Keywords
chromosomal anomalies - aneuploidy - trisomy 21 - nuchal translucency measurement
- PAPP-A - β−HCG - pregnancy complications - biomarkers - screen tests on maternal
serum
Palavras-chave
anomalias cromossômicas - aneuploidia - trissomia 21 - medida da translucência nucal
- PAPP-A - β−HCG - complicações de gravidez - biomarcadores - testes de rastreio em
soro materno
Introduction
Chromosomal diseases are the leading cause of perinatal mortality and developmental
abnormalities.[1] In 1866, Langdon Down described for the first time the syndrome that today bears
his name, in reference to individuals affected by the trisomy of chromosome 21, the
most common chromosomal aneuploidy in humans (0.12% or 1 in 800 births).[1] The diagnosis of aneuploidies depends on invasive procedures that are associated
with risks of gestational loss. The total fetal loss for chorionic villus sampling
and amniocentesis ranges from 1.5 to 2.0%.[2] In an attempt to indicate these tests only to patients considered to be at high
risk, several screening strategies have been developed. Therefore, prenatal genetic
counseling is necessary. In addition, several gynecology and obstetrics societies
around the world recommend prenatal screening for aneuploidies in all pregnant women.[3]
First-trimester screening allows early diagnosis of aneuploidy. There are many strategies
that are available for chromosomal abnormality screening. The first-trimester combined
test was introduced by Wald and Hackshaw (1997)[4] and is one of the most popular and useful strategies. In this screening strategy,
the risk is calculated based on the sonographic findings and maternal serum levels
of free beta-human chorionic gonadotrophin (β-hCG) and pregnancy-associated plasma
protein A (PAPP-A).[4] The screening performance of the combined test has been reported to range from 82
to 95% detection rate, with a 5 to 7% false positive (FP) rate.[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15] However, it may vary between different ethnicities as well as by age group.[16]
[17]
[18]
According to the Fetal Medicine Foundation (FMF) proposed screening, patients with
a risk lower than 1:1,000 are classified as low risk and are reassured. If we include
the additional ultrasound marker of the presence of nasal bone and Doppler of the
venous duct, the cut-off point for high-risk patients will be 1:100. Patients with
risk between 1:100 and 1:1,000 are considered to have an intermediate risk.[19]
In Brazil, the first-trimester combined test has been widely used but little information
is available on the performance of this screening method in the Brazilian population.
The aim of this study was to examine the performance of the first-trimester combined
test in unselected pregnancies from Rio de Janeiro by analyzing the detection rates
(sensitivity), specificity, positive and negative predictive values, percentage of
FPs and relative risks for the occurrence of aneuploidy, as well as to estimate the
sensitivity and specificity for various risk cut-off points to construct a receiving
operator characteristics (ROC) curve.
Methods
The current study was based on a cohort of singleton gestation women who booked a
combined test for first-trimester risk assessment at our center from February 2009
to September 2015. Women with pregnancies resulting from ovum donation, multiple gestation
or without postpartum follow-up were excluded.
The fetal crown rump length (CRL) was measured, and if it was between 45 and 84 mm,
we evaluated the following fetal ultrasound parameters: nuchal translucency (NT),
nasal bone (NB), and ductus venosus (DV) flow. After that, the PAPP-A and free β-hCG
levels in the maternal serum were determined. The sample was analyzed by means of
a fluoroimmunometric assay using an automated AutoDelfia system (Perkin Elmer, Wallac,
Turku, Finland). Analysis of NT thickness, PAPPA-A and free β-hCG was performed using
the algorithm provided by the FMF, in London, UK, and was calculated using the Astraia
software (astraia software gmbh, Munich, Germany).
The ultrasound parameters were evaluated only by experienced sonographers who had
been certificated by the FMF for 11–13 weeks' scan. The measurements were taken using
a transabdominal transducer (5 MHz curvilinear transducer, Voluson E6 [GE Healthcare, Milwaukee, WI, USA]).
