Prenatal Screening for Common Aneuploidies Before and After the Introduction of Cell-Free DNA-Based Prenatal Testing

Aneuploidies

Aneuploidies, the presence of an abnormal number of chromosomes, are conditions associated with significant morbidity and mortality. Aneuploidies affect approximately 1 in 160 live births , although the overall incidence is higher due to natural pregnancy losses and termination of affected pregnancies. The commonest aneuploidies affecting live births are trisomies 21, 18, and 13 and monosomy X. Trisomies describe an extra copy of a particular chromosome and usually occur due to maternal meiotic nondysjunction. Monosomy X describes a missing copy of the X chromosome in females (45X0, Turner syndrome) which also usually occurs due to maternal meiotic nondysjunction. Sex chromosome aneuploidies such as 45XO and 47XXY (Klinefelter syndrome) are not commonly screened for because in the absence of structural anomalies they would not satisfy the criteria for antenatal screening, and there is insufficient data on the accuracy of cfDNA-based testing for this indication . Sex chromosome aneuploidies will therefore not be discussed further in this chapter, but are extensively addressed in Chapter 15 .

Trisomy 21 (T21), Down's syndrome, is the commonest trisomy compatible with life. It affects 1.08 per 1000 live births in the United Kingdom and is associated with a number of defects which vary between people in their presence and severity. Possible features include cardiac anomalies, duodenal atresia, mild-to-moderate learning difficulties, hypothyroidism, hearing and vision disorders, seizures, increased risks of certain cancers, particularly leukemia, and Alzheimer's disease.

Trisomy 18 (T18), Edwards’ syndrome, affects 1 in 6000 to 1 in 8000 live births . The prevalence at the time of screening, 12 weeks, is 1 in 600 for a 35-year-old woman; between 12 weeks and 40 weeks the fetal death rate is around 80%. Possible features include cardiac defects, renal anomalies, severe learning disabilities, omphalocele, central nervous system defects, breathing and feeding difficulties, and physical deformities such as “rocker-bottom” feet, “clenched” hands, micrognathia, and low-set ears. The majority of live-born babies die in the first few days or weeks of life and less than 10% survive to 1 year of age .

Trisomy 13 (T13), Patau's syndrome, affects approximately 1 in 8000 to 1 in 12,000 live births. The prevalence at the time of screening, 12 weeks, is 1 in 1800 for a 35-year-old woman; again, there is a fetal death rate between 12 and 40 weeks of around 80%. Possible features include cardiac defects, renal anomalies, severe learning disabilities, omphalocele, microcephaly, holoprosencephaly, deafness, seizures, and cleft lip and palate. As with trisomy 18, the vast majority of fetuses die in utero, the majority of live-born babies die in the first few days or weeks of life and less than 10% survive to 1 year of age [5].

Screening by Maternal Age

The rate of autosomal aneuploidies increases significantly with advancing maternal age; the risk of trisomy 21 at 12 weeks of gestation rises from 1 in 1000 for a woman aged 20 years to 1 in 250 for a woman aged 35 years . Similarly, the risk of trisomy 18 increases from 1 in 2500 to 1 in 600 and the risk of trisomy 13 increases from 1 in 8000 to 1 in 1800, over the same time period. Prior to the advent of detailed antenatal ultrasonography and maternal serum biochemistry, maternal age was used as a screening tool for the detection of trisomies. However, maternal age is a poor screening test for trisomies as the majority of babies affected are born to women under the age of 35 years. During the 1970s and 1980s, the estimated detection rate with maternal age as a screening tool was 30% ( Fig. 1 ).

Once identified as “high risk” for trisomies by maternal age, the diagnostic option available to the mother was invasive testing, which less than half of women at the time opted for. Amniocentesis in the 1980s was estimated to carry a 1.0% additional risk of spontaneous pregnancy loss . As the vast majority of pregnancies conceived by women over the age of 35 years are not affected by trisomies, using maternal age alone as a screening tool to offer amniocentesis would therefore lead to a high loss of nonaffected fetuses with a low detection rate, therefore it is no longer recommended.

