In 1866 J Langdon Down made the observation of a subgroup of patients with particular facial features and mental handicap in the outpatient department of the London Hospital 1 . In the late 1950’s, it was shown that an extra acrocentric chromosome was present in persons with Down syndrome, resulting in a diploid chromosome number of 47. It is now known that Down syndrome results when either the whole or a segment of the long arm of chromosome 21 is present in three copies instead of two (Table 1). This can occur as a result of three separate mechanisms: nondysjunction (94% of cases), Robert sonian translocation (3.6%) and mosaicism (2.4%).
The live incidence of Down syndrome is about 1 in 700 births. Approximately 30% of Down syndrome fetuses miscarry between 12 weeks gestation and term, while it is estimated that 24% will miscarry between 16 weeks and term 2 . Affected babies are likely to suffer from severe mental disability and have a high chance of associated physical disabilities, affecting in particular the heart, gastrointestinal tract, eyes, and ears. Individuals with Down syndrome also have a higher incidence of Alzheimer’s disease and a 15 to 20 times higher risk of leukemia. Twenty percent die by the age of 5 years, usually from cardiac causes, but over half are expected to survive into their fifties.
Antenatal screening for Down syndrome was first performed in the 1970’s using advanced maternal age or a previous history of aneuploidy. In the 1980’s, the association of Down syndrome with abnormal levels of certain specific serum markers was discovered, and maternal serum screening was developed which further improved the detection rate 3 . Recent addition of ultrasound as a tool for screening offers several advantages:
1. Fetal nuchal translucency thickness in the first trimester becomes an independent screening tool for Down syndrome 4 .
2. Accurate dating of pregnancy facilitates more precise interpretation of serum screening.
3. Second trimester scanning can reveal ‘soft markers’ for Down syndrome, besides major structural anomalies.
Screening for trisomy 21 should be offered to all women as part of routine antenatal care. This offer should include detailed counseling about the implications and limitations of the test used in the screening program. Women should have the option as to whether they wish to have screening or not. Screening tests will not diagnose whether or not the fetus is affected by Trisomy 21, but simply place the woman in a highrisk or lowrisk category. Being in the lowrisk group does not exclude the possibility of Down syndrome. Women in the highrisk group should be offered diagnostic testing to establish whether or not the fetus is affected.
Invasive prenatal diagnosis is associated with a risk of fetal loss of 0.5-1%. Therefore information gained from the various screening technics (biochemical and ultrasound) is combined with maternal age to lower false positive rates and minimise unnecessary invasive testing.
Maternal age
The incidence of trisomy 21 rises with increasing maternal
age and falls with advancing gestational age (Figure 1,Table 2) 6,7. This knowledge could not be utilised until
prenatal diagnosis became available in the late 1960’s.
Unfortunately, prenatal diagnostic methods are associated
with a risk of miscarriage in the order of 0.5-1%. This
fact, combined with the cost implications, meant that
prenatal diagnosis was offered only to women aged 35
and over. This highrisk group constituted 5% of the
pregnant population.
Disappointingly, 20 years of screening in the UK using maternal age alone failed to produce a noticeable effect on the birth incidence of Down syndrome. There are several reasons for this failure:
1. The great majority of affected babies are born to women under 35 years of age because a much larger number of babies are born to women in this age group. Women over 35 years of age contribute only 20-30% of all babies born with Down syndrome.
2. The uptake of invasive fetal karyotyping in the high risk group was less than 50%.
3. The expected fall in the birth prevalence of trisomy 21 may have been counterbalanced by the rise in mean maternal age (from 26.1 years in 1970 to 29 years now) in the UK.
The poor performance of screening for Down syndrome on
the basis of advanced maternal age alone is now universally
accepted. This has led to the development of newer screening
program and the concept of Patient Specific Risk.
