*3.2.2.1. Trisomy 13*

Trisomy 13 is a severe disorder, characterized by the presence of different malformations in the brain (holoprosencephaly, agenesis of the corpus callosum, Dandy-Walker malformation, vermian agenesis and neural tube defects), face (cyclopia, hypotelorism, cleft lip and palate), kidneys (renal cystic dysplasia and hydronephrosis) and heart (aterial septal defect, ventricular septal defect and patent ductus arteriosus). Also, cystic hygroma, polydactyly and club or rocker-bottom feet could be identified. All these anomalies can be observed easily by ultrasound examination in the second trimester of pregnancy. Also in the cases of trisomy 13 some sonographic markers can be discovered, but they are nonspecific. Such markers are intrauterine growth restriction (IUGR), mild dilatation of the lateral cerebral ventricles, hyperechoic bowel and echogenic intracardiac foci. The most commonly identified marker is echogenic intracardiac foci. A specific association that can be considered is the one between echogenic intracardiac foci and the hypoplastic left side of the heart [62].

24 weeks of gestation performed by a competent specialist could discover around 50% of congenital anomalies (including subtle ventricular septal defects) in trisomy 21 fetuses [80]. The markers for trisomy 21 in the second trimester of gestation are nuchal thickening, hyperechoic bowel, shortened limbs, pyelectasis, echogenic intracardiac foci, widened pelvic angle, shortened frontal lobes, clinodactyly, pericardial effusion, right–left disproportion of the

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Each marker taken alone has a low sensitivity, but the presence of more than one marker is detected in minimum half of the pregnancies with trisomy 21. The presence of only one marker is discovered in around 22.6% of fetuses with trisomy 21 but also in 11% of normal fetuses. In opposition, identification of more than two markers is present in one-third of pregnancies with trisomy 21, while only 2% of normal pregnancies show such a characteristic. The risk for trisomy 21 is twofold higher if one marker is detected, but the risk becomes 10-fold higher when two markers are present and gets more than 100-fold when three markers are identified. The search of these markers is widely referred to as a "genetic sonogram". The results of a genetic sonogram depend on gestational age, quality of the apparatus and competence of the sonographer and the type of marker, but the minimum sensitivity of this method is around 60% [62]. The big inconvenience of this screening is the high false positive rate associated with the use of multiple sonographic markers that generates anxiety among low-risk patients and imposes an invasive procedure to deny the

To improve the use of sonographic markers in detection of trisomy 21, Bromley et al. proposed a scoring index, and amniocentesis is offered only to those with a score of 2 or higher. A major congenital anomaly, nuchal thickening and an age bigger than 40 years are noted with 2 points each. Hyperechoic bowel, shortened femur, shortened humerus, pyelectasis, echogenic intracardiac foci and age between 35 and 40 years are noted with 1 point. The authors showed that a score ≥ 2 is associated with 75.5% of 21 trisomic pregnancies, while in normal pregnancies such a score is discovered in only 5.7% of cases [84]. An improvement of sonographic screening is the combination of this with biochemical screening, because the sonographic fea-

Redundant skin on the neck is a clinical feature of infants with trisomy 21 and it was first reported by Benacerraf et al. as nuchal thickening during the second trimester [86]. Nuchal thickening is one of the most important markers of trisomy 21 during the second trimester. The sensitivity of this criterion is 20–40%. First, a threshold ≥6 mm after 15 weeks of pregnancy was associated with a high risk of trisomy 21, but now a nuchal thickening of greater than 5 mm up to 20 weeks is considered an adequate value [87]. The normal nuchal thickness varies with gestational age. Thus, the better choice is the use of multiple-of-the-median data, comparing the nuchal measurement with the expected measurement. In these conditions, the calculation of likelihood ratios would permit integration with maternal serum biochemical

heart and small ears [81, 82].

presence of trisomy 21 [83].

*3.2.2.4. Nuchal thickening*

markers for a combined risk [88].

tures are independent of biochemical analytes [85].

