- Protocol for Screening and Diagnosis of Fetal Down Syndrome in the First Trimester
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Down syndrome is the most common chromosomal abnormality in live- births with an overall incidence of about 1 in 650. Approximately 95 percent of Down syndrome cases are due to a meiotic nondisjunction leading to an extra copy of chromosome 21. About 2–3 percent are due to robertsonian translocations and 1–2 percent are due to mosaicism. Down syndrome is associated with a spectrum of physical and mental handicap. It is the most common chromosomal abnormality causing mental retardation requiring long-term care with considerable physical, psychological and financial burden on the parents and the society. In addition, chromosomal abnormalities are responsible for a significant proportion of neonatal deaths. There are various screening strategies that are available currently in order to identify pregnancies that are at increased risk for Down syndrome.
The risk of Down syndrome increases with advancing maternal age (Fig. 1.1):
Previously Affected Pregnancies
With nondisjunction trisomy 21, the recurrence risk at 12 weeks is 0.75 percent above the age-related risk. Approximately 40 percent of pregnancies with Down syndrome fetuses undergo an intrauterine demise at some stage. Therefore, the risk at term is 60 percent that of the risk at 11–14 weeks due to spontaneous losses in the remainder. The recurrence risk following trisomy due to a translocation is dependent on the type of translocation and which partner carries the translocation.
SCREENING VERSUS DIAGNOSIS
Screening is the systematic application of a test or inquiry, to identify individuals at sufficient risk of a specific disorder to benefit from further investigation. It is important to differentiate screening from diagnostic tests. A screening test can only give an estimate of the risk of carrying a baby with Down syndrome. Diagnostic testing would then be offered to confirm whether or not the condition is present. Screening for Down syndrome is justified because of the facts that:
- The condition has a relatively high prevalence (compared to other chromosomal abnormalities)
- Effective screening and diagnostic strategies are available and parents have the option of termination if the diagnosis is confirmed
- The condition has huge financial and social implications both to the parents and the society.
Invasive testing is associated with a risk of miscarriage. Therefore, the challenge of an antenatal screening program is to identify women in whom the risk of carrying a baby with Down syndrome is sufficiently high to justify an invasive test and to minimize the risk of miscarrying a healthy baby. An important feature of a screening test should be low risk of loss of a normal pregnancy per case of Down syndrome detected. It is also important for women to understand that not all affected pregnancies will have a ‘high-risk’ screening result and that a proportion will therefore remain undetected by any screening strategy.
Counseling Prior to Screening
All pregnant women
- Should have accurate and balanced information about Down syndrome and the fact that screening is entirely voluntary.
- Should understand what screen positive and screen negative means and the implications including the fact that a screen-negative result does not rule out the possibility of a pregnancy with the abnormality.
- Should have the information regarding the decisions that could be made with regards to continuing the pregnancy.
- Should be made aware of the presence of the diagnostic tests and the pregnancy loss rates associated with these tests.
Methods of Screening
There are four methods of screening for Down syndrome: Maternal age, nuchal translucency measurement, serum biochemistry (both in the first and second trimester), and second trimester anomaly scan. An ideal screening test should achieve a high detection rate (sensitivity) and a low false-positive rate. The UK National Screening Committee recommends that Down syndrome screening test should have a sensitivity of at least 75 percent at a false-positive rate of 3 percent or less.
The risk of Down Syndrome is directly proportional to advancing maternal age (MA). Even if all women over the age of 35 years accept the diagnostic test, only 30 percent of all cases of Down syndrome can be detected (sensitivity of 30%). Women aged 35 years or over, constitute 5–10 percent of the pregnant population (false-positive rate of 5–10%). So, maternal age is a poor screening test on its own.
The serum markers used for the first trimester screening are:
- Pregnancy associated plasma protein-A (PAPP-A): Produced by placental syncytiotrophoblasts. Levels are reduced in pregnancies affected by Down syndrome.
- Beta human chorionic gonadotrophin (β-hCG): Produced by placental syncytiotrophoblasts. Levels are raised in pregnancies affected by Down syndrome.
Maternal serum β-hCG and PAPP-A are tested from 10–14 weeks gestation. As these markers are affected by factors like gestational age, maternal weight, ethnicity, smoking habits, presence of vaginal bleeding and maternal insulin dependent diabetes mellitus, it is necessary to make adjustments to correct for these characteristics. Hence, each hormone level is converted to a multiple of the median (MoM) specific to pregnancy. This results in correcting for the effect of gestational age, maternal weight, ethnicity, smoking status, method of conception and parity. The sensitivity of a screening policy based on maternal age and the two biochemical markers is 65 percent for a 5 percent false-positive rate.
Fetal nuchal translucency (NT) screening uses ultrasound to measure the size of the nuchal translucency behind the fetal neck (Fig. 1.2). It is normally performed between 11+4 and 13+6 weeks of pregnancy. In a fetus with a given CRL, each nuchal translucency measurement can be converted into a factor which is multiplied by the age-related background risk to estimate a post-test risk. 84 percent of trisomy 21 fetuses would have a nuchal translucency >3 mm at 10–13 weeks' gestation. The risk of Down syndrome is directly proportional to the measurement of Nuchal translucency.