Every woman has some risk that her fetus may be affected by a chromosomal defect. In order to calculate this individual risk, it is necessary first to take into account the woman’s a priori risk based on her age and gestational age (Table 2). This a priori risk is then multiplied by a likelihood ratio, calculated from her ultrasound findings and/or serum biochemistry results obtained during the course of the current pregnancy. The product of the a priori risk and the likelihood ratio yields the patient specific risk.
Second trimester
In 1984 Merkatz et al 3 retrospectively analyzed the maternal
serum alpha fetoprotein (AFP) in 44 Down’s affected
pregnancies and found it to be low. Subsequently, Bogart et
al 8 found elevated levels of maternal serum human chorionic
gonadotrophin (hCG), and Canick et al 9,10 found low levels
of unconjugated estriol (uE3) in Down syndrome
pregnancies. The reason for these biochemical changes is not yet fully understood, but may relate to functional
immaturity, leading to a delay in the normal gestational rise
or fall.
The best combination of maternal serum markers is still
debated 11-13. Screening performance depends on the
combination of markers chosen and whether ultrasound has
been used to date the pregnancy accurately.
The optimal window for second trimester biochemical
screening is between 15 and 22 weeks gestation. Apart from
maternal age and gestation, other factors which affect the
expected levels of the biochemical markers must be taken
into account. These include maternal weight, ethnic origin,
the presence of insulin dependent diabetes mellitus, multiple
pregnancy, previous Down syndrome pregnancy, smoking,
and vaginal bleeding.
Serum markers
AFP was the first serum marker used in screening program
for trisomy 21 3,14. It is a fetal-specific protein produced by
the yolk sac and fetal liver. Traditionally, it was performed
between 15 and 21 weeks of gestation to screen for open
neural tube defects. AFP levels are reduced in pregnancies
affected by Down syndrome. Adding maternal serum AFP
to maternal age increased the detection rate of screening to
approximately 30%.
Other serum markers used in the second trimester include human chorionic gonadotrophin (hCG) or its free beta subunit (free β-hCG), both of which are increased in Down pregnancies; unconjugated estriol (uE3) which is decreased; and inhibin A which is increased.
Multiple of Median (MoM)
In screening program, marker levels are described in terms of Multiple of the Median (MoM). This is to allow for the fact that marker levels vary with gestational age. MoM values are calculated by dividing an individual’s marker level by the median level of that marker for the entire population at that gestational age in that laboratory. Using MoM values, rather than absolute levels, also allows results from different laboratories to be interpreted in a consistent way.
Second trimester biochemical screening tests
The performance of different screening tests can be compared
by evaluating their detection rates (DR) and false positive
rates (FPR). The DR is a measure of the proportion of
affected pregnancies which will be picked up by the test.
The FPR is a measure of the number of pregnancies incorrectly identified as highrisk. The higher the FPR the
greater the number of unnecessary invasive tests and
therefore the greater the number of unnecessary miscarriages
of normal fetuses.
The available second trimester screening tests are the Double, Triple and Quadruple Tests. They are compared in Table 3.
First trimester biochemical screening
Pregnancy-associated plasma protein A (PAPP-A) and free
β-hCG are two serum markers used in screening for Down
syndrome in the first trimester 15,16. PAPP-A levels are reduced
in affected pregnancies while free β-hCG levels are raised.
Adding maternal age to PAPP-A and free β-hCG gives a DR
of 60% and a FPR 5%, using a risk cut-off level of 1 in 250
(i.e. any woman with a risk greater than 1 in 250 is defined
as highrisk, and offered invasive testing).
Thirty percent of fetuses with trisomy 21 have a major
structural malformation. Congenital cardiac anomalies are
the commonest (up to 40%) and of these atrioventricular
canal defects and ventricular septal defects are the most
frequent.
Trisomy 21 in the second trimester is also associated with
nasal bone hypoplasia, increased nuchal fold thickness,
duodenal atresia, echogenic bowel, mild hydronephrosis,
shortening of the femur or humerus, sandal gap, and
clinodactyly or midphalanx hypoplasia of the fifth finger.