### *3.2.2.2. Trisomy 18*

Trisomy 18 is a severe disorder characterized by a lot of congenital anomalies that could be identified by ultrasound examination during the second trimester of pregnancy. These anomalies involve the central nervous system (hydrocephalus, spina bifida, vermian agenesis), lymphatic system (cystic hygroma, nonimmune hydrops), cardiovascular malformations (ventricular and atrial septal defects, patent ductus arteriosus and polyvalvular disease), thorax and abdomen (diaphragmatic hernia, tracheoesophageal fistula, omphalocele), genitourinary system and skeletal system (clenched hands, club feet, radial aplasia, limb shortening). Also, the ultrasound examination shows an intrauterine growth restriction associated with polyhydramnios [75, 76].

Some subtle anomalies discovered by ultrasound examination were considered markers for trisomy 18. Such anomalies are choroid plexus cysts, brachycephaly, "strawberry-shaped" head and single umbilical artery [62]. Choroid plexus cysts are a relatively common variant during the second trimester. It is transient, and if the karyotype is normal it does not have an adverse outcome. The median prevalence of choroid plexus cysts in the general population has been reported as 1–2%. The importance of this marker is limited. Snijders et al. discovered only two cases of chromosomal anomalies in a cohort of 107 fetuses with isolated choroid plexus cysts who had karyotyping. On the other hand, the same authors found no chromosomal anomaly among the 174 children with choroid plexus cysts who did not have amniocentesis [77]. However, Nyberg et al. noted that choroid plexus cysts are observed in 30–40% of fetuses with trisomy 18 before 20 weeks, but usually this marker was associated with other major anomalies specific to trisomy 18 [62]. The presence of isolated choroid plexus cysts is associated with higher likelihood ratios for trisomy 18. Thus, Ghidini et al. [78] found a likelihood ratio of 7.1 and Yoder et al. [79] found a likelihood ratio of 13.8 for trisomy 18 in the presence of isolated choroid plexus cysts. Nyberg et al. concluded that fetal karyotyping should be offered only in cases when all the following conditions are met: maternal age at delivery is over 36 years, the biochemical risk for trisomy 18 is more than 1/3000 and choroid plexus cysts are large. However, the detection of isolated choroid plexus cysts imposes an ultrasound follow-up that can show other abnormalities that were previously missed [62].

#### *3.2.2.3. Trisomy 21*

During the second trimester of pregnancy, congenital anomalies in trisomy 21 fetuses are not frequently discovered. Before 20 weeks, the detection of such anomalies in trisomy 21 is around 16% and this value reflects the low sensitivity of sonography for detection of cardiac defects. The most frequent anomalies are cardiac defects, hydrops, cystic hygroma and duodenal atresia associated with esophageal atresia [62]. However, a sonographic scan at 24 weeks of gestation performed by a competent specialist could discover around 50% of congenital anomalies (including subtle ventricular septal defects) in trisomy 21 fetuses [80].

The markers for trisomy 21 in the second trimester of gestation are nuchal thickening, hyperechoic bowel, shortened limbs, pyelectasis, echogenic intracardiac foci, widened pelvic angle, shortened frontal lobes, clinodactyly, pericardial effusion, right–left disproportion of the heart and small ears [81, 82].

Each marker taken alone has a low sensitivity, but the presence of more than one marker is detected in minimum half of the pregnancies with trisomy 21. The presence of only one marker is discovered in around 22.6% of fetuses with trisomy 21 but also in 11% of normal fetuses. In opposition, identification of more than two markers is present in one-third of pregnancies with trisomy 21, while only 2% of normal pregnancies show such a characteristic. The risk for trisomy 21 is twofold higher if one marker is detected, but the risk becomes 10-fold higher when two markers are present and gets more than 100-fold when three markers are identified. The search of these markers is widely referred to as a "genetic sonogram". The results of a genetic sonogram depend on gestational age, quality of the apparatus and competence of the sonographer and the type of marker, but the minimum sensitivity of this method is around 60% [62]. The big inconvenience of this screening is the high false positive rate associated with the use of multiple sonographic markers that generates anxiety among low-risk patients and imposes an invasive procedure to deny the presence of trisomy 21 [83].