In the absence of appropriate training and continuous auditing, NT screening has the potential disadvantage of poor reproducibility and technical difficulties. Used with maternal age (MA + NT), it has a detection rate (sensitivity) of 77 percent for a false-positive rate of 5 percent.
The other ultrasound markers are:
- Fetal nasal bone: In 65–70 percent of fetuses with Down syndrome, the fetal nasal bone is not visualized on ultrasound scan at 11–14 weeks of gestation. Hence this can be used as an adjunct to the screening strategies in the first trimester in order to improve the detection rate.
- Ductus venosus Doppler: Combined data from various studies show that increased impedance to the flow in ductus venosus (exhibited as reversed ‘a’ wave) was observed in 66 percent of fetuses with trisomy 21 and 3 percent of euploid fetuses at 11–13 weeks (Fig. 1.3). Hence the inclusion of this in the first trimester screening would improve the performance of first trimester screening.
- Tricuspid regurgitation: Tricuspid regurgitation, as determined by pulsed wave ultrasonography is a common finding in 56 percent of fetuses with trisomy 21 and 1 percent of euploid fetuses at 11–14 weeks gestation. Tricuspid regurgitation was diagnosed if it was found during at least half of the systole and with a velocity of over 80 cm/s.
All the above ultrasound markers have the problems of training, technical difficulties and reproducibility. Hence, an alternative approach of reserving these to the subgroup of pregnancies with an intermediate risk (1 in 101-1 in 1000) after combined screening, substantially improves the performance of screening.
PERFORMANCE OF SCREENING TESTS
None of the above tests on its own qualifies to be a good screening method according to the stipulations of the UK National Screening Committee. However, combining them improves the performance considerably (Table 1.1).
The process of contingent screening is to offer all women the first trimester combined screening which consists of nuchal translucency, β-hCG and PAPP-A, and on the basis of the result to stratify them into one of the three risk groups:
- High risk (risk of >1 in 100)- offer diagnostic test
- Low risk (risk of <1 in 1000)- no further testing
- Intermediate risk (risk between 1 in 100 − 1 in 1000) - offer further tests to improve sensitivity. These women are offered further ultrasound to look for other markers like absent nasal bone, ductus venosus Dopplers, tricuspid regurgitation, etc. Should the revised risk after the scan be 1 in 100 or close, they are then offered an invasive test.
With contingent screening about 90 percent of Down syndrome fetuses are identified with a false-positive rate of 2–3 percent.
Currently, the widely used diagnostic procedures are all invasive, with a risk of a miscarriage.
- Chorionic villus sampling (CVS): The most commonly used procedure in the first trimester, a CVS is usually performed after 10 weeks gestation. It can be performed transcervically or transabdominally. Ultrasound guidance is needed to improve accuracy of the procedure. It is associated with a miscarriage rate of 1 percent. It is technically more demanding than amniocentesis, requiring a higher level of training to acquire the appropriate skill. Chorionic villus sampling is sometimes complicated by confined placental mosaicism. Two populations of cells exist, one with normal, and the other with abnormal chromosomes. In confined placental mosaicism, cells containing a chromosomal abnormality are limited to the placenta. An amniocentesis is needed to clarify if the mosaicism is confined to the placenta or is also present in fetal cells (True mosaicism). The rate of confined placental mosaicism with CVS is under 1 percent.
- Amniocentesis: Amniocentesis is a diagnostic procedure of the second trimester, performed after 14 weeks of gestation. Early amniocentesis before 14 weeks has largely been discontinued for prenatal testing due to the increased rate of miscarriage (about 1%), talipes (clubfoot) and respiratory problems.
Following invasive diagnostic procedures, the techniques for chromosomal analysis that are commonly employed are-Quantitative fluorescent polymerase chain reaction (QF-PCR) and conventional karyotyping.
- QF-PCR: This is a laboratory marker analysis that does not require cell culture. Results can be obtained more rapidly, usually within 3–5 working days. A result from a rapid test is nearly 100 percent accurate in confirming whether the fetus does or does not have trisomy 21.
- Conventional karyotype: Chromosomes are only visible in the process of cell division. This procedure involves growing the fetal cells and making a preparation showing the chromosomes. These are then examined for changes in the number and appearance of all the chromosomes. The cells take about 10–15 days to grow in the laboratory. Results from karyotyping therefore take longer to obtain than results from PCR (usually 10–14 days).
The use of QF-PCR analysis for the detection of trisomy 21 is robust and is considered economical and has been validated. The potential disadvantage is that some cases where there is normal fetal karyotype and confined placental mosaicism, PCR could lead to an erroneous diagnosis in a very small proportion of cases (5/1000). Hence consideration should be given for back up culture in cases where the nuchal translucency measurement is more than 4.0 mm, as this is associated with a whole range of aneuploidies.