If the second trimester scan demonstrates major defects, it is advisable to offer fetal karyotyping, even if these defects are apparently isolated. The prevalence of such defects is low and therefore the cost implications are small. If the defects are either lethal or associated with severe handicap, such as hydrops or duodenal atresia, fetal karyotyping constitutes one of a series of investigations to determine the possible cause and thus the risk of recurrence. If the defect is potentially correctable by surgery (either intrauterine or postnatal) such as diaphragmatic hernia, it may be logical to exclude an underlying chromosomal abnormality. This is especially important as in many of these conditions, the associated chromosomal abnormality is trisomy 18 or 13.
Minor fetal defects or soft markers are common and not usually associated with any handicap unless there is an underlying chromosome abnormality 17-20. Routine karyotyping of all pregnancies with these markers, therefore, would have major implications, both in terms of miscarriage and financial cost. In this situation, it is best to base counseling on an individual estimated risk for chromosomal abnormality. The overall risk for chromosomal abnormalities increases with the total number of defects identified. It is therefore recommended that when a defect/marker is detected, a thorough ultrasound examination is done for other features of the chromosomal abnormality known to be associated with that defect, because the presence of additional defects increases the risk substantially.
One promising marker for Down syndrome, which was recently described, is nasal bone hypoplasia. This is defined as a nasal bone that is not visible or has a length of less than the 3rd centile for gestational age in the second trimester. One study examined 1046 pregnancies undergoing amniocentesis for fetal karyotyping at 15-22 weeks gestation 21. The nasal bone was hypoplastic in 62% of fetuses with trisomy 21, but in only 1% of chromosomally normal fetuses. Nasal bone hypoplasia was commoner in normal Afro-Caribbean fetuses (8.8%) than in normal Caucasian fetuses (0.5%). Although much more evidence needs to be gathered, it seems likely that this marker will have a major impact on Down syndrome screening, and should be incorporated into the detailed second trimester anomaly scan.
The estimated risk can be derived by multiplying the a priori maternal age related risk by the likelihood ratio of the specific defect. The best estimates of both the positive and negative likelihood ratios for each of the common markers of trisomy 21 are given in Table 4. On the basis of these data the likelihood ratio for trisomy 21 if there is no detectable defect or marker is 0.30.In each case the likelihood ratio is derived by dividing the incidence of a given marker in trisomy 21 pregnancies by its incidence in chromosomally normal pregnancies. For example, an intracardiac echogenic focus is found in 28.2% of trisomy 21 fetuses and in 4.4% chromosomally normal fetuses, resulting in a positive likelihood ratio of 6.41 (28.2 / 4.4) and a negative likelihood ratio of 0.75 (71.8 / 95.6). Consequently, the finding of an echogenic focus increases the background risk by a factor of 6.41, but at the same time absence of this marker reduces the risk by 25%. It is important to bear in mind that the same logic applies to each one of the six markers in Table 4. Thus, in a 25 year old woman undergoing an ultrasound scan at 20 weeks of gestation, the a priori risk is around 1 in 1000. If the scan demonstrates an intracardiac
First trimester
Nuchal translucency (NT) refers to the fluid-filled space between the fetal skin and the soft tissue overlying the cervical spine. It is measured between 11 and 13 weeks gestation, and the criteria for measurement include:
- crown-rump length between 45 and 84 mm
- midsagittal view
- neutral position
- measure of the maximal lucency
- the ultrasound machine with 0.1mm callipers
- ‘on-to-on’ measure
- fetal neck away from the amnion (it is important to distinguish between the fetal skin and the amnion).
A number of studies have demonstrated that an
increased nuchal translucency is associated with
abnormal karyotype 22,23. One large multicenter study
concluded that maternal age in combination with nuchal
translucency measurement achieved a 77% DR for a 5%
FPR 4
.
