Diagnosis of chromosomal abnormalities in fetus is one of the most important challenges in modern perinatology. The most common chromosomal abnormalities in newborns are trisomies 21, 18, 13, monosomy X and other sex chromosome aneuploidies. These aneuploidies can account for up to 95% of live-born chromosomal abnormalities.
Down’s syndrome is still the most common human chromosomal abnomality: the incidence of Down syndrome is estimated at 4.6 per 10,000 births. The risk of having a child with Down’s syndrome increase with advancing maternal age as shown in Table 1 below.
Table 1. Maternal age and risk of Down's syndrome
However, only 30% of Down’s syndrome pregnancies occur in women over 35 and screening based on maternal age alone has only resulted in a 15% reduction in the presence of births of Down’s syndrome babies. Prenatal diagnosis employs a variety of techniques to determine the health and condition of an unborn fetus. Methods of prenatal diagnosis can be divided into non-invasive and invasive techniques.
Non-invasive methods include ultrasound and biochemical screening from maternal blood. Maternal serum screening in the second trimester has now been available for over two decades. More recently, first trimester screening tests offer women the opportunity of early screening for fetal aneuploidy and the option of earlier diagnosis.
Invasive testing is advised for pregnancies that bear a high risk of being affected by a chromosomal aberration from family and individual history.
According to the maternity guidelines in some countries, pregnant women of the age of 35 years and over must be informed about the possibilities of prenatal diagnostics. Pregnant women of this age, who do not favour the performance of an amniocentesis, which is accompanied by a rate of abortion of 0,5 - 1 %, can first have a triple test (or other similar test) performed to obtain a risk estimation. In Italy, a triple test for Down syndrome is recommended to all women at risk and for those aged over 35 years. The triple test and amniocentesis are free for woman over 35 years and:
-parents who had children with chromosomal abnormalities;
-parent carrier of a structural chromosome rearrangement;
.parent with sex chromosome aneuploidy compatible with fertility;
-fetal malformation observed by ultrasound;
In women under 35 years of age the probability for the occurrence of a trisomy 21 is smaller than the risk of losing a healthy child by an amniocentesis, thus this invasive interference and with it the diagnosis are rarely performed.
The prenatal screening (available from the first and second trimester) is the first step towards a prenatal diagnosis of the congenital abnormalities. The goal of the screening is to identify the fetuses at high risk to have a congenital abnormality; after the screening they will be further investigated using invasive methods such as amniocentesis and the biopsy of the chorial villi (invasive method).
The big breakthrough in first trimester screening was the advent of the nuchal translucency (NT) measurement. Between 11 and 14 weeks, a clearly demarcated fluid-filled space can be seen behind the fetal neck. This space is present in all fetuses.
An increased NT measurement is significantly associated with trisomy 21 and other forms of aneuploidy, as shown in Figure 1 below.
NT measurement alone has a detection rate for Down Syndrome (DS) of 70% with a 5% false positive rate. Ultrasonography may also be used for screening in the second trimester, either alone or as an adjunct to maternal serum testing. The use of ultrasound for prenatal diagnosis is appealing for many reasons. Its safety and noninvasive characteristics are certainly two of its most desirable traits. Second trimester ultrasonography may identify fetal anatomic defects, such as congenital heart defect or markers suggestive of fetal aneuploidy like a thickened nuchal fold, absent nasal bone, renal pyelectasis, or echogenic bowel.
The advantages of this non invasive method are the aiming to reduce the number of women undergoing invasive prenatal diagnosis, as well as increase the proportion of Down’s syndrome detection.
Serum screening: combined and integrated test
In the first trimester of pregnancy, screening by a combination of ultrasound markers (the nuchal translucency, NT) and maternal serum β-hCG (human chorionic gonadotropin) and PAPP-A (pregnancy associated plasma protein A) can identify up to 97% of fetuses with trisomy 21 and other major chromosomal abnormalities. This first approach is defined “Combined Test”. Collection of blood for the combined test is performed between 9 and 13 weeks’ gestation. In trisomy 21, during the first trimester of pregnancy, the maternal serum concentration of free β-hCG is increased and PAPP-A is decreased. In trisomies 18 and 13 maternal serum free β-hCG and PAPP-A are decreased.
If a result of the combined test is borderline, the results was confirmed in the second trimester by triple or quadruple test: this complete approach is defined “Integrated Test”.
If a result of the combined test is positive, you may be offered an biopsy of the chorial villi (10-13 gestation week) that can provide more information about your pregnancy, anticipating the choice of abortion.
Second-trimester maternal serum testing includes the triple and quadruple screens. Multiple marker screening is used in the second trimester (15–20 weeks) to screen for trisomies 21 and 18 as well as open neural tube defects.
The triple screen is the measurement of alpha fetoprotein (AFP), human chorionic gonadotropin (hCG), unconjugated estriol (uE3), levels in maternal serum. This combination of markers can detect approximately 60% of cases of fetal Down syndrome with a false positive rate of approximately 4%. The addition of inhibin A testing to the triple screen yielded the quadruple screen. The values of these parameters can be influenced by the presence of maternal diabetes type 1, smoking and pregnancy-related weight gain. In most cases of Down Sindrome, the AFP and uE3 levels are lower, whereas hCG and dimeric inhibin-A levels are higher.