Around 2 percent of pregnancies affected by Down syndrome are twins. If the twins are dizygotic, the maternal age-related risk of Down syndrome for each baby individually is the same as for a single baby. The risk of at least one affected fetus is thus twice (age-risk for twin 1 + age-risk for twin 2) that of a singleton. If the twins are monozygotic, the risk to both of having Down syndrome is equal to the age-related risk of the mother. In monochorionic (and therefore monozygotic) twins, the average of the two NT measurements can be used to calculate the pregnancy risk. In dichorionic twins, the individual NT measurement of each fetus can be used to calculate the fetus specific risk. Allowing for gestation and chorionicity, twin pregnancies affected with trisomy 21 have higher levels of free β-hCG and lower levels of PAPP-A. Generation of chorionicity-specific medians for the biochemical markers and their use in risk assessment has improved the performance of first-trimester screening for chromosomal abnormalities in twins to a level comparable with that in singleton pregnancies. Even then, the sensitivity of these tests is lower. Invasive procedures in multiple pregnancies are more risky and there are potential problems if one twin is affected with a chromosomal abnormality. Hence referral of such patients to a fetal medicine expert is recommended.
Previous History of Down Syndrome
Traditionally, these women were offered a diagnostic testing without risk-assessment. If the woman has had a previous pregnancy with a trisomy, the normal age-specific risk will not apply. In a small proportion of cases, there will be a parental structural chromosome rearrangement and the recurrence risk can be high, depending on the specific parental genotype. In the remaining vast majority of cases of previous trisomy, the risk is modest. It can be readily be calculated by adding 0.77, 0.54 and 0.42 percent for risks calculated by screening tests in the first, second trimester and at term respectively. Hence, these women need not necessarily have to undergo invasive procedures.
Dealing with abnormal results:
If the diagnostic test results confirm Down syndrome, the diagnosis should be explained as soon as possible to the patient and the partner. Whatever the outcome, the woman must feel reassured that the health professionals caring for her will support her decision to either:
- Continue the pregnancy and use the information received to help prepare for the birth and care of her baby by addressing the anticipated lifestyle changes
- Continue the pregnancy and consider adoption
- Pregnancy termination.
If the woman chooses to have a termination of pregnancy for an abnormal result, she may have the choice of having either a surgical or medically induced termination. If she chooses to continue with the pregnancy, it may also be helpful if she consults support groups and resources.
FUTURE OF THE FIRST TRIMESTER SCREENING
Currently noninvasive diagnosis has gained a lot of attention due to improved safety and earlier detection. But these methods are still in infancy and will need more research to prove their reliability. The potential sources of prenatal diagnosis are:
- Fetal cells in maternal circulation- Fetal erythroblasts and leukocytes have been isolated from maternal circulation but they are difficult to isolate and they may persist for years after a pregnancy.
- Cell free fetal DNA in the maternal circulation- Fetal DNA originates from the trophoblast. It is detectable from 5 weeks of gestation and is cleared within 30 minutes after delivery. At present, it can be used to identify fetal blood group, gender and some specific genes. Prenatal identification of fetal trisomies is more difficult, and has not reached accuracy comparable to testing on fetal tissue obtained using invasive tests.
- Cell-free fetal RNA in the maternal circulation- These can be fetal specific, maternal specific or expressed both in mother and fetus. They are found as early as 5 weeks of gestation and clear within 30 minutes of delivery.
IMPROVING THE SENSITIVITY AND REDUCING THE FALSE- POSITIVE RATES
The sensitivity of modern tests is in the nineties. Appreciable increase in the sensitivity is thus not possible. The main thrust of recent research is on reducing the false-positive rates for the Down syndrome screening. This includes incorporating various other ultrasound markers, offering an individual risk-oriented two stage screening, etc. The potential difficulties are continued training, cost and patient acceptability.
There have been major advances in the screening programs for Down syndrome recently with their own advantages and disadvantages. Regardless the method of screening, it is vital that the women are given the right information in order for them to decide whether or not to take up the screening and should they wish to proceed, they are fully aware of the limitations and the implications.
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- Dhallan R, Guo X, Emche S, et al. A non-invasive test for prenatal diagnosis based on fetal DNA present in maternal blood: A preliminary study. Lancet 2007;369:474–81.
- Kagan KO, Wright D, Baker A, et al. Screening for trisomy 21 by maternal age, fetal nuchal translucency thickness, free beta-human chorionic gonadotropin and pregnancy-associated plasma protein-A. Ultrasound. Obstet Gynecol 2008;31:618–24.
- Nicolaides KH. Screening for chromosomal defects. Ultrasound Obstet Gynecol 2003;21:313–21.
- Nicolaides KH, Spencer K, Avgidou K, et al. Multicenter study of first-trimester screening for trisomy 21 in 75, 821 pregnancies: results and estimation of the potential impact of individual risk-orientated two-stage first-trimester screening. Ultrasound Obstet Gynecol 2005;25:221–6.