Concerns have been expressed that the measurement of nuchal
translucency may be difficult or time-consuming if the fetal
position is incorrect. It is clear that adequate training is
essential to ensure that the measurement is reproducible in
different centres 24.
echogenic focus, but the nuchal fold is not increased, the
humerus and femur are not short and there is no
hydronephrosis, hyperechogenic bowel or major defect,
the combined likelihood ratio should be 1.1 (6.41 x 0.67 x
0.68 x 0.62 x 0.85 x 0.87 x 0.79) and consequently her
risk remains at around 1 in 1,000.
Recent advances include using a combination of NT and biochemical markers. The combination of first trimester free β-hCG, PAPP-A, NT and maternal age is known as the Combined Test, and is measured between 11 and 13 weeks. This has been reported in some studies to have a DR of 80- 89% with a FPR of 5% 25, 26 .
The Integrated Test is the most recent screening test for Down syndrome 27. This combines maternal age with the following:
1. 11-14 weeks: NT + PAPP-A
2. 15-22 weeks: AFP + hCG + uE3 + Inhibin A
The performance of this test is reported to be better than that of all others. The model of screening described by Wald and Hackshaw 25 has the major theoretical advantage of a high DR of 94% for a FPR of 5% or alternatively 85% DR with a 1% FPR. The SURUSS trial 27 found that for a fixed DR of 85% the FPR for the integrated test was 1.2%. However the data available at present is from a single retrospective study and large prospective trials are currently being conducted.
The integrated test requires two stage screening and a proportion of women may fail to attend for the second stage test. In addition, for women who complete the test, the result is not obtained until after 16 weeks. Termination of pregnancy is more traumatic at this stage since it usually requires a medical abortion rather than surgical, and the mother may have already felt the fetal movements. The same argument could of course be applied to the second trimester anomaly scan at 18 to 23 weeks. Although the combined test may have a lower DR than the integrated test, it does yield a result in the first trimester, thus allowing an early surgical (or medical) termination, which may be less traumatic for the mother.
First trimester ultrasound markers other than NT
More recent studies have examined the role of first
trimester ultrasound markers other than NT. They suggest
that absence of the nasal bone, increased impedance to
flow in the ductus venosus (DV) and tricuspid
regurgitation (TR) are highly sensitive and specific firsttrimester markers for trisomy 21. 28-30.
Two stage screening process
In 2005, Nicolaides et al 31 proposed a two-stage screening process in the first trimester.They suggested using the combined test to triage women into high risk (1 in 100 or greater), intermediate risk (between 1 in 101 and 1 in 1000) and low risk (less than 1 in 1000). Intermediate risk women were offered further assessment of risk by first trimester ultrasound examination to determine the presence or absence of the nasal bone, presence or absence of TR and normal/ abnormal doppler velocity waveform in the DV. They concluded that using this approach, more than 90% of trisomy 21 fetuses can potentially be identified in the first trimester, for a FPR rate of 2-3%.
The concept of sequential screening
As discussed earlier, a woman will start with an a priori risk
of Down syndrome, based on her age and gestation
period.This can then be modified to a more patient specific
risk by serum screening – the combined or integrated test.
The risk obtained can then be further modified by a second
trimester scan looking for major fetal malformations (such
as cardiac defects) or soft markers for Down syndrome
(such as short femur or humerus). The information from
this second trimester scan, including major and minor defects
(particularly nasal bone hypoplasia and nuchal edema), will
contribute to identification of further cases.
It is of utmost importance to consider the results of any previous screening during the 18-23 weeks scan, as this will help to accurately calculate the adjusted patient specific risk. There are some exceptions to this process of sequential screening results. It seems obvious that the findings of increased nuchal edema or a cardiac defect at the second trimester scan cannot be considered as risk factors independent from an increased NT in the first trimester. Similarly, hyperechogenic bowel (which may be due to intraamniotic bleeding) and relative shortening of the femur (which may be due to placental insufficiency) may well be related to abnormal serum biochemistry (high hCG and inhibin A, and low uE3 may be markers of placental damage). They cannot, therefore, be considered as independent risk factors while screening for the risk of trisomy 21.