First-trimester risk assessment was provided for trisomy 21, trisomy 18 and trisomy
13. The risk was calculated using a previously described algorithm.[20] The cut-off point for high risk for aneuploidies was defined as greater than 1:100,
intermediate risk was defined to be between 1:100 and 1:1,000 and low risk was defined
as less than 1:1,000.[4] The variable aneuploidy was considered as a result not only of trisomy of chromosome
21 but also the trisomy of chromosomes 13 and 18. Patients classified as high or intermediate
risk were referred for fetal medicine counseling. Chorionic villus sampling or amniocentesis
was performed for karyotype analysis in women who expressed their wish and signed
an informed consent.
Information about patient characteristics, chromosomal abnormalities and the pregnancy
outcome was obtained by the personnel, hospital registry or the postpartum routine
follow-up registry.
The statistical analysis was performed using the SPSS software version 20.0 (IBM Corp.,
Armonk, NY, USA). A descriptive analysis of the population was performed in the form
of mean and median with standard deviation (SD) for quantitative variables and the
proportions, percentages and ratios by calculating the 95% confidence intervals (95%CIs)
for categorical variables.
We calculated detection rates (sensitivity), specificity, positive and negative predictive
values and percentage of FP for aneuploidies. P-values < 0.05 were considered statistically significant. For quantitative variables,
the Mann-Whitney test was used for comparison between two independent groups, and
for categorical variables, we used the Chi-square test. A ROC curve was constructed
to estimate the sensitivity and specificity of aneuploidy screening for various risk
cut-off points. The Spearman coefficient was calculated to evaluate the correlation
of four factors (age, NT, β-hCG and PAPP-A) with the risk for aneuploidies in the
first trimester.
Results
Among 3,639 pregnant women who underwent a combined test in the first-trimester risk
assessment at the perinatal group, a total of 2,748 hospital registries were analyzed.
Among the patients that were excluded, there were 775 that were lost to follow-up
after delivery or the pregnancy resulted from ovum donation, and 116 cases of multiple
gestation. The ethnic origin of the pregnant women was almost all Latin American.
The median CRL was 62.5 mm (with a range from 45.0 to 84.0 mm). The median maternal
age was 33 years (with a range from 18 to 46 years). A total of 1,142 (41.6%) of the
women were aged 35 years or older, and 1,606 (58,45%) women were aged between 18 and
34 years old. A total of 173 (6.3%) of the women were aged 41 years or older.
Considering the cut-off point for high risk for aneuploidies (risk greater than 1:100),
62 (2.3%) women were classified as high risk, 155 (5.6%) as intermediate risk (between
1:100 and 1:1,000), and 2.531 (92.1%) as low risk (less than 1:1,000). In the population
that was screened, there were 21 pregnancies (0.76%) detected for aneuploidy.
The Spearman coefficient was calculated in our population for four algorithm composition
factors, age, TN, β-hCG and PAPP-A, to analyze the influence of these factors in the
risk assessment. Among the factors that influence the risk for aneuploidies, age is
the one with the greatest weight ([Table 1]). Also, if we analyze these four variables separately, we can see that all of them
influence the risk classification with a statistically significant p-value ([Table 2]). In the groups aged under 34 years, between 35 and 40 years and over 41 years,
71 (4.4%), 77 (7.9%) and 69 (39.9%) patients, respectively, were classified as intermediate
and high risk and were referred for fetal medicine counseling ([Table 3]). When stratifying by age group, we found that the chance of being classified as
high/medium risk is 1.8 times higher in the age group between 35 and 40 years and
up to 9 times higher in the age group over 41 years, in relation to the age group
up to 34 years ([Table 3]).
Table 1
Influence of algorithm composition factors in the studied population's first-trimester
risk assessment
|
Algorithm Factor
|
Risk for aneuploidies correlation
|
|
Spearman
|
p
|
|
Maternal Age
|
–0.770
|
< 0.001
|
|
NT
|
–0.111
|
< 0.001
|
|
Free β-HCG
|
–0.122
|
< 0.001
|
|
PAPP-A
|
0.163
|
< 0.001
|
Abbreviations: β-HCG, beta-human chorionic gonadotropin; NT, nuchal translucency;
PAPP-A, pregnancy-associated plasma protein A.
