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744748, 1998
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Introduction
Merkatz et al. (1984) reported that pregnancies complicated
by fetal autosomal trisomies are characterized by low maternal
serum -fetoprotein (MSAFP) concentrations. In 1987, Cuckle
et al. published the first results on screening for Downs
syndrome using AFP and maternal age. Numerous other
pregnancy-associated maternal serum markers for fetal trisomy
21 (Downs syndrome) have been evaluated since. It has
been established that human chorionic gonadotrophin (HCG)
concentrations are higher and oestriol concentrations are lower
in Downs syndrome pregnancies. These three parameters
(AFP, HCG and free oestriol), in addition to maternal age, are
used routinely in risk calculation programmes for second
744
trimester Downs syndrome screening. Multiple-marker screening tests for fetal Downs syndrome detect ~60% of all Downs
syndrome cases in women aged ,35 years (screen-positive
rate 5 3.8%) and 7589% in women aged 35 years (screenpositive rate 5 25%) (Haddow et al., 1992; Cheng et al.,
1993). It is not surprising, therefore, that prenatal screening
for Downs syndrome has become an important and established
part of modern prenatal care in Belgium.
Efforts to improve biochemical screening have centred on
the search for better markers in order to either improve the
Downs syndrome detection rate or reduce the false-positive
rate. Numerous pregnancy-associated substances analysed in
serum have been evaluated. Additional markers, such as
progesterone (Kratzer et al., 1991), pregnancy-associated
plasma protein A (Brambati et al., 1993), schwangerschaftsprotein 1 (Brock et al., 1990) and placental alkaline phosphatase
(Ind et al., 1993), have been evaluated but without success.
Inhibin, a heterodimeric protein with a molecular weight of
32 000, has emerged recently as a promising candidate. It is
composed of one -subunit and one of two related -subunits
(-A or -B), and is produced by the syncytiotrophoblast of
human placenta (Petraglia et al., 1987). Of the bioactive
dimeric inhibins, only inhibin-A is present in maternal serum
during pregnancy (Riley et al., 1996). Three early studies,
depending on assays using antibodies directed against epitopes
only on the -subunit, reported conflicting immunoreactive
inhibin concentrations in maternal serum from second trimester
as well as first trimester pregnancies affected by Downs
syndrome (Van Lith et al., 1992; Spencer et al., 1993; Cuckle
et al., 1994a). However, an estimation of dimeric inhibin-A
concentrations using a two-site immunoassay with monoclonal
antibodies has reported an additional value in the detection of
Downs syndrome in two recent studies (Aitken et al., 1996;
Wallace et al., 1996).
These studies are all retrospective and have been performed
on small series. In a preliminary trial we analysed 260 serum
samples for dimeric inhibin-A and 1156 serum samples in a
second trial, in parallel with our routine second trimester
Downs syndrome screening programme. We attempted to
confirm the promising preliminary inhibin-A data for second
trimester Downs syndrome screening in a larger series and in
routine day-to-day screening procedures.
use routine daily assays rather than specially designed ones in the
screening programme for Downs syndrome. AFP and HCG were
measured with an immunoradiometric assay respectively from
Diagnostic Products Corporation (Los Angeles, CA, USA) and
BioSource Europe SA (formerly Medgenix-Belgium, Fleurus,
Belgium). Results were expressed in multiple of the median (MoM)
for the appropriate gestation and the log 10 value was used for risk
calculations. Unconjugated oestriol was measured with a sensitive
radioimmunoassay from Diagnostic Systems Laboratories Inc.
(Webster, TX, USA); results were also expressed in MoM without
logarithmic transformation.
Screening for Downs syndrome is performed twice a week (~80
samples each run) and all assays are performed in duplicate. For risk
calculations we used the algorithm published by Palomaki et al. (1992;
Foundation for Blood Research, Box 190, Scarborough, ME 04074,
USA), which can be used freely if acknowledged as such. After each
run, a quality control program calculated the means and SD for each
parameter. MoM values were improved and controlled each month
with an exponential regression analysis. Correction was performed for
weight and smoking. In cases of multiple pregnancy (twins), we used
the correction factors published by Wald (1991). The obstetric population from an ethnic point of view was considered to be homogeneous
Caucasian because most other ethnic groups refused triple testing for
religious reasons. Mean 6 SD maternal age distribution was 32 6 1
years. In the affected pregnancies group (n 5 18) all mothers were
multiparous. In the unaffected (control) group the mean 6 SD maternal
age was 28 6 1 years. The group consisted of 20% nullipara and 80%
multipara. In this control group 2% of newborn babies suffered from
major or minor neonatal complications. Therefore it was highly unlikely
that the control group was biased for factors that would raise inhibin-A
or HCG concentrations at 16 weeks of pregnancy, as was suggested
recently for hypertensive pregnancies at 29 weeks (Muttukrishna
et al., 1997).
To evaluate the discriminative power of dimeric inhibin-A, we analysed 260 serum samples stored at 20C, including 14 samples from
Downs syndrome pregnancies, in the first retrospective pilot trial.
