ADAM12sconcentration to be unrelated to maternal age (Laigaardet al., 2003, 2006b).

Figure 1 shows the regression equation for ADAM12sconcentration in the controls against the gestationalage alongside the concentrations in the ten trisomy 21cases. Converting the ADAM12s concentration in thecases to MoM using the regression equation resultsin a median MoM of 0.61 at a median gestation of9.3 weeks, this was significantly lower than in thecontrols (Mann–Whitney test p = 0.0116). Previousstudies have shown the need for log10 transformation ofADAM12s MoM in order to fit a gaussian distribution(Laigaard et al., 2003, 2006b). The mean log10 MoMwas −0.2624 with an sd of 0.2460. The median PAPP-A MoM in the same series was 0.30 and that forfree β-hCG was 2.02 with log10 sd’s of 0.3081 and0.2544, respectively. With only ten cases, it was feltinappropriate to provide estimates of correlation with

y = 54.538x - 185.26

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0

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7.5 8 8.5 9 9.5 10 10.5weeks pregnancy

AD

AM

12s

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/ml

Figure 1—Regression of ADAM12s concentration with gestationalage in controls (o) showing the regression line and the 95% confidenceinterval. Also plotted are the concentrations of the ten trisomy 21 cases(�)

0.1

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8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2

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Figure 2—Variation of ADAM12s MoM in ten trisomy 21 pregnan-cies between 8 and 10 weeks of gestation

these other markers. In the controls, the correlationwith free β-hCG and PAPP-A were 0.051 and 0.285,respectively, similar to that in a previous larger series(Laigaard et al., 2006b). Figure 2 shows the distributionof ADAM12s MoM with gestation and shows thesuggestion of an increase of MoM with gestational age.

DISCUSSION

The results of this small study have shown that mater-nal serum levels of ADAM12s are reduced in preg-nancy prior to 10 weeks but not to the levels observedpreviously in the study by Laigaard et al. (2003). Inthe Laigaard et al. (2003) study, the median MoMADAM12s prior to 10 weeks in 13 cases was 0.04.

While the present data might support a possible rolefor ADAM12s prior to 10 weeks, the early promise fromthe Laigaard et al. (2003) study has not been realised.

Copyright 2008 John Wiley & Sons, Ltd. Prenat Diagn 2008; 28: 209–211.DOI: 10.1002/pd

MATERNAL SERUM ADAM12S—POTENTIAL EARLY MARKER OF TRISOMY 21 211

Further studies are required of cases prior to 10 weeksto assess the value of ADAM12s at this time.

ACKNOWLEDGEMENTS

This study was supported in part by a grant from NHSR&D (RF4: Risk Assessment in Pregnancy) to ProfessorKevin Spencer. PerkinElmer Life Science gratefullyprovided the equipment and reagents for this study.

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Copyright 2008 John Wiley & Sons, Ltd. Prenat Diagn 2008; 28: 209–211.DOI: 10.1002/pd