Table 2
Correlation between four variables (age, nuchal translucency, β-hCG and PAPP-A) and
the risk classification of aneuploidy
|
Variables and risk for aneuploidy
|
n
|
Average
|
SD
|
Minimum
|
Median
|
Maximum
|
P-value*
|
|
Age (years)
|
|
|
|
|
|
|
|
|
High/intermediate risk
|
217
|
37.1
|
5.5
|
22.0
|
38.0
|
50.0
|
< 0.001
|
|
Low risk
|
2,531
|
33.3
|
4.2
|
18.0
|
33.0
|
49.0
|
|
Nuchal translucency (mm)
|
|
|
|
|
|
|
|
|
High/intermediate risk
|
217
|
2.04
|
1.13
|
0.70
|
1.80
|
7.20
|
< 0.001
|
|
Low risk
|
2,531
|
1.50
|
0.47
|
0.50
|
1.50
|
9.00
|
|
β-hCG (MoM)
|
|
|
|
|
|
|
|
|
High/intermediate risk
|
216
|
1.71
|
1.40
|
0.09
|
1.25
|
8.34
|
< 0.001
|
|
Low risk
|
2,529
|
2.06
|
24.42
|
0.01
|
1.01
|
933.00
|
|
PAPP-A (MoM)
|
|
|
|
|
|
|
|
|
High/intermediate risk
|
215
|
0.915
|
0.702
|
0.029
|
0.680
|
3.964
|
< 0.001
|
|
Low risk
|
2,499
|
1.437
|
0.960
|
0.108
|
1.228
|
9.987
|
Abbreviations:β-hCG, beta-human chorionic gonadotropin; MoM, Multiple of the Median;
PAPP-A, pregnancy-associated plasma protein A; SD, standard deviation.
Table 3
Combined test risk classification for aneuploidies in the first trimester by maternal
age group (n = 2,748)
|
Patient Age
|
Low risk for aneuploidies
n (%)
|
Intermediate and high risk for aneuploidies
n (%)
|
Total
n (%)
|
Intermediate and high risk
PR (CI95%)
|
|
≤ 34 years
|
1,535 (95.6)
|
71 (4.4)
|
1,606 (100)
|
1
|
|
35–40 years
|
892 (92.1)
|
77 (7.9)
|
969 (100)
|
1.8 (1.3–2.5)
|
|
≥ 41 years
|
104 (60.1)
|
69 (39.9)
|
173 (100)
|
9.0 (6.7–12.1)
|
|
Total
|
2,531 (92.1)
|
217 (7.9)
|
2,748 (100)
|
|
Abbreviations: CI, confidence interval; PR, prevalence ratio.
In the present report, the detection rate for aneuploidy or sensitivity was 42.9%,
specificity was 98.1%, for a FP rate of 1.9%, positive predictive value (PPV) of 14.52%
and negative predictive value (NPV) of 99.55% for a 1:100 cut-off point. The first-trimester
combined test achieved 71.4% sensitivity with a 7.4% FP rate, specificity of 92.6%,
PPV of 6.91% and NPV of 99.76%, when the cut-off point was adjusted to greater than
1:1,000 ([Table 4]).