Reagents for dimeric inhibin-A were commercially available from
Serotec Ltd (Kidlington, UK; product code MCA 127 3KZZ). Dimeric
inhibin-A was measured by a two-site enzyme-linked immunosorbent
assay which utilised an immobilized anti-A inhibin subunit monoclonal antibody as the capture antibody. The second or detection
antibody was a monoclonal antibody specific for the -subunit of
inhibin and was coupled to alkaline phosphatase. The assay was
rather cumbersome and time-consuming (2.5 days), and therefore not
ideal for routine day-to-day large-scale population screening purposes.
In the second trial on 1156 serum samples we modified the original
assay (with the support of N.Groome) so that results were obtained
on the same working day (duration time of the assay 66 h). In this
partially prospective trial, the former affected serum samples, as well
as four new affected ones, were reintroduced and reanalysed at random,
along with incoming routine screening samples. The sensitivity of
the modified assay was 0.3 pg/tube (4 pg/ml); the variation within
and between assays was 3.9 and 18.7% respectively.
Our reference values in the second trimester were in agreement
with those in the leaflet of the kit (Table I).
Statistical analyses were performed using a software program
(NCCS 6.0; NCSS, Kaysville, UT, USA). The results were presented
as means 6 SD. An unpaired t-test was used to compare pairs of
means; when necessary, the unequal variance of data was taken
into account.
Results
In the preliminary pilot trial, 260 samples from second trimester
pregnancies (1220 weeks), including 14 samples from trisomy
Groome et al.a
Our laboratory
13
14
15
16
17
18
328
253
175
174
185
167
167
203
154
146
148
132
aCited
(193612)
(172543)
(67642)
(82424)
(96391)
(50324)
(11365)
(44662)
(60267)
(59260)
(52259)
(48239)
Unaffected (n 5 246)
Trisomy 21 affected (n 5 14)
Trial 2
Unaffected (n 5 1138)
Trisomy 21 affected (n 5 18)
0.93
2.15
P5
0.97
1.78
P,
6 0.64
6 0.75
0.0154
6 0.65
6 0.84
0.0001
Range
0.291.57
1.402.90
0.035.39
0.533.79
Triple test
AFP/
inhibin-A
AFP/
inhibin-A/HCG
9/14
64.3
89.9
12/14
85.7
90
11/14
78.6
93.7
11/14
78.6
87.7
1/250
11/18
61.5
95.2
13/14
93
91
1/160
7/18
38.9
94.6
11/14
78.6
93.7
1/16
12/18
66.7
94.7
M.A.Renier et al.
Figure 1. Multiple of the median (MoM) inhibin-A concentrations in normal (u) and affected (d) pregnancies.
Discussion
Results of the second trial are highly representative of results
from day to day practice. From a statistical viewpoint, however,
ideally a cohort of at least 18 000 samples is needed when 18
Downs syndrome pregnancies are included (a risk of 1/1000
in the second trimester of pregnancy). Compared with the
literature, our number of controls is high in comparison with
the number of affected pregnancies included (Table IV).
Most studies in the literature are retrospective and could
tend to overestimate performance because the ideal conditions
of a study may not apply in practice. One study (Wallace
et al., 1996) obtained a higher prediction rate (75% at a 5%
false-positive rate) by adding inhibin-A to the AFP and -HCG
combination in the first trimester screening. They found a
strong correlation between inhibin-A and intact HCG, which
excluded the use of intact HCG 1 inhibin-A in the algorithm
(two non-independent variables). We confirmed this correlation
(r 5 0.507) in the second trimester and thus were forced to
omit HCG from the algorithm, which decreased the detection
ratio (P 5 0.095) significantly in our risk calculation.
Wald et al. (1997) recently reduced their estimated detection
rate from 78 to 75% for a 5% false-positive rate using
the quadruple test (AFP, unconjugated oestriol, free -HCG,
inhibin-A with maternal age, maternal weight adjustment and
an ultrasound scan examination to estimate gestational age).
They revised their underestimated SD of inhibin-A in pregnancies with Downs syndrome. This highlights the importance
of a laboratorys own assay standards for inhibin and explains
why our inhibin serum concentrations may have slightly
different values especially for our modified short assay.
No. of
trisomy 21
No. of
controls
MoM
inhibin-A
% False-positive
rate (%)
% Sensitivity
21
14
23
19
44
150
206
89
95
202
2.20
1.79
2.46
1.60
2.24
5.3
4.0
5.0
62
65
75
Yes
No
69
627
Vaginal bleeding
Yes
No
67
1088
Ovulation induction
Yes
No
142
1014
Inhibin-A MoM
1.442 6 0.908
0.958 6 0.647
P , 0.0001
0.994 6 0.506
0.986 6 0.674
NS
0.935 6 0.532
0.993 6 0.681
NS
NS 5 not significant.
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Received on May 28, 1997; accepted on November 19, 1997
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