Screening by Maternal Biochemistry

Measurement of maternal serum biochemistry was initially intended to screen for neural tube defects; however, it was shown that alpha-fetoprotein (AFP) was low in fetuses with trisomy 21, leading to further research in this area. Serum biochemistry was initially suggested as a screening option for women under 35 years of age but, as it was increasingly shown to be more sensitive than maternal age alone, it also became an option for women over 35 years of age . As several maternal biochemical blood markers are affected by trisomic pregnancies, these can be measured and converted to a gestational age-specific multiple of the median (MoM), which is compared to a level at which one would expect an unaffected pregnancy. Markers investigated during the 1980s and 1990s for second trimester screening included AFP, human chorionic gonadotropin (hCG), unconjugated estriol and inhibin A.

Trisomy 21 is typically associated with low levels of AFP and estriol, and high levels of hCG and inhibin A. Adding the results of these biomarkers to maternal age increases the sensitivity of second trimester screening in a stepwise fashion, as follows :

• Double test (AFP and hCG): 55%–60% detection, 5% false positive rate.

• Triple test (AFP, hCG and E3): 60%–65% detection, 5% false positive rate.

• Quadruple test (AFP, hCG, inhibin A, unconjugated estriol): 65%–70% detection, 5% false positive.

Using free instead of total hCG increases the maximum detection rate by about 5% in all the earlier tests.

In the first trimester, hCG and PAPP-A can be used for serum screening. Trisomy 21 is typically associated with high levels of hCG, as in the second trimester, but low levels of PAPP-A. Combining maternal age with hCG and PAPP-A levels in the first trimester gives a detection rate of approximately 65% with a false positive rate of 5% . This detection rate decreases as the first trimester progresses, as PAPP-A is a more powerful marker at earlier gestations.

Screening by Ultrasound

Maternal serum biochemistry alters with gestational age and calculation of the MoM requires accurate pregnancy dating. Ultrasound was initially introduced to date the pregnancy prior to measurement of AFP. In addition, ultrasound scanning equipment, image resolution, and technique were also advancing. In the 1990s, evidence accumulated that increased fluid at the back of the fetal neck (nuchal translucency, NT) ( Fig. 2 ) in the first trimester was associated with fetal trisomies , as well as other chromosomal and structural abnormalities. Screening for trisomy 21 using maternal age and NT together gives a detection rate of 75%, with a false positive rate of 5% . Adding in first trimester maternal serum biochemistry increases the detection rate further, and so the combined test (maternal age, NT, hCG, and PAPP-A) has a detection rate for trisomy 21 of 85%–90% with a 5% false positive rate ( Table 1 ).

Comparisons of first trimester screening by the combined test and second trimester screening by the quadruple test have shown the combined test to be superior. However, performing both sets of screening in a sequential fashion, that is, maternal age, nuchal translucency, and maternal serum biochemistry in both first and second trimesters, may have a higher detection rate (90%–95%) for trisomy 21 . However, sequential screening introduces diagnostic delay and requires a second blood test and has not been widely accepted by women or healthcare professionals.

It is important to note that all the rates discussed previously are for the detection of trisomy 21. Trisomies 18 and 13 are also associated with an increased NT and low PAPP-A in the first trimester but are instead associated with low levels of hCG. The use of the previous combined screening protocol will identify approximately 75% of fetuses with trisomy 18 or 13 when using algorithms for trisomy 21 but over 90% when using trisomy 18/13 algorithms . As in over two-thirds of fetuses with these trisomies, there are associated anatomical defects that can be detected early in pregnancy (e.g. holoprosencephaly, exomphalos), their overall detection rate is likely to be significantly increased with the use of routine first trimester ultrasound scanning.

In the United Kingdom and in many parts of the world, the current trisomy screening recommendations are to use the combined test in the first trimester and, if that results in a low risk (less than 1 in 150 at term), no further invasive testing is performed. For women presenting in the second trimester (between 14 + 2 and 20 + 0 weeks’ gestation), the quadruple test is performed with the same cutoff of 1 in 150. All women are subsequently offered a detailed anomaly scan at 18–20 + 6 weeks of gestation, at which time the detection of fetal anomalies will result in the offer of invasive testing.