Table 4
Detection rate or sensitivity, specificity, positive and negative predictive values
for aneuploidy at different cut-off points
|
Risk cut-off
|
Occurrence of aneuploidy
|
Total
|
Predictive values
|
|
Yes
|
No
|
Positive
|
Negative
|
|
n
|
(%)
|
n
|
(%)
|
n
|
(%)
|
|
1/100
|
Positive (> 1/100)
|
9
|
(42.9)
|
53
|
(1.9)
|
62
|
(2.3)
|
14.52%
|
99.55%
|
|
Negative (< 1/100)
|
12
|
(57.1)
|
2,674
|
(98.1)
|
2,686
|
(97.7)
|
|
1/1,000
|
Positive (> 1/1,000)
|
15
|
(71.4)
|
202
|
(7.4)
|
217
|
(7.9)
|
6.91%
|
99.76%
|
|
Negative (< 1/1,000)
|
6
|
(28.6)
|
2,525
|
(92.6)
|
2,531
|
(92.1)
|
|
1/1,860
|
Positive (> 1/1,860)
|
17
|
(81.0)
|
390
|
(14.3)
|
407
|
(14.8)
|
4.18%
|
99.83%
|
|
Negative (< 1/1,860)
|
4
|
(19.0)
|
2,337
|
(85,7)
|
2,341
|
(85.2)
|
When we plotted the ROC curve for our population, the cut-off point that maximized
the sensitivity and specificity for the diagnosis of aneuploidies was 1:1,860. If
we adjust the FP rate to 5%, the detection rate for this analysis is 72.7% with a
cut-off point of 1:610 ([Table 5]).
Table 5
Cutoff point defined by plotted ROC curve to detect aneuploidy: sensibility, specificity
and predictive values
|
Criteria defined by plotted ROC curve
|
Value
|
Sensibility
|
Specificity
|
PPV
|
NPV
|
|
Cut-off point to maximize sensitivity and specificity
|
1/1,860
|
90.91%
|
85.5%
|
2.46%
|
99.96%
|
|
Fixed FP ratio at 0.5%
|
1/610
|
72.7%
|
95.0%
|
5.5%
|
99.88%
|
Abbreviations: FP, false positive; NPV, negative predictive value; PPV, positive predictive
value; ROC, receiving operating characteristic.
Discussion
Many authors have reported that a combined screening test for aneuploidy makes sense
because it results in a sensibility of 80% and higher.[12]
[21]
[22]
[23]
[24]
[25]
[26] However, many critics of the methodology used by these authors cast doubt on these
results.[14]
[27]
[28] Therefore, it is important to analyze the combined screening test performance in
our own population.
In our study, the FP rate for the first-trimester combined test for chromosomal abnormalities
altogether, considering the cut-off of 1:1,000, was 7.4%, which was similar to the
range of 5 to 7% found in other studies. It was somewhat higher than the usual set
value of 5%. The detection rate was 71.4%, which is less than the values of 75.9 to
95% produced by other studies.[6]
[7]
[8]
[14]
[28]
The detection rate was much lower (42.9%) if we consider the cut-off to be 1:100,
for a FP rate of 1.9%. The best result that maximized the sensitivity and specificity
for the diagnosis of aneuploidies in our population was 1:1,860. Most likely, our
results were influenced by maternal age, because 41.6% of the women were aged 35 years
or older. The influence of maternal age on combined screening test performance was
also described in the Chinese population. Pan et al[17] demonstrated that the FP rate increases with increasing maternal age.
The limitation of this study is a small sample size in comparison with larger studies.
Additionally, because this study was conducted in only one center, the results cannot
fully represent the screening performance in all of the Brazilian population. In addition,
the overall screening performance might be affected by the test timing. In this study,
the first-trimester combined test was performed between 11 + 0 and 13 + 6 weeks of
gestation. Both serum markers and NT are affected by the gestational age of the fetus.
The discrimination of PAPP-A is greatest at 10 weeks and declines afterwards, whereas
screening performance of free β-hCG improves with increasing gestational age until
13 weeks. Additionally, there might be a difference in screening power depending on
the gestational age of serum marker measurements. The NT was measured by multiple
observers; therefore, there is a possibility of error as a result of inter-observer
variation. Finally, there were 775 women who were lost to follow-up at the center.
There is a possibility that an error has occurred with these women who had no records
on aneuploidy screening.
Conclusion
In conclusion, the combined test of aneuploidies screening showed a detection rate
inferior to those described in the literature for a higher FP rate. It may suggest
that we have to consider a different cut-off point (1:610) as a reference for the
population assisted at our maternity center to achieve similar performance to the
literature.