To improve the use of sonographic markers in detection of trisomy 21, Bromley et al. proposed a scoring index, and amniocentesis is offered only to those with a score of 2 or higher. A major congenital anomaly, nuchal thickening and an age bigger than 40 years are noted with 2 points each. Hyperechoic bowel, shortened femur, shortened humerus, pyelectasis, echogenic intracardiac foci and age between 35 and 40 years are noted with 1 point. The authors showed that a score ≥ 2 is associated with 75.5% of 21 trisomic pregnancies, while in normal pregnancies such a score is discovered in only 5.7% of cases [84]. An improvement of sonographic screening is the combination of this with biochemical screening, because the sonographic features are independent of biochemical analytes [85].

#### *3.2.2.4. Nuchal thickening*

are intrauterine growth restriction (IUGR), mild dilatation of the lateral cerebral ventricles, hyperechoic bowel and echogenic intracardiac foci. The most commonly identified marker is echogenic intracardiac foci. A specific association that can be considered is the one between

Trisomy 18 is a severe disorder characterized by a lot of congenital anomalies that could be identified by ultrasound examination during the second trimester of pregnancy. These anomalies involve the central nervous system (hydrocephalus, spina bifida, vermian agenesis), lymphatic system (cystic hygroma, nonimmune hydrops), cardiovascular malformations (ventricular and atrial septal defects, patent ductus arteriosus and polyvalvular disease), thorax and abdomen (diaphragmatic hernia, tracheoesophageal fistula, omphalocele), genitourinary system and skeletal system (clenched hands, club feet, radial aplasia, limb shortening). Also, the ultrasound examination shows an intrauterine growth restriction associated with

Some subtle anomalies discovered by ultrasound examination were considered markers for trisomy 18. Such anomalies are choroid plexus cysts, brachycephaly, "strawberry-shaped" head and single umbilical artery [62]. Choroid plexus cysts are a relatively common variant during the second trimester. It is transient, and if the karyotype is normal it does not have an adverse outcome. The median prevalence of choroid plexus cysts in the general population has been reported as 1–2%. The importance of this marker is limited. Snijders et al. discovered only two cases of chromosomal anomalies in a cohort of 107 fetuses with isolated choroid plexus cysts who had karyotyping. On the other hand, the same authors found no chromosomal anomaly among the 174 children with choroid plexus cysts who did not have amniocentesis [77]. However, Nyberg et al. noted that choroid plexus cysts are observed in 30–40% of fetuses with trisomy 18 before 20 weeks, but usually this marker was associated with other major anomalies specific to trisomy 18 [62]. The presence of isolated choroid plexus cysts is associated with higher likelihood ratios for trisomy 18. Thus, Ghidini et al. [78] found a likelihood ratio of 7.1 and Yoder et al. [79] found a likelihood ratio of 13.8 for trisomy 18 in the presence of isolated choroid plexus cysts. Nyberg et al. concluded that fetal karyotyping should be offered only in cases when all the following conditions are met: maternal age at delivery is over 36 years, the biochemical risk for trisomy 18 is more than 1/3000 and choroid plexus cysts are large. However, the detection of isolated choroid plexus cysts imposes an ultrasound follow-up that can show other abnormalities that were previously missed [62].

During the second trimester of pregnancy, congenital anomalies in trisomy 21 fetuses are not frequently discovered. Before 20 weeks, the detection of such anomalies in trisomy 21 is around 16% and this value reflects the low sensitivity of sonography for detection of cardiac defects. The most frequent anomalies are cardiac defects, hydrops, cystic hygroma and duodenal atresia associated with esophageal atresia [62]. However, a sonographic scan at

echogenic intracardiac foci and the hypoplastic left side of the heart [62].

*3.2.2.2. Trisomy 18*

378 Congenital Anomalies - From the Embryo to the Neonate

polyhydramnios [75, 76].

*3.2.2.3. Trisomy 21*

Redundant skin on the neck is a clinical feature of infants with trisomy 21 and it was first reported by Benacerraf et al. as nuchal thickening during the second trimester [86]. Nuchal thickening is one of the most important markers of trisomy 21 during the second trimester. The sensitivity of this criterion is 20–40%. First, a threshold ≥6 mm after 15 weeks of pregnancy was associated with a high risk of trisomy 21, but now a nuchal thickening of greater than 5 mm up to 20 weeks is considered an adequate value [87]. The normal nuchal thickness varies with gestational age. Thus, the better choice is the use of multiple-of-the-median data, comparing the nuchal measurement with the expected measurement. In these conditions, the calculation of likelihood ratios would permit integration with maternal serum biochemical markers for a combined risk [88].