If a result indicates your are at 'high risk', you may be offered an amniocentesis or ultrasound in order to provide more information about your pregnancy (Figure 2).
Figure 2. Screening strategies in the first and second trimester of pregnancy
However, it is worth noting that many factors can influence the result of the tests e.g. if the mother is overweight, or there has been recent vaginal bleeding it can cause abnormal hormone levels.
Biochemichal marker and reference value
The concentrations of the markers vary with gestational age. In the first trimester PAPP-A and NT increase, while free ß-hCG decreases. In the second trimester AFP and uE3 increase, hCG decreases, and inhibin decreases before 17 weeks and increases after 17 weeks. Also, the measurement of serum markers may vary between laboratories. In order to take account of this variation, the concentration of each marker is expressed as a multiple of the median for unaffected pregnancies of the same gestational age (MoM).
The table 2 shows reference values, expressed in MoM. A marker is considered suspicious if its values are greater (>) or lower (<) than these reference values (Table 2).
Table 2. Reference values of the biochemical marker used in screening strategies
The nuchal translucency (NT) is the most important ultrasound marker and the most influential on risk calculation. No pathologies associated with a NT below 1 MoM are known. The thicker the NT is, the worse foetal prognosis is. Pathological values of NT usually range between 1.8 - 2 MoM or a measure greater than 3 mm (regardless of the MoM).
PAPP-A (pregnancy-associated plasma protein A)
Overall, foetuses with chromosomal abnormalities have low PAPP-A values. Values lower than 0.4 MoM increase the risk of chromosomal anomaly.
Free beta hCG or hCG (total human chorionic gonadotropin or its free beta fraction)
Foetuses with chromosomal abnormalities can present altered free beta hCG or hCG values. Overall, in Trisomy 21 these values are higher, those higher than 2.5 MoM indicating a possible pathology. In Trisomies 13 and 18, these values are generally low, with suspicious values being those below 0.4 MoM.
AFP (alpha-feto protein)
High levels of AFP, higher than 2.5 MoM, may indicate foetal malformation (spina bifida, anencephaly, etc.). Low levels of AFP, lower than 0.4 MoM, are observed in certain chromosomopathies. Hence, in trisomies 21 and 18 AFP may present low values whereas in trisomy 13 it can be slightly higher than normal.
uE3 (unconjugated estriol)
Values lower than 0.5 MoM can indicate possible chromosomopathy of chromosomes 21, 13 or 18.
When its values are greater than 2.5 MoM, they may be indicative of trisomy 21 or 13.
The triple test is one of a range of screening tests that are used to identify pregnant women whose fetus is likely to be affected by trisomy 21 (Down syndrome) and who should then be offered a diagnostic test. All of the tests similar to the triple test are based on the same mathematical principle (Bayes theorem) and work by combining a prior probability derived from maternal age at expected date of delivery with a likelihood ratio usually based on two multivariate Gaussian distribution functions.
This combination results in a reasonably accurate risk estimate of the probability that the fetus has Down syndrome. Women whose risk exceeds a specified cutoff are then offered a diagnostic test (ie, amniocentesis or chorionic villus biopsy), which allows a cytogenetic diagnosis to be determined.
The triple test is used only in the second trimester of pregnancy and now has a range of competitors (Table 3).
Table 3. Strategies for antenatal Down's syndrome screening
There are a number of factors that affect the decision about which screening test to use: screening test effectiveness; cost-effectiveness; cost-benefit/cost-hazard.
Prenatal diagnosis of chromosomal abnormalities is currently accomplished by invasive techniques, such as amniocentesis and chorionic villus sampling (CVS).
CVS is performed in the first trimester from 10 through 13 weeks’ gestation, whereas amniocentesis can be performed starting at 15 weeks’ gestation.
Fetal chromosome analysis has been traditionally performed using Giemsa banding (G-banding) of cultured cells in metaphase and is considered the gold standard detection method (Figure 3).
Figure 3. Karyotype of Down's syndrome
This technique is accurate and reliable allowing the detection of a variety of numerical and structural aberrations. The diagnostic accuracy of karyotyping with amniocentesis is 99.4–99.8% and for CVS 97.5–99.6%.
The primary disadvantage of the conventional cytogenetics is that the prenatal tissue must be cultured for several days prior to analysis. It takes 10 days to obtain results and has a culture failure rate of about 1%. Advances in molecular genetics, using either fluorescence in situ hybridization (FISH) or quantitative fluorescence-polymerase chain reaction (QF-PCR), can be applied to give karyotype results within one or two days. Fluorescence in situ hybridization on uncultured amniotic fluid cells using chromosome-specific DNA probes offers the opportunity for rapid screening of aneuploidies and has become an integral part of the current practice in many clinical cytogenetics laboratories.
The triple test as a screening technique for Down syndrome: reliability and relevance. Reynolds.Int J Womens Health. 2010.
Gestione test integrato/combinato/tritest per valutazione rischio sindrome di Down. A.O. OIRM Sant'Anna TORINO, 2010.
Use of a DNA method, QF-PCR, in the prenatal diagnosis of fetal aneuploidies. Langlois S et al. J Obstet Gynaecol Can. 2011
Prenatal screening for fetal aneuploidy. Summers AM et al. J Obstet Gynaecol Can. 2007
Diagnosis and management of fetal nuchal translucency. Jackson et al.1998.