#### *3.2.2.5. Hyperechoic bowel*

Hyperechoic bowel is detected with increased frequency in cases with aneuploidy (including trisomy 21) but it is nonspecific and could be observed also in about 0.5% of normal fetuses. In addition, this marker was identified also in other disorders like bowel atresia, congenital infection, meconium ileus secondary and cystic fibrosis. The presence of hyperechoic bowel also represents a risk factor for intrauterine growth retardation, fetal death and placentarelated complications [62]. To standardize this feature, a scale with three values was proposed: grade 1—mildly diffuse echogenic, grade 2—moderately focal echogenic and grade 3—very echogenic (like in a bone). The echogenicity of normal bowel increases with transducer frequency, and to minimize the subjectivity, is preferable to take in consideration only the cases with moderate and markedly hyperechoic bowel. In these situations, the risk for fetal aneuploidy is high, but the sensitivity remains acceptable [89].

disappears during the third trimester of pregnancy [93]. The EIF is not an efficient marker for detection of trisomy 21 because it occurs only in 16% of fetuses with this anomaly and the false positive rate is 17%. In contrast, EIF is present in 29% of those with trisomy 13% [94, 95]. The likelihood ratio of EIF in trisomy 21 has been estimated in the range of 1.8–4.2 [62]. Usually, the EIF is detected in the left ventricle, but right-sided or bilateral EIF had an

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The lateral cerebral ventricles normally show a mean diameter of 6.1 ± 1.3 mm. The presence of ventriculomegaly is suspected when diameter reaches 10 mm and such change has been associated with trisomy 21 (and also with other aneuploidies) [97]. The prevalence of this marker in trisomy 21 fetuses varies between 2.8 and 5% [98–100]. On the other hand, van der Hof et al. showed that mild ventriculomegaly is present in 0.15% of euploid fetuses compared with 1.4% of the fetuses with trisomy 21, with a likelihood of aneuploidy 9 times greater in

The prenatal diagnosis is based on the chromosomal or molecular analyses of embryonic or

The methods for obtaining embryonic or fetal cells are chorionic villus sampling, amniocen-

**Chorionic villus sampling** (CVS) is a method that allows prenatal diagnosis in the first trimester of pregnancy, typically at 10–12 weeks of gestation. It is done transabdominal or transcervical, under ultrasound guidance. The samples obtained contain trophoblast cells (derived from fetus) and maternal decidua cells. The last must be removed before analysis. The major advantages of this technique are reduced maternal risk generated by termination of pregnancy and a limited emotional trauma on the patient. The risks of this method are injury of the embryo (especially limb abnormalities in early procedures), miscarriage, bleeding or embryonic infection [102, 103]. The risk of miscarriage after performing chorionic villus sampling was initially estimated at 2–3%. Recent data disproves this risk, showing that the real risk is

**Amniocentesis** is performed from the 16th week of gestation (rarely sooner) and involves the transabdominal extraction of 10–20 ml of amniotic fluid, under ultrasound guidance. The method has a low risk of miscarriage and other incidents. The risk of miscarriage was initially estimated at 1%, but recent data disproves this risk, showing that the real risk is about 0.11%. There is also a risk of amniotic fluid leakage and some rare complications (placental hemorrhage, intra-amniotic infection, abdominal wall hematoma and fetal lesion). A major disadvantage of this method is that it provides the final results of prenatal diagnosis in the second

approximately twofold greater risk of aneuploidy compared with left-sided foci [96].

*3.2.2.9. Mild ventricular dilatation*

**3.3. Prenatal diagnosis methods**

tesis and cordocentesis.

about 0.22% [104].

half of the pregnancy [103–105].

aneuploid fetuses versus normal fetuses [101].

fetal cells obtained using an invasive method.

*3.3.1. Invasive methods to obtain embryo-fetal cells*
